Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system

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1 Journal of Thrombosis and Haemostasis, 2: IN FOCUS Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system G. A. ALLEN,* A. S. WOLBERG, J. A. OLIVER,** M. HOFFMAN, H. R. ROBERTSà and D. M. MONROEà *Department of Pediatrics, Department of Pathology & Laboratory Medicine, àdepartment of Medicine and Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, NC, USA; and Durham VA Medical Center and Department of Pathology and **Department of Immunology, Duke University, Durham, NC, USA To cite this article: Allen GA, Wolberg AS, Oliver JA, Hoffman M, Roberts HR, Monroe DM. Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system. J Thromb Haemost 2004; 2: See also Hemker HC, Béguin S. The love of the artist for his model of thrombin generation. This issue, pp Summary. 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. Keywords: coagulation factors, platelet, thrombin. Correspondence: G. A. Allen, Department of Pediatrics, University of North Carolina, Chapel Hill, NC, USA Tel.: ; fax: ; [email protected] Received 9 July 2003, accepted 20 November 2003 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 [1 8]. 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 [9]. 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.

2 Procoagulant concentration and thrombin generation 403 Experimental procedures Materials 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. Proteins Prothrombinwaspurifiedusingbariumcitrate, DEAE-cellulose, and a copper chelate column, and was obtained from Hematologic Technologies (Essex Junction, Vt, USA). FIX was purified as described previously [10]. FX was purchased from Enzyme Research Labs (South Bend, IN, USA). FV and FXI were purchased from Hematologic Technologies. FVIII was repurifiedfromkoate(fromtheuniversityofnorthcarolinahospital 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 [11]. All zymogen coagulation factors were treated with an inhibitor mixture (tosyl-lysyl chloromethyl ketone, tosylphenyl chloromethyl ketone, phenylmethyl sulphonyl fluoride, Phe-Pro-Arg chloromethyl ketone, and dansyl Glu-Gly-Arg chloromethylketone) for1 h, thenrepurifiedonqsepharosefast 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 peroxidaselabeled 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). Cell isolation One individual served as the monocyte donor for all assays. Monocytes were isolated as described [12] 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 [13]. 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. Cell-based model of coagulation 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 L )1. Final concentrations of proteins approximated normal plasma levels unless otherwise noted: AT (180 lg ml )1 ;3.0lM), TFPI (0.1 lg ml )1 ;3nM), prothrombin (100 lg ml )1 ;1.4lM), FV (7 lg ml )1 ;20nM), FVIII (0.1 lg ml )1 ; 0.3 nm), FIX (4 lg ml )1 ; 70 nm), FX (8 lg ml )1 ; 135 nm), and FXI (5 lg ml )1 ;25nM). 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 2h 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 2h 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 (0.2nM) and calcium chloride (3 mm) were added to initiate the clotting reaction in the microtiter wells. Thrombin assay At timed intervals after initiating the coagulation reactions, 10-lL samples of the reaction mixture were removed and assayed for thrombin activity by addition to 90 ll of a solution of 5 mm EDTA, 0.5 mm Chromozyme Th, and 50 lm TenStop. As the Km of Chromozyme Th for thrombin is approximately 3 lm, > 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 a granule marker CD62(P-selectin, GMP140) on their surface using a

3 404 G. A. Allen et al FACScan flow cytometer. Samples (10 ll) were removed for platelet activation studies and added to 50 ll paraformaldehyde, incubated for at least 30 min, then diluted with Tyrodes/ albumin. Samples were then stained with the phycoerythrinconjugated anticd62for at least 60 min at 25 C. The time to 50% of maximal platelet activation was designated as the t 50. Results Thrombin generation in a model system 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 12min 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 [14,15]. 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.). Effect of varied FVIII levels on thrombin generation 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. Effect of varied FIX levels on thrombin generation 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 Fig. 1. Rate of thrombin generation. The rate of thrombin generation was calculated for each concentration of the varied factor from the slope of a straight line fitted to the points on the upswing of the thrombin curve. The values for each individual s assay were normalized to that measured at 100% levels of the varied factor and then averaged with those of the other individuals. *The factor (F)V data shown above is from an assay using FV-deficient platelets.

