A novel recombinant fibrinogenase of Agkistrodon acutus venom protects against hyperacute rejection via degradation of complements.

Hyperacute rejection (HAR) is a main barrier in xenotransplantation, which is mediated by the combination of natural antibody to the xenograft and complement activation. Current therapies have focus on the inhibition of complement by development of complement inhibitor and transgenic animal organ. Here, we investigated the effects of rFII, a recombinant fibrinogenase from Agkistrodon acutus venom, on complement and HAR. The degradation effect of rFII on complement was tested by SDS-PAGE, CH50 examination, ELISA Kit and cofocal immunofluorescence microscopy in vitro and in vivo. An ex-vivo rat-to-human perfusion model and a vivo guinea-pig-to-rat heat HAR model were used to determine the protection of rFII against HAR. Our investigation indicated that rFII could significantly degrade human C5, C6, and C9, decrease the activity of complement, and inhibit the MAC deposition on HUVECs membrane in vitro. In addition, serum levels of C1q, C3 and C4 in rat were gradually reduced after infusion of rFII. Importantly, in an ex vivo rat-to-human perfusion model, the survival of rat hearts perfused with human serum treated with rFII (83.36 ± 16.63 min) were significantly longer than that of hearts perfused with fresh human serum(15.94 ± 4.75 min). At the time of 15 minutes after perfusion, functions of hearts added with 50 ug/ml rFII sustained well with heart rates at 283 ± 65.32 beats/minute and LVDP at 13.70 ± 5.45 Kpa, while that of hearts perfused with fresh human serum were severely damaged by HAR with heart rates at 107.77 ± 40.31 beats/minute and LVDP at 1.01 ± 0.83 Kpa. We also found that rFII significantly decreased the levels of C1q, C3 and C4 in human fresh serum perfusate. In a vivo guinea-pig-to-rat heat HAR model, the survival of rat hearts treated with rFII were significantly longer than that of hearts perfused with normal saline; and relieved heart damage by complete activation. Our finding demonstrates the anti-complement property of rFII and its protection against HAR, indicating that rFII might be as a potential therapeutic agent for xenotransplantation.

A novel fibrinogenase from Agkistrodon acutus venom protects against DIC via direct degradation of thrombosis and activation of protein C.

The incidence of disseminated intravascular coagulation (DIC), which leads to multiple organ dysfunction and high mortality, has remained constant in recent years. At present, treatments of DIC have focused on preventing cytokine induction, inhibiting coagulation processes and promoting fibrinolysis. Recent clinical trials have supported the use of antithrombin and activated protein C supplementation in DIC. To better understand the mechanism of treatment on DIC, we here report a novel fibrinogenase from Agkistrodon acutus (FIIa) that effectively protected against LPS-induced DIC in a rabbit model, and detected the tissue factors expression in HUVE cells after using FIIa. In vivo, administration of FIIa reduced hepatic and renal damage, increased the concentration of fibrinogen, the activities of protein C, the platelet count, APTT, PT, FDP, the level of AT-III and t-PA, decreased the level of PAI-1, and increased survival rate in LPS-induced DIC rabbits. In vitro experiments, we further confirmed that FIIa up-regulated the expression of t-PA and u-PA, down-regulated the expression of PAI-1, and directly activated protein C. Our findings suggest that FIIa could effectively protect against DIC via direct degradation of microthrombi and activation of protein C as well as provide a novel strategy to develop a single proteinase molecule for targeting the main pathological processes of this disease.