Platelet Collagen Receptor Glycoprotein VI Research Articles

Restrained glycoprotein VI-induced platelet signaling by tyrosine protein phosphatases independent of phospholipase Cγ2

The platelet collagen receptor glycoprotein VI (GPVI) signals to activation of phospholipase Cγ2 (PLCγ2) and phosphoinositide 3-kinases (PI3K), causing platelet activation and aggregation. The non-receptor Src homology tyrosine phosphatases Shp1/2 modulate GPVI signaling in partly opposite ways, both of which are targeted by the potential drug NSC87877. Effect measurements of the Shp1/2 inhibitor NSC87877 on platelet activation via GPVI using light transmission aggregometry, Ca2+ flux assay, western blotting and flow cytometry. Effect measurements of selective PI3K inhibitor TGX221. Inhibition of Shp1/2 with NSC87877 enhanced platelet aggregation induced by the GPVI agonist, collagen-related peptide (CRP). Furthermore, NSC87877 antagonized the effects of PI3Kb inhibition, but not of Btk inhibition. Both NSC87877 and TGX221 suppressed the CRP-induced phosphorylation of PLCγ2 at activation site Tyr759. These findings indicate that drug interference of the two phosphatases Shp1/2 subtly enhances GPVI-induced platelet responses via a mechanism not involving PLCγ2 activation, even upon PI3K inhibition.

The Subtilisin-Like Protease Furin Regulates Hemin-Dependent Ectodomain Shedding of Glycoprotein VI.

Hemolysis results in release of free hemoglobin and hemin liberation from erythrocytes. Hemin has been described to induce platelet activation and to trigger thrombosis. We evaluated the effect of hemin on platelet function and surface expression of the platelet collagen receptor glycoprotein VI (GPVI). Isolated platelets were stimulated with increasing concentrations of hemin. We found that hemin strongly enhanced platelet activation, aggregation, and aggregate formation on immobilized collagen under flow. In contrast, we found that surface expression of GPVI was significantly reduced upon hemin stimulation with high hemin concentrations indicating that hemin-induced loss of surface GPVI does not hinder platelet aggregation. Loss of hemin-induced surface expression of GPVI was caused by shedding of the ectodomain of GPVI as verified by immunoblotting and is independent of the GPVI or CLEC-2 mediated ITAM (immunoreceptor-tyrosine-based-activation-motif) signaling pathway as inhibitor studies revealed. Hemin-induced GPVI shedding was independent of metalloproteinases such as ADAM10 or ADAM17, which were previously described to regulate GPVI degradation. Similarly, concentration-dependent shedding of CD62P was also induced by hemin. Unexpectedly, we found that the subtilisin-like proprotein convertase furin controls hemin-dependent GPVI shedding as shown by inhibitor studies using the specific furin inhibitors SSM3 and Hexa-D-arginine. In the presence of SSM3 and Hexa-D-arginine, hemin-associated GPVI degradation was substantially reduced. Further, SSM3 inhibited hemin-induced but not CRP-XL-induced platelet aggregation and thrombus formation, indicating that furin controls specifically hemin-associated platelet functions. In summary, we describe a novel mechanism of hemin-dependent GPVI shedding and platelet function mediated by furin.

Platelet Activation by Antisense Oligonucleotides (ASOs) in the Göttingen Minipig, including an Evaluation of Glycoprotein VI (GPVI) and Platelet Factor 4 (PF4) Ontogeny.

Antisense oligonucleotide (ASO) is a therapeutic modality that enables selective modulation of undruggable protein targets. However, dose- and sequence-dependent platelet count reductions have been reported in nonclinical studies and clinical trials. The adult Göttingen minipig is an acknowledged nonclinical model for ASO safety testing, and the juvenile Göttingen minipig has been recently proposed for the safety testing of pediatric medicines. This study assessed the effects of various ASO sequences and modifications on Göttingen minipig platelets using in vitro platelet activation and aggregometry assays. The underlying mechanism was investigated further to characterize this animal model for ASO safety testing. In addition, the protein abundance of glycoprotein VI (GPVI) and platelet factor 4 (PF4) was investigated in the adult and juvenile minipigs. Our data on direct platelet activation and aggregation by ASOs in adult minipigs are remarkably comparable to human data. Additionally, PS ASOs bind to platelet collagen receptor GPVI and directly activate minipig platelets in vitro, mirroring the findings in human blood samples. This further corroborates the use of the Göttingen minipig for ASO safety testing. Moreover, the differential abundance of GPVI and PF4 in minipigs provides insight into the influence of ontogeny in potential ASO-induced thrombocytopenia in pediatric patients.

