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.

Platelet Receptor Glycoprotein VI-Dimer Is Overexpressed in Patients with Atrial Fibrillation at High Risk of Ischemic Stroke.

Introduction Atrial fibrillation (AF) increases the risk of ischemic stroke (IS). We hypothesized that the functional form of platelet receptor glycoprotein (GP) VI, GPVI-dimer, which binds to collagen and fibrin causing platelet activation, is overexpressed in patients with AF who have not had a stroke. Methods A total of 75 inpatients with AF were recruited. None were admitted with or had previously had thrombotic events, including IS or myocardial infarction. Platelet surface expression of total GPVI, GPVI-dimer, and the platelet activation marker P-selectin were quantitated by whole blood flow cytometry. Serum biomarkers were collected in AF patients. Results were compared against patients contemporaneously admitted to hospital with similar age and vascular risk-factor profiles without AF (noAF, n = 30). Results Patients with AF have similar total GPVI surface expression ( p = 0.58) and P-selectin exposure ( p = 0.73) on their platelets compared with noAF patients but demonstrate significantly higher GPVI-dimer expression ( p = 0.02 ). Patients with paroxysmal AF express similar GPVI-dimer levels compared with permanent AF and GPVI-dimer levels were not different between anticoagulated groups. Serum N-terminal pro b-type natriuretic peptide ( p < 0.0001 ) and high sensitivity C-reactive protein ( p < 0.0001 ) were significantly correlated with GPVI-dimer expression in AF platelets. AF was the only vascular risk factor that was independently associated with higher GPVI-dimer expression in the whole population ( p = 0.02 ) . Conclusion GPVI inhibition is being explored in clinical trials as a novel target for IS treatment. As GPVI-dimer is elevated in AF patients' platelets, the exploration of targeted GPVI-dimer inhibition for stroke prevention in patients at high risk of IS due to AF is supported.

Role of heat shock protein 47 in platelet glycoprotein VI dimerization and signaling.

Background: Heat shock protein 47 (HSP47) is an intracellular chaperone protein with an indispensable role in collagen biosynthesis in collagen-secreting cells. This chaperone has also been shown to be released and present on the surface of platelets. The inhibition of HSP47 in human platelets or its ablation in mouse platelets reduces platelet function in response to collagen and the glycoprotein (GP) VI collagen receptor agonist CRP-XL. Objectives: In this study, we sought, through experiments, to explore cellular distribution, trafficking, and influence on GPVI interactions to understand how HSP47 modulates collagen receptor signaling. Methods: HSP47-deficient mouse platelets and SMIH- treated human platelets were used to study the role of HSP47 in collagen mediated responses and signaling. Results: Using subcellular fractionation analysis and immunofluorescence microscopy, HSP47 was found to be localized to the platelet-dense tubular system. Following platelet stimulation, HSP47 mobilization to the cell surface was shown to be dependent on actin polymerization, a feature common to other dense tubular system resident platelet proteins that are released to the cell surface during activation. In this location, HSP47 was found to contribute to platelet adhesion to collagen or CRP-XL but not to GFOGER peptide (an integrin α2ß1-binding sequence within collagens), indicating selective effects of HSP47 on GPVI function. Dimerization of GPVI on the platelet surface increases its affinity for collagen. GPVI dimerization was reduced following HSP47 inhibition, as was collagen and CRP-XL-mediated signaling. Conclusion: The present study identifies a role for cell surface-localized HSP47 in modulating platelet responses to collagen through dimerization of GPVI, thereby enhancing platelet signaling and activation.

Factor XIII is a newly identified binding partner for platelet collagen receptor GPVI-dimer-An interaction that may modulate fibrin crosslinking.

