High inhibitory activity on proteases in a reptile plasma ( Bothrops jararaca snake) impairs its intrinsic fibrinolytic-like mechanism

Abstract

Summary 1. A mammal-like fibrinolytic system was investigated in the blood of male and female poisonous snakes Bothrops jararaca (Bj). 2. Conventional and alternative methodologies were used to study this mechanism in plasma and euglobulin fraction (EUF) since it was previously reported that classes Aves and Reptilia did not have this system. 3. EUFs from male and female snakes showed activity on fibrin-agarose plates when diluted plasma (1:20) was precipitated at pH 5.9. No activity was seen at pH 5.5, and very little or occasional at pH 5.7. 4. A 79 KDa activator of fibrinolysis was detected in EUF of both sexes after SDS-PAGE and zymography independently of the pH of precipitation. 5. Activity of EUF measured on conventional human fibrin plates was variable and increased by overnight standing at 4°C and/or addition of flufenamates. 6. The difference in behaviour described in items 3 and 5 may be due to a sieving effect in the fibrin-agarose plates which by separating activators and inhibitors allows the expression of fibrinolytic activity. The same principle could explain the results after SDS-PAGE and zymography. 7. Bj plasma showed high inhibitory activity on fibrin lysis by the proteases trypsin and plasmin and, moderate on the same activity by plasminogen activators, urokinase (UK) and tissue activator (t-PA). Reverse zymography of plasma on fibrin agarose plates showed an inhibition area in the range of 45 to 69 KDa as compared to rabbit PAI-1 in a single band of 42 KDa. 8. Bj plasma was more inhibitory to UK when compared to human, bovine and rabbit plasma on fibrin-agarose plates. Bj female plasma was more inhibitory than male. 9. Euglobulin clot lysis time (ECLT) for Bj EUFs was very prolongated even in high dilution (1:30) when compared to the human system. Correction of the ionic strength in higher dilutions did not improve the ECLT of Bj EUFs, which was decreased only by further addition of UK (30 IU) to EUF from dilution 1:60. 10. It is suggested that a mammal-like fibrinolytic system operates on Bj snake plasma. However, a different balance of activator-inhibitor levels seems to hinder the detection of the lytic activity.