Resuscitation of patients with trauma-related bleeding remains challenging despite recent advances in treatment strategies.1 Coagulopathy may contribute to massive postinjury hemorrhage and remains a major cause of death after trauma2,3 even if surgical bleeding control can be achieved.4 Trauma-induced coagulopathy is characterized by acute impairment of all components of the coagulation system with an imbalance of pro- and anticoagulant factors, disturbed platelet function, endothelial damage, hypofibrinogenemia, and hyperfibrinolysis.5
The clinical importance of treating coagulopathy early to achieve hemostasis has been demonstrated.6–8 In recent years, a shift from empirical treatment by plasma transfusion toward targeted therapy including the use of coagulation factor concentrates such as fibrinogen and prothrombin complex concentrate (PCC) has been advocated.8–10 In major bleeding, fibrinogen is the first coagulation factor to reach critically low levels.11 Low fibrinogen levels have been associated with increased bleeding tendency,12,13 increased transfusion,14 and increased mortality in this setting.15 Consequently, fibrinogen supplementation is recommended in European guidelines as first-line hemostatic therapy for patients with trauma-related bleeding.1 Patients with ongoing, life-threatening bleeding after fibrinogen supplementation may develop inadequate thrombin generation, in which case PCC has the potential to be used as hemostatic therapy.16 Administration of coagulation factor concentrates instead of allogeneic blood products may be preferable for a short administration time, consistent dosing, reliable availability, and favorable safety/tolerability.9,10,17 Aside from the supplementation of coagulation factors, the antifibrinolytic agent tranexamic acid (TXA) is increasingly administered as early treatment for trauma since the CRASH-2 (Clinical Randomization of an Antifibrinolytic in Significant Haemorrhage 2) study reported survival benefits with this treatment.18
There are little data on whether the combination of TXA with coagulation factor concentrates reduces blood loss and improves hemostasis in trauma patients. Using a porcine model of blunt trauma, we hypothesized that human fibrinogen concentrate (FC) and PCC, administered as combined therapy with TXA, would provide additive effects in correcting coagulopathy and reducing blood loss in trauma.
This study was conducted according to German legislation based on the Principles of Laboratory Animal Care. Official permission was granted from the appropriate governmental animal care and use office (Landesamt für Natur, Umwelt und Verbraucherschutz, Recklinghausen; No: 84-02.04.2011.A318).19 Thirty-six male German Landrace pigs from a disease-free breeding facility (body weight 36–42 kg) were housed in ventilated rooms for at least 5 days to acclimatize. They were fasted overnight before surgery with free access to water.
The animals were first given IM injections of azaperone (4 mg*kg−1; Stresnil; Janssen, Neuss, Germany) and atropine (0.1 mg*kg−1; Atropinsulfate; B. Braun Melsungen AG, Melsungen, Germany). General anesthesia was then induced with propofol (3 mg*kg−1; Disoprivan; AstraZeneca, Wedel, Germany) administered by IV injection via an 18-G cannula into the right ear vein. The animals’ lungs were ventilated using a pressure-controlled mode at 16 to 18 breaths per minute with a tidal volume of 8 mL*kg−1 to keep Paco2 between 34 and 40 mm Hg with an oxygen fraction of 1.0 in a closed circuit (PhysioFlex; Dräger, Lübeck, Germany). Anesthesia was maintained with isoflurane (Forane; Abbott, Wiesbaden, Germany) at an end-tidal concentration of 1.2% to 1.4% and constant infusion of fentanyl (2 μg*kg−1*h−1; Fentanyl-Rotexmedica; Rotexmedica, Trittau, Germany).
Initial fluid therapy was composed of Ringer’s solution (4 mL*kg−1*h−1; Sterofundin Iso, B. Braun AG, Melsungen, Germany). Blood temperature; arterial, central venous, and pulmonary arterial pressure; tail pulse oximetry; and electrocardiography were monitored constantly using a standard anesthesia monitor (AS/3; Datex Ohmeda, Helsinki, Finland).
