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Ex vivo effect of hemostatic therapy in subarachnoid and intracerebral hemorrhage
Thrombosis Research 189 (2020) 42–47
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Full Length Article
Ex vivo effect of hemostatic therapy in subarachnoid and intracerebral hemorrhage
T
Christine Lodberg Hvasa, Signe Voigt Lauridsena, Emilie Sandgaard Pedersenb, Tua Gyldenholmb, ⁎ Anne-Mette Hvasb,c, a
Department of Anaesthesiology and Intensive Care, Aarhus University Hospital; Aarhus, Denmark Thrombosis and Hemostasis Research Unit, Department of Clinical Biochemistry; Aarhus University Hospital, Aarhus, Denmark c Department of Clinical Medicine, Aarhus University; Aarhus, Denmark b
A R T I C LE I N FO
A B S T R A C T
Keywords: Blood coagulation Subarachnoid hemorrhage Intracerebral hemorrhage Ex vivo Hemostatic agents
Background: Rebleeding and hematoma growth are serious complications in subarachnoid hemorrhage (SAH) and intracerebral hemorrhage (ICH). As treatment options are sparse, a mechanistic approach may reveal new therapeutic targets. Aim: Firstly, to evaluate hemostasis using a sensitive low tissue factor thromboelastometry (ROTEM®) assay in patients with SAH or ICH and compare them with healthy controls. Secondly, to investigate the ex vivo effect of hemostatic or antifibrinolytic medications in blood from patients with SAH or ICH. Methods: Blood was drawn on admission to hospital in patients with SAH (n = 39) or ICH (n = 35). We included 41 sex and age matched healthy controls for comparison. A low tissue factor (diluted 1:100,000) ROTEM® assay was run in patients and healthy controls. In parallel, coagulation factor XIII, fibrinogen concentrate, prothrombin complex concentrate, and recombinant soluble thrombomodulin were added in concentrations equivalent to doses used in clinical practice. Results: Patients with SAH or ICH demonstrated a hypercoagulable profile indicated by significantly shorter clotting time, faster maximum velocity, shorter time to maximum velocity, and higher maximum clot firmness than healthy controls (all p-values < .0001). Ex vivo addition of coagulation factor XIII, fibrinogen concentrate, prothrombin complex concentrate, and recombinant soluble thrombomodulin, respectively, did not improve the hemostatic potential in patients with SAH or ICH. Conclusion: Patients with SAH or ICH demonstrated a hypercoagulable state in the systemic circulation as evaluated by a sensitive low tissue factor assay. Ex vivo addition of hemostatic medication did not further improve coagulation.
1. Introduction Hematoma growth and rebleeding are serious complications in subarachnoid hemorrhage (SAH) and intracerebral hemorrhage (ICH) affecting both morbidity and mortality [1,2]. Currently, a combination of surgery and blood pressure control are the mainstay of treatment. Hemostatic and antifibrinolytic therapy have been tested in randomized trials in both SAH and ICH, but results have pointed in different directions [3–6].
In SAH, much focus has been on tranexamic acid. However, European guidelines and the most recent Cochrane review, do not advice routine use of tranexamic acid in SAH [4,7]. Tranexamic acid reduces the risk of rebleeding in the acute phase, but does not affect mortality, possibly due to an increase in ischemic events in the studies, where long-term treatment is applied (> 72 h), or due to lack of power [8]. In ICH, the focus regarding improved hemostasis has primarily been on recombinant coagulation factor VIIa (rFVIIa) [6] and reversal of
Abbreviations: aPTT, activated partial thromboplastin time; CT, clotting time; FXIII, coagulation factor XIII; ICH, intracerebral hemorrhage; INR, international normalized ratio; IQR, interquartile range; MCF, maximum clot firmness; MaxVel, maximum velocity; PCC, prothrombin complex concentrate; rFVIIa, recombinant coagulation factor VIIa; ROTEM®, thromboelastometry; SAH, subarachnoid hemorrhage; SD, standard deviation; TAFI, thrombin activatable fibrinolysis inhibitor; tMaxVel, time to maximum velocity ⁎ Corresponding author at: Thrombosis and Hemostasis Research Unit, Department of Clinical Biochemistry, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark. E-mail address: [email protected] (A.-M. Hvas). https://doi.org/10.1016/j.thromres.2020.02.012 Received 24 November 2019; Received in revised form 25 January 2020; Accepted 14 February 2020 Available online 26 February 2020 0049-3848/ © 2020 Published by Elsevier Ltd.
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The Regional Ethics Committee of Central Denmark approved the study (incompetent patients case no.: 1-10-72-95-14, version 3, 05052014 and competent patients case no.: 1-10-72-94-14, version 4, 27042014). All patients or their legal proxies provided written informed consent before study enrolment. The Danish Data Protection Agency also approved the study (incompetent patients case no.: 1-1602-225-14 and competent patients case no.: 1-16-02-224-14). The Helsinki Declaration was followed in all aspects. In total 41 age- and sex-matched healthy controls were recruited among healthy blood donors in the blood bank, Department of Clinical Immunology, Aarhus University Hospital, Denmark.