Evaluation of the effect of eltrombopag therapy on the platelet collagen receptor glycoprotein VI (GPVI) expression and soluble GPVI levels in young patients with immune thrombocytopenia

BackgroundPlatelet glycoprotein VI (GPVI) receptor is essential for platelet adhesion and aggregation. Eltrombopag is as an effective treatment for chronic immune thrombocytopenia (ITP); yet, its effect on platelet function is not fully characterized.AimThis prospective study investigated the effect of eltrombopag therapy on platelet function through assessment of GPVI receptor expression and soluble GPVI levels among pediatric patients with persistent or chronic ITP.MethodsThirty-six children and adolescents with persistent or chronic ITP were divided equally into two groups either to receive eltrombopag therapy or the standard of care. All patients were followed-up for 12 months with assessment of bleeding score and complete blood count (CBC). Evaluation of GPVI expression using flow cytometry and measurement of its soluble form by ELISA was done at baseline and at 6 months.ResultsITP patients on eltrombopag had significantly lower bleeding score after 6 months of therapy while the quality of life has significantly improved. Platelet count was significantly increased throughout the study. GPVI expression by flow cytometry and soluble GPVI levels were significantly increased after eltrombopag therapy. After 12 months, ITP patients on eltrombopag were able to maintain a good quality of life and low bleeding score.ConclusionOur data suggest that eltrombopag, through its effect on the GPVI receptor expression and its soluble form, might reduce bleeding manifestations and improve the quality of life of chronic and persistent ITP children independent of its effect on the platelet count.

Chronic edible dosing of Δ9-tetrahydrocannabinol (THC) in nonhuman primates reduces systemic platelet activity and function.

Cannabis usage has steadily increased as acceptance is growing for both medical and recreational reasons. Medical cannabis is administered for treatment of chronic pain based on the premise that the endocannabinoid system signals desensitize pain sensor neurons and produce anti-inflammatory effects. The major psychoactive ingredient of cannabis is Δ9-tetrahydrocannabinol (THC) that signals mainly through cannabinoid receptor-1 (CBr), which is also present on nonneuron cells including blood platelets of the circulatory system. In vitro, CBr-mediated signaling has been shown to acutely inhibit platelet activation downstream of the platelet collagen receptor glycoprotein (GP)VI. The systemic effects of chronic THC administration on platelet activity and function remain unclear. This study investigates the effects of chronic THC administration on platelet function using a nonhuman primate (NHP) model. Our results show that female and male NHPs consuming a daily THC edible had reduced platelet adhesion, aggregation, and granule secretion in response to select platelet agonists. Furthermore, a change in bioactive lipids (oxylipins) was observed in the female cohort after THC administration. These results indicate that chronic THC edible administration desensitized platelet activity and function in response to GPVI- and G-protein coupled receptor-based activation by interfering with primary and secondary feedback signaling pathways. These observations may have important clinical implications for patients who use medical marijuana and for providers caring for these patients.

Dimers of the platelet collagen receptor glycoprotein VI bind specifically to fibrin fibers during clot formation, but not to intact fibrinogen

