Cleavage of human high molecular weight kininogen markedly enhances its coagulant activity. Evidence that this molecule exists as a procofactor.
Cleavage of human high molecular weight kininogen markedly enhances its coagulant activity. Evidence that this molecule exists as a procofactor.
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人高分子量激肽原的裂解显着增强其凝血活性。
DOI：
10.1172/jci111319
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Colman,RW
中科院分区：
文献类型：
--
作者：
Scott,CF;Silver,LD;Schapira,M;Colman,RW
高分子量激肽原（HMW）-激肽原，接触活化凝血的辅因子，加速带负电荷表面上因子XII、前激肽释放酶和因子XI的活化。尽管前激肽释放酶和因子XI作为与HMW-激肽原的复合物循环，但在因子XII和HMW-激肽原之间没有证明物理缔合，也没有完全阐明这些蛋白质表面的吸附顺序。在这份报告中，我们探讨的要求吸附HMW-激肽原凝块促进表面（高岭土），在纯化的系统，以及在正常血浆和血浆遗传缺陷的接触系统的每一种蛋白质。通过测量初始样品与高岭土孵育后上清液之间的凝血活性差异，或通过直接定量与高岭土沉淀物相关的125 I-HMW-激肽原的量，测定与高岭土沉淀物相关的每种凝血蛋白的分数。在正常血浆中，随着孵育混合物中高岭土量的增加，高分子量激肽原对高岭土的吸附增加。然而，HMW-激肽原因子XII缺乏血浆中没有明显吸收高岭土。此外，与正常血浆相比，从前激肽释放酶缺乏血浆吸附到高岭土的HMW-激肽原的量减少。这些观察结果表明，血浆中的HMW-激肽原必须通过涉及因子XII和前激肽释放酶的反应来改变，以使HMW-激肽原吸附到高岭土上，并表达其凝血活性。随后，HMW-激肽原不能与带负电荷的表面缔合的结果导致表面活化降低。该评估来源于进一步观察到缺乏前激肽释放酶吸附和因子XII缺乏和HMW-激肽原缺乏血浆中因子XI吸附减少，因为这两种酶原（前激肽释放酶和因子XI）被转运至与HMW-激肽原复合的带负电荷表面。高岭土对高分子量激肽原的吸附量与高分子量激肽原的吸附量呈显著正相关（r = 0.98，slope = 0.97），表明高分子量激肽原的吸附导致其凝血活性的表达。由于激肽释放酶，这是已知的切割HMW-激肽原，产生时，高岭土被添加到血浆中，我们测试的假设，蛋白水解的激肽释放酶是负责的HMW-激肽原的高岭土的吸附增强。当纯化的HMW-激肽原与纯化的激肽释放酶一起孵育时，其吸收高岭土的能力随着消化时间的增加而增加，直到达到最大值。此外，（125）I-HMW-激肽原，经激肽释放酶裂解后，与未裂解的起始材料相比，对高岭土的亲和力显著增加。此外，纤维蛋白原，在血浆浓度（3毫克/毫升），显着减少的混合物的切割和未切割的HMW-激肽原吸附高岭土，但不能阻止完全切割的HMW-激肽原吸附到高岭土。将纯化的激肽释放酶添加到因子XII缺陷血浆中，其绕过因子XII依赖性接触激活，放大了其HMW-激肽原吸附到高岭土上的能力。这些观察结果表明，HMW-激肽原是一种由激肽释放酶激活的前辅因子，激肽释放酶是它加速的反应产物。这种裂解增强了其与血浆环境中促凝表面的结合，是表达其辅因子活性所必需的事件。这些相互作用将允许HMW的协调。
High molecular weight kininogen (HMW)-kininogen, the cofactor of contact-activated blood coagulation, accelerates the activation of Factor XII, prekallikrein, and Factor XI on a negatively charged surface. Although prekallikrein and Factor XI circulate as a complex with HMW-kininogen, no physical association has been demonstrated between Factor XII and HMW-kininogen, nor has the order of adsorption to surfaces of these proteins been fully clarified. In this report we explore the requirements for adsorption of HMW-kininogen to a clot-promoting surface (kaolin), in purified systems, as well as in normal plasma and plasma genetically deficient in each of the proteins of the contact system. The fraction of each coagulant protein associated with the kaolin pellet was determined by measuring the difference in coagulant activity between the initial sample and supernatants after incubation with kaolin, or by directly quantifying the amount of 125I-HMW-kininogen that was associated with the kaolin pellet. In normal plasma, the adsorption of HMW-kininogen to kaolin increased as the quantity of kaolin was increased in the incubation mixture. However, the HMW-kininogen in Factor XII-deficient plasma did not absorb appreciably to kaolin. Furthermore, the quantity of HMW-kininogen from prekallikrein-deficient plasma that adsorbed to kaolin was decreased as compared with normal plasma. These observations suggested that HMW-kininogen in plasma must be altered by a reaction involving both Factor XII and prekallikrein in order for HMW-kininogen to adsorb to kaolin, and to express its coagulant activity. Subsequently, the consequence of the inability of HMW-kininogen to associate with a negatively charged surface results in decreased surface activation. This assessment was derived from the further observation of the lack of prekallikrein adsorption and the diminished Factor XI adsorption in both Factor XII-deficient and HMW-kininogen-deficient plasmas, since these two zymogens (prekallikrein and Factor XI) are transported to a negatively charged surface in complex with HMW-kininogen. The percentage of HMW-kininogen coagulant activity that adsorbed to kaolin closely correlated (r = 0.98, slope = 0.97) with the amount of 125I-HMW-kininogen adsorbed, suggesting that adsorption of HMW-kininogen results in the expression of its coagulant activity. Since kallikrein, which is known to cleave HMW-kininogen, is generated when kaolin is added to plasma, we tested the hypothesis that proteolysis by kallikrein was responsible for the enhanced adsorption of HMW-kininogen to kaolin. When purified HMW-kininogen was incubated with purified kallikrein, its ability to absorb to kaolin increased with time of digestion until a maximum was reached. Moreover, (125)I-HMW-kininogen, after cleavage by kallikrein, had markedly increased affinity for kaolin than the uncleaved starting material. Furthermore, fibrinogen, at plasma concentration (3 mg/ml), markedly curtailed the adsorption of a mixture of cleaved and uncleaved HMW-kininogen to kaolin, but was unable to prevent fully cleaved HMW-kininogen from adsorbing to the kaolin. Addition of purified kallikrein to Factor XII-deficient plasma, which bypasses Factor XII-dependent contact-activation amplified the ability of its HMW-kininogen to adsorb to kaolin. These observations indicate that HMW-kininogen is a procofactor that is activated by kallikrein, a product of a reaction which it accelerates. This cleavage, which enhances its association with a clot-promoting surface in a plasma environment, is an event that is necessary for expression of its cofactor activity. These interactions would allow coordination of HMW …