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Keywords:

  • factor IX;
  • factor XI;
  • prothrombin;
  • thrombin generation;
  • tissue factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Summary.  The influence of plasma and platelet factor (F)XI on thrombin generation initiated with 10 pm tissue factor (TF) in a synthetic coagulation model was evaluated in the presence of either 2 × 108 mL−1 platelets or the equivalent (2 µm) phospholipids. In either system, with all proteins present at physiological concentrations, FXI (30 nm) had no effect on thrombin generation. With phospholipids in the absence of FXI, an increase in vitamin K-dependent proteins (VKDP) (up to 500%) significantly prolonged the initiation phase of thrombin generation and decreased maximum thrombin levels. The inhibition was principally caused by the elevated prothrombin and FIX concentrations. When 30 nm FXI was added with elevated VKDP and phospholipids, the initiation phase was decreased and the maximum thrombin levels generated substantially increased. In experiments with platelets (with and without plasma FXI), an increase in VKDP had little effect on the initiation phase of thrombin generation. These data indicate that (i) FXI has no effect on thrombin generation at 10 pm TF and physiological concentrations of VKDP; (ii) platelets and plasma FXI are able to compensate for the inhibitory effects of elevated VKDP.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Factor (F)XI is a serine protease precursor present in plasma and platelets and involved in the process of blood coagulation [1]. Although it is well established that activated FXI is one of FIX activators [2], the role of this protein in the scheme of blood coagulation is not entirely clear. Traditionally, FXI has been assigned as a protein central to the intrinsic (contact) pathway of coagulation. This assignment was made based upon the observation that FXI plays a crucial role in the in vitro clot formation process initiated by the contact pathway proteins in the presence of negatively charged surfaces [3,4]. However, bleeding pathology does not seem to be associated with deficiency states of the proteins responsible for triggering the contact pathway, i.e. FXII, high-molecular-weight kininogen and prekallikrein [5,6]. On the other hand, in FXI deficiency, while spontaneous bleeding tendencies are rarely observed, many deficient individuals are prone to excessive bleeding upon hemostatic challenge [7–11]. This apparent paradox between the clinical manifestations of FXI deficiency and the absence of pathology associated with defects of proteins involved in contact activation of FXI is hypothesized to be due to the presence of another, contact pathway-independent, activation of this zymogen by thrombin [12–15].

In in vitro coagulation models, no alterations in thrombin generation or clot formation were observed with deficiencies in FXI when relatively high concentrations (≥10 pm) of tissue factor (TF) were used to initiate thrombin generation [16–19]. However, at very low TF concentrations (<5 pm), thrombin generation and clot formation in FXI-deficient blood or plasma were delayed, and maximum thrombin levels were reduced [16–18].

The major fraction of FXI circulating in blood is represented by the plasma protein, whereas blood platelets are reported to contain approximately 0.5% of total FXI [20,21]. However, the platelet pool of FXI is reported to display relatively high activity, exceeding that of the plasma [1,20,21].

In previous studies we observed a somewhat paradoxical inhibitory effect of elevated FIX on thrombin generation in the synthetic coagulation model expressed in the presence of phospholipids [22,23]. We also established that an increase in prothrombin to 150% of mean physiological concentration in both synthetic and whole blood coagulation models increases thrombin generation and accelerates clot formation [22].

In the present study we evaluate the influence of FXI and platelets on TF-initiated thrombin generation in the synthetic coagulation model and minimally altered whole blood at elevated concentrations of FIX, prothrombin, and other vitamin K-dependent proteins (VKDP). These studies provide some insight into the role of FXI in TF-initiated thrombin generation.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Materials

