Comparable effect of recombinant and plasma‐derived human fibrinogen concentrate on ex vivo clot formation after cardiac surgery

Abstract

There is a renewed interest in fibrinogen and its importance in hemostasis [1Levy J.H. Szlam F. Tanaka K.A. Sniecienski R.M. Review article: fibrinogen and hemostasis: a primary hemostatic target for the management of acquired bleeding.Anesth Analg. 2012; 114: 261-74Crossref PubMed Scopus (204) Google Scholar, 2Kozek‐Langenecker S. Sorensen B. Hess J.R. Spahn D.R. Clinical effectiveness of fresh frozen plasma compared with fibrinogen concentrate: a systematic review.Crit Care. 2011; 15: R239Crossref PubMed Scopus (181) Google Scholar]. The use of fibrinogen concentrate has been associated with improved outcome compared with transfusion of plasma [2Kozek‐Langenecker S. Sorensen B. Hess J.R. Spahn D.R. Clinical effectiveness of fresh frozen plasma compared with fibrinogen concentrate: a systematic review.Crit Care. 2011; 15: R239Crossref PubMed Scopus (181) Google Scholar] and peri‐operative supplementation with fibrinogen has reduced bleeding and transfusion requirements [3Fenger‐Eriksen C. Jensen T.M. Kristensen B.S. Jensen K.M. Tonnesen E. Ingerslev J. Sorensen B. Fibrinogen substitution improves whole blood clot firmness after dilution with hydroxyethyl starch in bleeding patients undergoing radical cystectomy: a randomized, placebo‐controlled clinical trial.J Thromb Haemost. 2009; 7: 795-802Crossref PubMed Scopus (209) Google Scholar, 4Rahe‐Meyer N. Solomon C. Winterhalter M. Piepenbrock S. Tanaka K. Haverich A. Pichlmaier M. Thromboelastometry‐guided administration of fibrinogen concentrate for the treatment of excessive intraoperative bleeding in thoracoabdominal aortic aneurysm surgery.J Thorac Cardiovasc Surg. 2009; 138: 694-702Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 5Karlsson M. Ternstrom L. Hyllner M. Baghaei F. Flinck A. Skrtic S. Jeppsson A. Prophylactic fibrinogen infusion reduces bleeding after coronary artery bypass surgery. A prospective randomised pilot study.Thromb Haemost. 2009; 102: 137-44Crossref PubMed Scopus (203) Google Scholar]. Today, all available fibrinogen concentrates are derived from human plasma; however, there are development projects with the aim of producing recombinant human fibrinogen. The present study compares for the first time the functional properties of recombinant fibrinogen derived from a human cell line with fibrinogen derived from human plasma. Ten elective coronary artery bypass grafting (CABG) patients (five women, median age 60 years [range 54–78] with pre‐operative fibrinogen concentration < 3.5 g L−1) were included after written informed consent was obtained. All patients were treated with acetylsalicylic acid. No patient was treated with anticoagulants. The study was approved by the regional research ethics committee. All patients had activated partial thromboplastin time (APTT), prothrombin time (PT) and platelet count within the normal ranges before surgery. Human plasma‐derived fibrinogen (Haemocomplettan®; CSL Behring, Marburg, Germany) and wild‐type recombinant human fibrinogen (ProFibrix, Leiden, The Netherlands) were dissolved to 10 mg mL−1 in phosphate‐buffered saline (PBS) and frozen until analysis. Wild‐type recombinant fibrinogen was produced using the human PER.C6® cell line. In developing the recombinant human fibrinogen, optimized cDNA’s encoding the fibrinogen Aα‐, Bβ‐ and γ‐chains were cloned into pcDNA3.1(+) expression vectors carrying the cytomegalovirus (CMV) promoter and subsequently co‐transfected into human PER.C6® cells using electroporation. Clonal growing cell lines were selected and individually expanded. The culture supernatants were screened for intact recombinant fibrinogen using ELISA and Western blotting methods. The monoclonal cell lines that performed the best were used to develop a production platform in chemically defined medium in stirred bioreactors that resulted in expression levels of > 0.5 g L−1. Recombinant fibrinogen was purified from clarified tissue culture harvest using both cationic and anionic column chromatography. Characterization of the recombinant fibrinogen demonstrated fully assembled, intact Aα‐, Bβ‐ and γ‐chains (Fig. S1). Arterial whole blood samples were collected before anesthesia and 30 min after heparin reversal. Median activated clotting time (ACT) after heparin reversal was 117 s (range 