Efficacy of Fibrinogen Concentrate Compared With Cryoprecipitate for Reversal of the Antiplatelet Effect of Clopidogrel in an In Vitro Model, as Assessed by Multiple Electrode Platelet Aggregometry, Thromboelastometry, and Modified Thromboelastography Bruce Lloyd Cartwright, MBBS, FANZCA, PGDipEcho, PTEeXAM, Peter Kam, MBBS, FANZCA, and Kenny Yang, BSc Objectives: The management of dual-antiplatelet therapy when patients present for surgical revascularization is a clinical challenge. Whether increasing fibrinogen levels can restore hemostasis in this context is not established but may represent increased platelet glycoprotein fibrinogen binding, altered adenosine diphosphate (ADP)-dependent platelet activation, or an increase in formation of soluble fibrin as a component of whole blood clot. Design: The study hypothesis was that fibrinogen concentrate would normalize in vitro hemostatic parameters after clopidogrel loading. The effect was compared with cryoprecipitate. Setting: University Hospital Participants: Elective coronary catheter studies Interventions: Assessment of platelet aggregation was made using whole blood platelet impedance. Viscoelastic assessment also was made using whole blood rotational thromboelastometry and modified thromboelastography. Twenty patients presenting for cardiac catheterization on dual-antiplatelet therapy were studied. Whole blood was titrated with increasing amounts of cryoprecipitate and
fibrinogen concentrate. Samples then were diluted 40% with normal saline and further titrated. Measurement and Main Results: The principal finding of the study was that fibrinogen supplementation primarily improved assays of fibrin formation. Improvement in platelet aggregation response to ADP and TRAP was not observed. Neither cryoprecipitate nor fibrinogen concentrate, at the concentrations used, were able to improve the amplitude at 30 minutes (A30) in the modified TEG-ADP assay. Furthermore, they produced comparable amplitudes at 30 minutes despite a twofold difference in fibrinogen supplementation. Conclusions: Fibrinogen supplementation may play a role in the hemostatic resuscitation of patients on dual-antiplatelet therapy, but there is no evidence in this in vitro study that there is a specific platelet effect involved that would allow for platelet substitution. Crown Copyright & 2015 Published by Elsevier Inc. All rights reserved.
T
Finally, reversal of thienopyridine effect with strategies directed at increasing vonWillebrand factor (vWf), such as cryoprecipitate or desmopressin infusion,13 may be associated with thromboembolic cardiovascular events as vWf plays a central role in the pathogenesis of myocardial infarction.14 Recently, Schöchl et al reported the hemostatic utility of high-dose fibrinogen concentrate for a trauma patient on dualantiplatelet therapy,15 and it has been shown that fibrinogen supplementation can reverse glycoprotein IIb/IIIa inhibition.16 Furthermore, investigation of “clopidogrel resistance” by Ang et al found that elevated plasma fibrinogen level was associated with lower inhibition of platelet reactivity when measured by the VerifyNow ADP assay.17 However, they only demonstrated this association with diabetes and elevated body mass index, but importantly, indicators of inflammation (ie, the acute phase response) were absent. Whether fibrinogen concentrate can improve hemostasis in the context of dual-antiplatelet therapy has not been explored in isolation. Fibrinogen is a known component of the process of ADP-induced platelet aggregation18 and also may have a key role in acquired bleeding associated with thrombocytopenia.19 The authors decided to investigate the mechanisms by which this may occur, using patients presenting for cardiac catheterization in an in vitro model as a surrogate for patients presenting for surgical revascularization and to compare this to the effects of addition of cryoprecipitate, their current source of fibrinogen supplementation. In particular, the authors were interested in delineating whether (1) the mechanism of restoring hemostasis was related to adenosine diphosphate (ADP)-dependent platelet activation, (2) if it was related to fibrinogen-binding sites on the GP IIb/
HE INTRODUCTION of dual-antiplatelet therapy, commonly aspirin and the thienopyridine clopidogrel, has resulted in increased bleeding, transfusion, and reoperation in cardiac surgery.1,2 Different approaches to this clinical challenge include allowing the drug effect to wear off, platelet transfusion, cryoprecipitate transfusion, or desmopressin infusion; however, the best strategy and transfusion endpoint is yet to be established. Although timing of surgery in relation to drug cessation can be improved by assessment of platelet function,3–5 there are potential hazards associated with drug withdrawal; in particular, ischemic events especially in patients awaiting urgent surgical revascularization after failed percutaneous coronary intervention.6,7 The major alternative, platelet concentrate transfusion, is not without risk and is associated with complications such as febrile and allergic transfusion reactions, alloimmunization, and transfusion-related acute lung injury.8,9 Additionally, platelet transfusion readily reverses the beneficial effect of aspirin and is associated with cost.10–12
From the Department of Anaesthesia, University of Sydney, Royal Prince Alfred Hospital, Camperdown, Sydney, Australia. Address reprint requests to Bruce Lloyd Cartwright, MBBS, FANZCA, PGDipEcho, PTEeXAM, Department of Anaesthesia, University of Sydney, Royal Prince Alfred Hospital, Level 4, Building 87, Missenden Road, Camperdown, Sydney, New South Wales, NSW 2050 Australia. E-mail: [email protected] Crown Copyright © 2015 Published by Elsevier Inc. All rights reserved. 1053-0770/2601-0001$36.00/0 http://dx.doi.org/10.1053/j.jvca.2014.12.010 694
KEY WORDS: fibrinogen concentrate, clopidogrel, hemostasis, cardiac surgery
cryoprecipitate,
Journal of Cardiothoracic and Vascular Anesthesia, Vol 29, No 3 (June), 2015: pp 694–702
EFFICACY OF FIBRINOGEN CONCENTRATE
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IIIa complex by the surrogate of thrombin receptor activating peptide-6 (TRAP)-dependent platelet activation, or (3) whether it primarily represented an increase in the formation of the soluble fibrin as a component of whole blood clot. Because Li et al found that approximately 2,400 mg of fibrinogen were required to restore platelet aggregation to at least 50% of normal, 5 and 10 units of cryoprecipitate were chosen for titration, because this represents, on average, 1,250 mg and 2,500 mg of fibrinogen supplementation, which were the clinical doses used at the authors’ institution.16 This would represent 18 mg/kg and 35 mg/kg of fibrinogen, respectively, for the average 70-kg patient and be comparable to the 2-gm dose administered in the case report of von Heymann et al, in which fibrinogen concentrate was used in addition to allogeneic products to reverse refractory clopidogrel-associated bleeding.20 Furthermore, because the 85-kg male in the case report of Schochl et al had received a total of 12 g of fibrinogen concentrate without additional blood component therapy for complete hemostasis, 50 mg/kg and 100 mg/kg were chosen initially as titration amounts.15 METHODS
The study was approved by the Ethics Review Committee of the Sydney Local Health District as protocol X-12-0312 and HREC 12/RPAH/476, and written consent was obtained from all subjects. The study hypothesis was that fibrinogen concentrate (RiaStap, CSL Behring, Australia) would normalize in vitro hemostatic parameters after clopidogrel loading. The effect of 50 mg/kg and 100 mg/kg body-weight doses was compared with the response to 5- and 10-unit doses of cryoprecipitate. Subsequently, samples were diluted 40% with normal saline, to simulate the approximate level of hemodilution that occurs with institution of cardiopulmonary bypass, and a further titration of the equivalent of 10 units of cryoprecipitate and 100 mg/kg of fibrinogen concentrate was made (Fig 1). Assessment of platelet aggregation response to ADP and TRAP was made using the Multiplate (Dynabyte, Munich, Germany) whole blood platelet impedance aggregometer with area under the curve (AUC) reported as aggregation units (U, previously AU*min), which is representative of total aggregation and velocity of reaction. Viscoelastic assessment was made using whole blood rotational thromboelastometry (ROTEM, TEM International) using the ExTEM assay, which uses tissue factor activation to assess whole blood clot formation and fibrin polymerization (recalcification þ tissue factor), and the FibTEM assay, which adds cytochalasin-D to assess fibrin formation in the absence of platelet activation (ie, recalcification þ tissue factor þ cytochalasin-D). Modified thromboelastography ADP-Platelet Mapping (Hemonetics, Australia) also was performed. This test involves addition of reptilase to produce an activated assay (tissue factor þ reptilase þ XIIIa), representing formation of primarily fibrinopeptide A-based fibrin monomer and an ADP-activated assay (tissue factor þ reptilase þ XIIIa þ10μL ADP), resulting in the compound of activated fibrin monomer and platelets responsive to ADP. The citrated sample was used for the
Fig 1. Method flow chart. Abbreviations: ADP, adenosine diphosphate; TRAP, thrombin receptor-activating peptide-6; ROTEM, rotational elastometry. (Color version of figure is available online.)
