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Elevated factor VIII enhances thrombin generation in the presence of factor VIII-deficiency, factor XI-deficiency or fondaparinux
Thrombosis Research 127 (2011) 135–140
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Elevated factor VIII enhances thrombin generation in the presence of factor VIII-deficiency, factor XI-deficiency or fondaparinux☆ Fania Szlam a, Gautam Sreeram a, Cristina Solomon c,d , Jerrold H. Levy a, Ross J. Molinaro b, Kenichi A. Tanaka a,⁎ a
Department of Anesthesiology, Emory University School of Medicine, Atlanta, Georgia, USA Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA Department of Anesthesiology, Salzburger Landeskliniken SALK, Salzburg, Austria d Department of Intensive Care, Salzburger Landeskliniken SALK, Salzburg, Austria b c
a r t i c l e
i n f o
Article history: Received 19 April 2010 Received in revised form 2 September 2010 Accepted 19 October 2010 Available online 8 December 2010 Keywords: Factor VIII Hemodilution Thrombin Generation
a b s t r a c t Background: Increased levels of factor VIII occur as a response to vascular injury and/or inflammation, and may increase thrombotic risks. In contrast, factor VIII deficiency poses a major hemostatic challenge. The role of factor VIII in modulating hemostasis/thrombosis was investigated in plasma models of hypocoagulable and hypercoagulable state using thrombin generation (TG) assay. Methods: TG was performed in undiluted/diluted control, FVIII-deficient, FVIII-deficient with low antithrombin (AT activity, ~ 59%), and factor XI-deficient plasma samples using relipidated tissue factor (TF, 2 pM) or dilute Actin as activators. The impact of elevated FVIII on TG was simulated by adding Humate-P (0 to 3 U/ml) to the above plasma samples. In fondaparinux (1 μg/ml) treated plasma with normal or lower AT activity effects of Humate-P vs. 60 nM of recombinant activated factor VII (rFVIIa) were also evaluated. Results: Humate-P increased TG concentration dependently in undiluted and diluted control plasma with TF activation. With Actin activation, only the concentration dependent shortening of lag time, but no change in peak thrombin was observed. In FVIII-deficient, FVIII-deficient with low AT, and FXI-deficient samples, 3 U/ml of Humate-P increased TG, and decreased its onset with either activator. The reduced peak thrombin due to fondaparinux was reversed with Humate-P (3 U/ml) more than with rFVIIa. Elevated FVIII levels seem to favor intrinsic tenase formation and antagonize fondaparinux because anti-FIXa aptamer added to fondaparinux effectively attenuated TG. Conclusion: Elevated FVIII supports the propagation of TG via intrinsic tenase formation under low TF condition, factor XI deficiency or in the presence of fondaparinux. © 2010 Elsevier Ltd. All rights reserved.
Introduction Factor VIII (FVIII) is a glycoprotein, which circulates in plasma at a low concentration (0.0003 μM) in complex with von Willebrand factor (vWF). Upon vascular injury or during inflammation, plasma FVIII levels can rapidly increase due to the release of FVIII-vWF from endothelial cells [1]. As an acute phase reactant, FVIII levels usually increase during surgery/trauma, or tend to remain at nearly normal (hemostatic) level, contrary to other coagulation factors that are susceptible to hemodilution during major surgery [2,3]. In case of patients with hemophilia A, trauma and surgery pose a major
☆ Support: This study was supported in part by Department of Anesthesiology, Emory University School of Medicine, and CSL Behring, Marburg, Germany. ⁎ Corresponding author. Department of Anesthesiology, Emory University School of Medicine, 1364 Clifton Road, N.E., Atlanta, Georgia, USA 30322. Tel.: +1 404 778 3900; fax: +1 404 778 5194. E-mail address: [email protected] (K.A. Tanaka). 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.10.017
bleeding risk, which is usually mitigated by prophylactic FVIII replacement [4,5]. The presence of neutralizing antibodies against FVIII further increases the risks of recurrent muscle/joint bleeds and perioperative hemorrhage [5–7]. In contrast to FVIII deficiency, bleeding risks in individuals with FXI deficiency are considered mild to moderate, but highly variable after trauma and injury [8,9]. Such variability in hemostatic response can possibly play a role in the incidence of thromboembolism associated with congenital antithrombin (AT) deficiency because elevated FVIII levels can dynamically affect hemostatic activity after trauma/surgery [2,3,10] and during pregnancy [11]. Individual variability in FVIII levels could in part explain the differences in achieving hemostasis in FXI deficiency or thrombophilic tendency in AT deficiency. On the other hand it is difficult to predict a bleeding or thrombotic risk based on a single factor level because the amount of thrombin produced at a vascular injury site is influenced by complex interactions among various cell surface proteins, and procoagulant and anticoagulant elements. Therefore, we hypothesized that the impact of elevated
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FVIII levels in hypocoagulable and thrombophilic plasma could be quantified by the calibrated automated thrombin generation (TG) measurement. Because TG patterns are influenced by the activity of both procoagulant and anticoagulant proteins, TG is considered more useful than clotting time tests in evaluating hemostatic function and thrombotic tendency [12–14]. Therefore in the current investigation we evaluated the modulating effects of increased concentrations of FVIII-vWF on thrombin generation in the setting of i) hemophilia A, ii) FXI deficiency, iii) deficiency/decrease of FVIII/AT and iv) partial FIX deficiency as simulated by use of anti FIXa aptamer. We also determined the effects of increased concentrations of FVIII on the reversal of fondaparinux induced factor X inhibition in comparison to recombinant activated factor VII. Materials and methods All parts of the study were conducted according to the local IRB approved protocol at Emory University. Normal pooled plasma, FVIII deficient (FVIII-/-) and FXI deficient (FXI-/-) plasmas were obtained from George King Biomedical (Overland Park, KS). Double-deficient plasma for FVIII and antithrombin (AT) was obtained from Enzyme Research Laboratories (South Bend, IN), and it was mixed 1:1(v/v) with FVIII-/plasma to achieve approximately 50% of normal AT activity while maintaining b1% of FVIII activity denoted as FVIII-/-/AT+/-. Because FVIII circulates in complex with vWF, Humate-P (CSL Behring, Marburg, Germany) was used to simulate physiological increases in FVIII and vWF [2,3,15,16]. It contained FVIII 70.0 U and vWF 154.8 U per ml. The final concentrations of added Humate-P to all the plasma samples evaluated in the current study are expressed in terms of replaced FVIII activity. The following agents were used in experiments; fondaparinux (GlaxoSmithKline, Research Triangle Park, NC), recombinant activated factor VII (rFVIIa; NovoNordisk, Bagsvaerd, Denmark), and anti-FIXa aptamer (RB006; Regado Biosciences, Inc., Durham, NC). RB006 is a 31-nucleotide RNA aptamer that is a potent FIXa inhibitor that is currently being evaluated as a therapeutic agent in cardiovascular interventions [12]. For TG assay, tissue factor-based (TF) platelet-poor plasma (PPP, 1 and 5 pM) activators, thrombin calibrator and fluorogenic thrombin substrate were obtained from Diagnostica Stago (Parsippany, NJ), and Actin FS was from Dade Behring (Marburg, Germany). 