Thromb Haemost 2016; 116(01): 134-145
DOI: 10.1160/TH15-10-0801
New Technologies, Diagnostic Tools and Drugs
Schattauer GmbH

Simultaneous measurement of thrombin generation and fibrin formation in whole blood under flow conditions

Hilde Kelchtermans*
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, The Netherlands
2   Synapse BV, Maastricht, The Netherlands
,
Leonie Pelkmans*
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, The Netherlands
2   Synapse BV, Maastricht, The Netherlands
,
Anne Bouwhuis
3   Department of Anaesthesiology, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, The Netherlands
,
Evelien Schurgers
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, The Netherlands
2   Synapse BV, Maastricht, The Netherlands
,
Theo Lindhout
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, The Netherlands
2   Synapse BV, Maastricht, The Netherlands
,
Dana Huskens
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, The Netherlands
2   Synapse BV, Maastricht, The Netherlands
,
Adam Miszta
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, The Netherlands
2   Synapse BV, Maastricht, The Netherlands
,
H. Coenraad Hemker
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, The Netherlands
2   Synapse BV, Maastricht, The Netherlands
,
Marcus D. Lancé
3   Department of Anaesthesiology, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, The Netherlands
,
Bas de Laat
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, The Netherlands
2   Synapse BV, Maastricht, The Netherlands
4   Department of Clinical Chemistry and Haematology, University Medical Centre Utrecht, The Netherlands
› Author Affiliations
Further Information

Publication History

Received: 15 October 2015

Accepted after major revision: 28 March 2016

Publication Date:
27 November 2017 (online)

Summary

Assays based on the formation of thrombin and fibrin are frequently used, and results are considered exchangeable in research/clinical settings. However, thrombin generation and fibrin formation do not always go hand in hand and flow profoundly influences thrombus formation. We describe the technical/clinical evaluation of an assay to simultaneously measure thrombin generation and fibrin formation under conditions of flow. Introduction of a fluorometer into a ‘cone and base principle’-based rheometer allowed the measurement of thrombin generation (using a thrombin-sensitive substrate) and fibrin formation (changes in viscosity), while applying a linear shear flow. Increasing shear rates inversely related with thrombin generation and fibrin formation. Increasing fibrinogen concentrations in defibrinated plasma resulted in increased thrombin generation and fibrin formation. In pre-operative samples of 70 patients undergoing cardiothoracic surgery, fibrin formation and thrombin generation parameters correlated with fibrinogen content, rotational thromboelastometry (ROTEM) and whole blood Calibrated Automated Thrombinography (CAT) parameters, respectively. Upon dividing patients into two groups based on the median clot strength, a significant difference in perioperative/total blood loss was established. In conclusion, we clinically evaluated a method capable of simultaneously measuring thrombin generation and fibrin formation in plasma/whole blood under continuous flow, rendering our method one step closer to physiology. Importantly, our test proved to be indicative for the amount of blood loss during/after cardiothoracic surgery.

* These authors contributed equally to this work.