kaolin-activated baseline, recalcified according to the manufacturer’s method (20 μL of 0.2 M CaCl2 added to each 340-μL sample) and the sodium heparin tube for activated and ADP platelet mapping. Initially, 5 units of cryoprecipitate were analyzed (RPAH, Sydney South West Pathology Service, Sydney, Australia) to determine the average amount of fibrinogen in mg/mL (Clauss method) and von-Willebrand factor in IU (Latex antigen method) available in each unit.
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Thereafter, 20 patients presenting electively for cardiac catheterization who previously were established on dual-antiplatelet therapy, aspirin, and clopidogrel and who had a documented adenosine diphosphate (ADP) response on multiple electrode platelet aggregometry (ADPtest) o40 U were studied. Because of the variability in clopidogrel responsiveness, 29 patients were recruited; 9 patients did not reach the predetermined threshold for ADP response, and their blood samples were not used for analysis. Patients were excluded from the study if they were on other medications known to influence platelet function, if there was a history or family history of bleeding disorder, such as hemophilia or von Willebrand disease, if they were taking an oral anticoagulant, and if they were anemic, defined as o100 g/L for females and o110 g/L for males. A full blood count and coagulation studies consisting of internationalized ratio, activated partial thromboplastin time (aPTT), and fibrinogen level (Clauss method) were performed as well to ensure absence of co-existing coagulopathy. Blood was drawn after placement of a femoral arterial catheter (with an initial discard of 4 mL) and was placed into a single hirudin sodium tube (double-walled, vacuum, 15 μg/mL recombinant hirudin, Dynabyte), 6 citrate tubes (3.2% sodium citrate, Becton, Dickinson & Co, New South Wales, Australia) and a sodium heparin tube (Haemonetics) for analysis. The baseline platelet function of the hirudin sample was measured using the Multiplate ADPtest (20 μL of 0.2-mM adenosine diphosphate) to ensure clopidogrel response, together with assessment of aspirin response with the ASPItest (20 μL of 15-mM arachidonic acid) and an assessment to ensure normal glycoprotein IIb/IIIa activity with the TRAPtest (20 μL of 1-mM thrombin receptor-activating peptide-6). An assessment of platelet function then was conducted on the citrated sample with the Multiplate ADPtest and TRAPtest, which was recalcified 1:1 with Na-CaCl diluent in place of normal saline. During this evaluation, a unit of cryoprecipitate was thawed and analyzed for fibrinogen and vWf (RPAH, Sydney South West Pathology Service) and a calculation made to standardize a single unit of cryoprecipitate as a volume of 30 mL, 250 mg of fibrinogen, and approximately 180 IU of vWf. Thereafter, whole blood was titrated with the equivalent of 5 and 10 units of cryoprecipitate (1,250 mg and 2,500 mg of fibrinogen, respectively) and the equivalent of 50 mg/kg and 100 mg/kg of fibrinogen concentrate. The patient blood volume was calculated using Nadler’s formula.21 The sample volume was adjusted to account for change in blood volume that would have resulted from the volume expansion by cryoprecipitate (ie, 150 and 300 mL, respectively) and addition of fibrinogen concentrate (RiaStap, CSL Behring, Australia), which has a reconstituted volume of 50 mL per 1,000 mg of fibrinogen (average fibrinogen content of 900-1,300 mg, together with 400-700 mg of albumin, 375-660 mg of Larginine, and 50-100 mg of sodium citrate). Samples were incubated at 37oC for 15 minutes, during which time they were gently inverted twice. The samples then were analyzed using the Multiplate ADPtest and TRAPtest to assess platelet function and by Rotational Elastometry (ROTEM, TEM International).
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Subsequently, samples were diluted 40% with normal saline and further titrated with the equivalent of 10 units of cryoprecipitate and 100 mg/kg of fibrinogen concentrate using the method described above. The samples were incubated at 37 degrees for 15 minutes and gently inverted twice. These samples then were analyzed using the Multiplate ADPtest and TRAPtest and by Rotational Elastometry using the ExTEM and the FibTEM assays. The diluted samples additionally had assessment with an ADP platelet mapping assay (modified thromboelastography, Hemonetics, Australia). All analyses were performed 5 minutes after incubation, and the total number of titrations chosen such that a complete patient study could be completed within 2 hours. This was because of concerns about the degradation of aggregation response present after 2 hours when assessing platelet function with viscoelastic assays and the Multiplate Analyser.22,23 STATISTICAL ANALYSIS
Power and Sample Size Calculations In a previous study of subjects taking clopidogrel, the mean ADP platelet aggregation was 28 U with a standard deviation of 17 U.24 Based on these numbers, a sample size of 65 patients per group (total number of 130) would provide 80% power with a two-sided alpha of 0.05 to detect an absolute 10-U increase in platelet aggregation for patients who will receive fibrinogen concentrate. This calculation was based on a t test of platelet aggregation (using logtransformation). However, in a study using the related technology of light transmission aggregometry to assess normalization of platelet function after platelet transfusion to offset clopidogrel effect, only 11 patients were required because of the greater absolute increase required in aggregation for normalization.25 Because studies have documented equivalency in clopidogrel response between light transmission aggregometry and the Multiplate, it was expected that 20 patients would be required to document an increase in ADP agonist aggregometry from a mean of 28 U, to above the threshold of 31 U reported in a study using the Multiplate to assess bleeding risk in cardiac surgery patients receiving thienopyridines.26,27 This also, theoretically, would match with the desire to balance hemostasis by offsetting bleeding risk without unduly increasing the risk of thrombotic events.28 Data Handling Data analysis was performed using MedCalc Statistical software for Windows 7, version 9.6 (MedCalc Software, Ostend, Belgium). As data distribution was not normal, the Mann-Whitney U test was used to test the significance of difference between individual samples – data available as Appendix 1. Correlation of non-parametric data was analyzed using Spearman’s coefficient. RESULTS
The initial cryoprecipitate analysis showed that the average amount of fibrinogen found in each unit of approximately 30 mL was 274 mg, and the approximate vWf activity was 164 IU (100% ¼ 1 IU/mL) (Table 1). The median baseline fibrinogen
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EFFICACY OF FIBRINOGEN CONCENTRATE
Table 1. Cryoprecipitate Analysis Thus, Average 30 mL of Cryoprecipitate Contains Sample Fibrinogen g/L vWf %
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
9.9 5.6 9.6 7.5 13.1 7.1 7.8 8.9 10.0 11.1 12.2 13.3 14.4 15.6 16.7
650 512 423 750 392 665 867 1047 601 993 739 756 824 660 850
Fibrinogen mg
vWf (IU)
297 168 289 226 392 213 233 267 300 333 367 400 433 467 500
195 154 127 225 118 200 260 314 180 298 222 227 247 198 255
Abbreviations: vWf, vonWillebrand factor.
