Regulation of platelet guanylyl cyclase by collagen: evidence that Glycoprotein VI mediates platelet nitric oxide synthesis in response to collagen

Summary

The molecular regulation of nitric oxide synthase (NOS) in blood platelets is an uncharacterised area of platelet biology. We investigated the mechanism of collagen-stimulated NO synthesis in platelets. Our aim was to identify the key collagen receptor and downstream signalling mechanisms linking collagen to NOS activation. Collagen and the GpVI-specific platelet activator collagen-related peptide (CRP-XL) stimulated NO synthesis, as evidenced by increased [³H]L-citrulline production, and cyclic GMP (cGMP) formation. After platelet activation by collagen and CRP-XL was normalised, we found no differences in NOS activation or cGMP formation in response to these agonists. Blocking the interaction of collagen with integrin α2β1, a second collagen receptor, failed to affect NOS activation by collagen. These data indicate that collagen-induced NO synthesis is linked to GpVI activation. cGMP formation in response to collagen and CRP-XL required increased intracellular Ca²⁺, Src family kinases,phosphatidylinositol 3-kinase (PI3-K) and protein kinase C. By comparison, Gp VI-independent cGMP formation induced by thrombin was Src kinase-dependent, but was independent of PI3-K and PKC. Thus the mechanisms of collagen- and CRP-XL-induced NOS activation were identical, but distinct from that of thrombin. Platelet activation in response to collagen leads to secretion of adenosine diphosphate (ADP) and thromboxaneA2 (TxA₂). Our results demonstrate that collagenstimulated cGMP synthesis was enhanced significantly by platelet- derived ADP and TxA₂. These results reveal that collagen stimulates platelet NOS activation through a specific Ca²⁺-dependent GpVI receptor signalling cascade,and demonstrate that collagen-induced cGMP accrual requires the release of secondary platelet agonists.

