The l-arginine/NO pathway in the early phases of platelet stimulation by collagen

Nitric oxide production, l-arginine transport and intracellular [Ca2+] changes in human platelets stimulated without stirring by low doses of collagen have been evaluated. Collagen decreased in a dose-dependent manner the nitric oxide formation. A reduction of about 30% of the basal level was produced by 5 μg/mL. Aspirin did not change the collagen effect. The inhibition was reversed by EGTA. Moreover collagen reduced l-arginine uptake. The exposure of platelets to 5 μg/mL collagen diminished of about 30% l-arginine transport. The specific involvement of the system y+ is suggested. In addition in FURA 2-loaded platelets collagen induced a dose-dependent slow sustained [Ca2+] rise that was almost completely cancelled by EGTA. Finally the treatment of whole platelets with collagen affected in a dose-dependent manner the maximal nitric oxide formation, suggesting a direct effect at the level of nitric oxide synthase enzyme. The phosphorylation of specific serine/threonine residues regulated by protein kinase C could be involved. In conclusion during the early phases of platelet stimulation with collagen nitric oxide formation is diminished. This reduction can be due to a lower availability of l-arginine for cytosolic nitric oxide synthase and/or to a decreased activity related to modifications of the enzyme.