level (Clauss method) was 3.8 g/L and ranged from 2.5 to 7.2 g/L. Platelet Aggregation: Multiplate Whole Blood Impedance Aggregometry
reduced the ADPtest aggregation (p ¼ 0.0004), and the addition of fibrinogen concentrate at 100 mg/kg likewise reduced aggregation but to a lesser degree (p ¼ 0.0172). A similar effect was observed with the TRAPtest aggregation as shown in Figure 3, with impaired aggregation occurring in vitro with the addition of cryoprecipitate and fibrinogen. Viscoelastic Assessment: Rotational Thromboelastometry and Modified Thromboelastography Rotational Thromboelastometry: ExTEM Here the dilutional effect of normal saline was partially reversed (p ¼ 0.022) with the addition of 100 mg/kg of fibrinogen concentrate, but an addition of 10 units of cryoprecipitate resulted in a nonsignificant change (p ¼ 0.1632) in the A30 parameter (Fig 4). Likewise, with rate of clot formation, the dilutional effect of 40% normal saline was reversed by the addition of 10 units of cryoprecipitate and the addition of 100 mg/kg of fibrinogen concentrate generated a significantly supranormal response (p o 0.001) (data not shown). Rotational Thromboelastometry: FibTEM The results were consistent across both whole blood and diluted blood titration. The addition of cryoprecipitate had little impact until the blood was diluted and, thereafter, partially restored the induced abnormality; whereas the addition of fibrinogen concentrate produced a significantly (p o 0.05) supranormal response in a dose-dependent fashion (Fig 5). Comparable results were found in clot formation rate (data not shown).
Titration of whole and diluted blood with cryoprecipitate and fibrinogen concentrate and the effects on the ADPtest platelet aggregation are shown in Figure 2. Addition of cryoprecipitate and of fibrinogen concentrate reduced aggregation in the ADPtest results. By increasing the concentration of each, the effect of sample dilution was decreased but aggregation was still reduced overall. The effect was proportional to baseline level of aggregation. All results were significantly different (p o 0.05) except addition of 10 U of cryoprecipitate and 50 mg/kg of fibrinogen concentrate, which were comparable. Aggregation was further reduced when the sample was diluted 40% with normal saline. The addition of cryoprecipitate
Clot elasticity calculated as MCE ¼ (A30*100)/(100-A30) for the ExTEM and FibTEM assays allows derivation of the platelet elasticity component by subtraction of the fibrinogen component (ExTEM-MCE – FibTEM-MCE). Here the addition of cryoprecipitate and fibrinogen showed effects comparable to hemodilution (Fig 6).
Fig 2. Multiplate ADPtest: Titration. Abbreviations: ADP, adenosine diphosphate; AUC, area under the curve.
Fig 3. Multiplate TRAPtest: Diluted blood titration. Abbreviations: TRAP, thrombin receptor-activating peptide-6; AUC, area under the curve.
Rotational Thromboelastometry: Derived Platelet Elasticity Component
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Fig 4.
ROTEM ExTEM A30 diluted blood titration.
Modified Thromboelastography ADP-Activated Assay The ADP-activated assay (tissue factor þ reptilase þ XIIIa þ10 μL ADP) produces a compound of activated fibrin monomer and platelets responsive to ADP. The amplitude at 30 minutes (A30) represents the total amount of platelet-fibrinogen interaction, the angle measures rate of clot formation and elasticity is derived as the G function, ie, G ¼ (5000*MA)/(100-MA). Here addition of cryoprecipitate produced no significant changes in all parameters although the trend was toward reduction in A30 (near-total substrate) and Gf; whereas addition of fibrinogen concentrate was able to significantly improve the rate of clot formation, represented by clot angle without significant changes in A30 and G. Data are shown in Figure 7. Full data analysis is shown in Appendix 1. DISCUSSION
The principal finding of the study was that fibrinogen supplementation primarily improved assays of fibrin formation. Improvement in platelet aggregation response to ADP and TRAP was not observed.
Fig 5.
ROTEM FibTEM A30 titration.
Fig 6.
ROTEM-derived platelet elasticity component.
Fibrinogen supplementation at 100 mg/kg reduced platelet aggregation on the ADPtest less than cryoprecipitate or hemodilution but no therapy improved aggregation. A similar response was shown on the TRAPtest and derived ROTEM platelet elasticity assays, which is in contrast to the findings of Li et al, which found stepwise improvement in response to fibrinogen supplementation.16 The authors suggest that this is because small molecule glycoprotein IIb/IIIa receptor antagonists are “competitive” inhibitors, whereas clopidogrel binds in a “noncompetitive” fashion to the P2 receptor. Fibrinogen supplementation also resulted in supranormal fibrin polymerization in whole blood and restoration of fibrin polymerization between 50 mg/kg and 100 mg/kg in diluted blood as shown by the ExTEM and FibTEM assays. It also significantly improved the rate of clot formation, represented by the angle in the modified TEG-ADP assay. In contrast, neither cryoprecipitate nor fibrinogen concentrate, at the concentrations used, were able to improve the amplitude at 30 minutes in the modified TEG-ADP assay, beyond the threshold value used in the Target-CABG trial to delineate bleeding risk with clopidogrel.5
Fig 7. Modified TEG (ADP) angle and A30 titration. Abbreviations: TEG, ADP, adenosine diphosphate.
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EFFICACY OF FIBRINOGEN CONCENTRATE
Furthermore, addition of cryoprecipitate and fibrinogen concentrate produced comparable amplitudes on the TEGADP assay at 30 minutes, despite a twofold difference in fibrinogen supplementation. This presumably reflects an additional effect of cryoprecipitate on platelet-fibrinogen interaction from fibronectin or factor XIII. Whether the improvement in clot formation rate demonstrated is sufficient (ie, the ability to form sufficient amplitude of clot within a set time period) by the addition of fibrinogen concentrate alone in the context of platelets impaired by clopidogrel remains to be established. The improvement in fibrin polymerization seen would be significant in the context of cardiopulmonary bypass in which fibrin formation is impaired to a greater extent than thrombin generation or platelet activity.29 The threshold amount of ADP-activatable platelets required was not established by this study. A major limitation with this study was that only changes in in vitro assays were shown. Also, because of citrate being present in cryoprecipitate and fibrinogen concentrate, citrate tubes were used primarily for analysis and there are complex effects on platelet aggegation, which vary among hirudin, citrate, and sodium heparin.30 The lack of improvement in
platelet aggregation could have reflected this, issues of adequate recalcification, the lack of shear forces in the assays used, or be related to the dose of cryoprecipitate and fibrinogen concentrate used. Furthermore, in vitro assay results do not always reflect in vivo hemostasis. For example, the amplitude of clot formation in the ExTEM assay demonstrates primarily thrombin-mediated platelet interaction with fibrinogen, masking the effect of ADP-mediated platelet dysfunction. Additionally, in vitro titration assays in healthy patients may not reflect the response in patients bleeding clinically. CONCLUSIONS
Fibrinogen supplementation may play a role in the hemostatic resuscitation of patients on dual antiplatelet therapy, but there was no evidence in this in vitro study that there was a specific ADP-related platelet effect involved that would allow for platelet substitution. APPENDIX A
See below for Table A1