antithrombotic therapy [9]. Subgroup analysis revealed that patients under the age of 70 with large hematomas might benefit from hemostatic treatment with rFVIIa [10]. Recently, the Tranexamic acid for hyperacute primary IntraCerebral Hemorrhage (TICH-2 trial) tested tranexamic acid in the largest multi-center randomized trial to date [5]. Hematoma growth was reduced, but mortality and functional outcome at 90 days, measured by modified ranking scale, did not differ between the control and the treatment group. However, there were fewer early deaths by day 7 and fewer adverse effects in the group treated with tranexamic acid. A profound limitation of the trials evaluating hemostatic or antifibrinolytic therapy in ICH is that they pool data from patients receiving anticoagulant or antiplatelet therapy with non-anticoagulated patients. Taken together, these studies indicate that there is a potential for hemostatic or antifibrinolytic treatment in carefully selected patients with either SAH or ICH, and there is a need to investigate non-anticoagulated patients separately. Hemostatic and antifibrinolytic therapy is part of the treatment in major hemorrhage or trauma [11,12]. Fibrinogen concentrate and tranexamic acid now have a key role in the treatment of postpartum hemorrhage [13,14], while prothrombin complex concentrate (PCC) is used in case of hemorrhage due to warfarin treatment [15]. Coagulation factor XIII (FXIII) is clot stabilizing and prevents bleeding in congenital or acquired FXIII deficiency [16], while studies with recombinant soluble thrombomodulin (solulin) are only experimental [17,18]. Solulin may have clot stabilizing potential through activation of thrombin activatable fibrinolysis inhibitor (TAFI) [19]. None of the aforementioned agents is part of the standard treatment of SAH or ICH in the non-anticoagulated patient. By applying a mechanistic approach, it may be possible to reveal the specific way to stabilize hemostasis after SAH or ICH. The first step is to perform ex vivo testing of the medications, in blood from patients with SAH or ICH. Thromboelastometry (ROTEM®) is implemented in many laboratories as a standard tool to evaluate hemostasis in severe trauma and/or major hemorrhage. In previous studies, using the standard ROTEM® assays, we found increased clot strength following both SAH [20] and ICH [21], which is in accordance with others [22,23]. A low tissue factor ROTEM® assay, developed in our laboratory, reveals changes in hemostasis, which are otherwise concealed, when using higher concentrations of tissue factor [24]. Hence, this sensitive assay allows us to further investigate the coagulation in detail following SAH and ICH and to evaluate the effect of the different hemostatic modalities. We aimed to: 1) evaluate coagulation following SAH and ICH using a low tissue factor ROTEM® assay and compare both with healthy controls, 2) evaluate the ex vivo effect of FXIII, fibrinogen concentrate, PCC, and recombinant soluble thrombomodulin in blood from patients with SAH or ICH.
2.2. Laboratory analyses and ex-vivo experiments Blood samples were drawn from an antecubital vein or an arterial cannula. The first tube was discarded. Whole blood for ROTEM® (Instrumentation Laboratory, Bedford, USA) was sampled in 3.5 ml tubes containing sodium citrate 3.2% (Vacuette® Greiner bio-one GmbH, Austria) and the analysis was performed within two hours after blood sampling. The ROTEM® analyses were performed in healthy controls, and in SAH and ICH patients at baseline (without hemostatic agents) and after addition of hemostatic agents at a concentration corresponding to normal or slightly higher dosage used in clinical practice for a 70 kg individual. Prior to ROTEM®-analyses whole blood rested for 30 min at 37 °C. For ROTEM®-analyses of healthy controls and at baseline in SAH and ICH patients, 20 μl buffer with calcium (HEPES 20 nM, NaCl 150 mM, CaCl2 200 mM, pH = 7.4) and 20 μl tissue factor diluted in buffer (HEPES 20 nM, NaCl 150 nM, pH = 7.4) was mixed in the ROTEM®-cup and 300 μl whole blood was added, providing a final dilution of tissue factor (Innovin®) at 1:100,000. For analysis of the effect of hemostatic agents in whole blood from SAH and ICH patients, a mixture of hemostatic agent and tissue factor diluted in buffer (HEPES 20 nM, NaCl 150 nM, pH = 7.4), providing a volume of 20 μl (final dilution of Innovin® at 1:100,000), and 20 μl buffer with calcium (HEPES 20 nM, NaCl 150 mM, CaCl2 200 mM, pH = 7.4) was mixed, and 300 μl whole blood was added. The following hemostatic components were examined separately: FXIII (Cluvot, CSL Behring GmbH, Marburg, Germany) final concentration 0.25 IE/ml (corresponding to a dose of 20 IE/kg body weight); fibrinogen concentrate (Riastap, CSL Behring GmbH, Marburg, Germany) final concentration 0.38 mg/ml (corresponding to a dose of 30 mg/kg body weight); PCC (Octaplex, Octapharma Nordic AB, Roskilde, Denmark) final concentration 0.36 IE/ml (corresponding to a dose of 2000 IE); PCC with added protamine sulphate (Protaminsulfate “Leo Pharma”, Leo Pharma Nordic, Malmö, Sweden) in dosage according to neutralization of the amount of heparin in PCC used in our experiment (1 μl protamine sulphate); soluble thrombomodulin (Solulin, Paion Germany GmbH) final concentration 10 nM. The ROTEM®-analyses were performed in duplicate and the mean coefficient of variation was below 6% for healthy, baseline and all the different medications. The following parameters were registered for data analysis: clotting time, maximum velocity, time to maximum velocity, and maximum clot firmness. Measurements of hemoglobin, platelet count, international normalized ratio (INR), activated partial thromboplastin time (aPTT), fibrinogen, fibrin d-dimer, C-reactive protein, and FXIII were performed as described in previous publications [20,21,26], but because not all patients in the original study were included in this ex vivo study, numbers vary from these publications.
2. Methods 2.1. Study populations The study consisted of a cohort of prospectively included patients enrolled from June 18th 2014 until August 3th 2016. In total, 39 patients with SAH and 35 patients with ICH were enrolled at Department of Neurology and Neurosurgery, Aarhus University Hospital, Denmark. We have previously published other data on these SAH and ICH patients [20,21,25–27]. Blood samples were collected at admission to hospital. In brief, our inclusion criteria were hemorrhage diagnosed by cerebral Computed Tomography (CT) or Magnetic Resonance Imaging (MRI). Exclusion criteria were: age below 18 years, ischemic stroke within the past three months, pregnancy, bleeding disorder, active cancer or chemotherapy within the past three months, liver cirrhosis, current infection, treatment with antithrombotic or antiplatelet drugs, and hemorrhage triggered by arteriovenous malformation, brain tumor or trauma [15,16].
2.3. Statistical analysis The primary outcome was low tissue factor ROTEM® maximum clot firmness in SAH and ICH patients at admission compared with healthy 43
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Fig. 1. Low tissue factor ROTEM® analyses in healthy controls (n = 41) and in patients with subarachnoid hemorrhage (SAH) (n = 39) before (baseline) and after addition of hemostatic agents. Subtext: The box is placed at the median of the distribution. Top and bottom of the box represent 1st and 3rd quartiles of data, while whiskers length is 1.5 inter-quartile distances. Individual dots are outliers as they are beyond 1.5 inter-quartile distance.