Two 8.5-Fr catheters were inserted percutaneously into the right and left femoral veins for fluid administration and blood withdrawal, and a pulmonary artery catheter was placed in a wedge position through a third 8.5-Fr catheter that was implanted surgically in the right jugular vein. The wedged catheter position was confirmed by observing the change waveform. Hemodynamic variables were recorded through an 18-G catheter in the right femoral artery. After vascular cannulation, a midline laparotomy was performed and the crystalloid administration rate was increased to 8 mL*kg−1*h−1.
Liver Injury and Hemostatic Intervention
A standardized grade III blunt liver injury was inflicted as previously described using a custom-made instrument.20 With the base of the instrument’s plate positioned beneath the right middle lobe of the liver, the parenchyma was clamped with a force of 205 ± 23 N and then released. Five minutes afterward, controlled hemorrhage from the venous access was initiated at a rate of 100 mL*min−1. Crystalloids were infused as required to maintain the mean arterial blood pressure (MAP) >30 mm Hg. Withdrawn blood was collected and processed in an autologous cell saver system (Cell Saver 5; Haemonetics, Braintree, MA) for subsequent retransfusion. After a shock phase of 30 minutes, animals received 3 times the blood volume lost from the circulation (i.e., blood loss minus the volume infused to maintain minimal arterial blood pressure) as a crystalloid infusion over 10 minutes. After a stabilization phase of 10 minutes, a second blunt liver injury (grade IV) was inflicted, this time to the central lobe (force: 236 ± 82 N). Five minutes after infliction of the second injury, washed salvaged red blood cells and crystalloid solution were infused (10 mL*kg−1 each). The use of room temperature solutions was expected to decrease the animals’ body temperatures; a further decrease in temperature was avoided by using a forced-air warming blanket (WarmTouch; Covidien, Mansfield, MA).
Animals were prospectively randomized using a computer-generated list in a 1:1:1:1 format using sealed envelopes into 1 of the following treatment groups: control group, TXA group, TXA–FC (TXA + FC) group, or TXA–FC–PCC (TXA + FC + PCC) group. Treatment was initiated 15 minutes after the second injury. Animals in the treatment groups were first given TXA (15 mg*kg−1 IV bolus; Cyklokapron; Pfizer, Berlin, Germany). This was followed by FC (90 mg*kg−1 IV infusion over 10 minutes; Haemocomplettan P; CSL Behring, Marburg, Germany) in the TXA–FC group. Animals in the TXA–FC–PCC group received 4-factor PCC (20 IU*kg−1 IV bolus; Beriplex PN, CSL Behring) 30 minutes after trauma. Animals in groups not receiving PCC, FC, and/or TXA received the equivalent infusion volume of normal saline instead of active treatment. To ensure study blinding, hemostatic therapy and saline were administered using identical, opaque syringes, and blood samples were sent for laboratory analysis without identification of the study arm. The observation period ended 240 minutes after injury. Animals surviving for the entire study period were euthanized with pentobarbital (400 mg*kg−1). Immediately after death (whether this occurred at the end of the observation period or earlier), the abdomen was reopened, the vena cava was clamped cranial to the liver, and the intraperitoneal blood was collected to determine the total blood loss postinjury. The lungs, heart, liver, and kidneys were removed and prepared for histologic examination.
