Platelet collagen receptor Glycoprotein VI-dimer recognizes fibrinogen and fibrin through their D-domains, contributing to platelet adhesion and activation during thrombus formation.

Essentials Glycoprotein VI (GPVI) binds collagen, starting thrombogenesis, and fibrin, stabilizing thrombi. GPVI-dimers, not monomers, recognize immobilized fibrinogen and fibrin through their D-domains. Collagen, D-fragment and D-dimer may share a common or proximate binding site(s) on GPVI-dimer. GPVI-dimer-fibrin interaction supports spreading, activation and adhesion involving αIIbß3. SUMMARY: Background Platelet collagen receptor Glycoprotein VI (GPVI) binds collagen, initiating thrombogenesis, and stabilizes thrombi by binding fibrin. Objectives To determine if GPVI-dimer, GPVI-monomer, or both bind to fibrinogen substrates, and which region common to these substrates contains the interaction site. Methods Recombinant GPVI monomeric extracellular domain (GPVIex ) or dimeric Fc-fusion protein (GPVI-Fc2 ) binding to immobilized fibrinogen derivatives was measured by ELISA, including competition assays involving collagenous substrates and fibrinogen derivatives. Flow adhesion was performed with normal or Glanzmann thrombasthenic (GT) platelets over immobilized fibrinogen, with or without anti-GPVI-dimer or anti-αIIbß3. Results Under static conditions, GPVIex did not bind to any fibrinogen substrate. GPVI-Fc2 exhibited specific, saturable binding to both D-fragment and D-dimer, which was inhibited by mFab-F (anti-GPVI-dimer), but showed low binding to fibrinogen and fibrin under our conditions. GPVI-Fc2 binding to D-fragment or D-dimer was abrogated by collagen type III, Horm collagen or CRP-XL (crosslinked collagen-related peptide), suggesting proximity between the D-domain and collagen binding sites on GPVI-dimer. Under low shear, adhesion of normal platelets to D-fragment, D-dimer, fibrinogen and fibrin was inhibited by mFab-F (inhibitor of GPVI-dimer) and abolished by Eptifibatide (inhibitor of αIIbß3), suggesting that both receptors contribute to thrombus formation on these substrates, but αIIbß3 makes a greater contribution. Notably, thrombasthenic platelets showed limited adhesion to fibrinogen substrates under flow, which was further reduced by mFab-F, supporting some independent GPVI-dimer involvement in this interaction. Conclusion Only dimeric GPVI interacts with fibrinogen D-domain, at a site proximate to its collagen binding site, to support platelet adhesion/activation/aggregate formation on immobilized fibrinogen and polymerized fibrin.

Clustering of glycoprotein VI (GPVI) dimers upon adhesion to collagen as a mechanism to regulate GPVI signaling in platelets.

Essentials Dimeric high-affinity collagen receptor glycoprotein VI (GPVI) is present on resting platelets. Spatio-temporal organization of platelet GPVI-dimers was evaluated using advanced microscopy. Upon platelet adhesion to collagenous substrates, GPVI-dimers coalesce to form clusters. Clustering of GPVI-dimers may increase avidity and facilitate platelet activation SUMMARY: Background Platelet glycoprotein VI (GPVI) binding to subendothelial collagen exposed upon blood vessel injury initiates thrombus formation. Dimeric GPVI has high affinity for collagen, and occurs constitutively on resting platelets. Objective To identify higher-order oligomerization (clustering) of pre-existing GPVI dimers upon interaction with collagen as a mechanism to initiate GPVI-mediated signaling. Methods GPVI was located by use of fluorophore-conjugated GPVI dimer-specific Fab (antigen-binding fragment). The tested substrates include Horm collagen I fibers, soluble collagen III, GPVI-specific collagen peptides, and fibrinogen. GPVI dimer clusters on the platelet surface interacting with these substrates were visualized with complementary imaging techniques: total internal reflection fluorescence microscopy to monitor real-time interactions, and direct stochastic optical reconstruction microscopy (dSTORM), providing relative quantification of GPVI cluster size and density. Confocal microscopy was used to locate GPVI dimer clusters, glycoprotein Ib, integrin α2 ß1 , and phosphotyrosine. Results Upon platelet adhesion to all collagenous substrates, GPVI dimers coalesced to form clusters; notably clusters formed along the fibers of Horm collagen. dSTORM revealed that GPVI density within clusters depended on the substrate, collagen III being the most effective. Clusters on fibrinogen-adhered platelets were much smaller and more numerous; whether these are pre-existing oligomers of GPVI dimers or fibrinogen-induced is not clear. Some GPVI dimer clusters colocalized with areas of phosphotyrosine, indicative of signaling activity. Integrin α2 ß1 was localized to collagen fibers close to GPVI dimer clusters. GPVI clustering depends on a dynamic actin cytoskeleton. Conclusions Platelet adhesion to collagen induces GPVI dimer clustering. GPVI clustering increases both avidity for collagen and the proximity of GPVI-associated signaling molecules, which may be crucial for the initiation and persistence of signaling.

