Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system
2004; Elsevier BV; Volume: 2; Issue: 3 Linguagem: Inglês
10.1111/j.1538-7933.2003.00617.x
ISSN1538-7933
AutoresGeoffrey Allen, Alisa S. Wolberg, J.A. Oliver, Maureane Hoffman, Harold R. Roberts, Dougald M. Monroe,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoSummaryUsing a cell‐based model system of coagulation, we performed a systematic examination of the effect of varying individual procoagulant proteins (over the range of 0–200% of pooled plasma levels) on the characteristics of thrombin generation. The results revealed a number of features unique to the different coagulation factors, as well as common features allowing them to be grouped according to the patterns observed. Variation of those factors contributing to formation of the tenase complex, factor (F)VIII, factor (F)IX and factor (F)XI, primarily affected the rate and peak of thrombin production, but had little to no effect on total thrombin production. The effect of decreased FXI was milder than seen with decreased FVIII or FIX, and more variable between platelet donors. In contrast, varying the concentration of factors that contribute to formation of the prothrombinase complex, prothrombin or factor (F)V (with FV‐deficient platelets), significantly affected all three measures of thrombin production: rate, peak and total. Additionally, while no thrombin generation was observed with no factor X, only very small amounts (between 1% and < 10% of normal plasma levels) were required to normalize the measured parameters. Finally, our results with this cell‐based system highlight differences in thrombin generation on cell surfaces (platelets) compared with phospholipids, and suggest that platelets contribute more than simply a surface for the generation of thrombin. Using a cell‐based model system of coagulation, we performed a systematic examination of the effect of varying individual procoagulant proteins (over the range of 0–200% of pooled plasma levels) on the characteristics of thrombin generation. The results revealed a number of features unique to the different coagulation factors, as well as common features allowing them to be grouped according to the patterns observed. Variation of those factors contributing to formation of the tenase complex, factor (F)VIII, factor (F)IX and factor (F)XI, primarily affected the rate and peak of thrombin production, but had little to no effect on total thrombin production. The effect of decreased FXI was milder than seen with decreased FVIII or FIX, and more variable between platelet donors. In contrast, varying the concentration of factors that contribute to formation of the prothrombinase complex, prothrombin or factor (F)V (with FV‐deficient platelets), significantly affected all three measures of thrombin production: rate, peak and total. Additionally, while no thrombin generation was observed with no factor X, only very small amounts (between 1% and < 10% of normal plasma levels) were required to normalize the measured parameters. Finally, our results with this cell‐based system highlight differences in thrombin generation on cell surfaces (platelets) compared with phospholipids, and suggest that platelets contribute more than simply a surface for the generation of thrombin. Hemostasis requires appropriate thrombin generation. Thrombin generation in vivo, or in models designed to mimic aspects of in vivo coagulation, is influenced by a number of parameters including the nature of the surface for the reactions as well as the concentrations of the protein components. For example, our previous studies have demonstrated that thrombin generation is significantly influenced by the type of cell surface on which the thrombin generation takes place. Conversion of prothrombin to thrombin by the prothrombinase complex is much more efficient on the surface of the activated platelet than on the surface of the tissue factor‐bearing cell. Additionally, previous studies in models using lipid surfaces have suggested that thrombin generation is influenced by the concentrations of the procoagulant and inhibitory proteins present [1Andrew M. Schmidt B. Mitchell L. Paes B. Ofusu F. Thrombin generation in newborn plasma is critically dependent on the concentration of prothrombin.Thromb Haemost. 