Serine/threonine phosphatases regulate platelet αIIbβ3 integrin receptor outside-in signaling mechanisms and clot retraction.

Aims: We studied the role of serine/threonine phosphatases (PSTPs) on αIIbβ3 signaling and the potential selectivity of the level of PSTP inhibition with okadaic acid (OA) on αIIbβ3 signaling for regulation of platelet aggregation and clot retraction.

Main Methods: We used washed platelets from normal donors and OA as inhibitor of PSTPs. Clot retraction was induced by 1U/mL of thrombin. Reorganized cytoskeleton was isolated from Triton X-100 lysed platelets. The presence of proteins incorporated to the cytoskeleton was assayed by immunoblotting with specific antibodies.

Key Findings: We found that both 100 and 500 nM OA blocked platelet mediated clot retraction. In contrast, only 500 nM OA inhibited thrombin-induced inside-out αIIbβ3 activation, platelet aggregation, and cytoskeletal reorganization. Among markers of αIIbβ3 outside-in signaling, 500 nM OA inhibited the incorporation to the cytoskeleton of syk, src, and FAK (Focal Adhesion Kinase) tyrosine kinases and the incorporation and phosphorylation at Tyr(759) of the β3 chain of αIIbβ3, while 100 nM OA only inhibited the FAK translocation and its tyrosine phosphorylation.

Significance: The level of inhibition of PSTPs by low or high OA concentration (33% and 73% inhibition, respectively) in intact whole cells differentially regulates platelet aggregation and integrin signaling, but have a common effect in blocking clot retraction. The latter may be associated with the presence of phosphorylated FAK in the cytoskeleton. This study reveals a novel target for anti-platelet treatment to block clot retraction without affecting the platelet hemostatic function by a partial inhibition of PSTPs.