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.

Platelets perform a central role in haemostasis and thrombosis. They adhere to subendothelial collagens exposed at sites of blood vessel injury via the glycoprotein (GP) Ib-V-IX receptor complex, GPVI and integrin α₂β₁. These receptors perform distinct functions in the regulation of cell signalling involving non-receptor tyrosine kinases (e.g. Src, Fyn, Lyn, Syk and Btk), adaptor proteins, phospholipase C and lipid kinases such as phosphoinositide 3-kinase. They are also coupled to an increase in cytosolic calcium levels and protein kinase C activation, leading to the secretion of paracrine/autocrine platelet factors and an increase in integrin receptor affinities. Through the binding of plasma fibrinogen and von Willebrand Factor to integrin α_IIbβ₃, a platelet thrombus is formed. Although increasing evidence indicates that each of the adhesion receptors GPIb-V-IX and GPVI and integrins α₂β₁ and α_IIbβ₃ contribute to the signalling that regulates this process, the individual roles of each are only beginning to be dissected. By contrast, adhesion receptor signalling through platelet endothelial cell adhesion molecule 1 (PECAM-1) is implicated in the inhibition of platelet function and thrombus formation in the healthy circulation. Recent studies indicate that understanding of platelet adhesion signalling mechanisms might enable the development of new strategies to treat and prevent thrombosis.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1–deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1–deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1–deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1–deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation.

Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinase-dependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase C_γ2 are early events in collagen-induced activation. We recently proposed that collagen-signaling in platelets involves a receptor or a receptor-associated protein containing an immunoreceptor tyrosine-based activation motif (ITAM) enabling interaction with Syk. In this report we show that collagen stimulation of platelets causes rapid tyrosine phosphorylation of the ITAM containing Fc receptor γ-chain and that this is precipitated by the tandem Src homology 2 (SH2) domains of Syk expressed as a fusion protein. In addition we demonstrate an association between the Fc receptor γ-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor γ-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin α₂β₁ and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, FcγRIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor γ-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin α₂β₁ and involves phosphorylation of the Fc receptor γ-chain, its association with Syk and subsequent phosphorylation of phospholipase C_γ2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.