2 pM TF activator was prepared by mixing appropriate volumes of 1 pM and 5 pM reagents. Thrombin generation (TG) The calibrated automated TG assay (Thrombinoscope™, Synapse BV, Maastricht, The Netherlands) was used to measure the rate and the amount of TG in plasma according to the change in fluorescence produced by the hydrolysis of a fluorogenic peptide (Z-Gly-Gly-ArgAMC) by thrombin [17]. PPP samples were run in duplicate for the measurement of TG. Briefly, to each well of a 96 well microtiter plate (Microfluor black, ThermoLabsystems, Franklin, MA), we added 80 μl of various PPP samples. Calibrator wells, in which 20 μl of thrombin calibrator was added to 80 μl of plasma samples, were run in parallel for each plasma. TG was triggered with 20 μl of 2 pM tissue factor-based PPP reagent or 1:20 dilution of Actin FS (ellagic acid plus purified soy phosphatides). The reaction was started by adding 20 μl/well of CaCl2subtrate buffer and was continuously monitored for ~90 min. Thrombin parameters were calculated using Thrombinoscope software (Thrombinoscope BV, Maastricht, the Netherlands). Effects of FVIII on TG were evaluated using the following plasma samples; a) Normal (control), b) FVIII-/-, c) FVIII-/-/AT+/-, and d) FXI-/-. Both FVIII-/- and FXI-/- are associated with clinical bleeding tendency and reduced thrombin generation [6,13,18,19]. Because changes in AT levels affect thrombin generation assays in normal and FVIII-/plasmas, we also evaluated the effects of Humate-P (0-3 U/ml) in FVIII-/-/AT+/- samples [20]. To simulate hypocoaguable state, we
treated normal and FVIII-/-/AT+/- plasma with fondaparinux (1 μg/ml), with anti-FIXa aptamer (12 μg/ml) or combination of both agents [14,21]. Some of control PPP samples were also tested after 50% dilution with saline to simulate perioperative hemodilution [4]. Finally, in some plasma samples the recovery of thrombin generation with Humate-P was compared with the recovery afforded with 60 nM (3 μg/ml) of rFVIIa. Coagulation time measurements We performed prothrombin time (PT), and activated partial thromboplastin time (aPTT) testing and determined the levels of factor VIII in normal control (undiluted/diluted), FVIII-/-, FVIII-/-/AT+/and Humate-P supplemented samples. Plasma AT activity was determined using Diagnostica Stago Stachrom AT III kit (chromogenic method, Parsippany, NJ). All tests were run on Diagnostica Stago Compact® analyzer using manufacturer described protocols and kits (Diagnostica Stago, Parsippany, NJ). Data analyses Data are expressed as mean ± SD. After testing for normal distribution by Kolgomorov-Smirnov test, data from TG experiments were compared by one-way ANOVA for repeated measurement followed by Bonferroni's posthoc t test. Where appropriate different plasma groups were compared using paired t test. A P-value less than 0.05 was considered significant. All statistical analyses were performed using Sigma Plot 11 (Systat Software Inc., San Jose, CA). Results Thrombin generation in normal plasma Adding FVIII-vWF to normal plasma samples triggered with 2 pM TF increased peak thrombin in a concentration dependent manner (Table 1 and Fig. 1A, P b 0.001). The peak TG increased by ~95% relative to baseline at 3 U/ml of added FVIII. In contrast, in Actin activated
Table 1 Lag time and peak thrombin generation in control, diluted control, FVIII-/-FVIII-/-/AT+/and FXI-/- plasma samples supplemented with Humate-P. Plasma sample
Control + 0.5 U/ml Humate-P + 1.5 U/ml Humate-P + 3.0 U/ml Humate-P Diluted control + 0.5 U/ml Humate-P + 1.5 U/ml Humate-P + 3.0 U/ml Humate-P FVIII -/+ 0.5 U/ml Humate-P + 1.5 U/ml Humate-P + 3.0 U/ml Humate-P FVIII -/-/AT +/+ 0.5 U/ml Humate-P + 1.5 U/ml Humate-P + 3.0 U/ml Humate-P FXI -/+ 0.5 U/ml Humate-P + 1.5 U/ml Humate-P + 3.0 U/ml Humate-P
Lag time (min)
Peak thrombin (nM)
2pMTF
Actin
2pMTF
Actin
4.4 ± 0.5 4.2 ± 0.2 3.8 ± 0.3 3.4 ± 0.4⁎ 4.0 ± 0.1 3.6 ± 0.2 3.0 ± 0.4 2.6 ± 0.3⁎ 3.4 ± 0.3 3.1 ± 0.3 2.8 ± 0.5 2.7 ± 0.4⁎ 3.4 ± 0.3 3.0 ± 0.1 2.8 ± 0.1 2.7 ± 0.2⁎ 6.9 ± 0.5 6.6 ± 0.1 6.1 ± 0.5 5.7 ± 0.4⁎
4.6 ± 0.4 3.8 ± 0.4 2.7 ± 0.3 2.5 ± 0.3⁎ 5.0 ± 0.1 3.3 ± 0.5 2.6 ± 0.5 2.3 ± 0.4⁎ 16.5 ± 2.3 4.5 ± 0.5 3.4 ± 0.3 2.7 ± 0.2⁎ 21 ± 2.8 2.5 ± 0.1 1.6 ± 0.2 1.3 ± 0.1⁎ 24.7 ± 2.6 18.9 ± 1.5 15.1 ± 1.5 13.8 ± 0.4⁎
202 ± 14 262 ± 26 326 ± 40 394 ± 43⁎ 226 ± 9.0 266 ± 10 289 ± 7.0 296 ± 15⁎ 94 ± 14 227 ± 31 307 ± 19 340 ± 27⁎ 151 ± 16 417 ± 28 436 ± 27 472 ± 27⁎ 171 ± 21 296 ± 27 323 ± 35 360 ± 30⁎
505 ± 39 517 ± 18 506 ± 18 522 ± 36 322 ± 18 331 ± 25 336 ± 14 338 ± 16 66 ± 6.0 388 ± 43 380 ± 21 396 ± 37⁎ 73 ± 8.0 476 ± 50 474 ± 42 476 ± 44⁎ 22 ± 1.0 56 ± 2.0 105 ± 7.0 169 ± 16⁎
FVIII-/- = factor VIII deficient plasma. FVIII-/-/AT+/- = factor VIII deficient/decreased level of AT plasma. FXI-/- = factor XI deficient plasma. Humate-P = FVIII-vWF. Only differences between baseline and highest concentration of FVIII-vWF are shown. ⁎ P b 0.05 by one-way ANOVA for repeated measurement followed by Bonferroni's posthoc t test.
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onset of thrombin generation was notable in addition to the decreased peak TG (Table 1, Fig. 2B). Significant recovery of TG in terms of lag time and peak thrombin was already observed when missing FVIII was replaced with 0.5 U/ml of FVIII-vWF (Table 1, Fig. 2B-C, P b 0.001). Thrombin generation in FVIII
-/-
/AT
+/-
plasma
In FVIII-/-/AT+/- plasma samples AT levels were reduced by ~51% (AT level = 59%) vs. control plasma (120%, Table 2). After adding only 0.5 U/ml of FVIII to FVIII-/-/AT+/- samples and activating with dilute Actin peak thrombin increased to 476 ± 50 nM, thus there was no longer difference vs. normal control (505 ± 39 nM, no FVIII-vWF added, P = 0.249)(Table 1). Similarly, the peak thrombin increased by N2.5 fold from 151 ± 16 nM to 417 ± 28 nM in TF (2 pM) activated FVIII-/-/AT+/- plasma samples supplemented with FVIII-vWF (0.5 U/ ml). Fondaparinux, added at a concentration of 1 μg/ml, was effective in reducing the peak thrombin level in FVIII-/-/AT+/- plasma samples, although the efficiency of inhibition was slightly lower in comparison to normal control plasma supplemented with 1 μg/ml of fondaparinux; ~62% vs. 80%, respectively (P b 0.001). This observation suggests the therapeutic effect of fondaparinux despite a moderate (~51%) decrease in AT activity. On the other hand, anticoagulant effect of fondaparinux was efficiently reversed with 3 U/ml of FVIII-vWF (Figs. 2D, 3), but not by 60 nM rFVIIa. No significant additive inhibitory effect on TG was observed after adding anti-FIXa aptamer (12 μg/ml) to plasma samples containing 1 μg/ml fondaparinux. However, FVIII-vWF (3 U/ml) was no longer capable of restoring TG in plasma treated with anti-FIXa aptamer and fondaparinux (Fig. 3). Thrombin generation in FXI Fig. 1. Effects of FVIII-vWF (Humate-P) on thrombin generation in control (normal) plasma with 2 pM tissue factor (panel A) and Actin activation (panel B).
