INCORPORATION OF PLASMINOGEN ACTIVATOR INHIBITOR INTO FIBRIN, AN ALTERNATIVE REGULATORY PATHWAY OF FIBRINOLYSIS

A plasminogen activator inhibitor (PAI-1) Mj, 50 kd is normally found in plasma at low concentrations. Plasma levels increase sharply upon stimulation of endothelial cells with endotoxin or monokines and activated platelets secrete significant quantities of PAI-1. It is possible that high levels of PAI-1 may be achieved at the local sites of intravascular thrombi. Semipurified PAI-1 was therefore prepared from human platelets to study its affinity for fibrin (F). Approximately 50% PAI-1 adsorbed to F monomer immobilized on sepharose and desorbed under conditions of acidic pH and high ionic strength suggesting hydrogen bonding as the mode of interaction. Wells of 96-well microtiter plates were each coated with 50 yg [¹²⁵I] plasminogen (P)-free fibrinogen and clotted with thrombin in the presence and absence of different concentrations of PAI-1. After extensive washing of the wells, they were incubated with 5 mU of tissue plasminogen activator (t-PA) and 5 mU of P for 6 h. Appropriate calibration curves utilizing different concentrations of t-PA and different concentrations of PAI-1 added to the supernatant rather than to F established that 8-15% of 21-166 mU PAI-1 incorporated into crosslinked (XL) F or noncrosslinked (NXL) F. Incorporated PAI strikingly inhibited fibrinolysis (FL). Percent inhibition of FL of XL or NXLF (Mean±S.D., N=5) plotted in the presence of 166, 83, 42 and 21 mU of PAI were: 83±3.3, 59.5±1.8, 29.7±5.2 and 15.2±6.14 for XLF and 78±5.3, 31±8.1, 14.5±10.5 and 0 Tor NXL F. As demonstrated by radioautography on SDS PAGE PAI-1 incorporated into F readily formed complexes with [¹²⁵I] urokinase (u-PA). In these experiments, no evidence for crosslinking of PAI-1 into F has been obtained to date. In experiments utilizing agarose immobilized proteins, it was evident that not only F but also fibrinogen binds PAI-1; PAI-1 associated with F as well as fibrinogen is capable of forming complexes with [1251] u-PA.

In contrast, fibronectin, collagen, gelatin and albumin did not bind PAI-1. Thus, PAI-1 in analogy with alpha-2 plasmin inhibitor may modulate physiological fibrinolysis through incorporation into fibrin.