The kinetics of tPA inhibition by C1-inhibitor in vivo and in vitro

Tissue plasminogen activator (TPA) activity in blood is regulated in part by a series of inhibitors including plasminogen activator inhibitor 1 (PAI-1) and Cl-inhibitor. In this study we: 1) estimated the rate of the reaction between TPA and Cl-inhibitor in vivo based on measured levels of active TPA, C1 -inhibitor and TPA/C1 -inhibitor complex and a simple model of TPA/C 1-inhibitor complex formation versus clearance and 2) evaluated the effect of various factors and cells in the vascular system on the rate of the TPA versus Cl-inhibitor reaction in vitro. In vivo TPA reacted with Cl-inhibitor with an apparent secondorder rate constant of approximately 500 M^-1s^-1. In contrast, the reaction rate in vitro using pure components was 2 to 3 M^-1s^-1, at least a 100 fold lower. Heparin, heparan sulfate, plasma proteins, red cells, white cells, and platelets had no apparent effect on the rate of the reaction between TPA and Cl-inhibitor in vitro. Further, when 1000 ng/mL of exogenous TPA was added with Cl-inhibitor to human umbilical vein endothelial cells (HUVEC) in culture there was no detectable acceleration. In contrast, when HUVEC in culture were stimulated to secrete high levels of TPA using sodium butyrate, the apparent rate of the reaction between secreted TPA and Cl-inhibitor was approximately 389 M^-1s^-1, a 100 fold acceleration. This suggests that secretion of TPA from endothelial cells may play a role in the apparent acceleration of the TPA versus Cl-inhibitor reaction in vivo.