A Study of the Protection of Plasmin from Antiplasmin Inhibition within an Intact Fibrin Clot during the Course of Clot Lysis

Abstract

Previous work using soluble fibrin surrogates or very dilute fibrin indicate that inhibition of plasmin by antiplasmin is attenuated by fibrin surrogates; however, this phenomenon has not been quantified within intact fibrin clots. Therefore, a novel system was designed to measure plasmin inhibition by antiplasmin in real time within an intact clot during fibrinolysis. This was accomplished by including the plasmin substrate S2251 and a recombinant fluorescent derivative of plasminogen (S741C-fluorescein) into clots formed from purified components. Steady state plasmin levels were estimated from the rates of S2251 hydrolysis, the rates of plasminogen activation were estimated by fluorescence decrease over time, and residual antiplasmin was deduced from residual fluorescence. From these measurements, the second order rate constant could be inferred at any time during fibrinolysis. Immediately after clot formation, the rate constant for inhibition decreased 3-fold from 9.6 ؋ 10 6 M ؊1 s ؊1 measured in a soluble buffer system to 3.2 ؋ 10 6 M ؊1 s ؊1 in an intact fibrin clot. As the clot continued to lyse, the rate constant for inhibition continued to decrease by 38-fold at maximum. To determine whether this protection was the result of plasmin exposure of carboxyl-terminal lysine residues, clots were formed in the presence of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). In the presence of TAFIa, the initial protective effect associated with clot formation occurred; however, the secondary protective effect associated with lysine residue exposure was delayed in a TAFIa concentration-dependent manner. This latter effect represents another mechanism whereby TAFIa attenuates fibrinolysis.As a fibrin clot forms in response to a vascular injury, it regulates its own degradation by serving as cofactor for plasminogen (Pgn) 1 activation to plasmin (Pn) (1). Once formed, Pn directly catalyzes clot breakdown by cleaving fibrin. In the process, carboxyl-terminal lysine residues are exposed that serve to enhance Pgn activation through a positive feedback mechanism for Pn formation (2). Once formed, Pn is quickly inhibited by the potent serine protease inhibitor antiplasmin (AP), which forms a tight nearly irreversible complex with Pn. Therefore, Pn levels and consequently the rate of fibrinolysis are determined by the balance between the kinetics of Pn formation and inhibition.Thrombin-activatable fibrinolysis inhibitor (TAFI) is a recently identified plasma zymogen (3-5) that can be activated to the carboxypeptidase B-like enzyme TAFIa by thrombin/ thrombomodulin (6), free thrombin (3, 7), or plasmin (3,8). In the context of a fibrin clot, TAFIa removes carboxyl-terminal lysine residues from Pn-modified fibrin (9). Although no naturally occurring inhibitors of TAFIa have been identified in human plasma, TAFIa is thermally unstable and this instability is probably how TAFIa is regulated once it is activated (10, 11). Previously, a naturally occurring polymorphism at amino acid position 325 (Thr/Ile) was identi...

Reduced Plasminogen Binding and Delayed Activation Render γ′-Fibrin More Resistant to Lysis than γA-Fibrin

“…The corrected absorbance values were plotted against time-squared, with time 0 set as the time when Fg was added to the wells. Activation rates were then determined from the linear portions of the plots (24). Some conditions produced nonlinear plots, suggesting a delay in the initiation of Pg activation.…”

Measurement of Procarboxypeptidase U (TAFI) in Human Plasma: A Laboratory Challenge

“…In the following years the proCPU/CPU pathway was identified as an important coagulation-dependent pathway in the control of the fibrinolytic rate. By removing the C-terminal lysine residues on partially degraded fibrin, CPU prevents the acceleration of plasminogen binding and activation and promotes the inhibition of plasmin by ␣ 2 -antiplasmin (7)(8)(9)(10). As long as the plasma CPU activity remains above a certain threshold value, fibrinolysis does not accelerate but stays in its initial phase (11,12 ).…”

Fibrin Clot Structure and Function

“…Plasmin that is bound to fibrin is relatively protected from inhibition by circulating ␣ 2 -antiplasmin. 27 However, ␣ 2 -antiplasmin also binds to fibrin and helps to protect the clot against fibrinolysis. 20,21 A key inhibitor of tPA, plasminogen activator inhibitor-1 (PAI-1), is released from platelets and the endothelium on stimulation with cytokines or thrombin.…”