Blood Sampling and Analytical Methods
Blood was collected and arterial blood gas analysis was performed at baseline, 30, and 50 minutes after the first injury as well as 15, 45, 90, 180, and 240 minutes after the second liver injury. A schematic representation of the study time line is shown in Figure 1. For animals dying before 240 minutes after injury, the last assessment was performed immediately after death. Arterial pH, Pao2, and Paco2 were measured with a blood gas analyzer (ABL725; Radiometer GmbH, Willich, Germany). A standard hematology analyzer (MEK-6108; Nihon Kohden, Tokyo, Japan) was used to assess platelet count and hemoglobin concentration. Other testing included prothrombin time (PT; Dade® Innovin; Siemens, Marburg, Germany), activated partial thromboplastin time (aPTT; Dade Actin FS; Siemens), FC (Dade Thrombin reagent; Siemens), and D-dimers (Innovance® D-Dimer; Siemens) measured on a steel-ball coagulometer (MC 4 plus; Merlin Medical, Lemgo, Germany). Thrombin–antithrombin (TAT; Enzygnost; Dade Behring, Marburg, Germany) complexes and fibrinopeptide A (FPA; Zymutest FPA; HyphenBiomed, Neuville-sur-Oise, France) were quantified by enzyme-linked immunosorbent assay.
Thromboelastometry and Thrombin Generation
A ROTEM device (Tem International GmbH, Munich, Germany) was used to perform the EXTEM assay in whole blood. The following variables were measured: clotting time (CT; seconds) and maximum clot firmness (MCF; millimeters). We devised an assay with ex vivo tissue plasminogen activator to investigate fibrinolysis. Briefly, ROTEM plastic cups were loaded with 300 μL of whole blood and spiked with 20 μL of buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 150 mM NaCl, pH 7.4) and 10 µL tissue plasminogen activator (LOXO GmbH, Dossenheim, Germany). The hemostatic process was activated with tissue factor (Innovin; Dade Behring) and recalcified with 0.2 M CaCl. The lysis index 30 (LI30, which shows clot lysis 30 minutes after CT as a percentage of MCF) was analyzed in this modified ex vivo assay.
Thrombin generation was measured in plasma using the calibrated automated thrombogram (Thrombinoscope BV, Maastricht, The Netherlands).21 Assessments were conducted in 80 μL samples of plasma with 20 μL of fluorogenic substrate and 20 μL of trigger reagent. The trigger reagent was 1 pM tissue factor with 4 μM phospholipids. Each thrombin generation analysis was calibrated against the fluorescence curve obtained in the same plasma with a fixed amount of calibrator (Thrombin Calibrator; Thrombinoscope BV). Fluorescence was measured using an Ascent Reader (Thermo Labsystems OY, Helsinki, Finland), and thrombin generation curves were calculated using Thrombinoscope version 4 software (Thrombinoscope BV). The following variables were measured: peak height (peak thrombin generation), endogenous thrombin potential (capacity of plasma to generate thrombin over time), and lag time (time taken for thrombin generation to increase above the horizontal baseline).
Platelet activation was assessed by flow cytometry using fluorescently labeled antibodies to determine activation markers on platelets, as described elsewhere.22 Citrated whole blood was diluted (1:50) in Tyrode’s buffer, and 50 μL was added to 10 μL of vehicle or 2-methylthioadenosine diphosphate (Santa Cruz Biotechnology, Santa Cruz, CA), 10 μL Alexa Fluor 488-labeled antihuman P-selectin (AbD Serotec, Oxford, UK), and 10 μL Alexa Fluor 647-conjugated human fibrinogen (Invitrogen Molecular Probes, Eugene, OR). After incubation for 20 minutes at 37°C, the samples were fixed with 100 μL CellFix (BD Biosciences, San Jose, CA) and stored at 4°C until analysis. Binding of the fluorescent labels to the platelets was assessed using a 3-channel Accuri C6 flow cytometer (BD Biosciences). Platelet activation after adenosine diphosphate stimulation was evaluated by calculating the ratio of the mean fluorescence intensity of stimulated platelets to the mean fluorescence intensity of unstimulated platelets.