Human coagulation factor (F)VII, FIX, FX, FXI and FV, prothrombin, AT-III and protein C were isolated from fresh frozen plasma [24–26], and purged of trace contaminants and traces of active enzymes as described [27]. Recombinant FVIII (rFVIII) and recombinant TF (residues 1–242) were provided as gifts by Drs Shu Len Liu and Roger Lundblad (Hyland Division, Baxter Healthcare Corp, Duarte, CA, USA). Recombinant human FVIIa was purchased from Novo Pharmaceuticals (Copenhagen, Denmark). Recombinant full-length tissue factor pathway inhibitor (TFPI) produced in Escherichia coli was provided as a gift by Dr K. Johnson (Chiron Corp, Emeryville, CA, USA). Recombinant soluble thrombomodulin (TM) (Solulin) was provided as a gift by Dr J. Morser (Berlex, Richmond, CA, USA). Washed platelets were prepared by the procedure of Mustard et al. [28]. Prothrombin fragment 1 was generated and purified as described [29]. Monoclonal anti-FXI antibody (α-FXI-2) was developed by the Biochemistry Antibody Core Laboratory (University of Vermont). 1,2-Dioleolyl-sn-Glycero-3-[Phospho-L-Serine] (PS), 1,2-Dioleolyl-sn-Glycero-3-Phosphocholine (PC), and EDTA were purchased from Sigma (St Louis, MO, USA). Phospholipid vesicles (PCPS) composed of 25% PS and 75% PC were prepared as described [30]. The TF/lipid reagent (2 nm TF/10 µm PCPS) and corn trypsin inhibitor (CTI) were prepared as described [16]. Spectrozyme TH was purchased from American Diagnostica, Inc. (Greenwich, CT, USA). The ELISA thrombin-AT-III (TAT) kit (Enzygnost) was purchased from Behring (Marburg, Germany).

Synthetic coagulation model

The procedure used is a modification of Lawson et al. [31] and van ‘t Veer et al. [27]. In all experiments thrombin generation was initiated with 10 pm relipidated TF in the presence of either 2 µm PCPS (PCPS at 1–2 µm are equivalent in this model to 2 × 108 mL−1 platelets in supporting thrombin generation [32]) or 2 × 108 mL−1 platelets (mean physiological concentration). Concentrations of all proteins, platelets and PCPS in the reaction are indicated in Table 1.

Table 1.  Concentrations of components in solutions I and II of the synthetic coagulation model
ComponentConc. in solution I or IIFinal conc. in the reaction
100%300%500%100%300%500%
  • *

    Membrane proteins.

  • 2 µm PCPS are equivalent in this model to 2 × 108 mL−1 platelets in supporting thrombin generation [32].

I. Procofactor solution
TF*20 pm10 pm
PCPS4 µm2 µm
Platelets4 × 108 mL−12 × 108 mL−1
Factor V40 nm20 nm
Factor VIII1.4 nm0.7 nm
TM*2.0 nm1.0 nm
II. Zymogen-inhibitor solution
Prothrombin2.8 µm8.4 µm14 µm1.4 µm4.2 µm7.0 µm
Factor VII20 nm60 nm100 nm10 nm30 nm50 nm
Factor VIIa0.2 nm0.6 nm1.0 nm0.1 nm0.3 nm0.5 nm
Factor IX180 nm540 nm90 nm270 nm
Factor X340 nm1020 nm1700 nm170 nm510 nm850 nm
Protein C120 nm360 nm600 nm60 nm180 nm300 nm
Factor XI60 nm30 nm
TFPI5.0 nm2.5 nm
AT-III6.8 µm3.4 µm
α-factor XI-2100 µg mL−150 µg mL−1
  • I Procofactor solution. Relipidated TF was incubated with PCPS (omitted in platelet experiments) in HBS (20 mm HEPES and 150 mm NaCl), 2 mm CaCl2 for 10 min at 37 °C. FV, FVIII, TM and platelets (in platelet experiments only) were added to the relipidated TF prior to the initiation of the reaction.

  • II Zymogen-inhibitor solution. FVII, FVIIa, FX and FIX, protein C, prothrombin FXI (when desired), α-FXI-2 (when desired), TFPI, and AT-III at 2 × desired concentrations were preheated in HBS, 2 mm CaCl2 at 37 °C for 3 min.

The reaction was started by mixing equal volumes of both solutions resulting in physiological concentrations of FV, FVIII and FXI, TFPI, AT-III and platelets, 100–500% physiological concentrations of FVII, FVIIa, FIX and FX, protein C and prothrombin. Following initiation of the reaction, at selected time points, 10-µL aliquots were withdrawn from the reaction mixture and quenched in 20 mm EDTA in HBS pH 7.4 containing 0.2 mm Spectrozyme TH and assayed immediately for thrombin activity. The hydrolysis of the substrate was monitored by the change in absorbance at 405 nm using a Molecular Devices Vmax spectrophotometer. Thrombin generation was calculated from a standard curve prepared by serial dilutions of α-thrombin.

