The mechanism by which occupancy of collagen receptors is coupled to platelet activation has been uncertain. Our group previously demonstrated that glycoprotein (GP) VI, an uncharacterized platelet membrane protein, is specifically required for collagen-platelet interaction leading to activation of protein-tyrosine kinase Syk. Since collagen stimulation of platelets has recently been found to induce tyrosine phosphorylation of Fc receptor (FcR) ␥-chain, a signal-generating subunit of FcR, we further investigated the relationships between FcR ␥-chain and GPVI in human platelets. Our present study revealed the following. FcR ␥-chain was physically and stably associated with GPVI in human platelets; both FcR ␥-chain and GPVI were proportionally absent in GPVI-deficient platelets; GPVI cross-linking or collagen stimulation of platelets resulted in tyrosine phosphorylation of GPVI-associated FcR ␥-chain accompanied by Syk association and activation. These findings strongly suggest that the associated complex of GPVI and FcR ␥-chain is a collagen receptor featuring the signaling through immune receptors.Despite the widely accepted consensus that the interactions between the extracellular matrix protein collagen and platelets are vital for the maintenance of hemostasis, the exact nature of collagen receptor on platelets has been a great enigma to date except for well characterized integrin heterodimer ␣ 2 ␤ 1 (1, 2), also called glycoprotein (GP) 1 Ia-IIa, as a principal adhesion receptor for collagen. Among several candidates that have been proposed to be platelet collagen receptors (3-7), we have recently provided biochemical evidence that GPVI, as yet an unidentified 62-kDa platelet membrane protein (6, 7), is specifically required for collagen-induced platelet activation other than GPIa-IIa (integrin ␣ 2 ␤ 1 ). GPVI cross-linking with the F(abЈ) 2 fragments of anti-GPVI IgG (F(abЈ) 2 ␣GPVI) induces cAMP-insensitive activation of protein-tyrosine kinase Syk accompanied by tyrosine phosphorylation of phospholipase C␥2 (PLC␥2) in a manner similar to collagen stimulation (8); GPVIdeficient platelets (6, 7, 9) expressing a normal amount of GPIa-IIa exhibit lack of collagen-stimulated Syk activation and tyrosine phosphorylation of PLC␥2 (10). However, the question of how GPVI is involved in collagen receptor and transduces signals leading to Syk activation accompanied by tyrosine phosphorylation of PLC␥2 still remains unsolved.One of the mechanisms by which Syk is activated is achieved via interaction between its tandem Src homology 2 (SH2) domains and a tyrosine-phosphorylated activation motif, termed the immunoreceptor tyrosine-based activation motif, found in receptors of the immune system or their associated chains (11). In platelets, this mechanism of Syk activation is a prerequisite for the activation through a low affinity Fc receptor for IgG (Fc␥R) (12, 13). Among known Fc receptors belonging to the immunoglobulin superfamily (14), human platelets express only a single Fc␥R encoded by the Fc␥RIIA gene (15). Rec...

Activation of circulating platelets by subendothelial collagen is an essential event in vascular hemostasis. In human platelets, two membrane glycoprotein (GP) abnormalities, integrin alpha2 beta1 deficiency and GPVI deficiency, have been reported to result in severe hyporesponsiveness to fibrillar collagen. Although it has been well established that integrin alpha2 beta1, also known as the GPIa-IIa complex, functions as a primary platelet adhesion receptor for collagen, the mechanism by which GPVI contributes to collagen-platelet interaction has been ill defined to date. However, our recent observation that GPVI cross-linking couples to cyclic AMP-insensitive activation of c-Src and Syk tyrosine kinases suggested a potential role for GPVI in regulating protein-tyrosine phosphorylation by collagen (Ichinohe, T., Takayama, H., Ezumi, Y., Yanagi, S., Yamamura, H., and Okuma, M. (1995) J. Biol. Chem. 270, 28029-28036). To further investigate this hypothesis, here we examined the collagen-induced protein-tyrosine phosphorylation in GPVI-deficient platelets expressing normal amounts of alpha2 beta1. In response to collagen, these platelets exhibited alpha2 beta1-dependent c-Src activation accompanied by tyrosine phosphorylation of several substrates including cortactin. In contrast, severe defects were observed in collagen-stimulated Syk activation and tyrosine phosphorylation of phospholipase C-gamma2, Vav, and focal adhesion kinase, implicating a specific requirement of GPVI for recruiting these molecules to signaling cascades evoked by collagen-platelet interaction.

Platelet glycoprotein VI (GPVI), a 62kD membrane protein, has been identified as one of the platelet receptors for collagen, since GPVI-deficient platelets exhibit abnormal responses to collagen and an abnormal bleeding tendency. We report a female patient with a mild bleeding history whose platelets expressed 10% GPVI of normal platelets. Shape change, aggregation and ATP release of the patient's platelets were completely absent in response to 1-5 micrograms/ml collagen but present normally in response to ADP and Ca2+ ionophore A23187. Adhesion of the patient's platelets to coated collagen was mildly affected (40-60% of normal platelets) in spite of only 10% expression of GPVI. Flow cytometrical studies revealed that the patient's platelets expressed normal amounts of the GPIa/IIa complex. These results suggest that platelet GPVI is less involved in adhesion to collagen than shape change and aggregation induced by collagen.