1990; 63: 27-30Crossref PubMed Scopus (160) Google Scholar, 2Butenas S. Van't Veer C. Mann KG. ‘Normal’ thrombin generation.Blood. 1999; 94: 2169-78Crossref PubMed Google Scholar, 3Glueck HI. The utilization of a synthetic substrate (TAMe) to measure the plasma prothrombin in coagulation disorders.J Lab Clin Med. 1957; 49: 41-60PubMed Google Scholar, 4Xi M. Beguin S. Hemker HC. The relative importance of the factors II, VII, IX and X for the prothrombinase activity in plasma of orally anticoagulated patients.Thromb Haemost. 1989; 62: 788-91Crossref PubMed Scopus (99) Google Scholar, 5Van‘t Veer C. Mann KG. Regulation of tissue factor initiated thrombin generation by the stoichiometric inhibitors tissue factor pathway inhibitor, antithrombin‐III, and heparin cofactor‐II.J Biol Chem. 1997; 272: 4367-77Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 6Ibbotson S.H. Tate G.M. Davies JA. Effect of high physiological levels of factor VIII:C and factor V on rate of generation of thrombin activity in vitro.Blood Coagul Fibrinolysis. 1993; 4: 415-9Crossref Scopus (4) Google Scholar, 7Al Dieri R. Peyvandi F. Santagostino E. Giansily M. Mannucci P.M. Schved J.F. Beguin S. Hemker HC. The thrombogram in rare inherited coagulation disorders: its relation to clinical bleeding.Thromb Haemost. 2002; 88: 576-82Crossref PubMed Scopus (245) Google Scholar, 8Kyrle P.A. Mannhalter C. Beguin S. Stumpflen A. Hirschl M. Weltermann A. Stain M. Brenner B. Speiser W. Pabinger I. Lechner K. Eichinger S. Clinical studies and thrombin generation in patients homozygous or heterozygous for the G20210A mutation in the prothrombin gene.Arterioscler Thromb Vasc Biol. 1998; 18: 1287-91Crossref PubMed Scopus (156) Google Scholar]. The purpose of this study was to determine the influence of each of the procoagulant proteins on thrombin generation on the platelet surface. To accomplish this, we employed a previously described cell‐based model system of coagulation, in which coagulation is initiated by tissue factor‐bearing cells in the presence of activated factor (F)VII, with physiological numbers of platelets and physiological concentrations of procoagulant and inhibitory proteins [9Monroe D.M. Roberts H.R. Hoffman M. Platelet procoagulant complex assembly in a tissue factor‐initiated system.Br J Haematol. 1994; 88: 364-71Crossref PubMed Scopus (105) Google Scholar]. The advantages of this cell‐based system are that the concentration of the elements comprising the system, including both procoagulant and inhibitory proteins, can be tightly controlled while the cellular components approximate the surface available in physiological circumstances. Thrombin generation was characterized by measurement of the rate and peak of thrombin generation, and calculation of the area under the thrombin curve (AUC). Using this model system, we measured thrombin generation in the presence of a constant number of tissue factor‐bearing cells and platelets while varying the concentrations of prothrombin and factor (F)V, factor (F)VIII, factor (F)IX, factor (F)X and factor (F)XI, from 0% to 200% of the plasma concentrations. Macrophage serum‐free medium (SFM) was purchased from Gibco (Grand Island, NY, USA). TenStop, a synthetic inhibitor of FXa with no detectable activity towards thrombin, was purchased from American Diagnostica (Greenwich, CT, USA). Chromozyme Th, a chromogenic substrate (tosyl‐Gly‐Pro‐Arg‐pNA) for thrombin, was purchased from Boehringer‐Mannheim (Indianapolis, IN, USA). All other reagents were of a high commercial grade. Prothrombin was purified using barium citrate, DEAE‐cellulose, and a copper chelate column, and was obtained from Hematologic Technologies (Essex Junction, Vt, USA). FIX was purified as described previously [10McCord D.M. Monroe D.M. Smith K.J. Robert HR. Characterization of the functional defect in factor IX Alabama.J Biol Chem. 1990; 265: 10250-4Abstract Full Text PDF PubMed Google Scholar]. FX was purchased from Enzyme Research Labs (South Bend, IN, USA). FV and FXI were purchased from Hematologic Technologies. FVIII was repurified from Koate (from the University of North Carolina Hospital Pharmacy) by gel filtration on Sepharose CL‐2B. FVIIa and recombinant full‐length tissue factor pathway inhibitor (TFPI) were the generous gifts of U. Hedner (Novo Nordisk, Gentofte, Denmark). Antithrombin (AT) was prepared as described previously [11Church F.C. Meade J.B. Treanor R.E. Whinna HC. Antithrombin activity fucoidan: the interaction of fucoidan with heparin cofactor II, antithrombin III, and thrombin.J Biol Chem. 1989; 264: 3618-23Abstract Full Text PDF PubMed Google Scholar]. All zymogen coagulation factors were treated with an inhibitor mixture (tosyl‐lysyl chloromethyl ketone, tosyl‐phenyl chloromethyl ketone, phenylmethyl sulphonyl fluoride, Phe‐Pro‐Arg chloromethyl ketone, and dansyl Glu‐Gly‐Arg chloromethyl ketone) for 1 h, then repurified on Q Sepharose fast flow using calcium chloride elution. Protein preparations were examined for contamination via both ELISA and functional assays. No significant contamination by other procoagulant or anticoagulant proteins were found. Polyclonal rabbit anti‐FX antibody, peroxidase‐labeled polyclonal rabbit antiprothrombin antibody, and peroxidase‐labeled polyclonal rabbit antiAT antibody were from Dako (Carpinteria, CA, USA) and sheep anti‐FVIII from Affinity Biologicals (Hamilton, Ontario, Canada). Anti‐FIX antibody was the generous gift of M. Blackburn (SmithKline Beecham, King of Prussia, PA, USA). One individual served as the monocyte donor for all assays. Monocytes were isolated as described [12Hoffman M. Monroe D.M. Roberts HR. Human monocytes support factor X activation by factor VIIa, independent of tissue factor: implications for the therapeutic mechanism of high‐dose factor VIIa in hemophilia.Blood. 1994; 83: 38-42Crossref PubMed Google Scholar] and cultured in 96‐well plates in lipopolysaccharide (LPS)‐containing media for 18 h to induce tissue factor expression, on the order of 1 pm. Platelets were prepared as described previously using Accu‐Prep Lymphocyte Isolation Medium (Accurate Chemicals, Westbury, NY, USA), followed by gel filtration on Sepharose CL‐2B [13Hoffman M. Monroe D.M. Roberts HR. A rapid method to isolate platelets from human blood by density gradient centrifugation.Am J Clin Pathol. 1992; 98: 531-3Crossref PubMed Scopus (43) Google Scholar]. Platelets were obtained from normal healthy volunteers, except in the assays using FV‐deficient platelets, which were obtained from a FV‐deficient individual. Informed consent was obtained from all donors. For each experiment unactivated platelets and LPS‐activated monocytes expressing tissue factor were combined with purified coagulation proteins and calcium chloride. In the final reaction mixture, monocytes were present at approximately 5000 per well, while platelet concentration was approximately 75 × 109 L−1. Final concentrations of proteins approximated normal plasma levels unless otherwise noted: AT (180 µg mL−1; 3.0 µm), TFPI (0.1 µg mL−1; 3 nm), prothrombin (100 µg mL−1; 1.4 µm), FV (7 µg mL−1; 20 nm), FVIII (0.1 µg mL−1; 0.3 nm), FIX (4 µg mL−1; 70 nm), FX (8 µg mL−1; 135 nm), and FXI (5 µg mL−1; 25 nm). Levels of prothrombin and FV, FVIII, FIX, FX and FXI were varied in sequential experiments from 0% to 200% of normal plasma levels. When 0% levels of FVIII, FIX or FX were to be achieved, an inhibitory antibody to FVIII, FIX or FX, respectively, was added to the final mixture of platelets and proteins. The amount of antibody used was that required to prolong the clotting time of normal plasma to that of plasma with < 1% levels of the factor being varied. Prior to their use in all assays, zymogen proteins were incubated with 10‐fold plasma concentrations of AT and TFPI for at least 2 h to inhibit any activated proteases contaminating the zymogen preparations. Similarly, prior to all assays, FXI was incubated with C‐1‐esterase inhibitor for at least 2 h to ensure no contamination by activated factor. Combining platelet and concentrated protein fractions at the beginning of the assay resulted in dilution of procoagulant and inhibitory proteins to physiological levels. FVIIa (0.2 nm) and calcium chloride (3 mm) were added to initiate the clotting reaction in the microtiter wells. At timed intervals after initiating the coagulation reactions, 10‐µL samples of the reaction mixture were removed and assayed for thrombin activity by addition to 90 µL of a solution of 5 mm EDTA, 0.5 mm Chromozyme Th, and 50 µm TenStop. As the Km of Chromozyme Th for thrombin is approximately 3 µm, > 99% of thrombin should be bound to this substrate