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
References
1
Colman RW,
Hirsh J,
Marder VC,
Salzman EW.
Hemostatis and Thrombosis. Basic Principles and Clinical Practice. Philadelphia: J. B. Lippincott Company; 1994
3
Morrissey JH,
Macik BG,
Neuenschwander PF,
Comp PC.
Quantation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation. Blood 1993; 81: 734-744
7
Shigematsu Y,
Miyata T,
Higashi S,
Miki T,
Sadler JE,
Iwanga S.
Expression of human soluble tissue factor in yeast and enzymatic properties of its complex with factor Vila. J Biol Chem 1992; 267: 21329-21337
9
Lawson JH,
Kalafatis M,
Stram S,
Mann KG.
A model for the tissue factor pathway to thrombin II. An empirical study. J Biol Chem 1994; 269: 23357-23366
12
Bajaj SP,
Rapaport SI,
Brown SF.
Isolation and characterization of human factor VII. Activation of factor VII by factor Xa. J Biol Chem 1981; 256: 253-259
14
Darras V,
Thienpont M,
Stump DC,
Collen D.
Measurement of urokinase- type plasminogen activator (u-PA) with an enzyme-linked immunosorbent assay (ELISA) based on three murine monoclonal antibodies. Thromb Haemost 1986; 56: 411-414
15
Lawson JH,
Mann KG.
Cooparative activation of human factor IX by the human extrinsic pathway of blood coagulation. J Biol Chem 1991; 266: 11317-11327
18
Seligsohn U,
Østerud B,
Rapaport SI.
Coupled amidolytic assay for factor VII: its use with a clotting assay to determine the activity state of factor VH. Blood 1978; 52: 978-988
19
Ruf W,
Edgington TS.
An anti-tissue factor monoclonal antibody which inhibits TF-VIIa complex is a potent anticoagulant in plasma. Thromb Haemost 1991; 66: 529-533
21
Barlow GH,
Francis CW,
Marder VJ.
On the conversion of high molecular weight urokinase to the low molecular weight form by plasmin. Thromb Res 1981; 23: 54-61
22
Gyzander E,
Eriksson E,
Teger-Nilsson AC.
A sensitive assay for tissue plasminogen activator activity in plasma using adsorption on lysine-Sepha- rose. Thromb Res 1984; 35: 547-558
23
Verheijen JH,
de JongYF,
Chang GTG.
Quantitative analysis of mixtures of one-chain and two-chain tissue-type plasminogen activator with a spectrophotometric method. Thromb Res 1985; 39: 281-288
26
Lottenberg R,
Christensen U,
Jackson CM,
Coleman PL.
Assay of coagulation proteases using peptide chromogenic and fluorogenic substrates. Methods Enzymol 1981; 80: 341-361
27
Butenas S,
Orfeo T,
Lawson JH,
Mann KG.
Aminonaphthalenesulfona-mides, a new class of modifiable fluorescent detecting groups and their use in substrates for serine protease enzymes. Biochemistry 1992; 31: 5399-5411
31
Zushi M,
Gomi K,
Yamamoto S,
Maruyama I,
Hayashi T,
Suzuki K.
The last three consecutive epiderman growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C- activating cofactor activity and anticoagulant activity. J Biol Chem 1989; 264: 10351-10353
32
Horie S,
Ishi H,
Hara H,
Kazama M.
Enhancement of thrombin-thrombomodulin-catalyzed protein C activation by phosphatidylethanolamine containing unsaturated fatty acids: possible physiological significance of phosphatidylethanolamine in anticoagulant activity of thrombomodulin. Biochem J 1994; 301: 683-691
33
Miyata T,
Funatsu A,
Kato H.
Chemical cross-linking of activated coagulation factor VH with soluble tissue factor calcium ions are not essential for full amidolytic activity of the factor VDa-tissue factor complex after complex formation. J Biochem 1995; 117: 836-844
35
Higgins DL,
Mann KG.
The interaction of bovine factor V and factor V-de- rived peptides with phospholipid vesicles. J Biol Chem 1983; 258: 6503-6508
39
Weinstein MJ,
Doolittle RF.
specificities of thrombin, plasmin and trypsin with regard to synthetic and natural substrates and inhibitors. Biochim Biophys Acta 1972; 258: 577-590
42
Kiss I,
Aurell L,
Pozsgay M,
Elodi P.
Investigation on the substrate specificity of human plasmin using tripeptidyl-p-nitroanilide substrates. Biochem Biophys Res Commun 1985; 131: 928-934
44
Butenas S,
Kalafatis M,
Mann KG.
Analysis of tissue plasminogen activator specificity using peptidyl fluorogenic substrates. Biochemistry 1997; 36: 2123-2131
45
Lottenberg R,
Hall JA,
Blinder EP,
Jackson CM.
The action of thrombin on peptide p-nitroenilide substrates. Substrate selectivity and examination of hydrolysis under different reaction conditions. Biochim Biophys Acta 1983; 742: 539-557
46
Rezaie WR,
Esmon CT.
Convertion of glutamic acid 192 to glutamine in activated protein C changes the substrate specificity and increases reactivity toward macromolecular inhibitors. J Biol Chem 1993; 268: 19943-19948
47
Qiu X,
Padmanabhan KP,
Carperos VE,
Tulinsky A,
Kline T,
Maraganore JM,
Fenton II JW.
Structure of the hirulog 3-thrombin complex and nature of the S' subsites of substrates and inhibitors. Biochemistry 1992; 31: 11689-11697
48
Le BonniecBF,
Myles T,
Johnson T,
Knight CG,
Tapparelli C,
Stone SR.
Characterization of the P2' and P3' specificities of thrombin using fluorescence-quenched substrates and mapping of the subsites by mutagenesis. Biochemistry 1996; 35: 7114-7122
51
von demBorne PA,
Koppelman SJ,
Bouma BN,
Meijers JCM.
Surface independent factor XI activation by thrombin in the presence of high molecular weight kininogen. Thromb Haemostasis 1994; 72: 397-402
52
Andreasen PA,
Petersen LC,
Dana K.
Diversity in catalytic properties of single chain and two chain tissue-type plasminogen activator. Fibrinolysis 1991; 5: 207-215