Comparative effects of nitroxil (HNO) and NO on platelet function and tyrosine phosphorilation pattern of activated human platelets.

BERMEJO, E; ALBERTO, F; SAENZ, D; GAMBERALLE, R; BARI, S; ROSENSTEIN, R; LAZZARI, M

Sydney, Australia

Congreso; XXth Congress of the International Society on Thrombosis and Haemostasis; 2005

International Society of Thrombosis & Haemostasis

Comparative Effects of Nitroxil (HNO) and NO on Platelet Function and Tyrosine Phosphorilation Pattern of Activated Human Platelets Bermejo, E 1, Alberto, MF1, Saenz, DA2, Gamberalle1,  R, Bari, S3, Rosenstein, R2, Lazzari, MA1. 1Instituto de Investigaciones Hematologicas, Academia Nacional de Medicina,Buenos Aires,Argentina 2Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos Aires, Argentina 3Departamento de Química Inorgánica, Analítica y Química Física, Universidad de Buenos Aires,Argentina Recently, interest has developed in the functional roles of an alternative redox form of NO, namely, nitroxyl (HNO).The aim of the present report was to analyze comparatively, the effect of HNO and NO on pattern of tyrosine phosphorilated proteins (Tyr-PP) of human platelets upon thrombin (Thr) stimulation. Platelet aggregation, CD62P expression, PAC1 binding and cGMP accumulation, were evaluated as complementary tests. Methods: All the tests were assessed with washed platelets (WP) (300 109plat/l). Angeli´s salt (AS) and sodium nitroprusside (SNP) were used as a HNO donor, and a NO donor, respectively at 10µM. The samples (n= 5) were incubated with buffer, AS or SNP at 37ºC for 2 min. After adding 0.5 U/ml Thr we performed: a) platelet aggregation, b) immunobloting, analyzed by scanning densitometry at 0, 30, 60 and 180 sec; c) expression of platelet membrane markers, analyzed by flow cytometry using mean fluorescence intensities (MFI) and d) cGMP accumulation by RIA. Results: Both donors significantly inhibited: platelet aggregation (p<0.001), the expression of CD62P (p<0.05) and PAC-1(p<0.05) and increased cGMP levels (p<0.01).The Tyr-PP of 130-100 kD bands were delayed for SA and SNP. The 50 kD band is strongly reduced only for SA (p<0.05). Conclusion: An approach to investigating the mechanisms provoked by HNO we compared its effects with NO on several platelets parameters. Both donors produce inhibition of platelet aggregation and PAC-1/CD62P expression, a delayed phosphorilation of tyrosine residues 130-100kD and increased cGMP levels. As NO, HNO would activate guanylate-cyclase but the finding observed on 50kD band would suggest differences in the mechanisms of action. Taken together, HNO may constitute a novel target for future antithrombotic drug development.