Presence of platelet-associated anti-glycoprotein (GP)VI autoantibodies and restoration of GPVI expression in patients with GPVI deficiency.

BACKGROUND: Glycoprotein (GP)VI deficiency is a rare platelet disorder with a mild bleeding tendency. However, its pathophysiology remains unclear. OBJECTIVES: We characterized a novel GPVI-deficient patient with immune thrombocytopenic purpura and searched for the presence of anti-GPVI autoantibodies in this and another patient with GPVI deficiency. METHODS AND RESULTS: A 12-year-old Japanese girl (case 1) with moderate thrombocytopenia and mild bleeding showed selectively impaired collagen-induced platelet aggregation. Flow cytometric analysis indicated that the patient had a defect in the expression of GPVI-FcRgamma. An eluate of her platelet-associated IgG contained anti-alpha(IIb)beta3 autoantibodies. Moreover, using GPVI-FcRgamma-transfected cells, we unexpectedly identified anti-GPVI antibodies against the soluble ectodomain of GPVI in the eluate, despite the patient's GPVI deficiency. In contrast, anti-GPVI antibodies were not detectable in her plasma. In another case of GPVI deficiency (case 2) without detectable plasma anti-GPVI antibodies, we again detected platelet-associated anti-GPVI antibodies. In a 2-year follow-up of case 1, the platelet count increased to within the normal range and the bleeding tendency improved. Interestingly, GPVI was again expressed on her platelets, in association with a decrease in the relative amount of anti-GPVI antibodies. CONCLUSIONS: This is the first demonstration of platelet-associated anti-GPVI antibodies in GPVI-deficient subjects, in one case with spontaneous restoration of GPVI expression. These results strongly suggest an autoimmune mechanism in GPVI deficiency.

Glycoprotein (GP) VI dimer as a major collagen-binding site of native platelets: direct evidence obtained with dimeric GPVI-specific Fabs.

BACKGROUND: The platelet collagen receptor glycoprotein (GP) VI is suggested to exist as a dimer on the platelet surface, but no direct proof of the functional importance of dimer formation has been provided. OBJECTIVES: To obtain direct evidence for GPVI dimers on the platelet membrane and their functional importance, Fab antibodies were developed that bind to GPVI dimer (GPVI-Fc2) but not to GPVI monomer (GPVIex) through a phage display method. RESULTS: Six Fabs were found: B-F, only reactive with GPVI-Fc2, and A, mainly reactive with GPVI-Fc2, with some reactivity towards GPVIex; each Fab (Fab-dHLX-MH) forms a bivalent dimer (b-Fab) by dimerizing the dHLX domains from two Fab molecules. Fab F was subcloned to a monovalent format by deleting its dHLX domain. All b-Fabs induced platelet aggregation, but the monomeric form of Fab F (m-Fab-F) specifically inhibited collagen-induced aggregation. All b-Fabs and m-Fab-F inhibited GPVI-Fc2 binding to fibrous collagen. Immunoblotting showed that b-Fab-F and m-Fab-F bound weakly to GPVI-Fc2. Adding the anti-GPVI monoclonal antibody 204-11 increased the B(max) of m-Fab-F binding to GPVI-Fc2, suggesting that 204-11 binds to GPVI-Fc2 molecules not already in the appropriate conformation to recognize the Fab, converting them to a conformation reactive to the Fab. CONCLUSIONS: GPVI forms a specific structure by dimerization that is necessary for the binding of this receptor to collagen fibrils. The binding of m-Fab-F to platelets directly demonstrates that GPVI is present as a functionally relevant dimer on the platelet surface.

Compromised ITAM-based platelet receptor function in a patient with immune thrombocytopenic purpura.

