NO‐synthase‐/NO‐independent regulation of human and murine platelet soluble guanylyl cyclase activity

Objectives: Platelets, specialized adhesive cells, play key roles in normal and pathological hemostasis through their ability to rapidly adhere to subendothelial matrix proteins (adhesion) and to other activated platelets (aggregation), functions which are inhibited by nitric oxide (NO). Platelets have been reported to be regulated not only by exogenous endothelium‐derived NO, but also by two isoforms of NO synthase, endothelial (eNOS) and inducible (iNOS), endogenously expressed in platelets. However, data concerning expression, regulation and function of eNOS and iNOS in platelets remain controversial. Methods and results: Using important positive (endothelial cells, stimulated macrophages) and negative (eNOS/iNOS knock‐out mouse) controls, as well as human platelets highly purified by a newly developed protocol, we now demonstrate that human and mouse platelets do not contain eNOS/iNOS proteins or mRNA. NOS substrate (l‐arginine), NOS inhibitors (L‐NAME, L‐NMMA), and eNOS/iNOS deficiency did not produce detectable functional effects on human and mouse platelets. von Willebrand factor (VWF)/ristocetin treatment of platelets increased cGMP by NO‐independent activation of soluble guanylyl cyclase (sGC) which correlated with Src kinase‐dependent phosphorylation of sGC β1‐subunit‐Tyr192. Conclusions: Human and mouse platelets do not express eNOS/iNOS. VWF/ristocetin‐mediated activation of the sGC/cGMP signaling pathway may contribute to feedback platelet inhibition.