Inhibition of clotting factor XIII activity by nitric oxide.
The plasma factor XIII (FXIII) is a transglutaminase which catalyzes the cross-linking of fibrin monomers during blood coagulation. S-nitrosylation of protein sulfhydryl groups has been shown to regulate protein function. Therefore, to establish whether nitric oxide (NO) affects the enzymatic activity of FXIII, we studied the effect of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) in a blood coagulation test in vitro. High concentrations of SNAP were found to have inhibitory effects on clot formation. Moreover, specific formation of gamma-dimers through the action of FXIII is selectively inhibited by high concentrations of SNAP, as revealed by Western blot. Purified activated FXIII and plasma preparations were then exposed to NO-donor compounds and the enzyme activity was assayed by measuring the incorporation of [3H] putrescine into dimethylcasein. The NO donors, SNAP, spermine-NO (SPER-NO) and 3-morpholinosydnonimine (SIN-1), and the NO-carrier, S-nitrosoglutathione (GSNO), inhibited FXIII activity in a dose-dependent manner, in both purified enzyme and plasma preparations. Titration of -SH groups of FXIII with [14C] iodoacetamide has shown that the number of titratable cysteines per monomer of FXIII decreased from 1 (in absence of NO donors) to 0 (in the presence of NO donors). These results demonstrate that blood coagulation FXIII is a target for NO both in vitro and in vivo, and that inhibition occurs by S-nitrosylation of a highly reactive cysteine residue. In conclusion, we show that inhibition of FXIII activity by NO may represent an additional regulatory mechanism for the formation of blood clot with physio-pathological implications.
IDI-IRCCS, Biochemistry Laboratory, Department of Experimental Medicine, University of Rome Tor Vergata, Italy.
M V
MV
Catani
F
F
Bernassola
A
A
Rossi
G
G
Melino
D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005176:Factor XIII / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism* / Q000494:pharmacology*; D009603:Nitroso Compounds / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D026422:S-Nitrosoglutathione; D013096:Spermine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D009603:Nitroso Compounds; D010975:Platelet Aggregation Inhibitors; C110881:S-nitro-N-acetylpenicillamine; D013096:Spermine; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; C002385:linsidomine; D005176:Factor XIII; D008981:Molsidomine; D005978:Glutathione; D010396:Penicillamine