BACKGROUND: Receptors on platelets that contain immunoreceptor tyrosine-based activation motifs (ITAMs) include collagen receptor glycoprotein (GP) VI, and FcgammaRIIa, a low affinity receptor for immunoglobulin (Ig) G. OBJECTIVES: We examined the function of GPVI and FcgammaRIIa in a patient diagnosed with immune thrombocytopenic purpura (ITP) who had unexplained pathological bruising despite normalization of the platelet count with treatment. METHODS AND RESULTS: Patient platelets aggregated normally in response to ADP, arachadonic acid and epinephrine, but not to GPVI agonists, collagen or collagen-related peptide, or to FcgammaRII-activating monoclonal antibody (mAb) 8.26, suggesting ITAM receptor dysfunction. Plasma contained an anti-GPVI antibody by MAIPA and aggregated normal platelets. Aggregating activity was partially (approximately 60%) blocked by FcgammaRIIa-blocking antibody, IV.3, and completely blocked by soluble GPVI ectodomain. Full-length GPVI on the patient platelet surface was reduced to approximately 10% of normal levels, and a approximately 10-kDa GPVI cytoplasmic tail remnant and cleaved FcgammaRIIa were detectable by western blot, indicating platelet receptor proteolysis. Plasma from the patient contained approximately 150 ng mL(-1) soluble GPVI by ELISA (normal plasma, approximately 15 ng mL(-1)) and IgG purified from patient plasma caused FcgammaRIIa-mediated, EDTA-sensitive cleavage of both GPVI and FcgammaRIIa on normal platelets. CONCLUSIONS: In ITP patients, platelet autoantibodies can curtail platelet receptor function. Platelet ITAM receptor dysfunction may contribute to the increased bleeding phenotype observed in some patients with ITP.

A mechanism to safeguard platelet adhesion under high shear flow: von Willebrand factor-glycoprotein Ib and integrin alphabeta-collagen interactions make complementary, collagen-type-specific contributions to adhesion.

BACKGROUND: Blood vessels contain different types of collagen, with types I and III being the major components of vascular collagen. Platelet adhesion under high shear stress has been suggested to depend on the binding of von Willebrand factor (VWF) to collagen. OBJECTIVE: We analyzed the collagen type specificity for the interaction with VWF and high shear stress platelet adhesion. METHODS: VWF binding to different types of immobilized collagen and effects of antibodies against glycoprotein Ib (gpIb) and integrin alpha(2)beta(1) on platelet adhesion to type I and III collagens under high shear were analyzed. RESULTS: VWF showed high-affinity, selective binding to human and bovine type III collagens, but weak or no affinity for types I, II, IV and V under static conditions. Anti-integrin alpha(2)beta(1) markedly inhibited adhesion to type I collagen, but did not affect that to type III collagen. Anti-gpIb antibody significantly inhibited adhesion to type III collagen. Adding both antibodies abrogated the adhesion to either type I or III collagen. CONCLUSIONS: Both the gpIb-VWF interaction and the integrin alpha(2)beta(1)-collagen interaction contribute to platelet adhesion to collagen under high shear stress, and integrin alpha(II)beta(1) makes a greater contribution to adhesion to type I collagen because less VWF is bound to it.

Characterization of a patient with glycoprotein (GP) VI deficiency possessing neither anti-GPVI autoantibody nor genetic aberration.

BACKGROUND: There have been only seven reported cases of glycoprotein (GP) VI deficiency. However, the pathogenesis of this disorder has not been well-elucidated. OBJECTIVES: We characterized a novel patient with GPVI deficiency and used these platelets to investigate the role of GPVI in normal hemostasis. PATIENT: A 31-year-old female with immune thrombocytopenic purpura who had been suffering from mild bleeding diathesis even after recovery from thrombocytopenia. RESULTS AND CONCLUSION: The patient's platelets did not aggregate in response to either convulxin or collagen-related peptide. Immunoblotting revealed complete absence of the GPVI molecule, whereas a significantly reduced but substantial amount of Fc receptor (FcR) gamma-chain was expressed. Platelet stimulation with convulxin did not induce tyrosine-phosphorylation of FcR gamma-chain, indicating a defect in GPVI-mediated signaling. Concerning the underlying pathogenesis, we found normal level of GPVI-mRNA expression, no aberration of the sequence of the entire coding region of GPVI, and presence of degraded GPVI in her plasma. However, no anti-GPVI autoantibody was detected either by the binding assay to GPVI-Fc2 fusion protein or by immunoblotting/immunoprecipitation using the patient's immunoglobulin. We thus consider that either a short-time exposure to anti-GPVI autoantibody or a continuous exposure to low titers of the autoantibody has resulted in persistent GPVI deficiency. Under high shear flow, the patient's platelets could not form large aggregates, although initial platelet attachment was obviously observed. These results suggest that GPVI deficiency in this patient resulted in defective platelet thrombi development, manifesting as bleeding diathesis. Furthermore, our observations indicate that coordination of GPVI with integrin alpha2beta1 is essential for physiological platelet thrombus formation.