control plasma samples, peak TG was minimally affected, but the lag time was shortened from 4.6 ± 0.4 min to 2.5 ± 0.3 min (P b 0.001) when exogenous FVIII was increased from 0 to 3 U/ml (Table 1 and Fig. 1B) After 50% saline dilution of control plasma (2 pM TF activation), the effect of FVIII-vWF on TG was obtunded resulting in only ~ 31% increase in peak thrombin at the highest concentration tested. With Actin activation, the shortening of lag time was significant at all 3 levels of added FVIII-vWF regardless whether tested plasma was diluted or not (P b 0.001) but only at two highest concentrations of added FVIII with 2 pM TF activation (Table 1). In control PPP spiked with 1 μg/ml (final concentration) of fondaparinux to simulate hypocoagulable state, peak thrombin decreased from 202 ± 14 nM (TF activation) at baseline to 40 ± 5 nM. The addition of FVIII-vWF (3 U/ml) reversed fondaparinuxinduced decreases in TG by 5 fold; thus peak thrombin increased to 207 ± 27 nM (Figs. 2A, 3). Notably, adding 60 nM rFVIIa shortened the lag time of TG, from 10.5 ± 0.7 to 4.7 ± 0.4 min and increased the rate of TG. However, the change in the peak thrombin level (40 ± 5 nM without rFVIIa to 68 ± 8 with rFVIIa (60 nM) was significantly smaller than the effect of Humate-P (Figs. 2A, 3). Addition of anti FIXa (12 μg/ml) aptamer to control plasma inhibited thrombin generation by ~ 65%. Supplementing these samples with 3 U/ml FVIII-vWF and triggering with 2 pM TF resulted in only ~ 42% recovery of thrombin peak (Fig. 3). Thrombin generation in FVIII
-/-
plasma
Peak thrombin levels were significantly decreased in FVIII-/plasma compared to normal plasma after either dilute Actin or TF trigger (Table 1, Fig. 2B-C). In Actin-triggered plasma, much delayed
-/-
plasma
FXI deficiency caused profound changes in TG when Actin was used as an activator. The lag time was very much prolonged from 4.6 ± 0.4 min (normal plasma) to 24.7 ± 2.6 min, a 5-fold increase, and peak thrombin level was decreased by ~96% (Table 1). The recovery of TG parameters was observed with increasing concentrations of FVIII (Table 1, Fig. 2E), although even with 3 U/ml of FVIIIvWF, the peak thrombin and lag time with Actin activation did not normalize to the control level; the peak thrombin rose only to ~ 33% of normal plasma (supplemented with Humate-P) and lag time decreased to 13.8 ± 0.4 min (still a 3-fold prolongation vs. baseline). Coagulation time measurements As expected, factor VIII was profoundly decreased in FVIII-/- and FVIII-/-/AT+/- samples (1% vs. 76% in normal plasma) (Table 2). Deficiency of FVIII prolonged aPTT from 31 sec to 86 sec, but had no effect on PT. After 50% dilution of control PPP samples, both PT and aPTT were prolonged (Table 2). Addition of increasing concentrations of FVIII-vWF (0–3 U/ml) to normal control, FVIII-/- and FVIII-/-/AT+/plasma samples progressively shortened aPTTs but not the PT values. Further, adding incremental FVIII-vWF to diluted normal plasma samples normalized the aPTT but did not affect the PT results, which stayed prolonged. The decrease in AT (~59% in FVIII-/-/AT+/- plasma vs. 120% in normal plasma) was not reflected in PT or aPTT results. Discussion In the present study, we demonstrated that elevated FVIII levels improve the rate and peak level of TF-initiated TG in hypocoagulable FVIII-/-, FXI-/-, and fondaparinux-treated plasma samples (Fig. 2A-E). We also evaluated concomitant changes in procoagulant and anticoagulant proteins using plasma with reduced AT, and in salinediluted plasma. In the presence of low AT activity, TG was more rapidly restored at a small increment of FVIII in FVIII-/- plasma
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Fig. 2. Panel A. Effects of FVIII-vWF (Humate-P) and recombinant activated factor VII (rFVIIa) in control (normal) plasma treated with fondaparinux (2 pM tissue factor activation); Panels B-C. Recovery of thrombin generation in FVIII-/- plasma with increasing concentrations of FVIII-vWF (Humate-P). Actin (B) and 2 pM tissue factor (C) activation; Panel D. Recovery of thrombin generation with FVIII-vWF and rFVIIa in FVIII-/-AT+/- plasma treated with fondaparinux. Fonda = fondaparinux 1 μg/ml. rFVIIa= recombinant activated factor VII 60 nM (3 μg/ml). Humate-P = FactorVIII-vWF (3 U/ml); Panel E. Effects of supplemental factor VIII on recovery of thrombin peak and lag time in FXI-/- plasma. Thrombin generation in control (normal) plasma (PPP) is shown for comparison. Actin activation. Humate-P = FVIII-vWF (3 U/ml).
(Table 1, Fig. 3). Thus slower FXa and thrombin inhibition due to low AT potentiates procoagulant activity of FVIII even in the presence of fondaparinux (Figs. 2D, 3). When normal (control) plasma was diluted by 50% with saline to simulate the general effect of hemodilution in trauma/surgery, incremental changes of thrombin peak were obtunded relative to non-diluted plasma (Table 1). In contrast to an isolated decrease of AT, diluted plasma incurs decreases in procoagulant proteins, especially prothrombin, which result in lower peak thrombin levels [10,22,23]. Hemostatic function of FVIII is conventionally evaluated by aPTT using a contact activator such as Actin FS. In normal undiluted and saline-diluted plasma samples triggered with dilute Actin FS, lag times of TG were progressively shortened by increasing concentrations of FVIII. However, no additional increase in peak thrombin levels was observed when FVIII level was increased above 57% (Table 2). The ceiling effects of FVIII on peak thrombin level were also seen after adding 0.5 U/ml of FVIII (Table 1, 2) in FVIII-/- and in FVIII-/-/AT+/samples. The robustness of restoring TG with contact activation may be in part explained by lack of efficient physiological inhibitors against FXIIa and FXIa, resulting in full activation of intrinsic tenase (FIXaFVIIIa) to support TG [24]. Indeed, the ceiling effect was not seen in FXI-/- plasma supplemented with FVIII-vWF because the lack of FXI was a limiting step of Actin-mediated activation of plasma coagulation (Table 1, Fig 2E). Based on these results, TG triggered with TF (1–2 pM) may be more suitable to evaluate hemostatic and possibly thrombotic potential of FVIII (Table 2) [25]. Plasma FVIII activity dynamically changes during and after major trauma and surgery [26,27]. While coagulation factor(s) levels decrease progressively according to the extent of blood loss and hemodilution, plasma vWF and FVIII activities are relatively well maintained [3,27,28]. Assuming normal vWF activity (1 U/ml) at
baseline, Weinstein, et al. estimates that even after a 50% hemodilution that reduced vWF activity to 0.5 U/ml, post-surgical vWF increases to 1.3 U/ml due to an influx of 0.8 U/ml during cardiopulmonary bypass. The release of FVIII-vWF complex is stimulated by
Fig. 3. Changes in peak thrombin generation with FVIII-vWF and rFVIIa in control and FVIII-/-/AT+/- plasma triggered with 2pM tissue factor. The impact of FIX inhibition using anti-FIXa aptamer in addition to FXa inhibition by fondaparinux is also shown. Statistical analyses by one-way ANOVA for repeated measurement followed by Bonferroni's posthoc t test. *P b 0.05 vs. control plasma with fondaparinux. #P b 0.05 vs. FVIII-/-AT+/- plasma with fondaparinux. ^P b 0.05 vs. combination of fondaparinux and anti-FIXa aptamer. The results of statistical analyses of anti-FIXa aptamer alone or in combination with Humate-P are not shown. Fonda = fondaparinux 1 μg/ml. rFVIIa = Recombinant activated factor VII 60 nM (3 μg/ml). Aptamer = Anti-FIXa aptamer (12 μg/ml). Humate-P = FVIII-vWF (3 U/ml).
