Studies on the Different Modes of Action of the Anticoagulant Protease Inhibitors DX-9065a and Argatroban

The present study began with mathematical modeling of how inhibitors of both factor Xa (fXa) and thrombin affect extrinsic pathway-triggered blood coagulation. Numerical simulation demonstrated a stronger inhibition of thrombin generation by a thrombin inhibitor than a fXa inhibitor, but both prolonged clot time to a similar extent when they were given an equal dissociation constant (30 nm) for interaction with their respective target enzymes. These differences were then tested by comparison with the real inhibitors DX-9065a and argatroban, specific competitive inhibitors of fXa and thrombin, respectively, with similar K ivalues. Comparisons were made in extrinsically triggered human citrated plasma, for which endogenous thrombin potential and clot formation were simultaneously measured with a Wallac multilabel counter equipped with both fluorometric and photometric detectors and a fluorogenic reporter substrate. The results demonstrated stronger inhibition of endogenous thrombin potential by argatroban than by DX-9065a, especially when coagulation was initiated at higher tissue factor concentrations, while argatroban appeared to be slightly less potent in its ability to prolong clot time. This study demonstrates differential inhibition of thrombin generation by fXa and thrombin inhibitors and has implications for the pharmacological regulation of blood coagulation by the anticoagulant protease inhibitors. The present study began with mathematical modeling of how inhibitors of both factor Xa (fXa) and thrombin affect extrinsic pathway-triggered blood coagulation. Numerical simulation demonstrated a stronger inhibition of thrombin generation by a thrombin inhibitor than a fXa inhibitor, but both prolonged clot time to a similar extent when they were given an equal dissociation constant (30 nm) for interaction with their respective target enzymes. These differences were then tested by comparison with the real inhibitors DX-9065a and argatroban, specific competitive inhibitors of fXa and thrombin, respectively, with similar K ivalues. Comparisons were made in extrinsically triggered human citrated plasma, for which endogenous thrombin potential and clot formation were simultaneously measured with a Wallac multilabel counter equipped with both fluorometric and photometric detectors and a fluorogenic reporter substrate. The results demonstrated stronger inhibition of endogenous thrombin potential by argatroban than by DX-9065a, especially when coagulation was initiated at higher tissue factor concentrations, while argatroban appeared to be slightly less potent in its ability to prolong clot time. This study demonstrates differential inhibition of thrombin generation by fXa and thrombin inhibitors and has implications for the pharmacological regulation of blood coagulation by the anticoagulant protease inhibitors. factor Xa Z-Gly-Gly-Arg-aminomethylcoumarin 7-amino-4-methylcoumarin tissue factor endogenous thrombin potential clot time The purpose of the current study is to investigate the differences in the modes of action of two anticoagulant protease inhibitors, DX-9065a (1Hara T. Yokoyama A. Ishihara H. Yokoyama Y. Nagahara T. Iwamoto M. Thromb. Haemost. 1994; 71: 314-319Crossref PubMed Scopus (178) Google Scholar) and argatroban (2Kikumoto R. Tamao Y. Tezuka T. Tonomura S. Hara H. Ninomiya K. Hijikata A. Okamoto S. Biochemistry. 1984; 23: 85-90Crossref PubMed Scopus (294) Google Scholar). DX-9065a is a synthetic fXa1 inhibitor currently under clinical development. Argatroban is a synthetic thrombin inhibitor, which is already approved in Japan and the United States as an anticoagulant agent. Despite targeting different enzymes, the two inhibitors are similar in that they are synthetic, low molecular weight compounds, are monospecific to one of the coagulation cascade proteases, and can inhibit their target enzymes directly and competitively. Moreover, they have similar K i values for inhibition of the respective target enzymes: K ivalues are 41 nm for fXa with DX-9065a (1Hara T. Yokoyama A. Ishihara H. Yokoyama Y. Nagahara T. Iwamoto M. Thromb. Haemost. 