 
  • References

  • 1 Wolberg AS, Campbell RA. Thrombin generation, fibrin clot formation and haemostasis. Transfus Apher Sci 2008; 38: 15-23.
  • 2 Ryan EA, Mockros LF, Weisel JW. et al. Structural origins of fibrin clot rheology. Biophys J 1999; 77: 2813-2826.
  • 3 Glover CJ, McIntire LV, Brown 3rd CH. et al. Rheological properties of fibrin clots. Effects of fibrinogen concentration, Factor XIII deficiency, and Factor XIII inhibition. J Lab Clin Med 1975; 86: 644-656.
  • 4 Brenni M, Worn M, Bruesch M. et al. Successful rotational thromboelastometry-guided treatment of traumatic haemorrhage, hyperfibrinolysis and coagulopathy. Acta Anaesth Scand 2010; 54: 111-117.
  • 5 Tirosh-Wagner T, Strauss T, Rubinshtein M. et al. Point of care testing in children undergoing cardiopulmonary bypass. Ped Blood Cancer 2011; 56: 794-798.
  • 6 Wegner J, Popovsky MA. Clinical utility of thromboelastography: one size does not fit all. Semin Thromb Haemost 2010; 36: 699-706.
  • 7 Shah GA, Nair CH, Dhall DP. Physiological studies on fibrin network structure. Thromb Res 1985; 40: 181-188.
  • 8 Hemker HC, Al Dieri R, De Smedt E. et al. Thrombin generation, a function test of the haemostatic-thrombotic system. Thromb Haemost 2006; 96: 553-561.
  • 9 Konings J, Govers-Riemslag JW, Philippou H. et al. Factor XIIa regulates the structure of the fibrin clot independently of thrombin generation through direct interaction with fibrin. Blood 2011; 118: 3942-3951.
  • 10 Orvim U, Roald HE, Stephens RW. et al. Tissue factor-induced coagulation triggers platelet thrombus formation as efficiently as fibrillar collagen at arterial blood flow conditions. Arterioscler Thromb 1994; 14: 1976-1983.
  • 11 Weiss HJ, Turitto VT, Baumgartner HR. Role of shear rate and platelets in promoting fibrin formation on rabbit subendothelium. Studies utilizing patients with quantitative and qualitative platelet defects. J Clin Invest 1986; 78: 1072-1082.
  • 12 Hathcock JJ. Flow effects on coagulation and thrombosis. Arterioscl Thromb Vasc Biol 2006; 26: 1729-1737.
  • 13 Campbell RA, Aleman M, Gray LD. et al. Flow profoundly influences fibrin network structure: implications for fibrin formation and clot stability in haemostasis. Thromb Haemost 2010; 104: 1281-1284.
  • 14 Gersh KC, Edmondson KE, Weisel JW. Flow rate and fibrin fibre alignment. J Thromb Haemost 2010; 08: 2826-2828.
  • 15 Hemker HC, Giesen P, Al Dieri R. et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33: 4-15.
  • 16 Wagenvoord R, Hemker PW, Hemker HC. The limits of simulation of the clotting system. J Thromb Haemost 2006; 04: 1331-1338.
  • 17 Richards FJ. A flexible growth function for empirical use. J Exp Bot 1959; 10: 290-301.
  • 18 Hemker HC, Kremers R. Data management in thrombin generation. Thromb Res 2013; 131: 3-11.
  • 19 Ninivaggi M, Apitz-Castro R, Dargaud Y. et al. Whole-blood thrombin generation monitored with a calibrated automated thrombogram-based assay. Clin Chem 2012; 58: 1252-1259.
  • 20 Schols SE, Lance MD, Feijge MA. et al. Impaired thrombin generation and fibrin clot formation in patients with dilutional coagulopathy during major surgery. Thromb Haemost 2010; 103: 318-328.
  • 21 Evans PA, Hawkins K, Lawrence M. et al. Rheometry and associated techniques for blood coagulation studies. Med Engin Phys 2008; 30: 671-679.
  • 22 Scrutton MC, Ross-Murphy SB, Bennett GM. et al. Changes in clot deformability--a possible explanation for the epidemiological association between plasma fibrinogen concentration and myocardial infarction. Blood Coagul Fibrinol 1994; 05: 719-723.
  • 23 Tynngard N, Lindahl TL, Ramstrom S. et al. Free oscillation rheometry detects changes in clot properties in pregnancy and thrombocytopenia. Platelets 2008; 19: 373-378.
  • 24 Ganter MT, Hofer CK. Coagulation monitoring: current techniques and clinical use of viscoelastic point-of-care coagulation devices. Anesthesia and analgesia 2008; 106: 1366-1375.
  • 25 Ortmann E, Rubino A, Altemimi B. et al. Validation of viscoelastic coagulation tests during cardiopulmonary bypass. J Thromb Haemost 2015; 13: 1207-1216.
