Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRγ deficiency

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4346-4353 ◽  
Author(s):  
Pierre Mangin ◽  
Cindy L. Yap ◽  
Christelle Nonne ◽  
Sharelle A. Sturgeon ◽  
Isaac Goncalves ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Thrombus Formation ◽  
Chain Complex ◽  
Concurrent Administration ◽  
Glycoprotein Vi ◽  
Thrombin Inhibition ◽  
Arterial Thrombus ◽  
Major Defect

AbstractFibrillar 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.

scholarly journals Mice Lacking the Inhibitory Collagen Receptor LAIR-1 Exhibit a Mild Thrombocytosis and Hyperactive Platelets

2017 ◽  
Vol 37 (5) ◽  
pp. 823-835 ◽  
Author(s):  
Christopher W. Smith ◽  
Steven G. Thomas ◽  
Zaher Raslan ◽  
Pushpa Patel ◽  
Maxwell Byrne ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Thrombus Formation ◽  
Physiological Role ◽  
Src Family Kinase ◽  
Glycoprotein Vi ◽  
Collagen Receptor ◽  
Deficient Mice

Objective— Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a collagen receptor that belongs to the inhibitory immunoreceptor tyrosine-based inhibition motif–containing receptor family. It is an inhibitor of signaling via the immunoreceptor tyrosine-based activation motif–containing collagen receptor complex, glycoprotein VI-FcRγ-chain. It is expressed on hematopoietic cells, including immature megakaryocytes, but is not detectable on platelets. Although the inhibitory function of LAIR-1 has been described in leukocytes, its physiological role in megakaryocytes and in particular in platelet formation has not been explored. In this study, we investigate the role of LAIR-1 in megakaryocyte development and platelet production by generating LAIR-1–deficient mice. Approach and Results— Mice lacking LAIR-1 exhibit a significant increase in platelet counts, a prolonged platelet half-life in vivo, and increased proplatelet formation in vitro. Interestingly, platelets from LAIR-1–deficient mice exhibit an enhanced reactivity to collagen and the glycoprotein VI–specific agonist collagen-related peptide despite not expressing LAIR-1, and mice showed enhanced thrombus formation in the carotid artery after ferric chloride injury. Targeted deletion of LAIR-1 in mice results in an increase in signaling downstream of the glycoprotein VI–FcRγ-chain and integrin αIIbβ3 in megakaryocytes because of enhanced Src family kinase activity. Conclusions— Findings from this study demonstrate that ablation of LAIR-1 in megakaryocytes leads to increased Src family kinase activity and downstream signaling in response to collagen that is transmitted to platelets, rendering them hyper-reactive specifically to agonists that signal through Syk tyrosine kinases, but not to G-protein–coupled receptors.

Local delivery of soluble platelet collagen receptor glycoprotein VI inhibits thrombus formation in vivo

2006 ◽  
Vol 95 (05) ◽  
pp. 763-766 ◽  
Author(s):  
Andreas Bültmann ◽  
Christian Herdeg ◽  
Zhongmin Li ◽  
Götz Münch ◽  
Christine Baumgartner ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Vascular Injury ◽  
Carotid Arteries ◽  
Thrombus Formation ◽  
Critical Role ◽  
Local Delivery ◽  
Computer Assisted ◽  
Glycoprotein Vi ◽  
Collagen Receptor

SummaryPlatelet-mediated thrombus formation at the site of vascular injury isa major trigger for thrombo-ischemic complications after coronary interventions. The platelet collagen receptor glycoprotein VI (GPVI) plays a critical role in the initiation of arterial thrombus formation. Endothelial denudation of the right carotid artery in rabbits was induced through balloon injury. Subsequently, local delivery of soluble, dimeric fusion protein of GPVI (GPVI-Fc) (n=7) or control Fc (n=7) at the site of vascular injury was performed with a modified double-balloon drugdelivery catheter.Thrombus area within the injured carotid artery was quantified using a computer-assisted image analysis and was used as index of thrombus formation.The extent of thrombus formation was significantly reduced in GPVI-Fc- compared with control Fc-treated carotid arteries (relative thrombus area, GPVI-Fc vs. Fc: 9.3 ± 4.2 vs. 2.3 ± 1.7, p<0.001). Local delivery of soluble GPVI resulted in reduced thrombus formation after catheter-induced vascular injury.These data suggest a selective pharmacological modulation of GPVI-collagen interactions to be important for controlling onset and progression of pathological arterial thrombosis, predominantly or even exclusively at sites of injured carotid arteries in the absence of systemic platelet therapy.

