血浆蛋白C抑制剂的纯化与特性鉴定

Purification and characterization of plasma protein C inhibitor.

作者信息

España F, Berrettini M, Griffin J H

机构信息

Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA 92037.

出版信息

Thromb Res. 1989 Aug 1;55(3):369-84. doi: 10.1016/0049-3848(89)90069-8.

DOI:10.1016/0049-3848(89)90069-8

PMID:2551064

Abstract

Plasma protein C inhibitor (PCI) was purified to homogeneity (greater than 95%) with good recovery (greater than 25%) and reproducibility, and the inhibition of a number of blood clotting and fibrinolytic enzymes by purified PCI was studied. PCI inhibited activated protein C (APC), two-chain urokinase (2c-uPA), two-chain tissue plasminogen activator (2c-tPA), thrombin, factor Xa, plasma kallikrein and factor XIa, and this inhibition was accelerated by heparin. The inhibition of each enzyme was accompanied by formation of enzyme inhibitor complexes and by degradation of the inhibitor to lower molecular weight derivatives. Plasma kallikrein and factor XIa cleaved PCI of native Mr = 57,000 into two products with Mr = 54,000 and 52,000 whereas the other enzymes converted the PCI to a product with Mr = 54,000. PCI did not detectably inhibit alpha-factor XIIa or plasmin. Kinetic studies using PCI yielded the following second-order rate constants for inhibition of human APC, 2c-uPA, 2c-tPA, thrombin, factor Xa, kallikrein and factor XIa respectively: 0.65 x 10(4), 0.22 x 10(4), 0.08 x 10(4), 0.61 x 10(4), 2.01 x 10(4), 6.50 x 10(4), and 9.03 x 10(4) M-1s-1 in the absence of heparin and 1.58 x 10(6), 0.43 x 10(6), 0.03 x 10(6), 0.52 x 10(6), 0.09 x 10(6), 0.18 x 10(6) and 0.74 x 10(6) M-1s-1 in the presence of optimal concentrations of heparin. The rate constants for the inhibition of factor XIa and 2c-uPA by PCI suggest a possible role of PCI in the physiologic regulation of these enzymes. The second order rate constants for inhibition of bovine APC and Gla-domainless bovine APC by human PCI were 0.61 x 10(4) and 0.26 x 10(4) M-1s-1 in the absence of heparin and 0.54 x 10(6) and 0.71 x 10(6) M-1s-1 in the presence of heparin, respectively. Calcium ions (0.05 to 4 mM) did not affect these rate constants. The results obtained with normal and Gla-domainless APC indicate that the Gla domain of APC is not required for inactivation by PGI and is not essential for the heparin stimulation of this reaction.

摘要

血浆蛋白C抑制剂（PCI）被纯化至均一性（大于95%），回收率良好（大于25%）且具有可重复性，并研究了纯化后的PCI对多种凝血和纤溶酶的抑制作用。PCI抑制活化蛋白C（APC）、双链尿激酶（2c - uPA）、双链组织型纤溶酶原激活剂（2c - tPA）、凝血酶、因子Xa、血浆激肽释放酶和因子XIa，并且肝素可加速这种抑制作用。对每种酶的抑制均伴随着酶抑制剂复合物的形成以及抑制剂降解为低分子量衍生物。血浆激肽释放酶和因子XIa将天然Mr = 57,000的PCI裂解为Mr = 54,000和52,000的两种产物，而其他酶则将PCI转化为Mr = 54,000的产物。PCI未检测到对α - 因子XIIa或纤溶酶的抑制作用。使用PCI进行的动力学研究得出了以下分别针对人APC、2c - uPA、2c - tPA、凝血酶、因子Xa、激肽释放酶和因子XIa抑制作用的二级速率常数：在无肝素时分别为0.65×10⁴、0.22×10⁴、0.08×10⁴、0.61×10⁴、2.01×10⁴、6.50×10⁴和9.03×10⁴ M⁻¹s⁻¹，在存在最佳浓度肝素时分别为1.58×10⁶、0.43×10⁶、0.03×10⁶、0.52×10⁶、0.09×10⁶、0.18×10⁶和0.74×10⁶ M⁻¹s⁻¹。PCI对因子XIa和2c - uPA抑制作用的速率常数表明PCI在这些酶的生理调节中可能发挥作用。人PCI对牛APC和无Gla结构域的牛APC抑制作用的二级速率常数在无肝素时分别为0.61×10⁴和0.26×10⁴ M⁻¹s⁻¹，在有肝素时分别为0.54×10⁶和0.71×10⁶ M⁻¹s⁻¹。钙离子（0.05至4 mM）不影响这些速率常数。用正常APC和无Gla结构域的APC获得的结果表明，APC的Gla结构域对于被PGI灭活不是必需的，并且对于肝素刺激此反应也不是必需的。