TF-initiated clotting of fresh human blood

Single-tube clotting time test The protocol used was that of Holmes et al. [33]. Fresh human blood (1 mL) was added to a K-resin ACT tube (International Technidyne, Edison, NJ, USA) without the glass beads used to activate the contact pathway containing 100 µg mL−1 CTI (CTI prevents contact pathway of blood coagulation by inhibiting FXIIa), 10 or 20 pm relipidated TF and selected amounts of prothrombin, FIX or all VKDP. The tube was placed in a Hemochron ACT instrument (International Technidyne). The time to clot was detected by the displacement of a magnet within the rotating tube when fibrin strands formed.

Multiple-tube experiments The protocol used was a modification of Rand et al. [34]. Experiments were performed in 32 tubes placed on a rocking table enclosed in a 37 °C temperature-controlled glove box using fresh CTI-inhibited (100 µg mL−1 CTI) blood. Blood was drawn by venepuncture and immediately delivered into the reagent-loaded tubes. Thirty of 32 tubes (two series per experiment—16 tubes/series) were loaded with CTI and 5 pm relipidated TF in HBS, 2 mm CaCl2. Two phlebotomy control tubes (one tube/series) contained no TF. Selected amounts of prothrombin or FIX (all tubes, experiment series only) and equivalent volume of their dilution buffer (HBS, 2 mm CaCl2, all tubes control series only) were loaded. The zero-time tube of each series was pretreated with 1 mL of 50 mm EDTA and 10 µL of 10 mmd-Phe-Pro-ArgCH2Cl (FPRck) (diluted in 10 mm HCl). After blood was delivered, the tubes were periodically (1–20 min) quenched with EDTA and FPRck. No more than 35 µL of reagents were loaded in each tube. The clotting time was observed visually by two observers and was called when ‘clumps’ were observed on the side of the tube. After the experiment, tubes were centrifuged and the supernatants aliquoted for the TAT (thrombin generation) analysis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In the presence of all proteins in the synthetic coagulation mixture at mean physiological concentrations, thrombin generation initiated with 10 pm TF was marginally affected by the presence of 30 nm plasma FXI. This observation is valid for experiments conducted in the presence of 2 µm PCPS as well as for those conducted in the presence of platelets at mean physiological concentrations (2 × 108 mL−1) (compare ▪ in Figs 1A,B and 2A,B; ▪ and ● in Fig. 3A,C). However, when the concentrations of VKDP were significantly increased above their mean physiological concentrations, FXI began to play a pronounced role in thrombin generation, especially in the reactions conducted in the presence of phospholipids.

image

Figure 1. Thrombin generation in the synthetic coagulation model in the presence of normal and elevated concentrations of vitamin K-dependent proteins (VKDP) and 2 µm phospholipid vesicles (PCPS). Thrombin generation was initiated with 10 pm relipidated tissue factor (TF) in the absence (A) and in the presence (B) of 30 nm plasma factor XI. Factors V and VIII, AT-III and TFPI were present at physiological concentrations, thrombomodulin (TM) at 1 nm, and VKDP (factors VII, VIIa, IX and X, prothrombin and protein C) at 100% (▪), 300% (□) and 500% (▵) of their physiological concentrations.

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image

Figure 2. Thrombin generation in the synthetic coagulation model in the presence of normal and elevated concentrations of vitamin K-dependent proteins (VKDP) and 2 × 108 mL−1 platelets. Thrombin generation was initiated with 10 pm relipidated tissue factor (TF) in the absence (A) and in the presence (B) of 30 nm plasma factor XI. Factors V and VIII, AT-III and TFPI were present at physiological concentrations, thrombomodulin (TM) at 1 nm, and VKDP (factors VII, VIIa, IX and X, prothrombin and protein C) at 100% (▪), 300% (□) and 500% (▵) of their physiological concentrations.