  • 26 Meesters MI, Lance MD, van der Steeg R. et al. The value of the thromboelastometry heparinase assay (HEPTEM) in cardiac surgery. Thromb Haemost 2015; 114: 1058-1063.
  • 27 Nagler M, ten Cate H, Kathriner S. et al. Consistency of thromboelastometry analysis under scrutiny: results of a systematic evaluation within and between analysers. Thromb Haemost 2014; 111: 1161-1166.
  • 28 Cosemans JM, Angelillo-Scherrer A, Mattheij NJ. et al. The effects of arterial flow on platelet activation, thrombus growth, and stabilisation. Cardiovasc Res 2013; 99: 342-352.
  • 29 Slack SM, Cui Y, Turitto VT. The effects of flow on blood coagulation and thrombosis. Thromb Haemost 1993; 70: 129-134.
  • 30 Weiss HJ, Baumgartner HR, Tschopp TB. et al. Correction by factor VIII of the impaired platelet adhesion to subendothelium in von Willebrand disease. Blood 1978; 51: 267-279.
  • 31 Baskurt OK, Meiselman HJ. Blood rheology and hemodynamics. Semin Thromb Haemost 2003; 29: 435-450.
  • 32 Kremers RM, Wagenvoord RJ, Hemker HC. The effect of fibrin(ogen) on thrombin generation and decay. Thromb Haemost 2014; 112: 486-494.
  • 33 Berny MA, Munnix IC, Auger JM. et al. Spatial distribution of factor Xa, thrombin, and fibrin(ogen) on thrombi at venous shear. PloS one 2010; 05: e10415.
  • 34 Onasoga-Jarvis AA, Puls TJ, O’Brien SK. et al. Thrombin generation and fibrin formation under flow on biomimetic tissue factor-rich surfaces. J Thromb Haemost 2014; 12: 373-382.
  • 35 Wilhelmsen L, Svardsudd K, Korsan-Bengtsen K. et al. Fibrinogen as a risk factor for stroke and myocardial infarction. N Engl J Med 1984; 311: 501-505.
  • 36 Weitz JI, Leslie B, Hudoba M. Thrombin binds to soluble fibrin degradation products where it is protected from inhibition by heparin-antithrombin but susceptible to inactivation by antithrombin-independent inhibitors. Circulation 1998; 97: 544-552.
  • 37 Romlin BS, Wahlander H, Synnergren M. et al. Earlier detection of coagulopathy with thromboelastometry during pediatric cardiac surgery: a prospective observational study. Paed Anaesth 2013; 23: 222-227.
  • 38 Bolliger D, Tanaka KA. Roles of thrombelastography and thromboelastometry for patient blood management in cardiac surgery. Transfusion Med Rev 2013; 27: 213-220.
  • 39 Ogawa S, Szlam F, Chen EP. et al. A comparative evaluation of rotation thromboelastometry and standard coagulation tests in hemodilution-induced coagulation changes after cardiac surgery. Transfusion 2012; 52: 14-22.
  • 40 Bosch Y, Al Dieri R, ten Cate H. et al. Preoperative thrombin generation is predictive for the risk of blood loss after cardiac surgery: a research article. J Cardiothor Surg 2013; 08: 154.
  • 41 Coakley M, Hall JE, Evans C. et al. Assessment of thrombin generation measured before and after cardiopulmonary bypass surgery and its association with postoperative bleeding. J Thromb Haemost 2011; 09: 282-292.
  • 42 Theusinger OM, Schroder CM, Eismon J. et al. The influence of laboratory coagulation tests and clotting factor levels on Rotation Thromboelastometry (ROTEM(R)) during major surgery with hemorrhage. Anesth Analg 2013; 117: 314-321.
  • 43 Ghavidel AA, Toutounchi Z, Shahandashti FJ. et al. Rotational thromboelastometry in prediction of bleeding after cardiac surgery. Asian Cardiovasc Thorac Ann. 2015 Epub ahead of print..
  • 44 Karlsson M, Ternstrom L, Hyllner M. et al. Plasma fibrinogen level, bleeding, and transfusion after on-pump coronary artery bypass grafting surgery: a prospective observational study. Transfusion 2008; 48: 2152-2158.
  • 45 Walden K, Jeppsson A, Nasic S. et al. Low preoperative fibrinogen plasma concentration is associated with excessive bleeding after cardiac operations. Ann Thoracic Surg 2014; 97: 1199-1206.
  • 46 Faraoni D, Willems A, Savan V. et al. Plasma fibrinogen concentration is correlated with postoperative blood loss in children undergoing cardiac surgery. A retrospective review. Eur J Anaesthesiol 2014; 31: 317-326.
  • 47 Solomon C, Baryshnikova E, Tripodi A. et al. Fibrinogen measurement in cardiac surgery with cardiopulmonary bypass: analysis of repeatability and agreement of Clauss method within and between six different laboratories. Thromb Haemost 2014; 112: 109-117.