4 Procoagulant concentration and thrombin generation 405 Fig. 2. Thrombin production with varied factor (F)VIII levels. In separate assays using platelets from four healthy, normal subjects (designated A, B, C, D), levels of FVIII were varied (none; 1%; 3%; 10%; 100%; 200% of pooled plasma levels) in a model system with otherwise normal plasma levels of factors II, V, IX, X and XI, antithrombin and tissue factor pathway inhibitor, catalytic amounts of factor VIIa, tissue factor-bearing monocytes and unactivated platelets. Timed samples were taken for determination of thrombin generation. j ¼ none, n ¼ 1%, m ¼ 3%, s ¼ 10%, d ¼ 100%, h ¼ 200%. 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). Effect of varied FXI levels on thrombin generation 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). Effect of varied FX levels on thrombin generation 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).

5 406 G. A. Allen et al Fig. 3. Cumulative thrombin production. The area under the thrombin curve was calculated for each concentration of the varied factor, using the trapezoidal method. The values for each individual s assay were normalized to that measured at 100% levels of the varied factor and then averaged with those of the other individuals. *The AUC values for all levels of factor (F)V, using normal platelets, were similar, the FV data shown above are from an assay using FV-deficient platelets. Effect of varied FV levels on thrombin generation 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 (Figs 3 and 8). Effect of varied prothrombin levels on thrombin generation 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 (Figs 1, 3 and 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 [5,16 18]. 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 [19]. This is due, in part, to inactivation of AT by thrombin [20]. 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

6 Procoagulant concentration and thrombin generation 407 Fig. 4. Thrombin production with varied factor (F)IX levels. In separate assays using platelets from four healthy, normal subjects (designated A, B, C, D), levels of FIX were varied (none; 1%; 3%; 10%; 100%; 200% of pooled plasma levels) in a model system of coagulation with otherwise normal plasma levels of factors II, V, VIII, X and XI, antithrombin and tissue factor pathway inhibitor, catalytic amounts of factor VIIa, tissue factor-bearing monocytes and unactivated platelets. Timed samples were taken for determination of thrombin generation. j ¼ none, n ¼ 1%, m ¼ 3%, s ¼ 10%, d ¼ 100%, h ¼ 200%. insufficient AT to inactivate all thrombin produced in assays containing prothrombin levels of 150% and 200%. Platelet activation in the model system 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 t 50 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 t 50 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 t 50,withno 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. Discussion 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

7 408 G. A. Allen et al Fig. 5. Thrombin production with varied factor (F)XI levels. In separate assays using platelets from four healthy, normal subjects (designated A, B, C, D), levels of FXI were varied (0%; 3%; 10%; 30%; 100%; 200% of pooled plasma levels) in a model system of coagulation with otherwise normal plasma levels of factors II, V, VIII, IX, and X, antithrombin and tissue factor pathway inhibitor, catalytic amounts of factor VIIa, tissue factor-bearing monocytes and unactivated platelets. Timed samples were taken for determination of thrombin generation. j ¼ 0%, n ¼ 3%, m ¼ 10%, s ¼ 30%, d ¼ 100%, h ¼ 200%. 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 [21 23]. 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 [6]. Similarly, Butenas reported a 2-fold increase in peak thrombin generation when FVIII levels were increased from 50% to 150% [2]. 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% and35%ofmaximalpeakthrombingenerationwithfix levels of approximately 3% and 5%, respectively [4]. We observed a more rapid increase in peak thrombin generation,

8 Procoagulant concentration and thrombin generation 409 Fig. 6. Thrombin production with varied factor (F)X levels. In separate assays using platelets from four healthy, normal subjects (designated A, B, C, D), levels of FX were varied (1%; 3%; 10%; 50%; 100%; 150%; 200% of pooled plasma levels) in a model system of coagulation with otherwise normal plasma levels of factors II, V, VIII, IX, and XI, antithrombin and tissue factor pathway inhibitor, catalytic amounts of factor VIIa, tissue factor-bearing monocytes and unactivated platelets. Timed samples were taken for determination of thrombin generation. n ¼ 1%, ¼ 3%, m ¼ 10%, s ¼ 50%, d ¼ 100%, h ¼ 150%, j ¼ 200%. with 60 70% of maximal thrombin generation observed at FIX concentrations of 3%. Butenas reported a ÔparadoxicalÕ 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% [2,24]. In our assays, maximal peak thrombin generation was achieved at FIX concentrations between 10% and 30%, and remained relatively constant as the concentration was increased to 200%. Such discrepancies are probably due, in part, to the different characteristics of the synthetic phospholipid surfaces used by other investigators, whereas in this study the surface was provided by platelets. Our findings demonstrate that the effects of varying FXI concentration are unique among the factors tested. The primary role of FXI in coagulation is thought to be the augmentation of FIX activation on the platelet surface, leading to increased thrombin generation. Even in the event of severe FXI deficiency, some tenase complexes can be formed and lead to formation of the prothrombinase complex on the platelet surface. However, clinical observation reveals that some individuals with FXI deficiency have bleeding episodes while others do not, and the severity of the bleeding symptoms do not correlate well with the plasma FXI levels. Our results reflect both these facts. While the effect of decreased FXI concentration on the rate of thrombin generation is similar to that seen with decreased FIX, the changes in thrombin generation are not of the same magnitude. In addition, there was considerable interindividual variability noted in the effect on peak and rate of thrombin generation in assays with no added FXI. The degree of variable response to added FXI may be an inherent feature of the individual s platelets, either the amount of FXI activity or their ability to support the activity of plasma FXI on the platelet surface. In contrast to our findings with FXI, Keularts et al. observed significant decreases in the ETP at FXI concentrations < 10%, in platelet-rich plasma and low tissue factor concentrations [25]. Conversely, it has been previously reported that at relatively high tissue factor concentrations, the AUC and rate of thrombin generation in FXI-deficient and normal whole blood were found to be equivalent [26]. The effect of varied FV on thrombin generation was significantly different from that observed with variation of FVIII, FIX or FXI. This is the first examination of the role of