ObjectiveThe platelet collagen receptor glycoprotein VI (GPVI) has an independent role as a receptor for fibrin produced via the coagulation cascade. However, various reports of GPVI binding to immobilized fibrin(ogen) are not consistent. As a collagen receptor, GPVI‐dimer is the functional form, but whether GPVI dimers or monomers bind to fibrin remains controversial. To resolve this, we analyzed GPVI binding to nascent fibrin clots, which more closely approximate physiological conditions. Methods and resultsELISA using biotinyl‐fibrinogen immobilized on streptavidin‐coated wells indicated that GPVI dimers do not bind intact fibrinogen. Clots were formed by adding thrombin to a mixture of near‐plasma level of fibrinogen and recombinant GPVI ectodomain: GPVI dimer (GPVI‐Fc2 or Revacept) or monomer (GPVI‐His: single chain of Revacept GPVI domain, with His tag). Clot‐bound proteins were analyzed by SDS‐PAGE/immunoblotting. GPVI‐dimer bound to noncrosslinked fibrin clots with classical one‐site binding kinetics, with µM‐level KD, and to crosslinked clots with higher affinity. Anti‐GPVI‐dimer (mFab‐F) inhibited the binding. However, GPVI‐His binding to either type of clot was nonsaturable and nearly linear, indicating very low affinity or nonspecific binding. In clots formed in the presence of platelets, clot‐bound platelet‐derived proteins were integrin αIIbβ3, present at high levels, and GPVI. ConclusionsWe conclude that dimeric GPVI is the receptor for fibrin, exhibiting a similar KD to those obtained for its binding to fibrinogen D‐fragment and D‐dimer, suggesting that fibrin(ogen)'s GPVI‐binding site becomes exposed after fibrin formation or cleavage to fragment D. Analysis of platelets bound to fibrin clots indicates that platelet GPVI binds to fibrin fibers comprising the clot.

Annexin A7 is a critical regulator of Ca2+ mobilization and lipid metabolism during platelet activation and arterial thrombosis

Abstract Background Platelet activation after contact to subendothelial collagen following atherosclerotic plaque rupture can lead to arterial thrombosis with acute thrombotic vascular occlusion. Annexin A7 (AnxA7) is an intracellular Ca2+- and phospholipid-binding protein that participates in the regulation of prostaglandin production in inflammatory diseases, but also in cell survival and tumor growth. Objective In the present study, we aimed to determine the role of AnxA7 for platelet Ca2+ signaling and lipid metabolism in platelet activation and arterial thrombosis in gene-targeted mice lacking annexin A7 (Anxa7−/−). Results AnxA7 is strongly expressed in platelets of platelet-rich human coronary thrombi aspirated from patients with acute ST elevation myocardial infarction. Functionally, platelet aggregation and dense granule secretion were significantly abrogated in Anxa7−/− platelets as compared to wildtype platelets (Anxa7+/+) after activation with collagen or collagen-related peptide (CRP), a specific agonist of the major platelet collagen receptor glycoprotein VI (GPVI). Further, in vitro thrombus formation on a collagen-coated surface under high arterial shear rates was significantly diminished in Anxa7-deficient platelets, and thrombotic vascular occlusion after FeCl3-induced injury in vivo was blunted in Anxa7−/−bone marrow chimeric mice, but no prolongation of bleeding time was observed. Moreover, Anxa7−/− platelets showed a significant reduction of IP3 production due to an abolished phospholipase C (PLC) gamma2 phosphorylation resulting in an abolished increase of [Ca2+]i after platelet activation with CRP. Moreover, we could show by quantitative lipidomics analysis that annexin A7 critically affects platelet oxylipid metabolism following activation of GPVI-dependent platelet signalling since Anxa7−/− platelets showed a significant reduction of the bioactive metabolites thromboxane A2 and 12(S)-hydroxy-eicosatetraenoic acid (12(S)-HETE) levels as well as significantly reduced levels of several other prostaglandins following stimulation with collagen or CRP. Finally, defective PLCgamma2 phosphorylation, IP1 production and blunted increase of [Ca2+]i in Anxa7−/− platelets could be rescued by exogenous addition of 12(S)-HETE indicating that AnxA7 is a critical regulator of the platelet oxygenase 12-lipoxygenase (12-LOX) in GPVI-dependent platelet Ca2+ signalling during arterial thrombosis following activation by collagen. Conclusions The present study reveals annexin A7 as a critical regulator of oxylipid metabolism and Ca2+ signaling in GPVI-dependent platelet activation. Anxa7-deficiency further results in decreased in vitro and in vivo thrombus formation, but does not affect bleeding time. In conclusion, annexin A7 plays an important role in platelet signaling during arterial thrombosis and thus, may reflect a promising target for novel antiplatelet strategies. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (Deutsche Forschungsgemeinschaft, DFG)

Antidepressants: bleeding or thrombosis?