Background: In the fibrin-forming process, thrombin cleaves fibrinogen to fibrin, which form fibrils and then fibers, producing a gel-like clot. Thrombin also activates coagulation factor XIII (FXIII), which crosslinks fibrin γ-chains and α-chains, stabilizing the clot. Many proteins bind to fibrin, including FXIII, an established regulation of clot structure, and platelet glycoprotein VI (GPVI), whose contribution to clot function is largely unknown. FXIII is present in plasma, but the abundant FXIII in platelet cytosol becomes exposed to the surface of strongly activated platelets. Objectives: We determined if GPVI interacts with FXIII and how this might modulate clot formation. Methods: We measured interactions between recombinant proteins of the GPVI extracellular domain: GPVI-dimer (GPVI-Fc2) or monomer (GPVIex) and FXIII proteins (nonactivated and thrombin-activated FXIII, FXIII subunits A and B) by ELISA. Binding to fibrin clots and fibrin γ-chain crosslinking were analyzed by immunoblotting. Results: GPVI-dimer, but not GPVI-monomer, bound to FXIII. GPVI-dimer selectively bound to the FXIII A-subunit, but not to the B-subunit, an interaction that was decreased or abrogated by the GPVI-dimer-specific antibody mFab-F. The GPVI-dimer-FXIII interaction decreased the extent of γ-chain crosslinking, indicating a role in the regulation of clot formation. Conclusions: This is the first report of the specific interaction between GPVI-dimer and the A-subunit of FXIII, as determined in an in vitro system with defined components. GPVI-dimer-FXIII binding was inhibitory toward FXIII-catalyzed crosslinking of fibrin γ-chains in fibrin clots. This raises the possibility that GPVI-dimer may negatively modulate fibrin crosslinking induced by FXIII, lessening clot stability.

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

OBJECTIVE: The 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 RESULTS: ELISA 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 αIIbß3, present at high levels, and GPVI. CONCLUSIONS: We 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.

Are integrin alpha(2)beta(1), glycoprotein Ib and vWf levels correlated with their contributions to platelet adhesion on collagen under high-shear flow?

Platelets in flowing blood at high-shear stress are recruited to exposed subendothelial collagen of injured vessels by GPIb-von Willebrand factor (vWf) and integrin alpha(2)beta(1) (alpha(2)beta(1))-collagen interactions. Platelet adhesion to type I collagen depends mainly on the alpha(2)beta(1)-collagen interaction and that to type III collagen depends on the GPIb-vWf interaction due to vWf's weak affinity for type I collagen. Contributions of these two interactions would differ depending on expressions of alpha(2)beta(1), vWf, or GPIb. We quantitated platelet adhesion to low- and high-density collagen under high-shear flow conditions in the presence of anti-alpha(2)beta(1) (Gi9) and anti-GPIb (NNKY5-5) antibodies to determine if their inhibitory effects were correlated with the amounts of alpha(2)beta(1), GPIb and vWf. Gi9 inhibition of adhesion to type I collagen was decreased in platelets with more integrin alpha(2)beta(1). Gi9 and NNKY5-5 are more inhibitory against adhesion to low-density type III and I, respectively. Higher alpha(2)beta(1) expression decreases adhesion to low-density type III and increases Gi9 inhibition of adhesion to high-density type III, suggesting crosstalk between the alpha(2)beta(1)-collagen and GPIb-vWf interactions in adhesion to type III. Integrin alpha(2)beta(1)-collagen and GPIb-vWf interactions both contribute to platelet adhesion to collagen under high-shear flow. In adhesion under high-shear stress, the two interactions would compensate for each other, when there is a deficiency in one or the other. The alpha(2)beta(1)-collagen interaction was also suggested to have an inhibitory effect on platelet adhesion to type III collagen, through a yet undefined mechanism.

Activation-induced changes in platelet surface receptor expression and the contribution of the large-platelet subpopulation to activation.

