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Thromb Haemost 1997; 78(04): 1193-1201
DOI: 10.1055/s-0038-1657714
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Schattauer GmbH Stuttgart

Ultrasensitive Fluorogenic Substrates for Serine Proteases[*]

Saulius Butenas
The Department of Biochemistry, Health Science Complex, University of Vermont, Burlington, VT, USA
,
Maria E DiLorenzo
The Department of Biochemistry, Health Science Complex, University of Vermont, Burlington, VT, USA
,
Kenneth G Mann
The Department of Biochemistry, Health Science Complex, University of Vermont, Burlington, VT, USA
› Author Affiliations
Further Information

Publication History

Received 28 1997

Accepted after resubmission 13 June 1997

Publication Date:
12 July 2018 (online)

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Summary

Selective, sensitive assays for the quantitation of serine proteases involved in coagulation and fibrinolysis have been developed employing fluorogenic substrates containing a 6-amino-1-naphthalenesulfonamide leaving group (PNS-substrates). Over one hundred substrates were evaluated for hydrolysis by the serine proteases of blood coagulation and fibrinolysis, and substrate structure-efficiency correlations were examined. PNS-substrates which contain Lys in the P1 position are specific for Lys-plasmin and are either not hydrolyzed or hydrolyzed at a relatively 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 kcat value of 170 s-1 and allowing quantitation of factor XIa at 10 fM. Eighteen of almost 90 PNS-substrates tested display high specificity for thrombin, some exceeding that for factor Xa by > 10,000-fold and > 100-fold for activated protein C (APC). Seven of these substrates have a over 100 s-1 and three of them have a KM below 1 μM. They allow the quantitation of thrombin at concentrations as low as 20 fM. For APC, uPA and the factor Vila/tissue factor complex, 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-substrates presented in this study may be employed for the development of direct and sensitive serine protease assays.

This work was supported by Grant from the National Institute of Health HL46703


 

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