Identification of a Hydrophobic Exosite on Tissue Type Plasminogen Activator That Modulates Specificity for Plasminogen

Abstract

A wide variety of important biological processes, including both the formation and dissolution of blood clots, depend on specific cleavage of individual target proteins by serine proteases. For example, tissue type plasminogen activator (t-PA), a trypsin-like enzyme that catalyzes the rate-limiting step of the endogenous fibrinolytic cascade, has only one known substrate in vivo, a single peptide bond (Arg 561 -Val 562 ) in the proenzyme plasminogen. We have previously suggested that the specificity of t-PA for plasminogen is mediated in part by direct protein-protein interactions between the protease domain of t-PA and plasminogen that are distinct from those occurring within t-PA's active site. We demonstrate in this study that residues 420 -423 of t-PA, which form a fully solvent-exposed, hydrophobic region of a surface loop mapping near one edge of the active site of t-PA, form, or are essential for the integrity of, an important, secondary site of interaction between t-PA and plasminogen that significantly modulates the rate of plasminogen activation in the absence, but not the presence, of fibrin. Identification of this secondary site of interaction between t-PA and plasminogen provides new insight into molecular details of the evolution of stringent substrate specificity by t-PA and suggests a novel strategy to enhance the fibrin dependence of plasminogen activation by t-PA. While the activity of wild type t-PA is stimulated by fibrin by a factor of approximately 650, the activity of two variants characterized in this study, t-PA/R275E,P422G and t-PA/R275E,P422E, is stimulated by a factor of approximately 39,000 or 61,000, respectively. It is therefore possible that, compared with wild type t-PA, the two variants would display enhanced "clot selectivity" in vivo due to reduced activity in the circulation but full activity at a site of fibrin deposition.Tissue type plasminogen activator (t-PA) 1 has been widely and successfully used as a therapeutic agent to treat acute myocardial infarction (1). t-PA does not, however, dissolve blood clots directly; instead, the enzyme catalyzes conversion of the zymogen plasminogen into the active protease plasmin, which efficiently degrades the fibrin mesh forming the core of a thrombus (2). Both of these critical fibrinolytic enzymes, t-PA and plasmin, are members of the chymotrypsin family of serine proteases (3, 4). Unlike plasmin, however, t-PA is a remarkably specific enzyme. Because such highly restricted substrate specificity is in striking contrast to the broad specificity of well studied serine proteases such as trypsin, chymotrypsin, and elastase (5), the molecular basis of the selectivity of t-PA for plasminogen is of considerable interest. A detailed understanding of the mechanisms employed by t-PA to ensure selectivity would provide new insight into the evolution of the endogenous fibrinolytic cascade and might suggest effective new strategies for the rational design of novel, highly selective proteases with unique specificities as well as novel plasminogen...

Distinct Contributions of Residue 192 to the Specificity of Coagulation and Fibrinolytic Serine Proteases

“…Measurement of Second Order Rate Constants for Inhibition by PAI-1-Second order rate constants (k i ) for the inhibition of wild type and mutated t-PA were measured under pseudo-first order conditions as described previously (32)(33)(34)(35)(36). Inhibitor was present in at least a 20-fold molar excess over enzyme.…”

“…Kinetic Analysis of Plasminogen Activation Using Indirect Chromogenic Assays-Indirect chromogenic assays of t-PA utilized the substrates Lys-plasminogen (American Diagnostica) and Spectrozyme PL (American Diagnostica) and were performed as described previously (32,33). Assays were performed both in the presence and absence of the co-factor DESAFIB (American Diagnostica).…”

Elimination of the Cys558‐Cys566 bond in Lys78‐plasminogen

“…6), the absence of the Cys558 Cys566 bond in plasmin appears also to result in a distinct effect on the degragen activation loop. Studies with tPA variants in which the plasminogen interaction site was modified [23] confirm this assump-dation pattern during more advanced stages of fibrin clot dissolution. These stages are characterised by dissociation of fibres tion, which was further substantiated by the results obtained with our [Ser558, Ser566]Lys78-plasminogen variant.…”

“…A recent study of substitution of these amino acid leads to an increased activity with respect to cleavage of the AC domain in fibrin(ogen). Since [23] has identified a hydrophobic exosite (residues 420Ϫ423) on the edge of the active site of tPA which, together with the a fast initial plasmin degradation is supposed to result in early appearance of C-terminal lysine residues and an increased bindprimary site, is involved in the interaction with plasminogen. Also, the isolated protease domain of uPA is as active as full-ing of plasminogen to fibrin, this might also explain the increased tPA activity observed with the [Ser558, Ser566]Lys78-length uPA in the absence of stimulator [24].…”

Variants of Tissue-type Plasminogen Activator That Display Extraordinary Resistance to Inhibition by the Serpin Plasminogen Activator Inhibitor Type 1

“…However, recent demonstrations that, compared with wild type t-PA, PAI-1-resistant variants possess enhanced potency toward platelet-rich thrombi but normal potency toward platelet-poor clots, both in vitro and in vivo, strongly suggests an important role for platelet derived PAI-1 during thrombolytic therapy (15,38,39). We have previously suggested that the recruitment of residues in surface loops mapping near the active site of an enzyme to form specific enzyme-inhibitor and/or enzyme-substrate interactions was likely to be an important and recurring theme during the evolution of enzyme specificity (13,21,22,40). The properties of variants of t-PA carrying mutations in the 296 -304 region clearly demonstrate that this mechanism played an essential role during the co-evolution of specificity between t-PA and PAI-1, individual members of two very large and medically relevant gene families.…”