OBJECTIVE: Platelet surface receptors are also present subcellularly in organelle membranes and can be expressed on the surface upon platelet activation. However, some receptors were reported to be decreased after activation. We analyzed the mechanism of activation-dependent expression for different receptors. METHODS: Flow cytometry using platelet-rich plasma or washed platelets was used to analyze receptor-expression changes after platelet activation by glycoprotein (GP) VI-specific agonists, crosslinked collagen-related peptide (CRP-XL) and convulxin (Cvx), and thrombin. Platelets prelabeled with fluorescent antibody specific for a receptor were allowed to adhere on immobilized collagen or fibrinogen and post-stained with antibody against the same receptor labeled with another fluorophore, allowing us to differentiate preexisting receptors from newly expressed receptors. RESULTS: Surface expression of αIIbß3 increased in CRP-XL-, Cvx-, or thrombin-stimulated platelets, but GPIb decreased due to shedding and internalization. Both total and dimeric GPVI increased in thrombin-induced platelets, but decreased in platelets stimulated by Cvx, as a result of internalization. The larger platelets showed a greater increase in surface receptor (α2ß1, αIIbß3, GPVI, GPIb) expression upon activation compared to the smaller ones. Pre- and postlabeling with antibody specific for the same receptor, but conjugated with different fluorophores, allowed us to differentiate the receptors expressed on the surface of resting platelets from receptors newly exposed to the surface upon platelet activation. CONCLUSIONS: Increased receptor expressions after activation are mainly manifested in the larger platelets. On platelets adhered on fibrinogen, the newly expressed receptors, especially GPVI, are localized in the lamellipodia of the spread platelets.

GPVI surface expression and signalling pathway activation are increased in platelets from obese patients: Elucidating potential anti-atherothrombotic targets in obesity.

BACKGROUND AND AIMS: Platelets play a fundamental role in the increased atherothrombotic risk related to central obesity since they show hyperactivation and lower sensitivity to antiplatelet therapy in obese patients. The main goal of this study was to identify platelet biomarkers related to the risk of atherothrombosis in obese patients, confirm platelet activation levels in these patients, and identify altered activation pathways. METHODS: Platelets were obtained from cohorts of obese patients and age- and sex-matched lean controls. Biochemical and proteome analyses were done by two-dimensional differential in-gel electrophoresis (2D-DIGE), mass spectrometry, and immunoblotting. Functional and mechanistic studies were conducted with aggregation assays and flow cytometry. RESULTS: We confirmed an up-regulation of αIIb and fibrinogen isoforms in platelets from obese patients. A complementary platelet aggregation approach showed platelets from obese patients are hyper-reactive in response to collagen and collagen-related peptide (CRP), revealing the collagen receptor Glycoprotein VI (GPVI) signalling as one of the altered pathways. We also found the active form of Src (pTyr418) is up-regulated in platelets from obese individuals, which links proteomics to aggregation data. Moreover, we showed that CRP-activated platelets present higher levels of tyrosine phosphorylated PLCγ2 in obese patients, confirming alterations in GPVI signalling. In line with the above, flow cytometry studies show higher surface expression levels of total GPVI and GPVI-dimer in obese platelets, both correlating with BMI. CONCLUSIONS: Our results suggest a higher activation state of SFKs-mediated signalling pathways in platelets from obese patients, with a primary involvement of GPVI signalling.

Data on hyper-activation of GPVI signalling in obese patients: Towards the identification of novel antiplatelet targets in obesity.

This data article is associated with the manuscript "GPVI surface expression and signalling pathway activation are increased in platelets from obese patients: elucidating potential anti-atherothrombotic targets in obesity" [1]. The study refers to a combination of different approaches in order to identify platelet-derived biomarkers in obesity. A total of 34 obese patients and their lean-matched controls were included in the study. We carried out a proteomic and functional (aggregation assays) analysis to find alterations in platelet-derived signalling pathways. After that, biochemical and mechanistic (flow cytometry assays) approaches were done in order to confirm a hyperactivation of the GPVI-related signalling pathway.