The contribution of depression to the pathogenesis of cardiovascular disease includes autonomic disturbances, endothelial dysfunction, inflammation, smoking, sedentary lifestyle, carbohydrate craving, and impaired fibrinolysis. There is evidence that serotonergic antidepressants (selective serotonin reuptake inhibitors and serotonin and noradrenaline reuptake inhibitors) restore the fibrinolytic profile. Contrary to common belief, such antidepressants do not affect platelet aggregation induced by adenosine diphosphate or adrenaline but reduce platelet adhesion to collagen. Since platelet collagen receptor glycoprotein VI binds to fibrin, it is possible that fibrinolytic properties of serotonergic antidepressants could impair platelet adhesion to collagen. The profibrinolytic and antiplatelet properties of serotonergic antidepressants help explain the increased risk of gastrointestinal, intracranial, and surgical bleeding in patients using these medications. Studies evaluating the impact of antidepressants on thrombotic and cardiovascular risk have yielded contradictory results. Corroborating the hypothesis that serotonergic antidepressants have profibrinolytic and antiplatelet properties, some authors showed that these medications prevent both cardiovascular and thromboembolic events. Others showed an increased risk of ischemic stroke, cardiac events and thromboembolic disease. Silent brain infarction may present in some elders with depressive symptoms, so it is presumed that antidepressants are prescribed for subclinical stroke patients. Another explanation for the increased risk of cardiovascular and thromboembolic events reported by some authors in individuals taking antidepressants includes antidepressant side effects such as sedation and weight gain and depression comorbidities such as anxiety, obesity and hyperhomocysteinemia. In conclusion, we suggest that serotonergic antidepressants be considered weak anticoagulants. We also suggest that depressed patients with comorbidities increasing the risk of cardiovascular and thromboembolic disease be recommended to follow a balanced diet and engage in physical activity, such as daily walking.

Assessing single-stranded oligonucleotide drug-induced effects in vitro reveals key risk factors for thrombocytopenia.

Single-stranded oligonucleotides (ON) comprise a promising therapeutic platform that enables selective modulation of currently undruggable targets. The development of novel ON drug candidates has demonstrated excellent efficacy, but in certain cases also some safety liabilities were reported. Among them are events of thrombocytopenia, which have recently been evident in late stage trials with ON drugs. The underlying mechanisms are poorly understood and the risk for ON candidates causing such events cannot be sufficiently assessed pre-clinically. We investigated potential thrombocytopenia risk factors of ONs and implemented a set of in vitro assays to assess these risks. Our findings support previous observations that phosphorothioate (PS)-ONs can bind to platelet proteins such as platelet collagen receptor glycoprotein VI (GPVI) and activate human platelets in vitro to various extents. We also show that these PS-ONs can bind to platelet factor 4 (PF4). Binding to platelet proteins and subsequent activation correlates with ON length and connected to this, the number of PS in the backbone of the molecule. Moreover, we demonstrate that locked nucleic acid (LNA) ribosyl modifications in the wings of the PS-ONs strongly suppress binding to GPVI and PF4, paralleled by markedly reduced platelet activation. In addition, we provide evidence that PS-ONs do not directly affect hematopoietic cell differentiation in culture but at higher concentrations show a pro-inflammatory potential, which might contribute to platelet activation. Overall, our data confirm that certain molecular attributes of ONs are associated with a higher risk for thrombocytopenia. We propose that applying the in vitro assays discussed here during the lead optimization phase may aid in deprioritizing ONs with a potential to induce thrombocytopenia.