100–156). Postoperative samples (10 mL) were collected before treatment with a procoagulant drug. Increasing doses of recombinant or plasma‐derived fibrinogen were added to the postoperative samples. Each study sample consisted of 1.2 mL of whole blood. PBS in various doses was used to maintain the same hemodilution in all study samples. Measurements were performed pre‐operatively (with and without the addition of PBS) and postoperatively without any additives and with the addition of: (i) 180 μL PBS only (baseline for effect studies of fibrinogen), (ii) 60 μL fibrinogen (either plasma derived or recombinant) + 120 μL PBS, (iii) 120 μL fibrinogen + 60 μL PBS and (iv) 180 μL fibrinogen + 0 PBS. All analyzes were performed in duplicate. The chosen doses increased the fibrinogen plasma concentration with 0.5, 1.0 and 1.5 g L−1, respectively. Pre‐ and post‐operative plasma fibrinogen concentrations were analyzed according to the Clauss method with STA®‐Fib2 reagent on a STA‐R Evolution® device (Diagnostica Stago, Asniéres sur Seine, France). Modified rotational whole blood thromboelastometry (ROTEM®; Pentapharm, Munich, Germany) analyzes (EXTEM and FIBTEM) were performed as previously described [6Lang T. Bauters A. Braun S.L. Potzsch B. von Pape K.W. Kolde H.J. Lakner M. Multi‐centre investigation on reference ranges for ROTEM thromboelastometry.Blood Coagul Fibrinolysis. 2005; 16: 301-10Crossref PubMed Scopus (422) Google Scholar]. EXTEM clotting time (CT), clot formation time (CFT), alpha angle and maximum clot firmness (MCF) and FIBTEM CT, alpha angle and MCF were registered. Data are reported as median and range. The effects of recombinant and plasma‐derived fibrinogen were compared with a general linear model using dose, treatment and dose × treatment as fixed effects. Statistical significance was defined as a P‐value < 0.05. The median pre‐operative and post‐operative fibrinogen plasma concentration was 2.9 g L−1 (1.6–3.4) and 2.0 g L−1 (1.3–2.8), respectively, P= 0.005. There were no significant differences between recombinant and plasma‐derived fibrinogen in any of the thromboelastometric analyzes at any concentration, Table 1. EXTEM‐CFT decreased dose dependently after the addition of both plasma‐derived and recombinant fibrinogen (P= 0.031 and P= 0.007 for trend, respectively) whereas EXTEM‐MCF (P< 0.001 both), EXTEM‐α (P= 0.002 and P< 0.001, respectively), FIBTEM‐A10 and FIBTEM‐MCF (P< 0.001 all) increased dose dependently, Table 1. EXTEM‐CT, FIBTEM‐CT, FIBTEM‐α and FIBTEM LI30 did not change significantly with any of the fibrinogens.Table 1Pre‐ and post‐operative dataPre‐opPre‐op + 180 uL PBSPost‐opPost‐op + 180 uL PBSPost‐op + 60 uL FGN + 120 uL PBSPost‐op + 120 uL FGN + 60 uL PBSPost‐op + 180 uL FGN P‐value pFGN vs. rhFGNEXTEM‐CT pFGN61 (53–70)62 (57–70)63 (58–82)74 (64–88)62 (58–73)61 (54–70)61 (53–66)0.98 rhFGN66 (61–72)63 (58–67)62 (54–67)EXTEM‐CFT pFGN92 (78–147)106 (92–104)120 (90–192)141 (102–228)118 (92–163)98 (72–174)90 (68–112)0.83 rhFGN117 (84–156)97 (78–152)89 (64–111)EXTEM‐α pFGN72 (62–77)68 (56–72)67 (56–72)66 (52–72)72 (59–74)78 (58–80)78 (75–81)0.25 rhFGN70 (62–74)74 (62–78)75 (68–78)EXTEM‐MCF pFGN62 (56–66)59 (50–62)58 (51–62)54 (46–58)58 (52–62)60 (52–64)62 (57–65)0.38 rhFGN58 (52–62)62 (52–66)64 (61–68)FIBTEM‐CT pFGN52 (46–58)55 (46–81)59 (49–98)60 (53–84)55 (45–65)52 (46–59)51 (45–58)0.83 rhFGN56 (50–64)52 (48–63)48 (44–60)FIBTEM‐α pFGN70 (62–77)68 (62–74)67 (63–70)61 (60–71)72 (61–78)78 (62–82)80 (76–83)0.99 rhFGN72 (54–75)75 (66–78)78 (74–80)FIBTEM‐A10 pFGN13 (7–16)13 (6–16)10 (5–17)10 (6–16)13 (8–17)18 (10–21)20 (17–22)0.26 rhFGN13 (9–17)16 (9–20)20 (16–24)FIBTEM‐MCF pFGN14 (8–16)13 (6–16)10 (5–17)11 (6–16)14 (10–18)19 (10–24)21 (18–26)0.82 rhFGN12 (10–18)18 (10–23)23 (19–28)FIBTEM‐LI30 pFGN98 (84–100)100 (92–100)100 (100–100)100 (100–100)100 (94–100)100 (92–100)100 (92–100)0.43 rhFGN100 (100–100)100 (93–100)100 (91–100)CFT, clot formation time; CT, clotting Time; LI30, lysis index at 30 min; MCF, maximum clot firmness; A10, clot amplitude after 10 min; PBS, phosphate‐buffered saline; pFGN, plasma‐derived fibrinogen; rhFGN, recombinant human fibrinogen. Open table in a new tab CFT, clot formation time; CT, clotting Time; LI30, lysis index at 30 min; MCF, maximum clot firmness; A10, clot amplitude after 10 min; PBS, phosphate‐buffered saline; pFGN, plasma‐derived fibrinogen; rhFGN, recombinant human fibrinogen. The results indicate that recombinant fibrinogen and plasma‐derived fibrinogen have comparable effects on ex vivo clot formation as measured by thromboelastometry. All available fibrinogen concentrates today are derived from human plasma and licensed for treatment of congenital a‐ and hypofibrinogenemia in US and Europe, whereas use for acquired fibrinogen deficiency is granted only in some countries [1Levy J.H. Szlam F. Tanaka K.A. Sniecienski R.M. Review article: fibrinogen and hemostasis: a primary hemostatic target for the management of acquired bleeding.Anesth Analg. 