1994; 71: 314-319Crossref PubMed Scopus (178) Google Scholar) and 39 nm for thrombin with argatroban (2Kikumoto R. Tamao Y. Tezuka T. Tonomura S. Hara H. Ninomiya K. Hijikata A. Okamoto S. Biochemistry. 1984; 23: 85-90Crossref PubMed Scopus (294) Google Scholar).The coagulation cascade comprises multiple sequential steps of protease reactions, and it is not intuitively obvious how the kinetics of the activation of each step contributes to the overall cascade. In the current study, as an attempt to better understand the differences between fXa inhibitors and thrombin inhibitors, a mathematical model of blood coagulation has been developed to simulate actions of these compounds. Computer modeling shows that a thrombin inhibitor gives a stronger inhibitory effect on thrombin generation than a fXa inhibitor. However, the two types of inhibitor prolong clot time to a similar extent when coagulation is triggered with an extrinsic pathway stimulus of the same intensity.In the experimental portions of the current study, a microplate method was developed to examine thrombin generation and clot formation simultaneously during the course of plasma coagulation. The method utilized a fluorogenic reporter substrate, Z-GGR-AMC (3Hemker H.C. Giesen P.L. Ramjee M. Wagenvoord R. Beguin S. Thromb. Haemost. 2000; 83: 589-591Crossref PubMed Scopus (250) Google Scholar, 4Ramjee M. Anal. Biochem. 2000; 277: 11-18Crossref PubMed Scopus (37) Google Scholar), in combination with a Wallac multilabel counter equipped with both fluorometric and photometric detectors. This methodology was used to compare the abilities of DX-9065a and argatroban to inhibit thrombin generation as well as to prolong clot time during extrinsically initiated plasma coagulation. It was shown that, while the two compounds prolonged clot time to a comparable extent, argatroban exerted a much stronger inhibitory effect on thrombin generation than did DX-9065a. The results were in good agreement with those predicted by the numerical simulation described herein.DISCUSSIONWhile thrombin generation initiated by extrinsic stimuli occurs in an explosive fashion during the propagation phase of blood coagulation, the thrombin concentration is very low during the initiation phase,i.e. before the propagation phase. The duration of the initiation phase normally coincides with the clot time of coagulation and, therefore, represents anticoagulant potencies if it is assessed in the presence of anticoagulants. The results of numerical simulation in the present study have predicted that fXa inhibitors and thrombin inhibitors might display comparable anticoagulant potencies as long as they are presumed to be competitive inhibitors with the same kinetic properties for interaction with their respective target enzymes. This was demonstrated experimentally by comparing DX-9065a and argatroban for their ability to prolong the clot time in human plasma (Fig. 3). It has been shown by these experiments that DX-9065a and argatroban are virtually equipotent in maintaining a subthreshold thrombin concentration during the initiation phase of extrinsically triggered plasma coagulation.As predicted for the hypothetical inhibitors in the mathematical model, however, DX-9065a and argatroban exerted a differential influence on thrombin generation during the propagation phase of plasma coagulation. This was typically shown at the highest TF concentration examined, where DX-9065a reduced ETP much less effectively than did argatroban (Fig. 4 A). The previous comparative studies on DX-9065a and other anticoagulants also demonstrated that DX-9065a was effective in prolonging clot time, but was less potent in reducing ETP than other anticoagulants (11Prasa D. Svendsen L. Stürzebecher J. Tromb. Haemost. 1997; 78: 1215-1220Crossref PubMed Scopus (39) Google Scholar, 12Peyrou V. Lormeau J.C. Hérault J.P. Gaich C. Pfliegger A.M. Herbert J.M. Thromb. Haemost. 