Phospholipid composition controls thromboplastin sensitivity to individual clotting factors

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1 Journal of Thrombosis and Haemostasis, : ORIGINAL ARTICLE Phospholipid composition controls thromboplastin sensitivity to individual clotting factors S. A. SMITH,* P. C. COMP and J. H. MORRISSEY* *Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL; and Veterans Administration Medical Center, Oklahoma City, OK, USA To cite this article: Smith SA, Comp PC, Morrissey JH. Phospholipid composition controls thromboplastin sensitivity to individual clotting factors. J Thromb Haemost 006; : Summary. Background: Tissue factor is the active ingredient in thromboplastin reagents used to perform prothrombin time (PT) clotting tests to monitor oral anticoagulant therapy and to screen for clotting factor deficiencies. Thromboplastins are complex mixtures prepared from extracts of brain or placenta, although newer thromboplastins contain recombinant tissue factor incorporated into phospholipid vesicles. Thromboplastins can vary widely in their sensitivity to reductions in the levels of vitamin K-dependent clotting factors. A system to compensate for this, the International Sensitivity Index (ISI) and International Normalized Ratio (INR), has revolutionized the monitoring of oral anticoagulant therapy. The INR system is also sometimes used to monitor coagulopathies in patients with sepsis or liver failure, applications for which it was not originally designed and for which it has not been rigorously validated. Objectives: To better understand thromboplastin performance, we systematically investigated which properties of recombinant thromboplastins influence their sensitivities to changes in the levels of specific clotting factors. Results: We now report that relative sensitivities to changes in the plasma levels of factors V, VII, X (FV, FVII, FX) and prothrombin are differentially influenced by a recombinant thromboplastin s content of phospholipid and sodium chloride. Furthermore, thromboplastins of similar ISI values may exhibit quite different sensitivities to each of these clotting factors. Conclusions: Differing sensitivities of thromboplastin reagents to individual clotting factor levels have implications for monitoring of oral anticoagulant therapy and interpreting results of the PT assay. Keywords: international normalized ratio, international sensitivity index, prothrombin time, tissue factor. Correspondence: James H. Morrissey, Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, 7 Medical Sciences Bldg. MC-7, 06 S. Mathews, Urbana, IL 680, USA. Tel.: ; fax: ; jhmorris@ uiuc.edu Received 7 November 00, accepted January 006 Introduction Successful anticoagulant therapy with oral vitamin K antagonists such as coumarins requires careful monitoring of the patient s prothrombin time (PT) to achieve effective anticoagulation while minimizing the bleeding risk. The PT clotting test is also widely used for general screening of the plasma clotting system to assess preoperative bleeding risk and to evaluate coagulopathies because of liver failure, sepsis, and other illnesses []. Thromboplastin reagents used in PT tests contain tissue factor to trigger coagulation. Originally made from crude tissue extracts of animal or human origin, newer generation thromboplastins contain recombinant human tissue factor (rtf), an integral membrane protein which must be incorporated into phospholipid vesicles for optimal clotting activity [ ]. Thromboplastins can vary widely in their responsiveness to the coagulation deficits induced by oral anticoagulation, which, if left uncorrected, may lead to unacceptable differences in dosing of oral anticoagulation []. A calibration system, the International Sensitivity Index (ISI), was developed to compensate for differences in thromboplastin sensitivity []. The lower the ISI value, the more sensitive the thromboplastin, with ISI values near.0 considered most desirable. A patient s PT value is converted to the International Normalized Ratio (INR) for reporting to the physician using this equation: INR ¼ (PT patient /PT normal ) ISI. The ISI/INR system has vastly improved the standardization of oral anticoagulant therapy and is widely credited with enhancing efficacy and reducing bleeding complications []. Although ISI/INR has improved the monitoring and dosing of oral anticoagulation, some studies have reported discrepant INR values of various patient plasmas even using thromboplastins with similar ISI values [,6 ]. Recombinant thromboplastins have also been criticized for being overly sensitive to plasma FVII levels [7 9]. FVII has a short plasma half-life so its levels may fluctuate, especially during the onset of oral anticoagulant therapy [ ]. The success of the ISI/INR system for monitoring oral anticoagulation has led to its widespread use in correcting PT values from patients whose PT is prolonged because of sepsis, liver failure, and other coagulopathies for which the INR system was not originally designed, and for which it has not been rigorously validated [6 0]. Some studies of liver failure Ó 006 International Society on Thrombosis and Haemostasis

2 Clotting factor sensitivities of thromboplastin 8 have reported relatively poor correlations between INR values obtained with different rabbit brain thromboplastins [] or when comparing rabbit brain, human placental, and human recombinant thromboplastins [7]. Another reported that deficiencies in FV and FVII were generally greater in patients with liver disease than in oral anticoagulation even when INR values were similar, and pointed out that, while INR values obtained with low ISI thromboplastins can be used to quantify coagulopathies in liver patients, they should not be interpreted as having the same meaning as INR values from patients on warfarin [6]. For these reasons, it is important to understand how thromboplastins respond to various clotting factor deficiencies. We previously showed that ISI values of recombinant human thromboplastins depend on NaCl concentration and the phospholipid composition of rtf-liposomes []. We now report that sensitivities of rtf-based thromboplastins to changes in the plasma levels of FV, FVII, FX and prothrombin are differentially influenced by thromboplastin composition. Furthermore, thromboplastins of nearly identical ISI values can have divergent sensitivities to these clotting factors. Materials and methods Materials Purified egg phosphatidylcholine (PC), bovine brain phosphatidylserine (PS), and bovine liver phosphatidylethanolamine (PE) were from Avanti Polar Lipids (Alabaster, AL, USA). Human rtf was expressed in Escherichia coli and purified as described []. FVII, FX, and prothrombin (from human plasma) were from Haematologic Technologies, Inc. (Essex Junction, VT, USA). Hemoliance RecombiPlasTin Ò was from Instrumentation Laboratory (Lexington, MA, USA); Innovin Ò and Thromborel-S Ò were from Dade Behring (Newark, DE, USA); and Neoplastine Cl Plus Ò was from Diagnostica Stago (Parsippany, NJ, USA). Plasmas immunodepleted of individual coagulation factors were from Biopool International (Ventura, CA, USA). Normal plasma pooled from individuals (6 males, females, age 8 6 years) and plasmas from stably anticoagulated patients (receiving Coumadin > months) were from George King Bio-Medical (Overland Park, KS, USA). Platelet-poor plasma was also collected from stably anticoagulated patients at the University of Oklahoma Health Sciences Center GCRC. Recombinant thromboplastins Recombinant human tissue factor was reconstituted into liposomes as described [,] using an 8700: molar ratio of phospholipid to rtf. (Phospholipid compositions are given as mol%.) The synthetic thromboplastins contained 0.0% (w/v) bovine serum albumin,. mm CaCl, 0.0% (w/v) NaN, mm Tris HCl, ph 7., and varying concentrations of relipidated rtf and NaCl (Table ). rtf concentrations were adjusted to yield 0 s clotting times with pooled normal plasma. ISI values were determined via a simplified version of the World Health Organization (WHO) method (relative ISI values) [], using RecombiPlasTin as the reference (machinespecific ISI, 0.9). Tissue factor concentrations of commercial thromboplastins were determined by ELISA []. Clotting assays Prothrombin time clotting assays were performed using a STart coagulometer (Diagnostica Stago): after 0 ll plasma Table Thromboplastin reagents Reagent number Symbol Fig. Phospholipids Phosphatidylserine (PS) (%) Phosphatidylethanolamine (PE) (%) Phosphatidylcholine (PC) (%) NaCl (mm) Tissue factor (TF) (nm) Prothrombin times (PT) (s) International Sensitivity Index (ISI) d (0.) 