After death, internal organs (heart, lungs, liver, and kidneys) were removed immediately and fixed in 10% buffered formalin. Injured parts of the liver were cut into 3-mm-thick slices and examined macroscopically and microscopically by a pathologist blinded to therapy to assess the degree of injury. In addition, representative tissue sections of all 4 organs (thickness: 5 mm) were examined macroscopically to determine the occurrence of thromboembolic events via the detection of dilated, clotted vessels (limit of detection: approximately 1 mm diameter in liver, heart, and kidney and approximately 2 mm in lung tissue). All samples were embedded in paraffin and stained, both by hematoxylin/eosin and by a standard elastica van Gieson protocol, for histologic examination under light microscopy (Microscope: Eclipse 50i, Nikon, Tokyo, Japan). Sections of lung and liver tissue from regions with a high likelihood of thrombus formation were immunostained to test for fibrinogen (antibody and detection kit from Dako, Glostrup, Denmark) as described elsewhere.23
The primary endpoint was blood loss. Based on data from previous animal experiments performed by our group, we considered that a reduction of approximately 25% to 50% in blood loss would be significant.23,24 Considering our previous data, the inclusion of 4 groups in this study, an estimated high effect size (d = 0.8), and a significance level of 0.05, a sample size of 9 animals per group was sufficient to achieve a power of 0.96. Assumptions for a repeated-measures analysis of variance were tested by P–P plots of the residuals and Mauchly sphericity test. For comparison of variables with normally distributed residuals, a repeated-measure analysis of variance was used with intervention as group factor and time as repeated factor. The group × time interaction was also included to allow the group differences to vary over time. The Sidak method was used post hoc for significant effects. For variables not meeting the assumptions of the repeated-measure analysis of variance (i.e., fibrinogen, FPA, and thrombin generation variables), a Kruskal-Wallis test for multiple group comparisons was used. In case of significant differences in the Kruskal-Wallis test, pairwise between-group comparisons with Wilcoxon-Mann-Whitney tests and Bonferroni adjustment were performed. Pairwise log-rank tests were used for survival analysis. Data are presented as mean with SD or, in case of values not following the normal distribution, as median and interquartile range. Statistical tests were performed 2-tailed; P values <0.05 were considered statistically significant. P values for all analyses are provided as Supplemental Digital Content (http://links.lww.com/AA/B430). Statistical analysis was performed using SPSS 22 (SPSS, Chicago, IL), and GraphPad Prism (GraphPad Software, La Jolla, CA) was used for graphing purposes.
Hematological, coagulation, metabolic, and hemodynamic data obtained before liver injury and treatment are shown in Tables 1 and 2. There were no significant differences among the 4 treatment groups in any of the study variables at baseline or any of the other time points before administration of study treatment.
Blood Loss and Survival
As shown in Figure 2, total blood loss after the second liver injury, the primary endpoint of the study, was lower in the TXA–FC and TXA–FC–PCC groups (1012 ± 86 mL and 1037 ± 118 mL, respectively) than in the TXA and control groups (1579 ± 306 mL and 2376 ± 478 mL, respectively; P < 0.001 for all 4 comparisons). Total blood loss in the TXA group was lower than in the control group (P < 0.001).
All animals in the TXA–FC and TXA–FC–PCC groups survived the complete observational period, whereas 5 of 9 (56%) and 2 of 9 animals (22%) died after the second trauma in the control and TXA-only groups, respectively (for both these groups, P < 0.05 versus TXA–FC and TXA–FC–PCC).
Hemoglobin Levels, Platelet Counts, Hemodynamic Variables, and Shock
Decreasing values for MAP and cardiac output were observed in the control group after the second liver injury (Table 1). MAP was higher in the TXA–FC and TXA–FC–PCC groups than in the control and TXA groups from 45 to 240 minutes after the second trauma (P < 0.05). Cardiac output was higher in the TXA–FC and TXA–FC–PCC groups than in the control group from 90 to 240 minutes after the second trauma. Lactate increased over time in the control and TXA groups (Table 1). At 180 and 240 minutes after the second trauma, lower levels of lactate were observed in the TXA–FC and TXA–FC–PCC groups than in the TXA and control groups (P < 0.05).