Background-We studied the role of glycoprotein (GP) VI in platelet adhesion and thrombus formation on the immobilized collagen and von Willebrand factor (vWF) surface under flow conditions. Methods and Results-Whole blood obtained from 2 patients with GP VI-deficient platelets and the effects of the Fab of anti-GP VI antibody (Fab/anti-GP VI) were tested. Blood containing platelets rendered fluorescent by mepacrine was perfused on immobilized type I collagen or vWF under controlled wall shear rate. Platelet adhesion and thrombus formation were detected by epifluorescent videomicroscopy. The percentage of surface coverage by the platelets was calculated. Fc receptor ␥-chain and spleen tyrosine kinase (Syk) were immunoprecipitated from the lysate of platelets stimulated by vWF plus ristocetin and then analyzed by antiphosphotyrosine immunoblotting. No platelet attachment was seen on the surface of collagen even after 9 minutes of perfusion of blood at relatively low (100 s Ϫ1 ) or high (1500 s

Most antibodies to factor VIII have recently been shown to react with discrete regions of the factor VIII light chain (within the C2 domain) and/or the factor VIII heavy chain (within the amino-terminal segment of the A2 domain). The mechanism by which these antibodies, usually designated "factor VIII inhibitors," interfere with factor VIII function has been examined by determining their effect on factor VIII binding to a phospholipid. Factor VIII-phosphatidylserine binding was prevented by all seven factor VIII inhibitors that had strong factor VIII light chain reactivity and reduced by two inhibitors with weak anti-light chain reactivity. None of four inhibitors with heavy chain reactivity prevented factor VIII-phosphatidylserine interaction, though a partial reduction (< 50%) was noted for the intact IgG preparations. However, when Fab' fragments were substituted, no detectable reduction in factor VIII-phosphatidylserine binding was noted for the anti-heavy chain inhibitors and complete inhibition was retained by the anti-light chain inhibitors. These data suggest that a subset of factor VIII inhibitors, those that bind to light chain determinants, inactivate factor VIII by preventing its effective interaction with phospholipid.

To date it has been difficult to characterize completely a genetic disorder, such as hemophilia A, in which the involved gene is large and unrelated affected individuals have different mutations, most of which are point mutations. Toward this end, we analyzed the DNA of 29 patients with mild-to-moderate hemophilia A in which the causative mutation is likely to be a missense mutation. Using computer analysis, we determined the melting properties of factor VIII gene sequences to design primer sets for PCR amplification and subsequent denaturing gradient gel electrophoresis (DGGE). A total of 45 primer sets was chosen to amplify 99% of the coding region of the gene and 41 of 50 splice junctions. To facilitate detection of point mutations, we mixed DNA from two male patients, and both homoduplexes and heteroduplexes were analyzed. With these 45 primer sets, 26 DNAs containing previously identified point mutations in the factor VIII gene were studied, and all 26 mutations were easily distinguishable from normal. After analyzing the 29 patients with unknown mutations, we identified the disease-producing mutation in 25 (86%). Two polymorphisms and two rare normal variants were also found. Therefore, DGGE after computer analysis is a powerful method for nearly complete characterization of disease-producing mutations and polymorphisms in large genes such as that for factor VIII.

Key Points• Clot retraction of sphingomyelin-rich raftdepleted platelets from sphingomyelin synthase knockout mouse is delayed.• Translocation of fibrin to sphingomyelin-rich rafts in platelet membrane is induced by thrombin in the presence of FXIII crosslinking activity.Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen g-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunitdeficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-b-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-aIIbb3-myosin complex is formed as a primary axis to promote platelet contraction. (Blood. 2013;122(19):3340-3348) IntroductionMembrane rafts are dynamic assemblies of sphingolipids, cholesterol, and proteins that can be stabilized into platforms involved in the regulation of a number of vital cellular processes. 1 The important role of rafts at the cell surface may be their function in signal transduction. A number of studies provide considerable evidence that rafts are integral to the regulation of immune and neuronal signaling. Membrane rafts are also involved in hemostasis and thrombosis. Among blood cells, platelets are critical for maintaining the integrity of the blood coagulation system. Platelet rafts are critical membrane domains in physiological responses such as adhesion and aggregation. 2 The localization of the adhesion receptor glycoprotein (GP)Ib-IX-V complex to membrane rafts is required for platelet adhesion to the vessel wall by binding the von Willebrand factor. 3 Membrane rafts are also required for platelet aggregation via the collagen receptor GPVI, 4 the adenosine 59-diphosphate (ADP) receptor P2Y12, 5 the Fcg receptor FcgRIIa, 6 and the C-type lectinlike receptor CLEC-2.7 Detergent-resistant membrane (DRM) rafts of platelets show round vesicles of heterogeneous sizes ranging from 20 to 500 nm, which are enriched in CD36 (GPIV). 8,9 Recent reports have demonstrated that membrane rafts are ...

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