1999; 81: 400-406Crossref PubMed Scopus (141) Google Scholar). Thus these results are in good agreement with those obtained at higher TF concentrations in the current study.One explanation for the different abilities of the selective inhibitors to affect thrombin generation is that fXa inhibitors are able to inhibit de novo production of thrombin but not the ongoing actions of preformed thrombin. In contrast, thrombin inhibitors are able to inhibit not only the production of thrombin but also the actions of thrombin directly. The previous experimental studies, however, are not consistent with such a mechanism-based, straightforward rationalization. Hirudin and hirulog are known to be specific and direct inhibitors of thrombin, which delay thrombin peaks effectively during coagulation (12Peyrou V. Lormeau J.C. Hérault J.P. Gaich C. Pfliegger A.M. Herbert J.M. Thromb. Haemost. 1999; 81: 400-406Crossref PubMed Scopus (141) Google Scholar, 13Prasa D. Svendsen L. Stürzebecher J. Tromb. Haemost. 1997; 77: 498-503Crossref PubMed Scopus (42) Google Scholar), yet they do not reduce ETP as effectively as other anticoagulants (12Peyrou V. Lormeau J.C. Hérault J.P. Gaich C. Pfliegger A.M. Herbert J.M. Thromb. Haemost. 1999; 81: 400-406Crossref PubMed Scopus (141) Google Scholar) and display properties similar to those of DX-9065a. Although hirudin is a very potent inhibitor of thrombin, its tight but rather slow binding to thrombin (14Kuzmiè P. Anal. Biochem. 1999; 267: 17-23Crossref PubMed Scopus (13) Google Scholar) might somehow affect its overall anticoagulant properties in the plasma milieu. Furthermore, a recombinant tick anticoagulant peptide (11Prasa D. Svendsen L. Stürzebecher J. Tromb. Haemost. 1997; 78: 1215-1220Crossref PubMed Scopus (39) Google Scholar) and a pentasaccharide, SR-90107A (12Peyrou V. Lormeau J.C. Hérault J.P. Gaich C. Pfliegger A.M. Herbert J.M. Thromb. Haemost. 1999; 81: 400-406Crossref PubMed Scopus (141) Google Scholar), are two examples of fXa inhibitors reported to suppress ETPs more efficiently than DX-9065a. The reasons for these unpredictable observations are not clear at present. However, it should be pointed out that, for a given anticoagulant protease inhibitor, not only which enzyme is inhibited but also its precise mechanism of inhibition are important factors influencing its impact on the overall dynamic process during blood coagulation. Fortunately, both DX-9065a (1Hara T. Yokoyama A. Ishihara H. Yokoyama Y. Nagahara T. Iwamoto M. Thromb. Haemost. 1994; 71: 314-319Crossref PubMed Scopus (178) Google Scholar) and argatroban (2Kikumoto R. Tamao Y. Tezuka T. Tonomura S. Hara H. Ninomiya K. Hijikata A. Okamoto S. Biochemistry. 1984; 23: 85-90Crossref PubMed Scopus (294) Google Scholar) are simple, competitive, reversible inhibitors, with which one can simply postulate a rapid equilibrium for the dissociation-association interactions with their respective target enzymes. This might be a reason why numerical simulation models the dynamic effects of these compounds quite well. Thus, the findings presented in this report can be applied to other inhibitors of the same mechanism but only when they display similar kinetic properties.The differential effects of the inhibitors in the simulation results are best perceived when thrombin activity curves are viewed with a proper time scale as shown in Fig. 2. If ETP, a time-integral of a thrombin activity curve, is calculated for an infinite time interval for a given set of numerical simulation data, the resultant ETP values approach invariable ones that are no longer dependent on inhibitor concentrations (data not shown). Thus, the differential effect presented in Fig. 2 should be regarded as an apparent one that does not necessarily represent the mathematical characteristics of the model. It is not known, however, whether the same explanation is applicable to the experimental results from the thrombin generation assays where ETP can be obtained only within a limited time scale.Interestingly, the inhibitory effect of DX-9065a on ETP varied depending on the concentration of TF used to stimulate coagulation. The initiation of thrombin generation at the highest thromboplastin concentration resulted in up to more than a 5-fold in the ETP for DX-9065a with that at the thromboplastin concentration a effect was only when plasma was with DX-9065a. Argatroban did not effect on ETP The two compounds only a limited in the ability to inhibit ETP at the thromboplastin concentration (Fig. 4 and Moreover, no effect was with 2 of whether plasma was with DX-9065a argatroban the of inhibitors, it was also shown that ETP with TF concentrations the values inhibitors in This have to the effect of DX-9065a on the numerical simulation not of these effects on thrombin generation when the concentration of was varied a of concentrations (data not shown). The mechanism for the regulation is However, it to that the effects might have important in the regulation of blood coagulation. The purpose of the current study is to investigate the differences in the modes of action of two anticoagulant protease inhibitors, DX-9065a (1Hara T. Yokoyama A. Ishihara H. Yokoyama Y. Nagahara T. Iwamoto M. Thromb. Haemost. 