PROTEOLYTIC PROCESSING OF HUMAN FACTOR-VIII - CORRELATION OF SPECIFIC CLEAVAGES BY THROMBIN, FACTOR XA, AND ACTIVATED PROTEIN-C WITH ACTIVATION AND INACTIVATION OF FACTOR-VIII COAGULANT ACTIVITY
PROTEOLYTIC PROCESSING OF HUMAN FACTOR-VIII - CORRELATION OF SPECIFIC CLEAVAGES BY THROMBIN, FACTOR XA, AND ACTIVATED PROTEIN-C WITH ACTIVATION AND INACTIVATION OF FACTOR-VIII COAGULANT ACTIVITY
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DOI：
10.1021/bi00350a035
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发表时间：
1986-01-28
期刊：
影响因子：
2.9
通讯作者：
VEHAR, GA
中科院分区：
文献类型：
--
作者：
EATON, D;RODRIGUEZ, H;VEHAR, GA
人因子VIII是从商业因子VIII浓缩物中分离出来的，发现它由多个分子量在8万到21万之间的多肽组成。免疫学和氨基酸序列数据确定这些多肽是因子VIII的亚基。N端氨基酸序列分析确定Mr 210,000和80,000蛋白分别来自因子VIII的N端和c端部分；Mr 90,000-180,000多肽由Mr 210,000多肽通过c -末端切割得到。用凝血酶处理纯化的VIII因子导致Mr 80,000-210,000蛋白的蛋白水解，并产生Mr 73,000, 50,000和43,000的多肽。凝血酶活化因子VIII的最大凝血活性与这些多肽的产生有关。凝血酶的蛋白水解和因子VIII的活化被发现明显依赖于CaCl2浓度。因子VIII与活化蛋白C （APC）的蛋白水解导致Mr 90,000-210,000蛋白的降解，并产生Mr 45,000片段。这种分裂与APC使因子VIII失活有关。Mr 80000蛋白未被APC降解。因子Xa切割Mr为80,000-210,000的因子VIII蛋白，产生Mr为73,000、67,000、50,000、45,000和43,000的片段。发现因子Xa最初激活因子VIII，随后使其失活。Xa因子的激活与Mr 73,000, 50,000和43,000多肽的产生相关，而失活与Mr 73,000和50,000多肽分别裂解为Mr 67,000和45,000片段相关。凝血酶因子VIII、因子Xa和APC的裂解位点通过对这些蛋白酶裂解因子VIII后产生的片段进行氨基酸测序确定。有趣的是，Xa因子被发现与APC和凝血酶在相同的位点切割因子VIII。这也许可以解释为什么Xa因子激活和灭活因子VIII。
Human factor VIII was isolated from commercial factor VIII concentrates and found to consist of multiple polypeptides with molecular weights ranging from 80,000 to 210,000. Immunological and amino acid sequence data identified these polypeptides as subunits of factor VIII. N-Terminal amino acid sequence analysis determined that the Mr 210,000 and 80,000 proteins are derived from the N- and C-terminal portions of factor VIII, respectively; Mr 90,000-180,000 polypeptides are derived from the Mr 210,000 polypeptide by C-terminal cleavages. Treatment of purified factor VIII with thrombin resulted in proteolysis of Mr 80,000-210,000 proteins and the generation of polypeptides of Mr 73,000, 50,000, and 43,000. Maximum coagulant activity of thrombin-activated factor VIII was correlated with the generation of these polypeptides. The proteolysis as well as activation of factor VIII by thrombin was found to be markedly dependent on CaCl2 concentration. Proteolysis of factor VIII with activated protein C (APC) resulted in degradation of the Mr 90,000-210,000 proteins with the generation of an Mr 45,000 fragment. This cleavage correlated with inactivation of factor VIII by APC. The Mr 80,000 protein was not degraded by APC. Factor Xa cleaves the Mr 80,000-210,000 factor VIII proteins, resulting in the generation of fragments of Mr 73,000, 67,000, 50,000, 45,000, and 43,000. Factor Xa was found to initially activate and subsequently inactivate factor VIII. Activation by factor Xa correlated with the generation of Mr 73,000, 50,000, and 43,000 polypeptides while inactivation correlated with the cleavage of Mr 73,000 and 50,000 polypeptides to fragments of Mr 67,000 and 45,000, respectively. The cleavage sites in factor VIII of thrombin, factor Xa, and APC were identified by amino acid sequencing of the fragments generated after cleavage of factor VIII by these proteases. Interestingly, factor Xa was found to cleave factor VIII at the same sites as APC and thrombin. This may explain why factor Xa activates as well as inactivates factor VIII.