Background: Adhesion of platelets to subendothelial collagen after plaque rupture results in a phospholipase C (PLC) evoked hydrolysis of inositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) with subsequent rising cytosolic Ca2+ concentrations ([Ca2+]i). Increasing [Ca2+]i is a prerequisite for platelet activation, which is essential for the development of thrombotic vascular occlusions. Janus kinase 3 (JAK3) is a member of the Janus family protein-tyrosine kinases which is strongly expressed in platelets and affects pseudopodia formation, degranulation and aggregation in human platelets. Although STAT3, a downstream target of JAK3, plays a crucial role in Ca2+ signaling and platelet aggregation, less is known about the impact of JAK3 in IP3-dependent platelet activation and thrombus formation.
Objective: The present study explored the role of JAK3 in PIP2 and IP3 signaling mechanisms during Ca2+-dependent platelet activation culminating in thrombotic vascular occlusions in JAK3-deficient (jak3-/-) mice.
Methods and Results: Upon stimulation of the major platelet receptor glycoprotein VI (GPVI) by means of collagen-related peptide (CRP)-dependent activation, fura-2-AM spectrofluorometric Ca2+ measurements revealed a diminished increase of [Ca2+]i in platelets from jak3-/- mice when compared to platelets from corresponding wildtype littermates. Consequently, in comparison to naïve platelets, flow cytometry and light transmission aggregometry revealed a significantly blunted activation-dependent integrin αIIbβ3 activation, aggregation and degranulation in JAK3-deficient platelets. Genetic perturbation of JAK3 also resulted in impaired platelet spreading and in vitro thrombus formation on a collagen-coated surface under high arterial shear rates. More importantly, thrombotic vascular occlusion after FeCl3-induced injury in vivo was blunted in jak3-/- chimeric mice, whereas no prolongation of bleeding time was observed in these mice. To address the mechanisms of CRP-triggered Ca2+-dependent platelet activation in jak3-/- mice, we analyzed PIP2 and downstream IP3 levels. By employing ELISA assays, lipidomic analysis as well as immunoblots, the observed significantly decreased PIP2 and IP3 levels in platelets from jak3-/- mice resulted from diminished levels of upstream phosphatidylinositol-4-phosphate 5-kinase (PIP5K).
Conclusions: The present study reveals JAK3 as a regulator of PIP2 and IP3 generation in platelets with subsequent Ca2+-dependent activation, dense granule secretion and aggregation upon GPVI stimulation. Finally, JAK3-deficiency results in decreased in vitro and in vivo thrombus formation, without affecting bleeding time. Therefore, JAK3 could play a decisive role in arterial thrombosis underlying myocardial infarction or ischemic stroke and may represent a druggable target in future improved treatment regimens of cardiovascular diseases.
