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Johan W. M. Heemskerk - One of the best experts on this subject based on the ideXlab platform.
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atherosclerotic geometries exacerbate pathological Thrombus Formation poststenosis in a von willebrand factor dependent manner
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Erik Westein, Marijke J. E. Kuijpers, Andries D Van Der Meer, Jeanphilippe Frimat, Albert Van Den Berg, Johan W. M. HeemskerkAbstract:Rupture of a vulnerable atherosclerotic plaque causes Thrombus Formation and precipitates cardiovascular diseases. In addition to the thrombogenic content of a plaque, also the hemodynamic microenvironment plays a major role in Thrombus Formation. How the altered hemodynamics around a plaque promote pathological Thrombus Formation is not well understood. In this study, we provide evidence that plaque geometries result in fluid mechanical conditions that promote platelet aggregation and Thrombus Formation by increased accumulation and activity of von Willebrand factor (vWF) at poststenotic sites. Resonant-scanning multiphoton microscopy revealed that in vivo arterial stenosis of a damaged carotid artery markedly increased platelet aggregate Formation in the stenotic outlet region. Complementary in vitro studies using microfluidic stenotic chambers, designed to mimic the flow conditions in a stenotic artery, showed enhanced platelet aggregation in the stenotic outlet region at 60-80% channel occlusion over a range of input wall shear rates. The poststenotic Thrombus Formation was critically dependent on bloodborne vWF and autocrine platelet stimulation. In stenotic chambers containing endothelial cells, flow provoked increased endothelial vWF secretion in the stenotic outlet region, contributing to exacerbated platelet aggregation. Taken together, this study identifies a role for the shear-sensitive protein vWF in transducing hemodynamic forces that are present around a stenosis to a prothrombogenic microenvironment resulting in spatially confined and exacerbated platelet aggregation in the stenosis outlet region. The developed stenotic microfluidic chamber offers a realistic platform for in vitro evaluation of shear-dependent Thrombus Formation in the setting of atherosclerosis.
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measurement of whole blood Thrombus Formation using parallel plate flow chambers a practical guide
Platelets, 2012Co-Authors: Roger Van Kruchten, Judith M E M Cosemans, Johan W. M. HeemskerkAbstract:Custom-made and commercial parallel-plate flow chambers are widely used for studies of platelet activation and Thrombus Formation in whole blood at defined shear rates. When used in a reproducible way, such flow chamber devices give valuable inFormation on the thrombogenic potential of human, mouse, or rat blood. This article aims to provide a practical guide for the use of parallel-plate flow chambers in combination with routine microscopic imaging techniques. The following methodological aspects are addressed: preparation of surface coatings, calculation of blood flow and shear rate, control of pre-analytical variables, protocols for routine performing of flow chamber tests with non-coagulating or coagulating blood, and procedures for real-time and end-point analysis of Thrombus Formation. Frequently encountered experimental problems and artifacts are discussed, as well as possibilities for using flow chamber devices as a diagnostic tool to test antithrombotic medication.
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protein kinase c mediates platelet secretion and Thrombus Formation through protein kinase d2
Blood, 2011Co-Authors: O Konopatskaya, Judith M E M Cosemans, Johan W. M. Heemskerk, Karen Gilio, Sharon A Matthews, Maria N Navarro, Matthew T Harper, Christopher Williams, Deborah A Carter, Michael LeitgesAbstract:Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in Thrombus Formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and Thrombus Formation through its selective regulation of dense granule secretion.
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functional divergence of platelet protein kinase c pkc isoforms in Thrombus Formation on collagen
Journal of Biological Chemistry, 2010Co-Authors: Karen Gilio, Judith M E M Cosemans, Imke C. A. Munnix, Lenneke Prinzen, O Konopatskaya, Matthew T Harper, Michael Leitges, Jeffery D Molkentin, Qinghang Liu, Johan W. M. HeemskerkAbstract:Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and Thrombus Formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates Thrombus Formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit Thrombus Formation on collagen.
