The Study Of A Novel Serine Proteinase From The Venom Of Deinagkistrodon Acutus And The Hypotensive Mechanism Of ACF Ⅱ

The thesis is divided into two parts. The first part is about the purification and partial characterization of a novel fibrinogenase, named DAnase, from the venom of Deinagkistrodon acutus. The second part is about the exploration of the hypotensive mechanism of anticoagulation factor II (ACF II), isolated from the venom of Agkistrodon acutus. ACF II is a factor IX/factor X-binding protein with both anticoagulation and hypotensive activities. However, up to now, the hypotensive mechanism of ACF II is still unclear. In this part, ACF II was purified from the venom of Deinagkistrodon acutus and its hypotensive mechanism was explored. The whole thesis includes three chapters.The first chapter gives a brief overview of fibrin(ogen)olytic enzymes, metalloproteases, serine proteases, blood coagulation, hypertension, nitric oxide (NO) and ACF II.In the second chapter, a novel fibrinogenase, DAnase, was purified from the venom of Deinagkistrodon acutus by a combination of anion and cation exchange chromatography. Unlike other fibrinogenases which are usually single polypeptide chain proteins, the enzyme was a disulfide-linked dimer with an isoelectric point of6.03and an apparent molecular weight of25kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). DAnase showed a-fibrinogenase activity devoid of fibrinolytic activity. It hydrolyzed rapidly the Aa-chain of fibrinogen and followed by the β-chain and did not cleave the y-chain. It also exhibited arginine esterase activity. The fibrinogenolytic and arginine esterase activities were completely inhibited by phenylmethanesulfonyl fluoride (PMSF) or tris-(2-carboxyethyl) phosphine hydrochloride (TCEP), but not by EDTA, indicating that DAnase is a serine protease requiring disulfide bridge(s) for its activity. The protease strongly inhibited ADP-induced platelet aggregation in human platelet-rich plasma but was lack of ADPase activity, indicating that its fibrinogenolytic activity is involved in its inhibition of ADP-induced platelet aggregation. DAnase was devoid of hemorrhagic activity and Factor XIII activation activity. In the third chapter, ACF II was purified from the venom of Deinagkistrodon acutus by a combination of anion and cation exchange chromatography. It has been reported that ACF II was a member of the coagulation factor IX/coagulation factor X-binding protein family and with both anticoagulant and hypotensive effects. We have investigated the effect of ACF II on the NO concentration in the plasma of rats. The results indicate that ACF II induces a dose-dependent increase in NO concentration. However, the amount of NO decreased with the time on and finally reached a plateau. ACF H-induced increase in NO concentration is significantly blocked by pretreated with the nitric oxide synthase inhibitor N-omega-L-arginine methyl ester (L-NAME) and the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), but not by the Isatin, an inhibitor of atrial natriuretic peptide (ANP), suggesting that nitric oxide synthase and calmodulin may be involved in ACF II-induced hypotension. ACF II does not bind with calmodulin, indicating that calmodulin is not the target protein of ACF II. It has hypotensive effect through a different pathway. These observations demonstrate that ACF II induces hypotension through the activation of a NO pathway. The target protein of ACF II for the hypotension may be nitric oxide synthase or the upstream component of the NO signaling pathway.