Role of platelet endothelial form of nitric oxide synthase in collagen-platelet interaction: regulation by phosphorylation.
Different pathways have been reported to be involved in platelet-collagen interaction. We have reported that the platelet endothelial form of nitric oxide synthase (eNOS) and the platelet receptor for type I collagen, p65, are closely associated. But the controlling mechanism underlying the generation of nitric oxide (NO) by the eNOS has not been fully explored. In this investigation, Western blot analyses of time course samples with anti-phosphorylated tyrosine, and anti-serine/threonine showed a marked increase in serine/threonine phosphorylation of eNOS during type I collagen-induced platelet aggregation. Meanwhile, the eNOS activity measured by the conversion of [3H]-arginine to [3H]-citrulline is significantly decreased. Correlation of type I collagen-induced platelet aggregation and the activity of eNOS in the presence of the serine/threonine phosphatase inhibitor, okadiac acid and the tyrosine phosphatase inhibitor, vanadate were performed with PRP. Results show the decrease in eNOS activity by adding okadiac acid correlated with the inhibitory effect on platelet aggregation in a dose-dependent manner. On the other hand, vanadate significantly inhibits platelet aggregation and also inhibits eNOS activity when the concentration of vanadate is greater than 2 mM. These results suggest that phosphorylation of serine/threonine and tyrosine residues control the activity of eNOS through different mechanisms to affect collagen-induced platelet aggregation.
Chiang
Thomas M
TM
Veterans Affairs Medical Center, 1030 Jefferson Ave., Memphis, TN 38104, USA. TChiang@utmem.edu
Woo-Rasberry
Virginia
V
Cole
Flecia
F
D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism*; D015153:Blotting, Western; D024042:Collagen Type I / Q000494:pharmacology*; D003598:Cytoskeletal Proteins; D004727:Endothelium / Q000378:metabolism*; D006801:Humans; D008840:Microfilament Proteins; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019319:Okadaic Acid; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010974:Platelet Aggregation; D014638:Vanadates
D024042:Collagen Type I; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; C066559:Lcp1 protein, mouse; D019319:Okadaic Acid; D014638:Vanadates; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III