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...

We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor γ chain (FcRγ) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRγ, Syk, and phospholipase Cγ2 (PLCγ2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI–FcRγ complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI–FcRγ complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRγ, Syk, and PLCγ2 and recruited tyrosine-phosphorylated Syk to the GPVI–FcRγ complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRγ and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI–FcRγ complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family–specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRγ, Syk, and PLCγ2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI–FcRγ complex.

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.

We investigated in vitro effects of recombinant human thrombopoietin (TPO), or c-Mpl ligand, on human platelets. TPO induced rapid dose-dependent tyrosine phosphorylation of several proteins. We identified Janus tyrosine kinases, Tyk2 and JAK2, and a member of STAT (signal transducers and activators of transcription) family, STAT3, as the tyrosine-phosphorylated proteins in response to TPO. TPO by itself did not cause platelet aggregation and shape change, but augmented ADP-indnced aggregation in a dose-dependent manner. Acetyisalicylic acid inhibited the secondary aggregation enhanced by TPO, but not the TPO-induced potentiation of the primary aggregation. TPO modulates platelet activation possibly through protein-tyrosine phosphorylation.

The major platelet integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) has been implicated in the regulation of tyrosine phosphorylation and dephosphorylation in activated platelets. To investigate the mechanisms of the alpha IIb beta 3-dependent tyrosine dephosphorylation, normal platelets or thrombasthenic platelets lacking alpha IIb beta 3 were stimulated with thrombin and fractionated into Triton X-100-soluble or -insoluble subcellular matrices. We then examined the kinetics of the tyrosine-phosphorylated proteins and distribution of protein-tyrosine phosphatases in these fractions and whole cell lysates. First, alpha IIb beta 3-dependent tyrosine dephosphorylation was recovered mainly in the cytoskeleton with similar kinetics to the whole cell lysate. Second, protein-tyrosine phosphatase (PTP) 1B and its cleaved 42-kDa form were associated with the cytoskeleton in an aggregation-dependent manner, whereas association of PTP1C with the cytoskeleton was regulated differentially both by thrombin stimulation and by alpha IIb beta 3-mediated aggregation. Several calpain inhibitors did not affect either tyrosine phosphorylation and dephosphorylation or relocation of PTP1B, but they did inhibit cleavage of PTP1B. Cytochalasin D blocked relocation of both PTP1B and PTP1C but not PTP1B cleavage. SH-PTP2 was distributed in the other fractions than the cytoskeleton and showed no relocation on thrombin stimulation. Finally, the cytoskeleton-associated PTP1C became tyrosine-phosphorylated in an alpha IIb beta 3-mediated aggregation-dependent manner. Thus, integrin alpha IIb beta 3 was involved differentially in the regulation of PTP1B and PTP1C.