Schmaier A H, Kuo A, Lundberg D, Murray S, Cines D B
Department of Medicine, Temple University, Philadelphia, Pennsylvania 19140.
J Biol Chem. 1988 Nov 5;263(31):16327-33.
High molecular weight kininogen (HMWK) functions as a cofactor for activation of plasma serine zymogens and as an inhibitor of tissue cysteine proteases. Cell surfaces to which HMWK binds may provide sites for regulation of these systems. Localization of these HMWK-dependent processes at sites of vascular injury may depend on its binding to specific receptors on endothelial cells. In culture, passaged human umbilical vein endothelial cells (HUVEC) bind anti-HMWK antibody to the cell surface and contain 171 +/- 75 ng of HMWK/10(8) cells. [35S]Methionine-labeled HUVEC in culture synthesize a 120-kDa protein immunoisolated using an anti-kininogen antibody, and a 3500-nucleotide message for human HMWK was detected by Northern blot in RNA extracted from HUVEC. HUVEC also express unoccupied binding sites for HMWK on their surface. 125I-HMWK specifically binds to HUVEC in a reaction requiring Zn2+. 125I-HMWK binding to HUVEC is saturable at 4 degrees C but not at 23 degrees C. 125I-HMWK binds to HUVEC with equal affinity as unlabeled HMWK. Kallikrein, factor XII, fibrinogen, fibronectin, and thrombin do not inhibit 125I-HMWK binding to HUVEC. 125I-HMWK-HUVEC binding remains fully reversible at 60 min following the addition of a 50-fold molar excess HMWK. HUVEC express 9.3 +/- 2.0 X 10(5) (mean +/- S.E.) HMWK binding sites/cell (Kd = 52 +/- 13 nM). Both added and cell-bound 125I-HMWK migrate at 120 kDa on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein remains uncleaved upon binding to the HUVEC surface. These studies indicate that HUVEC synthesize HMWK and the HUVEC surface has a site for its expression. By synthesizing and localizing HMWK to the cell surface, endothelial cells may contribute to the activation of plasma's contact serine zymogens and regulation of tissue cysteine proteases.
高分子量激肽原（HMWK）作为血浆丝氨酸酶原激活的辅因子以及组织半胱氨酸蛋白酶的抑制剂发挥作用。HMWK结合的细胞表面可能为这些系统的调节提供位点。这些依赖HMWK的过程在血管损伤部位的定位可能取决于其与内皮细胞上特定受体的结合。在培养中，传代的人脐静脉内皮细胞（HUVEC）将抗HMWK抗体结合到细胞表面，并且每10⁸个细胞含有171±75 ng的HMWK。培养中的[³⁵S]甲硫氨酸标记的HUVEC合成一种使用抗激肽原抗体免疫分离的120 kDa蛋白质，并且通过Northern印迹在从HUVEC提取的RNA中检测到人类HMWK的3500个核苷酸的信息。HUVEC在其表面也表达未被占据的HMWK结合位点。¹²⁵I-HMWK在需要Zn²⁺的反应中特异性结合到HUVEC。¹²⁵I-HMWK与HUVEC的结合在4℃下是可饱和的，但在23℃下不是。¹²⁵I-HMWK与HUVEC的结合亲和力与未标记的HMWK相同。激肽释放酶、因子XII、纤维蛋白原、纤连蛋白和凝血酶不抑制¹²⁵I-HMWK与HUVEC的结合。在加入50倍摩尔过量的HMWK后60分钟，¹²⁵I-HMWK-HUVEC结合仍完全可逆。HUVEC表达9.3±2.0×10⁵（平均值±标准误）个HMWK结合位点/细胞（Kd = 52±13 nM）。添加的和细胞结合的¹²⁵I-HMWK在十二烷基硫酸钠凝胶电泳上以120 kDa迁移，表明该蛋白质在与HUVEC表面结合时未被切割。这些研究表明HUVEC合成HMWK并且HUVEC表面有其表达位点。通过合成HMWK并将其定位到细胞表面，内皮细胞可能有助于血浆接触丝氨酸酶原的激活和组织半胱氨酸蛋白酶的调节。