REFERENCES 1. Mahla E, Metzler H, Tantry US, et al: Controversies in oral antiplatelet therapy in patients undergoing aortocoronary bypass surgery. Ann Thorac Surg 90:1040-1051, 2010 2. Morici N, Moja L, Rosato V, et al: Time from adenosine diphosphate receptor antagonist discontinuation to coronary bypass surgery in patients with acute coronary syndrome: Meta-analysis and meta-regression. Int J Cardiol 168:1955-1964, 2013 3. Gurbel PA, Mahla E, Tantry US: Peri-operative platelet function testing: The potential for reducing ischaemic and bleeding risks. Thromb Haemost 106:248-252, 2011 4. Di Dedda U, Ranucci M, Baryshnikova E, et al: Thienopyridines resistance and recovery of platelet function after discontinuation of thienopyridines in cardiac surgery patients. Eur J Cardiothorac Surg 45: 165-170, 2013 5. Mahla E, Suarez TA, Bliden KP, et al: Platelet function measurement-based strategy to reduce bleeding and waiting time in clopidogrel-treated patients undergoing coronary artery bypass graft surgery the timing based on platelet function strategy to reduce clopidogrel-associated bleeding related to cabg (target-cabg) study. Circ Cardiovasc Interv 5:261-269, 2012 6. Sibbing D, Stegherr J, Braun S, et al: A double-blind, randomized study on prevention and existence of a rebound phenomenon of platelets after cessation of clopidogrel treatment. J Am Coll Cardiol 55: 558-565, 2010 7. Sibbing D, Schulz S, Braun S, et al: Antiplatelet effects of clopidogrel and bleeding in patients undergoing coronary stent placement. J Thromb Haemost 8:250-256, 2010 8. Spiess BD: Platelet transfusions: The science behind safety, risks and appropriate applications. Best Pract Res Clin Anaesthesiol 24:65-83, 2010 9. Koch C, Li L, Figueroa P, et al: Transfusion and pulmonary morbidity after cardiac surgery. Ann Thorac Surg 88:1410-1418, 2009 10. Bachelani AM, Bautz JT, Sperry JL, et al: Assessment of platelet transfusion for reversal of aspirin after traumatic brain injury. Surgery 150:836-843, 2011 11. Gao C, Ren C, Li D, et al: Clopidogrel and aspirin versus clopidogrel alone on graft patency after coronary artery bypass grafting. Ann Thorac Surg 88:59-62, 2009
12. Bielby L, Hunt R, Roxby D, et al: Australian Red Cross. Available at: http://resources.transfusion.com.au/cdm/ref/collection/p16691coll1/id/58. 13. Leithäuser B, Zielske D, Seyfert UT, et al: Effects of desmopressin on platelet membrane glycoproteins and platelet aggregation in volunteers on clopidogrel. Clin Hemorheol Microcirc 39:293-302, 2008 14. Spiel AO, Gilbert JC, Jilma B: von Willebrand factor in cardiovascular disease: focus on acute coronary syndromes. Circulation 117:1449-1459, 2008 15. Schöchl H, Posch A, Hanke A, et al: High-dose fibrinogen concentrate for haemostatic therapy of a major trauma patient with recent clopidogrel and aspirin intake. Scand J Clin Lab Invest 70:453-457, 2010 16. Li YF, Spencer FA, Becker RC: Comparative efficacy of fibrinogen and platelet supplementation on the in vitro reversibility of competitive glycoprotein IIb/IIIa (αIIb/β3) receptor-directed platelet inhibition. Am Heart J 142:204-210, 2001 17. Ang L, Palakodeti V, Khalid A, et al: Elevated plasma fibrinogen and diabetes mellitus are associated with lower inhibition of platelet reactivity with clopidogrel. J Am Coll Cardiol 52:1052-1059, 2008 18. Marguerie GA, Edgington TS, Plow EF: Interaction of fibrinogen with its platelet receptor as part of a multistep reaction in ADP-induced platelet aggregation. J Biol Chem 255:154-161, 1980 19. Velik-Salchner C, Haas T, Innerhofer P, et al: The effect of fibrinogen concentrate on thrombocytopenia. J Thromb Haemost 5: 1019-1025, 2007 20. Heymann von C, Spies C, Grubitzsch H, et al: Bleeding after cardiac surgery: The role of recombinant factor VIIa [article in German]. Hamostaseologie 26(suppl):S77-S87, 2006 (suppl) 21. Nadler SB, Hidalgo JH, Bloch T: Prediction of blood volume in normal human adults. Surgery 51:224-232, 1962 22. Johnston LR, Larsen PD, La Flamme AC, et al: Methodological considerations for the assessment of ADP induced platelet aggregation using the Multiplates analyser. Platelets 24:303-307, 2013 23. Zambruni A, Thalheimer U, Leandro G, et al: Thromboelastography with citrated blood: comparability with native blood, stability of citrate storage and effect of repeated sampling. Blood Coagul Fibrinolysis 15:103-107, 2004
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24. Diehl P, Olivier C, Halscheid C, et al: Clopidogrel affects leukocyte dependent platelet aggregation by P2Y12 expressing leukocytes. Basic Res Cardiol 105:379-387, 2009 25. Vilahur G, Choi BG, Zafar MU, et al: Normalization of platelet reactivity in clopidogrel-treated subjects. J Thromb Haemost 5:82-90, 2007 26. Velik-Salchner C, Maier S, Innerhofer P, et al: Point-of-care whole blood impedance aggregometry versus classical light transmission aggregometry for detecting aspirin and clopidogrel: The results of a pilot study. Anesth Analg 107:1798-1806, 2008 27. Ranucci M, Baryshnikova E, Soro G, et al: Multiple electrode whole-blood aggregometry and bleeding in cardiac
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surgery patients receiving thienopyridines. Ann Thorac Surg 91: 123-129, 2011 28. Tantry US, Gurbel PA: Assessment of oral antithrombotic therapy by platelet function testing. Nat Rev Cardiol 8:572-579, 2011 29. Solomon C, Rahe-Meyer N, Sørensen B: Fibrin formation is more impaired than thrombin generation and platelets immediately following cardiac surgery. Thromb Res 128:277-282, 2011 30. Kaiser AF, Neubauer H, Franken CC, et al: Which is the best anticoagulant for whole blood aggregometry platelet function testing? Comparison of six anticoagulants and diverse storage conditions. Platelets 23:359-367, 2011
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Table A1. Significance Table: Two-Tailed Probability (p value): Mann-Whitney U 1. Whole Blood ADPtest titration
þ5 U cryoprecipitate (cryo) þ10 U cryo þ50 mg/kg fibrinogen concentrate (FC) þ100 mg/kg fibrinogen (FC)
Whole blood
þ5 U cryo
þ10 U cryo
þ50 mg/kg FC
0.0024 0.0118 0.0159 0.1013
0.1437 0.1401 0.0294
0.7046 0.2387
0.424
2. Diluted 40% ADPtest titration
40% dilution 40% dilution þ10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ10 U cryo
0.0514 0.0001 0.0002
0.0004 0.0581
0.0172
3. Diluted 40% TRAPtest titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
0.0179 o0.0001 o0.0001
0.0001 0.0004
0.2976
4. Diluted 40% ExTEM A30 titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
o0.0001 o0.0001 0.0006
0.1632 0.022
0.0007
5. Diluted 40% ExTEM Alpha titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
0.0007 0.0716 o0.0001
0.1129 o0.0001
o0.0001
6. Whole Blood FibTEM A30 titration
þ5 U cryo þ10 U cryo þ50 mg/kg fibrinogen (FC) þ100 mg/kg fibrinogen (FC)
Whole
þ5 U cryo
þ10 U cryo
þ50 mg/kg FC
0.9459 0.8709 0.0086 0.0002
0.9892 0.0047 0.0001
0.0105 0.005
0.074
6a Diluted 40% FibTEM A30 titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
o0.0001 0.0009 0.0002
0.0029 o0.0001
o0.0001
7. Whole Blood FibTEM Alpha titration
þ5 U cryo þ10 U cryo þ50 mg/kg fibrinogen (FC) þ100 mg/kg fibrinogen (FC)
Whole
þ5 U cryo
þ10 U cryo
þ50 mg/kg FC
0.2317 0.4647 0.0067 0.0054
0.8388 0.0463 0.013
0.0367 0.0076
0.4562
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Table A1 (continued ) 7a. Diluted 40% FibTEM Alpha titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
0.009 0.1836 o0.0001
0.0009 o0.0001
o0.0001
8. Derived Platelet MCE – titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
o0.0001 o0.0001 o0.0001
0.0373 0.8392
0.0565
9. Modified TEG – ADP – A30 titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
0.7573 0.1237 0.2482
0.1489 0.3123
0.6744
10. Modified TEG – ADP – Angle titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
0.1449 0.9616 0.0269
0.3603 0.3606
40% dilution þ 10 U cryo
0.0587
11. Modified TEG – ADP – G titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
0.8946 0.3123 0.5317
0.1489 0.312
0.6744
fibrinogen concentrate. Samples then were diluted 40% with normal saline and further titrated. Measurement and Main Results: The principal finding of the study was that fibrinogen supplementation primarily improved assays of fibrin formation. Improvement in platelet aggregation response to ADP and TRAP was not observed. Neither cryoprecipitate nor fibrinogen concentrate, at the concentrations used, were able to improve the amplitude at 30 minutes (A30) in the modified TEG-ADP assay. Furthermore, they produced comparable amplitudes at 30 minutes despite a twofold difference in fibrinogen supplementation. Conclusions: Fibrinogen supplementation may play a role in the hemostatic resuscitation of patients on dual-antiplatelet therapy, but there is no evidence in this in vitro study that there is a specific platelet effect involved that would allow for platelet substitution. Crown Copyright & 2015 Published by Elsevier Inc. All rights reserved.
