Molecular assembly of plasminogen and tissue-type plasminogen activator on an evolving fibrin surface

Abstract

A well characterized model of an intact and a degraded surface of fibrin that represents the states of fibrin during the initiation and the progression of fibrinolysis was used to quantitatively characterize the molecular interplay between tissue-type plasminogen activator (t-PA), plasminogen and fibrin. The molecular assembly of t-PA and plasminogen on these surfaces was investigated using combinations of proteins that preclude complications due to side reactions caused by generated plasmin: native plasminogen with di-isopropylphosphofluoridate-inactivated t-PA, and a recombinant human plasminogen with the active-site Ser741 mutagenized to Ala which renders the catalytic site inactive. Under these conditions, neither the affinity nor the maximal number of binding sites for plasminogen were modified by the presence of t-PA, indicating that binding sites for plasminogen pre-exist in intact fibrin and are not dependent on the presence of t-PA. In contrast, when plasminogen activation is allowed, increasing binding of plasminogen to the progressively degraded fibrin surface is directly correlated (r = 0.98) to the appearance of the fibrin E-fragment as shown using a monoclonal antibody (FDP-14) that has its epitope in the E domain of fibrin. t-PA was shown to bind with a high affinity to both the intact (Kd = 3.3 +/- 0.6 nM) and the degraded surface of fibrin (Kd = 1.2 +/- 0.4 nM). Binding of t-PA to carboxy-terminal lysine residues of degraded fibrin was shown to be efficiently competed by physiological concentrations of plasminogen (2 microM), indicating that the affinity of t-PA for these residues was lower than that of plasminogen (Kd = 0.66 +/- 0.22 microM) and unrelated to the high affinity of t-PA for specific binding sites on intact fibrin. These data confirm and establish that the generation of carboxy-terminal lysine residues on fibrin during ongoing fibrinolysis, and the binding of plasminogen to these sites, is an important pathway in the acceleration of clot dissolution.