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image

Figure 3. Thrombin generation in the synthetic coagulation model in the presence of normal and elevated concentrations of factor IX. Thrombin generation was initiated with 10 pm relipidated tissue factor (TF) in the presence of 2 µm phospholipid vesicles (PCPS) (A) or 2 × 108 mL−1 platelets (B,C) and in the absence (B) or in the presence (C) of 30 nm plasma factor XI. Factors V, VIII, VII, VIIa, and X, prothrombin, protein C, AT-III and TFPI were present at physiological concentrations, and thrombomodulin (TM) at 1 nm. Factor IX was present either at 100% of mean physiological concentration in the absence (▪) or in the presence (●) of 30 nm plasma factor XI or at 300% of mean physiological concentration in the absence of plasma factor XI (□), in the presence of 30 nm plasma factor XI (○) and in the presence of plasma factor XI and 50 µg mL−1α-factor XI-2 (▵).

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The effect of FXI on thrombin generation at increased VKDP concentrations

Figure 1A illustrates thrombin generation in the synthetic coagulation model in the absence of FXI and in the presence of 10 pm TF and 2 µm PCPS. In a control experiment [▪; all proteins were present at their mean physiological concentrations (Table 1)], thrombin generation entered the propagation phase [19] approximately 4–5 min after reagent mixing. Maximum rates of thrombin generation reached 2.3–2.9 nm s−1 and maximum levels of active thrombin achieved were 300–400 nm. In a similar experiment performed in the presence of the VKDP (FVII, FVIIa, FIX and FX, prothrombin and protein C) at 300% mean physiological concentrations (□ and Table 1), the initiation phase of thrombin generation was prolonged to 12 min. The maximum rate of thrombin generation was suppressed to 0.68 nm s−1 and the maximum level of thrombin observed decreased to 130 nm. When the VKDP concentration was increased to 500% (▵ and Table 1), no detectable thrombin generation was observed after 20 min of reaction.

At mean physiological VKDP concentrations (▪; Fig. 1B), 30 nm plasma FXI (mean physiological concentration) had no apparent effect on the reaction (compare ▪ in Fig. 1A and B; note vertical scale change). In marked contrast, the addition of FXI with elevated VKDP altered thrombin generation dramatically. At 300% VKDP, the initiation phase was prolonged by 1 min only (compare with prolongation by 8 min in the absence of FXI), whereas the maximum rate of thrombin generation and maximum level of active thrombin were increased from 2.9 to 11.9 nm s−1 and 400 nm to 1.5 µm, respectively (□; Fig. 1B). An increase in the VKDP to 500% (▵), led to a further prolongation of the initiation phase (to 10 min) and an increase in the maximum concentration of active thrombin (to 2.3 µm), whereas the maximum rate of thrombin generation (approximately 10 nm s−1) remained similar to that observed at 300% VKDP. At both 300 and 500% VKDP, the concentration of active thrombin remained high until the end of the experiment (20 min).

In the presence of washed platelets at physiological concentration (2.0 × 108 mL−1), the initiation phase duration of thrombin generation at 100% VKDP (Fig. 2A; ▪) was 2 min. The maximum level of active thrombin achieved was 280 nm and the maximum rate of thrombin generation reached 1.8 nm s−1. In contrast to the result when phospholipids were used in the absence of FXI, thrombin generation in the presence of platelets was only slightly influenced by elevated VKDP concentrations. At both 300% (□) and 500% (▵) VKDP, the initiation phase was prolonged by approximately 1 min from that observed at mean plasma concentrations (▪). Similarly, the maximum rates of thrombin generation (1.8 nm s−1 at 300% VKDP and 2.7 nm s−1 at 500%) and maximum thrombin levels (310 nm at 300% VKDP and 410 nm at 500%) were not altered to a large extent by elevated concentrations of the VKDP.

In the synthetic coagulation model with normal levels of plasma FXI, thrombin generation in the presence of platelets (Fig. 2B) somewhat resembled that observed on PCPS (compare Figs 1B and 2B). In the presence of all proteins at their mean physiological concentrations (▪), thrombin generation was not altered substantially by the presence of plasma FXI (compare ▪ in Fig. 2A,B; note vertical scale change). At 300% VKDP, the maximum rate of thrombin generation in the presence of platelets and plasma FXI was increased to 12.8 nm s−1 and maximum level to 1.0 µm (Fig. 2B; □). In the presence of 500% VKDP (▵), these parameters for thrombin generation were increased to 17.6 nm s−1 and 1.9 µm. The duration of the initiation phase was almost not affected by the presence of elevated concentrations of VKDP, in contrast to the effect observed in the presence of PCPS.