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molecular mri of early Thrombus Formation using a bimodal α2 antiplasmin based contrast agent
Jacc-cardiovascular Imaging, 2009Co-Authors: Robbertjan J H M Miserus, Johan W. M. Heemskerk, Veronica Herias, Lenneke Prinzen, Marc B I Lobbes, Robertjan Van Suylen, Anouk Dirksen, Tilman Mathias Hackeng, Jos M A Van Engelshoven, Mat J A P DaemenAbstract:Objectives We aimed to investigate whether early Thrombus Formation can be visualized with in vivo magnetic resonance imaging (MRI) by the use of a novel bimodal α2-antiplasmin–based contrast agent (CA). Background Thrombus Formation plays a central role in several vascular diseases. During the early phases of Thrombus Formation, activated factor XIII (FXIIIa) covalently cross-links α2-antiplasmin to fibrin, indicating the potential of α2-antiplasmin–based CAs in the detection of early Thrombus Formation. Methods A bimodal CA was synthesized by coupling gadolinium-diethylene triamine pentaacetic acid and rhodamine to an α2-antiplasmin–based peptide. For the control CA, a glutamine residue essential for cross-linking was replaced by alanine. In vitro-generated thrombi were exposed to both CAs and imaged by MRI and 2-photon laser-scanning microscopy. Immunohistochemistry was performed on human pulmonary thromboemboli sections to determine the presence of α2-antiplasmin and FXIII in different Thrombus remodeling phases. In vivo feasibility of the CA in detecting early Thrombus Formation specifically was investigated with MRI. Results In vitro–generated thrombi exposed to the α2-antiplasmin–based CA showed hyperintense magnetic resonance signal intensities at the Thrombus edge. No hyperintense signal was observed when we used the α2-antiplasmin–based CA in the presence of FXIII inhibitor dansylcadaverine nor when we used the control CA. Two-photon laser-scanning microscopy demonstrated that the α2-antiplasmin–based CA bound to fibrin. Immunohistochemistry demonstrated substantial α2-antiplasmin staining in fresh compared with lytic and organized thrombi. The administration of CA in vivo within seconds after inducing Thrombus Formation increased contrast-to-noise ratios (CNRs 2.28 ± 0.39, n=6) at the site of Thrombus Formation compared with the control CA (CNRs −0.14 ± 0.55, p = 0.003, n = 6) and α2-antiplasmin–based CA administration 24 to 48 h after Thrombus Formation (CNRs 0.11 ± 0.23, p = 0.006, n = 6). Conclusions A bimodal CA was developed, characterized, and validated. Our results showed that this bimodal CA enabled noninvasive in vivo magnetic resonance visualization of early Thrombus Formation.
Bruce Furie - One of the best experts on this subject based on the ideXlab platform.
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protein disulfide isomerase secretion following vascular injury initiates a regulatory pathway for Thrombus Formation
Nature Communications, 2017Co-Authors: Sheryl R Bowley, Barbara C Furie, Glenn Merrillskoloff, Chao Fang, Bruce FurieAbstract:Protein disulfide isomerase (PDI), secreted by platelets and endothelial cells on vascular injury, is required for Thrombus Formation. Using PDI variants that form mixed disulfide complexes with their substrates, we identify by kinetic trapping multiple substrate proteins, including vitronectin. Plasma vitronectin does not bind to αvβ3 or αIIbβ3 integrins on endothelial cells and platelets. The released PDI reduces disulfide bonds on plasma vitronectin, enabling vitronectin to bind to αVβ3 and αIIbβ3. In vivo studies of Thrombus generation in mice demonstrate that vitronectin rapidly accumulates on the endothelium and the platelet Thrombus following injury. This process requires PDI activity and promotes platelet accumulation and fibrin generation. We hypothesize that under physiologic conditions in the absence of secreted PDI, Thrombus Formation is suppressed and maintains a quiescent, patent vasculature. The release of PDI during vascular injury may serve as a regulatory switch that allows activation of proteins, among them vitronectin, critical for Thrombus Formation.