Collagen-type specificity of glycoprotein VI as a determinant of platelet adhesion.

Of the two physiologically important platelet collagen receptors, glycoprotein (GP) VI is the receptor responsible for platelet activation. However, its reactivities towards different types of vascular collagen have not been directly and quantitatively analysed with collagen preparations of defined composition, although the other major platelet collagen receptor integrin alpha(2)beta(1) was shown to react with collagen types I-VI and VIII under either static or flow conditions. We analysed the collagen type specificity of GPVI binding to identify the physiological contribution of the various vascular collagens and how platelet reactivity towards the various collagens may be affected by fibril size. We used two methods to analyse the binding of recombinant GPVI (GPVI-Fc(2)) to different types of bovine collagen: binding to collagen microparticles in suspension and binding to immobilized collagen. GPVI-Fc(2) bound to type I-III collagens that can form large fibrils, but not to type V that only forms small fibrils. The apparent GPVI binding to types IV and V could be ascribed to type I collagen that was a contaminant in each of these preparations. Kinetic analyses of the binding data showed that type III collagen fibrils have both a higher Kd and Bmax than types I and II. Flow adhesion studies demonstrated that type III collagen supports the formation of larger platelet aggregates than type I. Our present results suggest that the physiological importance of type III collagen is to induce thrombus formation. Furthermore, these studies indicate that GPVI mainly binds to collagen types that can form large collagen fibrils.

Platelet glycoprotein VI.

Glycoprotein VI (GPVI) is a membrane glycoprotein unique to platelets and has been identified as a physiological receptor for collagen. Damage to a vessel wall exposes the subendothelial component collagen to platelets in the blood flow. Interaction of platelets with collagen via the receptor GPVI results in platelet activation and adhesion--the processes that are essential for thrombus formation. On the platelet surface, GPVI is present as a complex with the homodimeric Fc receptor y-chain (FcRgamma with a possible stoichiometry of two GPVI molecules and one FcRgamma dimer). When collagen binds to GPVI, a platelet activation cascade is initiated by tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif of FcRgamma and this phosphorylation induces the formation of a large complex composed from many signal-transducing proteins. In flow adhesion experiments that closely approximate physiological conditions, GPVI is essential for the formation of large platelet aggregates on collagen. However, GPVI-deficient patients or mice do not show any severe bleeding tendency. This suggests that a GPVI inhibitor would be able to inhibit thrombus formation but still not cause a significant bleeding tendency. Such an inhibitor would show promise as an anti-thrombotic agent for clinical use.

MMP-13 binds to platelet receptors αIIbß3 and GPVI and impairs aggregation and thrombus formation.

BACKGROUND: Acute thrombotic syndromes lead to atherosclerotic plaque rupture with subsequent thrombus formation, myocardial infarction and stroke. Following rupture, flowing blood is exposed to plaque components, including collagen, which triggers platelet activation and aggregation. However, plaque rupture releases other components into the surrounding vessel which have the potential to influence platelet function and thrombus formation. OBJECTIVES: Here we sought to elucidate whether matrix metalloproteinase-13 (MMP-13), a collagenolytic metalloproteinase up-regulated in atherothrombotic and inflammatory conditions, affects platelet aggregation and thrombus formation. RESULTS: We demonstrate that MMP-13 is able to bind to platelet receptors alphaIIbbeta3 (αIIbß3) and platelet glycoprotein (GP)VI. The interactions between MMP-13, GPVI and αIIbß3 are sufficient to significantly inhibit washed platelet aggregation and decrease thrombus formation on fibrillar collagen. CONCLUSIONS: Our data demonstrate a role for MMP-13 in the inhibition of both platelet aggregation and thrombus formation in whole flowing blood, and may provide new avenues of research into the mechanisms underlying the subtle role of MMP-13 in atherothrombotic pathologies.

Two novel, putative mechanisms of action for citalopram-induced platelet inhibition.