F. Szlam et al. / Thrombosis Research 127 (2011) 135–140 Table 2 Effect of Increasing Concentrations of Humate-P on Coagulation Parameters in Control, dilute Control, FVIII-/-, FVIII-/-/AT+/-and FXI-/-plasma samples. Plasma Sample
aPTT (sec)
PT (sec)
ATIII %
FVIII %
Control + Humate-P 0.5 U/ml + Humate-P 1.5 U/ml + Humate-P 3.0 U/ml FVIII -/+ Humate-P 0.5 U/ml + Humate-P 1.5 U/ml + Humate-P 3.0 U/ml FVIII -/-/AT+/+ Humate-P 0.5 U/ml + Humate-P 1.5 U/ml + Humate-P 3.0 U/ml Diluted Control + Humate-P 0.5 U/ml + Humate-P 1.5 U/ml + Humate-P 3.0 U/ml
31.0 28.0 25.8 23.4 85.8 31.5 26.4 24.1 79.8 33.9 28.8 25.9 47.9 42.1 37.7 34.9
13.1 12.5 12.9 12.6 11.8 11.7 11.6 11.9 11.9 11.9 12.1 12.3 15.7 16.2 16.2 16.3
120 116 113 113 119 115 114 117 59 61 61 55 71 66 67 65
76 138 223 392 1 61 159 336 1 58 169 298 57 134 228 396
FVIII-/- = factor VIII deficient plasma. FVIII-/-/AT+/- = factor VIII deficient/decreased level of AT plasma. FXI-/- = factor XI deficient plasma. Humate-P = FVIII-vWF.
elevated stress hormones such as epinephrine and vasopressin [29]. Desmopressin (1-deamino-8-D-arginine vasopressin; DDAVP) can be therapeutically used to treat mild hemophilia A and von Willebrand disease [15,16]. In surgical patients without congenital bleeding disorders, the benefit of desmopressin is limited to a small decrease in perioperative blood loss, and the rate of allogeneic blood use does not seem to be reduced [30]. It is plausible that hemostatic efficacy of FVIII-vWF is reduced by concomitant release of tissue plasminogen activator (tPA, 2.5-fold increase in 30–45 min after desmopressin) and enhanced fibrinolytic tendency in perioperative hemodilution [31]. As purified FVIII-vWF, Humate-P should be more easily titrated to increase FVIII-vWF, without concomitant release of tPA, to optimize platelet adhesion and TG. Additional clinical studies are necessary to investigate a potential target population, efficacy and safety of FVIIIvWF therapy in acquired perioperative bleeding. Thrombophilia after major surgery is an important clinical problem, but perioperative bleeding complication from antithrombotic therapy is always a concern. The use of fondaparinux seems to reduce in-hospital mortality, but patients who had major bleeding had a 7-fold higher 30-day mortality compared to those without bleeding [32]. The modulation of fondaparinux anticoagulation by FVIII demonstrated in our study has clinical implications in terms of antithrombotic efficacy and reversal of anticoagulation. Fondaparinux was found to be effective even when AT was moderately decreased (Fig. 2D), while its anticoagulant effects could be reversed after increasing FVIII by 3 U/ml (Figs. 2D, 3). In addition, Humate-P added to fondaparinux-treated control plasma resulted in approximately 100% recovery of thrombin peak while only 33% was recovered by 60 nM of rFVIIa (Figs. 2A and 3). Our results are in agreement with Desmurs-Clave, et al. who observed the minimal recovery of thrombin peak with rFVIIa in fondaparinux-treated platelet-rich plasma [14]. Fondaparinux reduces TG by enhancing AT-mediated FXa neutralization, but it does not obliterate thrombin activity [33]. Anticoagulant efficacy of fondaparinux is reduced after thrombin activates FV to form prothrombinase (FXa-FVa complex) [34]. Further, FIXa is generated by either TF-FVIIa or FXIa, and intrinsic tenase (FIXa-FVIIIa complex) can be rapidly formed when thrombin-mediated FVIII activation is facilitated by elevated FVIII concentrations [25]. We demonstrated the pivotal role of intrinsic tenase to antagonize fondaparinux in FVIII-/-/AT+/- plasma samples. When FIXa activity was inhibited with 12 μg/ml of anti-FIXa aptamer [21] procoagulant activity of FVIII-vWF in fondaparinux-treated plasma was obtunded (105 ± 11 nM with anti-FIXa vs. 365 ± 32 nM without) (Fig. 3). This finding underlies the importance of intrinsic tenase system in
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supporting hemostasis when the trigger for TG is limited due to reduced activity of TF pathway. It partly explains the limited efficacy in reducing hemophilia-related joint bleeds in patients with inhibitors using rFVIIa [35,36]. Elevated FVIII levels are not reflected in PT due to supraphysiological TF levels used to activate plasma during the testing (Table 2). At a more physiological level (2 pM) used in TG assay, the effect of elevated FVIII on TG was clearly demonstrated in normal and FXIdeficient plasma (Table 1, Figs. 1A, 2E). Conversely, lag times of Actinactivated TG and aPTTs were shortened according to FVIII levels (Table 1, 2). The latter findings are in agreement with Tripodi, et al. who observed an association between high FVIII levels and shortened aPTT [37]. Interestingly, in saline diluted normal plasma repleted with increasing concentrations of FVIII-vWF, aPTT values were normalized with 3 U/ml of FVIII, but there was no change in PT results, which stayed elevated. Elevated FVIII levels up to 3.9 U/ml had been observed in patients under acute stress (e.g., trauma), resulting in shortened aPTT [2]. Recently, thrombin-mediated FXI activation was shown to be important for sustained TG under low TF or low FVII condition [13,38]. The contribution of intrinsic pathway to sustained TG is also supported by our experiments in which elevated FVIII partially compensates for FXI deficiency in terms of TG (Table 1, Fig. 2E) although the FVIII efficiency was much lower than in other plasma samples used in the current study. We speculate that heterogeneous bleeding and thrombosis patterns found in congenital FXI deficiency may be in part attributed to local FVIII-vWF levels achieved in a target organ [39,40]. There are several limitations in this study. First, platelets and other rheological elements of coagulation were not included in the present models, and thus the involvement of vWF in TG was not evaluated [41]. The presence of phospholipids (as in this study) versus plateletrich plasma on TG seems to affect the response to FVIII. Keularts et al. reported only a limited increase of peak thrombin when FVIII levels were between 50% and 150% of normal in platelet-rich plasma [16]. Similar to our study, Ibbotson, et al., and Butenas, et al., respectively, observed increases in peak thrombin levels when FVIII levels were increased from 100% to 350% and 50% to 100%, respectively [42,43]. More recently, in a study by Machlus et al., increased concentrations of FVIII (100-400% range) were found to increase TG (both peak and endogenous thrombin potential) in tissue factor triggered platelet poor plasma [25]. These data are supported by the longitudinal observational study which indicated the association of high peak TG (4th quartile of the cohort) using PPP, FVIII activity and the risk of thromboembolism [44]. In conclusion, we demonstrate that elevated FVIII supports the propagation of thrombin generation under low TF (2 pM) state, FXI deficiency, or in the presence of fondaparinux. Taken together, changes in plasma FVIII can dynamically modulate hemostatic and even thrombotic responses, and purified FVIII-vWF may represent a potential therapeutic agent in excess anticoagulation due to fondaparinux. Further clinical studies are needed to establish the efficacy and safety of purified FVIII-vWF in a perioperative setting. Conflict of interest statement Drs. Solomon and Tanaka have received consultant fees and research support from CSL Behring (Marburg, Germany). References [1] Jaffe E. Synthesis of factor VIII by endothelial cells. Ann NY Acad Sci 1982;401: 163–70. [2] Yuan S, Ferrell C, Chandler WL. Comparing the prothrombin time INR versus the APTT to evaluate the coagulopathy of acute trauma. Thromb Res 2007;120:29–37. [3] Harker LA, Malpass TW, Branson HE, Hessel II EA, Slichter SJ. Mechanism of abnormal bleeding in patients undergoing cardiopulmonary bypass: acquired transient platelet dysfunction associated with selective alpha-granule release. Blood 1980;56:824–34.
