High-molecular-weight kininogen is exclusively membrane bound on endothelial cells to influence activation of vascular endothelium

Abstract

An important biologic function of high-molecular-weight kininogen (HK) is to deliver bradykinin (BK) to its cellular receptors. Internalization and degradation of HK may provide a mechanism by which endothelial cells modulate the production of BK and control its activities. Therefore, we investigated the binding and subsequent distribution of biotinylated-HK (biotin-HK) associated with human umbilical vein endothelial cells (HUVEC). HUVEC bound 3 to 4 times more HK and with greater avidity at 1 to 3 hours at 37 degrees C than at 4 degrees C (Bmax = 1.0 +/- 0.02 x 10(7) molecules/cell, kd = 7 +/- 3 nmol/L v Bmax = 2.6 +/- 0.2 x 10(6) molecules/cell, kd = 46 +/- 8 nmol/L). However, there was no evidence that the difference was caused by internalization of HK at the higher temperature. First, the same amount of biotin-HK was associated with nonpermeabilized and permeabilized HUVEC using buffers containing 20 to 50 mumol/L zinc ion in the absence or presence of 2 mmol/L calcium ion. Second, binding of biotin-HK to HUVEC was approximately 92% reversible at 1 hour when the cells were maintained at both 37 degrees C and 4 degrees C. Third, neither chloroquine nor primaquine altered the amount of biotin-HK bound to HUVEC. Fourth, biotin-HK bound to HUVEC was almost completely removed by pronase. Fifth, the nonpermeable dye, crystal violet, almost completely quenched the fluorescence signal emitted by HUVEC-associated fluorescein isothiocyanate (FITC) HK. Finally, the localization of HUVEC-bound FITC-HK was restricted to the membrane as shown by laser scanning confocal microscopy. The expression of HK binding sites had an absolute requirement for metabolic energy, but was not dependent on new protein synthesis. Membrane-bound HK contributed to the anticoagulant nature of endothelial cells by blocking human alpha-thrombin binding and its resultant induction of prostacyclin formation. These studies indicate that HK is not internalized by HUVEC, but remains primarily on cell surfaces to be accessible for BK liberation and to modulate the binding and actions of alpha-thrombin.

The contact activation and kallikrein/kinin systems: pathophysiologic and physiologic activities

“…Domains 5 and 6 are necessary for the coagulant activity of HK [32]. Domain 5 (D5) is the artificial surface-binding region of HK and domain 6 binds PK and FXI in order to initiate intrinsic coagulation [33][34][35]. The majority of recognized physiologic functions of HK, with the exception of BK delivery, are associated with D5.…”

Assembly and activation of HK-PK complex on endothelial cells results in bradykinin liberation and NO formation

“…The interaction of kininogens with human umbilical vein endothelial cells (HUVEC) in culture has been described by binding studies using buffers that contain bovine serum albumin (BSA) (2,10,23,28,31). Kininogen binding to HUVEC in buffers containing BSA requires the addition of 25-50 M Zn 2ϩ (28,31).…”

High molecular weight kininogen inhibition of endothelial cell function on biomaterials

“…Several HMWK domains have been identified as endothelial cell binding sites. 22,[32][33][34][35] One site on domain 3, near the carboxyl terminus of the SC-HMWK, has been localized to a 13 amino-acid sequence containing a disulfide loop. A second high-affinity binding site, located on domain 5 of the DC-HMWK (histidine-rich aminoacid sequence), was found to be the binding site of HMWK to anionic artificial surfaces.…”

“…17 Interestingly, the binding of HMWK to cytokeratin 1 requires the cellbinding regions on domains 2, 4, and 5. 33,34 Cytokeratin belongs to the family of intermediate filaments known to participate in cellular actin filament assembly. Our present results suggest a potential mechanism whereby actin filament disassembly on HMWKcoated biomaterials may be related to the binding of cytokeratin 1 by HMWK.…”