Baird T Regan, Walsh Peter N
The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
J Biol Chem. 2003 Jun 6;278(23):20618-23. doi: 10.1074/jbc.M300224200. Epub 2003 Mar 27.
Previous studies on the interaction of high molecular weight kininogen (HK) with endothelial cells have reported a large number of binding sites (106-107 sites/cell) with differing relative affinities (KD = 7-130 nm) and have implicated various receptors or receptor complexes. In this study, we examined the binding of HK to human umbilical vein endothelial cells (HUVEC) with a novel assay system utilizing HUVEC immobilized on microcarrier beads, which eliminates the detection of the high affinity binding sites found nonspecifically in conventional microtiter well assays. We report that HK binds to 8.5 x 104 high affinity (KD = 21 nm) sites per HUVEC, i.e. 10-100-fold fewer than previously reported. Although HK binding is unaffected by the presence of a physiological concentration of prekallikrein, factor XI abrogates HK binding to HUVEC in a concentration-dependent manner. Disruption of the naturally occurring complex between factor XI and HK by the addition of a 31-amino acid peptide mimicking the factor XI-binding site on HK restored HK binding to HUVEC. Furthermore, HK inhibited thrombin-stimulated von Willebrand factor release by HUVEC but not thrombin receptor activation peptide (SFLLRN-amide)-stimulated von Willebrand factor release. Factor XI restored the ability of thrombin to stimulate von Willebrand factor release in the presence of low HK concentrations. These results suggest that free HK, or HK in complex with prekallikrein but not in complex with factor XI, interacts with the endothelium and can maintain endothelial cell quiescence by preventing endothelial stimulation by thrombin.
先前关于高分子量激肽原(HK)与内皮细胞相互作用的研究报道了大量具有不同相对亲和力(KD = 7 - 130 nM)的结合位点(106 - 107个位点/细胞),并涉及多种受体或受体复合物。在本研究中,我们使用一种新型检测系统,该系统利用固定在微载体珠上的人脐静脉内皮细胞(HUVEC)来检测HK与HUVEC的结合,这消除了在传统微量滴定板检测中非特异性发现的高亲和力结合位点的检测。我们报告称,每个HUVEC上HK与8.5×104个高亲和力(KD = 21 nM)位点结合,即比先前报道的少10 - 100倍。尽管HK的结合不受生理浓度前激肽释放酶存在的影响,但因子XI以浓度依赖的方式消除HK与HUVEC的结合。通过添加模拟HK上因子XI结合位点的31个氨基酸的肽破坏因子XI与HK之间天然存在的复合物,可恢复HK与HUVEC的结合。此外,HK抑制凝血酶刺激的HUVEC释放血管性血友病因子,但不抑制凝血酶受体激活肽(SFLLRN - 酰胺)刺激的血管性血友病因子释放。在低HK浓度存在下,因子XI恢复了凝血酶刺激血管性血友病因子释放的能力。这些结果表明,游离的HK或与前激肽释放酶结合而非与因子XI结合的HK与内皮相互作用,并可通过防止凝血酶刺激内皮来维持内皮细胞的静止状态。