3. Results
controls. The sample size calculation was based on data from healthy controls with a mean maximum clot firmness level of 54 mm and a standard deviation (SD) of 4.4 mm. We estimated the SD to be ± 5 mm as we expected it to be higher in SAH and ICH patients than in healthy controls. The minimal relevant difference was chosen to be 4 mm. With a significance level of 5% (2α) and a test power of 90% (1-β), a minimum of 33 patients in each group and 33 healthy controls should be included as a minimum. Data were checked for normal distribution using histograms and QQ-plots. Data are presented as mean ± SD if data followed normal distribution or median with interquartile range (IQR) if data did not follow normal distribution. For testing difference between healthy controls and patients, an unpaired t-test was used if data followed normal distribution and Mann-Whitney test if not. To test the effect of hemostatic agent, the results obtained at baseline versus results after addition of the different hemostatic agents were tested by a paired t-test if data followed normal distribution and by Wilcoxon matched-pairs signed rank test if not. Figs. 1 and 2 present data in a Tukey outlier box plot. The statistical analyses and graphs were performed using Prism 8.0 (GraphPad, La Jolla, CA, USA).
3.1. Characteristics of the study populations A summary of the demographic and clinical characteristics of the patient groups are shown in Table 1. Patients and healthy controls were similar according to age (years, mean ± SD, 59 ± 5 in healthy controls; 58 ± 10 in SAH patients (p = .56); 61 ± 15 in ICH patients (p = .36)). In addition, the sex distribution across groups was similar (females: 66% in healthy controls, 72% in SAH and 64% in ICH patients). Both hemoglobin, platelet count, INR, aPTT, fibrinogen, FXIII, and C-reactive protein were within the reference interval at admission in the study populations. Fibrin d-dimer was slightly increased. The mortality was substantially higher among SAH patients (31%) than among ICH patients (17%). 3.2. Patients compared with healthy controls As shown in Figs. 1 and 2, patients with SAH or ICH demonstrated significantly shorter clotting time, faster maximum velocity, shorter time to maximum velocity, and higher maximum clot firmness than healthy controls (all p-values < .0001). Data are outlined in Table 2. 44
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Fig. 2. Low tissue factor ROTEM® analyses in healthy controls (n = 41) and in patients with intracerebral hemorrhage (n = 36) before (baseline) and after addition of hemostatic agents. Subtext: The box is placed at the median of the distribution. Top and bottom of the box represent 1st and 3rd quartiles of data, while whiskers length is 1.5 inter-quartile distances. Individual dots are outliers as they are beyond 1.5 inter-quartile distance.
physiological initiator of coagulation [28]. Thereby, we reduced the risk of overlooking even subtle effects after addition of hemostatic agents. Together with Ramchand et al. [23], Kawano-Castillo et al. [22] and our previously presented data [20,21], we confirm the presence of hypercoagulation following both SAH and ICH. However, the studies differ in whether clot formation, clot amplification or clot strength is increased. When using a highly sensitive low tissue factor assay, we here find a hypercoagulant state in both SAH and ICH patients, affecting both clot formation, amplification as well as clot strength. Moreover, the present data indicate that hypercoagulation is present on admission, while Ramchand et al. do not find any affected TEG parameters until post-bleed day 3 in SAH patients [23]. In ICH patients, Kawano-Castillo et al. report faster clot formation on admission (within 6 h after ictus), but clot strength is not increased until 36 h after ictus [22]. None of the here investigated hemostatic agents are used in standard treatment of non-anticoagulated patients with SAH or ICH. The present experimental study is the first to investigate the ex vivo effect in blood obtained in the acute phase following SAH or ICH. The rationale
3.3. Ex vivo addition with hemostatic agents None of the hemostatic agents provided any substantial changes in hemostasis after addition to whole blood obtained from patients with SAH of ICH, Figs. 1 and 2. As shown in more detail in Table 2, both fibrinogen concentrate and PCC demonstrated subtle, though significantly, anticoagulant effect. This was indicated by shorter maximum velocity, longer time to maximum velocity, and lower maximum clot firmness than at baseline, Table 2.
4. Discussion Using a highly sensitive ROTEM® assay, the present study demonstrated that both SAH and ICH patients display a systemic hypercoagulable state at admission to hospital. Ex vivo addition of several different hemostatic agents did not have any substantial effect on coagulation in these patients. In the present study, we evaluated the entire coagulation process employing a low concentration of tissue factor, which represents the 45
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Table 1 Demographics and laboratory analyses at admission in 39 patients with subarachnoid hemorrhage and 35 patients with intracerebral hemorrhage (ICH). Data are presented as mean ± standard deviation or median (interquartile range) as appropriate. Variables Demographic data Age, years Females, n (%) Laboratory variables Hemoglobin (mmol/l)a Platelet count (109/l)a Fibrinogen (μmol/l) aPTT (s) before 5 April 2016 aPTT (s) after 5 April 2016 INR Fibrin D-dimer (mg/ml) Factor XIII (IU/l) Fibrinogen (μmol/l) C-reactive protein (mg/l)
Reference interval 7.3–10.5 145–400 5.5–12.0 25–38 20–29 < 1.2 < 0.5 0.61–1.77 5.5–12.0 <8
SAH (n = 39)
ICH (n = 35)
58 ± 10 28 (72%)
61 ± 15 23 (64%)
8.2 ± 0.8 222 ± 56 8.7 ± 1.9b 27 (26;30) (n = 35) 26 (21;27) (n = 4) 1.0 (1.0;1.1)b 1.5 (0.6;5.2)b 1.78 ± 0.38d 8.7 ± 1.9b 1.9 (0.8;3.4)
8.7 ± 1.1c 228 ± 61c 10.4 ± 2.5c 29 (26;32) (n = 27)c 28 (27;30) (n = 6) 1.1 (1.0;1.2)c 1.2 (0.6;1.9)c 1.09 ± 0.27e 10.4 ± 2.5c 3.8 (1.1;10.5)b
Abbreviations: aPTT: Activated partial thromboplastin time; INR: International Normalized Ratio. a For both genders combined. b 1 missing value. c 2 missing values. d 3 missing values. e 6 missing values.
patients. The recombinant soluble thrombomodulin, solulin, is able to increase clot stability through the activation of thrombin-activatable fibrinolysis inhibitor (TAFI) [19]. Solulin has lower affinity to thrombin than full-length thrombomodulin, which reduces its anticoagulant function, but it still adequately promotes TAFI activation and thereby increases clot stability [19]. This may be beneficial in case of hyperfibrinolysis, as hypothesized in SAH patients [31]. However, the ex vivo addition of solulin did not induced any changes in blood obtained from SAH and ICH patients. Both administration of fibrinogen concentrate and PCC demonstrated a subtle anticoagulant effect. As regards to fibrinogen concentrate, this finding might be caused by the fact that it contains citrate in very small concentrations. As regards to PCC all four-factor PCCs contain heparin to bind activated coagulation factors in order to reduce thrombogenicity [32]. Thus, this also applies for the PCC used in the present study. This low content of citrate and heparin implies no clinical significance, but will influence very sensitive coagulation assays.
