C-type lectin-like proteins (CLPs) have similar structures to that of C-type lectins, but they have no lectin activity. The CLPs from snake venom have a variety of biological activities, including anticoagulant- and platelet-modulating activities. A family of coagulation factor IX/factor X-binding protein (IX/X-bp) which belongs to CLPs has been identified from the venoms of different snake species. IX/X-bps interact with theγ-carboxyglutamic acid (Gla) domain of factor IX/IXa and/or factor X/Xa in a Ca2+-dependent manner and thereby block the amplification of the coagulation cascade. Anticoagulation factor I (ACFâ… ) and anticoagulation factor II (ACFâ…¡) purified from the venom of Agkistrodon acutus are two members of the IX/X-bp protein family. In this thesis, the effects of metal ions on the structural stability and function of ACFâ… and ACFâ…¡, the hypotensive effects of ACFâ… and ACF II on the cardiovascular system, and their application in the purification of coagulation factor IX and coagulation factor X have been investigated. The whole thesis is divided into four chapters.The first chapter gives a brief overview of the molecular mechanism of coagulation process, the character and function of the important coagulation factors, especially on coagulation factor IX and coagulation factor X, the purification technology of coagulation factor IX/X, C-type lectin-like proteins, and coagulation factor IX/X-binding protein family.In the second chapter, the anticoagulant activity of ACFâ… and in vivo, the thermodynamics of the binding of alkaline earth metal ions to ACFâ…¡and their effects on the stability of ACFâ…¡and the binding of ACFâ…¡to FXa were investigated by isothermal titration calorimetry, fluorescence, differential scanning calorimetry and surface plasmon resonance, respectively. Both ACFâ… and ACFâ…¡exhibit high anticoagulation activity in vivo. The binding of ACFâ…¡to FXa does not have an absolute requirement for Ca2+. Mg2+, Sr2+ and Ba2+ can induce the binding of ACFâ…¡to FXa. The radii of the cations bound in ACFâ…¡crucially affect the binding affinity of ACFâ…¡to cations and the structural stability of ACFâ…¡against GdnHCl and thermal denaturation, while the radii of cations bound in FXa markedly affect on the binding affinity between ACFâ…¡and FXa. The binding affinities of ACFâ…¡for cations and the capacities of metal-induced stabilization of ACFâ…¡follow the same trend Ca2+ > Sr2+ > Ba2+. The metal-induced binding affinities of ACFâ…¡to FXa follow the trend Mg2+ > Ca2+ > Sr2+ > Ba2+. Although Mg2+ shows significantly low binding affinity with ACFâ…¡, Mg2+ is the most effective to induce the binding of ACFâ…¡with FXa. Our observations suggest that in blood, the bindings of Ca2+ in two sites of ACFâ…¡increase the structural stability of ACFâ…¡, but these bindings are not essential for the binding of ACFâ…¡with FXa, and that the binding of Mg2+ and Ca2+ to FXa may be essential for the recognition between FXa and ACFâ…¡. Like Ca2+, the abundant Mg2+ in blood also plays an important role in the anticoagulation of ACFâ…¡.In the third chapter, we have developed an rapid affinity chromatography procedure using coagulation factor IX/factor X-binding proteins as novel affinity ligands to isolate coagulation factor IX and coagulation factor X from plasma. The native affinity ligands efficiently bind FIX/FX and release them under very mild conditions. FIX and FX can be simultaneously purified from unclarified plasma by IX/X-bps affinity chromatography followed by HPLC with a purification factor and yield similar to that of monoclonal antibody immunoaffinity chromatography. In contrast to monoclonal antibody that is cumbersome, expensive and suffer from its instability, the novel affinity ligands IX/X-bps are long-term stable and cheap to prepare in large scale. In addition, monoclonal antibody immunoaffinity chromatography is used to purify FIX only from the specific plasma, while the new affinity chromatography can be used to purify both FIX and FX from any unclarified plasma or solutions. This new affinity chromatography has potential application for the industrial scale purification of FIX and FX for specific replacement therapy in hemophilia B and patients with FX deficiency.In the fourth chapter, the effects of ACFâ… and ACFâ…¡on the mean arterial blood pressure (MABP) and heart rate (HR) in anesthetized rats have been investigated. The results indicate that ACFâ…¡induces a dose-dependent response in rats with a short fast drop of MABP followed by an increase and then a longer lasting slight decrease in MABP, but does not obviously affect HR. ACFâ…¡-induced hypotension is significantly blocked by the NO synthase inhibitor N-omega-L-arginine methyl ester (L-NAME). ACFâ…¡produces a concentration-dependent relaxation of rat aortic rings with functional-endothelium. The ACFâ…¡-induced vasodilatation is completely inhibited by removal of endothelium and significantly inhibited by pretreatment with L-NAME. These observations demonstrate that ACFâ…¡induces hypotension through an endothelium-dependent vasodilation, which is strongly mediated by the release of NO from endothelium. Therefore, ACFâ…¡is so far identified as the first unique bifunctional protein in the IX/X-bp family that has both anticoagulant and hypotensive effects on the blood of rats through different pathways. However, ACFâ… does not show any hypotensive effect in rats.
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