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

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 convulxln (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.