at this concentration. Cleavage of the synthetic substrate Chromozyme Th was stopped by the addition of 50% acetic acid and the absorbance at 405 nm was measured. Thrombin generation curves were plotted and fitted with curves using a Gaussian or modified Gaussian equation: a0 + a1 × exp[− 0.5 × [(t − a2)/(a3 + t/a4)]^2]. The rate of thrombin generation was calculated from the slope of the straight line fitted to the points on the upswing of the thrombin curve. Total thrombin generation was estimated by calculating the area under the curve (AUC) utilizing the trapezoidal method, analogous to the endogenous thrombin potential (ETP). Only thrombin actually produced during the assay was accounted for; no attempt was made to extrapolate beyond the end of the experiment. Platelet activation was determined by measuring the percentage of platelets expressing the activation‐specific α granule marker CD62 (P‐selectin, GMP140) on their surface using a FACScan flow cytometer. Samples (10 µl) were removed for platelet activation studies and added to 50 µL paraformaldehyde, incubated for at least 30 min, then diluted with Tyrodes/albumin. Samples were then stained with the phycoerythrin‐conjugated antiCD62 for at least 60 min at 25 °C. The time to 50% of maximal platelet activation was designated as the t50. A characteristic pattern of thrombin generation was observed after the addition of physiological concentrations of the plasma procoagulants, inhibitors and platelets to the tissue factor‐bearing monocytes. No measurable thrombin production was observed in the system until the time of detectable platelet activation (on the order of 6–12 min with normal plasma factor levels). This was succeeded by a period of very rapid thrombin production, followed by a return to baseline values as thrombin production ceased and the residual thrombin was inhibited by AT. The pattern of thrombin generation may be characterized by the rate of the thrombin burst, the peak thrombin level and by the AUC, which reflects both the amount and persistence of free thrombin. As has been demonstrated previously, these parameters vary significantly from individual to individual in the model system, due to differences in the platelet characteristics [14Sumner W.T. Monroe D.M. Hoffman M. Variability in platelet procoagulant activity in healthy volunteers.Thromb Res. 1996; 81: 533-43Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 15Oliver J.A. Monroe D.M. Roberts H.R. Hoffman M. Thrombin activates factor XI on activated platelets in the absence of factor XII.Arterioscler Thromb Vasc Biol. 1999; 19: 170-7Crossref PubMed Scopus (161) Google Scholar]. Therefore, four individuals were used as donors for platelets. For each platelet source, six assays, each with varied levels of a single procoagulant, were performed. In the thrombin generation figures, each panel consistently represents the same one of the four individuals (panel A from subject A, panel B from subject B, etc.). The effect of FVIII concentration on the rate of thrombin generation was biphasic (Fig. 1). With no added FVIII, the average rate of thrombin generation was 10% of that observed with 100% plasma levels. As the concentration of FVIII was increased from 0% to 10%, there was a rapid increase in rate. Above levels of 10% the rate increased more slowly, but continued increases in rate were observed to levels of 200% FVIII. Over the range of added FVIII, from 0% to 200% of normal, the peak thrombin level increased, on average, 6‐fold, with the most significant increase between levels of 0% and 10% (Fig. 2). Thrombin generation at the lowest concentrations of FVIII demonstrated significant variability. The AUC appeared similar at all concentrations, except at the lowest levels of FVIII, at which the AUC decreased on average 30% (Fig. 3). Values for the lowest concentrations do not reflect the total thrombin generation as the thrombin generation curves had not returned to baseline within the time frame of the experiment. The effect of FIX concentration on the rate of thrombin generation was similar to that of FVIII, except for slight differences (Fig. 1). Again, as the concentration of FIX was increased from 0% to 10% of plasma levels, there was a rapid increase in rate. However, in contrast to FVIII, above levels of 10% there was