ORCID Profile
0000-0002-4750-1991
Current Organisations
Heart Research Institute Ltd
,
National Health and Medical Research Council
,
Monash University
,
Scripps Research Institute
,
Royal Prince Alfred Hospital
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biochemistry and Cell Biology | Biomechanical Engineering | Cellular Interactions (Incl. Adhesion, Matrix, Cell Wall) | Fluidisation and Fluid Mechanics | Biomedical Instrumentation | Optical Properties of Materials | Biomedical Engineering | Fluidization And Fluid Mechanics | Synchrotrons; Accelerators; Instruments and Techniques | Signal Transduction | Cellular Interactions (incl. Adhesion, Matrix, Cell Wall) | Cardiorespiratory Medicine and Haematology | Other Physical Sciences | Protein Targeting And Signal Transduction | Membrane Biology | Haematology | Cell Development, Proliferation and Death
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Blood disorders | Cardiovascular system and diseases | Expanding Knowledge in Technology | Cardiovascular System and Diseases | Expanding Knowledge in the Physical Sciences | Expanding Knowledge in Engineering | Scientific Instruments |
Related Links
Publications
Megakaryocyte buds are distinct from microvesicles and likely to represent platelet precursors
Publisher: American Society of Hematology
Date: 17-03-2023
Valproic acid selectively increases vascular endothelial tissue-type plasminogen activator production and reduces thrombus formation in the mouse
Publisher: Elsevier BV
Date: 12-2016
DOI: 10.1111/JTH.13527
Abstract: Essentials Stimulating endogenous fibrinolysis could be a novel antithrombotic strategy. The effect of valproic acid on endothelial tissue plasminogen activator in mice was investigated. Valproic acid increased tissue plasminogen activator expression in vascular endothelium. Valproic acid reduced fibrin deposition and thrombus formation after vascular injury. Background The endogenous fibrinolytic system has rarely been considered as a target to prevent thrombotic disease. Tissue-type plasminogen activator (t-PA) production is potently increased by histone deacetylase (HDAC) inhibitors in endothelial cells in vitro, but whether this translates into increased vascular t-PA production and an enhanced fibrinolytic capacity in vivo is unknown. Objectives To determine whether the HDAC inhibitor valproic acid (VPA) stimulates production of t-PA in the vasculature of mice, and whether VPA pretreatment affects fibrin deposition and clot formation after mechanical vessel injury. Methods Mice were injected with VPA twice daily for up to 5 days. t-PA mRNA, and antigen expression in the mouse aorta and the circulating levels of t-PA were determined. Fibrin and thrombus dynamics after mechanical vessel injury were monitored with intravital confocal microscopy. Potential effects of VPA on platelets and coagulation were investigated. Results and Conclusions We found that VPA treatment increased vascular t-PA production in vivo and, importantly, that VPA administration was associated with reduced fibrin accumulation and smaller thrombi in response to vascular injury, but still was not associated with an increased risk of bleeding. Furthermore, we observed that higher concentrations of VPA were required to stimulate t-PA production in the brain than in the vasculature. Thus, this study shows that VPA can be dosed to selectively manipulate the fibrinolytic system in the vascular compartment and reduce thrombus formation in vivo.
Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRγ deficiency
Publisher: American Society of Hematology
Date: 06-2006
DOI: 10.1182/BLOOD-2005-10-4244
Abstract: Fibrillar collagens are among the most potent activators of platelets and play an important role in the initiation of thrombosis. The glycoprotein VI (GPVI)/FcRγ-chain complex is a central collagen receptor and inhibitors of GPVI produce a major defect in arterial thrombogenesis. In this study we have examined arterial thrombus formation in mice lacking the GPVI/FcRγ-chain complex (FcRγ–/–). Using 3 distinct arterial thrombosis models involving deep vascular injury, we demonstrate that deficiency of GPVI/FcRγ is not associated with a major defect in arterial thrombus formation. In contrast, with milder vascular injury deficiency of GPVI/FcRγ was associated with a 30% reduction in thrombus growth. Analysis of FcRγ–/– platelets in vitro, using thrombin-dependent and -independent thrombosis models, demonstrated a major role for thrombin in overcoming the thrombosis defect associated with GPVI/FcRγ deficiency. Inhibition of thrombin in vivo produced a much greater defect in thrombus formation in mice lacking GPVI/FcRγ compared with normal controls. Similarly, thrombin inhibition produced a marked prolongation in bleeding time in FcRγ–/– mice relative to wild-type mice. Our studies define an important role for thrombin in overcoming the hemostatic and thrombotic defect associated with GPVI/FcRγ deficiency. Moreover, they raise the interesting possibility that the full antithrombotic potential of GPVI receptor antagonists may only be realized through the concurrent administration of anticoagulant agents.
Procoagulant platelets: are they necrotic?
Publisher: American Society of Hematology
Date: 23-09-2010
DOI: 10.1182/BLOOD-2010-01-261669
Abstract: Apoptosis and necrosis represent distinct cell death processes that regulate mammalian development, physiology and disease. Apoptosis characteristically leads to the silent destruction and removal of cells in the absence of an inflammatory response. In contrast, necrotic cell death can induce physiologic inflammatory responses linked to tissue defense and repair. Although anucleate, platelets undergo programmed cell death, with apoptosis playing an important role in clearing effete platelets from the circulation. While it has long been recognized that procoagulant platelets exhibit characteristic features of dying cells, recent studies have demonstrated that platelet procoagulant function can occur independent of apoptosis. A growing body of evidence suggest that the biochemical, morphologic and functional changes underlying agonist-induced platelet procoagulant function are broadly consistent with cell necrosis, raising the possibility that distinct death pathways regulate platelet function and survival. In this article, we will discuss the mechanisms underlying apoptotic and necrotic cell death pathways and examine the evidence linking these pathways to the platelet procoagulant response. We will also discuss the potential contribution of these pathways to the platelet storage lesion and propose a simplified nomenclature to describe procoagulant platelets.
Platelet Thrombus Formation in Flowing Blood
Publisher: Elsevier
Date: 2013
Endoplasmic reticulum protein 5 attenuates platelet endoplasmic reticulum stress and secretion in a mouse model
Publisher: American Society of Hematology
Date: 26-04-2023
DOI: 10.1182/BLOODADVANCES.2022008457
Abstract: Extracellular protein disulfide isomerases (PDIs), including PDI, endoplasmic reticulum protein 57 (ERp57), ERp72, ERp46, and ERp5, are required for in vivo thrombus formation in mice. Platelets secrete PDIs upon activation, which regulate platelet aggregation. However, platelets secrete only ∼10% of their PDI content extracellularly. The intracellular role of PDIs in platelet function is unknown. Here, we aim to characterize the role of ERp5 (gene Pdia6) using platelet conditional knockout mice, platelet factor 4 (Pf4) Cre+/ERp5floxed (fl)/fl. Pf4Cre+/ERp5fl/fl mice developed mild macrothrombocytopenia. Platelets deficient in ERp5 showed marked dysregulation of their ER, indicated by a twofold upregulation of ER proteins, including PDI, ERp57, ERp72, ERp46, 78 kilodalton glucose-regulated protein (GRP78), and calreticulin. ERp5-deficient platelets showed an enhanced ER stress response to ex vivo and in vivo ER stress inducers, with enhanced phosphorylation of eukaryotic translation initiation factor 2A and inositol-requiring enzyme 1 (IRE1). ERp5 deficiency was associated with increased secretion of PDIs, an enhanced response to thromboxane A2 receptor activation, and increased thrombus formation in vivo. Our results support that ERp5 acts as a negative regulator of ER stress responses in platelets and highlight the importance of a disulfide isomerase in platelet ER homeostasis. The results also indicate a previously unanticipated role of platelet ER stress in platelet secretion and thrombosis. This may have important implications for the therapeutic applications of ER stress inhibitors in thrombosis.
Corrigendum: 14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function
Publisher: Springer Science and Business Media LLC
Date: 30-08-2017
DOI: 10.1038/NCOMMS16125
Abstract: Nature Communications 7: Article number: 12862 (2016) Published: 27 September 2016 Updated: 30 August 2017 In Supplementary Fig. 7 of this Article, graphs presenting 6-PFK kinetics in panel d were inadvertently duplicated from those in panel c. The correct version of Supplementary Fig. 7 appears below as Fig.
A functional 14-3-3ζ–independent association of PI3-kinase with glycoprotein Ibα, the major ligand-binding subunit of the platelet glycoprotein Ib-IX-V complex
Publisher: American Society of Hematology
Date: 05-2008
DOI: 10.1182/BLOOD-2007-09-111096
Abstract: Engagement of the adhesion receptor glycoprotein (GP) Ib-IX-V by von Willebrand factor (VWF) mediates platelet adhesion to damaged vessels and triggers platelet activation and thrombus formation in heart attack and stroke. GPIb-IX-V contains distinct 14-3-3ζ–binding sites at the GPIbα C-terminus involving phosphorylation of Ser609, an upstream site involving phosphorylated Ser587/Ser590, and a protein kinase A (PKA)–dependent site on GPIbβ involving Ser166. 14-3-3ζ regulates the VWF-binding affinity of GPIb-IX-V and inhibiting 14-3-3ζ association blocks receptor signaling, suggesting a key functional role for 14-3-3ζ. We used deletion mutants of GPIbα expressed in Chinese hamster ovary (CHO) cells to define the relationship of 14-3-3ζ binding to another GPIb-IX-V–associated signaling protein, phosphoinositide 3-kinase (PI3-kinase). Pull-down experiments involving glutathione S-transferase (GST)–PI3-kinase 85-subunit and GST–14-3-3ζ indicated that both proteins interacted with contiguous GPIbα sequences 580 to 590/591 to 610. Deleting these, but not upstream sequences of GPIbα expressed in CHO cells, inhibited VWF/ristocetin-dependent Akt phosphorylation, relative to wild-type receptor, confirming this region encompassed a functional PI3-kinase–binding site. Pull-down experiments with GST-p85 truncates indicated the GPIbα-binding region involved the p85 breakpoint cluster region (BCR) domain, containing RSXSXP. However, pull-down of GPIb-IX was unaltered by mutation/deletion hosphorylation of this potential 14-3-3ζ–binding sequence in mutant constructs of GST-p85, suggesting PI3-kinase bound GPIbα independently of 14-3-3ζ 14-3-3ζ inhibitor peptide R18 also blocked pull-down of receptor by GST-14-3-3ζ but not GST-p85, and GST-p85 pull-downs were unaffected by excess 14-3-3ζ. Together, these data suggest the GPIbα C-terminus regulates signaling through independent association of 14-3-3ζ and PI3-kinase.
Bidirectional integrin αIIbβ3 signalling regulating platelet adhesion under flow: Contribution of protein kinase C
Publisher: Portland Press Ltd.
Date: 15-05-2003
DOI: 10.1042/BJ20020868
Abstract: Platelet adhesion on von Willebrand factor (vWf) requires the co-ordinated adhesive function of glycoprotein Ib/V/IX and integrin αIIbβ3. Recent evidence [Nesbitt, Kulkarni, Giuliano, Gonclaves, Dopheide, Yap, Harper, Salem and Jackson (2002) J. Biol. Chem. 277, 2965–2972] suggests that outside-in signals from both receptors play important roles in regulating platelet-adhesion dynamics under flow. In the present study, we have examined the mechanisms utilized by protein kinase C (PKC) to promote irreversible platelet adhesion on vWf. We demonstrate that PKC is primarily activated downstream of integrin αIIbβ3, not glycoprotein Ib, during platelet adhesion on vWf. This integrin αIIbβ3-dependent PKC activation establishes a positive-feedback loop that promotes further integrin αIIbβ3 activation, calcium mobilization and firm platelet adhesion. This feedback loop appears to be most relevant at relatively low cytosolic calcium concentrations (mean Δ[Ca2+]i~100 nM) as artificially elevating calcium (mean Δ[Ca2+]i & 500 nM) induces integrin αIIbβ3 activation and irreversible platelet adhesion independent of PKC. Our studies demonstrate the existence of a complex signalling relationship operating between PKC, cytosolic calcium and integrin αIIbβ3 that serves to regulate platelet-adhesion dynamics under flow. Furthermore, we have established the existence of PKC-dependent and -independent pathways regulating integrin αIIbβ3 activation and stable platelet adhesion on vWf.
Shear-dependent tether formation during platelet translocation on von Willebrand factor
Publisher: American Society of Hematology
Date: 2002
Abstract: The adhesion and aggregation of platelets at sites of vascular injury is dependent on the initial binding of the GP Ib/V/IX receptor complex to immobilized von Willebrand factor (VWF). Under flow conditions, this interaction supports platelet translocation that is characteristically stop-start in nature. High resolution imaging of platelets during surface translocation on immobilized VWF revealed that thin membrane tethers (length: 0.91 μm-47.90 μm) were pulled from the surface of these cells. Membrane tethers were dynamic structures that extended from small, localized adhesion contacts under the influence of flow. Perfusion of platelets in the presence of blocking antibodies against integrin αIIbβ3, or over isolated A1 domains, demonstrated that the VWF–GP Ib interaction was sufficient to induce membrane tether formation. The rate and extent of tether elongation was shear-dependent (shear range: 150 s−1-10 000 s−1), with mean tether length ranging from 3.23 μm to 16.55 μm, tether frequency from 2.67% to 97.33%, and tether growth rate from 0.04 μm/sec to 8.39 μm/sec. Tether formation and retraction did not require platelet activation however, the growth rate, lifetime, and dimensions were significantly affected by the actin polymerization inhibitor, cytochalasin D, and by chelating intracellular calcium. Single-cell analysis revealed that formation of membrane tethers regulates the stop-start phases of platelet translocation on VWF, suggesting a potentially important role for this phenomenon in regulating the dynamics of the platelet-VWF interaction under flow.
Intercellular calcium communication regulates platelet aggregation and thrombus growth
Publisher: Rockefeller University Press
Date: 31-03-2003
Abstract: The ability of platelets to form stable adhesion contacts with other activated platelets (platelet cohesion or aggregation) at sites of vascular injury is essential for hemostasis and thrombosis. In this study, we have examined the mechanisms regulating cytosolic calcium flux during the development of platelet–platelet adhesion contacts under the influence of flow. An examination of platelet calcium flux during platelet aggregate formation in vitro demonstrated a key role for intercellular calcium communication (ICC) in regulating the recruitment of translocating platelets into developing aggregates. We demonstrate that ICC is primarily mediated by a signaling mechanism operating between integrin αIIbβ3 and the recently cloned ADP purinergic receptor P2Y12. Furthermore, we demonstrate that the efficiency by which calcium signals are propagated within platelet aggregates plays an important role in dictating the rate and extent of thrombus growth.
Identification of a Unique Co-operative Phosphoinositide 3-Kinase Signaling Mechanism Regulating Integrin αIIbβ3 Adhesive Function in Platelets
Publisher: Elsevier BV
Date: 09-2007
Apolipoprotein A-IV binds αIIbβ3 integrin and inhibits thrombosis
Publisher: Springer Science and Business Media LLC
Date: 06-09-2018
DOI: 10.1038/S41467-018-05806-0
Abstract: Platelet αIIbβ3 integrin and its ligands are essential for thrombosis and hemostasis, and play key roles in myocardial infarction and stroke. Here we show that apolipoprotein A-IV (apoA-IV) can be isolated from human blood plasma using platelet β3 integrin-coated beads. Binding of apoA-IV to platelets requires activation of αIIbβ3 integrin, and the direct apoA-IV-αIIbβ3 interaction can be detected using a single-molecule Biomembrane Force Probe. We identify that aspartic acids 5 and 13 at the N-terminus of apoA-IV are required for binding to αIIbβ3 integrin, which is additionally modulated by apoA-IV C-terminus via intra-molecular interactions. ApoA-IV inhibits platelet aggregation and postprandial platelet hyperactivity. Human apoA-IV plasma levels show a circadian rhythm that negatively correlates with platelet aggregation and cardiovascular events. Thus, we identify apoA-IV as a novel ligand of αIIbβ3 integrin and an endogenous inhibitor of thrombosis, establishing a link between lipoprotein metabolism and cardiovascular diseases.
