Role of the adapter protein SLP-76 in GPVI-dependent platelet procoagulant responses to collagen

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Abstract
The adapter protein SLP-76 is a critical mediator of signal transduction via the platelet collagen receptor glycoprotein VI (GPVI) and its coreceptor FcRγ. We tested the hypothesis that SLP-76 is required for collagen-induced procoagulant responses in murine platelets. Platelets from SLP-76 null (SLP-76−/−) or heterozygous (SLP-76+/−) mice were activated with the GPVI agonist convulxin, and surface expression of P-selectin (a marker of granule release) and annexin V binding (a marker of procoagulant phospholipid) were determined by flow cytometry. Convulxin induced surface expression of P-selectin in SLP-76+/− platelets, but not SLP-76−/− platelets (P < .01), and failed to stimulate annexin V binding to either SLP-76+/−or SLP-76−/− platelets. Platelet procoagulant activity was measured in a prothrombinase assay. Convulxin did not stimulate procoagulant activity in either SLP-76+/− or SLP-76−/− platelets, but fibrillar collagen produced a 1.9-fold increase in procoagulant activity in both SLP-76+/− and SLP-76−/− platelets (P < .001 versus unstimulated platelets). Similar results were obtained with platelets from FcRγ null mice, for which collagen, but not convulxin, induced procoagulant activity (P < .01). Costimulation with thrombin and collagen produced a further (2.3-fold) increase in procoagulant activity in SLP-76+/− platelets (P < .05), but not in SLP-76−/− platelets. SLP-76−/− platelets also exhibited less annexin V binding than SLP-76+/−platelets after costimulation with thrombin and convulxin (P < .05). These findings demonstrate that an intact GPVI/FcRγ/SLP-76 signal transduction pathway is not essential for platelet procoagulant activity induced by collagen but is necessary for maximal procoagulant response to costimulation with thrombin plus collagen. Thus, both GPVI-dependent and GPVI-independent pathways contribute to collagen-induced platelet procoagulant activity.