1994; 71: 314-319Crossref PubMed Scopus (178) Google Scholar) and argatroban (2Kikumoto R. Tamao Y. Tezuka T. Tonomura S. Hara H. Ninomiya K. Hijikata A. Okamoto S. Biochemistry. 1984; 23: 85-90Crossref PubMed Scopus (294) Google Scholar). DX-9065a is a synthetic fXa1 inhibitor currently under clinical development. Argatroban is a synthetic thrombin inhibitor, which is already approved in Japan and the United States as an anticoagulant agent. Despite targeting different enzymes, the two inhibitors are similar in that they are synthetic, low molecular weight compounds, are monospecific to one of the coagulation cascade proteases, and can inhibit their target enzymes directly and competitively. Moreover, they have similar K i values for inhibition of the respective target enzymes: K ivalues are 41 nm for fXa with DX-9065a (1Hara T. Yokoyama A. Ishihara H. Yokoyama Y. Nagahara T. Iwamoto M. Thromb. Haemost. 1994; 71: 314-319Crossref PubMed Scopus (178) Google Scholar) and 39 nm for thrombin with argatroban (2Kikumoto R. Tamao Y. Tezuka T. Tonomura S. Hara H. Ninomiya K. Hijikata A. Okamoto S. Biochemistry. 1984; 23: 85-90Crossref PubMed Scopus (294) Google Scholar). The coagulation cascade comprises multiple sequential steps of protease reactions, and it is not intuitively obvious how the kinetics of the activation of each step contributes to the overall cascade. In the current study, as an attempt to better understand the differences between fXa inhibitors and thrombin inhibitors, a mathematical model of blood coagulation has been developed to simulate actions of these compounds. Computer modeling shows that a thrombin inhibitor gives a stronger inhibitory effect on thrombin generation than a fXa inhibitor. However, the two types of inhibitor prolong clot time to a similar extent when coagulation is triggered with an extrinsic pathway stimulus of the same In the experimental portions of the current study, a microplate method was developed to examine thrombin generation and clot formation simultaneously during the course of plasma coagulation. The method utilized a fluorogenic reporter substrate, Z-GGR-AMC (3Hemker H.C. Giesen P.L. Ramjee M. Wagenvoord R. Beguin S. Thromb. Haemost. 2000; 83: 589-591Crossref PubMed Scopus (250) Google Scholar, 4Ramjee M. Anal. Biochem. 2000; 277: 11-18Crossref PubMed Scopus (37) Google Scholar), in combination with a Wallac multilabel counter equipped with both fluorometric and photometric detectors. This methodology was used to compare the abilities of DX-9065a and argatroban to inhibit thrombin generation as well as to prolong clot time during extrinsically initiated plasma coagulation. It was shown that, while the two compounds prolonged clot time to a comparable extent, argatroban exerted a much stronger inhibitory effect on thrombin generation than did DX-9065a. The results were in good agreement with those predicted by the numerical simulation described thrombin generation initiated by extrinsic stimuli occurs in an explosive fashion during the propagation phase of blood coagulation, the thrombin concentration is very low during the initiation phase,i.e. before the propagation phase. The duration of the initiation phase normally coincides with the clot time of coagulation and, therefore, represents anticoagulant potencies if it is assessed in the presence of anticoagulants. The results of numerical simulation in the present study have predicted that fXa inhibitors and thrombin inhibitors might display comparable anticoagulant potencies as long as they are presumed to be competitive inhibitors with the same kinetic properties for interaction with their respective target enzymes. This was demonstrated experimentally by comparing DX-9065a and argatroban for their ability to prolong the clot