little if any increase in the rate of thrombin generation. Over the range of added FIX, from 0% to 200% of normal, the peak thrombin level increased, on average, 4‐fold, with the most significant increase between levels of 0% and 3% (Fig. 4). Although peak thrombin was significantly decreased with no added FIX, it was never completely eliminated. The AUC appeared similar at all concentrations, except at the lowest levels, at which the AUC decreased on average 30% (Fig. 3). The effect of varied FXI levels demonstrated the greatest variability between individuals. Compared with FVIII and FIX, the average rate of thrombin generation with no added FXI was greater. In individual assays using platelets from different subjects, the rate of thrombin generation with no added FXI ranged from 6% to 99% of that observed at 100% levels. However, as the concentration of FXI increased from 0% to 200% of plasma levels, there was a steady increase in average rate, with no definitive plateau (Fig. 1). Although average peak thrombin was significantly decreased with no added FXI, it was never completely eliminated. As the amount of added FXI increased from 0% to 200% of normal, the average peak thrombin level increased slightly more than 2‐fold (Fig. 5). Considering each assay separately, the increase in peak thrombin ranged from 20% to 500%. The AUC was equivalent at all concentrations (Fig. 3). The rate of thrombin generation with no added FX was undetectable. However, as the concentration of FX was increased from 0% to 10% of plasma levels, there was a rapid increase in rate. Above levels of 10% there was little if any increase in the rate of thrombin generation (Fig. 1). While there was no thrombin generation with no added FX and the addition of an anti‐FX antibody, the peak thrombin generation observed at 1% levels was approximately 70% of maximum, reaching a maximum at 10% levels (Fig. 6). The AUC remained constant down to levels of 1% added FX (Fig. 3). Variation of plasma FV levels had a minimal effect on all parameters of thrombin generation in experiments with platelets from normal individuals, as normal platelets contain FV and appear to obviate the effect of plasma FV. Increasing the amount of added FV from 0% to 200% increased the rate of thrombin by an average amount of only 25% and the peak (Fig. 7) and AUC by approximately 10%. For better assessment of the role of plasma FV on thrombin generation, similar assays were performed with FV‐deficient platelets from a FV‐deficient subject. With no added FV, no thrombin generation was observed. Increasing the FV levels from 1% to 200% resulted in a progressive, nearly 2‐fold increase in the rate of thrombin production (Fig. 1). This increase was biphasic, with a rapid increase in rate observed from 1% to 30%, followed by a slower but steady increase in rate over the range from 30% to 200% FV levels. Changes in peak thrombin generation and AUC paralleled those seen in the rate, with rapid increases from zero over the range from 0% to 30% levels, followed by continued increase at a slower rate from 30% to 200% levels (Figure 3, Figure 8). The influence of prothrombin concentration on thrombin generation was unique. With no added prothrombin, no thrombin production was observed. The rate and peak of thrombin production and AUC increased in a near‐linear fashion as the concentration of prothrombin in the system was increased (Figure 1, Figure 3, Figure 9). No evidence of saturation of the prothrombinase complex was observed. The possibility that this represented complete conversion of ever increasing amounts of prothrombin added to the system was examined. Samples were removed from the model system reaction, boiled in SDS buffer, separated by gel electrophoresis and analyzed in a Western blot using a polyclonal antibody that detects prothrombin, thrombin, and thrombin in complex with AT. These results showed that residual prothrombin remains after cessation of thrombin generation with only about 30–50% of the initial prothrombin being converted to thrombin. This finding is not new, and has been observed in experimental systems currently in use, employing either phospholipids or whole blood [5Van‘t Veer C. Mann KG. Regulation of tissue factor initiated thrombin generation by the stoichiometric inhibitors tissue factor pathway inhibitor, antithrombin‐III, and heparin cofactor‐II.J Biol Chem. 1997; 272: 4367-77Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 16Brinkhous KM. A study of the clotting defect in hemophilia: the delayed formation of thrombin.Am J Med Sci. 1939; 198: 509-16Crossref Google Scholar, 17Rand M.D. Lock J.B. Van’t Veer C. Gaffney D.P. Mann KG. Blood clotting in minimally altered whole blood.Blood. 