An insight into platelet function and activation
Publisher: Elsevier BV
Date: 09-1997
An Investigation on Platelet Transport during Thrombus Formation at Micro-Scale Stenosis
Publisher: Public Library of Science (PLoS)
Date: 23-10-2013
Focal adhesion kinase (pp125FAK) cleavage and regulation by calpain
Publisher: Portland Press Ltd.
Date: 15-08-1996
DOI: 10.1042/BJ3180041
Abstract: Focal adhesion kinase (125 kDa form pp125FAK) is a widely expressed non-receptor tyrosine kinase that is implicated in integrin-mediated signal transduction. We have identified a novel means of pp125FAK regulation in human platelets, in which this kinase undergoes sequential proteolytic modification from the native 125 kDa form to 90, 45 and 40 kDa fragments in thrombin-, collagen- and ionophore A23187-stimulated platelets. The proteolysis of pp125FAK was prevented by pretreating platelets with the calpain inhibitors calpeptin or calpain inhibitor-1, and was reproduced in vitro by incubating immunoprecipitated pp125FAK with purified calpain. Proteolysis of pp125FAK resulted in a dramatic reduction in its autokinase activity and led to its dissociation from the cytoskeletal fraction of platelets. These studies define a novel signal-terminating role for calpain, wherein proteolytic modification of pp125FAK attenuates its autokinase activity and induces its subcellular relocation within the cell.
SHIP1 and Lyn Kinase Negatively Regulate Integrin αIIbβ3 Signaling in Platelets
Publisher: Elsevier BV
Date: 07-2004
The catalytic class IA PI3K isoforms play divergent roles in breast cancer cell migration
Publisher: Elsevier BV
Date: 03-2011
DOI: 10.1016/J.CELLSIG.2010.10.021
Abstract: Transforming growth factor-β (TGFβ) plays an important role in breast cancer metastasis. Here phosphoinositide 3-kinase (PI3K) signalling was found to play an essential role in the enhanced migration capability of fibroblastoid cells (FibRas) derived from normal mammary epithelial cells (EpH4) by transduction of oncogenic Ras (EpRas) and TGFβ1. While expression of the PI3K isoform p110δ was down-regulated in FibRas cells, there was an increase in the expression of p110α and p110β in the fibroblastoid cells. The PI3K isoform p110β was found to specifically contribute to cell migration in FibRas cells, while p110α contributed to the response in EpH4, EpRas and FibRas cells. Akt, a downstream targets of PI3K signalling, had an inhibitory role in the migration of transformed breast cancer cells, while Rac, Cdc42 and the ribosomal protein S6 kinase (S6K) were necessary for the response. Together our data reveal a novel specific function of the PI3K isoform p110β in the migration of cells transformed by oncogenic H-Ras and TGF-β1.
Integrin αIIbβ3-dependent Calcium Signals Regulate Platelet-Fibrinogen Interactions under Flow
Publisher: Elsevier BV
Date: 09-2003
Reconstituted High-Density Lipoprotein Attenuates Platelet Function in Individuals With Type 2 Diabetes Mellitus by Promoting Cholesterol Efflux
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 24-11-2009
DOI: 10.1161/CIRCULATIONAHA.109.870709
Abstract: Background— In iduals with diabetes mellitus have an increased risk of cardiovascular disease and exhibit platelet hyperreactivity, increasing their resistance to antithrombotic therapies such as aspirin and clopidogrel. Reconstituted high-density lipoprotein (rHDL) has short-term beneficial effects on atherosclerotic plaques, but whether it can effectively reduce the reactivity of diabetic platelets is not known. Methods and Results— In iduals with type 2 diabetes mellitus were infused with placebo or rHDL (CSL-111 20 mg · kg −1 · h −1 ) for 4 hours, resulting in an ≈1.4-fold increase in plasma HDL cholesterol levels. rHDL infusion was associated with a % reduction in the ex vivo platelet aggregation response to multiple agonists, an effect that persisted in washed platelets. In vitro studies in platelets from healthy in iduals revealed that the inhibitory effects of rHDL on platelet function were time and dose dependent and resulted in a widespread attenuation of platelet function and a 50% reduction in thrombus formation under flow. These effects could be recapitulated, in part, by the isolated phospholipid component of rHDL, which enhanced efflux of cholesterol from platelets and reduced lipid raft assembly. In contrast, the apolipoprotein AI component of rHDL had minimal effect on platelet function, cholesterol efflux, or lipid raft assembly. Conclusion— These findings suggest that rHDL therapy is highly effective at inhibiting the heightened reactivity of diabetic platelets, partly through reducing the cholesterol content of platelet membranes. These properties, combined with the known short-term beneficial effects of rHDL on atherosclerotic lesions, suggest that rHDL infusions may be an effective approach to reduce atherothrombotic complications in diabetic in iduals. Clinical Trial Registration Information— URL: www.clinicaltrials.gov. Unique identifier: NCT00395148.
The growing complexity of platelet aggregation
Publisher: American Society of Hematology
Date: 15-06-2007
DOI: 10.1182/BLOOD-2006-12-027698
Abstract: Platelet aggregation, the process by which platelets adhere to each other at sites of vascular injury, has long been recognized as critical for hemostatic plug formation and thrombosis. Until relatively recently, platelet aggregation was considered a straightforward process involving the noncovalent bridging of integrin αIIbβ3 receptors on the platelet surface by the dimeric adhesive protein fibrinogen. However, with recent technical advances enabling real-time analysis of platelet aggregation in vivo, it has become apparent that this process is much more complex and dynamic than previously anticipated. Over the last decade, it has become clear that platelet aggregation represents a multistep adhesion process involving distinct receptors and adhesive ligands, with the contribution of in idual receptor-ligand interactions to the aggregation process dependent on the prevailing blood flow conditions. It now appears that at least 3 distinct mechanisms can initiate platelet aggregation, with each of these mechanisms operating over a specific shear range in vivo. The identification of shear-dependent mechanisms of platelet aggregation has raised the possibility that vascular-bed–specific inhibitors of platelet aggregation may be developed in the future that are safer and more effective than existing antiplatelet agents.
Continuous signaling via PI3K isoforms β and γ is required for platelet ADP receptor function in dynamic thrombus stabilization
Publisher: American Society of Hematology
Date: 11-2006
DOI: 10.1182/BLOOD-2006-03-006338
Abstract: Signaling from collagen and G protein–coupled receptors leads to platelet adhesion and subsequent thrombus formation. Paracrine agonists such as ADP, thromboxane, and Gas6 are required for platelet aggregate formation. We hypothesized that thrombi are intrinsically unstable structures and that their stabilization requires persistent paracrine activity and continuous signaling, maintaining integrin αIIbβ3 activation. Here, we studied the disassembly of human and murine thrombi formed on collagen under high shear conditions. Platelet aggregates rapidly disintegrated (1) in the absence of fibrinogen-containing plasma (2) by blocking or inhibiting αIIbβ3 (3) by blocking P2Y12 receptors (4) by suppression of phosphoinositide 3-kinase (PI3K) β. In murine blood, absence of PI3Kγ led to formation of unstable thrombi, leading to dissociation of multiplatelet aggregates. In addition, blocking PI3Kβ delayed initial thrombus formation and reduced in idual platelet-platelet contact. Similarly without flow, agonist-induced aggregation was reversed by late suppression of P2Y12 or PI3K isoforms, resulting in single platelets that had inactivated αIIbβ3 and no longer bound fibrinogen. Together, the data indicate that continuous outside-in signaling via P2Y12 and both PI3Kβ and PI3Kγ isoforms is required for perpetuated αIIbβ3 activation and maintenance of a platelet aggregate. This novel concept of intrinsic, dynamic thrombus instability gives possibilities for the use of antiplatelet therapy.
Differentiating haemostasis from thrombosis for therapeutic benefit
Publisher: Georg Thieme Verlag KG
Date: 2013
DOI: 10.1160/TH13-05-0379
Abstract: The central role of platelets in the formation of the primary haemostatic plug as well as in the development of arterial thrombosis is well defined. In general, the molecular events underpinning these processes are broadly similar. Whilst it has long been known that disturbances in blood flow, changes in platelet reactivity and enhanced coagulation reactions facilitate pathological thrombus formation, the precise details underlying these events remain incompletely understood. Intravital microscopy studies have highlighted the dynamic and heterogeneous nature of thrombus development and demonstrated that there are considerable spatiotemporal differences in the activation states of platelets within a forming thrombus. In this review we will consider the factors regulating the activation state of platelets in a developing thrombus and discuss how specific prothrombotic factors may influence this process, leading to excessive thrombus propagation. We will also discuss some potentially novel therapeutic approaches that may reduce excess thrombus development whilst minimising bleeding risk.
Boosting platelet production
Publisher: Springer Science and Business Media LLC
Date: 09-2008
DOI: 10.1038/NM0908-917
Dual Biomembrane Force Probe enables single-cell mechanical analysis of signal crosstalk between multiple molecular species
Publisher: Springer Science and Business Media LLC
Date: 27-10-2017
DOI: 10.1038/S41598-017-13793-3
Abstract: Conventional approaches for studying receptor-mediated cell signaling, such as the western blot and flow cytometry, are limited in three aspects: 1) The perturbing preparation procedures often alter the molecules from their native state on the cell 2) Long processing time before the final readout makes it difficult to capture transient signaling events ( min) 3) The experimental environments are force-free, therefore unable to visualize mechanical signals in real time. In contrast to these methods in biochemistry and cell biology that are usually population-averaged and non-real-time, here we introduce a novel single-cell based nanotool termed dual biomembrane force probe (dBFP). The dBFP provides precise controls and quantitative readouts in both mechanical and chemical terms, which is particularly suited for juxtacrine signaling and mechanosensing studies. Specifically, the dBFP allows us to analyze dual receptor crosstalk by quantifying the spatiotemporal requirements and functional consequences of the up- and down-stream signaling events. In this work, the utility and power of the dBFP has been demonstrated in four important dual receptor systems that play key roles in immunological synapse formation, shear-dependent thrombus formation, and agonist-driven blood clotting.
Multi-scale spatial ecology analyses: a Kullback information approach
Publisher: Springer Science and Business Media LLC
Date: 08-09-2023
Synergistic adhesive interactions and signaling mechanisms operating between platelet glycoprotein Ib/IX and integrin αIIb β3 : Studies in human platelets and transfected Chinese hamster ovary cells
Publisher: Elsevier BV
Date: 12-2000
Thromboinflammation: challenges of therapeutically targeting coagulation and other host defense mechanisms
Publisher: American Society of Hematology
Date: 28-02-2019
DOI: 10.1182/BLOOD-2018-11-882993
Abstract: Thrombosis with associated inflammation (thromboinflammation) occurs commonly in a broad range of human disorders. It is well recognized clinically in the context of superficial thrombophlebitis (thrombosis and inflammation of superficial veins) however, it is more dangerous when it develops in the microvasculature of injured tissues and organs. Microvascular thrombosis with associated inflammation is well recognized in the context of sepsis and ischemia-reperfusion injury however, it also occurs in organ transplant rejection, major trauma, severe burns, the antiphospholipid syndrome, preecl sia, sickle cell disease, and biomaterial-induced thromboinflammation. Central to thromboinflammation is the loss of the normal antithrombotic and anti-inflammatory functions of endothelial cells, leading to dysregulation of coagulation, complement, platelet activation, and leukocyte recruitment in the microvasculature. α-Thrombin plays a critical role in coordinating thrombotic and inflammatory responses and has long been considered an attractive therapeutic target to reduce thromboinflammatory complications. This review focuses on the role of basic aspects of coagulation and α-thrombin in promoting thromboinflammatory responses and discusses insights gained from clinical trials on the effects of various inhibitors of coagulation on thromboinflammatory disorders. Studies in sepsis patients have been particularly informative because, despite using anticoagulant approaches with different pharmacological profiles, which act at distinct points in the coagulation cascade, bleeding complications continue to undermine clinical benefit. Future advances may require the development of therapeutics with primary anti-inflammatory and cytoprotective properties, which have less impact on hemostasis. This may be possible with the growing recognition that components of blood coagulation and platelets have prothrombotic and proinflammatory functions independent of their hemostatic effects.
Dok-2 adaptor protein regulates the shear-dependent adhesive function of platelet integrinαIIb β3 in mice
Publisher: Elsevier BV
Date: 05-0002
Evidence of Platelet Activation at Medically Used Hypothermia and Mechanistic Data Indicating ADP as a Key Mediator and Therapeutic Target
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-2011
DOI: 10.1161/ATVBAHA.111.226373
Abstract: Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia. Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28°C/18°C). Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl 3 -injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P 2 Y 12 antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively. The platelet agonist ADP plays a key role in hypothermia-induced platelet activation. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of novel antithrombotic strategies in clinically applied hypothermia.
Identification of a novel 14-3-3ζ binding site within the cytoplasmic tail of platelet glycoprotein Ibα
Publisher: American Society of Hematology
Date: 15-07-2004
DOI: 10.1182/BLOOD-2003-08-2881
Abstract: The glycoprotein Ib-V-IX (GPIb-V-IX) complex interacts with subendothelial von Willebrand factor (VWF) to ensure recruitment of platelets at sites of vascular injury, a process that culminates in integrin αIIbβ3-dependent stable adhesion and spreading. Interaction of the 14-3-3ζ adaptor protein with the C-terminal 606-610 phosphoserine motif of the GPIbα subunit has been implicated in the control of αIIbβ3 activation and cell spreading. In this study, we have examined potentially novel 14-3-3ζ binding sites by expressing mutant forms of GPIbα in Chinese-hamster-ovary (CHO) cells. Analysis of a series of neighboring 11-12 residue deletions identified a critical role for the 580-LVAGRRPSALS-590 sequence in promoting GPIbα-14-3-3ζ interaction. Development of a phosphospecific antibody demonstrated high levels of phosphorylation of the Ser587 and Ser590 residues in resting platelets (which became dephosphorylated during platelet spreading on VWF), and peptides containing these phosphorylated residues effectively displaced 14-3-3ζ from GPIbα. Analysis of single and double alanine substitutions of Ser587 and Ser590 demonstrated a major role for these residues in promoting GPIbα-14-3-3ζ binding. Moreover, these cell lines exhibited a defect in cell spreading on immobilized VWF. These studies demonstrate the existence of a second major 14-3-3ζ binding site within the cytoplasmic tail of GPIbα that has an important functional role in regulating integrin-dependent cell spreading. (Blood. 2004 :420-427)
Interaction between Platelet Glycoprotein Ibα and Filamin-1 Is Essential for Glycoprotein Ib/IX Receptor Anchorage at High Shear
Publisher: Elsevier BV
Date: 2002
Calpain cleavage of focal adhesion proteins regulates the cytoskeletal attachment of integrin α(IIb)β3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin clots
Publisher: Elsevier BV
Date: 1997
MouseMove: an open source program for semi-automated analysis of movement and cognitive testing in rodents
Publisher: Springer Science and Business Media LLC
Date: 04-11-2015
DOI: 10.1038/SREP16171
Abstract: The Open Field (OF) test is one of the most commonly used assays for assessing exploratory behaviour and generalised locomotor activity in rodents. Nevertheless, the vast majority of researchers still rely upon costly commercial systems for recording and analysing OF test results. Consequently, our aim was to design a freely available program for analysing the OF test and to provide an accompanying protocol that was minimally invasive, rapid, unbiased, without the need for specialised equipment or training. Similar to commercial systems, we show that our software—called MouseMove —accurately quantifies numerous parameters of movement including travel distance, speed, turning and curvature. To assess its utility, we used MouseMove to quantify unilateral locomotor deficits in mice following the filament-induced middle cerebral artery occlusion model of acute ischemic stroke. MouseMove can also monitor movement within defined regions-of-interest and is therefore suitable for analysing the Novel Object Recognition test and other field-related cognitive tests. To the best of our knowledge, MouseMove is the first open source software capable of providing qualitative and quantitative information on mouse locomotion in a semi-automated and high-throughput fashion and hence MouseMove represents a sound alternative to commercial movement analysis systems.