time in human plasma (Fig. 3). It has been shown by these experiments that DX-9065a and argatroban are virtually equipotent in maintaining a subthreshold thrombin concentration during the initiation phase of extrinsically triggered plasma coagulation.As predicted for the hypothetical inhibitors in the mathematical model, however, DX-9065a and argatroban exerted a differential influence on thrombin generation during the propagation phase of plasma coagulation. This was typically shown at the highest TF concentration examined, where DX-9065a reduced ETP much less effectively than did argatroban (Fig. 4 A). The previous comparative studies on DX-9065a and other anticoagulants also demonstrated that DX-9065a was effective in prolonging clot time, but was less potent in reducing ETP than other anticoagulants (11Prasa D. Svendsen L. Stürzebecher J. Tromb. Haemost. 1997; 78: 1215-1220Crossref PubMed Scopus (39) Google Scholar, 12Peyrou V. Lormeau J.C. Hérault J.P. Gaich C. Pfliegger A.M. Herbert J.M. Thromb. Haemost. 1999; 81: 400-406Crossref PubMed Scopus (141) Google Scholar). Thus these results are in good agreement with those obtained at higher TF concentrations in the current study.One explanation for the different abilities of the selective inhibitors to affect thrombin generation is that fXa inhibitors are able to inhibit de novo production of thrombin but not the ongoing actions of preformed thrombin. In contrast, thrombin inhibitors are able to inhibit not only the production of thrombin but also the actions of thrombin directly. The previous experimental studies, however, are not consistent with such a mechanism-based, straightforward rationalization. Hirudin and hirulog are known to be specific and direct inhibitors of thrombin, which delay thrombin peaks effectively during coagulation (12Peyrou V. Lormeau J.C. Hérault J.P. Gaich C. Pfliegger A.M. Herbert J.M. Thromb. Haemost. 1999; 81: 400-406Crossref PubMed Scopus (141) Google Scholar, 13Prasa D. Svendsen L. Stürzebecher J. Tromb. Haemost. 1997; 77: 498-503Crossref PubMed Scopus (42) Google Scholar), yet they do not reduce ETP as effectively as other anticoagulants (12Peyrou V. Lormeau J.C. Hérault J.P. Gaich C. Pfliegger A.M. Herbert J.M. Thromb. Haemost. 1999; 81: 400-406Crossref PubMed Scopus (141) Google Scholar) and display properties similar to those of DX-9065a. Although hirudin is a very potent inhibitor of thrombin, its tight but rather slow binding to thrombin (14Kuzmiè P. Anal. Biochem. 1999; 267: 17-23Crossref PubMed Scopus (13) Google Scholar) might somehow affect its overall anticoagulant properties in the plasma milieu. Furthermore, a recombinant tick anticoagulant peptide (11Prasa D. Svendsen L. Stürzebecher J. Tromb. Haemost. 1997; 78: 1215-1220Crossref PubMed Scopus (39) Google Scholar) and a pentasaccharide, SR-90107A (12Peyrou V. Lormeau J.C. Hérault J.P. Gaich C. Pfliegger A.M. Herbert J.M. Thromb. Haemost. 1999; 81: 400-406Crossref PubMed Scopus (141) Google Scholar), are two examples of fXa inhibitors reported to suppress ETPs more efficiently than DX-9065a. The reasons for these unpredictable observations are not clear at present. However, it should be pointed out that, for a given anticoagulant protease inhibitor, not only which enzyme is inhibited but also its precise mechanism of inhibition are important factors influencing its impact on the overall dynamic process during blood coagulation. Fortunately, both DX-9065a (1Hara T. Yokoyama A. Ishihara H. Yokoyama Y. Nagahara T. Iwamoto M. Thromb. Haemost. 1994; 71: 314-319Crossref PubMed Scopus (178) Google Scholar) and argatroban (2Kikumoto R. Tamao Y. Tezuka T. Tonomura S. Hara H. Ninomiya K. Hijikata A. Okamoto S. Biochemistry. 