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Elevated factor VIII enhances thrombin generation in the presence of factor VIII-deficiency, factor XI-deficiency or fondaparinux☆ Fania Szlam a, Gautam Sreeram a, Cristina Solomon c,d , Jerrold H. Levy a, Ross J. Molinaro b, Kenichi A. Tanaka a,⁎ a
Department of Anesthesiology, Emory University School of Medicine, Atlanta, Georgia, USA Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA Department of Anesthesiology, Salzburger Landeskliniken SALK, Salzburg, Austria d Department of Intensive Care, Salzburger Landeskliniken SALK, Salzburg, Austria b c
a r t i c l e
i n f o
Article history: Received 19 April 2010 Received in revised form 2 September 2010 Accepted 19 October 2010 Available online 8 December 2010 Keywords: Factor VIII Hemodilution Thrombin Generation
a b s t r a c t Background: Increased levels of factor VIII occur as a response to vascular injury and/or inflammation, and may increase thrombotic risks. In contrast, factor VIII deficiency poses a major hemostatic challenge. The role of factor VIII in modulating hemostasis/thrombosis was investigated in plasma models of hypocoagulable and hypercoagulable state using thrombin generation (TG) assay. Methods: TG was performed in undiluted/diluted control, FVIII-deficient, FVIII-deficient with low antithrombin (AT activity, ~ 59%), and factor XI-deficient plasma samples using relipidated tissue factor (TF, 2 pM) or dilute Actin as activators. The impact of elevated FVIII on TG was simulated by adding Humate-P (0 to 3 U/ml) to the above plasma samples. In fondaparinux (1 μg/ml) treated plasma with normal or lower AT activity effects of Humate-P vs. 60 nM of recombinant activated factor VII (rFVIIa) were also evaluated. Results: Humate-P increased TG concentration dependently in undiluted and diluted control plasma with TF activation. With Actin activation, only the concentration dependent shortening of lag time, but no change in peak thrombin was observed. In FVIII-deficient, FVIII-deficient with low AT, and FXI-deficient samples, 3 U/ml of Humate-P increased TG, and decreased its onset with either activator. The reduced peak thrombin due to fondaparinux was reversed with Humate-P (3 U/ml) more than with rFVIIa. Elevated FVIII levels seem to favor intrinsic tenase formation and antagonize fondaparinux because anti-FIXa aptamer added to fondaparinux effectively attenuated TG. Conclusion: Elevated FVIII supports the propagation of TG via intrinsic tenase formation under low TF condition, factor XI deficiency or in the presence of fondaparinux. © 2010 Elsevier Ltd. All rights reserved.
Introduction Factor VIII (FVIII) is a glycoprotein, which circulates in plasma at a low concentration (0.0003 μM) in complex with von Willebrand factor (vWF). Upon vascular injury or during inflammation, plasma FVIII levels can rapidly increase due to the release of FVIII-vWF from endothelial cells [1]. As an acute phase reactant, FVIII levels usually increase during surgery/trauma, or tend to remain at nearly normal (hemostatic) level, contrary to other coagulation factors that are susceptible to hemodilution during major surgery [2,3]. In case of patients with hemophilia A, trauma and surgery pose a major
☆ Support: This study was supported in part by Department of Anesthesiology, Emory University School of Medicine, and CSL Behring, Marburg, Germany. ⁎ Corresponding author. Department of Anesthesiology, Emory University School of Medicine, 1364 Clifton Road, N.E., Atlanta, Georgia, USA 30322. Tel.: +1 404 778 3900; fax: +1 404 778 5194. E-mail address: [email protected] (K.A. Tanaka). 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.10.017
bleeding risk, which is usually mitigated by prophylactic FVIII replacement [4,5]. The presence of neutralizing antibodies against FVIII further increases the risks of recurrent muscle/joint bleeds and perioperative hemorrhage [5–7]. In contrast to FVIII deficiency, bleeding risks in individuals with FXI deficiency are considered mild to moderate, but highly variable after trauma and injury [8,9]. Such variability in hemostatic response can possibly play a role in the incidence of thromboembolism associated with congenital antithrombin (AT) deficiency because elevated FVIII levels can dynamically affect hemostatic activity after trauma/surgery [2,3,10] and during pregnancy [11]. Individual variability in FVIII levels could in part explain the differences in achieving hemostasis in FXI deficiency or thrombophilic tendency in AT deficiency. On the other hand it is difficult to predict a bleeding or thrombotic risk based on a single factor level because the amount of thrombin produced at a vascular injury site is influenced by complex interactions among various cell surface proteins, and procoagulant and anticoagulant elements. Therefore, we hypothesized that the impact of elevated
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FVIII levels in hypocoagulable and thrombophilic plasma could be quantified by the calibrated automated thrombin generation (TG) measurement. Because TG patterns are influenced by the activity of both procoagulant and anticoagulant proteins, TG is considered more useful than clotting time tests in evaluating hemostatic function and thrombotic tendency [12–14]. Therefore in the current investigation we evaluated the modulating effects of increased concentrations of FVIII-vWF on thrombin generation in the setting of i) hemophilia A, ii) FXI deficiency, iii) deficiency/decrease of FVIII/AT and iv) partial FIX deficiency as simulated by use of anti FIXa aptamer. We also determined the effects of increased concentrations of FVIII on the reversal of fondaparinux induced factor X inhibition in comparison to recombinant activated factor VII. Materials and methods All parts of the study were conducted according to the local IRB approved protocol at Emory University. Normal pooled plasma, FVIII deficient (FVIII-/-) and FXI deficient (FXI-/-) plasmas were obtained from George King Biomedical (Overland Park, KS). Double-deficient plasma for FVIII and antithrombin (AT) was obtained from Enzyme Research Laboratories (South Bend, IN), and it was mixed 1:1(v/v) with FVIII-/plasma to achieve approximately 50% of normal AT activity while maintaining b1% of FVIII activity denoted as FVIII-/-/AT+/-. Because FVIII circulates in complex with vWF, Humate-P (CSL Behring, Marburg, Germany) was used to simulate physiological increases in FVIII and vWF [2,3,15,16]. It contained FVIII 70.0 U and vWF 154.8 U per ml. The final concentrations of added Humate-P to all the plasma samples evaluated in the current study are expressed in terms of replaced FVIII activity. The following agents were used in experiments; fondaparinux (GlaxoSmithKline, Research Triangle Park, NC), recombinant activated factor VII (rFVIIa; NovoNordisk, Bagsvaerd, Denmark), and anti-FIXa aptamer (RB006; Regado Biosciences, Inc., Durham, NC). RB006 is a 31-nucleotide RNA aptamer that is a potent FIXa inhibitor that is currently being evaluated as a therapeutic agent in cardiovascular interventions [12]. For TG assay, tissue factor-based (TF) platelet-poor plasma (PPP, 1 and 5 pM) activators, thrombin calibrator and fluorogenic thrombin substrate were obtained from Diagnostica Stago (Parsippany, NJ), and Actin FS was from Dade Behring (Marburg, Germany). 