At sites of vascular injury, platelets come into contact with subendothelial collagen, which triggers their activation and the formation of a hemostatic plug. Besides glycoprotein Ib (GPIb) and α_IIbβ₃ integrin, which indirectly interact with collagen via von Willebrand factor (VWF), several collagen receptors have been identified on platelets, most notably α₂β₁ integrin and the immunoglobulin (Ig) superfamily member GPVI. Within the last few years, major advances have been made in understanding platelet-collagen interactions including the molecular cloning of GPVI, the generation of mouse strains lacking individual collagen receptors, and the development of collagen receptor–specific antibodies and synthetic peptides. It is now recognized that platelet adhesion to collagen requires prior activation of integrins through “inside-out” signals generated by GPVI and reinforced by released second-wave mediators adenosine diphosphate (ADP) and thromboxane A₂. These developments have led to revision of the original “2-site, 2-step” model, which now places GPVI in a central position in the complex processes of platelet tethering, activation, adhesion, aggregation, degranulation, and procoagulant activity on collagen. This review discusses these recent developments and proposes possible mechanisms for how GPVI acts in concert with other receptors and signaling pathways to initiate hemostasis and arterial thrombosis.

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The snake venom rhodocytin has been reported to bind to integrin α2β1 and glycoprotein (GP) Ibα on platelets, but it is also able to induce activation independent of the 2 receptors and of GPVI. Using rhodocytin affinity chromatography, we have identified a novel C-type lectin receptor, CLEC-2, in platelets that confers signaling responses to rhodocytin when expressed in a cell line. CLEC-2 has a single tyrosine residue in a YXXL motif in its cytosolic tail, which undergoes tyrosine phosphorylation upon platelet activation by rhodocytin or an antibody to CLEC-2, but not to collagen, thrombin receptor agonist peptide (TRAP), or convulxin. Tyrosine phosphorylation of CLEC-2 and other signaling proteins by rhodocytin is inhibited by the Src family kinase inhibitor PP2. Further, activation of murine platelets by rhodocytin is abolished in the absence of Syk and PLCγ2, and partially reduced in the absence of LAT, SLP-76, and Vav1/Vav3. These findings define a novel signaling pathway in platelets whereby activation of CLEC-2 by rhodocytin leads to tyrosine phosphorylation of its cytosolic tail, binding of Syk and initiation of downstream tyrosine phosphorylation events, and activation of PLCγ2. CLEC-2 is the first C-type lectin receptor to be found on platelets which signals through this novel pathway.

The Ca²⁺-sensitive 85-kDa cytosolic phospholipase A₂ (cPLA₂) is responsible for thrombin-stimulated mobilization of arachidonic acid for the synthesis of thromboxane A₂ in human platelets. We have previously shown that thrombin activates p38 kinase, a recently discovered new member of the mitogen-activated protein kinase family (Kramer, R. M., Roberts, E. F., Strifler, B. A., and Johnstone, E. M. (1995) J. Biol. Chem. 270, 27395-27398) and also induces phosphorylation of cPLA₂, thereby increasing its intrinsic catalytic activity. In the present study we have examined the role of p38 kinase in the phosphorylation and activation of cPLA₂ in stimulated platelets. We have observed that activation of p38 kinase accompanies receptor-mediated events in platelets and coincides with cPLA₂ phosphorylation. Furthermore, in the presence of inhibitors of p38 kinase, the proline-directed phosphorylation of cPLA₂ was completely blocked in platelets stimulated with the thrombin receptor agonist peptide SFLLRN and was suppressed during the early (up to 2 min) phase of platelet stimulation caused by thrombin. Unexpectedly, we found that prevention of proline-directed phosphorylation of cPLA₂ in stimulated platelets did not attenuate its ability to release arachidonic acid from platelet phospholipids. We conclude that: 1) cPLA₂ is a physiological target of p38 kinase; 2) p38 kinase is involved in the early phosphorylation of cPLA₂ in stimulated platelets; and 3) proline-directed phosphorylation of cPLA₂ is not required for its receptor-mediated activation.

Activation of mouse platelets by collagen is associated with tyrosine phosphorylation of multiple proteins including the Fc receptor γ‐chain, the tyrosine kinase Syk and phospholipase Cγ2, suggesting that collagen signals in a manner similar to that of immune receptors. This hypothesis has been tested using platelets from mice lacking the Fc receptor γ‐chain or Syk. Tyrosine phosphorylation of Syk and phospholipase Cγ2 by collagen stimulation is absent in mice lacking the Fc receptor γ‐chain. Tyrosine phosphorylation of phospholipase Cγ2 by collagen stimulation is also absent in mice platelets which lack Syk, although phosphorylation of the Fc receptor γ‐chain is maintained. In contrast, tyrosine phosphorylation of platelet proteins by the G protein‐coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor γ‐chain or Syk. The absence of Fc receptor γ‐chain or Syk is accompanied by a loss of secretion and aggregation responses in collagen‐ but not thrombin‐stimulated platelets. These observations provide the first direct evidence of an essential role for the immunoreceptor tyrosine‐based activation motif (ITAM) in signalling by a non‐immune receptor stimulus.