Biochemical and hemostatic mechanism of a novel thrombin-like enzyme.
Thrombin-like enzyme (TLE) plays a significant role in vessel injury hemostasis. A novel snake venom TLE (Agacutin) was purified from Agkistrodon Acutus snake venom. Structural analysis indicated that Agacutin is a heterodimer that has a MW of 29,402 Da, a pI value of 5.39, and optimum activity at 35 degrees C and pH 7.5. The N-terminal 15 amino acid sequences of Agacutin are DSSGWSSYEGHEYYV (small subunit) and DCSSGWSSYEEHQYY (large subunit). In vitro studies indicated that the coagulation activity of Agacutin was activated by Ca(+2) or inhibited by phenylmethanesulfonyl fluoride, but not influenced by heparin or hirudin. The arginine esterase activity and fibrinogen hydrolysis result showed that Agacutin only cleaves alpha-subunit and releases fibrinopeptide A. In vivo studies indicated that Agacutin iv (0.01-0.05 U/kg) shortened 30.2-49% of the rabbit blood clotting time, or ip (0.5-2.0 U/kg) shortened 29.7-73.1% of the mouse tail bleeding time. Agacutin does not influence APTT, platelet or euglobulin clotting time, and activate Factor II or XIII. It converts fibrinogen into the soluble fibrin that accelerates hemostasis at wound.
Tang
Song-Shan
SS
Department of Biochemistry & Molecular Biology, School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou 510006, China. songstang@gmail.com
Zhang
Juan-Hui
JH
Tang
Bai-Shan
BS
Tang
Zhi-Hua
ZH
Li
Hong-Zhi
HZ
Yuan
Hao-Jia
HJ
Chui
Shu-Lian
SL
Zhao
Er-Yao
EY
D017836:Agkistrodon / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D003435:Crotalid Venoms / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D006487:Hemostasis / Q000187:drug effects*; D006493:Heparin / Q000494:pharmacology; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D013917:Thrombin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*
D003435:Crotalid Venoms; D006629:Hirudins; D005340:Fibrinogen; D006493:Heparin; D013917:Thrombin; C530991:thrombin-like protein, Agkistrodon acutus; D002118:Calcium