2 pM TF activator was prepared by mixing appropriate volumes of 1 pM and 5 pM reagents. Thrombin generation (TG) The calibrated automated TG assay (Thrombinoscope™, Synapse BV, Maastricht, The Netherlands) was used to measure the rate and the amount of TG in plasma according to the change in fluorescence produced by the hydrolysis of a fluorogenic peptide (Z-Gly-Gly-ArgAMC) by thrombin [17]. PPP samples were run in duplicate for the measurement of TG. Briefly, to each well of a 96 well microtiter plate (Microfluor black, ThermoLabsystems, Franklin, MA), we added 80 μl of various PPP samples. Calibrator wells, in which 20 μl of thrombin calibrator was added to 80 μl of plasma samples, were run in parallel for each plasma. TG was triggered with 20 μl of 2 pM tissue factor-based PPP reagent or 1:20 dilution of Actin FS (ellagic acid plus purified soy phosphatides). The reaction was started by adding 20 μl/well of CaCl2subtrate buffer and was continuously monitored for ~90 min. Thrombin parameters were calculated using Thrombinoscope software (Thrombinoscope BV, Maastricht, the Netherlands). Effects of FVIII on TG were evaluated using the following plasma samples; a) Normal (control), b) FVIII-/-, c) FVIII-/-/AT+/-, and d) FXI-/-. Both FVIII-/- and FXI-/- are associated with clinical bleeding tendency and reduced thrombin generation [6,13,18,19]. Because changes in AT levels affect thrombin generation assays in normal and FVIII-/plasmas, we also evaluated the effects of Humate-P (0-3 U/ml) in FVIII-/-/AT+/- samples [20]. To simulate hypocoaguable state, we
treated normal and FVIII-/-/AT+/- plasma with fondaparinux (1 μg/ml), with anti-FIXa aptamer (12 μg/ml) or combination of both agents [14,21]. Some of control PPP samples were also tested after 50% dilution with saline to simulate perioperative hemodilution [4]. Finally, in some plasma samples the recovery of thrombin generation with Humate-P was compared with the recovery afforded with 60 nM (3 μg/ml) of rFVIIa. Coagulation time measurements We performed prothrombin time (PT), and activated partial thromboplastin time (aPTT) testing and determined the levels of factor VIII in normal control (undiluted/diluted), FVIII-/-, FVIII-/-/AT+/and Humate-P supplemented samples. Plasma AT activity was determined using Diagnostica Stago Stachrom AT III kit (chromogenic method, Parsippany, NJ). All tests were run on Diagnostica Stago Compact® analyzer using manufacturer described protocols and kits (Diagnostica Stago, Parsippany, NJ). Data analyses Data are expressed as mean ± SD. After testing for normal distribution by Kolgomorov-Smirnov test, data from TG experiments were compared by one-way ANOVA for repeated measurement followed by Bonferroni's posthoc t test. Where appropriate different plasma groups were compared using paired t test. A P-value less than 0.05 was considered significant. All statistical analyses were performed using Sigma Plot 11 (Systat Software Inc., San Jose, CA). Results Thrombin generation in normal plasma Adding FVIII-vWF to normal plasma samples triggered with 2 pM TF increased peak thrombin in a concentration dependent manner (Table 1 and Fig. 1A, P b 0.001). The peak TG increased by ~95% relative to baseline at 3 U/ml of added FVIII. In contrast, in Actin activated
Table 1 Lag time and peak thrombin generation in control, diluted control, FVIII-/-FVIII-/-/AT+/and FXI-/- plasma samples supplemented with Humate-P. Plasma sample
Control + 0.5 U/ml Humate-P + 1.5 U/ml Humate-P + 3.0 U/ml Humate-P Diluted control + 0.5 U/ml Humate-P + 1.5 U/ml Humate-P + 3.0 U/ml Humate-P FVIII -/+ 0.5 U/ml Humate-P + 1.5 U/ml Humate-P + 3.0 U/ml Humate-P FVIII -/-/AT +/+ 0.5 U/ml Humate-P + 1.5 U/ml Humate-P + 3.0 U/ml Humate-P FXI -/+ 0.5 U/ml Humate-P + 1.5 U/ml Humate-P + 3.0 U/ml Humate-P
Lag time (min)
Peak thrombin (nM)
2pMTF
Actin
2pMTF
Actin
4.4 ± 0.5 4.2 ± 0.2 3.8 ± 0.3 3.4 ± 0.4⁎ 4.0 ± 0.1 3.6 ± 0.2 3.0 ± 0.4 2.6 ± 0.3⁎ 3.4 ± 0.3 3.1 ± 0.3 2.8 ± 0.5 2.7 ± 0.4⁎ 3.4 ± 0.3 3.0 ± 0.1 2.8 ± 0.1 2.7 ± 0.2⁎ 6.9 ± 0.5 6.6 ± 0.1 6.1 ± 0.5 5.7 ± 0.4⁎
4.6 ± 0.4 3.8 ± 0.4 2.7 ± 0.3 2.5 ± 0.3⁎ 5.0 ± 0.1 3.3 ± 0.5 2.6 ± 0.5 2.3 ± 0.4⁎ 16.5 ± 2.3 4.5 ± 0.5 3.4 ± 0.3 2.7 ± 0.2⁎ 21 ± 2.8 2.5 ± 0.1 1.6 ± 0.2 1.3 ± 0.1⁎ 24.7 ± 2.6 18.9 ± 1.5 15.1 ± 1.5 13.8 ± 0.4⁎
202 ± 14 262 ± 26 326 ± 40 394 ± 43⁎ 226 ± 9.0 266 ± 10 289 ± 7.0 296 ± 15⁎ 94 ± 14 227 ± 31 307 ± 19 340 ± 27⁎ 151 ± 16 417 ± 28 436 ± 27 472 ± 27⁎ 171 ± 21 296 ± 27 323 ± 35 360 ± 30⁎
505 ± 39 517 ± 18 506 ± 18 522 ± 36 322 ± 18 331 ± 25 336 ± 14 338 ± 16 66 ± 6.0 388 ± 43 380 ± 21 396 ± 37⁎ 73 ± 8.0 476 ± 50 474 ± 42 476 ± 44⁎ 22 ± 1.0 56 ± 2.0 105 ± 7.0 169 ± 16⁎
FVIII-/- = factor VIII deficient plasma. FVIII-/-/AT+/- = factor VIII deficient/decreased level of AT plasma. FXI-/- = factor XI deficient plasma. Humate-P = FVIII-vWF. Only differences between baseline and highest concentration of FVIII-vWF are shown. ⁎ P b 0.05 by one-way ANOVA for repeated measurement followed by Bonferroni's posthoc t test.
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onset of thrombin generation was notable in addition to the decreased peak TG (Table 1, Fig. 2B). Significant recovery of TG in terms of lag time and peak thrombin was already observed when missing FVIII was replaced with 0.5 U/ml of FVIII-vWF (Table 1, Fig. 2B-C, P b 0.001). Thrombin generation in FVIII
-/-
/AT
+/-
plasma
In FVIII-/-/AT+/- plasma samples AT levels were reduced by ~51% (AT level = 59%) vs. control plasma (120%, Table 2). After adding only 0.5 U/ml of FVIII to FVIII-/-/AT+/- samples and activating with dilute Actin peak thrombin increased to 476 ± 50 nM, thus there was no longer difference vs. normal control (505 ± 39 nM, no FVIII-vWF added, P = 0.249)(Table 1). Similarly, the peak thrombin increased by N2.5 fold from 151 ± 16 nM to 417 ± 28 nM in TF (2 pM) activated FVIII-/-/AT+/- plasma samples supplemented with FVIII-vWF (0.5 U/ ml). Fondaparinux, added at a concentration of 1 μg/ml, was effective in reducing the peak thrombin level in FVIII-/-/AT+/- plasma samples, although the efficiency of inhibition was slightly lower in comparison to normal control plasma supplemented with 1 μg/ml of fondaparinux; ~62% vs. 80%, respectively (P b 0.001). This observation suggests the therapeutic effect of fondaparinux despite a moderate (~51%) decrease in AT activity. On the other hand, anticoagulant effect of fondaparinux was efficiently reversed with 3 U/ml of FVIII-vWF (Figs. 2D, 3), but not by 60 nM rFVIIa. No significant additive inhibitory effect on TG was observed after adding anti-FIXa aptamer (12 μg/ml) to plasma samples containing 1 μg/ml fondaparinux. However, FVIII-vWF (3 U/ml) was no longer capable of restoring TG in plasma treated with anti-FIXa aptamer and fondaparinux (Fig. 3). Thrombin generation in FXI Fig. 1. Effects of FVIII-vWF (Humate-P) on thrombin generation in control (normal) plasma with 2 pM tissue factor (panel A) and Actin activation (panel B).
