Reversible modification of tissue-type plasminogen activator by methylphosphonate esters

In spite of their rapid aqueous hydrolysis, 4-nitrophenyl 4-X-phenacyl methylphosphonates (X = H, (PMN) CH3, CH3O, Cl and NO2) inactivate many serine proteases of the pancreatic and blood coagulation systems efficiently. The rate constants, k(i)/K-i, for the inactivation of tissue-type plasminogen activator enzyme (t-PA) are 470-750 M(-1) s(-1) with PMN, 4-CH3-PMN, and 4-CH3O-PMN in pH 7.8, 0.05 M Tris buffer at 7.0 +/- 0.5 degrees C, but t-PA cannot be inhibited with the 4-Cl and NO2 derivatives due to rapid competing hydrolysis. Enzyme activity returns from each enzyme-adduct at a characteristic rate, due to a self-catalyzed intramolecular reactivation process. The rate constants for spontaneous reactivation of t-PA from the adducts formed with the three inhibitors are k = 0.25-12.3 x 10(-2) min(-1) at pH 7.4 and 25.0 +/- 0.1 degrees C and pH-dependent with an apparent pK similar to 8.3. The recovery of t-PA activity from the adducts in 40% human plasma buffered at pH 7.4 is the same or twice that in plain buffer. The presence of fibrin has a slight effect on inactivation but not on reactivation. The modulation of enzyme activity by reversible generation of the phosphonylated adducts has potential for medical application.