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 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.
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017304:Annexin A5 / Q000097:blood; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D003029:Coagulants / Q000378:metabolism; D003094:Collagen / Q000494:pharmacology*; D006579:Heterozygote; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D010750:Phosphoproteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D010974:Platelet Aggregation / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D017304:Annexin A5; D003029:Coagulants; D010750:Phosphoproteins; D010980:Platelet Membrane Glycoproteins; C092947:SLP-76 signal Transducing adaptor proteins; C098341:platelet membrane glycoprotein VI; D003094:Collagen