Bajzar L, Nesheim M
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
J Biol Chem. 1993 Apr 25;268(12):8608-16.
The effect of human activated protein C (APC) on tissue plasminogen activator (tPA)-induced fibrinolysis was studied in cell free plasma and in a system of purified components. Clots were produced by adding plasma or a solution of fibrinogen and plasminogen to the wells of a microtiter plate containing small separated aliquots of Ca2+, thrombin, and tPA, plus and minus APC. Initial clotting and subsequent fibrinolysis were monitored continuously by turbidity. The lysis time of dialyzed normal human plasma (NHP) was longer than that of dialyzed barium citrate-adsorbed plasma (BAP). APC had no effect on the lysis time of BAP but shortened the lysis time of NHP to that of BAP. Two fractions were produced from material eluted from the barium citrate pellet by precipitation of selective components with polyethylene glycol 8000 (PEG). One fraction comprised materials which precipitated at 5% PEG (5% PF) and the other materials which precipitated between 5 and 40% PEG (5-40% PF). Both fractions together, but neither alone, prolonged the lysis time of BAP, an effect which could be reversed by APC. Fractionation of the 5% PF showed that the component with the required activity has properties of the procoagulant surface and can be replaced with vesicles of phosphatidylcholine/phosphatidylserine (PCPS). In addition, the 5-40% PF can be replaced with either the combination of purified coagulation Factors II, IX, and X or Factor II plus the prothrombin activator Factor Xa. When Factor Xa was used as the activator in BAP plus PSPC vesicles, a dose-dependent saturable increase in lysis time was observed with a half-maximal increase occurring at 32 pM Factor Xa. This effect was eliminated by APC. In a system of purified components comprising PCPS vesicles, Factors V and II, protein S, plasminogen and fibrinogen; the prothrombin activators Factor Xa and ecarin both induced a prolongation of the lysis time. APC prevented prolongation by Factor Xa but not by ecarin. The time courses of the generation of thrombin and plasmin during fibrinolysis of clots produced from systems of purified components in the presence and absence of APC, and with Factor Xa as the prothrombin activator, were determined by standardized activity assays using chromogenic substrates. In the absence of APC the lysis time was 145 min, and prothrombin was quantitatively converted to thrombin. In the presence of APC, however, the lysis time was reduced to 100 min with no evidence for the activation of prothrombin.(ABSTRACT TRUNCATED AT 400 WORDS)
在无细胞血浆和纯化成分系统中研究了人活化蛋白C（APC）对组织型纤溶酶原激活剂（tPA）诱导的纤维蛋白溶解的影响。通过将血浆或纤维蛋白原与纤溶酶原溶液加入到含有小份分离的Ca2+、凝血酶和tPA（加或不加APC）的微量滴定板孔中来形成凝块。通过浊度连续监测初始凝血和随后的纤维蛋白溶解。透析正常人血浆（NHP）的溶解时间比透析柠檬酸钡吸附血浆（BAP）的长。APC对BAP的溶解时间没有影响，但将NHP的溶解时间缩短至BAP的溶解时间。通过用聚乙二醇8000（PEG）沉淀选择性成分从柠檬酸钡沉淀中洗脱的物质产生了两个级分。一个级分包含在5% PEG沉淀的物质（5% PF），另一个包含在5%至40% PEG之间沉淀的物质（5 - 40% PF）。两个级分一起，但单独一个都不能延长BAP的溶解时间，APC可以逆转这种作用。对5% PF的分级分离表明，具有所需活性的成分具有促凝表面的特性，可以用磷脂酰胆碱/磷脂酰丝氨酸（PCPS）囊泡替代。此外，5 - 40% PF可以用纯化的凝血因子II、IX和X的组合或因子II加凝血酶原激活剂因子Xa替代。当因子Xa用作BAP加PSPC囊泡中的激活剂时，观察到溶解时间呈剂量依赖性饱和增加，在32 pM因子Xa时出现半数最大增加。APC消除了这种作用。在包含PCPS囊泡、因子V和II、蛋白S、纤溶酶原和纤维蛋白原的纯化成分系统中；凝血酶原激活剂因子Xa和蛇毒凝血酶都诱导溶解时间延长。APC阻止因子Xa引起的延长，但不阻止蛇毒凝血酶引起的延长。在有和没有APC的情况下，以因子Xa作为凝血酶原激活剂，通过使用显色底物的标准化活性测定来确定由纯化成分系统产生的凝块在纤维蛋白溶解过程中凝血酶和纤溶酶生成的时间进程。在没有APC的情况下，溶解时间为145分钟，凝血酶原定量转化为凝血酶。然而，在有APC的情况下，溶解时间减少到100分钟，没有凝血酶原激活的证据。（摘要截断于400字）
