Silverstein R L, Harpel P C, Nachman R L
J Biol Chem. 1986 Jul 25;261(21):9959-65.
Thrombospondin (TSP) is a multifunctional platelet alpha-granule and extracellular matrix glycoprotein that binds specifically to plasminogen (Plg) via that protein's lysine-binding site and modulates activation by tissue activator (TPA). In this study we report that the plasminogen activators, TPA and urokinase, greatly influence the binding of Plg to TSP. Using an enzyme-linked immunosorbent assay and a TSP-Sepharose affinity bead-binding assay we have found that Plg-TSP complex formation was markedly enhanced (up to 5-fold) when catalytic concentrations of Plg activators were included in the reaction mixtures. The enhancement was dependent upon the generation of small amounts of active plasmin and was duplicated by pretreatment of the immobilized TSP with plasmin prior to addition of the Plg. The enhancement effect was associated with selective proteolysis of the immobilized TSP. Purified Lys-Plg (the plasmin modified form of native Glu-Plg) bound to TSP to a greater extent than Glu-Plg, and binding of both forms was augmented by Plg activators. The apparent KD values of complex formation were unchanged in the presence of Plg activators suggesting that the enhancement effect was due to the generation of additional binding sites. The increased amount of bound Plg was demonstrated to result in a similar increase in the amount of plasmin generated from the complexes by TPA. Plg activators did not influence binding of Plg to histidine-rich glycoprotein or of histidine-rich glycoprotein to TSP, demonstrating specificity. In addition when TSP was treated with other proteases (human thrombin or human leukocyte elastase) no augmentation of Plg binding was seen. Thus, the initial production of small amounts of plasmin from Plg immobilized on TSP in fibrin-free microenvironments could generate a positive feedback loop by enzymatically modifying both TSP and Plg, resulting in an increase in TSP-Plg complex formation leading to the localized production of substantially more plasmin.
血小板反应蛋白（TSP）是一种多功能的血小板α颗粒和细胞外基质糖蛋白，它通过纤溶酶原（Plg）的赖氨酸结合位点与其特异性结合，并调节组织纤溶酶原激活物（TPA）介导的激活作用。在本研究中，我们报告纤溶酶原激活物TPA和尿激酶对Plg与TSP的结合有很大影响。通过酶联免疫吸附测定和TSP-琼脂糖亲和珠结合测定，我们发现当反应混合物中包含催化浓度的Plg激活物时，Plg-TSP复合物的形成显著增强（高达5倍）。这种增强依赖于少量活性纤溶酶的产生，并且在添加Plg之前用纤溶酶预处理固定化TSP可重复此增强效果。增强作用与固定化TSP的选择性蛋白水解有关。纯化的赖氨酸-Plg（天然谷氨酸-Plg的纤溶酶修饰形式）比谷氨酸-Plg与TSP的结合程度更高，并且两种形式的结合都被Plg激活物增强。在存在Plg激活物的情况下，复合物形成的表观解离常数（KD）值未改变，这表明增强作用是由于产生了额外的结合位点。已证明结合的Plg量增加会导致TPA从复合物中产生的纤溶酶量有类似增加。Plg激活物不影响Plg与富含组氨酸糖蛋白的结合，也不影响富含组氨酸糖蛋白与TSP的结合，表明具有特异性。此外，当用其他蛋白酶（人凝血酶或人白细胞弹性蛋白酶）处理TSP时，未观察到Plg结合的增强。因此，在无纤维蛋白的微环境中，固定在TSP上的Plg最初产生少量纤溶酶，可通过对TSP和Plg进行酶促修饰产生正反馈回路，导致TSP-Plg复合物形成增加，从而导致局部产生更多的纤溶酶。
