Rap1 binding to the talin 1 F0 domain makes a minimal contribution to murine platelet GPIIb-IIIa activation

Activation of platelet glycoprotein IIb-IIIa (GPIIb-IIIa; integrin alpha IIb beta 3) leads to high-affinity fibrinogen binding and platelet aggregation during hemostasis. Whereas GTP-bound Rap1 GTPase promotes talin 1 binding to the beta 3 cytoplasmic domain to activate platelet GPIIb-IIIa, the Rap1 effector that regulates talin association with beta 3 in platelets is unknown. Rap1 binding to the talin 1 F0 subdomain was proposed to forge the talin 1-Rap1 link in platelets. Here, we report a talin 1 point mutant (R35E) that significantly reduces Rap1 affinity without a significant effect on its structure or expression. Talin 1 head domain (THD) (R35E) was of similar potency to wild-type THD in activating alpha IIb beta 3 in Chinese hamster ovary cells. Coexpression with activated Rap1b increased activation, and coexpression with Rap1GAP1 reduced activation caused by transfection of wild-type THD or THD(R35E). Furthermore, platelets from Tln1(R35E/R35E) mice showed similar GPIIb-IIIa activation to those from wild-type littermates in response to multiple agonists. Tln1(R35E/R35E) platelets exhibited slightly reduced platelet aggregation in response to low doses of agonists; however, there was not a significant hemostatic defect, as judged by tail bleeding times. Thus, the Rap1-talin 1 F0 interaction has little effect on platelet GPIIb-IIIa activation and hemostasis and cannot account for the dramatic effects of loss of Rap1 activity on these platelet functions.