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extracellular thiol isomerases and their role in Thrombus Formation
Antioxidants & Redox Signaling, 2016Co-Authors: Sol Schulman, Barbara C Furie, Robert Flaumenhaft, Pavan K Bendapudi, Anish V Sharda, Vivien M Chen, Lola Bellidomartin, Reema Jasuja, Bruce FurieAbstract:Abstract Significance: The mammalian endoplasmic reticulum (ER) houses a large family of twenty thioredoxin-like proteins of which protein disulfide isomerase (PDI) is the archetypal member. Although the PDI family is best known for its role in oxidative protein folding of secretory proteins in the ER, these thioredoxin-like proteins fulfill ever-expanding roles, both within the secretory pathway and beyond. Recent Advances: Secreted PDI family proteins have now been shown to serve a critical role in platelet Thrombus Formation and fibrin generation. Utilizing intravital microscopy to visualize Thrombus Formation in mice, we have demonstrated the presence of extracellular PDI antigen during Thrombus Formation following injury of the vascular wall. Inhibition of PDI abrogates Thrombus Formation in vivo (16, 26, 46, 55). These observations have been extended to other PDI family members, including ERp57 (39, 116, 118, 123) and ERp5 (77). The vascular thiol isomerases are those PDI family members secreted fro...
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mechanisms of Thrombus Formation
The New England Journal of Medicine, 2008Co-Authors: Bruce Furie, Barbara C FurieAbstract:This review is an account of recent advances in our understanding of the mechanisms of Thrombus Formation, with emphasis on two independent pathways: one involving primarily platelets and the other initiated by tissue factor.
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a critical role for extracellular protein disulfide isomerase during Thrombus Formation in mice
Journal of Clinical Investigation, 2008Co-Authors: Jaehyung Cho, Barbara C Furie, Shaun R Coughlin, Bruce FurieAbstract:Thiol isomerases, including protein disulfide isomerase (PDI), catalyze disulfide oxidation, reduction, and isomerization, thereby playing an important role in protein synthesis. To determine whether extracellular PDI mediates Thrombus Formation in an animal model, PDI expression, platelet accumulation, and fibrin generation were monitored in the blood vessels of mice by intravital fluorescence microscopy following laser-induced arteriolar injury. A time-dependent increase in PDI was observed in murine thrombi following injury. Infusion of the PDI inhibitor bacitracin or a blocking monoclonal antibody against PDI inhibited platelet Thrombus Formation and fibrin generation. Fibrin deposition is normal in mice lacking the G protein-coupled platelet receptor Par4, although there is no stable accumulation of platelets. Infusion of monoclonal antibodies against PDI into the circulation of Par4(-/-) mice prior to vessel wall injury inhibited fibrin generation. These results indicate that PDI is required in vivo in mice for both fibrin generation and platelet Thrombus Formation.
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glycoprotein vi dependent and independent pathways of Thrombus Formation in vivo
Blood, 2006Co-Authors: Christophe Dubois, Laurence Panicotdubois, Barbara C Furie, Glenn Merrillskoloff, Bruce FurieAbstract:The role of the collagen receptor glycoprotein VI (GPVI) in arteriolar Thrombus Formation was studied in FcRγ-null mice (FcRγ–/–) lacking platelet surface GPVI. Thrombi were induced with severe or mild FeCl3 injury. Collagen exposure was significantly delayed and diminished in mild compared with severe FeCl3 injury. Times to initial Thrombus Formation and vessel occlusion were delayed in FcRγ–/– compared with wild-type mice after severe injury. Platelet accumulation in wild-type mice was decreased after mild compared with severe injury. However, there was little difference between platelet accumulation after severe or mild injury in FcRγ–/–. These data indicate a significant role for GPVI in FeCl3-induced Thrombus Formation. Pretreatment of wild-type mice with lepirudin further impaired mild FeCl3-induced Thrombus Formation, demonstrating a role for thrombin. Laser-induced Thrombus Formation in wild-type and FcRγ–/– was comparable. Collagen exposure to circulating blood was undetectable after laser injury. Normalized for Thrombus size, Thrombus-associated tissue factor was 5-fold higher in laser-induced thrombi than in severe FeCl3-induced thrombi. Thus, platelet activation by thrombin appears to be more important after laser injury than platelet activation by GPVI-collagen. It may thus be important when considering targets for antithrombotic therapy to use multiple animal models with diverse pathways to Thrombus Formation.