PI3-kinase p110α mediates β1 integrin-induced Akt activation and membrane protrusion during cell attachment and initial spreading
Publisher: Elsevier BV
Date: 12-2010
DOI: 10.1016/J.CELLSIG.2010.07.011
Abstract: Integrin-mediated cell adhesion activates several signaling effectors, including phosphatidylinositol 3-kinase (PI3K), a central mediator of cell motility and survival. To elucidate the molecular mechanisms of this important pathway the specific members of the PI3K family activated by different integrins have to be identified. Here, we studied the role of PI3K catalytic isoforms in β1 integrin-induced lamellipodium protrusion and activation of Akt in fibroblasts. Real-time total internal reflection fluorescence imaging of the membrane-substrate interface demonstrated that β1 integrin-mediated attachment induced rapid membrane spreading reaching essentially maximal contact area within 5-10 min. This process required actin polymerization and involved activation of PI3K. Isoform-selective pharmacological inhibition identified p110α as the PI3K catalytic isoform mediating both β1 integrin-induced cell spreading and Akt phosphorylation. A K756L mutation in the membrane-proximal part of the β1 integrin subunit, known to cause impaired Akt phosphorylation after integrin stimulation, induced slower cell spreading. The initial β1 integrin-regulated cell spreading as well as Akt phosphorylation were sensitive to the tyrosine kinase inhibitor PP2, but were not dependent on Src family kinases, FAK or EGF/PDGF receptor transactivation. Notably, cells expressing a Ras binding-deficient p110α mutant were severely defective in integrin-induced Akt phosphorylation, but exhibited identical membrane spreading kinetics as wild-type p110α cells. We conclude that p110α mediates β1 integrin-regulated activation of Akt and actin polymerization important for survival and lamellipodia dynamics. This could contribute to the tumorigenic properties of cells expressing constitutively active p110α.
Bcl-xL–inhibitory BH3 mimetics (ABT-737 or ABT-263) and the modulation of cytosolic calcium flux and platelet function
Publisher: American Society of Hematology
Date: 02-02-2012
Improved analogs and novel delivery systems for somatostatin octapeptides
Publisher: Elsevier BV
Date: 08-1996
DOI: 10.1016/S0026-0495(96)90074-8
Abstract: Appropriate N-terminus modification can result in somatostatin (SRIF) octapeptide analogs that are both more potent and more selective in vitro for the human SRIF receptor type 2 (hsst2). In addition, these modifications can improve the duration of action and bioavailability of SRIF analogs following parenteral administration, as shown by both pharmacological and distribution studies in vivo with BIM-23190 and BIM-23197 in the rat.
Insulin-like growth factor-1 regulates platelet activation through PI3-Kα isoform
Publisher: American Society of Hematology
Date: 15-12-2007
DOI: 10.1182/BLOOD-2007-03-080804
Abstract: Platelets release insulin-like growth factor-1 (IGF-1) from α granules upon activation. We have investigated the regulation of IGF-1 in Gi-dependent pathways leading to Akt activation and the role of IGF-1 in platelet activation. IGF-1 alone failed to induce platelet aggregation, but IGF-1 potentiated 2-MeSADP–induced platelet aggregation in a concentration-dependent manner. IGF-1 triggered platelet aggregation in combination with selective P2Y1 receptor activation. IGF-1 also caused platelet aggregation without shape change when combined with selective Gz stimulation by epinephrine, suggesting the role of IGF-1 in platelet aggregation by supplementing Gi pathways. The potentiating effect of IGF-1 was not affected by intracellular calcium chelation. Importantly, IGF-1 was unable to potentiate platelet aggregation by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, suggesting a critical regulation by PI3-K. Moreover, the potentiating effect of IGF-1 was abolished by the presence of PI3-K p110α inhibitor PIK-75. Stimulation of platelets with IGF-1 resulted in phosphorylation of Akt, a downstream effector of PI3-K, which was completely inhibited by wortmannin. IGF-1-induced Akt phosphorylation was abolished by PIK-75 suggesting the contribution of PI3-K p110α for activation of Akt by IGF-1. These results demonstrate that IGF-1 plays a role in potentiating platelet aggregation by complementing Gi- but not Gq-signaling pathways via PI3-K p110α.
Platelet adhesive receptors and novel anti-thrombotics
Publisher: Elsevier BV
Date: 09-1997
The class II PI 3-kinase, PI3KC2α, links platelet internal membrane structure to shear-dependent adhesive function
Publisher: Springer Science and Business Media LLC
Date: 17-03-2015
DOI: 10.1038/NCOMMS7535
Abstract: PI3KC2α is a broadly expressed lipid kinase with critical functions during embryonic development but poorly defined roles in adult physiology. Here we utilize multiple mouse genetic models to uncover a role for PI3KC2α in regulating the internal membrane reserve structure of megakaryocytes (demarcation membrane system) and platelets (open canalicular system) that results in dysregulated platelet adhesion under haemodynamic shear stress. Structural alterations in the platelet internal membrane lead to enhanced membrane tether formation that is associated with accelerated, yet highly unstable, thrombus formation in vitro and in vivo. Notably, agonist-induced 3-phosphorylated phosphoinositide production and cellular activation are normal in PI3KC2α-deficient platelets. These findings demonstrate an important role for PI3KC2α in regulating shear-dependent platelet adhesion via regulation of membrane structure, rather than acute signalling. These studies provide a link between the open canalicular system and platelet adhesive function that has relevance to the primary haemostatic and prothrombotic function of platelets.
Autoregulation of von Willebrand factor function by a disulfide bond switch
Publisher: American Association for the Advancement of Science (AAAS)
Date: 02-02-2018
Abstract: We demonstrate mechanochemical regulation of platelet adhesion to von Willebrand factor in thrombosis and hemostasis.
Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth
Publisher: American Society of Hematology
Date: 07-2008
DOI: 10.1182/BLOOD-2007-12-127001
Abstract: A fundamental property of platelets is their ability to transmit cytoskeletal contractile forces to extracellular matrices. While the importance of the platelet contractile mechanism in regulating fibrin clot retraction is well established, its role in regulating the primary hemostatic response, independent of blood coagulation, remains ill defined. Real-time analysis of platelet adhesion and aggregation on a collagen substrate revealed a prominent contractile phase during thrombus development, associated with a 30% to 40% reduction in thrombus volume. Thrombus contraction developed independent of thrombin and fibrin and resulted in the tight packing of aggregated platelets. Inhibition of the platelet contractile mechanism, with the myosin IIA inhibitor blebbistatin or through Rho kinase antagonism, markedly inhibited thrombus contraction, preventing the tight packing of aggregated platelets and undermining thrombus stability in vitro. Using a new intravital hemostatic model, we demonstrate that the platelet contractile mechanism is critical for maintaining the integrity of the primary hemostatic plug, independent of thrombin and fibrin generation. These studies demonstrate an important role for the platelet contractile mechanism in regulating primary hemostasis and thrombus growth. Furthermore, they provide new insight into the underlying bleeding diathesis associated with platelet contractility defects.
Type I phosphoinositide 3-kinases: potential antithrombotic targets?
Publisher: Springer Science and Business Media LLC
Date: 05-2006
DOI: 10.1007/S00018-006-6001-2
Abstract: Arterial thrombosis is the single most common cause of death and disability in industrialized societies and is the primary pathogenic mechanism underlying acute myocardial infarction and ischemic stroke. Platelets play a central role in this process, and as a consequence, a great deal of effort has gone into identifying the mechanisms regulating the adhesive function of platelets. Platelet adhesion is controlled by intracellular signaling pathways, with growing evidence for a major role for phosphoinositide 3-kinases (PI3Ks) in this process. Platelets express all type I PI3K isoforms, including p110alpha, p110beta, p110delta and p110gamma, with recent evidence suggesting important roles for p110gamma and p110beta in regulating distinct phases of the platelet activation process. Deficiency of p110 gamma or inhibition of p110beta produces a marked defect in arterial thrombosis without a corresponding increase in bleeding time, raising the possibility that inhibition of one or more PI3K isoforms may represent an effective antithrombotic approach.
Role of the platelet integrin glycoprotein IIb-IIIa in intracellular signalling
Publisher: Elsevier BV
Date: 07-1993
An integrin αIIbβ3 intermediate affinity state mediates biomechanical platelet aggregation
Publisher: Springer Science and Business Media LLC
Date: 25-03-2019
High shear-dependent loss of membrane integrity and defective platelet adhesion following disruption of the GPIba-filamin interaction
Publisher: American Society of Hematology
Date: 03-03-2011
DOI: 10.1182/BLOOD-2010-07-296194
Abstract: Platelets have evolved a highly specialized membrane skeleton that provides stability to the plasma membrane and facilitates adhesion under high shear stress. The cytoskeletal anchorage of glycoprotein (GP) Ibα plays an important role in regulating the membrane skeleton. However, its role in regulating membrane stability remains unknown. To investigate this role, we have developed a new mouse model that expresses wild-type human GPIbα (hGPIbαWT), or a mutant form of human GPIbα that has a selective defect in its ability to bind filamin A and anchor to the membrane skeleton (hGPIbαFW–Phe568Ala and Trp570Ala substitutions). Our study demonstrates that the link between platelet GPIb and the cytoskeleton does not alter the intrinsic ligand binding function of GPIbα or the ability of the receptor to stimulate integrin αIIbβ3-dependent spreading. However, exposure of hGPIbαFW platelets to pathologic shear rate levels (5000 to 40 000 s−1) leads to the development of unstable membrane tethers, defective platelet adhesion, and loss of membrane integrity, leading to complete disintegration of the platelet cell body. These outcomes suggest that the GPIbα–filamin A interaction not only regulates the architecture of the membrane skeleton, but also maintains the mechanical stability of the plasma membrane under conditions of high shear.
Dissecting isoform selectivity of PI3K inhibitors: the role of non-conserved residues in the catalytic pocket
Publisher: Portland Press Ltd.
Date: 27-08-2008
DOI: 10.1042/BJ20080512
Abstract: The last few years have seen the identification of numerous small molecules that selectively inhibit specific class I isoforms of PI3K (phosphoinositide 3-kinase), yet little has been revealed about the molecular basis for the observed selectivities. Using site-directed mutagenesis, we have investigated one of the areas postulated as being critical to the observed selectivity. The residues Thr886 and Lys890 of the PI3Kγ isoform project towards the ATP-binding pocket at the entrance to the catalytic site, but are not conserved. We have made reciprocal mutations between those residues in the β isoform (Glu858 and Asp862) and those in the α isoform (His855 and Gln859) and evaluated the potency of a range of reported PI3K inhibitors. The results show that the potencies of β-selective inhibitors TGX221 and TGX286 are unaffected by this change. In contrast, close analogues of these compounds, particularly the α-isoform-selective compound (III), are markedly influenced by the point mutations. The collected data suggests two distinct binding poses for these inhibitor classes, one of which is associated with potent PI3Kβ activity and is not associated with the mutated residues, and a second that, in accord with earlier hypotheses, does involve this pair of non-conserved amino acids at the catalytic site entrance and contributes to the α-isoform-selectivity of the compounds studied.
Non-redundant Roles of Phosphoinositide 3-Kinase Isoforms α and β in Glycoprotein VI-induced Platelet Signaling and Thrombus Formation
Publisher: Elsevier BV
Date: 12-2009
Potent Trivalent Inhibitors of Thrombin through Hybridization of Salivary Sulfopeptides from Hematophagous Arthropods
Publisher: Wiley
Date: 28-01-2021
Abstract: Blood feeding arthropods, such as leeches, ticks, flies and mosquitoes, provide a privileged source of peptidic anticoagulant molecules. These primarily operate through inhibition of the central coagulation protease thrombin by binding to the active site and either exosite I or exosite II. Herein, we describe the rational design of a novel class of trivalent thrombin inhibitors that simultaneously block both exosites as well as the active site. These engineered hybrids were synthesized using tandem diselenide‐selenoester ligation (DSL) and native chemical ligation (NCL) reactions in one‐pot. The most potent trivalent inhibitors possessed femtomolar inhibition constants against α‐thrombin and were selective over related coagulation proteases. A lead hybrid inhibitor possessed potent anticoagulant activity, blockade of both thrombin generation and platelet aggregation in vitro and efficacy in a murine thrombosis model at 1 mg kg −1 . The rational engineering approach described here lays the foundation for the development of potent and selective inhibitors for a range of other enzymatic targets that possess multiple sites for the disruption of protein–protein interactions, in addition to an active site.
SR-BI and fatty platelets
Publisher: American Society of Hematology
Date: 16-09-2010
Rapid assembly and profiling of an anticoagulant sulfoprotein library
Publisher: Proceedings of the National Academy of Sciences
Date: 20-06-2019
Abstract: Cardiovascular disease represents a significant health challenge, with towering economic and social costs resulting from the high levels of associated morbidity and mortality. For conditions resulting from undesired blood clotting, treatment options remain limited and are accompanied by significant side effects. Using a rapid chemical ligation platform, 34 homogeneously modified variants of tick anticoagulant proteins were generated. Access to this synthetic protein library enabled key structure–activity relationships to be elucidated and revealed unexpected differences in the mechanism of thrombin inhibition by this group of otherwise closely related molecules. The synthetic platform reported here provides a unique means to expedite the generation and identification of polypeptide and protein therapeutic leads for clotting-associated diseases.
Bcl-xL–inhibitory BH3 mimetics can induce a transient thrombocytopathy that undermines the hemostatic function of platelets
Publisher: American Society of Hematology
Date: 11-08-2011
DOI: 10.1182/BLOOD-2011-04-347849
Abstract: BH3 mimetics are a new class of proapo-ptotic anticancer agents that have shown considerable promise in preclinical animal models and early-stage human trials. These agents act by inhibiting the pro-survival function of one or more Bcl-2–related proteins. Agents that inhibit Bcl-xL induce rapid platelet death that leads to thrombocytopenia however, their impact on the function of residual circulating platelets remains unclear. In this study, we demonstrate that the BH3 mimetics, ABT-737 or ABT-263, induce a time- and dose-dependent decrease in platelet adhesive function that correlates with ectodomain shedding of the major platelet adhesion receptors, glycoprotein Ibα and glycoprotein VI, and functional down-regulation of integrin αIIbβ3. Analysis of platelets from mice treated with higher doses of BH3 mimetics revealed the presence of a subpopulation of circulating platelets undergoing cell death that have impaired activation responses to soluble agonists. Functional analysis of platelets by intravital microscopy revealed a time-dependent defect in platelet aggregation at sites of vascular injury that correlated with an increase in tail bleeding time. Overall, these studies demonstrate that Bcl-xL–inhibitory BH3 mimetics not only induce thrombocytopenia but also a transient thrombocytopathy that can undermine the hemostatic function of platelets.
