ULTRASENSITIVE FLUOROGENIC SUBSTRATES FOR SERINE PROTEASES

Selective, sensitive assays for the quantitation of serine proteases i nvolved in coagulation and fibrinolysis have been developed employing fluorogenic substrates containing a 6-amino-1-naphthalenesulfonamide l eaving group (PNS-substrates). Over one hundred substrates were evalua ted for hydrolysis by the serine proteases of blood coagulation and fi brinolysis, and substrate structure-efficiency correlations were exami ned. PNS-substrates which contain Lys in the P-1 position are specific for Lys-plasmin and are either not hydrolyzed or hydrolyzed at a rela tively low rate by factor Xa, thrombin, or urokinase-type plasminogen activator (uPA). These substrates allow quantitation of Lys-plasmin at concentrations as low as 1 pM. Eighteen of over 90 substrates tested for factor XIa are hydrolyzed by this enzyme at a relatively high rate reaching a k(cat) value of 170 s(-1) and allowing quantitation of fac tor XIa at 10 fM. Eighteen of almost 90 PNS-substrates tested display high specificity for thrombin, some exceeding that for factor Xa by >1 0,000-fold and >100-fold for activated protein C (APC). Seven of these substrates have a k(cat) over 100 s(-1) and three of them have a K-M below 1 mu M. They allow the quantitation of thrombin at concentration s as low as 20 fM. For APC, uPA and the factor VIIa/tissue factor comp lex, quantitation is feasible at 1 pM concentration. For factor Xa and factor VIIa the limits are 0.4 pM and 40 pM respectively. The PNS-sub strates presented in this study may be employed for the development of direct and sensitive serine protease assays.