Role of exosites 1 and 2 in thrombin reaction with plasminogen activator inhibitor-1 in the absence and presence of cofactors.
Role of exosites 1 and 2 in thrombin reaction with plasminogen activator inhibitor-1 in the absence and presence of cofactors.
复制标题
在辅因子不存在和存在的情况下，外位点 1 和 2 在凝血酶与纤溶酶原激活剂抑制剂 1 的反应中的作用。
DOI：
10.1021/bi9913303
复制
发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Rezaie,AR
中科院分区：
文献类型：
--
作者：
Rezaie,AR
辅因子肝素、玻连蛋白（VN）和血栓调节蛋白（TM）调节α-凝血酶与纤溶酶原激活物抑制剂（派-1）的反应性。虽然肝素和VN使反应加速约102个数量级，但TM在VN存在下保护α-凝血酶免于派-1的快速失活。为了理解这些辅因子如何起作用，我们研究了在不存在和存在这些辅因子的情况下，α-凝血酶、外位点1变体γ-凝血酶、外位点2突变体R93、97、101 A凝血酶和重组甲藻凝血酶的派-1失活动力学。肝素和VN使α-凝血酶与派-1的二级结合速率常数[k2=（7.9 ± 0.5）× 102 M ~（-1）s ~（-1）]分别增加了200倍和240倍。γ-凝血酶的k2值[（7.9 ± 0.7）× 101 M ~（-1）s ~（-1）]降低了10倍，而肝素和VN分别使k2值提高了10 ~ 280倍和10 ~ 75倍。与γ-凝血酶的失活类似，与TM的表皮生长因子样结构域4−6（TM 4 −6）复合的α-凝血酶的派-1失活受损约10倍。外位点2突变体R93，97，101 A凝血酶（先前显示不与肝素结合）和美佐凝血酶（其中外位点2被掩蔽）在不存在和存在肝素的情况下以相似的速率与派-1反应[对于R93，97，101 A凝血酶，k2=（1.3−1.5）× 103 M-1 s-1;对于美佐凝血酶，k2 =（3.6−5.1）× 102 M-1 s-1]。然而，与肝素不同的是，VN使R93、97、101 A凝血酶和甲藻凝血酶的k2分别增加了80倍和30倍。连续动力学分析以及在S195 A凝血酶的存在下的竞争动力学研究表明，VN或肝素的加速作用主要是通过降低形成非共价米氏型复合物的解离常数（Kd）来发生的。分析这些结果表明：（1）肝素通过模板机制与α-凝血酶的外部位点2结合以加速反应，（2）VN通过降低初始复合物形成的Kd来加速α-凝血酶的派-1失活，该机制未知，不需要与α-凝血酶的外部位点1或外部位点2结合，（3）α-凝血酶可能在外位点1内或附近具有派-1的结合位点，（4）外位点1的TM占据部分地解释了在玻连蛋白存在下凝血酶免受派-1快速失活的保护。
The cofactors heparin, vitronectin (VN), and thrombomodulin (TM) modulate the reactivity of α-thrombin with plasminogen activator inhibitor (PAI-1). While heparin and VN accelerate the reaction by ∼2 orders of magnitude, TM protects α-thrombin from rapid inactivation by PAI-1 in the presence of VN. To understand how these cofactors function, we studied the kinetics of PAI-1 inactivation of α-thrombin, the exosite 1 variant γ-thrombin, the exosite 2 mutant R93,97,101A thrombin, and recombinant meizothrombin in both the absence and presence of these cofactors. Heparin and VN accelerated the second-order association rate constant [k2= (7.9 ± 0.5) × 102M-1s-1] of α-thrombin with PAI-1 ∼200- and ∼240-fold, respectively. Thek2value for γ-thrombin [(7.9 ± 0.7) × 101M-1s-1] was impaired 10-fold, but was enhanced by heparin and VN ∼280- and ∼75-fold, respectively. Similar to inactivation of γ-thrombin, PAI-1 inactivation of α-thrombin in complex with the epidermal growth factor-like domains 4−6 of TM (TM4−6) was impaired ∼10-fold. The exosite 2 mutant R93,97,101A thrombin, which was previously shown not to bind heparin, and meizothrombin, in which exosite 2 is masked, reacted with PAI-1 at similar rates in both the absence and presence of heparin [k2= (1.3−1.5) × 103M-1s-1for R93,97,101A thrombin andk2= (3.6−5.1) × 102M-1s-1for meizothrombin]. Unlike heparin, however, VN enhanced thek2of R93,97,101A thrombin and meizothrombin inactivation ∼80- and ∼30-fold, respectively. Continuous kinetic analysis as well as competition kinetic studies in the presence of S195A thrombin suggested that the accelerating effect of VN or heparin occurs primarily by lowering the dissociation constant (Kd) for formation of a noncovalent, Michaelis-type complex. Analysis of these results suggest that (1) heparin binds to exosite 2 of α-thrombin to accelerate the reaction by a template mechanism, (2) VN accelerates PAI-1 inactivation of α-thrombin by lowering theKdfor initial complex formation by an unknown mechanism that does not require binding to either exosite 1 or exosite 2 of α-thrombin, (3) α-thrombin may have a binding site for PAI-1 within or near exosite 1, and (4) TM occupancy of exosite 1 partially accounts for the protection of thrombin from rapid inactivation by PAI-1 in the presence of vitronectin.