In whole blood clotting experiments initiated with 10 pm relipidated TF (50 nm PCPS), the addition of VKDP to produce 300% and 500% of their mean physiological concentrations had little effect on the clotting time of normal blood. CTI-inhibited normal blood without excess VKDP clotted in 6.2 min. The addition of VKDP to this blood to produce a final level of 500% led to only a marginal increase (to 6.6 min) in clotting time.

The influence of elevated FVII, FVIIa, FX and protein C on thrombin generation

In the presence of 2 µm PCPS and in the absence of FXI, the addition of 30–50 nm FVII to the synthetic coagulation model (the final concentrations of FVII in the reaction mixtures were 300 and 500% of mean physiological concentrations, respectively) prolonged the duration of the initiation phase by approximately 1 min and slightly suppressed maximum thrombin generation rate (from 2.5 nm s−1 to 2.0 nm s−1) and the maximum levels of thrombin (from 370 nm to 310 nm). When both FVII and FVIIa were added at 300 and 500% (final concentrations of FVIIa were 0.3 nm and 0.5 nm, respectively), the duration of the initiation phase of thrombin generation decreased in a concentration-dependent manner. The maximum rates of thrombin generation and thrombin levels were not influenced by simultaneous increases in FVII and FVIIa concentrations within the range studied. No alterations in thrombin generation were observed by the increased concentration (up to 500%) of FX.

Protein C at elevated concentrations in the presence of 10 pm TF, 2 µm PCPS and 1 nm thrombomodulin (no FXI) decreased the thrombin generation rate in the synthetic coagulation model during the first minute of the propagation phase. The rate of thrombin generation within this minute was inversely related to the protein C concentration. Thus, in the presence of 100% protein C the rate was 3.3 nm s−1, in the presence of 200% protein C it decreased to 1.2 nm s−1 and at 500% to 0.2 nm s−1. The maximum rate of thrombin generation during the following part of the propagation phase was almost not affected by the elevated protein C. At 20 pm TF and no added TM, the addition of 15 µg mL−1 protein C to normal blood (final protein C concentration was 500% of mean physiological) had no effect on the clotting time (4.3 min in both cases).

The influence of FXI on thrombin generation at elevated FIX concentrations

The presence of 30 nm FXI with FIX at 100% (90 nm) in the synthetic coagulation model with either 2 µm PCPS (Fig. 3A) or 2 × 108 mL−1 platelets (Fig. 3C) and initiated with 10 pm TF, had almost no effect on thrombin generation during both initiation and propagation phases (compare ▪ and ●). At 270 nm FIX (300% of mean physiological concentration) with 2 µm PCPS in the absence of FXI (□; Fig. 3A), the duration of the initiation phase was prolonged from approximately 4 min to almost 9 min, while the rate of thrombin generation during the propagation phase was not substantially affected by the elevated FIX concentration (1.4 nm s−1 at 100% FIX and 1.2 nm s−1 at 300% FIX). In contrast, for experiments performed in the presence of 30 nm FXI (mean physiological concentration) and 300% FIX (○; Fig. 3A), the initiation phase was shortened to approximately 5 min.

To test whether the reduction in initiation phase duration is related to FXIa activity, 50 µg mL−1 of an inhibitory monoclonal anti-FXI(a) antibody α-FXI-2 were added to the synthetic coagulation model. This antibody did not alter FXI activation but inhibited FIX activation by FXIa. In the presence of 2 µm PCPS, 300% FIX and 30 nm FXI, the addition of α-FXI-2 resulted in a thrombin generation profile (▵; Fig. 3A) similar to that observed in the absence of FXI (□). The antibody had no effect on thrombin generation when plasma FXI was not present in the reaction mixture. Similarly, thrombin generation was not altered by α-FXI-2 in the presence of both FIX and FXI at mean physiological concentrations.

When platelets were used in the synthetic coagulation model lacking plasma FXI (Fig. 3B), an increase in FIX from 100% (▪) to 300% (□) had no effect on thrombin generation.

