High molecular weight kininogen (HK) and factor XII are known to bind to human umbilical vein endothelial cells (HUVEC) in a zinc-dependent and saturable manner indicating that HUVEC express specific binding site(s) for those proteins. However, identification and immunochemical characterization of the putative receptor site (s) has not been previously accomplished. In this report, we have identified a cell surface glycoprotein that is a likely candidate for the HK (1,2). It possess six domains, the first three homologous to cysteine protease inhibitors (2 and 3 retain inhibitory activity) a fourth domain including the bradykinin moiety, and domains 5 and 6, which interact with surfaces (domain 5) and with prekallikrein or factor XII (domain 6). HK also functions as a coagulation cofactor (3, 4) by virtue of the interactions mediated by domains 5 and 6 (5-7). In addition, HK has been shown to bind to vascular endothelial cells in a zinc-dependent reaction (8, 9) requiring interaction with kininogen domains 3 and 5 (10, 11). The isolated heavy and light chains derived from kinin-free kininogen can each interact with endothelial cells (12) because they contain domains 3 and 5, respectively, although the two sites may act cooperatively in the intact protein (13).We have also shown that the initiating protein of the cascade, factor XII or Hageman factor, can also bind to endothelial cells with a Km (144 nM) and zinc requirement (50 ,tM) virtually identical to that of HK (14). Furthermore, HK and factor XII compete for binding (Ki of 98-152 nM), which suggests interaction with a common receptor (14).In the present report, we describe the isolation and partial characterization of the putative high molecular weight kininogen receptor from human umbilical vein endothelial cells (HUVEC). This molecule is a 33-kDa membrane protein that binds to HK or factor XII in a zinc-dependent reaction. Immunochemical and partial sequence analyses show this protein to be identical to the receptor that binds to the globular "heads" of Clq (gClq-R) described by Ghebrehiwet et al. (15).
MATERIALS AND METHODSEndothelial Cell Culture. HUVECs were isolated according to Jaffe (16) and cultured in gelatin-coated dishes in Medium 199 (GIBCO) with 20% fetal bovine serum (FBS), 20% NU-serum (Collaborative Research), 100 ,tg/ml penicillin, 100 ,ug/ml streptomycin, and 2.5 ,ug/ml amphotericin B. For cell-binding studies, the cells were subcultured into gelatincoated 96-well strip plates in 0.2 ml culture medium and the cells were washed 3x in phosphate-buffered saline (PBS) to remove bound serum proteins before each experiment. The cells were identified as endothelial cells by their distinct cobblestone morphology and interaction with antiserum to Von Willebrand's factor (16). All cells were used at the third cell passage.Preparation of the HK-Affinity Column. Purified HK (Enzyme Research Laboratories, South Bend, IN) was coupled with 3M Emphaze Biosupport Medium AB1 (Pierce) according to the manufacturer's directions. Briefly, 10 mg ...