Gerads I, Tans G, Zwaal R F, Rosing J
Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
Toxicon. 1992 Sep;30(9):1065-79. doi: 10.1016/0041-0101(92)90052-7.
The crude venom of many elapid snakes appeared to contain proteins that activated blood coagulation factor V. The factor V activator present in the venom of Naja naja oxiana was purified to homogeneity by chromatography on a mono-S column. The activator was a single chain protein with an apparent mol. wt of 48,000, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by gel permeation chromatography on Sephacryl S200. Activation of bovine factor V by the purified venom activator was accompanied by proteolytic cleavage of factor V and resulted in the formation of two major polypeptide chains with mol. wts of about 90,000 and 77,000. The final product obtained was compared with thrombin-activated factor V for its ability to function as cofactor in factor Xa-catalysed prothrombin activation in the presence of negatively charged phospholipid vesicles (5 mole% phosphatidylserine/95 mole% phosphatidylcholine). The Km for prothrombin obtained at a saturating amount of venom-activated factor Va was nine-fold higher than with thrombin-activated factor V (0.83 microM vs 0.09 microM, respectively) whereas both factor Va molecules stimulated the Vmax of thrombin formation some 6000-fold. Both forms of factor Va promoted the binding factor Xa to negatively charged phospholipid vesicles. However, the apparent Kd for factor Xa was less favorable in the presence of venom-activated factor V (0.67 x 10(-9) M) than in the presence of thrombin-activated factor V (0.043 x 10(-9) M). Thrombin cleaved a peptide bond in the 77,000 mol. wt polypeptide chain of venom-activated factor V, which resulted in the formation of a normal factor Va light chain. This peptide bond cleavage was, however, not associated with a change of cofactor activity. Venom treatment of thrombin-activated factor V, on the other hand, did remove a small fragment (mol. wt approximately 4000) from the heavy chain of factor Va (94,000), yielding a molecule with reduced cofactor activity. The diminished cofactor activity of venom-activated factor V is, therefore, likely due to the fact that a small peptide fragment, involved in the interaction with prothrombin and factor Xa, is missing from the heavy chain of venom-activated factor V.
许多眼镜蛇科蛇的粗毒液似乎含有能激活血液凝固因子V的蛋白质。通过在单-S柱上进行色谱分离，将中亚眼镜蛇毒液中的因子V激活剂纯化至同质。该激活剂是一种单链蛋白质，在十二烷基硫酸钠存在下通过聚丙烯酰胺凝胶电泳以及在Sephacryl S200上进行凝胶渗透色谱法判断，其表观分子量为48,000。纯化的毒液激活剂对牛因子V的激活伴随着因子V的蛋白水解裂解，并导致形成两条主要的多肽链，分子量约为90,000和77,000。将所得最终产物与凝血酶激活的因子V在带负电荷的磷脂囊泡（5摩尔％磷脂酰丝氨酸/95摩尔％磷脂酰胆碱）存在下作为因子Xa催化凝血酶原激活的辅因子的功能能力进行比较。在毒液激活的因子Va饱和量下获得的凝血酶原的Km比凝血酶激活的因子V高九倍（分别为0.83 microM对0.09 microM），而两种形式的因子Va分子都刺激凝血酶形成的Vmax约6000倍。两种形式的因子Va都促进因子Xa与带负电荷的磷脂囊泡结合。然而，在毒液激活的因子V存在下因子Xa的表观Kd（0.67×10⁻⁹ M）比在凝血酶激活的因子V存在下（0.043×10⁻⁹ M）更不利。凝血酶切割了毒液激活的因子V的77,000分子量多肽链中的一个肽键，这导致形成正常的因子Va轻链。然而，这种肽键切割与辅因子活性的变化无关。另一方面，用毒液处理凝血酶激活的因子V确实从因子Va的重链（94,000）中去除了一个小片段（分子量约4000），产生了一种辅因子活性降低的分子。因此，毒液激活的因子V辅因子活性降低可能是由于毒液激活的因子V重链中缺少一个参与与凝血酶原和因子Xa相互作用的小肽片段。
