Kindlin-3 phosphorylation is crucial for thrombosis and hemostasis in vivo.

BACKGROUND: A single phosphorylation in kindlin-3 at S(484) (human) or S(485) (mouse) has been shown to regulate its function in a variety of cells in vitro. However, whether this posttranslational modification of kindlin-3 plays a role in platelets and in hemostasis and thrombosis in vivo remains totally unknown. OBJECTIVES: To create a strain of mice expressing kindlin-3 that bears the S(485)A substitution and utilize these mice to determine if kindlin-3 phosphorylation influences platelet responses, hemostasis, and thrombosis in vivo. METHODS: By clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 gene editing, we generated a mouse strain that expressed a kindlin-3 mutant bearing the S(485)A substitution. S(485)A kindlin-3 mice were born normally, and the platelet and red blood cells numbers were similar to their wild-type counterparts. Wild-type and S(485)A kindlin-3 platelets showed similar expression of α(IIb)β(3) integrin, kindlin-3, and talin. RESULTS: Platelets isolated from S(485)A kindlin-3 mice did not undergo agonist-induced kindlin-3 phosphorylation. Functional analysis revealed that S(485)A kindlin-3 mice exhibited prolonged tail bleeding time, increased blood loss, and delayed thrombosis in the carotid artery injury model in vivo. Platelets isolated from S(485)A kindlin-3 mice showed defective platelet function, including impaired integrin α(IIb)β(3) activation, platelet aggregation, clot retraction, and adhesion. CONCLUSION: Our observations demonstrate that kindlin-3 phosphorylation on S(485) in mice is crucial for supporting activation of α(IIb)β(3) integrin and α(IIb)β(3) integrin-dependent platelet responses and consequently contributes to hemostasis and thrombosis in vivo.