cyclic-gmp and 5--(4-fluorosulfonylbenzoyl)adenosine

We have shown recently that the calcium-dependent phospholipid-binding protein annexin V (placental anticoagulant protein I) can be used to study the exposure of anionic phospholipid after platelet activation. In this study we have further examined the mechanism of this process. Collagen-induced exposure of annexin V binding sites correlated directly with increased ability to support activity of the reconstituted prothrombinase complex. The potency of annexin V as an inhibitor of platelet prothrombinase was the same as its Kd for platelets. Prior incubation of platelets with 5'-p-fluorosulfonylbenzoyladenosine or p-chloromercuribenzenesulfonate had no significant effect on annexin V binding. Similarly, inhibition of platelet cyclic endoperoxide synthesis by acetylsalicylic acid or indomethacin did not inhibit annexin V binding. Staurosporine inhibited collagen-induced, but not A23187-induced, annexin V binding. Agents that increase intraplatelet cyclic nucleotides partially inhibited collagen-induced annexin V binding. Thus, collagen-induced exposure of anionic phospholipid appears to depend primarily on increases in intraplatelet free calcium and may be independent of ADP- or endoperoxide-mediated pathways. Binding sites for annexin V on microparticles derived from collagen-stimulated platelets were demonstrated by flow cytometry and gel filtration. In addition, prior incubation of platelets with 100 nM annexin V inhibited factor Va binding to both platelets and platelet-derived microparticles. These results support the concept that the procoagulant effect of platelets and platelet-derived microparticles is mediated by calcium-induced exposure of anionic phospholipids.

Topics: 4-Chloromercuribenzenesulfonate; Adenosine; Affinity Labels; Alkaloids; Anions; Annexin A5; Binding Sites; Blood Platelets; Bucladesine; Calcimycin; Calcium-Binding Proteins; Colforsin; Collagen; Cyclic GMP; Factor Va; Flow Cytometry; Humans; Nitroprusside; Phospholipids; Platelet Aggregation; Pregnancy Proteins; Signal Transduction; Staurosporine; Theophylline; Thromboplastin

Bovine lung cyclic GMP-dependent protein kinase was covalently labeled with the ATP analog, 5-p-fluorosulfonylbenzoyladenosine. The inactivation reaction was pseudo-first order. The rate of kinase sulfonation exhibited saturation kinetics indicative of a rapid reversible binding of the reagent prior to enzyme modification. The enzyme could be protected by MgATP, MgADP, and Mg-adenylylimidodiphosphate but not by a synthetic peptide substrate. Cyclic GMP when bound to the kinase did not influence the rate of labeling. The reagent demonstrated competitive inhibition with respect to MgATP; the Ki was found to be 0.82 mM. Magnesium and cobalt ions when included in the reaction mixture accelerated the inactivation rate up to severalfold. Addition of basic polypeptides such as mixed histone, protamine sulfate, or poly-L-lysine HBr also markedly accelerated the sulfonation rate. Inactivation of the kinase with 5-'fluorosulfonyl[3H]benzoyladenosine resulted in a linear relationship between the residual phosphotransferase activity and the incorporation of up to 0.9 mol of reagent/mol of monomer.

Topics: Adenosine; Adenosine Triphosphate; Animals; Binding Sites; Cattle; Chelating Agents; Cobalt; Cyclic GMP; Kinetics; Lung; Magnesium; Protein Binding; Protein Kinases; Substrate Specificity