T
Finally, reversal of thienopyridine effect with strategies directed at increasing vonWillebrand factor (vWf), such as cryoprecipitate or desmopressin infusion,13 may be associated with thromboembolic cardiovascular events as vWf plays a central role in the pathogenesis of myocardial infarction.14 Recently, Schöchl et al reported the hemostatic utility of high-dose fibrinogen concentrate for a trauma patient on dualantiplatelet therapy,15 and it has been shown that fibrinogen supplementation can reverse glycoprotein IIb/IIIa inhibition.16 Furthermore, investigation of “clopidogrel resistance” by Ang et al found that elevated plasma fibrinogen level was associated with lower inhibition of platelet reactivity when measured by the VerifyNow ADP assay.17 However, they only demonstrated this association with diabetes and elevated body mass index, but importantly, indicators of inflammation (ie, the acute phase response) were absent. Whether fibrinogen concentrate can improve hemostasis in the context of dual-antiplatelet therapy has not been explored in isolation. Fibrinogen is a known component of the process of ADP-induced platelet aggregation18 and also may have a key role in acquired bleeding associated with thrombocytopenia.19 The authors decided to investigate the mechanisms by which this may occur, using patients presenting for cardiac catheterization in an in vitro model as a surrogate for patients presenting for surgical revascularization and to compare this to the effects of addition of cryoprecipitate, their current source of fibrinogen supplementation. In particular, the authors were interested in delineating whether (1) the mechanism of restoring hemostasis was related to adenosine diphosphate (ADP)-dependent platelet activation, (2) if it was related to fibrinogen-binding sites on the GP IIb/
HE INTRODUCTION of dual-antiplatelet therapy, commonly aspirin and the thienopyridine clopidogrel, has resulted in increased bleeding, transfusion, and reoperation in cardiac surgery.1,2 Different approaches to this clinical challenge include allowing the drug effect to wear off, platelet transfusion, cryoprecipitate transfusion, or desmopressin infusion; however, the best strategy and transfusion endpoint is yet to be established. Although timing of surgery in relation to drug cessation can be improved by assessment of platelet function,3–5 there are potential hazards associated with drug withdrawal; in particular, ischemic events especially in patients awaiting urgent surgical revascularization after failed percutaneous coronary intervention.6,7 The major alternative, platelet concentrate transfusion, is not without risk and is associated with complications such as febrile and allergic transfusion reactions, alloimmunization, and transfusion-related acute lung injury.8,9 Additionally, platelet transfusion readily reverses the beneficial effect of aspirin and is associated with cost.10–12
From the Department of Anaesthesia, University of Sydney, Royal Prince Alfred Hospital, Camperdown, Sydney, Australia. Address reprint requests to Bruce Lloyd Cartwright, MBBS, FANZCA, PGDipEcho, PTEeXAM, Department of Anaesthesia, University of Sydney, Royal Prince Alfred Hospital, Level 4, Building 87, Missenden Road, Camperdown, Sydney, New South Wales, NSW 2050 Australia. E-mail: [email protected] Crown Copyright © 2015 Published by Elsevier Inc. All rights reserved. 1053-0770/2601-0001$36.00/0 http://dx.doi.org/10.1053/j.jvca.2014.12.010 694
KEY WORDS: fibrinogen concentrate, clopidogrel, hemostasis, cardiac surgery
cryoprecipitate,
Journal of Cardiothoracic and Vascular Anesthesia, Vol 29, No 3 (June), 2015: pp 694–702
EFFICACY OF FIBRINOGEN CONCENTRATE
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IIIa complex by the surrogate of thrombin receptor activating peptide-6 (TRAP)-dependent platelet activation, or (3) whether it primarily represented an increase in the formation of the soluble fibrin as a component of whole blood clot. Because Li et al found that approximately 2,400 mg of fibrinogen were required to restore platelet aggregation to at least 50% of normal, 5 and 10 units of cryoprecipitate were chosen for titration, because this represents, on average, 1,250 mg and 2,500 mg of fibrinogen supplementation, which were the clinical doses used at the authors’ institution.16 This would represent 18 mg/kg and 35 mg/kg of fibrinogen, respectively, for the average 70-kg patient and be comparable to the 2-gm dose administered in the case report of von Heymann et al, in which fibrinogen concentrate was used in addition to allogeneic products to reverse refractory clopidogrel-associated bleeding.20 Furthermore, because the 85-kg male in the case report of Schochl et al had received a total of 12 g of fibrinogen concentrate without additional blood component therapy for complete hemostasis, 50 mg/kg and 100 mg/kg were chosen initially as titration amounts.15 METHODS
The study was approved by the Ethics Review Committee of the Sydney Local Health District as protocol X-12-0312 and HREC 12/RPAH/476, and written consent was obtained from all subjects. The study hypothesis was that fibrinogen concentrate (RiaStap, CSL Behring, Australia) would normalize in vitro hemostatic parameters after clopidogrel loading. The effect of 50 mg/kg and 100 mg/kg body-weight doses was compared with the response to 5- and 10-unit doses of cryoprecipitate. Subsequently, samples were diluted 40% with normal saline, to simulate the approximate level of hemodilution that occurs with institution of cardiopulmonary bypass, and a further titration of the equivalent of 10 units of cryoprecipitate and 100 mg/kg of fibrinogen concentrate was made (Fig 1). Assessment of platelet aggregation response to ADP and TRAP was made using the Multiplate (Dynabyte, Munich, Germany) whole blood platelet impedance aggregometer with area under the curve (AUC) reported as aggregation units (U, previously AU*min), which is representative of total aggregation and velocity of reaction. Viscoelastic assessment was made using whole blood rotational thromboelastometry (ROTEM, TEM International) using the ExTEM assay, which uses tissue factor activation to assess whole blood clot formation and fibrin polymerization (recalcification þ tissue factor), and the FibTEM assay, which adds cytochalasin-D to assess fibrin formation in the absence of platelet activation (ie, recalcification þ tissue factor þ cytochalasin-D). Modified thromboelastography ADP-Platelet Mapping (Hemonetics, Australia) also was performed. This test involves addition of reptilase to produce an activated assay (tissue factor þ reptilase þ XIIIa), representing formation of primarily fibrinopeptide A-based fibrin monomer and an ADP-activated assay (tissue factor þ reptilase þ XIIIa þ10μL ADP), resulting in the compound of activated fibrin monomer and platelets responsive to ADP. The citrated sample was used for the
Fig 1. Method flow chart. Abbreviations: ADP, adenosine diphosphate; TRAP, thrombin receptor-activating peptide-6; ROTEM, rotational elastometry. (Color version of figure is available online.)
kaolin-activated baseline, recalcified according to the manufacturer’s method (20 μL of 0.2 M CaCl2 added to each 340-μL sample) and the sodium heparin tube for activated and ADP platelet mapping. Initially, 5 units of cryoprecipitate were analyzed (RPAH, Sydney South West Pathology Service, Sydney, Australia) to determine the average amount of fibrinogen in mg/mL (Clauss method) and von-Willebrand factor in IU (Latex antigen method) available in each unit.