was the hypothesis that one of these products could hold the potential to reduce hemorrhage enlargement or risk of re-bleeding in patients with SAH or ICH. From a mechanistic point of view, the present results do not support this hypothesis. However, a potential clinical effect of the investigated hemostatic agents at the site of brain injury cannot be ruled out. During the last decade, the use of FXIII-products has attracted increasingly interest. A randomized clinical trial demonstrated a beneficial effect in terms of reduced postoperative bleeding in patients having reduced FXIII levels after coronary surgery [29]. In an observational study by Marti-Fabregas, the baseline FXIII activity was lower, however, non-significantly, in ICH patients experiencing hematoma growth compared to patients not experiencing hematoma growth [30]. In SAH patients, Larsen et al. also found lower FXIII activity at baseline, compared to controls, when blood sampling was performed within six hours after ictus [31]. We find FXIII levels within the reference interval for both SAH and ICH patients and the ex vivo addition of FXIII does not induce any changes in blood obtained from these
Table 2 ROTEM® results obtained by using a low tissue factor assay in healthy controls and before (baseline) and after addition of hemostatic agents in whole blood from 39 patients with subarachnoid hemorrhage (SAH) and 36 patients with intracerebral hemorrhage (ICH). Mean ± standard deviation or median and (interquartile range) are indicated as appropriate. p-Values represent test of differences between healthy and patients with SAH or ICH and between values before (baseline) and after addition of the hemostatic agent.
Healthy (n = 41) Baseline, no additive (vs healthy) After addition of hemostatic agents Factor XIII (vs baseline) Fibrinogen concentrate (vs baseline) PCC + protamine sulphate (vs baseline) Solulin (vs baseline)
CT, s
MaxVel, mm/s
tMaxVel, s
MCF, mm
SAH ICH
644 ± 82 427 ± 67 (p < .0001) 435 ± 87 (p < .0001)
8 ± 2 12 ± 3 (p < .0001) 12 ± 3 (p < .0001)
765 ± 107 510 ± 88 (p < .0001) 520 ± 115 (p < .0001)
54 ± 4 60 ± 5 (p < .0001) 61 ± 5 (p < .0001)
SAH ICH SAH ICH SAH ICH SAH ICH
433 455 445 451 441 472 414 445
11 ± 3a (p = .44) 11 ± 4a (p = .76) 10 ± 3 (p = .0002) 11 ± 4a (p = .0003) 9 (8–12) (p < .0001) 9 (8–11)a (p < .0001 12 ± 3b (p = .48) 12 ± 4c (p = .99)
520 519 528 538 517 558 489 522
± ± ± ± ± ± ± ±
80 (p = .37) 118a (p = .12) 64 (p = .03) 72a (p = .09) 83 (p = .11) 116a (p = .07) 72b (p = .07) 101c (p = .43)
± ± ± ± ± ± ± ±
93a (p = .58) 133a (p = .98) 80 (p = .04) 94a (p = .08) 102 (p = .53) 48a (p = .10) 89b (p = .01) 125c (p = .83)
60 60 59 60 57 58 60 60
± ± ± ± ± ± ± ±
5 (p = .16) 5a (p = .11) 5a (p = .04) 5a (p = .03) 6 (p < .0001) 6a (p < .0001) 5b (p = .16) 5c (p = .28)
Abbreviations: CT: Clotting time; MaxVel: Maximum Velocity; tMaxVel: Time to Maximum Velocity; MCF: Maximum Clot Firmness; PCC: prothrombin complex concentrate; SAH: subarachnoid hemorrhage; ICH: intracerebral hemorrhage. a One missing value. b Two missing values. c Three missing values. 46
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Thus, the present results indicate that we did not add sufficient protamine sulphate in order to neutralize the heparin.
[8]
4.1. Limitations and strengths [9]
The present study is limited by the fact that it includes only ex vivo experiments with changes in ROTEM® profiles as the endpoint. We spiked blood samples obtained in the acute phase following SAH or ICH aiming at concentrations a little higher than the one used in clinical practice; however, this does not necessarily reflect the clinical effect. Neither, did we measure the amount of tissue factor after addition of this. The study was strengthened by the fact that we included an ageand sex- matched healthy control group using the exactly same lot of tissue factor for the sensitive ROTEM® assay. In conclusion, this present study confirms a hypercoagulant state few hours after symptom onset in patients with SAH or ICH measured by a sensitive low tissue factor ROTEM® assay. The study suggests that administration of hemostatic agents will not improve systemic coagulation in these patients, but whether it could reduce the damage at the site of brain injury remains undetermined.
[10]
[11]
[12]
[13]
[14]
[15]
Acknowledgements [16]
We thank Mai Stenulm Veirup and Vivi Bo Mogensen for assistance in titration experiments and design of the ex vivo spiking experiments. We thank doctors, nurses and healthcare personnel at the Department of Neurology and the Department of Neurosurgery, Danish Stroke Centre at Aarhus University Hospital, Denmark, for their cooperation and assistance during enrolment procedures. We thank CLS Behring and Octapharma for financial research support and providing. FXIII concentrate (CSL Behring), fibrinogen concentrate (CSL Behring) and prothrombin complex concentrate (Octapharma), and PAION, Germany, for providing solulin for the ex-vivo studies. CSL Behring, Octapharma, and PAION had no influence on the design, analysis or interpretation of the results, or on the drafting of the article. The project was generously funded by Aarhus University, The Lippman Foundation, the Director Emil C. Hertz and Hustru Inger Hertz's Foundation, the Doctor Sofus Carl Emil Friis & Wife Olga Doris Friis Foundation, the Aase & Ejnar Danielsen's Foundation, the Director Werner Richter & Wife Foundation, the Danish Society of Anaesthesiology and Intensive Care Foundation (DASAIM), the Holger & Ruth Hesse's Memorial Foundation, the Lily Benthine Lund's Foundation of 1.6.1978, The Letterstedtske Foundation, King Christian the X's Foundation, CSL Behring and Octapharma.