FcγRIIB on liver sinusoidal endothelial cells is essential for antibody-induced GPVI ectodomain shedding in mice

AbstractThe activating platelet collagen receptor glycoprotein VI (GPVI) is a promising antithrombotic target because of its central role in arterial thrombosis and its minor relevance for normal hemostasis. The receptor can be specifically targeted by antibodies and irreversibly downregulated in circulating platelets in vivo, resulting in long-term antithrombotic protection in mice. This GPVI immunodepletion predominantly occurs through ectodomain shedding, which is accompanied by a transient drop in peripheral platelet counts. Mechanistic studies on this targeted GPVI loss have been hampered because it cannot be reproduced in isolated platelets in vitro. Here we show that both the transient thrombocytopenia and GPVI ectodomain shedding depend on the Fc portion of the anti-GPVI antibody and its interaction with the inhibitory Fcγ receptor (FcγR)IIB. In wild-type, but not Fcgr2b−/− mice, anti–GPVI-opsonized platelets became transiently trapped in the liver followed by the appearance of the soluble GPVI ectodomain in the plasma. Depletion of Kupffer cells neither affected anti–GPVI-induced platelet accumulation nor GPVI shedding, demonstrating that the other major FcγRIIB-expressing cell type, liver sinusoidal endothelial cells, is required for both processes to occur. These results reveal a novel and unexpected function of hepatic FcγRIIB in the targeted downregulation of GPVI in vivo.

The Novel Oral Syk Inhibitor, Bl1002494, Protects Mice From Arterial Thrombosis and Thromboinflammatory Brain Infarction.

Ischemic stroke, which is mainly caused by thromboembolic occlusion of brain arteries, is the second leading cause of death and disability worldwide with limited treatment options. The platelet collagen receptor glycoprotein VI (GPVI) is a key player in arterial thrombosis and a critical determinant of stroke outcome, making its signaling pathway an attractive target for pharmacological intervention. The spleen tyrosine kinase (Syk) is an essential signaling mediator downstream of not only GPVI but also other platelet and immune cell receptors. We sought to assess whether Syk might be an effective antithrombotic target. We demonstrate that mice lacking Syk in platelets specifically are protected from arterial thrombus formation and ischemic stroke but display unaltered hemostasis. Furthermore, we show that mice treated with the novel, selective, and orally bioavailable Syk inhibitor BI1002494 were protected in a model of arterial thrombosis and had smaller infarct sizes and a significantly better neurological outcome 24 hours after transient middle cerebral artery occlusion, also when BI1002494 was administered therapeutically, that is, after ischemia. These results provide direct evidence that pharmacological Syk inhibition might provide a safe therapeutic strategy to prevent arterial thrombosis and to limit infarct progression in acute stroke.

Expression of Platelet Collagen Receptor Glycoprotein VI in Coronary Heart Disease: Interaction between Platelets and Endothelial Cells.

1) To investigate the expression of platelet collagen receptor glycoprotein VI (GPVI) on the surface of platelets in patients with coronary heart disease (CHD). 2) To investigate the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the surface of endothelial cells after exposure to subjects' platelets. Platelets were extracted from blood samples of 97 subjects including 43 with acute coronary syndrome (ACS), 21 with stable angina pectoris (SAP), and 33 serving as controls in which CHD was excluded. Using flow cytometry, we measured the expression of GPVI and platelet degranulation markers CD62P and CD63. In addition we quantified expression of endothelial cell ICAM-1 and VCAM-1. A higher expression of platelet GPVI was observed in ACS compared with SAP and control groups. The expression level of platelet GPVI significantly correlated with CD62P and CD63 in the CHD population (r = 0.713 and 0.615, both p < 0.01). Significantly higher ICAM-1 expression was observed in endothelial cells after interaction with platelets from the ACS population. In the CHD subjects, the surface expression of platelet GPVI correlated with the level of ICAM-1 in endothelial cells (r = 0.371, p < 0.05). Platelet GPVI associated with platelet activation and endothelial inflammation is a vital index for predicting CHD occurrence and severity.

Targeting glycoprotein VI and the immunoreceptor tyrosine-based activation motif signaling pathway.

Coronary artery thrombosis and ischemic stroke are often initiated by the disruption of an atherosclerotic plaque and consequent intravascular platelet activation. Thus, antiplatelet drugs are central in the treatment and prevention of the initial, and subsequent, vascular events. However, novel pharmacological targets for platelet inhibition remain an important goal of cardiovascular research because of the negative effect of existing antiplatelet drugs on primary hemostasis. One promising target is the platelet collagen receptor glycoprotein VI. Blockade or antibody-mediated depletion of this receptor in circulating platelets is beneficial in experimental models of thrombosis and thrombo-inflammatory diseases, such as stroke, without impairing hemostasis. In this review, we summarize the importance of glycoprotein VI and (hem)immunoreceptor tyrosine-based activation motif signaling in hemostasis, thrombosis, and thrombo-inflammatory processes and discuss the targeting strategies currently under development for inhibiting glycoprotein VI and its signaling.

