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 x 10(6) m(-1) s(-1) measured in a soluble buffer system to 3.2 x 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.
Highlights
Directly catalyzes clot breakdown by cleaving fibrin
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
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)
Summary
Plasminogen; Pn, plasmin; tPA, tissue-type plasminogen activator; AP, antiplasmin; TAFI, thrombinactivatable fibrinolysis inhibitor; TAFIa, activated TAFI; TAFI-T, TAFI variant with Thr-325; TAFI-I, TAFI variant with Ile-325; 5IAF-Pgn, fluorescein-labeled recombinant (S741C) Pgn; 5IAF-Pn, fluorescein-labeled recombinant (S741C) Pn; rform, rate of formation; rinh, rate of inhibition; dF, change in fluorescence; dt, change in time. It was shown that TAFIa removal of these carboxyl-terminal lysine residues from fibrin removes the positive feedback for Pgn activation on a Pn modified fibrin surface [9]. By knowing the rate of Pn formation and the concentrations of Pn and AP, the second order rate constant for Pn inhibition by AP can be calculated during fibrinolysis With this model, it was found that modification of fibrin by Pn dramatically increased the ability of fibrin to protect Pn from inhibition. It was found that modification of fibrin by Pn dramatically increased the ability of fibrin to protect Pn from inhibition This protective effect was further characterized by forming clots in the presence of TAFIa, and it was found that TAFIa could effectively attenuate protection in a manner that depends on the concentration and thermal stability of the TAFIa isoform used. The rate of change in Pn concentration within a clot over time is as shown in Equation 2
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