control plasma samples, peak TG was minimally affected, but the lag time was shortened from 4.6 ± 0.4 min to 2.5 ± 0.3 min (P b 0.001) when exogenous FVIII was increased from 0 to 3 U/ml (Table 1 and Fig. 1B) After 50% saline dilution of control plasma (2 pM TF activation), the effect of FVIII-vWF on TG was obtunded resulting in only ~ 31% increase in peak thrombin at the highest concentration tested. With Actin activation, the shortening of lag time was significant at all 3 levels of added FVIII-vWF regardless whether tested plasma was diluted or not (P b 0.001) but only at two highest concentrations of added FVIII with 2 pM TF activation (Table 1). In control PPP spiked with 1 μg/ml (final concentration) of fondaparinux to simulate hypocoagulable state, peak thrombin decreased from 202 ± 14 nM (TF activation) at baseline to 40 ± 5 nM. The addition of FVIII-vWF (3 U/ml) reversed fondaparinuxinduced decreases in TG by 5 fold; thus peak thrombin increased to 207 ± 27 nM (Figs. 2A, 3). Notably, adding 60 nM rFVIIa shortened the lag time of TG, from 10.5 ± 0.7 to 4.7 ± 0.4 min and increased the rate of TG. However, the change in the peak thrombin level (40 ± 5 nM without rFVIIa to 68 ± 8 with rFVIIa (60 nM) was significantly smaller than the effect of Humate-P (Figs. 2A, 3). Addition of anti FIXa (12 μg/ml) aptamer to control plasma inhibited thrombin generation by ~ 65%. Supplementing these samples with 3 U/ml FVIII-vWF and triggering with 2 pM TF resulted in only ~ 42% recovery of thrombin peak (Fig. 3). Thrombin generation in FVIII
-/-
plasma
Peak thrombin levels were significantly decreased in FVIII-/plasma compared to normal plasma after either dilute Actin or TF trigger (Table 1, Fig. 2B-C). In Actin-triggered plasma, much delayed
-/-
plasma
FXI deficiency caused profound changes in TG when Actin was used as an activator. The lag time was very much prolonged from 4.6 ± 0.4 min (normal plasma) to 24.7 ± 2.6 min, a 5-fold increase, and peak thrombin level was decreased by ~96% (Table 1). The recovery of TG parameters was observed with increasing concentrations of FVIII (Table 1, Fig. 2E), although even with 3 U/ml of FVIIIvWF, the peak thrombin and lag time with Actin activation did not normalize to the control level; the peak thrombin rose only to ~ 33% of normal plasma (supplemented with Humate-P) and lag time decreased to 13.8 ± 0.4 min (still a 3-fold prolongation vs. baseline). Coagulation time measurements As expected, factor VIII was profoundly decreased in FVIII-/- and FVIII-/-/AT+/- samples (1% vs. 76% in normal plasma) (Table 2). Deficiency of FVIII prolonged aPTT from 31 sec to 86 sec, but had no effect on PT. After 50% dilution of control PPP samples, both PT and aPTT were prolonged (Table 2). Addition of increasing concentrations of FVIII-vWF (0–3 U/ml) to normal control, FVIII-/- and FVIII-/-/AT+/plasma samples progressively shortened aPTTs but not the PT values. Further, adding incremental FVIII-vWF to diluted normal plasma samples normalized the aPTT but did not affect the PT results, which stayed prolonged. The decrease in AT (~59% in FVIII-/-/AT+/- plasma vs. 120% in normal plasma) was not reflected in PT or aPTT results. Discussion In the present study, we demonstrated that elevated FVIII levels improve the rate and peak level of TF-initiated TG in hypocoagulable FVIII-/-, FXI-/-, and fondaparinux-treated plasma samples (Fig. 2A-E). We also evaluated concomitant changes in procoagulant and anticoagulant proteins using plasma with reduced AT, and in salinediluted plasma. In the presence of low AT activity, TG was more rapidly restored at a small increment of FVIII in FVIII-/- plasma
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F. Szlam et al. / Thrombosis Research 127 (2011) 135–140
Fig. 2. Panel A. Effects of FVIII-vWF (Humate-P) and recombinant activated factor VII (rFVIIa) in control (normal) plasma treated with fondaparinux (2 pM tissue factor activation); Panels B-C. Recovery of thrombin generation in FVIII-/- plasma with increasing concentrations of FVIII-vWF (Humate-P). Actin (B) and 2 pM tissue factor (C) activation; Panel D. Recovery of thrombin generation with FVIII-vWF and rFVIIa in FVIII-/-AT+/- plasma treated with fondaparinux. Fonda = fondaparinux 1 μg/ml. rFVIIa= recombinant activated factor VII 60 nM (3 μg/ml). Humate-P = FactorVIII-vWF (3 U/ml); Panel E. Effects of supplemental factor VIII on recovery of thrombin peak and lag time in FXI-/- plasma. Thrombin generation in control (normal) plasma (PPP) is shown for comparison. Actin activation. Humate-P = FVIII-vWF (3 U/ml).
(Table 1, Fig. 3). Thus slower FXa and thrombin inhibition due to low AT potentiates procoagulant activity of FVIII even in the presence of fondaparinux (Figs. 2D, 3). When normal (control) plasma was diluted by 50% with saline to simulate the general effect of hemodilution in trauma/surgery, incremental changes of thrombin peak were obtunded relative to non-diluted plasma (Table 1). In contrast to an isolated decrease of AT, diluted plasma incurs decreases in procoagulant proteins, especially prothrombin, which result in lower peak thrombin levels [10,22,23]. Hemostatic function of FVIII is conventionally evaluated by aPTT using a contact activator such as Actin FS. In normal undiluted and saline-diluted plasma samples triggered with dilute Actin FS, lag times of TG were progressively shortened by increasing concentrations of FVIII. However, no additional increase in peak thrombin levels was observed when FVIII level was increased above 57% (Table 2). The ceiling effects of FVIII on peak thrombin level were also seen after adding 0.5 U/ml of FVIII (Table 1, 2) in FVIII-/- and in FVIII-/-/AT+/samples. The robustness of restoring TG with contact activation may be in part explained by lack of efficient physiological inhibitors against FXIIa and FXIa, resulting in full activation of intrinsic tenase (FIXaFVIIIa) to support TG [24]. Indeed, the ceiling effect was not seen in FXI-/- plasma supplemented with FVIII-vWF because the lack of FXI was a limiting step of Actin-mediated activation of plasma coagulation (Table 1, Fig 2E). Based on these results, TG triggered with TF (1–2 pM) may be more suitable to evaluate hemostatic and possibly thrombotic potential of FVIII (Table 2) [25]. Plasma FVIII activity dynamically changes during and after major trauma and surgery [26,27]. While coagulation factor(s) levels decrease progressively according to the extent of blood loss and hemodilution, plasma vWF and FVIII activities are relatively well maintained [3,27,28]. Assuming normal vWF activity (1 U/ml) at
baseline, Weinstein, et al. estimates that even after a 50% hemodilution that reduced vWF activity to 0.5 U/ml, post-surgical vWF increases to 1.3 U/ml due to an influx of 0.8 U/ml during cardiopulmonary bypass. The release of FVIII-vWF complex is stimulated by
Fig. 3. Changes in peak thrombin generation with FVIII-vWF and rFVIIa in control and FVIII-/-/AT+/- plasma triggered with 2pM tissue factor. The impact of FIX inhibition using anti-FIXa aptamer in addition to FXa inhibition by fondaparinux is also shown. Statistical analyses by one-way ANOVA for repeated measurement followed by Bonferroni's posthoc t test. *P b 0.05 vs. control plasma with fondaparinux. #P b 0.05 vs. FVIII-/-AT+/- plasma with fondaparinux. ^P b 0.05 vs. combination of fondaparinux and anti-FIXa aptamer. The results of statistical analyses of anti-FIXa aptamer alone or in combination with Humate-P are not shown. Fonda = fondaparinux 1 μg/ml. rFVIIa = Recombinant activated factor VII 60 nM (3 μg/ml). Aptamer = Anti-FIXa aptamer (12 μg/ml). Humate-P = FVIII-vWF (3 U/ml).
