凝血因子 VIII 的膜结合动力学表明这是一个复杂的结合过程。

Membrane binding kinetics of factor VIII indicate a complex binding process.

作者信息

Bardelle C, Furie B, Furie B C, Gilbert G E

机构信息

Center for Hemostasis and Thrombosis Research, New England Medical Center, Boston, Massachusetts.

出版信息

J Biol Chem. 1993 Apr 25;268(12):8815-24.

PMID:8473326

Abstract

Factor VIII functions as a component of the tenase enzyme complex upon phospholipid membranes. Factor VIII binds to phosphatidylserine-containing membranes and apparently provides high affinity binding sites for factor IXa upon these membranes. We have characterized the binding kinetics of human factor VIII with phosphatidylserine-containing membranes and directly compared the measured properties with those of factor V. The initial phase of association was evaluated in a stopped-flow apparatus by fluorescence energy transfer from aromatic residues in the protein to dansyl-labeled phosphatidylethanolamine in the vesicles. Association proceeded at an apparent second-order rate of 0.12 microM-1 s-1 for extruded phospholipid vesicles and 0.42 microM-1 s-1 for sonicated vesicles under pseudo-first-order conditions in which the phospholipid concentration determined the rate. Increased temperature resulted in more rapid association, and the effect decreased in the order extruded vesicles > sonicated vesicles > extruded vesicles of dioleoylphospholipids, indicating that the structure of the phospholipid membrane contributes to the activation energy of binding. The binding of fluorescein-labeled factor VIII to membranes supported on glass microspheres (lipospheres) was monitored by flow cytometry. Under conditions in which the factor VIII concentration determined the rate there was rapid initial association at 6.9 microM-1 s-1, accounting for half of the bound factor VIII, and a slower component of 0.87 microM-1 s-1, accounting for the other half. Likewise, the dissociation of factor VIII from liposphere membranes was biphasic with a faster component of 0.010 s-1 and a slower component of 0.0012 s-1. Rates of association and dissociation for factor V were similar to those for factor VIII and were biphasic. These results allow estimation of the size of the phospholipid sites that interact with factors VIII and V and suggest that both proteins bind to membranes via a multistep process in which rapid association is followed by a slower step yielding higher affinity binding.

摘要

凝血因子 VIII 在磷脂膜上作为凝血酶原酶复合物的一个组成部分发挥作用。凝血因子 VIII 与含磷脂酰丝氨酸的膜结合，显然在这些膜上为凝血因子 IXa 提供高亲和力结合位点。我们已对人凝血因子 VIII 与含磷脂酰丝氨酸的膜的结合动力学进行了表征，并将测得的特性与凝血因子 V 的特性直接进行了比较。结合的初始阶段在停流装置中通过从蛋白质中的芳香族残基到囊泡中丹磺酰标记的磷脂酰乙醇胺的荧光能量转移来评估。在伪一级条件下，对于挤出的磷脂囊泡，结合以 0.12 μM⁻¹ s⁻¹ 的表观二级速率进行，对于超声处理的囊泡，结合速率为 0.42 μM⁻¹ s⁻¹，其中磷脂浓度决定速率。温度升高导致结合更快，其影响程度按挤出囊泡>超声处理囊泡>二油酰磷脂挤出囊泡的顺序降低，表明磷脂膜的结构有助于结合的活化能。通过流式细胞术监测荧光素标记的凝血因子 VIII 与玻璃微球（脂质球）上支持的膜的结合。在凝血因子 VIII 浓度决定速率的条件下，初始结合迅速，速率为 6.9 μM⁻¹ s⁻¹，占结合的凝血因子 VIII 的一半，较慢的组分速率为 0.87 μM⁻¹ s⁻¹，占另一半。同样，凝血因子 VIII 从脂质球膜上的解离是双相的，较快的组分为 0.010 s⁻¹，较慢的组分为 0.0012 s⁻¹。凝血因子 V 的结合和解离速率与凝血因子 VIII 相似，也是双相的。这些结果有助于估计与凝血因子 VIII 和 V 相互作用的磷脂位点的大小，并表明这两种蛋白质都通过多步过程与膜结合，其中快速结合之后是较慢的步骤，产生更高亲和力的结合。