Kornberg A, Francis C W, Marder V J
Department of Medicine, University of Rochester School of Medicine and Dentistry, NY.
Blood. 1992 Aug 1;80(3):709-17.
Plasma crosslinked fibrin polymers (XLFP) are formed as a result of in vivo hemostatic activation and are elevated in thrombotic disease. We have investigated the plasmic degradation of plasma XLFP in vitro to provide information regarding the pattern of crosslinking and the composition of degradation products. Plasma XLFP were identified by sodium dodecyl sulfate (SDS)-agarose electrophoresis and Western blotting and quantitated by gel scanning. D-dimer was measured by enzyme-linked immunosorbent assay and the results were verified by SDS-polyacrylamide gel electrophoresis and Western blotting of the digests. Complete degradation of XLFP occurred only after supplementation of plasma with plasminogen (5 U/mL) and incubation with recombinant tissue plasminogen activator (rt-PA), indicating that the normal plasma plasminogen concentration limits plasmic degradation in vitro. Gel electrophoresis showed that the principal terminal degradation products of XLDP were fragments D, DD, and E, indicating that crosslinking occurred primarily through gamma chain dimers. After adding a low concentration of thrombin to plasma in vitro, XLFP increased progressively before clotting, and the concentration correlated with the increase in the D-dimer concentration after degradation (r = .98). Plasma XLFP and D-dimer concentrations in plasmic digests were significantly elevated in patients with stroke (150 +/- 83 micrograms/mL and 88 +/- 32 micrograms/mL), myocardial infarction (217 +/- 110 micrograms/mL and 84 +/- 30 micrograms/mL), and venous thrombosis (187 +/- 80 micrograms/mL and 86 +/- 19 micrograms/mL) compared with normals (28 +/- 12 micrograms/mL and 25 +/- 7 micrograms/mL). There was a strong correlation between the plasma concentration of XLFP and the D-dimer immunoreactivity of plasma after plasmic degradation (r = .87). The results indicate that XLFP in plasma are crosslinked primarily through gamma chains and degrade to fragment DD with plasminogen activation. Also, the immunoreactivity of in vitro plasmic digests of plasma reflects the concentration of XLFP and may provide a useful indirect measure of in vivo hemostatic activation in patients with thrombotic disease.
血浆交联纤维蛋白聚合物（XLFP）是体内止血激活的产物，在血栓性疾病中会升高。我们研究了血浆XLFP在体外的血浆降解情况，以提供有关交联模式和降解产物组成的信息。通过十二烷基硫酸钠（SDS）-琼脂糖电泳和蛋白质印迹法鉴定血浆XLFP，并通过凝胶扫描进行定量。通过酶联免疫吸附测定法测量D-二聚体，并通过对消化产物的SDS-聚丙烯酰胺凝胶电泳和蛋白质印迹法验证结果。仅在向血浆中补充纤溶酶原（5 U/mL）并与重组组织型纤溶酶原激活剂（rt-PA）孵育后，XLFP才会完全降解，这表明正常血浆纤溶酶原浓度限制了体外的血浆降解。凝胶电泳显示，XLDP的主要末端降解产物是片段D、DD和E，表明交联主要通过γ链二聚体发生。在体外向血浆中加入低浓度凝血酶后，XLFP在凝血前逐渐增加，且该浓度与降解后D-二聚体浓度的增加相关（r = 0.98）。与正常人（28±12μg/mL和25±7μg/mL）相比，中风患者（150±83μg/mL和88±32μg/mL）、心肌梗死患者（217±110μg/mL和84±30μg/mL）和静脉血栓形成患者（187±80μg/mL和86±19μg/mL）血浆消化液中的血浆XLFP和D-二聚体浓度显著升高。血浆中XLFP的浓度与血浆降解后D-二聚体免疫反应性之间存在很强的相关性（r = 0.87）。结果表明，血浆中的XLFP主要通过γ链交联，并随着纤溶酶原激活降解为片段DD。此外，血浆体外消化液的免疫反应性反映了XLFP的浓度，并可能为血栓性疾病患者体内止血激活提供有用的间接测量方法。
