HIGH-MOLECULAR-WEIGHT KININOGEN IS EXCLUSIVELY MEMBRANE-BOUND ON ENDOTHELIAL-CELLS TO INFLUENCE ACTIVATION OF VASCULAR ENDOTHELIUM

An important biologic function of high-molecular-weight kininogen (HK) is to deliver bradykinin (BK) to its cellular receptors. Internalizat ion and degradation of HK may provide a mechanism by which endothelial cells modulate the production of BK and control its activities. There fore, we investigated the binding and subsequent distribution of bioti nylated-HK (biotin-HK) associated with human umbilical vein endothelia l cells (HUVEC). HUVEC bound 3 to 4 times more HK and with greater avi dity at 1 to 3 hours at 37 degrees C than at 4 degrees C (B-max = 1.0 +/- 0.02 x 10(7) molecules/cell, kd = 7 +/- 3 nmol/L v B-max = 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 a t the higher temperature. First, the same amount of biotin-HK was asso ciated with nonpermeabilized and permeabilized HUVEC using buffers con taining 20 to 50 mu mol/L zinc ion in the absence or presence of 2 mmo l/L calcium ion. Second, binding of biotin-HK to HUVEC was similar to 92% reversible at 1 hour when the cells were maintained at both 37 deg rees C and 4 degrees C. Third, neither chioroquine nor primaquine alte red the amount of biotin-HK bound to HUVEC. Fourth, biotin-HK bound to HUVEC was almost completely removed by pronase. Fifth, the nonpermeab le dye, crystal violet, almost completely quenched the fluorescence si gnal emitted by HUVEC-associated fluorescein isothiocyanate (FITC) HK. Finally, the localization of HUVEC-bound FITC-HK was restricted to th e membrane as shown by laser scanning confocal microscopy. The express ion of HK binding sites had an absolute requirement for metabolic ener gy, but was not dependent on new protein synthesis. Membrane-bound HK contributed to the anticoagulant nature of endothelial cells by blocki ng human alpha-thrombin binding and its resultant induction of prostac yclin 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. (C) 1995 by The American Society of Hematology.