Barbara C Furie - One of the best experts on this subject based on the ideXlab platform.
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protein disulfide isomerase secretion following vascular injury initiates a regulatory pathway for Thrombus Formation
Nature Communications, 2017Co-Authors: Sheryl R Bowley, Barbara C Furie, Glenn Merrillskoloff, Chao Fang, Bruce FurieAbstract:Protein disulfide isomerase (PDI), secreted by platelets and endothelial cells on vascular injury, is required for Thrombus Formation. Using PDI variants that form mixed disulfide complexes with their substrates, we identify by kinetic trapping multiple substrate proteins, including vitronectin. Plasma vitronectin does not bind to αvβ3 or αIIbβ3 integrins on endothelial cells and platelets. The released PDI reduces disulfide bonds on plasma vitronectin, enabling vitronectin to bind to αVβ3 and αIIbβ3. In vivo studies of Thrombus generation in mice demonstrate that vitronectin rapidly accumulates on the endothelium and the platelet Thrombus following injury. This process requires PDI activity and promotes platelet accumulation and fibrin generation. We hypothesize that under physiologic conditions in the absence of secreted PDI, Thrombus Formation is suppressed and maintains a quiescent, patent vasculature. The release of PDI during vascular injury may serve as a regulatory switch that allows activation of proteins, among them vitronectin, critical for Thrombus Formation.
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extracellular thiol isomerases and their role in Thrombus Formation
Antioxidants & Redox Signaling, 2016Co-Authors: Sol Schulman, Barbara C Furie, Robert Flaumenhaft, Pavan K Bendapudi, Anish V Sharda, Vivien M Chen, Lola Bellidomartin, Reema Jasuja, Bruce FurieAbstract:Abstract Significance: The mammalian endoplasmic reticulum (ER) houses a large family of twenty thioredoxin-like proteins of which protein disulfide isomerase (PDI) is the archetypal member. Although the PDI family is best known for its role in oxidative protein folding of secretory proteins in the ER, these thioredoxin-like proteins fulfill ever-expanding roles, both within the secretory pathway and beyond. Recent Advances: Secreted PDI family proteins have now been shown to serve a critical role in platelet Thrombus Formation and fibrin generation. Utilizing intravital microscopy to visualize Thrombus Formation in mice, we have demonstrated the presence of extracellular PDI antigen during Thrombus Formation following injury of the vascular wall. Inhibition of PDI abrogates Thrombus Formation in vivo (16, 26, 46, 55). These observations have been extended to other PDI family members, including ERp57 (39, 116, 118, 123) and ERp5 (77). The vascular thiol isomerases are those PDI family members secreted fro...
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mechanisms of Thrombus Formation
The New England Journal of Medicine, 2008Co-Authors: Bruce Furie, Barbara C FurieAbstract:This review is an account of recent advances in our understanding of the mechanisms of Thrombus Formation, with emphasis on two independent pathways: one involving primarily platelets and the other initiated by tissue factor.
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a critical role for extracellular protein disulfide isomerase during Thrombus Formation in mice
Journal of Clinical Investigation, 2008Co-Authors: Jaehyung Cho, Barbara C Furie, Shaun R Coughlin, Bruce FurieAbstract:Thiol isomerases, including protein disulfide isomerase (PDI), catalyze disulfide oxidation, reduction, and isomerization, thereby playing an important role in protein synthesis. To determine whether extracellular PDI mediates Thrombus Formation in an animal model, PDI expression, platelet accumulation, and fibrin generation were monitored in the blood vessels of mice by intravital fluorescence microscopy following laser-induced arteriolar injury. A time-dependent increase in PDI was observed in murine thrombi following injury. Infusion of the PDI inhibitor bacitracin or a blocking monoclonal antibody against PDI inhibited platelet Thrombus Formation and fibrin generation. Fibrin deposition is normal in mice lacking the G protein-coupled platelet receptor Par4, although there is no stable accumulation of platelets. Infusion of monoclonal antibodies against PDI into the circulation of Par4(-/-) mice prior to vessel wall injury inhibited fibrin generation. These results indicate that PDI is required in vivo in mice for both fibrin generation and platelet Thrombus Formation.
