Khan Sami U, Al-Saleh Saad S
aGomal Centre of Biochemistry & Biotechnology, Gomal, University, Dera Ismail Khan, Pakistan bDepartment of Clinical Laboratory Sciences, College of Applied, Medical Sciences, King Saud University, Riyadh, Saudi, Arabia.
Blood Coagul Fibrinolysis. 2015 Oct;26(7):772-7. doi: 10.1097/MBC.0000000000000336.
Factor X of blood coagulation cascade can be activated by both intrinsic and extrinsic activating complex, trypsin and some kind of snake venom. A factor X activator protein is reported in Elapidae snake venom. The aim of this study was to evaluate biochemical properties of factor X activator protein because of its prospective application in biochemical research and therapeutics. Crude venom was fractionated on a HPLC system Gold 126/1667 using a combination of Protein PAK 125 and Protein PAK 60 Columns. Molecular weight was determined using SDS-PAGE. Walterinnesia aegyptia venom was fractionated into several protein peaks, but procoagulant and factor X activation activity coexisted into peak no.6. It appeared as single band on native PAGE and molecular weight was 60,000 ± 3. Purified up to 37-fold over crude venom. It shortened recalcification time, effect was dose-dependent and strictly Ca(2++)-dependent. Factor X activator seems to be able to activate factor X specifically because it showed no activation activity on human prothrombin, plasminogen, or protein C. It did not hydrolyze factor Xa substrate S-2222, thrombin substrate S-2238, plasmin substrate S-2251 or S-2302 and kalikrein substrate S-2266. It did not hydrolyze synthetic ester benzoyl arginine ethyl ester. Procoagulant activity was completely inhibited by irreversible serine protease inhibitors phenylmethylsulphonyl fluoride and N-p-tosylphenylalanine chloromethyl ketone. This study illustrates that factor X activator from W. aegyptia is though different in many aspects from factor X activators of Viperidae and Crotalidae venoms, but shows several properties identical to factor X activators from Elapidae venoms.
血液凝固级联反应中的凝血因子X可被内源性和外源性激活复合物、胰蛋白酶及某些蛇毒激活。据报道，眼镜蛇科蛇毒中存在一种凝血因子X激活蛋白。由于该蛋白在生化研究和治疗方面具有潜在应用价值，本研究旨在评估其生化特性。粗毒液在配备Protein PAK 125和Protein PAK 60柱组合的HPLC系统Gold 126/1667上进行分离。通过SDS - PAGE测定分子量。埃及眼镜蛇毒液被分离成几个蛋白峰，但促凝活性和凝血因子X激活活性共存于第6号峰中。该蛋白在非变性聚丙烯酰胺凝胶电泳上呈现单一条带，分子量为60,000 ± 3。其纯化程度比粗毒液高37倍。它缩短了复钙时间，作用呈剂量依赖性且严格依赖Ca(2++)。凝血因子X激活剂似乎能够特异性激活凝血因子X，因为它对人凝血酶原、纤溶酶原或蛋白C没有激活活性。它不水解凝血因子Xa底物S - 2222、凝血酶底物S - 2238、纤溶酶底物S - 2251或S - 2302以及激肽释放酶底物S - 2266。它不水解合成酯苯甲酰精氨酸乙酯。不可逆丝氨酸蛋白酶抑制剂苯甲基磺酰氟和N -对甲苯磺酰苯丙氨酸氯甲基酮可完全抑制促凝活性。本研究表明，埃及眼镜蛇的凝血因子X激活剂虽然在许多方面与蝰蛇科和响尾蛇科毒液的凝血因子X激活剂不同，但具有一些与眼镜蛇科毒液的凝血因子X激活剂相同的特性。