scholarly journals Loss of talin1 in platelets abrogates integrin activation, platelet aggregation, and thrombus formation in vitro and in vivo

2007 ◽  
Vol 204 (13) ◽  
pp. 3113-3118 ◽  
Author(s):  
Bernhard Nieswandt ◽  
Markus Moser ◽  
Irina Pleines ◽  
David Varga-Szabo ◽  
Sue Monkley ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Thrombus Formation ◽  
Cellular Functions ◽  
High Affinity ◽  
Direct Binding ◽  
Normal Hemostasis ◽  
Necessary And Sufficient ◽  
Aggregation Of Platelets

Platelet adhesion and aggregation at sites of vascular injury are essential for normal hemostasis but may also lead to pathological thrombus formation, causing diseases such as myocardial infarction or stroke. Heterodimeric receptors of the integrin family play a central role in the adhesion and aggregation of platelets. In resting platelets, integrins exhibit a low affinity state for their ligands, and they shift to a high affinity state at sites of vascular injury. It has been proposed that direct binding of the cytoskeletal protein talin1 to the cytoplasmic domain of the integrin β subunits is necessary and sufficient to trigger the activation of integrins to this high affinity state, but direct in vivo evidence in support of this hypothesis is still lacking. Here, we show that platelets from mice lacking talin1 are unable to activate integrins in response to all known major platelet agonists while other cellular functions are still preserved. As a consequence, mice with talin-deficient platelets display a severe hemostatic defect and are completely resistant to arterial thrombosis. Collectively, these experiments demonstrate that talin is required for inside-out activation of platelet integrins in hemostasis and thrombosis.

CLEC-2 is an essential platelet-activating receptor in hemostasis and thrombosis

Blood ◽  
2009 ◽  
Vol 114 (16) ◽  
pp. 3464-3472 ◽  
Author(s):  
Frauke May ◽  
Ina Hagedorn ◽  
Irina Pleines ◽  
Markus Bender ◽  
Timo Vögtle ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Antibody Treatment ◽  
Cellular Activation ◽  
Primary Hemostasis ◽  
Arterial Thrombus ◽  
Normal Adhesion

Abstract Damage to the integrity of the vessel wall leads to exposure of the subendothelial extracellular matrix (ECM), triggering platelet activation and aggregation. This process is essential for primary hemostasis but it may also lead to arterial thrombosis. Although the mechanisms underlying platelet activation on the ECM are well explored, it is less clear which receptors mediate cellular activation in a growing thrombus. Here we studied the role of the recently identified C-type lectin-like receptor 2 (CLEC-2) in this process. We show that anti–CLEC-2 antibody treatment of mice leads to complete and highly specific loss of CLEC-2 in circulating platelets for several days. CLEC-2–deficient platelets displayed normal adhesion under flow, but subsequent aggregate formation was severely defective in vitro and in vivo. As a consequence, CLEC-2 deficiency was associated with increased bleeding times and profound protection from occlusive arterial thrombus formation. These results reveal an essential function of CLEC-2 in hemostasis and thrombosis.

Discrimination Between Prostglandin-Dependent And-Independent Functions Of Essential Fatty Acids In Arterial Throm- Bogenesis

1981 ◽  
Author(s):  
U M T Houtsmuller ◽  
G Hornstra ◽  
E Haddeman
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Linoleic Acid ◽  
Platelet Aggregation ◽  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Arterial Thrombus ◽  
Induced Aggregation