9 410 G. A. Allen et al Fig. 7. Thrombin production with varied factor (F)V levels. In separate assays using platelets from four healthy, normal subjects (designated A, B, C, D), levels of FV were varied (0%; 3%; 10%; 30%; 100%; 200% of pooled plasma levels) in a model system of coagulation with otherwise normal plasma levels of factors II, VIII, IX, X and XI, antithrombin and tissue factor pathway inhibitor, catalytic amounts of factor VIIa, tissue factor-bearing monocytes and unactivated platelets. Timed samples were taken for determination of thrombin generation. j ¼ 0%, n ¼ 3%, m ¼ 10%, s ¼ 30%, d ¼ 100%, h ¼ 200%. Fig. 8. Thrombin generation with factor (F)V-deficient platelets. Assays with varied concentrations of FV (0%; 1%; 3%; 10%; 30%; 50%; 100%; 200%) were performed using platelets from an otherwise healthy, FVdeficient patient. Thrombin generation data are shown for comparison with the assays with platelets from normal donors. j ¼ 0%,, ¼ 1%,. ¼ 3%, n ¼ 10%, m ¼ 30%, s ¼ 50%, d ¼ 100%, h ¼ 200%. plasma FV on thrombin generation on the surface of FVdeficient platelets. Assays with normal platelets, which have been estimated to carry 20% of total stores of FV [27], showed minimal dependence on FV for maximal thrombin generation. However, in assays using FV-deficient platelets from a FVdeficient individual, the rate and peak of thrombin production and AUC progressively declined as the concentration of added FV decreased, and no thrombin generation was observed with no added FV. The effect on the measured parameters was biphasic, with the most profound influence on thrombin generation seen with concentrations of FV < 30%. This profound effect on thrombin generation would seem incongruous with the clinical symptoms of our FV-deficient platelet donor, who has had little in the way of severe bleeding symptoms, other than menorrhagia. In assays using this individual s platelets, no thrombin generation is observed in the absence of FV. Thus, her platelets do not appear to have any functional form of the procoagulant, as measured by a functional assay. Her plasma levels of FV activity have been consistently < 1%. It has been reported that expression of a FV

