Studies On Factor IX/X Binding Protein From Agkistrodon Halys Pallas Venom (ahp Ix/x-bp)

The coagulation factor IX/X binding proteins with anticoagulant activity broadly exist in the venom of several viperidae snakes. They can bind to coagulation factor IX or factor X or both of factors IX and X. The purification of coagulation factor IX/X binding protein from Agkistrodon Halys Pallas (AHP IX/X-bp), and the effects of metal ions on its conformation, structural stability and functions have been investigated in this thesis. The whole thesis is divided into four chapters.The fist chapter is a brief review about the progress of coagulation factor IX/X binding proteins and blood coagulation system. After you read this chapter, you can have a whole understanding about blood coagulation system, the coagulation factors and the research history of coagulation factor IX/X binding proteins. Some research methods used in this study have been also reviewed in this chapter.The second chapter: AHP IX-bp was purified from the venom of Agkistrodon Halys Pallas by a simple two-step of chromatography procedure. We also used PAGE, MALDI-TOF-MS and cIEF methods to check the purity of AHP IX-bp and determine the molecular weight of AHP IX-bp.The third chapter: AHP IX-bp was reported to bind specifically with factor IX in a Zn²⁺-dependent manner. In this chapter, we have found that AHP IX-bp also binds factor X with high binding-affinity in a Mg²⁺-dependent manner as determined by native PAGE and surface plasmon resonance. For this reason, we renamed this protein as coagulation factor IX/X binding protein (AHP IX/X-bp). We also found that Mg(II) ions could not bind to AHP IX/X-bp. Mg²⁺ ions are required to maintain in vivo function of FX for its recognition of AHP IX-bp. Both Ca²⁺ and Zn²⁺ ions fail to induce the binding between apo-AHP IX-bp and FXa. The abundant Mg²⁺ ions in blood play an important role in the anticoagulation of AHP IX-bp.The fourth chapter: The binding properties of Ca(II) and Zn(II) to AHP IX/X-bp and their effects on its conformation and stability have been investigated. The results show that AHP IX/X-bp has two metal binding sites, one specific for Ca²⁺ with lower affinity for Zn²⁺ and one specific for Zn²⁺ with lower affinity for Ca²⁺. GdnHCl- and thermal-induced denaturations of 2Ca²⁺-AHP IX/X-bp, 2Zn²⁺-AHP IX/X-bp and Ca²⁺, Zn²⁺-AHP IX/X-bp are all a two-state process with no detectable intermediate state(s), indicating the tight packing of two chains. Ca²⁺ and Zn²⁺ increase the structural stability of AHP IX/X-bp against GdnHCl or thermal denaturation to the same extent. Although Ca²⁺ and Zn²⁺ have no obvious effect on the global structure of AHP IX/X-bp, they induce different rearrangements in local conformation. Zn²⁺-altered specific conformation of AHP IX/X-bp may be helpful to its recognition of the structure of FIX. This work suggests that in vitro, Ca²⁺ plays a structural rather than an active role in the anticoagulation of AHP IX/X-bp, while Zn²⁺ plays both structural and active roles in the anticoagulation. In vivo, Ca²⁺ binds to AHP IX/X-bp and stabilizes its structure, while Zn²⁺ can not binds to AHP IX/X-bp due to the low Zn²⁺ concentration in blood. AHP IX/X-bp prolongs the clotting time in vivo through its binding only with FX/FXa.