696
Thereafter, 20 patients presenting electively for cardiac catheterization who previously were established on dual-antiplatelet therapy, aspirin, and clopidogrel and who had a documented adenosine diphosphate (ADP) response on multiple electrode platelet aggregometry (ADPtest) o40 U were studied. Because of the variability in clopidogrel responsiveness, 29 patients were recruited; 9 patients did not reach the predetermined threshold for ADP response, and their blood samples were not used for analysis. Patients were excluded from the study if they were on other medications known to influence platelet function, if there was a history or family history of bleeding disorder, such as hemophilia or von Willebrand disease, if they were taking an oral anticoagulant, and if they were anemic, defined as o100 g/L for females and o110 g/L for males. A full blood count and coagulation studies consisting of internationalized ratio, activated partial thromboplastin time (aPTT), and fibrinogen level (Clauss method) were performed as well to ensure absence of co-existing coagulopathy. Blood was drawn after placement of a femoral arterial catheter (with an initial discard of 4 mL) and was placed into a single hirudin sodium tube (double-walled, vacuum, 15 μg/mL recombinant hirudin, Dynabyte), 6 citrate tubes (3.2% sodium citrate, Becton, Dickinson & Co, New South Wales, Australia) and a sodium heparin tube (Haemonetics) for analysis. The baseline platelet function of the hirudin sample was measured using the Multiplate ADPtest (20 μL of 0.2-mM adenosine diphosphate) to ensure clopidogrel response, together with assessment of aspirin response with the ASPItest (20 μL of 15-mM arachidonic acid) and an assessment to ensure normal glycoprotein IIb/IIIa activity with the TRAPtest (20 μL of 1-mM thrombin receptor-activating peptide-6). An assessment of platelet function then was conducted on the citrated sample with the Multiplate ADPtest and TRAPtest, which was recalcified 1:1 with Na-CaCl diluent in place of normal saline. During this evaluation, a unit of cryoprecipitate was thawed and analyzed for fibrinogen and vWf (RPAH, Sydney South West Pathology Service) and a calculation made to standardize a single unit of cryoprecipitate as a volume of 30 mL, 250 mg of fibrinogen, and approximately 180 IU of vWf. Thereafter, whole blood was titrated with the equivalent of 5 and 10 units of cryoprecipitate (1,250 mg and 2,500 mg of fibrinogen, respectively) and the equivalent of 50 mg/kg and 100 mg/kg of fibrinogen concentrate. The patient blood volume was calculated using Nadler’s formula.21 The sample volume was adjusted to account for change in blood volume that would have resulted from the volume expansion by cryoprecipitate (ie, 150 and 300 mL, respectively) and addition of fibrinogen concentrate (RiaStap, CSL Behring, Australia), which has a reconstituted volume of 50 mL per 1,000 mg of fibrinogen (average fibrinogen content of 900-1,300 mg, together with 400-700 mg of albumin, 375-660 mg of Larginine, and 50-100 mg of sodium citrate). Samples were incubated at 37oC for 15 minutes, during which time they were gently inverted twice. The samples then were analyzed using the Multiplate ADPtest and TRAPtest to assess platelet function and by Rotational Elastometry (ROTEM, TEM International).
CARTWRIGHT ET AL
Subsequently, samples were diluted 40% with normal saline and further titrated with the equivalent of 10 units of cryoprecipitate and 100 mg/kg of fibrinogen concentrate using the method described above. The samples were incubated at 37 degrees for 15 minutes and gently inverted twice. These samples then were analyzed using the Multiplate ADPtest and TRAPtest and by Rotational Elastometry using the ExTEM and the FibTEM assays. The diluted samples additionally had assessment with an ADP platelet mapping assay (modified thromboelastography, Hemonetics, Australia). All analyses were performed 5 minutes after incubation, and the total number of titrations chosen such that a complete patient study could be completed within 2 hours. This was because of concerns about the degradation of aggregation response present after 2 hours when assessing platelet function with viscoelastic assays and the Multiplate Analyser.22,23 STATISTICAL ANALYSIS
Power and Sample Size Calculations In a previous study of subjects taking clopidogrel, the mean ADP platelet aggregation was 28 U with a standard deviation of 17 U.24 Based on these numbers, a sample size of 65 patients per group (total number of 130) would provide 80% power with a two-sided alpha of 0.05 to detect an absolute 10-U increase in platelet aggregation for patients who will receive fibrinogen concentrate. This calculation was based on a t test of platelet aggregation (using logtransformation). However, in a study using the related technology of light transmission aggregometry to assess normalization of platelet function after platelet transfusion to offset clopidogrel effect, only 11 patients were required because of the greater absolute increase required in aggregation for normalization.25 Because studies have documented equivalency in clopidogrel response between light transmission aggregometry and the Multiplate, it was expected that 20 patients would be required to document an increase in ADP agonist aggregometry from a mean of 28 U, to above the threshold of 31 U reported in a study using the Multiplate to assess bleeding risk in cardiac surgery patients receiving thienopyridines.26,27 This also, theoretically, would match with the desire to balance hemostasis by offsetting bleeding risk without unduly increasing the risk of thrombotic events.28 Data Handling Data analysis was performed using MedCalc Statistical software for Windows 7, version 9.6 (MedCalc Software, Ostend, Belgium). As data distribution was not normal, the Mann-Whitney U test was used to test the significance of difference between individual samples – data available as Appendix 1. Correlation of non-parametric data was analyzed using Spearman’s coefficient. RESULTS
The initial cryoprecipitate analysis showed that the average amount of fibrinogen found in each unit of approximately 30 mL was 274 mg, and the approximate vWf activity was 164 IU (100% ¼ 1 IU/mL) (Table 1). The median baseline fibrinogen
697
EFFICACY OF FIBRINOGEN CONCENTRATE
Table 1. Cryoprecipitate Analysis Thus, Average 30 mL of Cryoprecipitate Contains Sample Fibrinogen g/L vWf %
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
9.9 5.6 9.6 7.5 13.1 7.1 7.8 8.9 10.0 11.1 12.2 13.3 14.4 15.6 16.7
650 512 423 750 392 665 867 1047 601 993 739 756 824 660 850
Fibrinogen mg
vWf (IU)
297 168 289 226 392 213 233 267 300 333 367 400 433 467 500
195 154 127 225 118 200 260 314 180 298 222 227 247 198 255
Abbreviations: vWf, vonWillebrand factor.
level (Clauss method) was 3.8 g/L and ranged from 2.5 to 7.2 g/L. Platelet Aggregation: Multiplate Whole Blood Impedance Aggregometry
reduced the ADPtest aggregation (p ¼ 0.0004), and the addition of fibrinogen concentrate at 100 mg/kg likewise reduced aggregation but to a lesser degree (p ¼ 0.0172). A similar effect was observed with the TRAPtest aggregation as shown in Figure 3, with impaired aggregation occurring in vitro with the addition of cryoprecipitate and fibrinogen. Viscoelastic Assessment: Rotational Thromboelastometry and Modified Thromboelastography Rotational Thromboelastometry: ExTEM Here the dilutional effect of normal saline was partially reversed (p ¼ 0.022) with the addition of 100 mg/kg of fibrinogen concentrate, but an addition of 10 units of cryoprecipitate resulted in a nonsignificant change (p ¼ 0.1632) in the A30 parameter (Fig 4). Likewise, with rate of clot formation, the dilutional effect of 40% normal saline was reversed by the addition of 10 units of cryoprecipitate and the addition of 100 mg/kg of fibrinogen concentrate generated a significantly supranormal response (p o 0.001) (data not shown). Rotational Thromboelastometry: FibTEM The results were consistent across both whole blood and diluted blood titration. The addition of cryoprecipitate had little impact until the blood was diluted and, thereafter, partially restored the induced abnormality; whereas the addition of fibrinogen concentrate produced a significantly (p o 0.05) supranormal response in a dose-dependent fashion (Fig 5). Comparable results were found in clot formation rate (data not shown).