10 Procoagulant concentration and thrombin generation 411 Fig. 9. Thrombin production with varied prothrombin levels. In separate assays using platelets from four healthy, normal subjects (designated A, B, C, D), levels of prothrombin were varied (10%; 50%; 75%; 100%; 150%; 200% of pooled plasma levels) in a model system of coagulation with otherwise normal plasma levels of factors V, VIII, IX, X and XI, antithrombin and tissue factor pathway inhibitor, catalytic amounts of factor VIIa, tissue factor-bearing monocytes and unactivated platelets. Timed samples were taken for determination of thrombin generation. n ¼ 10%, m ¼ 50%, s ¼ 75%, d ¼ 100%, h ¼ 150%, j ¼ 200%. Fig. 10. Platelet activation with varied factor (F)V or factor (F)X. Assays with varied concentrations of FV (d, using FV-deficient platelets) or FX (m, using normal platelets). Platelet activation was determined by measurement of the percentage of platelets expressing the activation-specific a granule marker CD62(P-selectin, GMP140) on their surface using a FACScan flow cytometer. The time to 50% of maximal platelet activation was designated as the t 50. ÔminigeneÕ, resulting in activity levels below the level of detection, is sufficient to rescue mice from an otherwise lethal knockout of their FV gene [28]. Such may be the case in our donor, that she expresses an undetectable amount of FV activity, but there is enough to prevent more serious bleeding symptoms. The effect of varied FX levels was distinctly different from its cofactor, FV. While no thrombin generation was observed in the absence of FX, only small amounts were required for significant thrombin generation; maximal rate and peak thrombin generation and AUC were achieved at concentrations between 1% and 10% of normal plasma levels. The significant thrombin generation observed at relatively low levels of FX may be explained by the observation that platelet stores of FV appear to account for the majority of the prothrombinase complex on the platelet surface. If platelet stores of FV account for 20% of total stores, then a plasma level of 3% FX would result in equimolar amounts of the factor and its cofactor. The significant thrombin generation observed at low levels in our system would appear inconsistent with the clinical symptoms described in mild-to-moderate FX deficiency, which are comparable to similar degrees of hemophilia [29,30].

11 412 G. A. Allen et al As in the case with FVIII and FIX, our results with varied FX in the cell-based model system are in contrast to those of other researchers using cell-free plasma and synthetic phospholipid systems. We observed a normalization of thrombin generation at a significantly lower FX level than previously described, between 1 and 10% with our model system vs % as previously reported [4,7]. Additionally, a significant increase in peak thrombin generation over the range of %, as reported by Butenas, was not observed [2]. The effect of the concentration of prothrombin in the system was unique. The rate of thrombin generation, as well as peak thrombin generation and AUC, increased in direct correlation with the prothrombin concentration. There was no evidence of saturation of the prothrombinase complex, even at levels of 200%. These results are in agreement with the results obtained by others, with varied model systems [1 4,7,8]. Prothrombin in this system was not completely converted to thrombin, even after long reaction times. In our system, prothrombin and FV, FVIII, and FXI all showed increases in thrombin generation when their levels were increased from 100% to 200% of plasma levels. However, increasing prothrombin to 200% levels had the most profound effect on thrombin generation parameters. While FV, FVIII and FXI have been described as correlating with thrombotic risk when higher than normal levels are present, the strongest clinical correlation with thrombotic risk seems to be high prothrombin levels associated with the G20210A mutation [31 35]. If our observation that there is a marked increase in thrombin production with high concentrations of prothrombin canbeextendedtothein vivo setting, it may explain the thrombotic risk associated with the prothrombin G20210A mutation. A significant effect on platelet activation was noted with only two of the factors varied in our assay, FV and FX. This reflects the current model of tissue factor-initiated coagulation, where the FXa/FVa complex formed on the tissue factor cell converts prothrombin to the small amount of thrombin that leads to platelet activation [36]. Impaired formation of the prothrombinase complex on the tissue factor-bearing cell would therefore affect initial platelet activation. Similar to the results observed in the thrombin generation assays, the greatest effect on platelet activation was observed below FV levels of 30% and FX levels of 10%. In summary, using a cell-based model system of coagulation, we have performed a comprehensive examination of effect of varied concentrations of procoagulant proteins on the characteristics of thrombin generation on the platelet surface. The results revealed a number of features unique to the different 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 (FVIII, FIX and FXI) primarily affected the rate and peak of thrombin production, while having little to no effect on the AUC. Our data reflect the mechanism underlying the milder and more variable bleeding manifestations in FXI deficiency, compared with severe hemophilia A and B, namely that even in severe FXI deficiency, some tenase complex is still able to be formed the presence of FXI only augments its formation. We know that thrombin production is not simply limited by the amount of prothrombin in the system, as residual prothrombin has been demonstrated after the cessation of thrombin production. In contrast, variation of the concentration of either prothrombin or FV (with FV-deficient platelets) in the system had significant effects on all three measures of thrombin production: rate, peak and AUC. Finally, while no thrombin generation was observed with no FX in the system, only very small amounts (between 1% and < 10% of normal plasma levels) were required for normalization of the parameters measured. Our findings may offer some insight as to the role of the individual proteins in the process of coagulation and perhaps the different clinical symptoms observed in persons with different factor deficiencies. Attempts to contrast our results with those of others are made difficult due to the numerous disparities between the model systems employed. In some instances, the inability of phospholipid particles to replace platelets is obvious, notably the primary role of platelet vs. plasma FV and platelet stores of procoagulant such as FVIII and FXI. 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