Citalopram, a selective serotonin reuptake inhibitor (SSRI), inhibits platelet function in vitro. We have previously shown that this action is independent of citalopram's ability to block serotonin uptake by the serotonin transporter and must therefore be mediated via distinct pharmacological mechanisms. We now report evidence for two novel and putative mechanisms of citalopram-induced platelet inhibition. Firstly, in platelets, citalopram blocked U46619-induced Rap1 activation and subsequent platelet aggregation, but failed to inhibit U46619-induced increases in cytosolic Ca2+. Similarly, in neutrophils, citalopram inhibited Rap1 activation and downstream functions but failed to block PAF-induced Ca2+ mobilisation. In a cell-free system, citalopram also reduced CalDAG-GEFI-mediated nucleotide exchange on Rap1B. Secondly, the binding of anti-GPVI antibodies to resting platelets was inhibited by citalopram. Furthermore, citalopram-induced inhibition of GPVI-mediated platelet aggregation was instantaneous, reversible and displayed competitive characteristics, suggesting that these effects were not caused by a reduction in GPVI surface expression, but by simple competitive binding. In conclusion, we propose two novel, putative and distinct inhibitory mechanisms of action for citalopram: (1) inhibition of CalDAG-GEFI/Rap1 signalling, and (2) competitive antagonism of GPVI in platelets. These findings may aid in the development of novel inhibitors of CalDAG-GEFI/Rap1-dependent nucleotide exchange and novel GPVI antagonists.

Analyzing the mechanism of Rap1 activation in platelets: Rap1 activation is related to the release reaction mediated through the collagen receptor GPVI.

The abundant Rap1 in platelets becomes activated when these cells are stimulated by various agonists, but its function has remained unknown. In view of this, we developed an assay to quantitatively measure activated Rap1 and used it to determine relationships between Rap1 activation and several platelet functions: integrin alpha2beta1 activation, tyrosine phosphorylation, and the release reaction. We looked at how these processes are affected by the protein kinase C inhibitor BIMI, tyrosine kinase inhibitor PP2, PI 3-kinase inhibitor wortmannin, and ADP scavenger apyrase. In CRP (collagen related peptide)-activated platelets, all the inhibitors severely inhibited Rap1 activation, but had little effect on integrin alpha2beta1 activation, indicating that the integrin activation mechanism is different from the Rap1 activation mechanism, at least in GPVI-dependent activation. With p85alpha-null mouse platelets, we demonstrated that Rap1 activation involves PI 3-kinase p85alpha-dependent tyrosine phosphorylation. All the inhibitors similarly decreased Rap1 activation and the serotonin release reaction, and the inhibition of Rap1 activation was not due to the lack of released ADP. Our results indicate that platelet Rap1 activation is closely related to the release reaction and not to integrin alpha2beta1 activation in GPVI-mediated platelet activation.

Beyond antiplatelets: The role of glycoprotein VI in ischemic stroke.

BACKGROUND: Platelets are essential to physiological hemostasis or pathological thrombus formation. Current antiplatelet agents inhibit platelet aggregation but leave patients at risk of systemic side-effects such as hemorrhage. Newer therapeutic strategies could involve targeting this cascade earlier during platelet adhesion or activation via inhibitory effects on specific glycoproteins, the thrombogenic collagen receptors found on the platelet surface. AIMS: Glycoprotein VI (GPVI) is increasingly being recognized as the main platelet-collagen receptor involved in arterial thrombosis. This review summarizes the crucial role GPVI plays in ischemic stroke as well as the current strategies used to attempt to inhibit its activity. SUMMARY OF REVIEW: In this review, we discuss the normal hemostatic process, and the role GPVI plays at sites of atherosclerotic plaque rupture. We discuss how the unique structure of GPVI allows for its interaction with collagen and creates downstream signaling that leads to thrombus formation. We summarize the current strategies used to inhibit GPVI activity and how this could translate to a clinically viable entity that may compete with current antiplatelet therapy. CONCLUSION: From animal models, it is clear that GPVI inhibition leads to an abolished platelet response to collagen and reduced platelet aggregation, culminating in smaller arterial thrombi. There is now an increasing body of evidence that these findings can be translated into the development of a bleeding free pharmacological entity specific to sites of plaque rupture in humans.