Platelets and Arterial Thrombosis: Evolving Role of Platelet Gpvi as an Antithrombotic Drug Target

Acute coronary syndrome is one of the leading causes of mortality and morbidity worldwide. Although the recent advancements in existing anti-platelet therapeutics have significantly reduced the burden of cardiovascular deaths but owing to their global impact on primary haemostasis, their clinical benefits are often limited by bleeding risk. Recent developments in the understanding of platelet biology have placed platelet-collagen interaction at the critical position in the sequence of events during arterial thrombosis. Thus there is considerable interest in developing novel therapeutic strategies against platelet collagen receptor glycoprotein VI (GPVI) because not only its selective blockade or deficiency holds promise for the reduced risk of bleeding complications as evidenced in mice and humans, but also the exclusive expression of GPVI on platelets and megakaryocytes offers to prevent the adverse off-target effects of antithrombotic drugs. The present review summarizes recent developments in understanding of platelet functions during arterial thrombosis and structural and functional attributes of platelet GPVI to identify it as a novel antithrombotic drug target. Moreover, it also emphasises on the development of GPVI antagonists using various experimental approaches as well as their prospective use in cardiovascular disorders.

The Relationship Between Platelet Collagen Receptor Glycoprotein VI and No-Reflow Phenomenon in Patients With Acute ST-Segment Elevation Myocardial Infarction

ObjectivesThe purpose of this study was to determine whether admission soluble glycoprotein VI (sGP-VI) level is associated with no-reflow phenomenon (NRP) after primary percutaneous coronary intervention (P-PCI) in patients with...

Plasma levels of soluble glycoprotein VI (sGPVI) are associated with ischemic stroke

Platelet collagen receptor glycoprotein VI (pGPVI) is elevated in patients with acute coronary syndrome (ACS) and ischemic stroke. Recently, we developed a novel bead-based sandwich immunoassay to determine soluble GPVI (sGPVI), which has been validated in ACS patients. This study aimed to evaluate the plasma levels of sGPVI and pGPVI expression in patients with suspected stroke. We consecutively evaluated 176 patients, who were admitted to the stroke unit. Surface expression of pGPVI was determined by flow cytometry, sGPVI concentrations were determined using our sandwich immunoassay. Unlike patients with TIA, patients with stroke showed significantly decreased plasma levels of sGPVI compared to patients with non-ischemic (NI) events (TIA: mean [µg/L] ± standard deviation): 6.1 ± 2.1 vs. NI: 8 ± 4; p = 0.192; stroke: 5.9 ± 2.3 vs. NI; p = 0.013), whereas for pGPVI, patients with TIA and ischemic stroke revealed a significantly increased platelet surface expression compared to NI patients (TIA: mean fluorescence intensity [MFI] ± standard deviation): 20.9 ± 5.4 vs. NI: 17.6 ± 5.2; p = 0.021; stroke: 20.3 ± 6.2 vs. NI; p = 0.016). Using logistic regression analysis, both sGPVI (p = 0.002) and pGPVI (p = 0.012) are independently associated with ischemic stroke compared to other laboratory markers. To predict the individual risk for ischemic stroke using the plasma levels of sGPVI, receiver operating characteristic (ROC) analysis determined an optimal cutoff value of sGPVI at 6.5 µg/l, thus, patients with decreased plasma levels (<6.5 µg/l) have a 1.5-fold adjusted odds ratio (95%confidence interval, 1.4–2.7). Lower plasma levels of sGPVI are associated with the slightly elevated risk of stroke and may be a promising novel biomarker.