Role of Phosphoinositide 3-Kinase β in Glycoprotein VI-mediated Akt Activation in Platelets
Publisher: Elsevier BV
Date: 12-2009
Rab8a interacts directly with PI3Kγ to modulate TLR4-driven PI3K and mTOR signalling
Publisher: Springer Science and Business Media LLC
Date: 15-07-2014
DOI: 10.1038/NCOMMS5407
Abstract: Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS) to mount innate immune responses. The TLR4-induced release of pro- and anti-inflammatory cytokines generates robust inflammatory responses, which must then be restrained to avoid disease. New mechanisms for the critical regulation of TLR-induced cytokine responses are still emerging. Here we find TLR4 complexes localized in LPS-induced dorsal ruffles on the surface of macrophages. We discover that the small GTPase Rab8a is enriched in these ruffles and recruits phosphatidylinositol 3-kinase (PI3Kγ) as an effector by interacting directly through its Ras-binding domain. Rab8a and PI3Kγ function to regulate Akt signalling generated by surface TLR4. Rab8a and PI3Kγ do not affect TLR4 endocytosis, but instead regulate mammalian target of rapamycin signalling as a mechanism for biasing the cytokine profile to constrain inflammation in innate immunity.
Platelets and the Injured Vessel Wall— “Rolling into Action” Focus on Glycoprotein Ib/V/IX and the Platelet Cytoskeleton
Publisher: Elsevier BV
Date: 07-2000
DOI: 10.1016/S1050-1738(00)00062-1
Abstract: Blood platelets play a key role in maintaining the integrity of the vascular system through their ability to arrest bleeding (haemostasis) and promote repair of injured blood vessels. Considerable progress has been made in the last few years in our understanding of the adhesion mechanisms utilized by platelets to adhere to sites of vascular injury. Studies have helped define the precise role of von Willebrand factor (vWf), and its platelet receptor, glycoprotein (GP) Ib/V/IX, in initiating platelet-vessel wall and platelet-platelet adhesion contacts. In addition to its adhesive role, recent studies have highlighted the importance of GPIb/V/IX in regulating the cytoskeleton of platelets. GPIb/V/IX not only maintains the normal cytoskeletal architecture of resting platelets but also induces cytoskeletal reorganization following engagement of vWf. Somewhat surprisingly, the physical link between GPIb/V/IX and the membrane skeleton does not appear necessary for GPIb/V/IX-induced cytoskeletal reorganization. In contrast, this linkage appears critical for GPIb/V/IX to maintain cell adhesion under high shear and also for the ability of the cytoskeleton to exert negative regulatory effects on the GPIb-vWf interaction. Thus a complex functional relationship appears to exist between GPIb/V/IX and the membrane skeleton that goes well beyond preserving the normal cytoskeletal architecture of resting platelets.
Platelet recruitment to the inflamed glomerulus occurs via an ?IIb ?3 /GPVI-dependent pathway
Publisher: Elsevier BV
Date: 09-2010
Compression force sensing regulates integrin αIIbβ3 adhesive function on diabetic platelets.
Publisher: Springer Science and Business Media LLC
Date: 14-03-2018
DOI: 10.1038/S41467-018-03430-6
Abstract: Diabetes is associated with an exaggerated platelet thrombotic response at sites of vascular injury. Biomechanical forces regulate platelet activation, although the impact of diabetes on this process remains ill-defined. Using a biomembrane force probe (BFP), we demonstrate that compressive force activates integrin α IIb β 3 on discoid diabetic platelets, increasing its association rate with immobilized fibrinogen. This compressive force-induced integrin activation is calcium and PI 3-kinase dependent, resulting in enhanced integrin affinity maturation and exaggerated shear-dependent platelet adhesion. Analysis of discoid platelet aggregation in the mesenteric circulation of mice confirmed that diabetes leads to a marked enhancement in the formation and stability of discoid platelet aggregates, via a mechanism that is not inhibited by therapeutic doses of aspirin and clopidogrel, but is eliminated by PI 3-kinase inhibition. These studies demonstrate the existence of a compression force sensing mechanism linked to α IIb β 3 adhesive function that leads to a distinct prothrombotic phenotype in diabetes.
Phosphoinositide 3-kinase p110βregulates integrin α IIb β3 avidity and the cellular transmission of contractile forces
Publisher: Elsevier BV
Date: 2010
Glycoprotein (GP) Ib-IX-transfected Cells Roll on a von Willebrand Factor Matrix under Flow
Publisher: Elsevier BV
Date: 03-1999
Essential role for phosphoinositide 3-kinase in shear-dependent signaling between platelet glycoprotein Ib/V/IX and integrin αIIbβ3
Publisher: American Society of Hematology
Date: 2002
Abstract: Platelet adhesion and aggregation at sites of vascular injury are critically dependent on the interaction between von Willebrand factor (VWF) and 2 major platelet adhesion receptors, glycoprotein (GP) Ib/V/IX and integrin αIIbβ3. GP Ib/V/IX binding to VWF mediates platelet tethering and translocation, whereas activation of integrin αIIbβ3 promotes cell arrest. To date, the signaling pathways used by the VWF-GP Ib/V/IX interaction to promote activation of integrin αIIbβ3, particularly under shear, have remained poorly defined. In this study, the potential involvement of type 1 phosphoinositide (PI) 3–kinases in this process was investigated. Results show that platelet adhesion and spreading on immobilized VWF results in a specific increase in the PI 3–kinase lipid product, PtdIns(3,4)P2. Under static conditions, inhibiting PI 3–kinase with LY294002 or wortmannin did not prevent platelet adhesion, integrin αIIbβ3activation, or platelet spreading although it significantly delayed the onset of these events. In contrast, PI 3–kinase inhibition under shear dramatically reduced both platelet adhesion and spreading. Real-time analysis of intracellular calcium demonstrated that under static conditions inhibiting PI 3–kinase delayed the onset of intracellular fluxes in adherent platelets, but did not affect the final magnitude of the calcium response. However, under shear, inhibiting PI 3–kinase dramatically reduced intracellular calcium mobilization and integrin αIIbβ3 activation, resulting in impaired thrombus growth. The studies demonstrate a shear-dependent role for PI 3–kinase in promoting platelet adhesion on immobilized VWF. Under static conditions, platelets appear to mobilize intracellular calcium through both PI 3–kinase–dependent and –independent mechanisms, whereas under shear PI 3–kinase is indispensable for VWF-induced calcium release.
Are Erk, Btk, and PECAM-1 major players in GPIb signaling? The challenge of unraveling signaling events downstream of platelet GPIb
Publisher: American Society of Hematology
Date: 15-01-2007
Application of a strain rate gradient microfluidic device to von Willebrand's disease screening
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7LC00498B
Abstract: We present the development of a strain rate gradient microfluidic device and apply it to the screening of von Willebrand's disease.
Multi-photon near-infrared emission saturation nanoscopy using upconversion nanoparticles
Publisher: Springer Science and Business Media LLC
Date: 17-08-2018
DOI: 10.1038/S41467-018-05842-W
Abstract: Multiphoton fluorescence microscopy (MPM), using near infrared excitation light, provides increased penetration depth, decreased detection background, and reduced phototoxicity. Using stimulated emission depletion (STED) approach, MPM can bypass the diffraction limitation, but it requires both spatial alignment and temporal synchronization of high power (femtosecond) lasers, which is limited by the inefficiency of the probes. Here, we report that upconversion nanoparticles (UCNPs) can unlock a new mode of near-infrared emission saturation (NIRES) nanoscopy for deep tissue super-resolution imaging with excitation intensity several orders of magnitude lower than that required by conventional MPM dyes. Using a doughnut beam excitation from a 980 nm diode laser and detecting at 800 nm, we achieve a resolution of sub 50 nm, 1/20th of the excitation wavelength, in imaging of single UCNP through 93 μm thick liver tissue. This method offers a simple solution for deep tissue super resolution imaging and single molecule tracking.
Distinct Substrate Specificities and Functional Roles for the 78- and 76-kDa Forms of μ-Calpain in Human Platelets
Publisher: Elsevier BV
Date: 10-1997
Is thrombin the problem or (dis)solution?
Publisher: American Society of Hematology
Date: 11-06-2009
Rücktitelbild: Potent Trivalent Inhibitors of Thrombin through Hybridization of Salivary Sulfopeptides from Hematophagous Arthropods (Angew. Chem. 10/2021)
Publisher: Wiley
Date: 04-02-2021
Raised Plasma Soluble P-Selectin in Peripheral Arterial Occlusive Disease Enhances Leukocyte Adhesion
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 06-01-2006
DOI: 10.1161/01.RES.0000199295.14073.69
Abstract: Raised levels of soluble P-selectin (sP-selectin) have been reported in the plasma of patients with vascular diseases however, the functional importance of this ligand remains unclear. In this study we have examined a potential role for plasma sP-selectin in regulating neutrophil adhesion in patients with peripheral arterial occlusive disease (PAOD). Patients with PAOD had significantly higher levels of sP-selectin (mean±SD: 73.3±13.0 versus 16.7±6.4 ng/mL) and enhanced whole blood leukocyte adhesion to platelets under shear. To examine whether the raised sP-selectin levels can directly influence leukocyte adhesion, isolated neutrophils were incubated with plasma from PAOD patients before and after immunodepletion of sP-selectin. Neutrophil adhesion to fibrinogen increased 2-fold following incubation with PAOD plasma, which was abrogated on sP-selectin immunodepletion. We subsequently demonstrated that recombinant sP-selectin dose-dependently (75 to 250 ng/mL) increased leukocyte adhesion to fibrinogen and platelet monolayers. This increase was PSGL-1 and Src kinase-dependent and correlated with an increase in sP-selectin-mediated Mac-1 activation. sP-selectin–stimulated neutrophil adhesion to platelet monolayers was inversely correlated with shear, such that at low shear (50 s −1 ) a 92.7%±15.7 increase in adhesion was observed decreasing to 38.5%±11.9 at 150 s −1 and 10.1%±7.4 at 300 s −1 . These studies suggest a potentially important role for sP-selectin in modulating neutrophil adhesion in patients with PAOD, particularly at sites of low shear, where it raises the possibility that raised plasma sP-selectin levels may enhance leukocyte recruitment to vascular injury and promote disease progression.
Potent Trivalent Inhibitors of Thrombin through Hybridization of Salivary Sulfopeptides from Hematophagous Arthropods
Publisher: Wiley
Date: 28-01-2021
Abstract: Blood feeding arthropods, such as leeches, ticks, flies and mosquitoes, provide a privileged source of peptidic anticoagulant molecules. These primarily operate through inhibition of the central coagulation protease thrombin by binding to the active site and either exosite I or exosite II. Herein, we describe the rational design of a novel class of trivalent thrombin inhibitors that simultaneously block both exosites as well as the active site. These engineered hybrids were synthesized using tandem diselenide‐selenoester ligation (DSL) and native chemical ligation (NCL) reactions in one‐pot. The most potent trivalent inhibitors possessed femtomolar inhibition constants against α‐thrombin and were selective over related coagulation proteases. A lead hybrid inhibitor possessed potent anticoagulant activity, blockade of both thrombin generation and platelet aggregation in vitro and efficacy in a murine thrombosis model at 1 mg kg −1 . The rational engineering approach described here lays the foundation for the development of potent and selective inhibitors for a range of other enzymatic targets that possess multiple sites for the disruption of protein–protein interactions, in addition to an active site.
Pathologic shear triggers shedding of vascular receptors: a novel mechanism for down-regulation of platelet glycoprotein VI in stenosed coronary vessels
Publisher: American Society of Hematology
Date: 03-05-2012
DOI: 10.1182/BLOOD-2011-10-386607
Abstract: Ligand-induced ectodomain shedding of glycoprotein VI (GPVI) is a metalloproteinase-dependent event. We examined whether shear force, in the absence of GPVI ligand, was sufficient to induce shedding of GPVI. Human-citrated platelet-rich plasma or washed platelets were subjected to increasing shear rates in a cone-plate viscometer, and levels of intact and cleaved GPVI were examined by Western blot and ELISA. Pathophysiologic shear rates (3000-10 000 seconds−1) induced platelet aggregation and metalloproteinase-dependent appearance of soluble GPVI ectodomain, and GPVI platelet remnant. Shedding of GPVI continued after transient exposure to shear. Blockade of αIIbβ3, GPIbα, or intracellular signaling inhibited shear-induced platelet aggregation but minimally affected shear-induced shedding of GPVI. Shear-induced GPVI shedding also occurred in platelet-rich plasma or washed platelets isolated from a von Willebrand disease type 3 patient with no detectable VWF, implying that shear-induced activation of platelet metalloproteinases can occur in the absence of GPVI and GPIbα ligands. Significantly elevated levels of sGPVI were observed in 10 patients with stable angina pectoris, with well-defined single vessel coronary artery disease and mean intracoronary shear estimates at 2935 seconds−1 (peak shear, 19 224 seconds−1). Loss of GPVI in platelets exposed to shear has potential implications for the stability of a forming thrombus at arterial shear rates.
Endogenous fibrinolysis facilitates clot retraction in vivo
Publisher: American Society of Hematology
Date: 07-12-2017
DOI: 10.1182/BLOOD-2017-06-789032
Abstract: Localized vascular injury with thrombin microinjection produces a fibrin network that undergoes myosin IIa–dependent retraction in vivo. Using this model, we demonstrate that endogenous fibrinolysis promotes fibrin clot retraction.
Back Cover: Potent Trivalent Inhibitors of Thrombin through Hybridization of Salivary Sulfopeptides from Hematophagous Arthropods (Angew. Chem. Int. Ed. 10/2021)
Publisher: Wiley
Date: 04-02-2021
Dual P2Y12 receptor signaling in thrombin‐stimulated platelets – involvement of phosphoinositide 3‐kinase β but not γ isoform in Ca2+ mobilization and procoagulant activity
Publisher: Wiley
Date: 14-12-2007
DOI: 10.1111/J.1742-4658.2007.06207.X
Abstract: During thrombus formation, thrombin, which is abundantly present at sites of vascular injury, activates platelets in part via autocrine-produced ADP. We investigated the signaling pathways by which thrombin and ADP in synergy induced platelet Ca(2+) elevation and procoagulant activity, and we monitored the consequences for the coagulation process. Even at high thrombin concentration, autocrine and added ADP enhanced and prolonged Ca(2+) depletion from internal stores via stimulation of the P2Y(12) receptors. This P2Y(12)-dependent effect was mediated via two distinct signaling pathways. The first is enhanced Ca(2+) mobilization by the inositol 1,4,5-trisphosphate receptors due to inhibition of protein kinase A. The second pathway concerns prolonged activation of phosphoinositide 3-kinase (PI3-K) and phospholipase C. Experiments with phosphoinositide 3-kinase isoform-selective inhibitors and p110gamma deficient platelets demonstrated that the phosphoinositide 3-kinase beta and not the phosphoinositide 3-kinase gamma isoform is responsible for the prolonged Ca(2+) response and for the subsequent increases in procoagulant activity and coagulation. Taken together, these results demonstrate a dual P2Y(12)-dependent signaling mechanism, which increases the platelet-activating effect of thrombin by prolongation of Ca(2+) elevation, thereby facilitating the coagulation process.
Arterial thrombosis—insidious, unpredictable and deadly
Publisher: Springer Science and Business Media LLC
Date: 11-2011
DOI: 10.1038/NM.2515
Abstract: The formation of blood clots--thrombosis--at sites of atherosclerotic plaque rupture is a major clinical problem despite ongoing improvements in antithrombotic therapy. Progress in identifying the pathogenic mechanisms regulating arterial thrombosis has led to the development of newer therapeutics, and there is general anticipation that these treatments will have greater efficacy and improved safety. However, major advances in this field require the identification of specific risk factors for arterial thrombosis in affected in iduals and a rethink of the 'one size fits all' approach to antithrombotic therapy.
Antiplatelet therapy: present status and future prospects
Publisher: Informa Healthcare
Date: 08-2007
DOI: 10.1517/17460441.2.8.1035
Abstract: The excessive accumulation of platelets at sites of vascular injury is a key event in the development of arterial thrombosis, the principal pathogenic mechanism underlying the acute coronary syndromes and ischaemic stroke. In combination, these disorders are the leading causes of morbidity and mortality in the industrialised world and, as a consequence, the platelet is a major therapeutic target in the management of cardiovascular disease. This perspective focuses on the present state of antiplatelet therapy and potential future strategies to improve the safety and efficacy of antiplatelet agents.