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

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.

A new monoclonal antibody, mAb 204-11, that influences the binding of platelet GPVI to fibrous collagen.

The newly identified platelet collagen receptor glycoprotein VI binds to fibrous collagen, inducing platelet activation. Several antibodies against GPVI have been reported, including a patient's auto-antibodies, that activates platelets through their ability to crosslink this glycoprotein. We have developed a monoclonal antibody (mAb) against GPVI using the recombinant extracellular domain of GPVI as an antigen. This antibody, mAb 204-11, induced platelet aggregation and tyrosine phosphorylation of proteins similar to those induced by GPVI-reactive proteins, collagen and convulxin. Its interaction with GPVI was analyzed by measuring the effect of the antibody on GPVI binding to collagen using a dimeric form of recombinant GPVI, GPVI-Fc2. MAb 204-11 inhibited the binding of GPVI-Fc2 to fibrous collagen particles, but enhanced the GPVI binding to immobilized collagen, suggesting that the antibody binds to a region near the collagen binding site of GPVI. MAb 204-11 also inhibited the GPVI binding to convulxin at a low concentration, but not completely. Since mAb 204-11 reacts specifically with GPVI and is applicable for immunoblotting and immunoprecipitation, this antibody would be useful for studies on GPVI.

Platelet glycoprotein VI: its structure and function.

Glycoprotein (GP) VI is a platelet membrane protein with a molecular weight of 62 kDa that was identified as a physiological collagen receptor from studies of patients deficient in this protein. GPVI-deficient platelets lacked specifically collagen-induced aggregation and the ability to form thrombi on a collagen surface under flow conditions, suggesting that GPVI makes an indispensable contribution to collagen-induced platelet activation. On the platelet surface, GPVI is present as a complex with the Fc receptor (FcR) gamma-chain, probably composed of two GPVI molecules and one FcR gamma-chain dimer. GPVI must form such a dimeric complex to exhibit high affinity binding to collagen. The GPVI-induced activation mechanism is initiated by tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of the FcR gamma-chain, and then this signal is transduced to many related proteins, mainly by tyrosine phosphorylation. GPVI is widely recognized as a requisite factor for the formation of platelet aggregates on a collagen surface under blood flow. However, individuals with GPVI-deficient or null platelets do not exhibit any strong bleeding tendency. Analyzing this apparent dichotomy should provide us with a more precise understanding of the mechanism of thrombus formation.

Novel approach for formation of platelet-like particles from mouse embryonic stem cells without using feeder cells.

Megakaryocytes (MKs) and platelet-like particles (PLPs) have generally been obtained by culturing embryonic stem (ES) cells over feeder cells. However, using feeder cells need many labor-consuming processes and the MK and PLP fractions obtained are often contaminated by such cells and their fragments. Here we describe our new culture system for differentiating mouse ES cells to MKs and PLPs without using feeder cells. ES cells are differentiated to cells with MK-like morphology and properties, including proplatelet formation, high ploidy (>8N), and CD41 expression. The culture medium contained PLPs expressing platelet glycoproteins, CD41 and GPIb. Integrin alpha(IIb)beta(3) of PLPs can be activated by thrombin. Addition of the metalloproteinase inhibitor TAPI-2 to the culture increased the surface expression of GPIbalpha and augmented the adhesion of PLPs to immobilized von Willebrand factor through decreasing the shedding of GPIbalpha. Thus our mouse ES cells culture system is a suitable and efficient method for obtaining MKs and functional PLPs that obviates the need for feeder cells.