Binding of thrombin to subendothelial extracellular matrix. Protection and expression of functional properties.
Binding of thrombin to subendothelial extracellular matrix. Protection and expression of functional properties.
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DOI：
10.1172/jci114272
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发表时间：
1989-10
期刊：
影响因子：
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通讯作者：
R. Bar-Shavit;A. Eldor;I. Vlodavsky
中科院分区：
文献类型：
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作者：
R. Bar-Shavit;A. Eldor;I. Vlodavsky
我们已经分析了凝血酶，一种丝氨酸蛋白酶，在止血中发挥重要作用，与培养的内皮细胞产生的内皮下细胞外基质（ECM）的结合。该基质提供了开始止血的血栓形成表面。结合是饱和的，与125 I-α-凝血酶孵育3小时后达到平衡。凝血酶结合的Scatchard分析显示，每平方毫米ECM存在5.1 × 10（9）个结合位点，表观Kd为13 nM。催化阻断的酶，二异氟磷酸（DIP）-α-凝血酶与125碘-α-凝血酶有效地竞争，表明结合是独立的催化位点。此外，高浓度的合成十四肽，代表凝血酶B链（凝血酶的巨噬细胞促有丝分裂结构域）的残基367-380，与凝血酶竞争结合ECM，表明结合位点可能位于“环B”区域附近。凝血酶与ECM中的硫酸皮肤素结合，如通过抑制125 I-α-凝血酶与用软骨素酶ABC预处理的ECM结合而不是用肝素酶或软骨素酶AC预处理的ECM结合所证明的。这与125 I-FGF（成纤维细胞生长因子）与ECM的结合形成对比，125 I-FGF（成纤维细胞生长因子）与ECM的结合被肝素酶抑制，但不被软骨素酶ABC抑制，ECM结合的凝血酶表现出暴露的蛋白水解位点，如通过Chromozyme TH测定监测的，并且通过其将纤维蛋白原转化为纤维蛋白凝块并诱导血小板活化的能力，如通过14 C-血清素释放所指示的。ECM结合的凝血酶未能形成一个复杂的主要循环转运蛋白-抗凝血酶III（AT III），与快速复合物形成可溶性凝血酶相比。我们认为凝血酶与内皮下ECM结合，在那里它保持功能活性，局部化，并受到循环抑制剂的保护免于失活。
We have analyzed the binding of thrombin, a serine protease with central roles in hemostasis, to the subendothelial extracellular matrix (ECM) produced by cultured endothelial cells. This substrate provides a thrombogenic surface where hemostasis is initiated. Binding was saturable and equilibrium was achieved after 3 h incubation with 125I-alpha-thrombin. Scatchard analysis of thrombin binding revealed the presence of 5.1 X 10(9) binding sites per squared millimeter ECM, with an apparent Kd of 13 nM. The catalytically blocked enzyme, diisofluorophosphate (DIP)-alpha-thrombin competed efficiently with 125I-alpha-thrombin, indicating that the binding was independent of its catalytic site. Moreover, high concentrations of the synthetic tetradecapeptide, representing residues 367-380 of thrombin B chain (the macrophage mitogenic domain of thrombin), competed with thrombin binding to ECM, indicating that the binding site may reside in the vicinity of "loop B" region. Thrombin binds to dermatan sulfate in the ECM, as demonstrated by the inhibition of 125I-alpha-thrombin binding to ECM pretreated with chondroitinase ABC, but not with heparitinase or chondroitinase AC. This stands in contrast to 125I-FGF (fibroblast growth factor) binding to ECM, which was inhibited by heparitinase but not by chondroitinase ABC, ECM-bound thrombin exhibits an exposed proteolytic site as monitored by the Chromozyme TH assay and by its ability to convert fibrinogen to a fibrin clot and to induce platelet activation as indicated by 14C-serotonin release. ECM-bound thrombin failed to form a complex with its major circulating inhibitor-antithrombin III (AT III), compared with rapid complex formation with soluble thrombin. We propose that thrombin binds to subendothelial ECM where it remains functionally active, localized, and protected from inactivation by circulating inhibitors.