Erythrocyte Hemolysis and Hemoglobin Oxidation Promote Ferric Chloride-induced Vascular Injury
Publisher: Elsevier BV
Date: 05-2009
Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function
Publisher: American Society of Hematology
Date: 16-07-2009
DOI: 10.1182/BLOOD-2009-01-200345
Abstract: Procoagulant platelets exhibit hallmark features of apoptotic cells, including membrane blebbing, microvesiculation, and phosphatidylserine (PS) exposure. Although platelets possess many well-known apoptotic regulators, their role in regulating the procoagulant function of platelets is unclear. To clarify this, we investigated the consequence of removing the essential mediators of apoptosis, Bak and Bax, or directly inducing apoptosis with the BH3 mimetic compound ABT-737. Treatment of platelets with ABT-737 triggered PS exposure and a marked increase in thrombin generation in vitro. This increase in procoagulant function was Bak/Bax- and caspase-dependent, but it was unaffected by inhibitors of platelet activation or by chelating extracellular calcium. In contrast, agonist-induced platelet procoagulant function was unchanged in Bak−/−Bax−/− or caspase inhibitor–treated platelets, but it was completely eliminated by extracellular calcium chelators or inhibitors of platelet activation. These studies show the existence of 2 distinct pathways regulating the procoagulant function of platelets.
Intrinsic apoptosis circumvents the functional decline of circulating platelets but does not cause the storage lesion
Publisher: American Society of Hematology
Date: 12-07-2018
DOI: 10.1182/BLOOD-2017-11-816355
Abstract: BAK/BAX depletion in murine platelets reveals that intrinsic apoptosis is not required for the development of the platelet storage lesion. Restriction of platelet life span by intrinsic apoptosis is pivotal to maintain a functional, hemostatically reactive platelet population.
Erratum: A microfluidics device to monitor platelet aggregation dynamics in response to strain rate micro-gradients in flowing blood (Lab on a Chip (2010) 10 (291-302) DOI: 10.1039/b916757a)
Publisher: Royal Society of Chemistry (RSC)
Date: 2010
DOI: 10.1039/C0LC90091E
RhoA Sustains Integrin αIIbβ3Adhesion Contacts under High Shear
Publisher: Elsevier BV
Date: 04-2002
The Role of Platelets in Atherothrombosis
Publisher: American Society of Hematology
Date: 10-12-2011
DOI: 10.1182/ASHEDUCATION-2011.1.51
Abstract: Platelets have evolved highly specialized adhesion mechanisms that enable cell-matrix and cell-cell interactions throughout the entire vasculature irrespective of the prevailing hemodynamic conditions. This unique property of platelets is critical for their ability to arrest bleeding and promote vessel repair. Platelet adhesion under conditions of high shear stress, as occurs in stenotic atherosclerotic arteries, is central to the development of arterial thrombosis therefore, precise control of platelet adhesion must occur to maintain blood fluidity and to prevent thrombotic or hemorrhagic complications. Whereas the central role of platelets in hemostasis and thrombosis has long been recognized and well defined, there is now a major body of evidence supporting an important proinflammatory function for platelets that is linked to host defense and a variety of autoimmune and inflammatory diseases. In the context of the vasculature, experimental evidence indicates that the proinflammatory function of platelets can regulate various aspects of the atherosclerotic process, including its initiation and propagation. The mechanisms underlying the proatherogenic function of platelets are increasingly well defined and involve specific adhesive interactions between platelets and endothelial cells at atherosclerotic-prone sites, leading to the enhanced recruitment and activation of leukocytes. Through the release of chemokines, proinflammatory molecules, and other biological response modulators, the interaction among platelets, endothelial cells, and leukocytes establishes a localized inflammatory response that accelerates atherosclerosis. These inflammatory processes typically occur in regions of the vasculature experiencing low shear and perturbed blood flow, a permissive environment for leukocyte-platelet and leukocyte-endothelial interactions. Therefore, the concept has emerged that platelets are a central element of the atherothrombotic process and that future therapeutic strategies to combat this disease need to take into consideration both the prothrombotic and proinflammatory function of platelets.
Ferric chloride thrombosis model: unraveling the vascular effects of a highly corrosive oxidant
Publisher: American Society of Hematology
Date: 10-12-2015
Generation and characterization of mice with null mutation of the chloride intracellular channel 1 gene
Publisher: Wiley
Date: 2010
DOI: 10.1002/DVG.20590
Abstract: CLIC1 belongs to a family of highly conserved and widely expressed intracellular chloride ion channel proteins existing in both soluble and membrane integrated forms. To study the physiological and biological role of CLIC1 in vivo, we undertook conditional gene targeting to engineer Clic1 gene knock-out mice. This represents creation of the first gene knock-out of a vertebrate CLIC protein family member. We first generated a Clic1 Knock-in (Clic1(FN)) allele, followed by Clic1 knock-out (Clic1(-/-)) mice by crossing Clic1(FN) allele with TNAP-cre mice, resulting in germline gene deletion through Cre-mediated recombination. Mice heterozygous or homozygous for these alleles are viable and fertile and appear normal. However, Clic1(-) (/-) mice show a mild platelet dysfunction characterized by prolonged bleeding times and decreased platelet activation in response to adenosine diphosphate stimulation linked to P2Y(12) receptor signaling.
A microfluidics device to monitor platelet aggregation dynamics in response to strain rate micro-gradients in flowing blood
Publisher: Royal Society of Chemistry (RSC)
Date: 2010
DOI: 10.1039/B916757A
Abstract: This paper reports the development of a platform technology for measuring platelet function and aggregation based on localized strain rate micro-gradients. Recent experimental findings within our laboratories have identified a key role for strain rate micro-gradients in focally triggering initial recruitment and subsequent aggregation of discoid platelets at sites of blood vessel injury. We present the design justification, hydrodynamic characterization and experimental validation of a microfluidic device incorporating contraction-expansion geometries that generate strain rate conditions mimicking the effects of pathological changes in blood vessel geometry. Blood perfusion through this device supports our published findings of both in vivo and in vitro platelet aggregation and confirms a critical requirement for the coupling of blood flow acceleration to downstream deceleration for the initiation and stabilization of platelet aggregation, in the absence of soluble platelet agonists. The microfluidics platform presented will facilitate the detailed analysis of the effects of hemodynamic parameters on the rate and extent of platelet aggregation and will be a useful tool to elucidate the hemodynamic and platelet mechano-transduction mechanisms, underlying this shear-dependent process.
Phosphoinositide 3-kinases and the regulation of platelet function
Publisher: Portland Press Ltd.
Date: 04-2004
DOI: 10.1042/BST0320387
Abstract: A clear understanding of the role of PI (phosphoinositide) 3-kinases in supporting the haemostatic function of platelets has been slow to evolve. In fact, insight into the roles of in idual PI 3-kinase isoforms in platelet function remains rudimentary. However, based on in vitro studies using wortmannin and LY294002, there is evidence for an important role for PI 3-kinases in regulating a broad range of functional platelet responses, including primary platelet adhesion, cytoskeletal remodelling and platelet aggregation. One of the critical platelet responses involves affinity regulation of the major platelet integrin αIIbβ3, the primary receptor mediating platelet aggregation and thrombus growth. The input signals regulating integrin αIIbβ3 can be ided into three main groups: (1) Gq-coupled receptors linked to the activation of PLCβ (phospholipase Cβ) (2) Gi-coupled receptors linked to the regulation of adenylate cyclase and Rap1b and (3) adhesion receptor signalling involving Src kinase-dependent activation of PLCγ isoforms. PI 3-kinases have not been demonstrated to play a critical role in Gq-dependent platelet activation however, one or more PI 3-kinase isoforms appears to be important for Gi-dependent activation of Rap1b and adhesion receptor activation of PLCγ isoforms. Thus distinct co-operative PI 3-kinase signalling mechanisms appear to play an important role in regulating the adhesive function of integrin αIIbβ3.
Importance of temporal flow gradients and integrin α IIbβ3 mechanotransduction for shear activation of platelets
Publisher: Elsevier BV
Date: 04-2005
Shear Induces a Unique Series of Morphological Changes in Translocating Platelets
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-2006
DOI: 10.1161/01.ATV.0000201931.16535.E1
Abstract: Objective— The platelet glycoprotein (GP) Ib/V/IX complex plays an important role in regulating the morphology of resting platelets and can induce shape change during adhesion to immobilized von Willebrand factor (vWf). In this study we have examined the effects of fluid shear stress on GPIb-dependent changes in platelet morphology during translocation on vWf. Methods and Results— We demonstrate that translocating platelets undergo a unique series of morphological changes in response to increasing fluid shear stress. Under moderately low shear conditions (600 s −1 ), initial shape change involved extension of membrane tethers and/or filopodia from the platelet surface. With increasing shear rate, platelets adopted a spherical morphology with numerous surface projections (1800 to 5000 s −1 ). At high wall shear rates (10000 to 20 000 s −1 ), translocating platelets retracted filopodia, developing a smooth ball-like appearance. These changes in morphology were dependent on reorganization of the actin and microtubule components of the cytoskeleton and were regulated by intracellular signaling processes linked to Src kinases. Functionally, alterations in platelet shape had a major effect on translocation dynamics in that conversion from discs to spheres resulted in a 3- to 8-fold increase in rolling velocity. Conclusions— These studies demonstrate that platelets undergo shear-specific morphological changes during surface translocation on vWf that may serve to regulate translocation dynamics under flow.
The Bioactive Phospholipid, Lysophosphatidylcholine, Induces Cellular Effects via G-Protein-dependent Activation of Adenylyl Cyclase
Publisher: Elsevier BV
Date: 10-1996
PLATELET AGGREGATING ACTIVITY IN THE PLASMA OF PATIENTS WITH ESTABLISHED THROMBOSIS
Publisher: Wiley
Date: 04-1989
DOI: 10.1111/J.1445-5994.1989.TB00220.X
Abstract: This study examines the incidence and significance of novel plasma derived platelet aggregating activity (PAA) in 190 consecutive patients admitted to the medical wards of a general hospital. Seventy five patients (39%) demonstrated this activity. The incidence was highest in patients with a history of thrombosis (52%) or in those with a heightened thrombotic tendency, for ex le, patients with diabetes or hypertension. In contrast, platelet aggregating activity was observed in six out of 62 patients (approximately 10%) in whom a current or past medical history of thrombosis could not be elicited and in only two out of 72 healthy volunteers examined (3%). A high frequency of PAA was also noted in a small group of patients with idiopathic thrombocytopenia and patients who had previously received platelet transfusions. In these patients, this activity presumably reflects the presence of antiplatelet antibodies. A good correlation between the presence of plasma derived platelet aggregating activity and the phenomenon of spontaneous platelet aggregation was observed. The platelet aggregating activity was not heparin dependent, but was completely abolished by EDTA (5 mM) and benzamidine (8 mM), or by pretreating the platelets with aspirin. A synergistic response was observed with subaggregatory concentrations of thrombin and adrenalin. Our results suggest that the presence of this platelet aggregating activity may provide a marker for vascular thrombosis. Furthermore we postulate that this plasma derived activity may be partly responsible for platelet hyperactivity previously observed in patients with thromboembolic disorders.
The von Willebrand factor-glycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells
Publisher: Elsevier BV
Date: 12-1999
Phosphatidylinositol(4,5)bisphosphate coordinates actin-mediated mobilization and translocation of secretory vesicles to the plasma membrane of chromaffin cells
Publisher: Springer Science and Business Media LLC
Date: 04-10-2011
DOI: 10.1038/NCOMMS1500
Abstract: Neurosecretory vesicles undergo docking and priming before Ca(2+)-dependent fusion with the plasma membrane. Although de novo synthesis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P(2)) is required for exocytosis, its precise contribution is still unclear. Here we show that inhibition of the p110δ isoform of PI3-kinase by IC87114 promotes a transient increase in PtdIns(4,5)P(2), leading to a potentiation of exocytosis in chromaffin cells. We then exploit this pathway to examine the effect of a transient PtdIns(4,5)P(2) increase on neurosecretory vesicles behaviour, outside the context of a secretagogue stimulation. Our results demonstrate that a rise in PtdIns(4,5)P(2) is sufficient to promote the mobilization and recruitment of secretory vesicles to the plasma membrane via Cdc42-mediated actin reorganization. PtdIns(4,5)P(2), therefore, orchestrates the actin-based conveyance of secretory vesicles to the plasma membrane.
The CXCR1/2 ligand NAP-2 promotes directed intravascular leukocyte migration through platelet thrombi
Publisher: American Society of Hematology
Date: 30-05-2013
DOI: 10.1182/BLOOD-2012-09-459636
Abstract: Identification of a distinct leukocyte recruitment mechanism by platelet thrombi. Leukocyte migration through thrombi is partially mediated by one or more CXCR1/2 ligands, including NAP-2.
Class I phosphoinositide 3-kinases
Publisher: Elsevier BV
Date: 07-2003
DOI: 10.1016/S1357-2725(02)00270-4
Abstract: Phosphoinositide 3-kinases (PI3-kinases) are a family of enzymes that 3'-phosphorylate the inositol head group of membrane phosphoinositides. They are sub ided into three major classes (I, II and III) based on their structural homology, regulation and substrate specificity. It is now becoming clear that PI3-kinase isoforms are subject to differential regulation and may play distinct roles within the cell. PI3-kinases and their second messenger lipid products have been implicated in a plethora of cellular responses with increasing evidence for involvement in the pathogenesis of human diseases. The future development of specific PI3-kinase isoform inhibitors may offer therapeutic benefit in a broad range of clinical settings, related to cancer, inflammatory and immunological diseases.
Distinct glycoprotein Ib/V/IX and integrin αIIb β3 -dependent calcium signals cooperatively regulate platelet adhesion under flow
Publisher: Elsevier BV
Date: 2002
A shear gradient-dependent platelet aggregation mechanism drives thrombus formation
Publisher: Springer Science and Business Media LLC
Date: 24-05-2009
DOI: 10.1038/NM.1955
Abstract: Platelet aggregation at sites of vascular injury is essential for hemostasis and arterial thrombosis. It has long been assumed that platelet aggregation and thrombus growth are initiated by soluble agonists generated at sites of vascular injury. By using high-resolution intravital imaging techniques and hydrodynamic analyses, we show that platelet aggregation is primarily driven by changes in blood flow parameters (rheology), with soluble agonists having a secondary role, stabilizing formed aggregates. We find that in response to vascular injury, thrombi initially develop through the progressive stabilization of discoid platelet aggregates. Analysis of blood flow dynamics revealed that discoid platelets preferentially adhere in low-shear zones at the downstream face of forming thrombi, with stabilization of aggregates dependent on the dynamic restructuring of membrane tethers. These findings provide insight into the prothrombotic effects of disturbed blood flow parameters and suggest a fundamental reinterpretation of the mechanisms driving platelet aggregation and thrombus growth.
Signaling Role for Phospholipase Cγ2 in Platelet Glycoprotein Ibα Calcium Flux and Cytoskeletal Reorganization
Publisher: Elsevier BV
Date: 08-2003
The impact of blood rheology on the molecular and cellular events underlying arterial thrombosis.