Arterial thrombus formation is reduced in essential fatty acid (EFA) deficiency. This goes together with an enhanced thrombin induced aggregation of platelets in vitro,whereas collagen-induced aggregation is definitely suppressed. A small amount of linoleic acid (18:2 (n-6)) is able to cure EFA-deficiency and to normalize arterial thrombogenesis. This latter effect may be due to either the structural function of this EFA or to its function as the ultimate dietary precursor of prostaglandins (PG). Columbinic acid, a stereo-isomer of γ-linolenic acid (18:3 (n-6)) was recently shown to possess all the structural functions of EFA, but not the PG-dependent ones. This fatty acid therefore presents a suitable tool to investigate the PG-dependence of arterial thrombogenesis and its underlying processes. We therefore compared the effect of small amounts of linoleic and columbinic acid (both as methylesters) on the water vapour release in vivo (which is a sensitive parameter for a non-PG dependent function of polyenoic fatty acids), arterial thrombosis tendency (time needed for the thrombotic obstruction of an aorta prosthesis) and platelet aggregation in vitro (aggregometry) induced by collagen and thrombin. In contrast to linoleic acid, columbinic acid did not normalize arterial thrombosis tendency and collagen induced platelet aggregation. Columbinic acid was equally effective as linoleic acid in the normalization of the water vapour release in vivo and of the thrombin-induced aggregation. We conclude that arterial thrombus formation and collagen- induced aggregation greatly depend on prostanoid formation, whereas thrombin-induced aggregation does not. The structural role of polyenoic fatty acids in thrombin-induced aggregation may provide a tool in the elucidation of factors determining the thrombin-sensitivity of blood platelets.

scholarly journals A Crucial Role of Glycoprotein VI for Platelet Recruitment to the Injured Arterial Wall In Vivo

2002 ◽  
Vol 197 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Steffen Massberg ◽  
Meinrad Gawaz ◽  
Sabine Grüner ◽  
Valerie Schulte ◽  
Ildiko Konrad ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
Arterial Occlusion ◽  
Arterial Injury ◽  
Platelet Glycoprotein ◽  
Glycoprotein Vi ◽  
Strict Requirement

Platelet adhesion and aggregation at sites of vascular injury is crucial for hemostasis but may lead to arterial occlusion in the setting of atherosclerosis and precipitate diseases such as myocardial infarction. A current hypothesis suggests that platelet glycoprotein (GP) Ib interaction with von Willebrand factor recruits flowing platelets to the injured vessel wall, where subendothelial fibrillar collagens support their firm adhesion and activation. However, so far this hypothesis has not been tested in vivo. Here, we demonstrate by intravital fluorescence microscopy of the mouse carotid artery that inhibition or absence of the major platelet collagen receptor, GPVI, abolishes platelet–vessel wall interactions after endothelial denudation. Unexpectedly, inhibition of GPVI by the monoclonal antibody JAQ1 reduced platelet tethering to the subendothelium by ∼89%. In addition, stable arrest and aggregation of platelets was virtually abolished under these conditions. Using different models of arterial injury, the strict requirement for GPVI in these processes was confirmed in GPVI-deficient mice, where platelets also failed to adhere and aggregate on the damaged vessel wall. These findings reveal an unexpected role of GPVI in the initiation of platelet attachment at sites of vascular injury and unequivocally identify platelet–collagen interactions (via GPVI) as the major determinant of arterial thrombus formation.

scholarly journals Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators

2015 ◽  
Vol 212 (2) ◽  
pp. 129-137 ◽  
Author(s):  
Ulrike Flierl ◽  
Tracy L. Nero ◽  
Bock Lim ◽  
Jane F. Arthur ◽  
Yu Yao ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Reactive Oxygen Species Generation ◽  
Modified Oligonucleotides ◽  
Development Programs ◽  
Drug Candidates ◽  
Glycoprotein Vi ◽  
Backbone Modification