1996; 88: 3432-45Crossref PubMed Google Scholar, 18Brummel K.E. Paradis S.G. Butenas S. Mann KG. Thrombin functions during tissue factor‐induced blood coagulation.Blood. 2002; 100: 148-52Crossref PubMed Scopus (325) Google Scholar]. In our system, the prothrombin that remained unconverted was not refractory to activation and could be completely converted to thrombin. In assays with prothrombin at 150% and 200% of plasma concentration, thrombin levels failed to return to baseline, although AT was present in excess of prothrombin. It has been previously reported that significantly greater than equimolar amounts of AT are required for complete inhibition of thrombin in vitro[19Bjork I. Nordenman B. Acceleration of the reaction between thrombin and antithrombin III by non‐stoichiometric amounts of heparin.Eur J Biochem. 1976; 68: 507-11Crossref PubMed Scopus (60) Google Scholar]. This is due, in part, to inactivation of AT by thrombin [20Fish W.W. Orre K. Bjork I. The production of an inactive form of antithrombin through limited proteolysis by thrombin.FEBS Lett. 1979; 98: 103-6Crossref PubMed Scopus (46) Google Scholar]. Samples taken from reactions with high levels of prothrombin and analyzed by Western blotting using polyclonal antibodies directed against AT confirmed the formation of this modified form of AT (data not shown). Thus, although present in amounts greater than that of the maximum amount of prothrombin added to the system, there appears to have been insufficient AT to inactivate all thrombin produced in assays containing prothrombin levels of 150% and 200%. Measurement of platelet activation revealed a pattern similar to that of thrombin generation. An initial quiescent period of zero platelet activation was observed, followed by a rapid rise from no platelet activation to a maximum level. Little effect on the t50 was noted with variation of prothrombin or FVIII, FIX or FXI concentration (data not shown). In assays with varied FV concentrations, using platelets from healthy volunteers, a small, progressive increase in t50 was observed as the concentrations of added FV were reduced to zero (data not shown). In assays performed with FV‐deficient platelets from a FV‐deficient individual, decreasing plasma FV concentrations below 50% resulted in a significant and progressive increase in t50, with no platelet activation observed in the absence of FV (Fig. 10). Similarly, decreasing concentrations of added FX below 10% also progressively delayed platelet activation. Visual inspection and flow cytometry did not demonstrate aggregation of platelets in the system. The study of the basic mechanisms of hemostasis is difficult, as is the interpretation of one's data and extrapolation to the true physiological state. Furthermore, the heterogeneity of the multitude of experimental systems currently in use makes comparisons with the findings of other investigators problematic. All experimental systems, whether in vitro, ex vivo or in vivo, are in their own way artificial, with limitations both known and unknown. Our own system, in comparison with humans, lacks the element of flow, has a finite supply of platelets, procoagulants and anticoagulants, and represents only a subset of those proteins normally found in plasma. However, our system also offers the advantage of the physiological surface of the platelet in place of the artificial phospholipid particle found in many current model systems and exquisite control over the amount of all constituents of the system. Thus, in the following discussion of our findings and comparison with the results from other groups employing other models, the reader is encouraged to be mindful of these points. Thrombin generation is influenced largely by the presence of cellular surfaces, the concentration of inhibitory proteins, and the concentrations of procoagulant proteins. If one holds the first two variables constant while