Publisher: Springer Science and Business Media LLC
Date: 30-09-2006
DOI: 10.1007/S00109-006-0101-1
Abstract: There is an increasing appreciation of the importance of disturbed blood flow, especially turbulent flow, in the pathogenesis of vascular disease. However, the precise mechanism(s) by which rheological changes accelerate the atherothrombotic process remains incompletely understood. Atherosclerotic lesions typically develop in vascular regions exhibiting bifurcated or curved architectures. Such regions exhibit complex blood flow profiles with considerable ergence from uniform laminar flow. These altered flow behaviours can promote deposition of pro-atherogenic lipids and proteins to the vessel wall and modulate the adhesive function of endothelial, platelets and leukocytes. Once developed, atherosclerotic lesions can further exacerbate flow disturbances, establishing a potential hazardous cycle of accelerated atherogenesis. At the cellular level, alterations in fluid flow can lead to significant changes in signal transduction, leading to a variety of functional and morphological changes. In particular, disturbed rheology has a significant impact on the adhesion and activation mechanisms utilised by platelets and leukocytes with high shear, playing an important role in accelerating platelet activation and thrombus growth. This review focuses on the impact of blood rheology on the cellular and molecular events underlying thrombosis, with particular emphasis on the role of platelets in this process.
Safety and efficacy of targeting platelet proteinase‐activated receptors in combination with existing anti‐platelet drugs as antithrombotics in mice
Publisher: Wiley
Date: 09-07-2012
Low adhesion receptor levels on circulating platelets in patients with lymphoproliferative diseases before receiving Navitoclax (ABT-263)
Publisher: American Society of Hematology
Date: 21-02-2013
Synthesis, biological evaluation and molecular modelling of sulfonohydrazides as selective PI3K p110α inhibitors
Publisher: Elsevier BV
Date: 12-2007
DOI: 10.1016/J.BMC.2007.08.062
Abstract: A series of 2-methyl-5-nitrobenzenesulfonohydrazides were prepared and evaluated as inhibitors of PI3K. An isoquinoline derivative shows good selectivity for the p110alpha isoform over p110beta and p110delta, and also demonstrates good in vitro activity in a cell proliferation assay. Molecular modelling provides a rationalisation for the observed SAR.
Caspase-9 mediates the apoptotic death of megakaryocytes and platelets, but is dispensable for their generation and function
Publisher: American Society of Hematology
Date: 03-05-2012
DOI: 10.1182/BLOOD-2011-11-394858
Abstract: Apoptotic caspases, including caspase-9, are thought to facilitate platelet shedding by megakaryocytes. They are known to be activated during platelet apoptosis, and have also been implicated in platelet hemostatic responses. However, the precise requirement for, and the regulation of, apoptotic caspases have never been defined in either megakaryocytes or platelets. To establish the role of caspases in platelet production and function, we generated mice lacking caspase-9 in their hematopoietic system. We demonstrate that both megakaryocytes and platelets possess a functional apoptotic caspase cascade downstream of Bcl-2 family-mediated mitochondrial damage. Caspase-9 is the initiator caspase, and its loss blocks effector caspase activation. Surprisingly, steady-state thrombopoiesis is unperturbed in the absence of caspase-9, indicating that the apoptotic caspase cascade is not required for platelet production. In platelets, loss of caspase-9 confers resistance to the BH3 mimetic ABT-737, blocking phosphatidylserine (PS) exposure and delaying ABT-737–induced thrombocytopenia in vivo. Despite this, steady-state platelet lifespan is normal. Casp9−/− platelets are fully capable of physiologic hemostatic responses and functional regulation of adhesive integrins in response to agonist. These studies demonstrate that the apoptotic caspase cascade is required for the efficient death of megakaryocytes and platelets, but is dispensable for their generation and function.
Platelet adhesion receptors: novel targets for anti‐thrombotic therapy
Publisher: Wiley
Date: 06-1999
DOI: 10.1111/J.1445-5994.1999.TB00742.X
Abstract: The critical role of platelets in the development of the acute coronary syndromes is now well recognised, and a great deal of effort has therefore focused on elucidating the key adhesion receptors mediating platelet-vessel wall and platelet-platelet interactions. The vascular adhesion protein von Willebrand factor (vWf) plays a key role in supporting platelet adhesion to the damaged vessel wall and binds to two adhesion receptors on the platelet surface, the glycoprotein (GP) Ib-V-IX complex and glycoprotein IIb-IIIa. The GP Ib-V-IX complex is a unique adhesion receptor which enables platelets to roll on a vWf matrix under conditions of rapid blood flow as well as transducing signals leading to the activation of GP IIb-IIIa. This latter receptor binds to a distinct site on vWf and is essential for stabilising platelet adhesion to the site of vessel wall injury. In addition to supporting platelet adhesion, GP IIb-IIIa plays a key role in a number of other platelet responses including platelet spreading, aggregation, the release of procoagulant-rich microvesicles, and clot retraction. Given its central role in platelet function GP IIb-IIIa has become an attractive target for the development of novel anti-thrombotic agents. In this paper, we consider the advantages of inhibitors of GP IIb-IIIa compared with other established anti-platelet drugs including aspirin and ticlopidine, and also discuss some potential problems associated with the inhibition of GP IIb/IIIa and other platelet adhesion receptors.
Development of a carotid artery thrombolysis stroke model in mice
Publisher: American Society of Hematology
Date: 23-09-2022
DOI: 10.1182/BLOODADVANCES.2021006008
Abstract: Recanalization with restored cerebral perfusion is the primary goal of thrombolytic therapy in acute ischemic stroke. The identification of adjunctive therapies that can be safely used to enhance thrombolysis in stroke remains an elusive goal. We report here the development of a mouse in situ carotid artery thrombolysis (iCAT) stroke model involving graded cerebral ischemia to induce unihemispheric infarction after thrombotic occlusion of the common carotid artery (CCA). Electrolytic-induced thrombotic occlusion of the left CCA enabled real-time assessment of recanalization and rethrombosis events after thrombolysis with recombinant tissue-type plasminogen activator (rtPA). Concurrent transient stenosis of the right CCA induced unihemispheric hypoperfusion and infarction in the left middle cerebral artery territory. Real-time assessment of thrombolysis revealed recanalization rates & % in rtPA-treated animals with high rates of rethrombosis. Addition of the direct thrombin inhibitor argatroban increased recanalization rates to 50% and reduced rethrombosis. Paradoxically, this was associated with increased cerebral ischemia and stroke-related mortality (25%-42%). Serial analysis of carotid and cerebral blood flow showed that coadministration of argatroban with rtPA resulted in a marked increase in carotid artery embolization, leading to distal obstruction of the middle cerebral artery. Real-time imaging of carotid thrombi revealed that adjunctive anticoagulation destabilized platelet-rich thrombi at the vessel wall, leading to dislodgement of large platelet emboli. These studies confirm the benefits of anticoagulants in enhancing thrombolysis and large artery recanalization however, at high levels of anticoagulation (∼3-fold prolongation of activated partial thromboplastin time), this effect is offset by increased incidence of carotid artery embolization and distal middle cerebral artery occlusion. The iCAT stroke model should provide important new insight into the effects of adjunctive antithrombotic agents on real-time thrombus dynamics during thrombolysis and their correlation with stroke outcomes.
Specific function of phosphoinositide 3-kinase beta in the control of DNA replication
Publisher: Proceedings of the National Academy of Sciences
Date: 05-05-2009
Abstract: Class I A phosphoinositide 3-kinase (PI3K) are enzymes comprised of a p85 regulatory and a p110 catalytic subunit that induce formation of 3-polyphosphoinositides, which activate numerous downstream targets. PI3K controls cell ision. Of the 2 ubiquitous PI3K isoforms, α has selective action in cell growth and cell cycle entry, but no specific function in cell ision has been described for β. We report here a unique function for PI3Kβ in the control of DNA replication. PI3Kβ regulated DNA replication through kinase-dependent and kinase-independent mechanisms. PI3Kβ was found in the nucleus, where it associated PKB. Modulation of PI3Kβ activity altered the DNA replication rate by controlling proliferating cell nuclear antigen (PCNA) binding to chromatin and to DNA polymerase δ. PI3Kβ exerted this action by regulating the nuclear activation of PKB in S phase, and in turn phosphorylation of PCNA negative regulator p21 Cip . Also, p110β associated with PCNA and controlled PCNA loading onto chromatin in a kinase-independent manner. These results show a selective function of PI3Kβ in the control of DNA replication.
Purification and characterisation of a snake venom phospholipase A2: A potent inhibitor of platelet aggregation
Publisher: Elsevier BV
Date: 06-1993
DOI: 10.1016/0049-3848(93)90089-7
Abstract: An inhibitor of human platelet aggregation was identified from the venom of an Australian Copperhead snake, Austrelaps superba, as a novel phospholipase A2. The inhibitor was purified to homogeneity by chromatography on Q-Sepharose, S-Sepharose and C8 reverse phase HPLC. The purified phospholipase A2 has a molecular weight of 15 kDa as assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequence analysis of the platelet inhibitor revealed 70-80% sequence identity to other previously described secretory phospholipase A2. Phospholipase activity of the purified protein was confirmed by the ability of the enzyme to hydrolyse lecithin. Pretreatment of the purified protein with the specific phospholipase A2 inhibitor p-bromophenacyl bromide, resulted in abrogation of both its enzyme and platelet inhibitory activity. The phospholipase A2 inhibited platelet aggregation and serotonin release, induced by a variety of platelet agonists, in a time and dose dependent manner.
Phenotype changes resulting in high-affinity binding of von Willebrand factor to recombinant glycoprotein Ib-IX: analysis of the platelet-type von Willebrand disease mutations
Publisher: American Society of Hematology
Date: 15-09-2001
Abstract: To maintain hemostasis under shear conditions, there must be an interaction between the platelet glycoprotein (GP) Ib-IX receptor and the plasma ligand von Willebrand factor (vWf). In platelet-type von Willebrand disease (Pt-vWD), hemostasis is compromised. Two mutations in the GPIbα polypeptide chain have been identified in these patients—a glycine-233 to valine change and a methionine-239 to valine change. For this investigation, these mutant proteins have been expressed in a Chinese hamster ovary cell model system. Ligand-binding studies were performed at various concentrations of ristocetin, and adhesion assays were performed under flow conditions. The Pt-vWD mutations resulted in a gain-of-function receptor. vWf binding was increased at all concentrations of ristocetin examined, and adhesion on a vWf matrix was enhanced in terms of cell tethering, slower rolling velocity, and decreased detachment with increasing shear rate. Two other mutations were also introduced into the GPIbα chain. One mutation, encompassing both the Pt-vWD mutations, created an increase in the hydrophobicity of this region. The second mutation, involving a valine-234 to glycine change, decreased the hydrophobicity of this region. Both mutations also resulted in a gain-of-function receptor, with the double mutation producing a hyperreactive receptor for vWf. These data further support the hypothesis that ligand binding is regulated by conformational changes in the amino-terminal region of GPIbα, thereby influencing the stability of the GPIbα–vWf interaction.
Novel role for insulin as an autocrine growth factor for malignant brain tumour cells
Publisher: Portland Press Ltd.
Date: 26-07-2007
DOI: 10.1042/BJ20070309
Abstract: AT/RTs (atypical teratoid/rhabdoid tumours) of the CNS (central nervous system) are childhood malignancies associated with poor survival rates due to resistance to conventional treatments such as chemotherapy. We characterized a panel of human AT/RT and MRT (malignant rhabdoid tumour) cell lines for expression of RTKs (receptor tyrosine kinases) and their involvement in tumour growth and survival. When compared with normal brain tissue, AT/RT cell lines overexpressed the IR (insulin receptor) and the IGFIR (insulin-like growth factor-I receptor). Moreover, insulin was secreted by AT/RT cells grown in serum-free medium. Insulin potently activated Akt (also called protein kinase B) in AT/RT cells, as compared with other growth factors, such as epidermal growth factor. Pharmacological inhibitors, neutralizing antibodies, or RNAi (RNA interference) targeting the IR impaired the growth of AT/RT cell lines and induced apoptosis. Inhibitors of the PI3K (phosphoinositide 3-kinase)/Akt pathway also impaired basal and insulin-stimulated AT/RT cell proliferation. Experiments using RNAi and isoform-specific pharmacological inhibitors established a key role for the class IA PI3K p110α isoform in AT/RT cell growth and insulin signalling. Taken together, our results reveal a novel role for autocrine signalling by insulin and the IR in growth and survival of malignant human CNS tumour cells via the PI3K/Akt pathway.
A Mechanistic Model for Paradoxical Platelet Activation by Ligand-Mimetic αIIbβ3(GPIIb/IIIa) Antagonists
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-2007
DOI: 10.1161/01.ATV.0000255307.65939.59
Abstract: Objective— Integrins are attractive therapeutic targets. Inhibition of integrin α IIb β 3 effectively blocks platelet aggregation. However, limitations with intravenous α IIb β 3 antagonists and failure of oral α IIb β 3 antagonists prompted doubts on the current concept of ligand-mimetic integrin blockade. Methods and Results— Evaluating P-selectin expression on platelets by flow cytometry, we report a mechanism of paradoxical platelet activation by ligand-mimetic α IIb β 3 antagonists and define three requirements: (1) Induction of ligand-bound conformation of α IIb β 3 , (2) receptor clustering, (3) prestimulation of platelets. Conformational change is inducible by clinically used ligand-mimetic α IIb β 3 antagonists, RGD-peptides, and anti-LIBS antibodies. In a mechanistic experimental model, clustering is achieved by crosslinking integrins via antibodies, and preactivation is induced by low-dose ADP. Finally, we demonstrate that platelet adhesion on collagen represents an in vivo correlate of platelet prestimulation and receptor clustering, in which the presence of ligand-mimetic α IIb β 3 antagonists results in platelet activation as detected by P-selectin, CD63, and CD40L expression as well as by measuring Ca 2+ -signaling. Blockade of the ADP receptor P2Y 12 by AR-C69931MX and clopidogrel inhibits α IIb β 3 antagonist-induced platelet activation. Conclusion— These findings can explain limitations of ligand-mimetic anti-α IIb β 3 therapy. They describe potential benefits of concomitant ADP receptor blockade and support a shift in drug development from ligand-mimetic toward allosteric or activation-specific integrin antagonists.
Antiplatelet therapy: in search of the 'magic bullet'
Publisher: Springer Science and Business Media LLC
Date: 10-2003
DOI: 10.1038/NRD1198
Platelet endothelial cell adhesion molecule-1 is a negative regulator of platelet-collagen interactions
Publisher: American Society of Hematology
Date: 09-2001
Abstract: The functional importance of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) in platelets is unclear. Because PECAM-1 represents a newly assigned immunoglobulin–ITIM superfamily member expressed on the surface of platelets, it was hypothesized that it may play an important regulatory role in modulating ITAM-bearing receptors such as collagen (GP)VI receptor and FcγRIIA. To examine the functional role of PECAM-1 in regulating platelet-collagen interactions, 2 different approaches were applied using recombinant human PECAM-1–immunoglobulin chimeras and platelets derived from PECAM-1–deficient mice. Stimulation of platelets by collagen-, (GP)VI-selective agonist, collagen-related peptide (CRP)–, and PECAM-1–immunoglobulin chimera induced tyrosine phosphorylation of PECAM-1 in a time- and dose-dependent manner. Activation of PECAM-1 directly through the addition of soluble wild-type PECAM-1–immunoglobulin chimera, but not mutant K89A PECAM-1–immunoglobulin chimera that prevents homophilic binding, was found to inhibit collagen- and CRP-induced platelet aggregation. PECAM-1–deficient platelets displayed enhanced platelet aggregation and secretion responses on stimulation with collagen and CRP, though the response to thrombin was unaffected. Under conditions of flow, human platelet thrombus formation on a collagen matrix was reduced in a dose-dependent manner by human PECAM-1–immunoglobulin chimera. Platelets derived from PECAM-1–deficient mice form larger thrombi when perfused over a collagen matrix under flow at a shear rate of 1800 seconds−1 compared to wild-type mice. Collectively, these results indicate that PECAM-1 serves as a physiological negative regulator of platelet-collagen interactions that may function to negatively limit growth of platelet thrombi on collagen surfaces.