Titration of whole and diluted blood with cryoprecipitate and fibrinogen concentrate and the effects on the ADPtest platelet aggregation are shown in Figure 2. Addition of cryoprecipitate and of fibrinogen concentrate reduced aggregation in the ADPtest results. By increasing the concentration of each, the effect of sample dilution was decreased but aggregation was still reduced overall. The effect was proportional to baseline level of aggregation. All results were significantly different (p o 0.05) except addition of 10 U of cryoprecipitate and 50 mg/kg of fibrinogen concentrate, which were comparable. Aggregation was further reduced when the sample was diluted 40% with normal saline. The addition of cryoprecipitate
Clot elasticity calculated as MCE ¼ (A30*100)/(100-A30) for the ExTEM and FibTEM assays allows derivation of the platelet elasticity component by subtraction of the fibrinogen component (ExTEM-MCE – FibTEM-MCE). Here the addition of cryoprecipitate and fibrinogen showed effects comparable to hemodilution (Fig 6).
Fig 2. Multiplate ADPtest: Titration. Abbreviations: ADP, adenosine diphosphate; AUC, area under the curve.
Fig 3. Multiplate TRAPtest: Diluted blood titration. Abbreviations: TRAP, thrombin receptor-activating peptide-6; AUC, area under the curve.
Rotational Thromboelastometry: Derived Platelet Elasticity Component
698
CARTWRIGHT ET AL
Fig 4.
ROTEM ExTEM A30 diluted blood titration.
Modified Thromboelastography ADP-Activated Assay The ADP-activated assay (tissue factor þ reptilase þ XIIIa þ10 μL ADP) produces a compound of activated fibrin monomer and platelets responsive to ADP. The amplitude at 30 minutes (A30) represents the total amount of platelet-fibrinogen interaction, the angle measures rate of clot formation and elasticity is derived as the G function, ie, G ¼ (5000*MA)/(100-MA). Here addition of cryoprecipitate produced no significant changes in all parameters although the trend was toward reduction in A30 (near-total substrate) and Gf; whereas addition of fibrinogen concentrate was able to significantly improve the rate of clot formation, represented by clot angle without significant changes in A30 and G. Data are shown in Figure 7. Full data analysis is shown in Appendix 1. DISCUSSION
The principal finding of the study was that fibrinogen supplementation primarily improved assays of fibrin formation. Improvement in platelet aggregation response to ADP and TRAP was not observed.
Fig 5.
ROTEM FibTEM A30 titration.
Fig 6.
ROTEM-derived platelet elasticity component.
Fibrinogen supplementation at 100 mg/kg reduced platelet aggregation on the ADPtest less than cryoprecipitate or hemodilution but no therapy improved aggregation. A similar response was shown on the TRAPtest and derived ROTEM platelet elasticity assays, which is in contrast to the findings of Li et al, which found stepwise improvement in response to fibrinogen supplementation.16 The authors suggest that this is because small molecule glycoprotein IIb/IIIa receptor antagonists are “competitive” inhibitors, whereas clopidogrel binds in a “noncompetitive” fashion to the P2 receptor. Fibrinogen supplementation also resulted in supranormal fibrin polymerization in whole blood and restoration of fibrin polymerization between 50 mg/kg and 100 mg/kg in diluted blood as shown by the ExTEM and FibTEM assays. It also significantly improved the rate of clot formation, represented by the angle in the modified TEG-ADP assay. In contrast, neither cryoprecipitate nor fibrinogen concentrate, at the concentrations used, were able to improve the amplitude at 30 minutes in the modified TEG-ADP assay, beyond the threshold value used in the Target-CABG trial to delineate bleeding risk with clopidogrel.5
Fig 7. Modified TEG (ADP) angle and A30 titration. Abbreviations: TEG, ADP, adenosine diphosphate.
699
EFFICACY OF FIBRINOGEN CONCENTRATE
Furthermore, addition of cryoprecipitate and fibrinogen concentrate produced comparable amplitudes on the TEGADP assay at 30 minutes, despite a twofold difference in fibrinogen supplementation. This presumably reflects an additional effect of cryoprecipitate on platelet-fibrinogen interaction from fibronectin or factor XIII. Whether the improvement in clot formation rate demonstrated is sufficient (ie, the ability to form sufficient amplitude of clot within a set time period) by the addition of fibrinogen concentrate alone in the context of platelets impaired by clopidogrel remains to be established. The improvement in fibrin polymerization seen would be significant in the context of cardiopulmonary bypass in which fibrin formation is impaired to a greater extent than thrombin generation or platelet activity.29 The threshold amount of ADP-activatable platelets required was not established by this study. A major limitation with this study was that only changes in in vitro assays were shown. Also, because of citrate being present in cryoprecipitate and fibrinogen concentrate, citrate tubes were used primarily for analysis and there are complex effects on platelet aggegation, which vary among hirudin, citrate, and sodium heparin.30 The lack of improvement in
platelet aggregation could have reflected this, issues of adequate recalcification, the lack of shear forces in the assays used, or be related to the dose of cryoprecipitate and fibrinogen concentrate used. Furthermore, in vitro assay results do not always reflect in vivo hemostasis. For example, the amplitude of clot formation in the ExTEM assay demonstrates primarily thrombin-mediated platelet interaction with fibrinogen, masking the effect of ADP-mediated platelet dysfunction. Additionally, in vitro titration assays in healthy patients may not reflect the response in patients bleeding clinically. CONCLUSIONS
Fibrinogen supplementation may play a role in the hemostatic resuscitation of patients on dual antiplatelet therapy, but there was no evidence in this in vitro study that there was a specific ADP-related platelet effect involved that would allow for platelet substitution. APPENDIX A
See below for Table A1