Thrombin generation in the synthetic coagulation model with platelets was also evaluated in the presence of plasma FXI at mean physiological concentrations. At 100% FIX, the addition of plasma FXI had almost no effect on thrombin generation (compare ▪ and ● in Fig. 3C). An increase in FIX concentration to 300% in the presence of 30 nm plasma FXI (○) accelerated thrombin generation from 1.5 nm s−1[at 100% FIX (▪)] to 2.5 nm s−1, while the duration of the initiation phase was not affected. The addition of 50 µg mL−1α-FIX-2 in the presence of 300% FIX and 100% plasma FXI (▵) extended the initiation phase of thrombin generation to 3 min.

The addition of purified FIX to whole blood to produce 200% of mean physiological concentration of this protein (5.1 µg mL−1) had almost no effect on thrombin generation (data not shown).

The influence of FXI on thrombin generation at elevated prothrombin concentrations

Elevated prothrombin concentrations in the synthetic coagulation model with 2 µm PCPS in the absence of FXI prolonged the initiation phase, decreased the maximum rate of thrombin generation and increased its maximum levels (Fig. 4). At 300% prothrombin (□) the initiation phase was extended from 4.5 min observed at 100% (▪) to 9 min, the maximum rate was decreased from 2.7 nm s−1 (at 100% prothrombin) to 1.6 nm s−1, and the maximum thrombin level was increased from 340 nm to 640 nm. A further increase in prothrombin concentration to 500% (▵) caused the extension of the initiation phase to 14 min, a decrease in thrombin generation rate to 1.1 nm s−1 and an increase in ultimate thrombin level to 870 nm. The addition of 30 nm plasma FXI to the reaction mixture containing 500% prothrombin did not increase thrombin generation (data not shown).

image

Figure 4. Thrombin generation in the synthetic coagulation model in the presence of normal and elevated concentrations of prothrombin and 2 µm phospholipid vesicles (PCPS). Thrombin generation was initiated with 10 pm relipidated tissue factor (TF) in the absence of factor XI. Factors V, VIII, VII, VIIa, IX and X, protein C, AT-III and TFPI were present at physiological concentrations, thrombomodulin (TM) at 1 nm, and prothrombin at 100% (▪), 300% (□) and 500% (▵) of the physiological concentration.

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When 2 × 108 mL−1 platelets replaced PCPS in the absence of plasma FXI (Fig. 5A), an increase in prothrombin from 100% (▪) to 300% (□) had little effect on the initiation phase duration. Thrombin generation during the beginning of the propagation phase was decreased from 1.1 nm s−1 to 0.45 nm s−1. Subsequently, the rate of thrombin generation at 300% prothrombin increased to 1.8 nm s−1. The maximum level of thrombin was increased from 210 nm at 100% prothrombin to 270 nm at 300%. An increase in prothrombin concentration to 500% (▵) prolonged the initiation phase from 2 min to 6 min, decreased the maximum rate of thrombin generation to 0.48 nm s−1 and maximum level of thrombin to 100 nm.

image

Figure 5. Thrombin generation in the synthetic coagulation model in the presence of normal and elevated concentrations of prothrombin and 2 × 108 mL−1 platelets. Thrombin generation was initiated with 10 pm relipidated tissue factor (TF) in the absence (A) and in the presence (B) of 30 nm plasma factor XI. Factors V, VIII, VII, VIIa, IX and X, protein C, AT-III and TFPI were present at physiological concentrations, thrombomodulin (TM) at 1 nm, and prothrombin at 100% (▪), 300% (□) and 500% (▵) of the physiological concentration.

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The addition of plasma FXI to the reaction with platelets at elevated prothrombin substantially altered thrombin generation (Fig. 5B). At 300% prothrombin (□), both the maximum rate of thrombin generation (from 0.9 nm s−1 at 100% prothrombin to 7.5 nm s−1 at 300%) and maximum levels (from 160 nm to 980 nm) were increased. The initiation phase of thrombin generation remained unaltered by this increase in prothrombin concentration. At 500% prothrombin (▵), the initiation phase was prolonged by approximately 1 min, the maximum thrombin generation rate was increased to 17.2 nm s−1, and maximum level of thrombin to 2.0 µm.