Antibody against platelet membrane glycoprotein VI in a patient with systemic lupus erythematosus.

Platelet-collagen interaction is important in primary hemostasis and collagen receptors on the platelet surface include membrane glycoprotein (GP) Ia/IIa and VI. Platelets from a 47-year-old woman with systemic lupus erythematosus (SLE) and a mild bleeding symptom showed a defective collagen-induced aggregation and an impaired adhesion to collagen surface. The patient's platelets had a markedly decreased content of GPVI. The patient had an antibody against GPVI in serum and the patient's plasma induced aggregation and release reaction of normal platelets. These findings indicate that GPVI is an important receptor for collagen on the platelet surface, and that anti-GPVI antibody activates the platelets, resulting in aggregation. This is the first documented case of SLE who acquired a platelet-aggregating anti-GPVI antibody.

Platelet collagen receptor integrin alpha2beta1 activation involves differential participation of ADP-receptor subtypes P2Y1 and P2Y12 but not intracellular calcium change.

In agonist-induced platelet activation, the collagen platelet receptor integrin alpha2beta1 is activated to high-affinity states through ADP involvement [Jung, S.M. & Moroi, M. (2000) J. Biol. Chem. 275, 8016-8026]. Here we determined the ADP-receptor subtypes involved and their relative contributions to alpha2beta1 activation (assessed by soluble-collagen binding) using the P2Y12 antagonist AR-C69931MX and P2Y1 antagonists adenosine 3',5'-diphosphate (Ado(3,5)PP) and adenosine 3'-phosphate 5'-phosphosulfate (AdoPPS). All three inhibited alpha2beta1 activation induced by low or high ADP, low thrombin, or low collagen-related peptide (CRP) concentrations; however, AR-C69931MX was markedly more inhibitory than the P2Y1 antagonists, suggesting the greater contribution of P2Y12. Inhibition patterns by various combinations of AR-C69931MX, AdoPPS, and wortmannin suggested that P2Y1 and P2Y12 mediate alpha2beta1 activation through different pathways, with possible involvement of phosphoinositide 3-kinase in both. Low concentrations of the acetoxy-methyl derivative of 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (calcium chelator) markedly decreased alpha2beta1 activation by low thrombin or CRP, but did not affect that by low or high ADP. Measurements of intracellular Ca2+ level (fluorimetric method) and alpha2beta1 activation (soluble-collagen binding) in the same platelet preparation indicated that alpha2beta1 activation via ADP receptors was independent of intracellular Ca2+ release. Our data indicate that integrin alpha2beta1 activation by ADP occurs through an inside-out signaling mechanism involving differential contributions by P2Y1 and P2Y12 wherein each contributes to some portion of the activation, with the stronger contribution of P2Y12. Furthermore, intracellular Ca2+ increase is not directly related to integrin alpha2beta1 activation, meaning that it is separate from the calcium mobilization pathways that these two ADP receptors are involved in.

Real-time analysis of the interaction of platelets with immobilized thrombospondin under flow conditions.

The platelet granule protein (TS) is extracellularly secreted upon platelet activation and then binds to the platelet surface where it can interact with various adhesive proteins. Here, we have analyzed platelet interactions with a TS-coated surface under flow conditions, a model for platelet adhesion onto surface-bound TS under physiological conditions. Platelets exhibited temporary, very short-time adhesion on the TS surface, but no firm adhesion. This adhesion was inhibited by NNKY5-5 (anti-glycoprotein (GP) Ib antibody) and AJvW-2 (anti-von Willebrand factor (vWF)), indicating that both platelet GP Ib and plasma vWF contribute to this interaction. Antibodies against platelet collagen receptor integrin alpha(2)beta(1) had no significant effect. These results suggested that binding of vWF to TS is the first step in platelet interaction with the TS surface. By surface plasmon resonance spectroscopy, a dissociation constant (K(d)) of 3.97x10(-7) M was obtained for the binding reaction between immobilized TS and vWF. These results suggest the following model for platelet interaction with the TS surface under flow: plasma vWF first binds to the immobilized TS and then platelets interact with the TS-bound vWF. A low density of bound vWF would account for the observed weak interaction between TS and platelets under flow.

[Influence of age on serial change in TL/BMIPP dual isotope SPECT images after direct PTCA in patients with acute myocardial infarction].