PI 3-kinase p110β: A new target for antithrombotic therapy
Publisher: Springer Science and Business Media LLC
Date: 17-04-2005
DOI: 10.1038/NM1232
Abstract: Platelet activation at sites of vascular injury is essential for the arrest of bleeding however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin alpha(IIb)beta(3) (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110beta isoform in regulating the formation and stability of integrin alpha(IIb)beta(3) adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110beta inhibitors have been developed which prevent formation of stable integrin alpha(IIb)beta(3) adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110beta as an important new target for antithrombotic therapy.
A multimode-TIRFM and microfluidic technique to examine platelet adhesion dynamics
Publisher: Humana Press
Date: 2013
DOI: 10.1007/978-1-62703-538-5_3
Abstract: Fluorescence microscopy techniques have provided important insights into the structural and signalling events occurring during platelet adhesion under both static and blood flow conditions. However, due to limitations in sectioning ability and sensitivity these techniques are restricted in their capacity to precisely image the adhesion footprint of spreading platelets. In particular, investigation of platelet adhesion under hemodynamic shear stress requires an imaging platform with high spatial discrimination and sensitivity and rapid temporal resolution. This chapter describes in detail a multimode imaging approach combining total internal reflection fluorescence microscopy (TIRFM) with high speed epifluorescence and differential interference contrast (DIC) microscopy along with a novel microfluidic perfusion system developed in our laboratory to examine platelet membrane adhesion dynamics under static and flow conditions.
A revised model of platelet aggregation
Publisher: American Society for Clinical Investigation
Date: 15-03-2000
DOI: 10.1172/JCI7569
14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function
Publisher: Springer Science and Business Media LLC
Date: 27-09-2016
DOI: 10.1038/NCOMMS12862
Abstract: The 14-3-3 family of adaptor proteins regulate erse cellular functions including cell proliferation, metabolism, adhesion and apoptosis. Platelets express numerous 14-3-3 isoforms, including 14-3-3ζ, which has previously been implicated in regulating GPIbα function. Here we show an important role for 14-3-3ζ in regulating arterial thrombosis. Interestingly, this thrombosis defect is not related to alterations in von Willebrand factor (VWF)–GPIb adhesive function or platelet activation, but instead associated with reduced platelet phosphatidylserine (PS) exposure and procoagulant function. Decreased PS exposure in 14-3-3ζ-deficient platelets is associated with more sustained levels of metabolic ATP and increased mitochondrial respiratory reserve, independent of alterations in cytosolic calcium flux. Reduced platelet PS exposure in 14-3-3ζ-deficient mice does not increase bleeding risk, but results in decreased thrombin generation and protection from pulmonary embolism, leading to prolonged survival. Our studies define an important role for 14-3-3ζ in regulating platelet bioenergetics, leading to decreased platelet PS exposure and procoagulant function.
New insights into the haemostatic function of platelets
Publisher: Wiley
Date: 27-10-2009
DOI: 10.1111/J.1365-2141.2009.07819.X
Abstract: Considerable progress has been made over the last two decades in delineating the key molecular events regulating the haemostatic function of platelets. Much of this new insight has been derived from the study of mouse models, in which the expression or structure of one or more platelet proteins has been genetically altered. Despite these advances on the research front, clinical progress in diagnosing patients with unexplained surgical bleeding or recurrent haemorrhage from mucocutaneous sites has been comparatively limited. There is a dearth of literature available to help physicians integrate and apply the burgeoning knowledge on platelet biology to diagnosing patients with atypical or unexplained platelet dysfunction. The purpose of this review is to summarise the major primary platelet disorders relevant to pathological bleeding in humans (excluding those primarily due to thrombocytopenia or acquired functional disorders), with a focus on lesions identified in mouse models that could represent candidate molecules for study in patients with impaired platelet function.
Collagen surfaces to measure thrombus formation under flow: possibilities for standardization
Publisher: Elsevier BV
Date: 04-2011
Dynamic Aspects Of Platelet Adhesion Under Flow
Publisher: Wiley
Date: 11-05-2001
DOI: 10.1046/J.1440-1681.2001.03468.X
Abstract: 1. Cell–cell and cell–matrix adhesive interactions are critical for a wide range of physiological processes, including embryogenesis, inflammation, immunity and haemostasis. 2. The ability of circulating blood cells, such as platelets and leucocytes, to adhere to sites of vascular injury is complicated by the presence of blood flow, which imposes hydrodynamic forces on adhesion contacts. 3. To overcome this problem, platelets and leucocytes have evolved specific adhesion receptors with unique biomechanical properties that enable these cells to adhere to the vessel wall under flow conditions. 4. Platelet adhesion in the normal circulation appears to be a multiple‐step process involving an initial reversible interaction between the platelet adhesion receptor glycoprotein Ib‐IX‐V and the vascular adhesion protein von Willebrand factor. Once tethered to the vessel wall, platelets form irreversible adhesion contacts through the binding of one or more platelet integrins to specific subendothelial matrix proteins. 5. There is now a wealth of evidence demonstrating that these receptors not only mediate platelet adhesion, but also transduce signals leading to platelet activation. 6. In the present review, we will briefly discuss the current understanding of the specific roles of in idual platelet receptors in supporting the haemostatic function of platelets and discuss mechanisms by which these receptors induce platelet activation.
Platelet Factor XIII and Calpain Negatively Regulate Integrin αIIbβ3 Adhesive Function and Thrombus Growth
Publisher: Elsevier BV
Date: 07-2004
Targeting the PI3K p110α Isoform Inhibits Medulloblastoma Proliferation, Chemoresistance, and Migration
Publisher: American Association for Cancer Research (AACR)
Date: 11-2008
DOI: 10.1158/1078-0432.CCR-08-0385
Abstract: Purpose: The phosphoinositide 3-kinase (PI3K)/Akt pathway is frequently activated in human cancer and plays a crucial role in medulloblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K/Akt signaling as a novel antiproliferative approach in medulloblastoma. Experimental Design: The expression pattern and functions of class IA PI3K isoforms were investigated in medulloblastoma tumour s les and cell lines. Effects on cell survival and downstream signaling were analyzed following down-regulation of p110α, p110β, or p110δ by means of RNA interference or inhibition with isoform-specific PI3K inhibitors. Results: Overexpression of the catalytic p110α isoform was detected in a panel of primary medulloblastoma s les and cell lines compared with normal brain tissue. Down-regulation of p110α expression by RNA interference impaired the growth of medulloblastoma cells, induced apoptosis, and led to decreased migratory capacity of the cells. This effect was selective, because RNA interference targeting of p110β or p110δ did not result in a comparable impairment of DAOY cell survival. Isoform-specific p110αinhibitors also impaired medulloblastoma cell proliferation and sensitized the cells to chemotherapy. Medulloblastoma cells treated with p110α inhibitors further displayed reduced activation of Akt and the ribosomal protein S6 kinase in response to stimulation with hepatocyte growth factor and insulin-like growth factor-I. Conclusions: Together, our data reveal a novel function of p110α in medulloblastoma growth and survival.
Conversion of platelets from a proaggregatory to a proinflammatory adhesive phenotype: role of PAF in spatially regulating neutrophil adhesion and spreading
Publisher: American Society of Hematology
Date: 15-09-2007
DOI: 10.1182/BLOOD-2006-08-040980
Abstract: The ability of platelets to provide a highly reactive surface for the recruitment of other platelets and leukocytes to sites of vascular injury is critical for hemostasis, atherothrombosis, and a variety of inflammatory diseases. The mechanisms coordinating platelet-platelet and platelet-leukocyte interactions have been well defined and, in general, it is assumed that increased platelet activation correlates with enhanced reactivity toward other platelets and neutrophils. In the current study, we demonstrate a differential role for platelets in supporting platelet and neutrophil adhesive interactions under flow. We demonstrate that the conversion of spread platelets to microvesiculated procoagulant (annexin A5–positive [annexin A5+ve]) forms reduces platelet-platelet adhesion and leads to a paradoxical increase in neutrophil-platelet interaction. This enhancement in neutrophil adhesion and spreading is partially mediated by the proinflammatory lipid, platelet-activating factor (PAF). PAF production, unlike other neutrophil chemokines (IL-8, GRO-α, NAP-2, IL-1β) is specifically and markedly up-regulated in annexin A5+ve cells. Physiologically, this spatially controlled production of PAF plays an important role in localizing neutrophils on the surface of thrombi. These studies define for the first time a specific proinflammatory function for annexin A5+ve platelets. Moreover, they demonstrate an important role for platelet-derived PAF in spatially regulating neutrophil adhesion under flow.
Antithrombotic phosphoinositide 3‐kinase β inhibitors in humans: a ‘shear’ delight!
Publisher: Elsevier BV
Date: 10-2012
Role of the intracellular domains of GPIb in controlling the adhesive properties of the platelet GPIb/V/IX complex
Publisher: American Society of Hematology
Date: 05-2003
DOI: 10.1182/BLOOD-2002-06-1847
Abstract: Glycoprotein (GP) Ib/V/IX complex–dependent platelet adhesion to von Willebrand factor (VWF) is supported by the 45-kd N-terminal extracellular domain of the GPIbα subunit. Recent results with an adhesion blocking antibody (RAM.1) against GPIbβ, which is disulfide linked to GPIbα, have suggested a novel function of this subunit in regulating VWF-mediated platelet adhesion, possibly involving its intracellular face. A putative cooperation between the GPIbα and GPIbβ cytoplasmic domains was investigated by measuring the adhesion under flow to immobilized VWF of K562 and Chinese hamster ovary (CHO) cells transfected with GPIb/(V)/IX containing mutations in this region. Adhesion of cells carrying a glycine substitution of the GPIbβ Ser166 phosphorylation site was 50% lower than normal and became insensitive to inhibition by RAM.1. In contrast, forskolin or PGE1 treatment increased both the phosphorylation of GPIbβ and adhesion of control cells, both effects being reversed by RAM.1, but had no influence on cells expressing the Ser166Gly mutation. A role of the GPIbα intracellular domain was also apparent as the VWF-dependent adhesion of cells containing deletions of the entire (Δ518-610) or portions (Δ535-568, Δ569-610) of the GPIbα cytoplasmic tail was insensitive to RAM.1 inhibition. Cells carrying progressive 11 amino acid deletions spanning the GPIbα 535-590 region were equally unresponsive to RAM.1, with the exception of those containing GPIbα Δ569-579, which behaved like control cells. These findings support a role of the GPIbβ intracellular domain in controlling the adhesive properties of the GPIb/V/IX complex through phosphorylation of GPIbβ Ser166 and point to the existence of cross-talk between the GPIbβ and GPIbα intracellular domains.
Mosquito-Derived Anophelin Sulfoproteins Are Potent Antithrombotics
Publisher: American Chemical Society (ACS)
Date: 28-03-2018
Identification of a unique filamin A binding region within the cytoplasmic domain of glycoprotein Ibα
Publisher: Portland Press Ltd.
Date: 26-04-2005
DOI: 10.1042/BJ20041836
Abstract: Binding of the platelet GPIb/V/IX (glycoprotein Ib/V/IX) receptor to von Willebrand factor is critical for platelet adhesion and aggregation under conditions of rapid blood flow. The adhesive function of GPIbα is regulated by its anchorage to the membrane skeleton through a specific interaction with filamin A. In the present study, we examined the amino acid residues within the cytoplasmic tail of GPIbα, which are critical for association with filamin A, using a series of 25-mer synthetic peptides that mimic the cytoplasmic tail sequences of wild-type and mutant forms of GPIbα. Peptide binding studies of purified human filamin A have demonstrated a major role for the conserved hydrophobic stretch L567FLWV571 in mediating this interaction. Progressive alanine substitutions of triple, double and single amino acid residues within the Pro561–Arg572 region suggested an important role for Trp570 and Phe568 in promoting GPIbα binding to filamin A. The importance of these two residues in promoting filamin A binding to GPIbα in vivo was confirmed from the study of Chinese-hamster ovary cells expressing GPIbα Trp570→Ala and Phe568→Ala substitutions. Phenotypic analysis of these cell lines in flow-based adhesion studies revealed a critical role for these residues in maintaining receptor anchorage to the membrane skeleton and in maintaining cell adhesion to a von Willebrand factor matrix under high-shear conditions. These studies demonstrate a novel filamin A binding motif in the cytoplasmic tail of GPIbα, which is critically dependent on both Trp570 and Phe568.
Generation and characterization of mice with null mutation of the chloride intracellular channel 1 gene
Publisher: Wiley
Date: 2010
DOI: 10.1002/DVG.20616
Dynamics of platelet thrombus formation
Publisher: Elsevier BV
Date: 07-2009
DOI: 10.1111/J.1538-7836.2009.03401.X
Abstract: Platelet aggregation and thrombus formation at sites of atherosclerotic plaque rupture is a dynamic process that can lead to intermittent or permanent obstruction to blood flow, resulting in ischemic tissue injury and organ dysfunction. There is a growing body of evidence suggesting that the dynamics of platelet aggregation and initial thrombus development are regulated by two distinct, complementary processes, involving: (i) rheological (biomechanical) and (ii) soluble-agonist-dependent mechanisms. Rheological-dependent platelet aggregation occurs between discoid platelets and requires the biomechanical adhesive and signaling function (mechanotransduction) of the major platelet adhesion receptors, GPIb and integrin alpha(IIb)beta3. Soluble agonists further potentiate platelet activation, stimulating global platelet shape change and degranulation, and play a major role in stabilizing formed aggregates. Unraveling the dynamics of platelet aggregation and thrombus formation in vivo requires consideration of the cooperative interplay between rheological- and soluble agonist-dependent platelet aggregation mechanisms.
The contribution of thrombin-induced platelet activation to thrombus growth is diminished under pathological blood shear conditions
Publisher: Georg Thieme Verlag KG
Date: 2012
DOI: 10.1160/TH11-06-0418
Abstract: Developing novel anti-platelet therapies is an important clinical strategy for the prevention of arterial thromboses which cause heart attacks and most strokes. Thrombin activates platelets via protease-activated receptors (PARs), and PAR antagonists are currently under investigation as antithrombotics. Yet despite these clinical advances, the importance of PARs to platelet activation during thromboses formed under pathological conditions has not been investigated. To this end, we examined the role of PAR-dependent platelet activation in thrombus formation in the presence of elevated blood shear rates. We used two in vivo thrombosis models and an ex vivo whole blood flow approach in PAR4-/-mice, whose platelets are unresponsive to thrombin, to show that the contribution of PAR-mediated platelet activation to thrombosis is diminished at pathological blood shear rates as a direct result of decreased incorporation of thrombin-activated platelets into growing thrombi. Our ex vivo observations were replicated in human whole blood treated with a PAR1 antagonist. These results define a novel, shear-regulated role for thrombin/PAR-dependent platelet activation during thrombosis and provide important insights into the conditions under which PAR antagonists may best be used for the prevention of acute coronary syndromes.
Pathophysiological levels of soluble P-selectin mediate adhesion of leukocytes to the endothelium through mac-1 activation
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-11-2008
DOI: 10.1161/CIRCRESAHA.108.180273
Abstract: Plasma soluble P-selectin (sP-selectin) levels are increased in pathologies associated with atherosclerosis, including peripheral arterial occlusive disease (PAOD). However, the role of sP-selectin in regulating leukocyte–endothelial adhesion is unclear. The aim of this study was to assess the ability of exogenous and endogenous sP-selectin to induce leukocyte responses that promote their adhesion to various forms of endothelium. In flow chamber assays, sP-selectin dose-dependently increased neutrophil adhesion to resting human iliac artery endothelial cells. Similarly, sP-selectin induced neutrophil adhesion to the endothelial surface of murine aortae and human radial venous segments in ex vivo flow chamber experiments. Using intravital microscopy to examine postcapillary venules in the mouse cremaster muscle, in vivo administration of sP-selectin was also found to significantly increase leukocyte rolling and adhesion in unstimulated postcapillary venules. Using a Mac-1–specific antibody and P-selectin knockout mouse, it was demonstrated that this finding was dependent on a contribution of Mac-1 to leukocyte rolling and endothelial P-selectin expression. This was confirmed in an ex vivo perfusion model using viable mouse aorta and human radial vessels. In contrast, with tumor necrosis factor-α–activated endothelial cells and intact endothelium, where neutrophil adhesion was already elevated, sP-selectin failed to further increase adhesion. Plasma s les from PAOD patients containing pathologically elevated concentrations of sP-selectin also increased neutrophil adhesion to the endothelium in a sP-selectin–dependent manner, as demonstrated by immunodepletion of sP-selectin. These studies demonstrate that raised plasma sP-selectin may influence the early progression of vascular disease by promoting leukocyte adhesion to the endothelium in PAOD, through Mac-1–mediated rolling and dependent on endothelial P-selectin expression.
