LETTER TO THE EDITORS-IN-CHIEF
EFFECT OF HEPARIN-BINDING TO THROMBIN ON THROMBIN ACTIVITY MEASURED WITH A CHROMOGENIC SIJBSTRATE Knut Bartl Boehringer Mannheim GmbH, Biochemica Werk Tutzing Forschungszentrum, D-s?32 Tutzing Federal Republic of Germany (Received
18.10.1978.
Accepted
by Editor
B. Blomb&ck)
I would like to contribute to the discussion in the publication of Nordenman and Bjoerk in Thrombosis Research (1). Their paper deals with the binding of heparin to thrombin and the effect of heparin-binding on thrombin activity measured by use of chromogenic substrates, We have obtained data with a different chromogenic substrate supporting their results and which also contradict those of Smith (2). Conflicting results exist in the literature as to the inhibitory effect of heparin on thrombin measured by the substrates Bz-Phe-Val-Arg-pNA (S 2160) and H-D-Phe-Pip-Arg-pNA (S 2238) (1,2). According to the results of Nordenman and Bjoerk inactivation of thrombin by heparin, which had been measured by use of S 2160 and had been attributed to a heparin/ thrombin interaction, was solely caused by turbidities. These turbidities were inferred to be due to the interaction between heparin and the substrate S 2160. During our own experiments on the determination of heparin we also tried to reproduce the work of Smith (2). He reported that heparin exerts an inhibitory effect on thrombin in the absence of antithrombin III, measured with S 2160. We used a different chromogenic substrate in our assay system, the tripeptide Tos-Gly-Pro-Arg-pNA (Chromozym TH). This substrate is known to be an easily soluble substrate for thrombin (3). In our experiments, reaction conditions corresponded to those'used by Smith. However, no inhibition of thrombin could be detected at all. These preliminary results have already been mentioned in a recent publication on the determination of heparin with the substrate Tos-GlyPro-Arg-pNA (4). These results strongly support the conclusions drawn by Nordenman suggesting that the thrombin activity is unaffected by heparin alone. Further our experiments show that the tripeptide TOS-GlyPro-Arg-pNA is highly suitable for the determination of coagulation parameters measurable by involvement of the proteolytic enzyme thrombin.
References 1. NORDENMAN, B. and BJOERK, I. Studies on the binding of heparin to prothrombin and thrombin and the effect of heparin-binding on thrombin 1141
1142
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vol.
13,x0.6
activity. Thromb. Res. 12, 755, 1978. SMITH, G.F. The heparin-thrombin complex in the mechanism of thrombin inactivation by heparin. Biochem. Biophys. Res. Common. 77, 111, 1977 3. WITT, I. Beue Methoden der GerinnungsanaIyee mit chromogenen Subetraten. J. Clin. Chem. Clin. Biochem. 15, 239, 1977. 4. BARTL, K., DORSCH, E. and ZIEGENHOBR, J. Determination of heparin using thrombin and the substrate Tos-Gly-Pro-Arg-pNA. Z. Anal. Chem. 290, 102, 1978. 2.
EFFECT OF HEPARIN-BINDING TO THROMBIN ON THROMBIN ACTIVITY MEASURED WITH A CHROMOGENIC SIJBSTRATE Knut Bartl Boehringer Mannheim GmbH, Biochemica Werk Tutzing Forschungszentrum, D-s?32 Tutzing Federal Republic of Germany (Received
18.10.1978.
Accepted
by Editor
B. Blomb&ck)
I would like to contribute to the discussion in the publication of Nordenman and Bjoerk in Thrombosis Research (1). Their paper deals with the binding of heparin to thrombin and the effect of heparin-binding on thrombin activity measured by use of chromogenic substrates, We have obtained data with a different chromogenic substrate supporting their results and which also contradict those of Smith (2). Conflicting results exist in the literature as to the inhibitory effect of heparin on thrombin measured by the substrates Bz-Phe-Val-Arg-pNA (S 2160) and H-D-Phe-Pip-Arg-pNA (S 2238) (1,2). According to the results of Nordenman and Bjoerk inactivation of thrombin by heparin, which had been measured by use of S 2160 and had been attributed to a heparin/ thrombin interaction, was solely caused by turbidities. These turbidities were inferred to be due to the interaction between heparin and the substrate S 2160. During our own experiments on the determination of heparin we also tried to reproduce the work of Smith (2). He reported that heparin exerts an inhibitory effect on thrombin in the absence of antithrombin III, measured with S 2160. We used a different chromogenic substrate in our assay system, the tripeptide Tos-Gly-Pro-Arg-pNA (Chromozym TH). This substrate is known to be an easily soluble substrate for thrombin (3). In our experiments, reaction conditions corresponded to those'used by Smith. However, no inhibition of thrombin could be detected at all. These preliminary results have already been mentioned in a recent publication on the determination of heparin with the substrate Tos-GlyPro-Arg-pNA (4). These results strongly support the conclusions drawn by Nordenman suggesting that the thrombin activity is unaffected by heparin alone. Further our experiments show that the tripeptide TOS-GlyPro-Arg-pNA is highly suitable for the determination of coagulation parameters measurable by involvement of the proteolytic enzyme thrombin.
References 1. NORDENMAN, B. and BJOERK, I. Studies on the binding of heparin to prothrombin and thrombin and the effect of heparin-binding on thrombin 1141
1142
HEP‘4RIN AXII THROMBIN
vol.
13,x0.6
activity. Thromb. Res. 12, 755, 1978. SMITH, G.F. The heparin-thrombin complex in the mechanism of thrombin inactivation by heparin. Biochem. Biophys. Res. Common. 77, 111, 1977 3. WITT, I. Beue Methoden der GerinnungsanaIyee mit chromogenen Subetraten. J. Clin. Chem. Clin. Biochem. 15, 239, 1977. 4. BARTL, K., DORSCH, E. and ZIEGENHOBR, J. Determination of heparin using thrombin and the substrate Tos-Gly-Pro-Arg-pNA. Z. Anal. Chem. 290, 102, 1978. 2.
