Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinasedependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase C ␥ 2 are early events in collageninduced activation. We recently proposed that collagensignaling in platelets involves a receptor or a receptorassociated 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 ␥-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 ␥-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor ␥-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin ␣ 2 ␤ 1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, Fc␥RIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor ␥-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin ␣ 2 ␤ 1 and involves phosphorylation of the Fc receptor ␥-chain, its association with Syk and subsequent phosphorylation of phospholipase C ␥ 2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.The adhesive and stimulatory properties of the extracellular matrix protein collagen on platelets are vital for the maintenance of hemeostasis. Upon vascular damage, platelets adhere to subendothelial collagen which stimulates a tyrosine kinase dependent pathway leading to platelet degranulation, aggregation and development of a hemeostatic plug. The mechanism of collagen stimulation of platelets is poorly understood, and the distinction between adhesion and stimulation ill defined. Several platelet glycoproteins have been implicated as potential collagen receptors, including the integrin ␣ 2 ␤ 1 (1), glycoprotein IV (GPIIIb, CD36) (2), glycoprotein VI (3), and uncharacterized 65-kDa (4) and 85-90-kDa glycoproteins (5). Patients whose platelets express abnormally low numbers of these proteins, or who possess autoantibodies to them, have limited bleeding defects (3, 5-9).Collagen stimulation of platelets activates tyrosine kinasedependent mechanisms which involve tyrosine phosphorylation of Syk and phospholipase C ␥ 2 (PLC ␥ 2) 1 (10 -12). Syk is a nonreceptor tyrosine kinase which is assembled into signaling complexes via interaction between its tandem Src homology 2 (SH2) domains and a tyrosine phosphorylated activation motif found in receptors of the immune system or their associated chains. The motif, termed the immunoreceptor tyrosine-based activation motif (ITAM), has the ...

The present study identifies the phosphorylation sites of the 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) in human platelets and HeLa cells. Tryptic digests of 32 Pphosphorylated and -immunoprecipitated cPLA 2 were analyzed by microbore high performance liquid chromatography and two-dimensional phosphopeptide mapping against synthetic phosphopeptide standards. Thrombin stimulated significant phosphorylation of platelet cPLA 2 at two sites, Ser-505 and Ser-727. Exclusive phosphorylation on these two sites was also seen in collagen-stimulated platelets and HeLa cells stimulated with interferon-␣ or arsenite; no tyrosine phosphorylation was detected. The inhibitor of the 38-kDa stressactivated protein kinase (p38 mapk ), SB 203580, reduced phosphorylation of both Ser-505 and Ser-727 by 50 and 60%, respectively, in thrombin-stimulated platelets. An additional p38 mapk inhibitor SB 202190 also partially (60%) inhibited the phosphorylation of cPLA 2 in arsenite-stimulated HeLa cells. These studies extend the previous work on the identification of multiple phosphorylation sites on cPLA 2 expressed in a baculovirus/insect cell system to cPLA 2 in mammalian cells stimulated with physiological agonists. They also underscore the necessity of high resolution phosphopeptide mapping combined with microbore high performance liquid chromatography for quantification of phosphorylation levels, which has lead to the conclusion that Ser-505 and Ser-727 are common phosphorylation sites on cPLA 2 in different mammalian cells stimulated with multiple agonists.Cytosolic phospholipase A 2 (cPLA 2 ) 1 catalyzes the cleavage of arachidonic acid from the sn-2 position of phospholipids (1, 2). The 85-kDa enzyme is present in many mammalian cells (3), and strong evidence is accumulating for the role of cPLA 2 in the generation of tissue mediators that are metabolites of arachidonic acid, such as prostaglandins, leukotrienes, and thromboxanes. In contrast to the small molecular weight phospholipase A 2 s that are secreted and are active on the outside of cells, cPLA 2 is regulated by intracellular signals that are propagated from surface receptors. One important regulatory mechanism appears to be a rise in the intracellular Ca 2ϩ concentration which causes translocation of cPLA 2 from the cytosol to internal membranes (4 -7) where it binds through a Ca 2ϩ -dependent lipid-binding domain (8). A second, well established mechanism of the regulation of cPLA 2 activity is by phosphorylation on Ser-505 through a mitogen-activated protein kinase (MAPK) (9) which modestly increases the intrinsic activity of the lipase measured in vitro (3, 10, 11). Phosphorylation of cPLA 2 together with release of arachidonic acid has been observed in a variety of cells (11)(12)(13)(14)(15)(16).A thorough characterization of the phosphorylation sites of human cPLA 2 heterologously expressed in Spodoptera frugiperda (Sf9) cells by high performance liquid chromatography (HPLC), mass spectrometry, and protein sequencing has revealed four sites of phosphorylation: Ser-...

Activation of platelets by collagen is mediated through a tyrosine kinase-dependent pathway that is associated with phosphorylation of the Fc receptor γ chain, the tyrosine kinase syk, and phospholipase Cγ2 (PLCγ2). We recently described a collagen-related triple-helical peptide (CRP) with the sequence GCP*(GPP*)GCP*G (single letter amino acid code: P* = hydroxyproline; Morton et al, Biochem J 306:337, 1995). The cross-linked peptide is a potent stimulus of platelet activation but, unlike collagen, does not support α2β1-mediated, Mg2+-dependent adhesion, suggesting that its action is independent of the integrin α2β1 . This finding suggests the existence of a platelet receptor other than α2β1 that underlies activation. In the present study, we show that CRP stimulates tyrosine phosphorylation of the same pattern of proteins in platelets as collagen, including syk and PLCγ2. Protein tyrosine phosphorylation induced by CRP is not altered in the absence of Mg2+ or the presence of monoclonal antibodies (MoAbs) to the integrin α2β1 (MoAb 6F1 and MoAb 13), conditions that prevent the interaction of collagen with the integrin. In contrast, phosphorylation of syk and PLCγ2 by collagen is partially reduced by MoAb 6F1 and MoAb 13 or by removal of Mg2+. This may reflect a direct role of α2β1 in collagen-induced signaling events or an indirect role in which the integrin facilitates the binding of collagen to its signaling receptor. The results show an α2β1-independent pathway of platelet activation by CRP that involves phosphorylation of syk and PLCγ2. This pathway appears to contribute to platelet activation by collagen.