0.77 h (0.) (0.) (0.) (0.) (0.) (0.) r (0.) (0.).8 Reagent number Symbol Fig. Commercial thromboplastins TF (nm) PT (s) ISI 0 d Innovin Ò (recombinant).6 (0.0) 9.9 (0.0) 0.9 RecombiPlasTin Ò (recombinant) 0.7 (0.07).6 (0.) 0.9 Thromborel S Ò (human placenta) 6.9 (0.8). (0.). Neoplastine Cl +Ò (rabbit brain) ND. (0.).6 Mean TF concentrations (±SE) for reagents 0 were determined by ELISA. PT values are mean clot times (±SE) of pooled normal plasma using three separate thromboplastin preparations. Relative ISI values are given for thromboplastins 9 while the manufacturer-supplied, machinespecific ISI values are reported for thromboplastins 0. ND, not determined. Ó 006 International Society on Thrombosis and Haemostasis

3 8 S. A. Smith et al samples were incubated at 7 C for min in coagulometer cuvettes, clotting was initiated by adding 00 ll prewarmed thromboplastin. Test samples were prepared as mixtures of pooled normal plasma and plasmas deficient in prothrombin, FV, FVII, or FX, resulting in 0%, %,0%, %, or % normal plasma. The is a sample s mean PT value divided by the mean PT value for pooled normal plasma clotted with the same thromboplastin. Supplemented test samples were also prepared from patient plasmas (receiving Coumadin for more than 6 weeks) by adding individual purified coagulation factors at % or 0% of their mean level in normal plasma(. lm for prothrombin, 0 nm for FVII, and 78 nm for FX []). Results Tissue factor concentration has little impact on ISI The rtf concentrations in synthetic thromboplastins were adjusted to obtain 0 s clotting times with normal plasma. We therefore examined if changing the rtf concentration per se influenced the ISI value. Figure A shows a very weak relationship between rtf concentration and ISI for the thromboplastins employed in this study (reagents of Table ). We also created variations on reagent with varying rtf concentrations but constant phospholipid concentration and composition: rtf concentration had no significant effect on ISI value (Fig. B). Phospholipid composition affects sensitivity to individual clotting factors We varied the phospholipid composition of rtf-liposomes (Table ) and used log log plots of s vs. % normal plasma to examine their responsiveness to changes in prothrombin, FV, FVII, and FX levels (Fig. ). In the absence of PE, rtfliposomes with the lowest PS content exhibited the highest PT ratios for all four clotting factor deficiencies (Fig. A,D,G,J). In particular, prothrombin exhibited a profound dependence on PS content, such that reagent (0% PS) exhibited little or no sensitivity to prothrombin deficiency (i.e. s were near ) when prothrombin levels spanned 0 0% (Fig. A). For thromboplastins containing 0% PE (Fig. B,E,H,K), changing the PS content from % to 0% had little or no impact on FVII or FX sensitivity (Fig. H,K), and limited impact on FV sensitivity (Fig. E). Sensitivity to prothrombin was strongly influenced by PS content: thromboplastin 6 (0% PS, 0% PE) had almost no sensitivity to prothrombin deficiency, while thromboplastin (0% PS, 0% PE) lacked sensitivity to prothrombin deficiency over the range of 0% (Fig. B). NaCl concentration affects sensitivity to individual clotting factors Increasing NaCl concentration increased the sensitivity to prothrombin deficiency most profoundly at low prothrombin A ISI TF (nmol L ) B ISI.0 PT reference (s) TF (nmol L ) PT local (s) Fig.. Recombinant human tissue factor (rtf) concentration has little effect on the International Sensitivity Index (ISI). (A) Relative ISI values for thromboplastins in Table were plotted vs. log (TF) concentration, to which a line was fitted by linear regression. (B) Log log plots of prothrombin time (PT) clotting times of pooled normal plasma and plasmas from four different patients stably anticoagulated on Coumadin. Clotting data were obtained using RecombiPlasTin (y-axis) and test thromboplastins (x-axis), to which lines were fitted by orthogonal regression. Test thromboplastins contained 0% PS, 90% PC, and the following rtf concentrations: nm (filled circles); nm (open squares), 0 nm (filled inverted triangles), or 0 nm (open triangles). Data are mean ± SEM obtained using three different preparations of relipidated rtf. Inset: relative ISI values are plotted vs. rtf concentration. concentrations (Fig. C). NaCl concentration had less effect on sensitivity to FV, although this was more pronounced at lower FV concentrations (Fig. F). NaCl concentration had essentially no effect on response to FVII (Fig. I) and little effect on FX (Fig. L). Clotting factor sensitivities of four commercial thromboplastins All four commercial thromboplastins tested exhibited similar sensitivities to prothrombin, FV, and FX (Fig. A,B,D). The rtf-based commercial thromboplastins (RecombiPlasTin and Innovin) were more sensitive to FVII than were the tissuederived reagents (Thromborel-S and Neoplastine Cl Plus), especially at low FVII concentrations (0% and below; Fig. C). Ó 006 International Society on Thrombosis and Haemostasis

4 Clotting factor sensitivities of thromboplastin 8 0 0% PS, 0% PE, 0 m mol NaCl 0% PS, 0% PE, 0 m mol NaCl 0% PS, 0% PE, 0 0 m mol NaCl (prothrombin deficiency) A B C (FV deficiency) D E F (FVII deficiency) G H I (FX deficiency) J K L % normal plasma Fig.. Phospholipid and NaCl content influence thromboplastin sensitivities to deficiencies in prothrombin, FV, FVII, or FX. s were determined using thromboplastins of varying composition (symbols given in Table ), and theindicatedmixturesofpoolednormal plasma and plasmas deficient in prothrombin, FV, FVII, or FX. Plasma mixtures used were: (A C) prothrombin-deficient; (D F) FV-deficient; (G I) FVII-deficient; and (J L) FXdeficient. They were clotted with: (A,D,G,J) thromboplastins (varying PS, 0% PE, 0 mm NaCl); (B,E,H,K) thromboplastins 6 (varying PS, 0% PE, 0 mm NaCl); or thromboplastins, 7, 8 and 9 (0% PS, 0% PE, and varying NaCl). Data are log log plots of mean ± SE (n ¼ ), although generally the error bars are smaller than the plotted symbols. Comparison of thromboplastin sensitivities Coumarins depress the plasma levels of FVII, FX, and prothrombin to differing degrees, ranging from about 0 0% of normal in therapeutically anticoagulated patients [,, 9]. To better compare thromboplastin properties using clotting factor deficiencies spanning the therapeutic range, we replotted data from Fig. at 0% and % normal plasma, grouping them in panels by thromboplastin (Fig. ). The vertical positions of data points and lengths of the connecting colored bars illustrate the relative clotting factor sensitivities of thromboplastins. As the PS content increases, thromboplastins dramatically lose sensitivity to prothrombin (blue bars), having essentially no sensitivity at all to prothrombin at the highest PS levels. Increasing the PS content had a much more modest effect on sensitivity to the other three clotting factors, although reagent is markedly more sensitive to FVII (red bars) than are the other five reagents in Fig.. Altering the PS content also affected sensitivity to FV (green bars), with thromboplastins containing PE generally exhibiting greater FV sensitivities (reagents 6). Effect of adding purified clotting factors The data above reveal the sensitivity of thromboplastins to single clotting factor deficiencies, but coumarin therapy decreases clotting factor activities because of both decreased antigen levels and undercarboxylation. We therefore examined the effects of adding purified prothrombin, FVII, or FX to Ó 006 International Society on Thrombosis and Haemostasis

5 8 S. A. Smith et al (prothrombin deficiency) A..0. No PE 0% PE 0% PS 0% PS 0% PS % PS 0% PS 0% PS 6 (0.77) (.6) (.) (0.98) (.7) (.7) (FV deficiency) (FVII deficiency) (FX deficiency) 0 0 % normal plasma Fig.. Sensitivities of four commercial thromboplastins to deficiencies in prothrombin, FV, FVII, or FX. s were determined as in Fig.. Plasma mixtures used were: (A) Prothrombin-deficient; (B) FV-deficient; (C) FVII-deficient; and (D) FX-deficient. They were clotted with commercial thromboplastins: RecombiPlasTin (filled circles); Innovin (open circles); Thromborel S (filled triangles); and Neoplastine Cl Plus (open triangles). plasmas from six patients stably anticoagulated with Coumadin (Fig. ), and focused on two thromboplastins (reagents 6 and 8) that differ dramatically in prothrombin sensitivity (Fig. ). With reagent 6, adding prothrombin had little or no impact on the, while adding either FVII or FX dramatically decreased the s (Fig. ). s using reagent 8 were more sensitive to addition of prothrombin, FVII, or FX than were