An Acquired Defect Associated with Abnormal Signaling of the Platelet Collagen Receptor Glycoprotein VI

Introduction: Ligands acting at the platelet collagen receptor, glycoprotein (GP)VI, induce intracellular FcRγ/Syk-dependent signaling pathways and Syk-dependent or Syk-independent generation of intracellular reactive oxygen species (ROS). Additional signaling-dependent or signaling-independent pathways lead to metalloproteinase-mediated shedding of GPVI. Aim: Analysis of platelet GPVI expression and signaling in a patient with a collagen-selective defect associated with myelodysplastic syndrome (MDS) uniquely demonstrates divergent pathways leading to ROS generation and Syk phosphorylation in human platelets. Methods: Surface expression of GPVI and ligand-induced ROS generation was quantitated by flow cytometry. GPVI shedding and Syk phosphorylation were analyzed by Western blot. Results: Despite platelet count/size and GPVI surface expression within normal ranges, platelet-rich plasma showed no aggregation in response to collagen or GPVI-selective agonist collagen-related peptide, but aggregated in response to other agonists, consistent with dysfunctional GPVI signaling. We observed rapid GPVI-dependent Syk-independent ROS generation and disulfide-dependent GPVI homodimerization, but not Syk-dependent ROS or ligand-induced shedding. Temporal analysis showed a gradual decline in platelet count and the appearance of ligand-induced phosphorylation of an ∼40-kDa Syk fragment. Conclusions: These studies show that GPVI ligation in platelets induces intracellular ROS production independent of either Syk activation or divergent pathways leading to platelet aggregation or ectodomain shedding.

Constitutive Dimerization of Glycoprotein VI (GPVI) in Resting Platelets Is Essential for Binding to Collagen and Activation in Flowing Blood

The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (K(d)) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)(10)-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ~29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ~39 and ~44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation.

Capture of endothelial progenitor cells by a bispecific protein/monoclonal antibody molecule induces reendothelialization of vascular lesions

Tissue injury is inevitably accompanied by disruption of the endothelium and exposure of the subendothelial matrix. To generate a guidance molecule directing progenitor cells to sites of vascular lesions, we designed a bifunctional protein. The protein consists of the soluble platelet collagen receptor glycoprotein VI and an antibody to CD133 (hereafter called GPVI-CD133). In vitro and in vivo, this construct substantially mediates endothelial progenitor cell (EPC) homing to vascular lesions. Exposure of EPCs to GPVI-CD133 did not impair their capability to differentiate toward mature endothelial cells as verified by the formation of colony-forming units, the upregulation of endothelial markers CD31 and CD146 analyzed by flow cytometry or von Willebrand factor and endoglin assessed by immunofluorescence microscopy, as well as the presence of Weibel-Palade bodies using transmission electron microscopy. In vivo, GPVI-CD133 augments reendothelialization of vascular lesions. Thus, this bifunctional protein could be a potential new therapeutic option for cardiovascular diseases.

Glycoprotein VI for diagnosis of acute coronary syndrome when ECG is ambiguous

BackgroundThe purpose of the study was to test whether an elevated surface expression of platelet collagen receptor glycoprotein VI (GPVI) is an appropriate marker for the diagnosis of the acute coronary syndrome (ACS), especially when the electrocardiogram (ECG) is ambiguous. MethodsBetween 2007 to 2008, we consecutively evaluated 378 patients with ACS and ambiguous ECG on hospital admission. In all patients, GPVI surface expression was determined by flow cytometry. In addition, the myocardial necrosis markers troponin-I (Tn-I) and creatine kinase-MB (CKMB) were measured. ResultsWe found that in patients with ACS and unclear ECG in whom GPVI levels (mean fluorescence intensity (MFI) ≥18.6) were elevated, the relative risk for ACS was 2.6-fold enhanced. Binary logistic regression analysis revealed that an elevated platelet GPVI level is indicating an ACS independent of biomarkers of myocardial necrosis including Tn-I, creatine kinase (CK), CKMB (GPVI: p=0.011; Tn-I: p=0.180; CKMB: p=0.250; CK: p=0.127). Patients with evident T-wave inversion and/or ST-depression showed a strong association between ACS and GPVI expression. ConclusionsPlatelet GPVI surface expression is enhanced in patients with ACS with unclear ECG findings and is strongly associated with myocardial ischemia. Additional to the classical markers of myocardial necrosis Tn-I and CK, GPVI is an early biomarker for the diagnosis of ACS, especially when the ECG is ambiguous.