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

Abstract

Prekallikrein (PK) activation on human umbilical endothelial cells (HUVEC) presumably leads to bradykinin liberation. On HUVEC, PK activation requires the presence of cell-bound high-molecular-weight kininogen (HK) and Zn2+. We examined the Zn2+requirement for HK binding to and the consequences of PK activation on endothelial cells. Optimal HK binding (14 pmol/106HUVEC) is seen with no added Zn2+in HEPES-Tyrode buffer containing gelatin versus 16–32 μM added Zn2+in the same buffer containing bovine serum albumin. The affinity and number of HK binding sites on HUVEC are a dissociation constant of 9.6 ± 1.8 nM and a maximal binding of 1.08 ± 0.26 × 107sites/cell (means ± SD). PK is activated to kallikrein by an antipain-sensitive mechanism in the presence of HK and Zn2+on HUVEC, human microvascular endothelial cells, umbilical artery smooth muscle cells, and bovine pulmonary artery endothelial cells. Simultaneous with kallikrein formation, bradykinin (5.0 or 10.3 pmol/106HUVEC in the absence or presence of lisinopril, respectively) is liberated from cell-bound HK. Liberated bradykinin stimulates the endothelial cell bradykinin B2 receptor to form nitric oxide. Assembly and activation of PK on endothelial cells modulates their physiological activities.