A Novel Anticoagulant Protein with High Affinity to BloodCoagulation Factor Va from Tegillarca granosa

A Novel Anticoagulant Protein with High Affinity to BloodCoagulation Factor Va from Tegillarca granosa

A novel inhibitory protein against blood coagulation factorVa (FVa) was purified from muscle protein of granulatedark (Tegillarca granosa, order Arcoida, marine bivalvia)by consecutive FPLC method using anion exchange andgel permeation chromatography. In the results of ESIQTOFtandem mass analysis and database research, it wasrevealed that the purified T. granosa anticoagulant protein(TGAP) has 7.7 kDa of molecular mass and its partialsequence, HTHLQRAPHPNALGYHGK, has a highidentity (64%) with serine/threonine kinase derived fromRhodopirellula baltica (order Planctomycetales, marinebacteria). TGAP could potently prolong thrombin time(TT), corresponding to inhibition of thrombin (FIIa)formation. Specific factor inhibitory assay showed thatTGAP inhibits FVa among the major components ofprothrombinase complex. In vitro assay for direct-bindingaffinity using surface plasmon resonance (SPR) spectrometerindicated that TGAP could be directly bound with FVa. Inaddition, the binding affinity of FVa to FII was decreasedby addition of TGAP in dose-dependant manner (IC50value = 77.9 nM). These results illustrated that TGAPmight interact with a heavy chain of FVa (FVaH) bound toFII in prothrombin complex. The present study elucidatedthat non-cytotoxic T. granosa anticoagulant protein(TGAP) bound to FVa can prolong blood coagulation timeby inhibiting conversion of FII to FIIa in blood coagulationcascade. In addition, TGAP did not significantly (P < 0.05)show fibrinolytic activity and cytotoxicity on venousendothelial cell line (ECV 304).

A novel inhibitory protein against blood coagulation factorVa (FVa) was purified from muscle protein of granulatedark (Tegillarca granosa, order Arcoida, marine bivalvia)by consecutive FPLC method using anion exchange andgel permeation chromatography. In the results of ESIQTOFtandem mass analysis and database research, it wasrevealed that the purified T. granosa anticoagulant protein(TGAP) has 7.7 kDa of molecular mass and its partialsequence, HTHLQRAPHPNALGYHGK, has a highidentity (64%) with serine/threonine kinase derived fromRhodopirellula baltica (order Planctomycetales, marinebacteria). TGAP could potently prolong thrombin time(TT), corresponding to inhibition of thrombin (FIIa)formation. Specific factor inhibitory assay showed thatTGAP inhibits FVa among the major components ofprothrombinase complex. In vitro assay for direct-bindingaffinity using surface plasmon resonance (SPR) spectrometerindicated that TGAP could be directly bound with FVa. Inaddition, the binding affinity of FVa to FII was decreasedby addition of TGAP in dose-dependant manner (IC50value = 77.9 nM). These results illustrated that TGAPmight interact with a heavy chain of FVa (FVaH) bound toFII in prothrombin complex. The present study elucidatedthat non-cytotoxic T. granosa anticoagulant protein(TGAP) bound to FVa can prolong blood coagulation timeby inhibiting conversion of FII to FIIa in blood coagulationcascade. In addition, TGAP did not significantly (P < 0.05)show fibrinolytic activity and cytotoxicity on venousendothelial cell line (ECV 304).