Anonick P K, Yoo J K, Webb D J, Gonias S L
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.
Biochem J. 1993 Feb 1;289 ( Pt 3)(Pt 3):903-9. doi: 10.1042/bj2890903.
These studies demonstrate relatively rapid association of plasmin with thrombospondin and the effects of this interaction on plasmin activity towards D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251) and the proteinase inhibitors alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M). Binding of plasmin to thrombospondin reached an apparent reversible equilibrium within 3 min at 22 degrees C. The amidase activity of bound plasmin was inhibited. An analysis of S-2251 hydrolysis indicated that thrombospondin is a linear mixed-type plasmin inhibitor. The dissociation constant (KD) for the binding of plasmin to thrombospondin was 0.5 microM, assuming one plasmin binding site per thrombospondin homotrimer. Plasmin and miniplasmin slowly cleaved thrombospondin, yielding products which were comparable with those generated by other proteinases. Tranexamic acid inhibited the digestion of thrombospondin by plasmin and miniplasmin, suggesting an important role for the kringle-5 domain in this process. When plasmin was incubated first with thrombospondin and then with alpha 2AP, plasmin that was apparently bound to thrombospondin reacted with alpha 2AP at a decreased rate; however, within 20 min, all of the plasmin was recovered in complex with alpha 2AP. Similar results were obtained with alpha 2M. Transfer of plasmin from thrombospondin to alpha 2AP or alpha 2M probably required plasmin-thrombospondin-complex dissociation. A low level of reaction of alpha 2AP with thrombospondin-associated plasmin could not be ruled out. These results demonstrate that the activity of plasmin, when bound to thrombospondin, is greatly diminished or eliminated. The plasmin-thrombospondin complex, which is formed within 3 min, is fully reversible and the associated plasmin is in a latent form protected from proteinase inhibitors. Therefore, thrombospondin may regulate plasmin activity in a manner which is distinct from conventional proteinase inhibitors and other extracellular-matrix proteins.
这些研究表明纤溶酶与血小板反应蛋白的结合相对迅速，以及这种相互作用对纤溶酶针对盐酸D-缬氨酰-L-亮氨酰-L-赖氨酸对硝基苯胺（S-2251）的活性以及蛋白酶抑制剂α2-抗纤溶酶（α2AP）和α2-巨球蛋白（α2M）的影响。在22℃下，纤溶酶与血小板反应蛋白的结合在3分钟内达到明显的可逆平衡。结合的纤溶酶的酰胺酶活性受到抑制。对S-2251水解的分析表明，血小板反应蛋白是一种线性混合型纤溶酶抑制剂。假设每个血小板反应蛋白同三聚体有一个纤溶酶结合位点，纤溶酶与血小板反应蛋白结合的解离常数（KD）为0.5微摩尔。纤溶酶和微型纤溶酶缓慢切割血小板反应蛋白，产生的产物与其他蛋白酶产生的产物相当。氨甲环酸抑制纤溶酶和微型纤溶酶对血小板反应蛋白的消化，表明kringle-5结构域在此过程中起重要作用。当纤溶酶先与血小板反应蛋白孵育，然后与α2AP孵育时，明显与血小板反应蛋白结合的纤溶酶与α2AP反应的速率降低；然而，在20分钟内，所有的纤溶酶都以与α2AP形成的复合物形式被回收。用α2M也得到了类似的结果。纤溶酶从血小板反应蛋白转移到α2AP或α2M可能需要纤溶酶-血小板反应蛋白复合物的解离。不能排除α2AP与血小板反应蛋白相关纤溶酶的低水平反应。这些结果表明，当纤溶酶与血小板反应蛋白结合时，其活性会大大降低或消除。在3分钟内形成且完全可逆的纤溶酶-血小板反应蛋白复合物，其相关的纤溶酶处于一种潜在形式，受到蛋白酶抑制剂的保护。因此，血小板反应蛋白可能以一种不同于传统蛋白酶抑制剂和其他细胞外基质蛋白的方式调节纤溶酶活性。
