NO-synthase-/NO-independent regulation of human and murine platelet soluble guanylyl cyclase activity.
OBJECTIVE
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.


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 beta(1)-subunit-Tyr(192).


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.
Gambaryan
S
S
Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Wuerzburg, Germany.
Kobsar
A
A
Hartmann
S
S
Birschmann
I
I
Kuhlencordt
P J
PJ
Müller-Esterl
W
W
Lohmann
S M
SM
Walter
U
U
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006152:Cyclic GMP / Q000097:blood; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000097:blood* / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D018345:Mice, Knockout; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000097:blood; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000097:blood* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II / Q000097:blood / Q000172:deficiency / Q000235:genetics; D052250:Nitric Oxide Synthase Type III / Q000097:blood / Q000235:genetics; D010766:Phosphorylation; D012333:RNA, Messenger / Q000097:blood / Q000235:genetics; D012310:Ristocetin / Q000494:pharmacology; D012995:Solubility; D019323:omega-N-Methylarginine / Q000494:pharmacology; D019061:src-Family Kinases / Q000097:blood; D014841:von Willebrand Factor / Q000494:pharmacology
D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D014841:von Willebrand Factor; D012310:Ristocetin; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496317:Nos2 protein, mouse; C496315:Nos3 protein, mouse; D019061:src-Family Kinases; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D019331:NG-Nitroarginine Methyl Ester