varying the latter, distinct patterns of factor concentration influence on thrombin generation can be observed. The effect of varying the concentration of either FVIII or FIX yielded similar results. Contrary to popular belief, the AUC was not the primary parameter affected when FVIII or FIX concentration was varied. Rather, as shown in Fig. 3, the AUC was relatively unaffected, even at the lower concentrations of added factor. This phenomenon is not due merely to total consumption of the prothrombin in the system, as substantial amounts of prothrombin remain after cessation of thrombin generation. Instead, the rate and peak of thrombin generation were the variables most affected. It would appear as though the bleeding tendency in hemophiliacs may be a function of their slower rate of thrombin generation (and the peak level of thrombin generated), rather than the quantity. Clot formation in vitro is observed at the very start of measurable thrombin generation, long before peak and total thrombin generation have occurred. These findings would complement previous reports that the rate of thrombin generation influences fibrin clot structure and stability [21Blomback B. Carlsson K. Fatah K. Hessel B. Procyk R. Fibrin in human plasma: gel architectures governed by rate and nature of fibrinogen activation.Thromb Res. 1994; 75: 521-38Abstract Full Text PDF PubMed Scopus (225) Google Scholar, 22Wolberg AS, Monroe DM, Hedner U, Roberts HR, Hoffman M. High dose factor VIIa enhances clot stability in a model of hemophilia. In: 43rd Meeting of the American Society of Hematology 2001, Orlando, Florida.Google Scholar, 23Wolberg A. Monroe D. Roberts H. Hoffman M. Elevated prothrombin results in clots with an altered fiber structure: a possible mechanism of the increased thrombotic risk.Blood. 2003; 101: 3008-13Crossref PubMed Scopus (141) Google Scholar]. We conclude that the rate of thrombin generation is the key variable to stable fibrin clot formation. It is interesting to note that other investigators using systems incorporating phospholipid vesicles rather than platelets have reported findings significantly different from our own. Ibbotson reported significant increases in peak thrombin generation as FVIII concentrations were increased from 100% to 350% of normal [6Ibbotson S.H. Tate G.M. Davies JA. Effect of high physiological levels of factor VIII:C and factor V on rate of generation of thrombin activity in vitro.Blood Coagul Fibrinolysis. 1993; 4: 415-9Crossref Scopus (4) Google Scholar]. Similarly, Butenas reported a 2‐fold increase in peak thrombin generation when FVIII levels were increased from 50% to 150%[2Butenas S. Van't Veer C. Mann KG. ‘Normal’ thrombin generation.Blood. 1999; 94: 2169-78Crossref PubMed Google Scholar]. In our own cell‐based model, peak thrombin generation was within 30% of maximum at a FVIII concentration of 10% and only a minimal increase in peak thrombin was observed as concentrations were increased from 100% to 200%. Previous findings with varied FIX on phospholipid surfaces as opposed to platelets also demonstrate some differences from our observations. At the lower concentrations, Xi found 20% and 35% of maximal peak thrombin generation with FIX levels of approximately 3% and 5%, respectively [4Xi M. Beguin S. Hemker HC. The relative importance of the factors II, VII, IX and X for the prothrombinase activity in plasma of orally anticoagulated patients.Thromb Haemost. 1989; 62: 788-91Crossref PubMed Scopus (99) Google Scholar]. We observed a more rapid increase in peak thrombin generation, with 60–70% of maximal thrombin generation observed at FIX concentrations of 3%. Butenas reported a ‘paradoxical’ effect of increasing FIX levels, with a progressive decline in peak thrombin observed as FIX levels increased over 50% of normal, with near elimination of thrombin generation at levels of 300%[2Butenas S. Van't Veer C. Mann KG. ‘Normal’ thrombin generation.Blood. 1999; 94: 2169-78Crossref PubMed Google Scholar, 24Butenas S. Mann K. Paradoxical effect of factor IX on tissue factor induced thrombin generation.Thromb Haemost. 1999; : 477Google Scholar]. In our assays, maximal peak thrombin generation was achieved at FIX concentrations between 10% and 30%, and remained relatively con
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