Calpain regulation of cytoskeletal signaling complexes in von Willebrand factor-stimulated platelets: Distinct roles for glycoprotein Ib-V-IX and glycoprotein IIb-IIIa (integrin α(IIb)β3 ) in von willebrand factor- induced signal transduction
Publisher: Elsevier BV
Date: 08-1997
Thrombin-dependent intravascular leukocyte trafficking regulated by fibrin and the platelet receptors GPIb and PAR4
Publisher: Springer Science and Business Media LLC
Date: 23-07-2015
DOI: 10.1038/NCOMMS8835
Abstract: Thrombin is a central regulator of leukocyte recruitment and inflammation at sites of vascular injury, a function thought to involve primarily endothelial PAR cleavage. Here we demonstrate the existence of a distinct leukocyte-trafficking mechanism regulated by components of the haemostatic system, including platelet PAR4, GPIbα and fibrin. Utilizing a mouse endothelial injury model we show that thrombin cleavage of platelet PAR4 promotes leukocyte recruitment to sites of vascular injury. This process is negatively regulated by GPIbα, as seen in mice with abrogated thrombin-platelet GPIbα binding (hGPIbα(D277N)). In addition, we demonstrate that fibrin limits leukocyte trafficking by forming a physical barrier to intravascular leukocyte migration. These studies demonstrate a distinct 'checkpoint' mechanism of leukocyte trafficking involving balanced thrombin interactions with PAR4, GPIbα and fibrin. Dysregulation of this checkpoint mechanism is likely to contribute to the development of thromboinflammatory disorders.
“Pulling strings”: shear, platelets, and microparticles
Publisher: American Society of Hematology
Date: 05-2006
Imaging signaling processes in platelets
Publisher: Elsevier BV
Date: 03-2006
DOI: 10.1016/J.BCMD.2005.12.009
Abstract: Unraveling the complex signaling processes regulating platelet adhesion has been a longstanding goal for those in the field of platelet research. Advances in high-speed live cell imaging techniques, taking advantage of developments in the area of fluorescent probe design hold considerable promise for the investigation of the dynamic signaling processes governing platelet activation and function, both physiologically and pathologically. This review broadly covers the application of existing imaging techniques to the investigation of platelet function and examines new developments in the area of live cell imaging that may have future applications in the field.
Leucine-rich repeats 2-4 (Leu60-Glu128) of platelet glycoprotein Ibalpha regulate shear-dependent cell adhesion to von Willebrand factor
Publisher: Elsevier BV
Date: 09-2006
Novel Role of Platelets in Mediating Inflammatory Responses and Ventricular Rupture or Remodeling Following Myocardial Infarction
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 04-2011
DOI: 10.1161/ATVBAHA.110.220467
Abstract: The goal of this study was to investigate the role of platelets in systemic and cardiac inflammatory responses and the development of postinfarct ventricular complications, as well as the efficacy of antiplatelet interventions. Using a mouse myocardial infarction (MI) model, we determined platelet accumulation and severity of inflammation within the infarcted myocardium by immunohistochemistry and biochemical assays, analyzed peripheral blood platelet-leukocyte conjugation using flow cytometry, and tested antiplatelet interventions, including thienopyridines and platelet depletion. Platelets accumulated within the infarcted region early post-MI and colocalized with inflammatory cells. MI evoked early increase in circulating platelet-leukocyte conjugation mediated by P-selectin/P-selectin glycoprotein ligand-1. Antiplatelet interventions inhibited platelet-leukocyte conjugation in peripheral blood, inflammatory infiltration, content of matrix metalloproteinases or plasminogen activation, and expression of inflammatory mediators in the infarcted myocardium (all P .05) and lowered rupture incidence ( P .01). Clopidogrel therapy alleviated the extent of chronic ventricular dilatation by serial echocardiography. Platelets play a pivotal role in promoting systemic and cardiac inflammatory responses post-MI. Platelets accumulate within the infarcted myocardium, contributing to regional inflammation, ventricular remodeling, and rupture. Antiplatelet therapy reduces the severity of inflammation and risk of post-MI complications, demonstrating a previously unrecognized protective action.
Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling
Publisher: Portland Press Ltd.
Date: 29-05-2007
DOI: 10.1042/BJ20070003
Abstract: Recent genetic knock-in and pharmacological approaches have suggested that, of class IA PI3Ks (phosphatidylinositol 3-kinases), it is the p110α isoform (PIK3CA) that plays the predominant role in insulin signalling. We have used isoform-selective inhibitors of class IA PI3K to dissect further the roles of in idual p110 isoforms in insulin signalling. These include a p110α-specific inhibitor (PIK-75), a p110α-selective inhibitor (PI-103), a p110β-specific inhibitor (TGX-221) and a p110δ-specific inhibitor (IC87114). Although we find that p110α is necessary for insulin-stimulated phosphorylation of PKB (protein kinase B) in several cell lines, we find that this is not the case in HepG2 hepatoma cells. Inhibition of p110β or p110δ alone was also not sufficient to block insulin signalling to PKB in these cells, but, when added in combination with p110α inhibitors, they are able to significantly attenuate insulin signalling. Surprisingly, in J774.2 macrophage cells, insulin signalling to PKB was inhibited to a similar extent by inhibitors of p110α, p110β or p110δ. These results provide evidence that p110β and p110δ can play a role in insulin signalling and also provide the first evidence that there can be functional redundancy between p110 isoforms. Further, our results indicate that the degree of functional redundancy is linked to the relative levels of expression of each isoform in the target cells.
Signaling events underlying thrombus formation
Publisher: Elsevier BV
Date: 07-2003
DOI: 10.1046/J.1538-7836.2003.00267.X
Abstract: Recent in vivo studies have highlighted the dynamic and complex nature of platelet thrombus growth and the requirement for multiple adhesive receptor-ligand interactions in this process. In particular, the importance of von Willebrand factor (VWF) in promoting both primary adhesion and aggregation under high shear conditions is now well established. In general, the efficiency with which platelets adhere and aggregate at sites of vessel wall injury is dependent on the synergistic action of various adhesive and soluble agonist receptors, with the contribution of each of the in idual receptors dependent on the prevailing blood flow conditions. In this review, we will discuss the major platelet adhesive interactions regulating platelet thrombus formation under high shear, with specific focus on the VWF (GPIb and integrin alphaIIbbeta3) and collagen receptors (GPVI and integrin alpha2beta1). We will also discuss the signaling mechanisms utilized by these receptors to induce platelet activation with specific emphasis on the role of cytosolic calcium flux in regulating platelet adhesion dynamics. The role of soluble agonists in promoting thrombus growth will be highlighted and a model to explain the synergistic requirement for adhesive and soluble stimuli for efficient platelet aggregation will be discussed.
Malaria's journey through the lymph node.
Publisher: Springer Science and Business Media LLC
Date: 09-2007
DOI: 10.1038/NM0907-1015
Thromboinflammatory Functions of Platelets in Ischemia-Reperfusion Injury and Its Dysregulation in Diabetes.
Publisher: Georg Thieme Verlag KG
Date: 02-01-2018
Abstract: Ischemia–reperfusion (IR) injury is a common complication of a variety of cardiovascular diseases, including ischemic stroke and myocardial infarction (MI). While timely re-establishment of blood flow in a thrombosed artery is the primary goal of acute therapy in these diseases, paradoxically, reperfusion of ischemic tissue can cause widespread microvascular dysfunction that significantly exacerbates organ damage. Reperfusion injury is associated with activation of the humoral and cellular components of the hemostatic and innate immune systems and also with excessive reactive oxygen species production, endothelial dysfunction, thrombosis, and inflammation. Platelets are critical mediators of thromboinflammation during reperfusion injury and a hyperactive platelet phenotype may contribute to an exaggerated IR injury response. This is particularly relevant to diabetes which is characteristically associated with hyperactive platelets, significantly worse IR injury, increased organ damage, and increased risk of death. However, the mechanisms underlying vulnerability to IR injury in diabetic in iduals is not well defined, nor the role of “diabetic platelets” in this process. This review summarizes recent progress in understanding the role of platelets in promoting microvascular dysfunction and inflammation in the context of IR injury. Furthermore, the authors discuss aspects of the thromboinflammatory function of platelets that are dysregulated in diabetes. They conclude that diabetes likely enhances the capacity of platelets to mediate microvascular thrombosis and inflammation during IR injury, which has potentially important implications for the future design of antiplatelet agents that can reduce microvascular dysfunction and inflammation.
Structural and hydrodynamic simulation of an acute stenosis-dependent thrombosis model in mice
Publisher: Elsevier BV
Date: 04-2011
DOI: 10.1016/J.JBIOMECH.2011.02.006
Abstract: Platelet activation under blood flow is thought to be critically dependent on the autologous secretion of soluble platelet agonists (chemical activators) such as ADP and thromboxane. However, recent evidence challenging this model suggests that platelet activation can occur independent of soluble agonist signalling, in response to the mechanical effects of micro-scale shear gradients. A key experimental tool utilized to define the effect of shear gradients on platelet aggregation is the murine intravital microscopy model of platelet thrombosis under conditions of acute controlled arteriolar stenosis. This paper presents a computational structural and hydrodynamic simulation of acute stenotic blood flow in the small bowel mesenteric vessels of mice. Using a homogeneous fluid at low Reynolds number (0.45) we investigated the relationship between the local hydrodynamic strain-rates and the severity of arteriolar stensosis. We conclude that the critical rates of blood flow acceleration and deceleration at sites of artificially induced stenosis (vessel side-wall compression or ligation) are a function of tissue elasticity. By implementing a structural simulation of arteriolar side wall compression, we present a mechanistic model that provides accurate simulations of stenosis in vivo and allows for predictions of the effects on local haemodynamics in the murine small bowel mesenteric thrombosis model.
A Sensitized RNA Interference Screen Identifies a Novel Role for the PI3K p110γ Isoform in Medulloblastoma Cell Proliferation and Chemoresistance
Publisher: American Association for Cancer Research (AACR)
Date: 07-2011
DOI: 10.1158/1541-7786.MCR-10-0200
Abstract: Medulloblastoma is the most common malignant brain tumor in children and is associated with a poor outcome. We were interested in gaining further insight into the potential of targeting the human kinome as a novel approach to sensitize medulloblastoma to chemotherapeutic agents. A library of small interfering RNA (siRNA) was used to downregulate the known human protein and lipid kinases in medulloblastoma cell lines. The analysis of cell proliferation, in the presence or absence of a low dose of cisplatin after siRNA transfection, identified new protein and lipid kinases involved in medulloblastoma chemoresistance. PLK1 (polo-like kinase 1) was identified as a kinase involved in proliferation in medulloblastoma cell lines. Moreover, a set of 6 genes comprising ATR, LYK5, MPP2, PIK3CG, PIK4CA, and WNK4 were identified as contributing to both cell proliferation and resistance to cisplatin treatment in medulloblastoma cells. An analysis of the expression of the 6 target genes in primary medulloblastoma tumor s les and cell lines revealed overexpression of LYK5 and PIK3CG. The results of the siRNA screen were validated by target inhibition with specific pharmacological inhibitors. A pharmacological inhibitor of p110γ (encoded by PIK3CG) impaired cell proliferation in medulloblastoma cell lines and sensitized the cells to cisplatin treatment. Together, our data show that the p110γ phosphoinositide 3-kinase isoform is a novel target for combinatorial therapies in medulloblastoma. Mol Cancer Res 9(7) 925–35. ©2011 AACR.
Corrigendum to “PI3-kinase p110α mediates β1 integrin-induced Akt activation and membrane protrusion during cell attachment and initial spreading” [Cell. Signal. 22 (2010) 1838–1848]
Publisher: Elsevier BV
Date: 04-2012
A live cell micro-imaging technique to examine platelet calcium signaling dynamics under blood flow
Publisher: Springer New York
Date: 17-11-2012
DOI: 10.1007/978-1-61779-307-3_6
Abstract: The platelet is a specialized adhesive cell that plays a key role in thrombus formation under both physiological and pathological blood flow conditions. Platelet adhesion and activation are dynamic processes associated with rapid morphological and functional changes, with the earliest signaling events occurring over a subsecond time-scale. The relatively small size of platelets combined with the dynamic nature of platelet adhesion under blood flow means that the investigation of platelet signaling events requires techniques with both high spatial discrimination and rapid temporal resolution. Unraveling the complex signaling processes governing platelet adhesive function under conditions of hemodynamic shear stress has been a longstanding goal in platelet research and has been greatly influenced by the development and application of microimaging-based techniques. Advances in the area of epi-fluorescence and confocal-based platelet calcium (Ca(2+)) imaging have facilitated the in vitro and in vivo elucidation of the early signaling events regulating platelet adhesion and activation. These studies have identified distinct Ca(2+) signaling mechanisms that serve to dynamically regulate activation of the major platelet integrin α(IIb)β(3) and associated adhesion and aggregation processes under flow. This chapter describes in detail a ratiometric calcium imaging protocol and associated troubleshooting procedures developed in our laboratory to examine live platelet Ca(2+) signaling dynamics. This technique provides a method for high-resolution imaging of the Ca(2+) dynamics underpinning platelet adhesion and thrombus formation under conditions of pathophysiological shear stress.
Identification of a 2-stage platelet aggregation process mediating shear-dependent thrombus formation
Publisher: American Society of Hematology
Date: 21-09-2007
DOI: 10.1182/BLOOD-2006-07-028282
Abstract: Disturbances of blood flow at sites of atherosclerotic plaque rupture are one of the key pathogenic events promoting platelet activation and arterial thrombus formation. Shear effects of platelets have been extensively investigated in vitro however, the mechanisms by which shear promotes platelet aggregation in vivo remain incompletely understood. By employing high-resolution imaging techniques to in vitro and in vivo thrombosis models, we demonstrate a unique mechanism initiating shear-dependent platelet aggregation involving aggregate formation between discoid platelets. These discoid platelet aggregates are initially unstable and result from the development of membrane tethers between coadhering platelets. Tether formation involves the adhesive function of GPIb/V/IX and integrin αIIbβ3, and conversion of discoid platelet aggregates into stable aggregates requires released ADP. The efficiency of this process is regulated by 3 independent variables, including the reactivity of the adhesive substrate, the level of shear flow, and the platelet density at the adhesive surface. These studies identify a new mechanism initiating platelet aggregation that is critically influenced by shear, physical proximity between translocating platelets, and membrane tether formation. Moreover, they provide a model to explain how the discoid morphology of platelets facilitates the maintenance of adhesive interactions with thrombogenic surfaces under high shear stress conditions.
Neutrophil macroaggregates promote widespread pulmonary thrombosis after gut ischemia
Publisher: American Association for the Advancement of Science (AAAS)
Date: 27-09-2017
DOI: 10.1126/SCITRANSLMED.AAM5861
Abstract: Neutrophil macroaggregates induce a distinct arterial-venous thrombotic response in the lung after gut ischemia.
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