A similar effect of increased prothrombin concentration on the clotting time (generally equivalent to the initiation phase duration) was observed in whole blood clotting experiments. In reactions initiated with 5 pm TF, the clotting time was prolonged from 4.5 min to 6.5 min when 0.4 mg mL−1 prothrombin (to achieve 500% of mean physiological concentration) was added. Thrombin generation during the propagation phase was almost not affected by the addition of prothrombin. A prolongation of the clotting time by approximately 2 min was also observed when 0.4 mg mL−1 prothrombin was added to whole blood in the Hemochron ACT experiment (data not shown).

To test the possibility that elevated prothrombin concentration inhibited thrombin generation primarily due to the occupancy of a limited surface provided by PCPS and platelets, the influence of prothrombin fragment 1 (which contains the phospholipid binding site) on thrombin generation was evaluated. The addition of 7 µm fragment 1 (equivalent of 500% prothrombin) to the reaction with 2 µm PCPS in the absence of FXI, increased the duration of the initiation phase by 3 min and suppressed the thrombin generation rate during the propagation phase from 2.5 nm s−1 to 1.5 nm s−1. In a similar experiment performed with platelets, the addition of 7 µm fragment 1 to the reaction in the absence of plasma FXI had no significant effect either on the duration of the initiation phase or on thrombin generation during the propagation phase (data not shown)—a result consistent with an earlier publication [35] indicating the presence of discrete binding sites on platelets.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The data of the current study indicate that (i) plasma FXI has no effect on thrombin generation induced with 10 pm TF when all proteins are present at their mean physiological concentrations; (ii) elevated concentrations of VKDP inhibit thrombin generation in the absence of FXI when phospholipids are used to provide the membrane surface; (iii) the inhibition is caused primarily by elevated concentrations of FIX and prothrombin; (iv) at their mean physiological concentrations, either FXI or platelets correct the thrombin generation in the presence of elevated FIX; (v) platelets compensate for the inhibition caused by the high level of VKDP; (vi) plasma FXI, alone or together with platelets, causes substantial increases in thrombin generation rates and maximum levels at elevated concentrations of VKDP.

The role of FXI in the blood coagulation process is not completely clear. Spontaneous bleeding among FXI-deficient individuals is rare, and most commonly occurs as a result of surgical intervention. The frequency and severity of bleeding manifestations, however, often do not correlate with the level of FXI. Some homozygotes may not bleed even after surgery, whereas some heterozygotes containing 50–70% FXI may have bleeding complications [7–11,36].

In our laboratory we have been evaluating the influence of FXI on thrombin generation and clot formation in a synthetic coagulation model and in minimally altered whole blood. FXI has a marginal (if any) effect on thrombin generation in the synthetic model initiated with ≥5 pm TF [19,22]. Similarly, the processes leading to blood coagulation are not affected by the FXI deficiency at TF concentrations exceeding 5 pm[16]. This lack of response to FXI is a consequence of relatively slow FXI activation by thrombin which occurs only late in the reaction [12,19]. Thus, the FXIa generated fails to support FIX activation substantially. When very low TF concentrations are added to the FXI-deficient blood or plasma and the initiation phase of thrombin generation is extended, FXI replacement has a pronounced effect on thrombin generation and clot formation [16–18]. As an extension of previous work [16,19], the influence of FXI on thrombin generation at elevated VKDP is evaluated in the current study.

An increase in the VKDP concentration leads to impaired thrombin generation in the presence of 2 µm phospholipids and in the absence of FXI. The most likely explanation of this phenomenon is that at the limited surface provided by 2 µm PCPS, the increase in the concentration of VKDP interferes with the assembly of procoagulant enzymatic complexes on the surface of phospholipids. As expected, prothrombin, which has the highest concentration and similar affinity for phospholipids to other VKDP [37], is the major inhibitor of thrombin generation at limiting phospholipid concentrations.

The inhibitory effect of prothrombin, however, is less than that observed when all VKDP are elevated. This leads to the conclusion that additional VKDP at elevated concentrations have pronounced inhibitory effect on thrombin generation. Data published from several laboratories have established that FIX inhibits FX activation by the FVIIa/TF complex due to the competition of these two zymogens for the same complex enzyme [23,38–42]. In this study a substantial increase in the duration of the initiation phase was observed when an elevated concentration of FIX was present in a reaction with phospholipids in the absence of FXI. A prolongation of the initiation phase is an indicator of decreased FX activation because FXa is essential for the generation of the initial thrombin required for procofactor activation [19,34].

