Identification of a hydrophobic exosite on tissue type plasminogen activator that modulates specificity for plasminogen.

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 (Arg561-Val562) 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.