Marciniak E, Gora-Maslak G
Thromb Haemost. 1984 Feb 28;51(1):27-31.
Interaction of human antithrombin III (AT III) with human alpha-thrombin coupled to Sepharose 4B was investigated. Despite markedly reduced esterolytic, amidolytic and especially coagulant activity, more than 90% of immobilized thrombin formed stable complexes with purified AT III. Presence of high affinity heparin did not facilitate the inhibition to the degree seen in reactions conducted with soluble thrombin. Instead, heparin induced proteolysis of up to 66% of the inhibitor that remained in solution. This led to the isolation of a homogeneous protein fragment which migrated in SDS-gel electrophoresis as a band of 50,000 Mr, cross-reacted with antibodies to human AT III but showed no biologic activity nor sufficient affinity for heparin. Out of the three major inhibitors capable of binding soluble thrombin in human plasma, only AT III reacted with immobilized thrombin. However, Sepharose-coupled thrombin mixed with plasma in the presence of heparin produced outstanding quantities of residual immunoreactive AT III devoid of inhibitory activity. These data suggest that presence of high affinity heparin in the environment of thrombin attached to a solid support may dramatically decrease the efficiency of enzyme inhibition.
研究了人抗凝血酶III（AT III）与偶联到琼脂糖4B上的人α-凝血酶之间的相互作用。尽管固定化凝血酶的酯解、酰胺解尤其是凝血活性显著降低，但超过90%的固定化凝血酶与纯化的AT III形成了稳定的复合物。高亲和力肝素的存在并未促进抑制作用达到与可溶性凝血酶反应时所观察到的程度。相反，肝素诱导溶液中高达66%的抑制剂发生蛋白水解。这导致分离出一种均一的蛋白质片段，其在SDS凝胶电泳中迁移为50,000 Mr的条带，与人AT III抗体发生交叉反应，但没有生物学活性，对肝素也没有足够的亲和力。在能够结合人血浆中可溶性凝血酶的三种主要抑制剂中，只有AT III与固定化凝血酶发生反应。然而，在肝素存在下，与血浆混合的琼脂糖偶联凝血酶产生了大量无抑制活性但仍具有免疫反应性的残留AT III。这些数据表明，在附着于固体支持物的凝血酶环境中高亲和力肝素的存在可能会显著降低酶抑制的效率。
