The adapter protein SLP-76 is a critical mediator of signal transduction via the platelet collagen receptor glycoprotein VI (GPVI) and its coreceptor FcRgamma. 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 FcRgamma 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/FcRgamma/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.