NEW FIBRINOGENASES ISOLATED FROM MARINE HYDROBIONT Adamussium colbecki

Aims: Enzymes that affect hemostasis have been isolated from different sources. Fibrinogenolytic enzymes because of their role in dissolving of blood clots as well as prevention their formation have attracted special medical and scientific attention. The main goal of current research was to analyze marine hydrobionts (an example of the Antarctic scallop Adamussium colbecki) from the Antarctic region on the presence of potential fibrinogenolytic enzymes.

Methodology: The fraction that consists of fibrinogenases was obtained by affinity chromatography on Blue-Sepharose column. SDS-PAGE was applied for the determination of protein composition in obtained fraction. The proteolytic activity was monitored by zymographic technique. Fibrinogenolytic activity and activity toward collagen were assessed by incubation of samples with fibrinogen or collagen, respectively followed by SDS-PAGE analysis. To test for substrate specificity, the fraction that consists of fibrinogenases was incubated with p-nitroanilide chromogenic peptides such as S-2366, S-2238, S-2251, S-2222. Further probes were preincubated with protease inhibitors EDTA, PMSF and activity toward S-2238 was measured.

Results: The obtained results clearly indicated the presence of enzymes with activity toward fibrinogen in extract of A. colbecki. Zymography analysis detected the presence of active enzymes at the region of 27-30 kDa. The fibrinogen cleavage pattern was analyzed by SDS-PAGE under reducing conditions revealed time- and dose-dependent hydrolysis of fibrinogen. Susceptibility of fibrinogen chains to proteolytic degradation by fibrinogenases from A. colbecki was different. These enzymes preferentially hydrolyzed the Aα-chain; they also cleaved the Bβ-chain but at slower rate. Collagen was found to be resistant to fibrinogenases under similar experimental condition. Fibrinogenases from A. colbecki hydrolyzed chromogenic substrates as S-2238, S-2251, S-2222 and S-2366 but with different specificity. Specific protease inhibitors PMSF and EDTA were used to identify the nature of fibrinogenases present in tissue of investigated hydrobiont. On the basis of this analysis, fraction of fibrinogenases from A. colbecki consisted of serine proteases as well as metalloproteases.