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thrombin initiated platelet activation in vivo is vwf independent during Thrombus Formation in a laser injury model
Journal of Clinical Investigation, 2007Co-Authors: Christophe Dubois, Laurence Panicotdubois, Justin F Gainor, Barbara C FurieAbstract:Adhesion of platelets to an injured vessel wall and platelet activation are critical events in the Formation of a Thrombus. Of the agonists involved in platelet activation, thrombin, collagen, and vWF are known to induce in vitro calcium mobilization in platelets. Using a calcium-sensitive fluorochrome and digital multichannel intravital microscopy to image unstimulated and stimulated platelets, calcium mobilization was monitored as a reporter of platelet activation (as distinct from platelet accumulation) during Thrombus Formation in live mice. In the absence of vWF, platelet activation was normal, but platelet adherence and aggregation were attenuated during Thrombus Formation following laser-induced injury in the cremaster muscle microcirculation. In WT mice treated with lepirudin, platelet activation was blocked, and platelet adherence and aggregation were inhibited. The kinetics of platelet activation and platelet accumulation were similar in FcRγ–/– mice lacking glycoprotein VI (GPVI), GPVI-depleted mice, and WT mice. Our results indicate that the tissue factor–mediated pathway of thrombin generation, but not the collagen-induced GPVI-mediated pathway, is the major pathway leading to platelet activation after laser-induced injury under the conditions employed. In the tissue factor–mediated pathway, vWF plays a role in platelet accumulation during Thrombus Formation but is not required for platelet activation in vivo.
Zaverio M Ruggeri - One of the best experts on this subject based on the ideXlab platform.
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the role of von willebrand factor in Thrombus Formation
Thrombosis Research, 2007Co-Authors: Zaverio M RuggeriAbstract:Abstract Von Willebrand factor (VWF) is a large multimeric glycoprotein produced in endothelial cells and megakaryocytes and present in subendothelial matrix, blood plasma and platelets. VWF mediates adhesion and aggregation of platelets at sites of vascular injury, processes that are critical for both haemostasis and thrombosis. Thrombus Formation involves complex events that are influenced by different environmental conditions. Progress in understanding the structure and function of VWF and the mechanisms that underlie its interactions with platelets has led to important insight into the differentiation between normal haemostasis and pathological arterial thrombosis. The conventional view of signalling-induced platelet aggregation has recently been extended to include activation-independent aggregation. A novel mechanism has been demonstrated for initiating Thrombus Formation under high haemodynamic forces that involves αIIbβ3-independent platelet aggregation at the interface between immobilised and soluble VWF. This VWF-mediated process may be a key determinant of platelet accumulation in stenotic arteries leading to acute thrombotic occlusion.
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the role of von willebrand factor in Thrombus Formation
Thrombosis Research, 2007Co-Authors: Zaverio M RuggeriAbstract:Von Willebrand factor (VWF) is a large multimeric glycoprotein produced in endothelial cells and megakaryocytes and present in subendothelial matrix, blood plasma and platelets. VWF mediates adhesion and aggregation of platelets at sites of vascular injury, processes that are critical for both haemostasis and thrombosis. Thrombus Formation involves complex events that are influenced by different environmental conditions. Progress in understanding the structure and function of VWF and the mechanisms that underlie its interactions with platelets has led to important insight into the differentiation between normal haemostasis and pathological arterial thrombosis. The conventional view of signalling-induced platelet aggregation has recently been extended to include activation-independent aggregation. A novel mechanism has been demonstrated for initiating Thrombus Formation under high haemodynamic forces that involves alpha(IIb)beta(3)-independent platelet aggregation at the interface between immobilised and soluble VWF. This VWF-mediated process may be a key determinant of platelet accumulation in stenotic arteries leading to acute thrombotic occlusion.