The purpose of this study was to investigate the influence of age on serial change in 201TlCl (TL) and 123I-BMIPP (BMIPP) dual isotope single photon emission computed tomography (SPECT) images after direct PTCA in patients (pts) with acute myocardial infarction (MI). Dual SPECT with TL and BMIPP at rest, radionuclide ventriculography for left ventricular ejection fraction (LVEF), and two-dimensional echocardiography for wall motion analysis were performed in 26 pts at the subacute and chronic phases after direct PTCA for acute MI. A defect score (DS) for SPECT images was interpreted as normal: 0, mildly decreased: 1, moderately or severely decreased: 2, complete defect: 3. The difference in DS between TL and BMIPP was defined as the mismatch score (MS). DS in BMIPP was greater than that in TL at the subacute phase in all pts. Significant improvement in the wall motion score was recognized in pts who showed TL/BMIPP discrepancy at the subacute phase. Pts were classified by age into two groups; group I: younger than 65 years old (n = 18); group II: 65 years and older (n = 8). Improvement of MS from the subacute to chronic phase was significant in group I (5.2 +/- 1.9 to 3.2 +/- 1.9, p = 0.0001), whereas not significant in group II (6.2 +/- 2.9 to 6.1 +/- 2.9, NS). There was a significant negative correlation between relative MS (ratio of subacute MS to chronic MS) and age (r = -0.78, p < 0.0001). No significant correlation was observed between age and improvement in LVEF. These results indicate that disordered myocardial fatty acid metabolism, reflected by TL/BMIPP discrepancy, persist longer in elderly pts than younger pts after acute MI.

Involvement of activated integrin alpha2beta1 in the firm adhesion of platelets onto a surface of immobilized collagen under flow conditions.

Recently, we demonstrated that agonist-induced activation of the platelet surface collagen-receptor integrin alpha1beta2 converts it to an active form that can bind soluble collagen with high affinity (Jung, SM, Moroi, M: J Biol Chem 1998; 273: 14827-37). Here, the involvement of alpha2beta1 activation and the high affinity binding property of activated alpha2beta1 in platelet adhesion to a collagen surface under flow conditions were analyzed. Platelet adhesion to immobilized collagen was measured in the presence of TS2/16, an activating anti-integrin alpha2beta1 antibody, and inhibiting antibodies, Gi9 and 6F1. TS2/16 decreased the moving velocity of platelets on the collagen surface, but Gi9 and 6F1 increased it, indicating that alpha2beta1 activation induces the tight binding of platelets to immobilized collagen under flow. Platelet adhesion, expressed as the surface area occupied by adhered platelets, in the presence of TS2/16 was similar to that in its absence. In contrast, adding Gi9 or 6F1 caused biphasic adhesion composed of a first phase, a lag phase whose length differed in each experiment, and a second phase adhesion with a rate similar to that of the control. This biphasic adhesion indicates that alpha2beta1 activity is inhibited and also suggests that some other factor(s) may contribute to the adhesion under flow. At concentrations where neither 6F1 nor Gi9 affected collagen-induced aggregation, these antibodies inhibited soluble collagen binding to thrombin-activated platelets. Only at much higher concentration did 6F1 inhibit collagen-induced aggregation. TS2/16 had no effect on the aggregation. The present results are evidence against the major involvement of integrin alpha2beta1 in platelet aggregation; instead, they indicate that integrin alpha2beta1 would be mainly associated with the tight binding of platelets to collagen.

Cloning and expression of the platelet-specific collagen receptor glycoprotein VI.

Platelet glycoprotein VI (GP VI) was purified from platelet membranes and its internal amino acid sequences were determined. The cloned cDNA of GP VI indicates an open reading frame coding for 20 amino acid signal sequences and a mature protein of 319 amino acids. Its extracellular region has two Ig-like domains and a mucin-like, Ser/Thr-rich region, suggesting that GP VI is a member of the paired Ig-like receptor family. GP VI-transfected cells contained convulxin-(reactive) and antibody against recombinant GP VI-reactive protein bands that migrated at the same position as platelet GP VI in SDS/PAGE-electroblotting. These data indicate that the protein deduced from the cloned cDNA corresponds to platelet GP VI.

Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1).

Evidence was obtained about the mechanism responsible for platelet integrin alpha(2)beta activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin alpha(2)beta(1) activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin alpha(2)beta(1), but those operating via glycoprotein Ib cannot. Activation of alpha(2)beta(1) induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin alpha(2)beta(1) activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin alpha(2)beta(1) activation, while at the high agonist concentrations, there would be several pathways through which integrin alpha(2)beta(1) activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B(max)) as thrombin-induced platelets, but their affinity (K(d)) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin alpha(2)beta(1) induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin alpha(2)beta(1); these activated forms of integrin alpha(2)beta(1) would have different conformations that determine their ligand affinity.