those using reagent 6 (Fig. ). B C D Fig.. Comparison of thromboplastin responsiveness to deficiencies in prothrombin, FV, FVII, or FX. Selected s from Fig. are individually plotted, grouped by thromboplastin. In each case, the upper data point is the mean at 0% normal plasma while the lower data point is the mean at % normal plasma, with error bars representing SE (n ¼ ). The distances spanned between the data points are shaded as follows: blue (prothrombin deficiency; filled circles), green (FV deficiency; open circles), red (FVII deficiency; filled squares), or orange (FX deficiency; open squares). Thromboplastin reagent numbers from Table are given inside the top of each panel with ISI values below in parentheses. The % PS and PE in the rtf-liposomes is indicated. Discussion We systematically examined how changes in composition of rtf-based thromboplastins affected their responses in clotting assays to deficiencies in prothrombin, FV, FVII, and FX, allowing us to determine the relative sensitivities of thromboplastins to each of these clotting factors. (Bader et al.[9] used a similar approach.) PT clotting times depend upon the sequential assembly of two clotting factor complexes on membrane surfaces the TF:FVIIa complex and the prothrombinase complex (FVa:FXa) which do not have identical optimal phospholipid requirements [0 ]. Altering the phospholipid composition of thromboplastins may therefore differentially affect the catalytic efficiencies of the TF:FVIIa and prothrombinase complexes, which translate into different relative impacts of changes in the plasma levels of FV, FVII, FX and prothrombin on the PT test. Changes in phospholipid composition affected a thromboplastin s response to prothrombin deficiency more profoundly than response to FVII or FX. At high PS content, synthetic thromboplastins exhibited little or no sensitivity to prothrombin over the range likely to be encountered in oral anticoagulant therapy (0 0%), which was exaggerated in the presence of PE. Changing the PS content generally altered the responses to FVII and FX deficiency to a lesser degree than for prothrombin. Very low PS content, however, increased FVII sensitivity dramatically (reagent in Fig. ). At more severe clotting factor deficiencies (<0%), differences in thromboplastin sensitivities to all the clotting factors tested became more pronounced. Slopes of the response curves to individual clotting factor deficiencies varied significantly. Thromboplastins lacking PE showed very steep slopes for FVII deficiency, somewhat steep slopes for FX deficiency, and much shallower slopes for either FV or prothrombin deficiency (Fig. A,D,G,J). Thromboplastins with properties like these may have somewhat similar sensitivities to prothrombin, FV, FVII and FX at moderate Ó 006 International Society on Thrombosis and Haemostasis

6 Clotting factor sensitivities of thromboplastin 8 (prothrombin deficiency) (FVII deficiency) (FX deficiency) Reagent 6 Reagent Added factor (%) Fig.. Effect on of adding purified prothrombin, FVII, or FX to plasmas from patients receiving Coumadin. s were obtained as in Fig. except that the test samples were plasmas from six individual patients stably anticoagulated on Coumadin to which purified prothrombin, FVII, or FX were added at % or 0% of their normal plasma levels. Each symbol indicates that the plasma sample came from a different patient. Clotting times were determined with two thromboplastins (reagents 6 and 8; Table ) chosen to have markedly different sensitivities to prothrombin deficiency. clotting factor deficiencies, but as coagulopathies become more severe their sensitivities are weighted progressively more toward FVII and FX and less to prothrombin and FV. On the other hand, thromboplastin (% PS, 0% PE) exhibited very similar slopes for deficiencies in prothrombin, FV, FVII, and FX (open inverted triangles in Fig. B,E,H,K), giving essentially equal weight to all four clotting factors no matter how severe the coagulopathy. Raising the NaCl concentration increased thromboplastin sensitivity to prothrombin but had little effect on FVII or FX sensitivity. Thrombin has a Na + -binding site, occupancy of which alters its catalytic activity []. The range of Na + concentrations employed in this study are sufficient to alter the fractional occupancy of