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mechanisms initiating platelet Thrombus Formation
Thrombosis and Haemostasis, 1997Co-Authors: Zaverio M RuggeriAbstract:The functions of platelets depend on their ability to interest with surface exposed at sites of tissue damage and then with one another after activation, thus aggregating into thrombi. This complex process, normally beneficial to arrest bleeding during hemostasis, may become a cause of catastrophic disease when it leads to thrombotic occlusion of atherosclerotic vessels curtailing arterial blood flow to vital organs. Fluid dynamic conditions modulate all aspects of platelet response to vascular injury. At higher levels of shear stress, encountered both in normal vessels during normal hemostasis or in pathological conditions of the vasculature during thrombosis, von Willebrand factor becomes the essential adhesive protein for both adhesion and aggregation. Two platelet membrane receptors, the glycoprotein complexes Ib-IX-V and IIb-IIIa (integrin alpha IIb beta 3), mediate the von Willebrand factor function in a coordinate and synergistic manner, each contributing unique biomechanical properties to support Thrombus Formation. The developing understanding of the structure and mechanism of action of the key adhesive domains of von Willebrand factor, as well as of their cognate cellular and extracellular binding sites, will provide solid pathophysiological foundation for the evaluation of novel anti-thrombotic strategies.
Imke C. A. Munnix - One of the best experts on this subject based on the ideXlab platform.
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functional divergence of platelet protein kinase c pkc isoforms in Thrombus Formation on collagen
Journal of Biological Chemistry, 2010Co-Authors: Karen Gilio, Judith M E M Cosemans, Imke C. A. Munnix, Lenneke Prinzen, O Konopatskaya, Matthew T Harper, Michael Leitges, Jeffery D Molkentin, Qinghang Liu, Johan W. M. HeemskerkAbstract:Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and Thrombus Formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates Thrombus Formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit Thrombus Formation on collagen.
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non redundant roles of phosphoinositide 3 kinase isoforms α and β in glycoprotein vi induced platelet signaling and Thrombus Formation
Journal of Biological Chemistry, 2009Co-Authors: Karen Gilio, Marion A H Feijge, Judith M E M Cosemans, Imke C. A. Munnix, Paola E. J. Van Der Meijden, Pierre Mangin, Serve Olieslagers, Magdalena Chrzanowskawodnicka, Rivka Lillian, Simone M SchoenwaelderAbstract:Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cγ2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent Thrombus Formation. The targeting of platelet PI3Kα or -β strongly and selectively suppressed GPVI-induced Ca2+ mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cγ2 by PI3Kα/β. That PI3Kα and -β have a non-redundant function in GPVI-induced platelet activation and Thrombus Formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca2+ increases and surface expression of phosphatidylserine under flow, and (iv) Thrombus Formation, under conditions where PI3Kα/β was blocked or p85α was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kδ or -γ. Furthermore, PI3Kα/β, but not PI3Kγ, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kα and -β isoforms are required for full GPVI-dependent platelet Ca2+ signaling and Thrombus Formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kα an interesting new target for anti-platelet therapy.
