Collagen fibers or a glycoprotein VI-specific collagenrelated peptide (CRP-XL) stimulated tyrosine phosphorylation of the focal adhesion kinase, p125 fak (FAK), in human platelets. An integrin ␣ 2 ␤ 1 -specific triple-helical peptide ligand, containing the sequence GFOGER (single-letter nomenclature, O ‫؍‬ Hyp) was without effect. Antibodies to the ␣ 2 and ␤ 1 integrin subunits did not inhibit platelet FAK tyrosine phosphorylation caused by either collagen fibers or CRP-XL. Tyrosine phosphorylation of FAK caused by CRP-XL or thrombin, but not that caused by collagen fibers, was partially inhibited by GR144053F, an antagonist of integrin ␣ IIb ␤ 3 . The intracellular Ca 2؉ chelator, BAPTA, and the protein kinase C inhibitor, Ro31-8220, were each highly effective inhibitors of the FAK tyrosine phosphorylation caused by collagen or CRP-XL. These data suggest that, in human platelets, 1) occupation or clustering of the integrin ␣ 2 ␤ 1 is neither sufficient nor necessary for activation of FAK, 2) the fibrinogen receptor ␣ IIb ␤ 3 is not required for activation of FAK by collagen fibers, and 3) both intracellular Ca 2؉ and protein kinase C activity are essential intermediaries of FAK activation.Hemostasis, prevention of blood loss from damaged blood vessels, is dependent upon the activation of platelets by subendothelial collagens (types I and III) of the vessel wall. Platelet activation involves shape change, adhesion, aggregation, and secretion of granule contents. These events lead to the formation of a clot at the site of injury (1).Platelets possess several receptors for collagen, recently reviewed (2-4), including the integrin ␣ 2 ␤ 1 (glycoproteins IaIIa), CD36 1 (glycoprotein IV), and glycoprotein VI (GpVI). Platelet activation by collagen is a two-stage process involving sites in collagen, which support platelet adhesion, and others, which support both platelet adhesion and activation (5, 6). At present, ␣ 2 ␤ 1 is considered primarily an adhesive co-receptor (7), whereas other collagen receptors, notably glycoprotein VI, activate platelets (8). This study was designed to clarify which collagen receptors transmit signals to the platelet interior, information which is crucial for the development of anti-platelet therapy based on collagen receptor antagonism (9).Recent evidence suggests that CD36, ␣ 2 ␤ 1 , and GpVI each contribute to both signaling and adhesion to collagen (10). GpVI, recently cloned (11), acts with the Fc receptor ␥-chain (12, 13) as a crucial signaling receptor complex, and platelets deficient in GpVI fail to aggregate in response to collagen, although the tyrosine kinase c-Src, but not p72 syk , is activated (14). The role of ␣ 2 ␤ 1 in platelet signaling is unclear: ␣ 2 ␤ 1 -reactive snake venoms fuel the debate on the integrin's role in platelet signaling (15, 16), and overexpression of ␣ 2 ␤ 1 has recently been advanced as a risk factor in myocardial infarction and stroke (17,18).We have synthesized a collagen-related peptide (CRP) recognized by GpVI, which shares both the triple-helica...

SummaryCalreticulin is an abundant protein in the endoplasmic reticulum of most cells. In this study, flow cytometry and immunoprecipitation from surface-biotinylated platelets each provided direct evidence that calreticulin is also expressed on the surface of human platelets. Anti-calreticulin antibodies caused platelet activation, inducing Fc RIIa-independent platelet aggregation. In addition, these antibodies inhibited platelet adhesion to the integrin α2β1-specific ligands, GFOGER-GPP and monomeric collagen I, and to the glycoprotein VI-specific ligand, CRP. Inhibition of platelet adhesion to these ligands was independent of integrin αIIbβ3. In resting platelets, calreticulin was shown to interact with integrin α2β1 and glycoprotein VI. Together, these data demonstrate that surface calreticulin is associated with collagen receptors on the platelet surface, where it may play a role in the modulation of the platelet-collagen interaction.Abbreviations: BSA: bovine serum albumin; cC1qR: complement component 1q receptor; CRP: collagen-related peptide; DTSSP: 3,3’-dithiobis (sulfosuccinimidyl propionate); ER: endoplasmic reticulum; GFOGER-GPP: GPC[GPP]5GFOGER[GPP]5GPC; GpVI: glycoprotein VI; HRP: horseradish peroxidase; PDI: protein disulfide isomerase.