Huang Xin, Liu Baoxin, Wei Yidong, Beyea Ryan, Yan Han, Olson Steven T
From the Center for Molecular Biology of Oral Diseases and Department of Periodontics, University of Illinois at Chicago, Chicago, Illinois 60612 and
the Departments of Cardiology and.
J Biol Chem. 2017 Sep 1;292(35):14625-14635. doi: 10.1074/jbc.M117.793901. Epub 2017 Jul 17.
Lipid oxidation due to oxidative stress plays an important role in the pathogenesis of inflammatory and thrombotic cardiovascular diseases. Several findings suggest that lipid peroxidation can alter the function of coagulation proteins and contribute to a hypercoagulable state, but the molecular mechanisms are unclear. Here, we report that oxidized phospholipids suppress the anticoagulant function of the serpin, protein Z-dependent protease inhibitor (ZPI), a specific inhibitor of membrane-associated factor Xa (FXa) that requires protein Z (PZ), phospholipid, and calcium as cofactors. We found that this suppression arises from a diminished ability of the oxidized membrane to function as a cofactor to promote ZPI inhibition of membrane-bound FXa, due fully or in part to the susceptibility of the bound ZPI-PZ complex to oxidative inactivation. Surprisingly, free ZPI was also susceptible to inactivation by oxidized membrane vesicles in the absence of calcium. Oxidized vesicles containing both phosphatidylserine and polyunsaturated fatty acids were required to promote inactivation of the ZPI-PZ complex or free ZPI, indicating that binding of the PZ-complexed or free ZPI to peroxide-modified phospholipid vesicles mediates the inactivation. Heparin protected the ZPI-PZ complex and free ZPI from inactivation, suggesting that blocking the heparin-binding site on ZPI interferes with ZPI binding to lipid or to PZ. This was confirmed by direct lipid-binding experiments. Native PAGE indicated that oxidization induced dissociation of the ZPI-PZ complex and increased the negative charge of ZPI. We conclude that compromised ZPI anticoagulant function could contribute to thrombus initiation and growth in oxidative stress-induced cardiovascular diseases.
氧化应激引起的脂质氧化在炎症性和血栓性心血管疾病的发病机制中起重要作用。多项研究结果表明，脂质过氧化可改变凝血蛋白的功能并导致高凝状态，但其分子机制尚不清楚。在此，我们报告氧化磷脂抑制丝氨酸蛋白酶抑制剂、蛋白Z依赖性蛋白酶抑制剂（ZPI）的抗凝功能，ZPI是膜相关因子Xa（FXa）的特异性抑制剂，它需要蛋白Z（PZ）、磷脂和钙作为辅因子。我们发现这种抑制源于氧化膜作为辅因子促进ZPI抑制膜结合FXa的能力减弱，这完全或部分归因于结合的ZPI-PZ复合物对氧化失活的敏感性。令人惊讶的是，在没有钙的情况下，游离ZPI也易被氧化膜囊泡失活。含有磷脂酰丝氨酸和多不饱和脂肪酸的氧化囊泡是促进ZPI-PZ复合物或游离ZPI失活所必需的，这表明PZ复合的或游离的ZPI与过氧化物修饰的磷脂囊泡的结合介导了失活。肝素可保护ZPI-PZ复合物和游离ZPI不被失活，这表明阻断ZPI上的肝素结合位点会干扰ZPI与脂质或PZ的结合。直接脂质结合实验证实了这一点。非变性聚丙烯酰胺凝胶电泳表明氧化诱导ZPI-PZ复合物解离并增加了ZPI的负电荷。我们得出结论，ZPI抗凝功能受损可能导致氧化应激诱导的心血管疾病中血栓的起始和生长。