thrombin s Na + -binding site. In addition to effects on ionic strength, this may contribute to NaCl effects on thromboplastin responsiveness. Thromboplastin composition (PS content, and to a lesser extent, NaCl concentration) also affected sensitivity to FV deficiency. The synthetic thromboplastins used in this study exhibited ranges of responsiveness to prothrombin, FV, FVII, or FX which spanned those of four widely used commercial thromboplastins. Both rtf-based thromboplastins (RecombiPlasTin and Innovin) were more sensitive to FVII deficiency than were tissue-based reagents (Thromborel S and Neoplastine Cl Plus), as previously noted [7 9]. The synthetic thromboplastins employed in this study were also more sensitive to FVII deficiency than were the tissue-based commercial thromboplastins. Discrepant INR values in patients with profound FVII deficiency have been reported for several commercially available recombinant human thromboplastins [6 9,]. Isolated factor depletions described in this study would not occur in patients on oral anticoagulant therapy, but this approach has allowed us to determine the relative sensitivities of thromboplastins to deficiencies in the three clotting factors suppressed by oral anticoagulation. Even in stably anticoagulated patients, the plasma levels of FVII, FX, and prothrombin may be depressed to different degrees [ 8]. Some studies have attempted to correlate the plasma activities of single coagulation factors with INR values from individual patients, variously concluding that INR correlates best with deficiency in FVII [,], a combination of FVII and prothrombin deficiency [9], or native prothrombin []. Our results suggest that differences in the apparent contributions of individual clotting factors to INR may result from using thromboplastins with differing relative sensitivities to FVII, FX, and prothrombin. Several studies have suggested that depression of plasma prothrombin levels (and to a lesser extent, FX levels) by coumarin therapy has the greatest relevance to reducing thrombosis [,, 9]. Indeed, some have recommended measuring plasma prothrombin levels as an alternative to PT assays for monitoring oral anticoagulant therapy [,7], although this approach has not been thoroughly assessed in clinical studies and may be impractical because of high cost and difficulties in standardization. Interestingly, we found that some synthetic thromboplastins (e.g. reagents and 6 in Table ) exhibited s near when the prothrombin concentration was 0% of normal or higher (Fig. ). Such reagents have virtually no sensitivity to prothrombin at any concentration likely to occur in properly anticoagulated patients. If prothrombin is the most important clotting factor in determining efficacy of oral anticoagulant therapy, then thromboplastins with properties like these would be essentially unable to detect changes in its level. We previously reported that rtf-based thromboplastins of identical ISI could be formulated from different combinations of phospholipid content and NaCl concentration []. It is now apparent that dissimilar thromboplastin composition is asso- Ó 006 International Society on Thrombosis and Haemostasis

7 86 S. A. Smith et al ciated with dissimilar relative sensitivities to deficiencies in prothrombin, FV, FVII, and FX, particularly as clotting factor deficiencies become more severe. Consequently, correcting PT clotting data by INR may not always produce equivalent results when clotting times are measured with different thromboplastins. This phenomenon has been reported for commercially available human recombinant thromboplastins, in which individuals who were over-anticoagulated (and thus had the lowest factor levels) were most likely to have INR values that were discrepant between thromboplastins [8,9,]. Results of the present study will allow the development of rtf-based thromboplastins with a desired combination of sensitivities to deficiencies in FVII, FX, and prothrombin. The relative sensitivities of various thromboplastins to deficiencies in these three clotting factors can be visualized using a threedimensional plot of s at % normal plasma obtained with plasmas deficient in FVII, FX, or prothrombin (Fig. 6). This graphically demonstrates that even thromboplastins of similar ISI do not necessarily respond in a similar