A novel anticoagulant protein with high affinity to blood coagulation factor Va from Tegillarca granosa.
A novel inhibitory protein against blood coagulation factor Va (FVa) was purified from muscle protein of granulated ark (Tegillarca granosa, order Arcoida, marine bivalvia) by consecutive FPLC method using anion exchange and gel permeation chromatography. In the results of ESIQTOF tandem mass analysis and database research, it was revealed that the purified T. granosa anticoagulant protein (TGAP) has 7.7 kDa of molecular mass and its partial sequence, HTHLQRAPHPNALGYHGK, has a high identity (64%) with serine/threonine kinase derived from Rhodopirellula baltica (order Planctomycetales, marine bacteria). TGAP could potently prolong thrombin time (TT), corresponding to inhibition of thrombin (FIIa) formation. Specific factor inhibitory assay showed that TGAP inhibits FVa among the major components of prothrombinase complex. In vitro assay for direct-binding affinity using surface plasmon resonance (SPR) spectrometer indicated that TGAP could be directly bound with FVa. In addition, the binding affinity of FVa to FII was decreased by addition of TGAP in dose-dependant manner (IC50 value = 77.9 nM). These results illustrated that TGAP might interact with a heavy chain of FVa (FVa(H)) bound to FII in prothrombin complex. The present study elucidated that non-cytotoxic T. granosa anticoagulant protein (TGAP) bound to FVa can prolong blood coagulation time by inhibiting conversion of FII to FIIa in blood coagulation cascade. In addition, TGAP did not significantly (P < 0.05) show fibrinolytic activity and cytotoxicity on venous endothelial cell line (ECV 304).
Jung
Won-Kyo
WK
Department of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea.
Jo
Hee-Yeon
HY
Qian
Zhong-Ji
ZJ
Jeong
Young-Ju
YJ
Park
Sae-Gwang
SG
Choi
Il-Whan
IW
Kim
Se-Kwon
SK
D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D049893:Arcidae / Q000737:chemistry*; D001777:Blood Coagulation / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D015943:Factor Va / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D008970:Molecular Weight; D011485:Protein Binding; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D020349:Surface Plasmon Resonance; D053719:Tandem Mass Spectrometry; D013696:Temperature; D013918:Thrombin Time
D000925:Anticoagulants; D015943:Factor Va