As a consequence of the interplay of two inhibitory processes, i.e. depletion of available surface by prothrombin and decreased FX activation, thrombin generation at elevated VKDP is impaired.

An addition of plasma FXI to the synthetic coagulation model with 2 µm phospholipids and elevated concentrations of VKDP substantially increases the rate of thrombin generation and maximum level produced. Presumably, under the conditions of the experiment, the thrombin produced during the extended initiation phase generated limited amounts of FXIa, which activated additional amounts of FIX. This led to an increased concentration of the FIXa–FVIIIa complex with a consequence of more efficient FX activation. Additionally, the efficiency of FIX activation by FXIa has to increase almost linearly with increased FIX concentration due to a relatively high Km for this reaction [43].

Activated platelets provide binding sites (receptors) for the components of the complex enzymes of blood coagulation [35,44–51]. Although the majority of binding sites are specific for particular proteins [35], some of them can be shared by more than one protein [51,52]. The observation that the lipid-binding protein annexin V inhibits prothrombin, FX(a) and FIXaβ binding to platelets [51,53,54] suggests that phospholipids present in the platelet membrane also mediate protein–membrane interactions.

In the synthetic coagulation model with platelets at mean physiological concentrations, elevated concentrations of VKDP in the absence of plasma FXI have a less pronounced effect on thrombin generation than in the reaction with phospholipids. The compensatory mechanism is presumably provided by both platelet FXI and specific binding sites present on platelets. The former conclusion is supported by the data, which show that elevated FIX does not affect thrombin generation in the presence of platelets. This result indicates that platelet FXI compensates for FX activation by a mechanism similar to that observed in the presence of phospholipids and plasma FXI. Platelets are reported to contain approximately 0.5% of total FXI [20,21]. The existence of platelet FXI is supported by the observation that patients with plasma FXI deficiency but normal platelet FXI have no hemostatic abnormality, whereas those with deficiency of both plasma and platelet FXI may display hemostatic problems [1,20,21].

Walsh and coworkers reported a competition between prothrombin and FIXa and prothrombin and FX for the binding sites on activated platelets [51,52]. In the latter case, they showed that prothrombin and prothrombin fragment 1 are equipotent inhibitors of FX binding. In this study, prothrombin fragment 1 at 7 µm concentration had only a marginal effect on thrombin generation in the synthetic coagulation model with platelets. Prothrombin, however, at the same concentration, significantly prolonged the initiation phase of thrombin generation and somewhat suppressed maximum thrombin levels. These data suggest that prothrombin may have a binding site outside of the Gla-domain, which interacts with shared binding site on platelets. Alternatively, prothrombin may more efficiently interfere with complex enzyme formation on the surface of platelets than prothrombin fragment 1 due to its larger size or steric orientation.

The addition of plasma FXI to reaction mixtures with platelets and elevated VKDP or platelets and elevated FIX alone further accelerates thrombin generation during the propagation phase due to more rapid FIX activation by increased FXIa according to the mechanism discussed above for the reaction in the presence of phospholipids.

The data of this study demonstrate that plasma FXI and platelets are essential to maintain normal hemostasis in the presence of elevated VKDP. On the other hand, at these conditions, elevated concentrations of VKDP cause substantial increases in total thrombin generated. This effect, however, will be undetectable in the conventional clotting assays because of limited sensitivity of the duration of the initiation phase of thrombin generation (corresponds to the clotting time [34]) to elevated concentrations of VKDP.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Dr Shu Len Liu and Dr Roger Lundblad for providing us with TF and factor VIII; Dr Kirk Johnson for providing TFPI; and Dr John Morser for providing Solulin. This work was supported by grants R01 HL34575 and PHS T32 HL07594 from the National Institutes of Health and by Baxter-Hyland Healthcare Corp. Portions of this work were presented at the XVII (August 14–21 1999, Washington, DC, USA) and XVIII (July 6–12 2001, Paris, France) Congresses of the International Society on Thrombosis and Haemostasis (abstracts ##1508 and 2390, respectively).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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