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

Abstract

Background

A single phosphorylation in kindlin-3 at S⁴⁸⁴ (human) or S⁴⁸⁵ (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 S⁴⁸⁵A 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 S⁴⁸⁵A substitution. S⁴⁸⁵A kindlin-3 mice were born normally, and the platelet and red blood cells numbers were similar to their wild-type counterparts. Wild-type and S⁴⁸⁵A kindlin-3 platelets showed similar expression of α_IIbβ₃ integrin, kindlin-3, and talin.

Results

Platelets isolated from S⁴⁸⁵A kindlin-3 mice did not undergo agonist-induced kindlin-3 phosphorylation. Functional analysis revealed that S⁴⁸⁵A 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 S⁴⁸⁵A kindlin-3 mice showed defective platelet function, including impaired integrin α_IIbβ₃ activation, platelet aggregation, clot retraction, and adhesion.

Conclusion

Our observations demonstrate that kindlin-3 phosphorylation on S⁴⁸⁵ in mice is crucial for supporting activation of α_IIbβ₃ integrin and α_IIbβ₃ integrin-dependent platelet responses and consequently contributes to hemostasis and thrombosis in vivo.