The role of subsite interactions in defining the stringent substrate specificity of tissue-type plasminogen activator (t-PA) has been examined by using an fd phage library that displayed random hexapeptide sequences and contained 2 x 108 independent recombinants. Forty-four individual hexapeptides were isolated and identified as improved substrates for t-PA. A peptide containing one of the selected amino acid sequences was cleaved by t-PA 5300 times more efficiently than a peptide that contained the primary sequence of the actual cleavage site in plasminogen. These results suggest that small peptides can mimic determinants that mediate specific proteolysis, emphasize the importance of subsite interactions in determining protease specificity, and have important implications for the evolution of protease cascades.plasminogen during catalysis, either to induce a conformational change in t-PA or to properly position a suboptimal sequence in the active site of t-PA. A third hypothesis, which does not necessarily require secondary sites of interaction between t-PA and plasminogen, is that t-PA recognizes an unusual, constrained conformation of the plasminogen target sequence that cannot be significantly populated by small, linear peptides. Only the second of these three hypotheses unequivocally predicts the existence of small peptides that could be efficiently cleaved by t-PA. Consequently, to distinguish among these hypotheses, we screened a library of random hexamers for peptide sequences that could be hydrolyzed by t-PA. A peptide containing one of the selected amino acid sequences was cleaved by t-PA 5300 times more efficiently than a peptide containing the primary sequence of the actual cleavage site in plasminogen.A wide variety of critical biological processes (1-3) depend on specific cleavage of individual target proteins by serine proteases. Enzymes capable of catalyzing this uniquely selective proteolysis have evolved while retaining high homology to related nonselective 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 bond (Arg560 -Val561) in the proenzyme plasminogen (4). Part of the specificity of t-PA for plasminogen is mediated by formation of a ternary complex between t-PA, plasminogen, and the cofactor fibrin (5). Even in the absence of fibrin, however, t-PA remains specific for cleavage of plasminogen. This stringent specificity is an inherent property of the protease domain of t-PA (6), in spite of its high homology to trypsin, an archetypal nonselective protease (7).Trypsin activates native plasminogen poorly, exhibiting only 10-30% of the catalytic efficiency of t-PA (6, 8). By contrast, trypsin cleaves small peptides containing the primary sequence of the activation site in plasminogen 29,000-200,000 times more rapidly than t-PA. The relative specificity of these two related enzymes for the same primary sequence, in two distinc...