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Full Length Article
Ex vivo effect of hemostatic therapy in subarachnoid and intracerebral hemorrhage
T
Christine Lodberg Hvasa, Signe Voigt Lauridsena, Emilie Sandgaard Pedersenb, Tua Gyldenholmb, ⁎ Anne-Mette Hvasb,c, a
Department of Anaesthesiology and Intensive Care, Aarhus University Hospital; Aarhus, Denmark Thrombosis and Hemostasis Research Unit, Department of Clinical Biochemistry; Aarhus University Hospital, Aarhus, Denmark c Department of Clinical Medicine, Aarhus University; Aarhus, Denmark b
A R T I C LE I N FO
A B S T R A C T
Keywords: Blood coagulation Subarachnoid hemorrhage Intracerebral hemorrhage Ex vivo Hemostatic agents
Background: Rebleeding and hematoma growth are serious complications in subarachnoid hemorrhage (SAH) and intracerebral hemorrhage (ICH). As treatment options are sparse, a mechanistic approach may reveal new therapeutic targets. Aim: Firstly, to evaluate hemostasis using a sensitive low tissue factor thromboelastometry (ROTEM®) assay in patients with SAH or ICH and compare them with healthy controls. Secondly, to investigate the ex vivo effect of hemostatic or antifibrinolytic medications in blood from patients with SAH or ICH. Methods: Blood was drawn on admission to hospital in patients with SAH (n = 39) or ICH (n = 35). We included 41 sex and age matched healthy controls for comparison. A low tissue factor (diluted 1:100,000) ROTEM® assay was run in patients and healthy controls. In parallel, coagulation factor XIII, fibrinogen concentrate, prothrombin complex concentrate, and recombinant soluble thrombomodulin were added in concentrations equivalent to doses used in clinical practice. Results: Patients with SAH or ICH demonstrated a hypercoagulable profile indicated by significantly shorter clotting time, faster maximum velocity, shorter time to maximum velocity, and higher maximum clot firmness than healthy controls (all p-values < .0001). Ex vivo addition of coagulation factor XIII, fibrinogen concentrate, prothrombin complex concentrate, and recombinant soluble thrombomodulin, respectively, did not improve the hemostatic potential in patients with SAH or ICH. Conclusion: Patients with SAH or ICH demonstrated a hypercoagulable state in the systemic circulation as evaluated by a sensitive low tissue factor assay. Ex vivo addition of hemostatic medication did not further improve coagulation.
1. Introduction Hematoma growth and rebleeding are serious complications in subarachnoid hemorrhage (SAH) and intracerebral hemorrhage (ICH) affecting both morbidity and mortality [1,2]. Currently, a combination of surgery and blood pressure control are the mainstay of treatment. Hemostatic and antifibrinolytic therapy have been tested in randomized trials in both SAH and ICH, but results have pointed in different directions [3–6].
In SAH, much focus has been on tranexamic acid. However, European guidelines and the most recent Cochrane review, do not advice routine use of tranexamic acid in SAH [4,7]. Tranexamic acid reduces the risk of rebleeding in the acute phase, but does not affect mortality, possibly due to an increase in ischemic events in the studies, where long-term treatment is applied (> 72 h), or due to lack of power [8]. In ICH, the focus regarding improved hemostasis has primarily been on recombinant coagulation factor VIIa (rFVIIa) [6] and reversal of
Abbreviations: aPTT, activated partial thromboplastin time; CT, clotting time; FXIII, coagulation factor XIII; ICH, intracerebral hemorrhage; INR, international normalized ratio; IQR, interquartile range; MCF, maximum clot firmness; MaxVel, maximum velocity; PCC, prothrombin complex concentrate; rFVIIa, recombinant coagulation factor VIIa; ROTEM®, thromboelastometry; SAH, subarachnoid hemorrhage; SD, standard deviation; TAFI, thrombin activatable fibrinolysis inhibitor; tMaxVel, time to maximum velocity ⁎ Corresponding author at: Thrombosis and Hemostasis Research Unit, Department of Clinical Biochemistry, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark. E-mail address: [email protected] (A.-M. Hvas). https://doi.org/10.1016/j.thromres.2020.02.012 Received 24 November 2019; Received in revised form 25 January 2020; Accepted 14 February 2020 Available online 26 February 2020 0049-3848/ © 2020 Published by Elsevier Ltd.
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The Regional Ethics Committee of Central Denmark approved the study (incompetent patients case no.: 1-10-72-95-14, version 3, 05052014 and competent patients case no.: 1-10-72-94-14, version 4, 27042014). All patients or their legal proxies provided written informed consent before study enrolment. The Danish Data Protection Agency also approved the study (incompetent patients case no.: 1-1602-225-14 and competent patients case no.: 1-16-02-224-14). The Helsinki Declaration was followed in all aspects. In total 41 age- and sex-matched healthy controls were recruited among healthy blood donors in the blood bank, Department of Clinical Immunology, Aarhus University Hospital, Denmark.
