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The regulation of integrin‐linked kinase in human platelets: evidence for involvement in the regulation of integrin α2β1

Summary.  Background: Activation of the platelet integrin α2β1 is closely regulated due to the high thrombogenicity of its ligand. As a β1 interacting kinase, ILK represents a candidate intracellular regulator of α2β1 in human platelets. Objectives We investigated the regulation of ILK in human platelets and the role of ILK in regulating α2β1 activation in HEL cells, a megakaryocytic cell line. Methods: An in‐vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with β1‐integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating α2β1 function assessed by overexpression studies in HEL cells. Results: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation‐independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin‐stimulated platelets and regulated by PI3K in collagen‐stimulated cells. ILK associates with the β1‐integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced α2β1‐mediated adhesion whereas overexpression of kinase‐dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of α2β1. Conclusions: Our findings that ILK regulates α2β1 in HEL cells, is activated in platelets and associates with β1‐integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.