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Dual role of collagen in factor XII-dependent Thrombus Formation
Blood, 2009Co-Authors: Paola E. J. Van Der Meijden, Judith M E M Cosemans, Thomas Renné, Imke C. A. Munnix, Jocelyn M. Auger, José W. P. Govers-riemslag, Marijke J. E. Kuijpers, Henri M. H. Spronk, Steve P. Watson, Johan W. M. HeemskerkAbstract:In vivo mouse models have indicated that the intrinsic coagulation pathway, initiated by factor XII, contributes to Thrombus Formation in response to major vascular damage. Here, we show that fibrillar type I collagen provoked a dose-dependent shortening of the clotting time of human plasma via activation of factor XII. This activation was mediated by factor XII binding to collagen. Factor XII activation also contributed to the stimulating effect of collagen on thrombin generation in plasma, and increased the effect of platelets via glycoprotein VI activation. Furthermore, in flow-dependent Thrombus Formation under coagulant conditions, collagen promoted the appearance of phosphatidylserine-exposing platelets and the Formation of fibrin. Defective glycoprotein VI signaling (with platelets deficient in LAT or phospholipase Cγ2) delayed and suppressed phosphatidylserine exposure and Thrombus Formation. Markedly, these processes were also suppressed by absence of factor XII or XI, whereas blocking of tissue factor/factor VIIa was of little effect. Together, these results point to a dual role of collagen in Thrombus Formation: stimulation of glycoprotein VI signaling via LAT and PLCγ2 to form procoagulant platelets; and activation of factor XII to stimulate thrombin generation and potentiate the Formation of platelet-fibrin thrombi.
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collagen mimetic peptides mediate flow dependent Thrombus Formation by high or low affinity binding of integrin alpha2beta1 and glycoprotein vi
Journal of Thrombosis and Haemostasis, 2008Co-Authors: Imke C. A. Munnix, Marion A H Feijge, Richard W Farndale, Hans Deckmyn, Pia Siljander, Karen Gilio, Nicolas Raynal, Tilman M Hackeng, P A Smethurst, Johan W. M. HeemskerkAbstract:Summary. Background: Collagen acts as a potent surface for platelet adhesion and Thrombus Formation under conditions of blood flow. Studies using collagen-derived triple-helical peptides have identified the GXX’GER motif as an adhesive ligand for platelet integrin α2β1, and (GPO)n as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). Objective: The potency was investigated of triple-helical peptides, consisting of GXX’GER sequences within (GPO)n or (GPP)n motifs, to support flow-dependent Thrombus Formation. Results: At a high-shear rate, immobilized peptides containing both the high-affinity α2β1-binding motif GFOGER and the (GPO)n motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co-immobilized VWF was needed for Thrombus Formation. The (GPO)n but not the (GPP)n sequence induced GPVI-dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low-affinity (GASGER, GAOGER) α2β1-binding motifs formed procoagulant thrombi only if both (GPO)n and VWF were present. At a low-shear rate, immobilized peptides with high- or low-affinity α2β1-binding motifs mediated Formation of thrombi with procoagulant platelets only in combination with (GPO)n. Conclusions: Triple-helical peptides with specific receptor-binding motifs mimic the properties of native collagen I in Thrombus Formation by binding to both platelet collagen receptors. At a high-shear rate, either GPIb or high-affinity (but not low-affinity) GXX’GER mediates GPVI-dependent Formation of procoagulant thrombi. By extension, high-affinity binding for α2β1 can control the overall platelet-adhesive activity of native collagens.
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segregation of platelet aggregatory and procoagulant microdomains in Thrombus Formation regulation by transient integrin activation
Arteriosclerosis Thrombosis and Vascular Biology, 2007Co-Authors: Imke C. A. Munnix, Jocelyn M. Auger, Marijke J. E. Kuijpers, Steve P. Watson, Christella M L G D Thomassen, Peter Panizzi, Marc A M J Van Zandvoort, Jan Rosing, Paul E Bock, Johan W. M. HeemskerkAbstract:Objective— Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced Thrombus Formation in vitro and in vivo. Methods and Results— High-resolution 2-photon fluorescence microscopy revealed that in Thrombus Formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine. The latter platelet population had inactivated αIIbβ3 integrins and displayed increased binding of coagulation factors. Coated platelets, expressing serotonin binding sites, were not identified as a separate population. Thrombin generation and coagulation favored the transFormation to phosphatidylserine-exposing platelets with inactivated integrins and reduced adhesion. Prolonged tyrosine phosphorylation in vitro resulted in secondary downregulation of active αIIbβ3. Conclusions— These results lead to a new sp...