antithrombotic therapy [9]. Subgroup analysis revealed that patients under the age of 70 with large hematomas might benefit from hemostatic treatment with rFVIIa [10]. Recently, the Tranexamic acid for hyperacute primary IntraCerebral Hemorrhage (TICH-2 trial) tested tranexamic acid in the largest multi-center randomized trial to date [5]. Hematoma growth was reduced, but mortality and functional outcome at 90 days, measured by modified ranking scale, did not differ between the control and the treatment group. However, there were fewer early deaths by day 7 and fewer adverse effects in the group treated with tranexamic acid. A profound limitation of the trials evaluating hemostatic or antifibrinolytic therapy in ICH is that they pool data from patients receiving anticoagulant or antiplatelet therapy with non-anticoagulated patients. Taken together, these studies indicate that there is a potential for hemostatic or antifibrinolytic treatment in carefully selected patients with either SAH or ICH, and there is a need to investigate non-anticoagulated patients separately. Hemostatic and antifibrinolytic therapy is part of the treatment in major hemorrhage or trauma [11,12]. Fibrinogen concentrate and tranexamic acid now have a key role in the treatment of postpartum hemorrhage [13,14], while prothrombin complex concentrate (PCC) is used in case of hemorrhage due to warfarin treatment [15]. Coagulation factor XIII (FXIII) is clot stabilizing and prevents bleeding in congenital or acquired FXIII deficiency [16], while studies with recombinant soluble thrombomodulin (solulin) are only experimental [17,18]. Solulin may have clot stabilizing potential through activation of thrombin activatable fibrinolysis inhibitor (TAFI) [19]. None of the aforementioned agents is part of the standard treatment of SAH or ICH in the non-anticoagulated patient. By applying a mechanistic approach, it may be possible to reveal the specific way to stabilize hemostasis after SAH or ICH. The first step is to perform ex vivo testing of the medications, in blood from patients with SAH or ICH. Thromboelastometry (ROTEM®) is implemented in many laboratories as a standard tool to evaluate hemostasis in severe trauma and/or major hemorrhage. In previous studies, using the standard ROTEM® assays, we found increased clot strength following both SAH [20] and ICH [21], which is in accordance with others [22,23]. A low tissue factor ROTEM® assay, developed in our laboratory, reveals changes in hemostasis, which are otherwise concealed, when using higher concentrations of tissue factor [24]. Hence, this sensitive assay allows us to further investigate the coagulation in detail following SAH and ICH and to evaluate the effect of the different hemostatic modalities. We aimed to: 1) evaluate coagulation following SAH and ICH using a low tissue factor ROTEM® assay and compare both with healthy controls, 2) evaluate the ex vivo effect of FXIII, fibrinogen concentrate, PCC, and recombinant soluble thrombomodulin in blood from patients with SAH or ICH.
2.2. Laboratory analyses and ex-vivo experiments Blood samples were drawn from an antecubital vein or an arterial cannula. The first tube was discarded. Whole blood for ROTEM® (Instrumentation Laboratory, Bedford, USA) was sampled in 3.5 ml tubes containing sodium citrate 3.2% (Vacuette® Greiner bio-one GmbH, Austria) and the analysis was performed within two hours after blood sampling. The ROTEM® analyses were performed in healthy controls, and in SAH and ICH patients at baseline (without hemostatic agents) and after addition of hemostatic agents at a concentration corresponding to normal or slightly higher dosage used in clinical practice for a 70 kg individual. Prior to ROTEM®-analyses whole blood rested for 30 min at 37 °C. For ROTEM®-analyses of healthy controls and at baseline in SAH and ICH patients, 20 μl buffer with calcium (HEPES 20 nM, NaCl 150 mM, CaCl2 200 mM, pH = 7.4) and 20 μl tissue factor diluted in buffer (HEPES 20 nM, NaCl 150 nM, pH = 7.4) was mixed in the ROTEM®-cup and 300 μl whole blood was added, providing a final dilution of tissue factor (Innovin®) at 1:100,000. For analysis of the effect of hemostatic agents in whole blood from SAH and ICH patients, a mixture of hemostatic agent and tissue factor diluted in buffer (HEPES 20 nM, NaCl 150 nM, pH = 7.4), providing a volume of 20 μl (final dilution of Innovin® at 1:100,000), and 20 μl buffer with calcium (HEPES 20 nM, NaCl 150 mM, CaCl2 200 mM, pH = 7.4) was mixed, and 300 μl whole blood was added. The following hemostatic components were examined separately: FXIII (Cluvot, CSL Behring GmbH, Marburg, Germany) final concentration 0.25 IE/ml (corresponding to a dose of 20 IE/kg body weight); fibrinogen concentrate (Riastap, CSL Behring GmbH, Marburg, Germany) final concentration 0.38 mg/ml (corresponding to a dose of 30 mg/kg body weight); PCC (Octaplex, Octapharma Nordic AB, Roskilde, Denmark) final concentration 0.36 IE/ml (corresponding to a dose of 2000 IE); PCC with added protamine sulphate (Protaminsulfate “Leo Pharma”, Leo Pharma Nordic, Malmö, Sweden) in dosage according to neutralization of the amount of heparin in PCC used in our experiment (1 μl protamine sulphate); soluble thrombomodulin (Solulin, Paion Germany GmbH) final concentration 10 nM. The ROTEM®-analyses were performed in duplicate and the mean coefficient of variation was below 6% for healthy, baseline and all the different medications. The following parameters were registered for data analysis: clotting time, maximum velocity, time to maximum velocity, and maximum clot firmness. Measurements of hemoglobin, platelet count, international normalized ratio (INR), activated partial thromboplastin time (aPTT), fibrinogen, fibrin d-dimer, C-reactive protein, and FXIII were performed as described in previous publications [20,21,26], but because not all patients in the original study were included in this ex vivo study, numbers vary from these publications.
2. Methods 2.1. Study populations The study consisted of a cohort of prospectively included patients enrolled from June 18th 2014 until August 3th 2016. In total, 39 patients with SAH and 35 patients with ICH were enrolled at Department of Neurology and Neurosurgery, Aarhus University Hospital, Denmark. We have previously published other data on these SAH and ICH patients [20,21,25–27]. Blood samples were collected at admission to hospital. In brief, our inclusion criteria were hemorrhage diagnosed by cerebral Computed Tomography (CT) or Magnetic Resonance Imaging (MRI). Exclusion criteria were: age below 18 years, ischemic stroke within the past three months, pregnancy, bleeding disorder, active cancer or chemotherapy within the past three months, liver cirrhosis, current infection, treatment with antithrombotic or antiplatelet drugs, and hemorrhage triggered by arteriovenous malformation, brain tumor or trauma [15,16].
2.3. Statistical analysis The primary outcome was low tissue factor ROTEM® maximum clot firmness in SAH and ICH patients at admission compared with healthy 43
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Fig. 1. Low tissue factor ROTEM® analyses in healthy controls (n = 41) and in patients with subarachnoid hemorrhage (SAH) (n = 39) before (baseline) and after addition of hemostatic agents. Subtext: The box is placed at the median of the distribution. Top and bottom of the box represent 1st and 3rd quartiles of data, while whiskers length is 1.5 inter-quartile distances. Individual dots are outliers as they are beyond 1.5 inter-quartile distance.