Nucleotide-based drug candidates such as antisense oligonucleotides, aptamers, immunoreceptor-activating nucleotides, or (anti)microRNAs hold great therapeutic promise for many human diseases. Phosphorothioate (PS) backbone modification of nucleotide-based drugs is common practice to protect these promising drug candidates from rapid degradation by plasma and intracellular nucleases. Effects of the changes in physicochemical properties associated with PS modification on platelets have not been elucidated so far. Here we report the unexpected binding of PS-modified oligonucleotides to platelets eliciting strong platelet activation, signaling, reactive oxygen species generation, adhesion, spreading, aggregation, and thrombus formation in vitro and in vivo. Mechanistically, the platelet-specific receptor glycoprotein VI (GPVI) mediates these platelet-activating effects. Notably, platelets from GPVI function–deficient patients do not exhibit binding of PS-modified oligonucleotides, and platelet activation is fully abolished. Our data demonstrate a novel, unexpected, PS backbone–dependent, platelet-activating effect of nucleotide-based drug candidates mediated by GPVI. This unforeseen effect should be considered in the ongoing development programs for the broad range of upcoming and promising DNA/RNA therapeutics.

Anti-Human VWF Monoclonal Antibody SZ-123 Prevents Arterial Thrombus Formation by Inhibiting VWF–collagen and VWF-Platelet Interactions in Rhesus Monkeys

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5194-5194
Author(s):  
Yiming Zhao ◽  
Changgeng Ruan
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Bleeding Time ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Significant Prolongation ◽  
Arterial Thrombus

Abstract Abstract 5194 Objective: To investigate the in vivo antithrombotic efficacy of an anti-VWF monoclonal antibody SZ-123, and its potential underlying mechanism. Methods and Results: Cyclic flow reductions (CFRs) were measured in the femoral artery of monkeys before and after intravenous administration of SZ-123. Ex vivo VWF binding to collagen, platelet aggregation, platelet count and template bleeding time were performed as measurements of antithrombotic activity. In addition, plasma VWF, SZ-123 levels, and VWF occupancy were measured by ELISA. Administration of 0. 1, 0. 3, and 0. 6 mg/kg SZ-123 resulted in 45. 3%, 78. 2%, and 100% reduction in CFRs, respectively. When 0. 3 and 0. 6 mg/kg SZ-123 were administrated, 100% of VWF was occupied by the antibody. Moreover, 100% ex vivo inhibition of VWF-collagen binding and 60–95% inhibition of platelet aggregation were observed from 15 min to 1h. None of the doses resulted in significant prolongation of bleeding time. In vitro experiment also revealed that SZ-123 not only blocks collagen-VWF A3 interaction but also inhibits indirectly VWF A1 binding to GPIba induced by ristocetin. Conclusions: SZ-123 prevents in vivo arterial thrombus formation under high shear conditions by inhibiting VWF A3–collagen and VWF A1-platelet interactions and does not prolong bleeding time. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Modifier of hemostasis (Mh) locus on chromosome 4 controls in vivo hemostasis of Gp6−/− mice

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1266-1273 ◽  
Author(s):  
Yann Cheli ◽  
Deborah Jensen ◽  
Patrizia Marchese ◽  
David Habart ◽  
Tim Wiltshire ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Chromosome 4 ◽  
Glycoprotein Vi ◽  
Genome Wide ◽  
Injury Model ◽  
A Genome ◽  
Platelet Thrombus

Abstract Platelet glycoprotein VI (GPVI) is a key receptor for collagens that mediates the propagation of platelet attachment and activation. Targeted disruption of the murine gene Gp6 on a mixed 129 × 1/SvJ × C57BL/6J background causes the expected defects in collagen-dependent platelet responses in vitro. The extent of this dysfunction in all Gp6−/− mice is uniform and is not affected by genetic background. However, the same Gp6−/− mice exhibit 2 diametrically opposed phenotypes in vivo. In some mice, tail bleeding times are extremely prolonged, and thrombus formation in an in vivo carotid artery ferric chloride-injury model is significantly impaired. In other littermates, tail bleeding times are within the range of wild-type mice, and in vivo thrombus formation is indistinguishable from that of control mice. Directed intercrosses revealed that these phenotypes are heritable, and a genome-wide single-nucleotide polymorphism scan revealed the most significant linkage to a single locus (8 megabases) on chromosome 4 (logarithm of the odds [LOD] score = 6.9, P < .0001) that we designate Modifier of hemostasis (Mh). Our results indicate that one or more modifier genes in Mh control the extent to which in vivo platelet thrombus formation is disrupted by the absence of platelet GPVI.

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