fashion to deficiencies in these three clotting factors. It also highlights the remarkable differences among these thromboplastins in their prothrombin sensitivity, with some exhibiting essentially no sensitivity to prothrombin at all while others have high prothrombin sensitivity. Sensitivity to FX deficiency likewise varies dramatically between these thromboplastins, even among those with ISI values in the range (dark blue data points). On the other hand, differences in FVII sensitivity were less pronounced among these thromboplastins, at least at the % clotting factor level. The success of the ISI/INR system for correcting PT values of patients receiving oral anticoagulant therapy has led to widespread use of this system to correct PT values from patients whose PT is prolonged because of sepsis, liver failure, -% (prothrombin deficiency) % (FVII deficiency) % (FX deficiency) ISI range < >.7 Fig. 6. Three-dimensional plot of the sensitivities of thromboplastins to deficiencies in FVII, FX, and prothrombin. Mean s at % normal plasma (PT Ratio-%) from Figs and were plotted for all thromboplastins examined in this study: circles, synthetic thromboplastins (reagents 9); triangles, recombinant commercial thromboplastins (reagents 0 and ); and squares, tissue-derived commercial thromboplastins (reagents and ). Data are color-coded according to ISI value (see key). and other coagulopathies for which the INR system was not originally designed and for which it has not been rigorously validated in clinical studies [6 0]. Plasmas from patients with consumptive coagulopathies or hepatic disease may have different profiles of coagulation factor deficiencies than are observed in oral anticoagulant therapy with coumarins [6,7], including FV and fibrinogen. Elevated thromboplastin response to FV deficiency could, therefore, contribute to thromboplastin-dependent discrepancies in INR values in patients with coagulopathies. An ISI is a one-dimensional description of thromboplastin sensitivity. The results of this study indicate that, in addition to ISI, it is possible to evaluate thromboplastins based on their relative sensitivities to deficiencies in FV, FVII, FX, and prothrombin. Furthermore, we now have a means by which to quantify and manipulate their responsiveness to individual clotting factors. This is the equivalent of placing the thromboplastin at a desired location within the three-dimensional space of Fig. 6. In summary, this work now allows the custom design of thromboplastins with any desired sensitivity to specific parts of the blood clotting cascade. Thromboplastins with decreased FVII sensitivity, for example, might be useful for long-term monitoring of oral anticoagulant therapy, as they may decrease day-to-day variability in PT values owing to the very short plasma half-life of FVII compared with the much longer halflives of FX and prothrombin. Thromboplastins with enhanced FV sensitivity might be beneficial in predicting the bleeding risk in patients with liver disease for example, bleeding risk following the liver biopsy. The optimal sensitivity profile of the ideal thromboplastin for monitoring anticoagulant therapy and/or evaluating the extrinsic pathway in patients with liver disease, sepsis, and coagulopathies remains to be determined in future clinical studies. Acknowledgements We thank Guoyu Li for excellent technical contributions to this work, and Brad Schwartz for helpful comments on this manuscript. This study was supported by NIH grants HL70 & HL0 (J.H.M) from the National Heart, Lung and Blood Institute, and also General Clinical Research Center Grant M0 RR-67 from the National Center for Research Resources (P.C.C). References Poller L. Prothrombin time (PT). In: Jespersen J, Bertina RM, Haverkate F, eds. Laboratory Techniques in Thrombosis. A Manual. nd revised edition of ECAT Procedures. Dordrecht: Kluwer Academic Publishers, 999: 6. Kitchen S, Jennings I, Woods TA, Walker ID, Preston FE. Two recombinant tissue factor reagents compared to conventional thromboplastins for determination of international normalised ratio: a thirtythree-laboratory collaborative study. The Steering Committee of the UK National External Quality Assessment Scheme for Blood Coagulation. Thromb Haemost 996; 76: 7 6. 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