3. Results
controls. The sample size calculation was based on data from healthy controls with a mean maximum clot firmness level of 54 mm and a standard deviation (SD) of 4.4 mm. We estimated the SD to be ± 5 mm as we expected it to be higher in SAH and ICH patients than in healthy controls. The minimal relevant difference was chosen to be 4 mm. With a significance level of 5% (2α) and a test power of 90% (1-β), a minimum of 33 patients in each group and 33 healthy controls should be included as a minimum. Data were checked for normal distribution using histograms and QQ-plots. Data are presented as mean ± SD if data followed normal distribution or median with interquartile range (IQR) if data did not follow normal distribution. For testing difference between healthy controls and patients, an unpaired t-test was used if data followed normal distribution and Mann-Whitney test if not. To test the effect of hemostatic agent, the results obtained at baseline versus results after addition of the different hemostatic agents were tested by a paired t-test if data followed normal distribution and by Wilcoxon matched-pairs signed rank test if not. Figs. 1 and 2 present data in a Tukey outlier box plot. The statistical analyses and graphs were performed using Prism 8.0 (GraphPad, La Jolla, CA, USA).
3.1. Characteristics of the study populations A summary of the demographic and clinical characteristics of the patient groups are shown in Table 1. Patients and healthy controls were similar according to age (years, mean ± SD, 59 ± 5 in healthy controls; 58 ± 10 in SAH patients (p = .56); 61 ± 15 in ICH patients (p = .36)). In addition, the sex distribution across groups was similar (females: 66% in healthy controls, 72% in SAH and 64% in ICH patients). Both hemoglobin, platelet count, INR, aPTT, fibrinogen, FXIII, and C-reactive protein were within the reference interval at admission in the study populations. Fibrin d-dimer was slightly increased. The mortality was substantially higher among SAH patients (31%) than among ICH patients (17%). 3.2. Patients compared with healthy controls As shown in Figs. 1 and 2, patients with SAH or ICH demonstrated significantly shorter clotting time, faster maximum velocity, shorter time to maximum velocity, and higher maximum clot firmness than healthy controls (all p-values < .0001). Data are outlined in Table 2. 44
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Fig. 2. Low tissue factor ROTEM® analyses in healthy controls (n = 41) and in patients with intracerebral hemorrhage (n = 36) before (baseline) and after addition of hemostatic agents. Subtext: The box is placed at the median of the distribution. Top and bottom of the box represent 1st and 3rd quartiles of data, while whiskers length is 1.5 inter-quartile distances. Individual dots are outliers as they are beyond 1.5 inter-quartile distance.
physiological initiator of coagulation [28]. Thereby, we reduced the risk of overlooking even subtle effects after addition of hemostatic agents. Together with Ramchand et al. [23], Kawano-Castillo et al. [22] and our previously presented data [20,21], we confirm the presence of hypercoagulation following both SAH and ICH. However, the studies differ in whether clot formation, clot amplification or clot strength is increased. When using a highly sensitive low tissue factor assay, we here find a hypercoagulant state in both SAH and ICH patients, affecting both clot formation, amplification as well as clot strength. Moreover, the present data indicate that hypercoagulation is present on admission, while Ramchand et al. do not find any affected TEG parameters until post-bleed day 3 in SAH patients [23]. In ICH patients, Kawano-Castillo et al. report faster clot formation on admission (within 6 h after ictus), but clot strength is not increased until 36 h after ictus [22]. None of the here investigated hemostatic agents are used in standard treatment of non-anticoagulated patients with SAH or ICH. The present experimental study is the first to investigate the ex vivo effect in blood obtained in the acute phase following SAH or ICH. The rationale
3.3. Ex vivo addition with hemostatic agents None of the hemostatic agents provided any substantial changes in hemostasis after addition to whole blood obtained from patients with SAH of ICH, Figs. 1 and 2. As shown in more detail in Table 2, both fibrinogen concentrate and PCC demonstrated subtle, though significantly, anticoagulant effect. This was indicated by shorter maximum velocity, longer time to maximum velocity, and lower maximum clot firmness than at baseline, Table 2.
4. Discussion Using a highly sensitive ROTEM® assay, the present study demonstrated that both SAH and ICH patients display a systemic hypercoagulable state at admission to hospital. Ex vivo addition of several different hemostatic agents did not have any substantial effect on coagulation in these patients. In the present study, we evaluated the entire coagulation process employing a low concentration of tissue factor, which represents the 45
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Table 1 Demographics and laboratory analyses at admission in 39 patients with subarachnoid hemorrhage and 35 patients with intracerebral hemorrhage (ICH). Data are presented as mean ± standard deviation or median (interquartile range) as appropriate. Variables Demographic data Age, years Females, n (%) Laboratory variables Hemoglobin (mmol/l)a Platelet count (109/l)a Fibrinogen (μmol/l) aPTT (s) before 5 April 2016 aPTT (s) after 5 April 2016 INR Fibrin D-dimer (mg/ml) Factor XIII (IU/l) Fibrinogen (μmol/l) C-reactive protein (mg/l)
Reference interval 7.3–10.5 145–400 5.5–12.0 25–38 20–29 < 1.2 < 0.5 0.61–1.77 5.5–12.0 <8
SAH (n = 39)
ICH (n = 35)
58 ± 10 28 (72%)
61 ± 15 23 (64%)
8.2 ± 0.8 222 ± 56 8.7 ± 1.9b 27 (26;30) (n = 35) 26 (21;27) (n = 4) 1.0 (1.0;1.1)b 1.5 (0.6;5.2)b 1.78 ± 0.38d 8.7 ± 1.9b 1.9 (0.8;3.4)
8.7 ± 1.1c 228 ± 61c 10.4 ± 2.5c 29 (26;32) (n = 27)c 28 (27;30) (n = 6) 1.1 (1.0;1.2)c 1.2 (0.6;1.9)c 1.09 ± 0.27e 10.4 ± 2.5c 3.8 (1.1;10.5)b
Abbreviations: aPTT: Activated partial thromboplastin time; INR: International Normalized Ratio. a For both genders combined. b 1 missing value. c 2 missing values. d 3 missing values. e 6 missing values.
patients. The recombinant soluble thrombomodulin, solulin, is able to increase clot stability through the activation of thrombin-activatable fibrinolysis inhibitor (TAFI) [19]. Solulin has lower affinity to thrombin than full-length thrombomodulin, which reduces its anticoagulant function, but it still adequately promotes TAFI activation and thereby increases clot stability [19]. This may be beneficial in case of hyperfibrinolysis, as hypothesized in SAH patients [31]. However, the ex vivo addition of solulin did not induced any changes in blood obtained from SAH and ICH patients. Both administration of fibrinogen concentrate and PCC demonstrated a subtle anticoagulant effect. As regards to fibrinogen concentrate, this finding might be caused by the fact that it contains citrate in very small concentrations. As regards to PCC all four-factor PCCs contain heparin to bind activated coagulation factors in order to reduce thrombogenicity [32]. Thus, this also applies for the PCC used in the present study. This low content of citrate and heparin implies no clinical significance, but will influence very sensitive coagulation assays.
