Sauls Derrick L, Lockhart Evelyn, Warren Maria Esteban, Lenkowski Angela, Wilhelm Susan E, Hoffman Maureane
Research Service, Durham Veteran's Affairs Medical Center, 508 Fulton Street, Durham, North Carolina 27705, USA.
Biochemistry. 2006 Feb 28;45(8):2480-7. doi: 10.1021/bi052076j.
We have previously shown functional differences in fibrinogen from hyperhomocysteinemic rabbits compared to that in control rabbits. This acquired dysfibrinogenemia is characterized by fibrin clots that are composed of abnormally thin, tightly packed fibers with increased resistance to fibrinolysis. Homocysteine thiolactone is a metabolite of homocysteine (Hcys) that can react with primary amines. Recent evidence suggests that Hcys thiolactone-lysine adducts form in vivo. We now demonstrate that the reaction of Hcys thiolactone with purified fibrinogen in vitro produces fibrinogen (Hcys fibrinogen) with functional properties that are strikingly similar to those we have observed in homocysteinemic rabbits. Fibrinogen purified from homocysteinemic rabbits and Hcys fibrinogen are similar in that (1) they both form clots composed of thinner, more tightly packed fibers than their respective control rabbit and human fibrinogens; (2) the clot structure could be made to be more like the control fibrinogens by increased calcium; and (3) they both form clots that are more resistant to fibrinolysis than those formed by the control fibrinogens. Further characterization of human fibrinogens showed that Hcys fibrin had similar plasminogen binding to that of the control and an increased capacity for binding tPA. However, tPA activation of plasminogen on Hcys fibrin was slower than that of the control. Mass spectrometric analysis of Hcys fibrinogen revealed twelve lysines that were homocysteinylated. Several of these are close to tPA and plasminogen binding sites. Lysines are major binding sites for fibrinolytic enzymes and are also sites of plasmin cleavage. Thus, modification of lysines in fibrinogen could plausibly lead to impaired fibrinolysis. We hypothesize that the modification of lysine by Hcys thiolactone might occur in vivo, lead to abnormal resistance of clots to lysis, and thereby contribute to the prothrombotic state associated with homocysteinemia.
我们之前已经表明，与对照兔相比，高同型半胱氨酸血症兔的纤维蛋白原存在功能差异。这种获得性异常纤维蛋白原血症的特征是纤维蛋白凝块由异常细、紧密堆积的纤维组成，对纤维蛋白溶解的抵抗力增加。同型半胱氨酸硫内酯是同型半胱氨酸（Hcys）的一种代谢产物，可与伯胺反应。最近的证据表明，Hcys硫内酯-赖氨酸加合物在体内形成。我们现在证明，Hcys硫内酯与纯化的纤维蛋白原在体外反应产生的纤维蛋白原（Hcys纤维蛋白原）具有与我们在高同型半胱氨酸血症兔中观察到的功能特性惊人相似的特性。从高同型半胱氨酸血症兔中纯化的纤维蛋白原和Hcys纤维蛋白原相似之处在于：（1）它们都形成由比各自对照兔和人纤维蛋白原更细、堆积更紧密的纤维组成的凝块；（2）通过增加钙可以使凝块结构更像对照纤维蛋白原；（3）它们形成的凝块比对照纤维蛋白原形成的凝块对纤维蛋白溶解更具抵抗力。对人纤维蛋白原的进一步表征表明，Hcys纤维蛋白与对照纤维蛋白具有相似的纤溶酶原结合能力，且结合组织型纤溶酶原激活物（tPA）的能力增强。然而，tPA对Hcys纤维蛋白上纤溶酶原的激活比对照纤维蛋白慢。对Hcys纤维蛋白原的质谱分析显示有十二个赖氨酸被同型半胱氨酸化。其中几个靠近tPA和纤溶酶原结合位点。赖氨酸是纤维蛋白溶解酶的主要结合位点，也是纤溶酶裂解的位点。因此，纤维蛋白原中赖氨酸的修饰可能会导致纤维蛋白溶解受损。我们假设，Hcys硫内酯对赖氨酸的修饰可能在体内发生，导致凝块对溶解产生异常抵抗力，从而促成与高同型半胱氨酸血症相关的血栓前状态。