Keywords

Platelet - nitric oxide - collagen - GpVI - cGMP

• References

• 1 Shaul PW. Regulation of endothelial nitric oxide synthase: location, location, location. Annu Rev Physiol 2002; 64: 749-74.
• 2 Radomski MW, Palmer RM, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 1987; 2: 1057-8.
• 3 Mellion BT, Ignarro LJ, Ohlstein EH. et al. Evidence for the inhibitory role of guanosine 3', 5'-monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood 1981; 57: 946-55.
• 4 Moro MA, Russel RJ, Cellek S. et al. cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase. Proc Natl Acad Sci U S A 1996; 93: 1480-5.
• 5 Sase K, Michel T. Expression of constitutive endothelial nitric oxide synthase in human blood platelets. Life Sci 1995; 57: 2049-55.
• 6 Freedman JE, Loscalzo J, Barnard MR. et al. Nitric oxide released from activated platelets inhibits platelet recruitment. J Clin Invest 1997; 100: 350-6.
• 7 Freedman JE, Ting B, Hankin B. et al. Impaired platelet production of nitric oxide predicts presence of acute coronary syndromes. Circulation 1998; 98: 1481-6.
• 8 Ikeda H, Takajo Y, Murohara T. et al. Plateletderived nitric oxide and coronary risk factors. Hypertension 2000; 35: 904-7.
• 9 Rabini RA, Staffolani R, Fumelli P. et al. Decreased nitric oxide synthase activity in platelets from IDDM and NIDDM patients. Diabetologia 1998; 41: 101-4.
• 10 Camilletti A, Moretti N, Giacchetti G. et al. Decreased nitric oxide levels and increased calcium content in platelets of hypertensive patients. Am J Hypertens 2001; 14: 382-6.
• 11 Radomski MW, Palmer RM, Moncada S. An L-arginine/ nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci U S A 1990; 87: 5193-7.
• 12 Freedman JE, Keaney JF. Jr. Nitric oxide and superoxide detection in human platelets. Methods Enzymol 1999; 301: 61-70.
• 13 Trovati M, Massucco P, Mattiello L. et al. The insulin- induced increase of guanosine-3',5'-cyclic monophosphate in human platelets is mediated by nitric oxide. Diabetes 1996; 45: 768-70.
• 14 Fleming I, Schulz C, Fichtlscherer B. et al. AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets. Thromb Haemost 2003; 90: 863-71.
• 15 Riddell DR, Graham A, Owen JS. Apolipoprotein E inhibits platelet aggregation through the L-arginine:nitric oxide pathway. Implications for vascular disease. J Biol Chem 1997; 272: 89-95.
• 16 Morton LF, Hargreaves PG, Farndale RW. et al. Integrin alpha 2 beta 1-independent activation of platelets by simple collagen-like peptides: collagen tertiary (triple-helical) and quaternary (polymeric) structures are sufficient alone for alpha 2 beta 1-independent platelet reactivity. Biochem J 1995; 306: 337-44.
• 17 Vargas JR, Radomski M, Moncada S. The use of prostacyclin in the separation from plasma and washing of human platelets. Prostaglandins 1982; 23: 929-45.
• 18 Asselin J, Gibbins JM, Achison M. et al. A collagenlike peptide stimulates tyrosine phosphorylation of syk and phospholipase C gamma2 in platelets independent of the integrin alpha2beta1. Blood 1997; 89: 1235-42.
• 19 Watson SP. Collagen receptor signaling in platelets and megakaryocytes. Thromb Haemost 1999; 82: 365-76.
• 20 Barry FA, Gibbins JM. Protein kinase B is regulated in platelets by the collagen receptor glycoprotein VI. J Biol Chem 2002; 277: 12874-8.
• 21 Quinton TM, Ozdener F, Dangelmaier C. et al. Glycoprotein VI-mediated platelet fibrinogen receptor ac- tivation occurs through calcium-sensitive and PKCsensitive pathways without a requirement for secreted ADP. Blood 2002; 99: 3228-34.
• 22 Russell KS, Haynes MP, Caulin-Glaser T. et al. Estrogen stimulates heat shock protein 90 binding to endothelial nitric oxide synthase in human vascular endothelial cells. Effects on calcium sensitivity and NO release. J Biol Chem 2000; 275: 5026-30.
• 23 Michell BJ, Chen Z, Tiganis T. et al. Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase. J Biol Chem 2001; 276: 17625-8.
• 24 Dimmeler S, Fleming I, Fisslthaler B. et al. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 1999; 399: 601-5.
• 25 Haynes MP, Li L, Sinha D. et al. Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen. J Biol Chem 2003; 278: 2118-23.
• 26 Achison M, Elton CM, Hargreaves PG. et al. Integrin-independent tyrosine phosphorylation of p125(fak) in human platelets stimulated by collagen. J Biol Chem 2001; 276: 3167-74.
• 27 Freedman JE, Li L, Sauter R. et al. alpha-Tocopherol and protein kinase C inhibition enhance platelet-derived nitric oxide release. Faseb J 2000; 14: 2377-9.
• 28 Onley DJ, Knight CG, Tuckwell DS. et al. Micromolar Ca2+ concentrations are essential for Mg2+- dependent binding of collagen by the integrin alpha 2beta 1 in human platelets. J Biol Chem 2000; 275: 24560-4.
• 29 Hirata K, Kuroda R, Sakoda T. et al. Inhibition of endothelial nitric oxide synthase activity by protein kinase C. Hypertension 1995; 25: 180-5.
• 30 Russo I, Doronzo G, Mattiello L. et al. The activity of constitutive nitric oxide synthase is increased by the pathway cAMP/cAMP-activated protein kinase in human platelets. New insights into the antiaggregating effects of cAMP-elevating agents. Thromb Res 2004; 114: 265-73.
• 31 Lantoine F, Brunet A, Bedioui F. et al. Direct measurement of nitric oxide production in platelets: relationship with cytosolic Ca2+ concentration. Biochem Biophys Res Commun 1995; 215: 842-8.