2012; 114: 261-74Crossref PubMed Scopus (204) Google Scholar]. Haemocomplettan® (CSL Behring), now licensed as RiaStap®, has been proven to be a safe product concerning potential thrombogenicity and transmission of pathogens [7Dickneite G. Pragst I. Joch C. Bergman G.E. Animal model and clinical evidence indicating low thrombogenic potential of fibrinogen concentrate (Haemocomplettan P).Blood Coagul Fibrinolysis. 2009; 20: 535-40Crossref PubMed Scopus (80) Google Scholar, 8Manco‐Johnson M.J. Dimichele D. Castaman G. Fremann S. Knaub S. Kalina U. Peyvandi F. Piseddu G. Mannucci P. for the Fibrinogen Concentrate Study GroupPharmacokinetics and safety of fibrinogen concentrate.J Thromb Haemost. 2009; 7: 2064-9Crossref PubMed Scopus (96) Google Scholar]. However, no plasma‐derived product can be considered completely safe, especially regarding diseases with a long incubation period or those yet unknown. There is therefore a great interest in developing products completely free from human or animal proteins, in any part of the manufacturing process. Furthermore, the increasing use and growing demand for fibrinogen products could, in future, overcome the fibrinogen fractionation capacities. This study has important limitations. The functional properties were only assessed with thromboelastometry and exact fibrinogen plasma concentrations after supplementation were not measured. However, the clinical utility of EXTEM‐MCF and FIBTEM‐MCF has previously been proven for monitoring fibrinogen substitution with strong correlations to fibrinogen activity and antigen [1Levy J.H. Szlam F. Tanaka K.A. Sniecienski R.M. Review article: fibrinogen and hemostasis: a primary hemostatic target for the management of acquired bleeding.Anesth Analg. 2012; 114: 261-74Crossref PubMed Scopus (204) Google Scholar, 9Solomon C. Cadamuro J. Ziegler B. Schöchl H. Varvenne M. Sørensen B. Hochleitner G. Rahe‐Meyer N. A comparison of fibrinogen measurement methods with fibrin clot elasticity assessed by thromboelastometry, before and after administration of fibrinogen concentrate in cardiac surgery patients.Transfusion. 2011; 51: 1695-706Crossref PubMed Scopus (86) Google Scholar]. Rotational thromboelastometry analyzes have acceptable repeatability, with an intra‐assay coefficient of variation for FIBTEM MCF of 6–13% and for EXTEM MCF < 5% [6Lang T. Bauters A. Braun S.L. Potzsch B. von Pape K.W. Kolde H.J. Lakner M. Multi‐centre investigation on reference ranges for ROTEM thromboelastometry.Blood Coagul Fibrinolysis. 2005; 16: 301-10Crossref PubMed Scopus (422) Google Scholar]. Furthermore, potential differences in the pharmacokinetic profile between recombinant and plasma‐derived fibrinogen were not assessed. In summary, recombinant human fibrinogen demonstrated in the present model comparable efficacy as plasma‐derived fibrinogen. The results indicate that from a functional aspect, recombinant fibrinogen may have a future application. Besides the functional efficacy, other factors regarding manufacturing, commercialization and safety will influence the future applicability of recombinant fibrinogen. The authors state that they have no conflict of interests. The study was supported by the Swedish Heart & Lung Foundation,Sahlgrenska University Hospital (ALF/LUA grant) and ProFibrix, Leiden, the Netherlands. The study sponsors had no influence on the analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. Figure S1. SDSpage gel (NuPage Novex 3–8%Tris Acetate/TrisAcetate buffer (Invitrogen, Carlsbad, CA, USA),comparing recombinant human fibrinogen (ProFibrix) and researchgrade plasma‐derived human fibrinogen (Enzyme ResearchLaboratories, South Bend, IN, USA). Gels were stained withInstantBlue Protein Gel Stain (Expedeon, San Diego, CA, USA) Thegel was provided by ProFibrix. Download .jpg (.03 MB) Help with files Supporting info item