1984; 23: 85-90Crossref PubMed Scopus (294) Google Scholar) are simple, competitive, reversible inhibitors, with which one can simply postulate a rapid equilibrium for the dissociation-association interactions with their respective target enzymes. This might be a reason why numerical simulation models the dynamic effects of these compounds quite well. Thus, the findings presented in this report can be applied to other inhibitors of the same mechanism but only when they display similar kinetic properties.The differential effects of the inhibitors in the simulation results are best perceived when thrombin activity curves are viewed with a proper time scale as shown in Fig. 2. If ETP, a time-integral of a thrombin activity curve, is calculated for an infinite time interval for a given set of numerical simulation data, the resultant ETP values approach invariable ones that are no longer dependent on inhibitor concentrations (data not shown). Thus, the differential effect presented in Fig. 2 should be regarded as an apparent one that does not necessarily represent the mathematical characteristics of the model. It is not known, however, whether the same explanation is applicable to the experimental results from the thrombin generation assays where ETP can be obtained only within a limited time scale.Interestingly, the inhibitory effect of DX-9065a on ETP varied depending on the concentration of TF used to stimulate coagulation. The initiation of thrombin generation at the highest thromboplastin concentration resulted in up to more than a 5-fold in the ETP for DX-9065a with that at the thromboplastin concentration a effect was only when plasma was with DX-9065a. Argatroban did not effect on ETP The two compounds only a limited in the ability to inhibit ETP at the thromboplastin concentration (Fig. 4 and Moreover, no effect was with 2 of whether plasma was with DX-9065a argatroban the of inhibitors, it was also shown that ETP with TF concentrations the values inhibitors in This have to the effect of DX-9065a on the numerical simulation not of these effects on thrombin generation when the concentration of was varied a of concentrations (data not shown). The mechanism for the regulation is However, it to that the effects might have important in the regulation of blood coagulation. thrombin generation initiated by extrinsic stimuli occurs in an explosive fashion during the propagation phase of blood coagulation, the thrombin concentration is very low during the initiation phase,i.e. before the propagation phase. The duration of the initiation phase normally coincides with the clot time of coagulation and, therefore, represents anticoagulant potencies if it is assessed in the presence of anticoagulants. The results of numerical simulation in the present study have predicted that fXa inhibitors and thrombin inhibitors might display comparable anticoagulant potencies as long as they are presumed to be competitive inhibitors with the same kinetic properties for interaction with their respective target enzymes. This was demonstrated experimentally by comparing DX-9065a and argatroban for their ability to prolong the clot time in human plasma (Fig. 3). It has been shown by these experiments that DX-9065a and argatroban are virtually equipotent in maintaining a subthreshold thrombin concentration during the initiation phase of extrinsically triggered plasma coagulation. predicted for the hypothetical inhibitors in the mathematical model, however, DX-9065a and argatroban exerted a differential influence on thrombin generation during the propagation phase of plasma coagulation. This was typically shown at the highest TF concentration examined, where DX-9065a reduced ETP much less effectively than did argatroban (Fig. 4 A). The previous comparative studies on DX-9065a and other anticoagulants also demonstrated that DX-9065a was effective in prolonging clot time, but was less potent in reducing ETP than other anticoagulants (11Prasa D. Svendsen L. Stürzebecher J. Tromb. Haemost. 1997; 78: 1215-1220Crossref PubMed Scopus (39) Google Scholar, 12Peyrou V. Lormeau J.C. Hérault J.P. Gaich C. Pfliegger A.M. Herbert J.M. Thromb. Haemost. 1999; 81: 400-406Crossref PubMed Scopus (141) Google Scholar). Thus these results are in good agreement with those obtained at higher TF concentrations in the current explanation for the different abilities of the selective inhibitors to affect thrombin generation is that fXa inhibitors are able to inhibit de novo production of thrombin but not the ongoing actions of preformed thrombin. In contrast, thrombin inhibitors are able to inhibit not only the production of thrombin but also the actions of thrombin directly. The previous experimental studies, however, are not consistent with such a mechanism-based, straightforward rationalization. Hirudin and hirulog are known to be specific and direct inhibitors of thrombin, which delay thrombin peaks effectively during coagulation (12Peyrou V. Lormeau J.C. Hérault J.P. Gaich C. Pfliegger A.M. Herbert J.M. Thromb. Haemost. 