REFERENCES 1. Mahla E, Metzler H, Tantry US, et al: Controversies in oral antiplatelet therapy in patients undergoing aortocoronary bypass surgery. Ann Thorac Surg 90:1040-1051, 2010 2. Morici N, Moja L, Rosato V, et al: Time from adenosine diphosphate receptor antagonist discontinuation to coronary bypass surgery in patients with acute coronary syndrome: Meta-analysis and meta-regression. Int J Cardiol 168:1955-1964, 2013 3. Gurbel PA, Mahla E, Tantry US: Peri-operative platelet function testing: The potential for reducing ischaemic and bleeding risks. Thromb Haemost 106:248-252, 2011 4. Di Dedda U, Ranucci M, Baryshnikova E, et al: Thienopyridines resistance and recovery of platelet function after discontinuation of thienopyridines in cardiac surgery patients. Eur J Cardiothorac Surg 45: 165-170, 2013 5. Mahla E, Suarez TA, Bliden KP, et al: Platelet function measurement-based strategy to reduce bleeding and waiting time in clopidogrel-treated patients undergoing coronary artery bypass graft surgery the timing based on platelet function strategy to reduce clopidogrel-associated bleeding related to cabg (target-cabg) study. Circ Cardiovasc Interv 5:261-269, 2012 6. Sibbing D, Stegherr J, Braun S, et al: A double-blind, randomized study on prevention and existence of a rebound phenomenon of platelets after cessation of clopidogrel treatment. J Am Coll Cardiol 55: 558-565, 2010 7. Sibbing D, Schulz S, Braun S, et al: Antiplatelet effects of clopidogrel and bleeding in patients undergoing coronary stent placement. J Thromb Haemost 8:250-256, 2010 8. Spiess BD: Platelet transfusions: The science behind safety, risks and appropriate applications. Best Pract Res Clin Anaesthesiol 24:65-83, 2010 9. Koch C, Li L, Figueroa P, et al: Transfusion and pulmonary morbidity after cardiac surgery. Ann Thorac Surg 88:1410-1418, 2009 10. Bachelani AM, Bautz JT, Sperry JL, et al: Assessment of platelet transfusion for reversal of aspirin after traumatic brain injury. Surgery 150:836-843, 2011 11. Gao C, Ren C, Li D, et al: Clopidogrel and aspirin versus clopidogrel alone on graft patency after coronary artery bypass grafting. Ann Thorac Surg 88:59-62, 2009
12. Bielby L, Hunt R, Roxby D, et al: Australian Red Cross. Available at: http://resources.transfusion.com.au/cdm/ref/collection/p16691coll1/id/58. 13. Leithäuser B, Zielske D, Seyfert UT, et al: Effects of desmopressin on platelet membrane glycoproteins and platelet aggregation in volunteers on clopidogrel. Clin Hemorheol Microcirc 39:293-302, 2008 14. Spiel AO, Gilbert JC, Jilma B: von Willebrand factor in cardiovascular disease: focus on acute coronary syndromes. Circulation 117:1449-1459, 2008 15. Schöchl H, Posch A, Hanke A, et al: High-dose fibrinogen concentrate for haemostatic therapy of a major trauma patient with recent clopidogrel and aspirin intake. Scand J Clin Lab Invest 70:453-457, 2010 16. Li YF, Spencer FA, Becker RC: Comparative efficacy of fibrinogen and platelet supplementation on the in vitro reversibility of competitive glycoprotein IIb/IIIa (αIIb/β3) receptor-directed platelet inhibition. Am Heart J 142:204-210, 2001 17. Ang L, Palakodeti V, Khalid A, et al: Elevated plasma fibrinogen and diabetes mellitus are associated with lower inhibition of platelet reactivity with clopidogrel. J Am Coll Cardiol 52:1052-1059, 2008 18. Marguerie GA, Edgington TS, Plow EF: Interaction of fibrinogen with its platelet receptor as part of a multistep reaction in ADP-induced platelet aggregation. J Biol Chem 255:154-161, 1980 19. Velik-Salchner C, Haas T, Innerhofer P, et al: The effect of fibrinogen concentrate on thrombocytopenia. J Thromb Haemost 5: 1019-1025, 2007 20. Heymann von C, Spies C, Grubitzsch H, et al: Bleeding after cardiac surgery: The role of recombinant factor VIIa [article in German]. Hamostaseologie 26(suppl):S77-S87, 2006 (suppl) 21. Nadler SB, Hidalgo JH, Bloch T: Prediction of blood volume in normal human adults. Surgery 51:224-232, 1962 22. Johnston LR, Larsen PD, La Flamme AC, et al: Methodological considerations for the assessment of ADP induced platelet aggregation using the Multiplates analyser. Platelets 24:303-307, 2013 23. Zambruni A, Thalheimer U, Leandro G, et al: Thromboelastography with citrated blood: comparability with native blood, stability of citrate storage and effect of repeated sampling. Blood Coagul Fibrinolysis 15:103-107, 2004
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24. Diehl P, Olivier C, Halscheid C, et al: Clopidogrel affects leukocyte dependent platelet aggregation by P2Y12 expressing leukocytes. Basic Res Cardiol 105:379-387, 2009 25. Vilahur G, Choi BG, Zafar MU, et al: Normalization of platelet reactivity in clopidogrel-treated subjects. J Thromb Haemost 5:82-90, 2007 26. Velik-Salchner C, Maier S, Innerhofer P, et al: Point-of-care whole blood impedance aggregometry versus classical light transmission aggregometry for detecting aspirin and clopidogrel: The results of a pilot study. Anesth Analg 107:1798-1806, 2008 27. Ranucci M, Baryshnikova E, Soro G, et al: Multiple electrode whole-blood aggregometry and bleeding in cardiac
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surgery patients receiving thienopyridines. Ann Thorac Surg 91: 123-129, 2011 28. Tantry US, Gurbel PA: Assessment of oral antithrombotic therapy by platelet function testing. Nat Rev Cardiol 8:572-579, 2011 29. Solomon C, Rahe-Meyer N, Sørensen B: Fibrin formation is more impaired than thrombin generation and platelets immediately following cardiac surgery. Thromb Res 128:277-282, 2011 30. Kaiser AF, Neubauer H, Franken CC, et al: Which is the best anticoagulant for whole blood aggregometry platelet function testing? Comparison of six anticoagulants and diverse storage conditions. Platelets 23:359-367, 2011
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EFFICACY OF FIBRINOGEN CONCENTRATE
Table A1. Significance Table: Two-Tailed Probability (p value): Mann-Whitney U 1. Whole Blood ADPtest titration
þ5 U cryoprecipitate (cryo) þ10 U cryo þ50 mg/kg fibrinogen concentrate (FC) þ100 mg/kg fibrinogen (FC)
Whole blood
þ5 U cryo
þ10 U cryo
þ50 mg/kg FC
0.0024 0.0118 0.0159 0.1013
0.1437 0.1401 0.0294
0.7046 0.2387
0.424
2. Diluted 40% ADPtest titration
40% dilution 40% dilution þ10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ10 U cryo
0.0514 0.0001 0.0002
0.0004 0.0581
0.0172
3. Diluted 40% TRAPtest titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
0.0179 o0.0001 o0.0001
0.0001 0.0004
0.2976
4. Diluted 40% ExTEM A30 titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
o0.0001 o0.0001 0.0006
0.1632 0.022
0.0007
5. Diluted 40% ExTEM Alpha titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
0.0007 0.0716 o0.0001
0.1129 o0.0001
o0.0001
6. Whole Blood FibTEM A30 titration
þ5 U cryo þ10 U cryo þ50 mg/kg fibrinogen (FC) þ100 mg/kg fibrinogen (FC)
Whole
þ5 U cryo
þ10 U cryo
þ50 mg/kg FC
0.9459 0.8709 0.0086 0.0002
0.9892 0.0047 0.0001
0.0105 0.005
0.074
6a Diluted 40% FibTEM A30 titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
o0.0001 0.0009 0.0002
0.0029 o0.0001
o0.0001
7. Whole Blood FibTEM Alpha titration
þ5 U cryo þ10 U cryo þ50 mg/kg fibrinogen (FC) þ100 mg/kg fibrinogen (FC)
Whole
þ5 U cryo
þ10 U cryo
þ50 mg/kg FC
0.2317 0.4647 0.0067 0.0054
0.8388 0.0463 0.013
0.0367 0.0076
0.4562
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Table A1 (continued ) 7a. Diluted 40% FibTEM Alpha titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
0.009 0.1836 o0.0001
0.0009 o0.0001
o0.0001
8. Derived Platelet MCE – titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
o0.0001 o0.0001 o0.0001
0.0373 0.8392
0.0565
9. Modified TEG – ADP – A30 titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
0.7573 0.1237 0.2482
0.1489 0.3123
0.6744
10. Modified TEG – ADP – Angle titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
0.1449 0.9616 0.0269
0.3603 0.3606
40% dilution þ 10 U cryo
0.0587
11. Modified TEG – ADP – G titration
40% dilution 40% dilution þ 10 U cryo 40% dilution þ100 mg/kg fibrinogen (FC)
Whole blood
40% dilution
40% dilution þ 10 U cryo
0.8946 0.3123 0.5317
0.1489 0.312
0.6744