Hemoglobin concentrations decreased below baseline levels after the administration of study treatment in all groups (Table 2). The most pronounced reduction was noted in control animals. At 180 and 240 minutes after the second trauma, higher levels of hemoglobin were observed in the TXA–FC and TXA–FC–PCC groups than in the TXA and control groups (P < 0.05). Similarly, the lowest platelet counts were observed in the control group. However, the only significant difference in platelet count was between the TXA–FC group and the control group, at 180 and 240 minutes after the second trauma (P < 0.05).
Fibrinogen Concentration, Coagulation Tests, and Thromboelastometry
After administration of study treatment, PT and aPTT increased in the control group (Table 2). In the TXA–FC and TXA–FC–PCC groups, PT was lower than in the control and TXA groups at 90, 180, and 240 minutes after the second trauma (P < 0.05). Lower values for aPTT were observed in the TXA–FC and TXA–FC–PCC groups than in the control group (P < 0.05) at the same 3 time points.
The administration of study intervention rapidly increased plasma fibrinogen levels in the TXA–FC and TXA–FC–PCC groups; no such increase was seen with control or TXA (Fig. 3A). Plasma fibrinogen levels were significantly higher in the TXA–FC–PCC group than in the control and TXA groups from 45 to 240 minutes after the second trauma (P < 0.05). The fibrinogen level in the TXA–FC group was significantly higher than in the control group at 45 and 90 minutes (P < 0.05). An increase in the FPA level was evident in the TXA–FC–PCC group compared with the control and TXA groups (P < 0.05; Fig. 3B). However, no significant difference in FPA level was evident between the TXA–FC group and the control or TXA group.
In the control group, EXTEM CT increased over time, and there was a gradual decline in MCF (Fig. 4, A and B). Treatment with TXA was associated with a lower EXTEM CT than in the control group at 180 minutes (P = 0.005), and MCF was higher than in the control group from 45 to 240 minutes after the second trauma (P < 0.05). In the fibrinogen-treated groups (TXA–FC and TXA–FC–PCC), CT was shorter than in the control group at 180 minutes (P < 0.05) and shorter than in the control and TXA groups at 240 minutes (P < 0.05). The fibrinogen-treated groups exhibited higher EXTEM MCF than in the control group at 45 and 90 minutes (P < 0.05) and higher MCF than in the control and TXA groups at 180 and 240 minutes (P < 0.05).
The tissue plasminogen activator ROTEM analysis showed low LI30 values (<10%) throughout the study in the control group. In all TXA-treated animals, LI30 was <35% before study intervention but exceeded 90% at all time points after study intervention (P < 0.05 versus control).
Thrombin Generation, Activation of Coagulation, and Platelet Activity
Thrombin generation data showed a clear response to study intervention in the TXA–FC–PCC group but not in any of the other groups. Peak height and endogenous thrombin potential were higher 45 minutes after the second trauma in the TXA–FC–PCC group than in any of the other 3 study groups (P < 0.05; Fig. 5, A and B). Values for these 2 variables remained higher in the TXA–FC–PCC group than in the TXA group and the control group from 90 to 240 minutes. No significant between-group differences were observed in lag time (data not shown).
TAT complex levels were higher in the TXA–FC–PCC group than in all the other study groups from 90 to 240 minutes after the second trauma (P < 0.05; Fig. 6A). At 180 and 240 minutes, higher D-dimer levels were observed in the control group than in all the other study groups (P < 0.05; Fig. 6B).
The binding of fluorescent labels to platelets (P-selectin and fibrinogen) after stimulation with adenosine diphosphate showed no significant between-group differences in platelet activation (data not shown).
The extent of tissue damage resulting from the liver injuries was not different among the 4 study groups. Macroscopic and histological evaluation of tissue samples from all organs showed no evidence of thromboembolic events.
Using a porcine model of severe trauma and trauma-induced coagulopathy, we have shown that combination therapy in the TXA and FC groups was effective for reducing blood loss and improving coagulation variables compared with either the TXA group or the control group. Total blood loss in the TXA–FC and TXA–FC–PCC groups was approximately half that in the control group. Additional treatment with PCC enhanced thrombin generation, but there was no further reduction of blood loss compared with the TXA–FC group. Coagulopathy worsened over time in the control group, and 56% of animals died before the end of the observation period. In contrast, all animals in the TXA–FC and TXA–FC–PCC groups survived to the end of the observation period and our data indicate a lack of deterioration in the coagulation status of these animals after the administration of study treatment.