1999; 81: 400-406Crossref PubMed Scopus (141) Google Scholar, 13Prasa D. Svendsen L. Stürzebecher J. Tromb. Haemost. 1997; 77: 498-503Crossref PubMed Scopus (42) Google Scholar), yet they do not reduce ETP as effectively as other anticoagulants (12Peyrou V. Lormeau J.C. Hérault J.P. Gaich C. Pfliegger A.M. Herbert J.M. Thromb. Haemost. 1999; 81: 400-406Crossref PubMed Scopus (141) Google Scholar) and display properties similar to those of DX-9065a. Although hirudin is a very potent inhibitor of thrombin, its tight but rather slow binding to thrombin (14Kuzmiè P. Anal. Biochem. 1999; 267: 17-23Crossref PubMed Scopus (13) Google Scholar) might somehow affect its overall anticoagulant properties in the plasma milieu. Furthermore, a recombinant tick anticoagulant peptide (11Prasa D. Svendsen L. Stürzebecher J. Tromb. Haemost. 1997; 78: 1215-1220Crossref PubMed Scopus (39) Google Scholar) and a pentasaccharide, SR-90107A (12Peyrou V. Lormeau J.C. Hérault J.P. Gaich C. Pfliegger A.M. Herbert J.M. Thromb. Haemost. 1999; 81: 400-406Crossref PubMed Scopus (141) Google Scholar), are two examples of fXa inhibitors reported to suppress ETPs more efficiently than DX-9065a. The reasons for these unpredictable observations are not clear at present. However, it should be pointed out that, for a given anticoagulant protease inhibitor, not only which enzyme is inhibited but also its precise mechanism of inhibition are important factors influencing its impact on the overall dynamic process during blood coagulation. Fortunately, both DX-9065a (1Hara T. Yokoyama A. Ishihara H. Yokoyama Y. Nagahara T. Iwamoto M. Thromb. Haemost. 1994; 71: 314-319Crossref PubMed Scopus (178) Google Scholar) and argatroban (2Kikumoto R. Tamao Y. Tezuka T. Tonomura S. Hara H. Ninomiya K. Hijikata A. Okamoto S. Biochemistry. 1984; 23: 85-90Crossref PubMed Scopus (294) Google Scholar) are simple, competitive, reversible inhibitors, with which one can simply postulate a rapid equilibrium for the dissociation-association interactions with their respective target enzymes. This might be a reason why numerical simulation models the dynamic effects of these compounds quite well. Thus, the findings presented in this report can be applied to other inhibitors of the same mechanism but only when they display similar kinetic The differential effects of the inhibitors in the simulation results are best perceived when thrombin activity curves are viewed with a proper time scale as shown in Fig. 2. If ETP, a time-integral of a thrombin activity curve, is calculated for an infinite time interval for a given set of numerical simulation data, the resultant ETP values approach invariable ones that are no longer dependent on inhibitor concentrations (data not shown). Thus, the differential effect presented in Fig. 2 should be regarded as an apparent one that does not necessarily represent the mathematical characteristics of the model. It is not known, however, whether the same explanation is applicable to the experimental results from the thrombin generation assays where ETP can be obtained only within a limited time the inhibitory effect of DX-9065a on ETP varied depending on the concentration of TF used to stimulate coagulation. The initiation of thrombin generation at the highest thromboplastin concentration resulted in up to more than a 5-fold in the ETP for DX-9065a with that at the thromboplastin concentration a effect was only when plasma was with DX-9065a. Argatroban did not effect on ETP The two compounds only a limited in the ability to inhibit ETP at the thromboplastin concentration (Fig. 4 and Moreover, no effect was with 2 of whether plasma was with DX-9065a argatroban In the of inhibitors, it was also shown that ETP with TF concentrations the values inhibitors in This have to the effect of DX-9065a on the numerical simulation not of these effects on thrombin generation when the concentration of was varied a of concentrations (data not shown). The mechanism for the regulation is However, it to that the effects might have important in the regulation of blood coagulation. with with