F. Szlam et al. / Thrombosis Research 127 (2011) 135–140 Table 2 Effect of Increasing Concentrations of Humate-P on Coagulation Parameters in Control, dilute Control, FVIII-/-, FVIII-/-/AT+/-and FXI-/-plasma samples. Plasma Sample
aPTT (sec)
PT (sec)
ATIII %
FVIII %
Control + Humate-P 0.5 U/ml + Humate-P 1.5 U/ml + Humate-P 3.0 U/ml FVIII -/+ Humate-P 0.5 U/ml + Humate-P 1.5 U/ml + Humate-P 3.0 U/ml FVIII -/-/AT+/+ Humate-P 0.5 U/ml + Humate-P 1.5 U/ml + Humate-P 3.0 U/ml Diluted Control + Humate-P 0.5 U/ml + Humate-P 1.5 U/ml + Humate-P 3.0 U/ml
31.0 28.0 25.8 23.4 85.8 31.5 26.4 24.1 79.8 33.9 28.8 25.9 47.9 42.1 37.7 34.9
13.1 12.5 12.9 12.6 11.8 11.7 11.6 11.9 11.9 11.9 12.1 12.3 15.7 16.2 16.2 16.3
120 116 113 113 119 115 114 117 59 61 61 55 71 66 67 65
76 138 223 392 1 61 159 336 1 58 169 298 57 134 228 396
FVIII-/- = factor VIII deficient plasma. FVIII-/-/AT+/- = factor VIII deficient/decreased level of AT plasma. FXI-/- = factor XI deficient plasma. Humate-P = FVIII-vWF.
elevated stress hormones such as epinephrine and vasopressin [29]. Desmopressin (1-deamino-8-D-arginine vasopressin; DDAVP) can be therapeutically used to treat mild hemophilia A and von Willebrand disease [15,16]. In surgical patients without congenital bleeding disorders, the benefit of desmopressin is limited to a small decrease in perioperative blood loss, and the rate of allogeneic blood use does not seem to be reduced [30]. It is plausible that hemostatic efficacy of FVIII-vWF is reduced by concomitant release of tissue plasminogen activator (tPA, 2.5-fold increase in 30–45 min after desmopressin) and enhanced fibrinolytic tendency in perioperative hemodilution [31]. As purified FVIII-vWF, Humate-P should be more easily titrated to increase FVIII-vWF, without concomitant release of tPA, to optimize platelet adhesion and TG. Additional clinical studies are necessary to investigate a potential target population, efficacy and safety of FVIIIvWF therapy in acquired perioperative bleeding. Thrombophilia after major surgery is an important clinical problem, but perioperative bleeding complication from antithrombotic therapy is always a concern. The use of fondaparinux seems to reduce in-hospital mortality, but patients who had major bleeding had a 7-fold higher 30-day mortality compared to those without bleeding [32]. The modulation of fondaparinux anticoagulation by FVIII demonstrated in our study has clinical implications in terms of antithrombotic efficacy and reversal of anticoagulation. Fondaparinux was found to be effective even when AT was moderately decreased (Fig. 2D), while its anticoagulant effects could be reversed after increasing FVIII by 3 U/ml (Figs. 2D, 3). In addition, Humate-P added to fondaparinux-treated control plasma resulted in approximately 100% recovery of thrombin peak while only 33% was recovered by 60 nM of rFVIIa (Figs. 2A and 3). Our results are in agreement with Desmurs-Clave, et al. who observed the minimal recovery of thrombin peak with rFVIIa in fondaparinux-treated platelet-rich plasma [14]. Fondaparinux reduces TG by enhancing AT-mediated FXa neutralization, but it does not obliterate thrombin activity [33]. Anticoagulant efficacy of fondaparinux is reduced after thrombin activates FV to form prothrombinase (FXa-FVa complex) [34]. Further, FIXa is generated by either TF-FVIIa or FXIa, and intrinsic tenase (FIXa-FVIIIa complex) can be rapidly formed when thrombin-mediated FVIII activation is facilitated by elevated FVIII concentrations [25]. We demonstrated the pivotal role of intrinsic tenase to antagonize fondaparinux in FVIII-/-/AT+/- plasma samples. When FIXa activity was inhibited with 12 μg/ml of anti-FIXa aptamer [21] procoagulant activity of FVIII-vWF in fondaparinux-treated plasma was obtunded (105 ± 11 nM with anti-FIXa vs. 365 ± 32 nM without) (Fig. 3). This finding underlies the importance of intrinsic tenase system in
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supporting hemostasis when the trigger for TG is limited due to reduced activity of TF pathway. It partly explains the limited efficacy in reducing hemophilia-related joint bleeds in patients with inhibitors using rFVIIa [35,36]. Elevated FVIII levels are not reflected in PT due to supraphysiological TF levels used to activate plasma during the testing (Table 2). At a more physiological level (2 pM) used in TG assay, the effect of elevated FVIII on TG was clearly demonstrated in normal and FXIdeficient plasma (Table 1, Figs. 1A, 2E). Conversely, lag times of Actinactivated TG and aPTTs were shortened according to FVIII levels (Table 1, 2). The latter findings are in agreement with Tripodi, et al. who observed an association between high FVIII levels and shortened aPTT [37]. Interestingly, in saline diluted normal plasma repleted with increasing concentrations of FVIII-vWF, aPTT values were normalized with 3 U/ml of FVIII, but there was no change in PT results, which stayed elevated. Elevated FVIII levels up to 3.9 U/ml had been observed in patients under acute stress (e.g., trauma), resulting in shortened aPTT [2]. Recently, thrombin-mediated FXI activation was shown to be important for sustained TG under low TF or low FVII condition [13,38]. The contribution of intrinsic pathway to sustained TG is also supported by our experiments in which elevated FVIII partially compensates for FXI deficiency in terms of TG (Table 1, Fig. 2E) although the FVIII efficiency was much lower than in other plasma samples used in the current study. We speculate that heterogeneous bleeding and thrombosis patterns found in congenital FXI deficiency may be in part attributed to local FVIII-vWF levels achieved in a target organ [39,40]. There are several limitations in this study. First, platelets and other rheological elements of coagulation were not included in the present models, and thus the involvement of vWF in TG was not evaluated [41]. The presence of phospholipids (as in this study) versus plateletrich plasma on TG seems to affect the response to FVIII. Keularts et al. reported only a limited increase of peak thrombin when FVIII levels were between 50% and 150% of normal in platelet-rich plasma [16]. Similar to our study, Ibbotson, et al., and Butenas, et al., respectively, observed increases in peak thrombin levels when FVIII levels were increased from 100% to 350% and 50% to 100%, respectively [42,43]. More recently, in a study by Machlus et al., increased concentrations of FVIII (100-400% range) were found to increase TG (both peak and endogenous thrombin potential) in tissue factor triggered platelet poor plasma [25]. These data are supported by the longitudinal observational study which indicated the association of high peak TG (4th quartile of the cohort) using PPP, FVIII activity and the risk of thromboembolism [44]. In conclusion, we demonstrate that elevated FVIII supports the propagation of thrombin generation under low TF (2 pM) state, FXI deficiency, or in the presence of fondaparinux. Taken together, changes in plasma FVIII can dynamically modulate hemostatic and even thrombotic responses, and purified FVIII-vWF may represent a potential therapeutic agent in excess anticoagulation due to fondaparinux. Further clinical studies are needed to establish the efficacy and safety of purified FVIII-vWF in a perioperative setting. Conflict of interest statement Drs. Solomon and Tanaka have received consultant fees and research support from CSL Behring (Marburg, Germany). References [1] Jaffe E. Synthesis of factor VIII by endothelial cells. Ann NY Acad Sci 1982;401: 163–70. [2] Yuan S, Ferrell C, Chandler WL. Comparing the prothrombin time INR versus the APTT to evaluate the coagulopathy of acute trauma. Thromb Res 2007;120:29–37. [3] Harker LA, Malpass TW, Branson HE, Hessel II EA, Slichter SJ. Mechanism of abnormal bleeding in patients undergoing cardiopulmonary bypass: acquired transient platelet dysfunction associated with selective alpha-granule release. Blood 1980;56:824–34.
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