Despite extensive clinical and laboratory research, the optimal approach to treating patients with trauma and massive hemorrhage and/or acute coagulopathy is under debate. This is attributable, in part, to the biological complexity resulting from injury, hemorrhagic shock, and hemostatic therapy. Hypothesized mechanisms for trauma-induced coagulopathy implicate a central role of upregulation of fibrinolysis and poor clot strength.3–5,25–28 Thus, one would speculate that antifibrinolytic therapy would lead to reduced coagulopathy in the setting of trauma. Published data on the incidence of hyperfibrinolysis in patients after severe trauma are variable, ranging between approximately 2% and 20%.29–33
In our study, we administered TXA to the massively bleeding pigs early (i.e., 15 minutes after the second trauma) in the absence of major changes from baseline in a ROTEM indicator of fibrinolysis or D-dimer levels. The resulting reduction in blood loss and the stabilization or improvement of ROTEM variables suggest that early administration of TXA may be beneficial in either occult or suspected hyperfibrinolysis. Systemic inflammation can make a significant contribution to the deleterious effects of trauma.34 In addition to its effects of fibrinolysis, TXA functions as an anti-inflammatory agent by inhibiting the effects of plasmin.35 It is possible that the impact of TXA on inflammation contributes to its effects on blood loss and mortality in patients with trauma.26,35
The benefits of early TXA treatment versus no treatment were increased when FC was administered as an additional therapy. This finding is concordant with the survival benefits of combined therapy with TXA and cryoprecipitate in the recent MATTERs II (Military Application of Tranexamic Acid in Trauma Emergency Resuscitation) study.36
Fibrinogen depletion occurs early after trauma.11,15,37–40 Low plasma levels of fibrinogen are associated with worsened injury severity score, shock, blood loss, transfusion requirements, and mortality.15,41,42 In our study, the administration of FC rapidly achieved plasma levels recommended by current European trauma guidelines.1 This was associated with enhancement of viscoelastic measurements and a reduction in blood loss compared with both control and TXA-treated animals. These data are consistent with a 2012 prospective cohort study investigating the time course of fibrinogen depletion in 517 trauma patients,15 where the authors advocated fibrinogen replacement as a means of improving outcomes. This is in line with the conclusions of previous studies.43–46
We observed that the administration of PCC in addition to TXA and FC produced no further reduction in blood loss. We suspect that thrombin generation is reduced only in the later stages of trauma-induced coagulopathy such as when coagulation factor II is decreased by two-thirds or more as is expected with blood loss of 150% to 200%.17,47,48 Nevertheless, previous studies have indicated a role for PCC in the treatment of trauma-induced coagulopathy when administered selectively to patients with prolonged EXTEM CT or high international normalized ratio and continued bleeding after fibrinogen supplementation.39,49,50 This use of PCC, in conjunction with fibrinogen supplementation, appears to reduce patients’ exposure to allogeneic blood products.49–51
It has been shown that thrombin generation may be upregulated as a physiological response to trauma.48 The potential risk of thromboembolic complications has been identified as a substantial shortcoming with PCC.48,52 For example, in a porcine model of liver injury, our group observed thromboembolism among animals receiving a PCC dose of 50 IU*kg−1, attributable to an imbalance of pro- and anticoagulant proteins.24 A clinical study has also reported a potential prothrombotic state lasting for several days after PCC therapy in trauma patients.53 In our study, there was no evidence of thromboembolic complications, but increased thrombin generation and enhanced fibrinogen activation indicated the potential for a prothrombotic state in the TXA–FC–PCC group. We would therefore advocate caution if considering the use of PCC for bleeding management in trauma patients.