was the hypothesis that one of these products could hold the potential to reduce hemorrhage enlargement or risk of re-bleeding in patients with SAH or ICH. From a mechanistic point of view, the present results do not support this hypothesis. However, a potential clinical effect of the investigated hemostatic agents at the site of brain injury cannot be ruled out. During the last decade, the use of FXIII-products has attracted increasingly interest. A randomized clinical trial demonstrated a beneficial effect in terms of reduced postoperative bleeding in patients having reduced FXIII levels after coronary surgery [29]. In an observational study by Marti-Fabregas, the baseline FXIII activity was lower, however, non-significantly, in ICH patients experiencing hematoma growth compared to patients not experiencing hematoma growth [30]. In SAH patients, Larsen et al. also found lower FXIII activity at baseline, compared to controls, when blood sampling was performed within six hours after ictus [31]. We find FXIII levels within the reference interval for both SAH and ICH patients and the ex vivo addition of FXIII does not induce any changes in blood obtained from these
Table 2 ROTEM® results obtained by using a low tissue factor assay in healthy controls and before (baseline) and after addition of hemostatic agents in whole blood from 39 patients with subarachnoid hemorrhage (SAH) and 36 patients with intracerebral hemorrhage (ICH). Mean ± standard deviation or median and (interquartile range) are indicated as appropriate. p-Values represent test of differences between healthy and patients with SAH or ICH and between values before (baseline) and after addition of the hemostatic agent.
Healthy (n = 41) Baseline, no additive (vs healthy) After addition of hemostatic agents Factor XIII (vs baseline) Fibrinogen concentrate (vs baseline) PCC + protamine sulphate (vs baseline) Solulin (vs baseline)
CT, s
MaxVel, mm/s
tMaxVel, s
MCF, mm
SAH ICH
644 ± 82 427 ± 67 (p < .0001) 435 ± 87 (p < .0001)
8 ± 2 12 ± 3 (p < .0001) 12 ± 3 (p < .0001)
765 ± 107 510 ± 88 (p < .0001) 520 ± 115 (p < .0001)
54 ± 4 60 ± 5 (p < .0001) 61 ± 5 (p < .0001)
SAH ICH SAH ICH SAH ICH SAH ICH
433 455 445 451 441 472 414 445
11 ± 3a (p = .44) 11 ± 4a (p = .76) 10 ± 3 (p = .0002) 11 ± 4a (p = .0003) 9 (8–12) (p < .0001) 9 (8–11)a (p < .0001 12 ± 3b (p = .48) 12 ± 4c (p = .99)
520 519 528 538 517 558 489 522
± ± ± ± ± ± ± ±
80 (p = .37) 118a (p = .12) 64 (p = .03) 72a (p = .09) 83 (p = .11) 116a (p = .07) 72b (p = .07) 101c (p = .43)
± ± ± ± ± ± ± ±
93a (p = .58) 133a (p = .98) 80 (p = .04) 94a (p = .08) 102 (p = .53) 48a (p = .10) 89b (p = .01) 125c (p = .83)
60 60 59 60 57 58 60 60
± ± ± ± ± ± ± ±
5 (p = .16) 5a (p = .11) 5a (p = .04) 5a (p = .03) 6 (p < .0001) 6a (p < .0001) 5b (p = .16) 5c (p = .28)
Abbreviations: CT: Clotting time; MaxVel: Maximum Velocity; tMaxVel: Time to Maximum Velocity; MCF: Maximum Clot Firmness; PCC: prothrombin complex concentrate; SAH: subarachnoid hemorrhage; ICH: intracerebral hemorrhage. a One missing value. b Two missing values. c Three missing values. 46
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Thus, the present results indicate that we did not add sufficient protamine sulphate in order to neutralize the heparin.
[8]
4.1. Limitations and strengths [9]
The present study is limited by the fact that it includes only ex vivo experiments with changes in ROTEM® profiles as the endpoint. We spiked blood samples obtained in the acute phase following SAH or ICH aiming at concentrations a little higher than the one used in clinical practice; however, this does not necessarily reflect the clinical effect. Neither, did we measure the amount of tissue factor after addition of this. The study was strengthened by the fact that we included an ageand sex- matched healthy control group using the exactly same lot of tissue factor for the sensitive ROTEM® assay. In conclusion, this present study confirms a hypercoagulant state few hours after symptom onset in patients with SAH or ICH measured by a sensitive low tissue factor ROTEM® assay. The study suggests that administration of hemostatic agents will not improve systemic coagulation in these patients, but whether it could reduce the damage at the site of brain injury remains undetermined.
[10]
[11]
[12]
[13]
[14]
[15]
Acknowledgements [16]
We thank Mai Stenulm Veirup and Vivi Bo Mogensen for assistance in titration experiments and design of the ex vivo spiking experiments. We thank doctors, nurses and healthcare personnel at the Department of Neurology and the Department of Neurosurgery, Danish Stroke Centre at Aarhus University Hospital, Denmark, for their cooperation and assistance during enrolment procedures. We thank CLS Behring and Octapharma for financial research support and providing. FXIII concentrate (CSL Behring), fibrinogen concentrate (CSL Behring) and prothrombin complex concentrate (Octapharma), and PAION, Germany, for providing solulin for the ex-vivo studies. CSL Behring, Octapharma, and PAION had no influence on the design, analysis or interpretation of the results, or on the drafting of the article. The project was generously funded by Aarhus University, The Lippman Foundation, the Director Emil C. Hertz and Hustru Inger Hertz's Foundation, the Doctor Sofus Carl Emil Friis & Wife Olga Doris Friis Foundation, the Aase & Ejnar Danielsen's Foundation, the Director Werner Richter & Wife Foundation, the Danish Society of Anaesthesiology and Intensive Care Foundation (DASAIM), the Holger & Ruth Hesse's Memorial Foundation, the Lily Benthine Lund's Foundation of 1.6.1978, The Letterstedtske Foundation, King Christian the X's Foundation, CSL Behring and Octapharma.
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