PLASMINOGEN ACTIVATION BY PROUROKINASE IN COMPLEX WITH ITS RECEPTOR -DEPENDENCE ON A TRIPEPTIDE (SPECTROZYME PLASMIN)

The intrinsic activity of single-chain pro-urinary-type plasminogen ac tivator (pro-uPA) and whether its receptor (uPAR) potentiates this act ivity remains controversial. In this report. the pro-uPAR/uPAR-(1-281) -peptide complex in solution is shown to have equivalent plasminogen-a ctivator activity to that of active two-chain uPA (tc-uPA). However, t he activity of the complex was dependent on a synthetic tripeptide, Sp ectrozyme plasmin (Sp1, H-D-2-aminohexanoic acid(Ahx)-hexatyrosyl-lysi ne-p-nitroanilide), which can also be used as a chromogenic substrate for plasmin. Furthermore, this activity could be completely suppressed by commonly used carrier proteins anti detergents. The pro-uPA/uPAR-( 1-281)-peptide complex at 1 nM displayed similar activity to that of t c-uPA for either [Glu 1]plasminogen or [Lys77]plasminogen in chromogen ic assays with Sp1 present as the plasmin substrate, When assayed with another plasmin substrate, S2251, the pro-uPA/uPAR-(1-281)-peptide co mplex was unable to activate plasminogen. The pro-uPA/uPAR-(1-281)-pep tide complex and tc-uPA also showed a similar extent of plasminogen ac tivation as measured by SDS/PAGE, when incubated with plasminogen and Spl in the presence of 100 mu M aprotinin, and plasminogen activation by pro-uPA alone was also stimulated in the presence of Spl in this as say. Activation of plasminogen by the pro-uPA/uPAR-(1-281)-peptide: st rictly required the presence of Sp1, and pro-uPA remained in single-ch ain form during these assays. This activity of the pro-uPA/uPAR-(1-281 )-peptide complex but not that of tc-uPA was completely inhibited by h uman serum albumin, bovine serum albumin, Tween-80, Triton X-100, and Pluronic-F68. Taken together, the data indicates that uPAR-(1-281)-pep tide itself is not sufficient to augment pro-uPA activity and the pres ence of an effector molecule (e.g. Sp1) is required to elicit the full plasminogen-activator activity of the pro-uPA/uPAR-(1-781)-peptide co mplex. It remains to be seen whether there is a physiological counterp art to this phenomenon.