A strength of our study is that promoters of coagulopathy such as hypothermia, acidosis, and hemodilution caused by intravascular volume replacement were included, mimicking the injury and treatment phases of severe trauma. There are also some limitations to our study. We investigated the effects of human and not porcine coagulation factors because of the unavailability of the latter. Although animal models enable a high degree of standardization, the results cannot necessarily be considered as fully transferable to humans because of species differences. In the clinical setting, coagulation factor concentrates are typically administered in response to clinical bleeding in the setting of abnormal coagulation test results. In contrast, in this study, these products were administered according to a fixed protocol independent of each animal’s coagulation data. Also, before the investigated treatment approach could be applied in humans, safety studies would be needed, particularly regarding the risk of thromboembolic events. The present study was too small to provide reliable insight into the risk of adverse events with the combinations of hemostatic agents administered. The posttreatment follow-up period was relatively short, and it is possible that prothrombotic complications might have occurred if the animals had been observed for a longer period of time. Finally, in accordance with clinical practice, we administered TXA, FC, and PCC sequentially. Consequently, our study provides no information regarding the possible benefits of administering these treatments simultaneously.
In conclusion, our data show that the early administration of TXA combined with FC reduces blood loss and improves coagulation test results after blunt liver injury and prolonged shock compared with no treatment and TXA administration alone. These effects could be related to the prevention of hyperfibrinolysis and rapid correction of hypofibrinogenemia. Sufficient thrombin generation was observed in this model, potentially explaining the lack of benefit with additional PCC therapy.
Name: Christian Zentai, MD.
Contribution: This author performed the experimental laboratory work.
Attestation: Christian Zentai approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.
Conflicts of Interest: None.
Name: Paola E. J. van der Meijden, PhD.
Contribution: This author performed the CAT assays and platelet analyses.
Attestation: Paola E. J. van der Meijden approved the final manuscript.
Conflicts of Interest: None.
Name: Till Braunschweig, MD.
Contribution: This author accomplished the pathological assessment.
Attestation: Till Braunschweig approved the final manuscript.
Conflicts of Interest: None.
Name: Nicolai Hueck, MD.
Contribution: This author performed the experimental laboratory work.
Attestation: Nicolai Hueck approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.
Conflicts of Interest: None.
Name: Markus Honickel, MD.
Contribution: This author performed the statistical analysis.
Attestation: Markus Honickel approved the final manuscript and attests to the integrity of the original data and the analyses reported in this manuscript.
Conflicts of Interest: Markus Honickel has received travel support from Boehringer Ingelheim (Germany).
Name: Henri M. H. Spronk, PhD.
Contribution: This author performed the CAT assays and platelet analyses.
Attestation: Henri M. H. Spronk approved the final manuscript.
Conflicts of Interest: Henri M. H. Spronk has received research funding from Boehringer Ingelheim (Germany) and honoraria for consultancy from Bayer (Germany).
Name: Rolf Rossaint, MD.
Contribution: This author participated in the study design.
Attestation: Rolf Rossaint approved the final manuscript.
Conflicts of Interest: Rolf Rossaint has received honoraria for lectures and consultancy from CSL Behring (Germany) and Novo Nordisk (Denmark).
Name: Oliver Grottke, MD, PhD, MPH.
Contribution: This author conceived and conducted the experimental laboratory work.
Attestation: Oliver Grottke approved the final manuscript, attests to the integrity of the original data and the analysis reported in this manuscript, and is also the archival author.
Conflicts of Interest: Oliver Grottke has received research funding from Novo Nordisk (Denmark), Biotest (Germany), CSL Behring (Germany), and Nycomed (Germany). He has also received honoraria for consultancy and/or travel support from CSL Behring (Germany), Boehringer Ingelheim (Germany), Bayer Healthcare (Germany), and Portola (USA).
Charles W. Hogue, MD.
The authors thank Renate Nadenau (Department of Anaesthesiology) for her excellent support.
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