All parts of this study were approved by the institutional review board at the University Medical School (Graz, Austria). Blood samples were obtained from eight healthy volunteers after obtaining a written, informed consent. Volunteers had not taken nonsteroidal antiinflammatory drugs in the preceding 2 wk. Blood was collected in 4.5 mL silicone-coated glass tubes containing 4.5 mL of 0.129 M buffered sodium citrate (VACUTAINER®; Becton Dickinson, Meylan, France). Platelet count was measured using Sysmex (Sysmex SF-3000, Sysmex GmBH, Norderstedt, Germany). Samples were aliquoted into three portions; the first part was used as whole blood, and the remaining aliquots were centrifuged at 500g and 3500g for 10 min to obtain PRP and platelet-poor plasma (PPP), respectively.
The second part of the study was a retrospective analysis of laboratory data from routine ROTEM in surgical patients with thrombocytopenia (<50 × 103 mm−3) between 1999 and 2005 at the University Medical School (Graz, Austria). EXTEM (extrinsic activation), FIBTEM, platelet count, and fibrinogen levels were obtained from laboratory records.
Thromboelastometric measurements were performed on the quad-channel ROTEM Coagulation Analyzer.5,13 EXTEM and FIBTEM were performed after the addition of 20 mm3 of 0.2 M CaCl2 and diluted tissue factor (extrinsic activator, 20 mm3) to the PRP samples. For FIBTEM, cytochalasin D was also added so that conformational changes of platelet glycoprotein (GP) IIb/IIIa receptors were inhibited.5,15 Thus, fibrin polymerization can be specifically evaluated in the absence of attachment to platelets, and the clot strength based on fibrinogen alone can be evaluated.5,12,15,16
The maximum clot firmness (MCF) is usually used to evaluate clot strength, but MCF does not reflect the actual physical properties of clot strength according to Hooke’s law (i.e., the relationship between MCF and MCE is curvilinear). Thus, the maximum clot elasticity (MCE) was calculated as follows: MCE = (MCF × 100)/(100 − MCF).
The “platelet component” of clot strength is expressed as the difference in clot strength between EXTEM and FIBTEM as previously reported for platelet GPIIb/IIIa inhibitors (Fig. 1)12,15,16:
In addition, MCEplatelet was adjusted to estimate the extent of interactions between fibrin(ogen) and platelets (1 × 103 mm−3) as “Platelet Index” (PI):
Normal ranges are MCF 54–72 mm for EXTEM, and MCF 9–25 mm for FIBTEM.17 Correspondingly, MCE varies from 117 to 257 for EXTEM, and from 9.9 to 33 for FIBTEM.
Effects of Platelet Count on Clot Strength (Experiment 1, Fig. 1)
To evaluate the influence of different platelet counts on clot strength, EXTEM was performed in PRP (n = 8), which was serially diluted with homologous PPP to achieve platelet counts between 4 × 103 mm−3 and 770 × 103 mm−3. To eliminate any platelet contribution to clot strength, the homologous PPP from each donor was also evaluated with FIBTEM for comparison.
Effects of Fibrinogen Levels on Clot Strength (Experiment 2, Fig. 1)
A stock solution of fibrinogen (111 mg/dL) was prepared by adding 1 g of fibrinogen (Haemocomplettan P®, CSL Behring, Marburg, Germany) in 9 mL isotonic saline. The effects of fibrinogen concentration on clot strength were studied in PRP (n = 8), which was adjusted to achieve platelet counts of 10 × 103 mm−3, 50 × 103 mm−3, or 100 × 103 mm−3. The adjusted PRP samples were subdivided and each aliquot was added to either normal saline (control) or to one of two different levels of fibrinogen to achieve calculated increments of 550 and 780 mg/dL. The target fibrinogen levels after the additions are 800–1000 mg/dL, which are observed clinically during pregnancy or in the inflammatory state.18 EXTEM and FIBTEM were performed for each sample. Fibrinogen levels were determined by the Clauss method using Multifibren U (Dade Behring, Marburg, Germany).
Whole Blood Clotting with Incremental Fibrinogen Levels (Experiment 3, Fig. 1)
The concentration-dependent increases in MCF and MCE were previously demonstrated by Nielsen et al.19 in fibrinogen-depleted plasma. In the present study, the effects of fibrinogen concentration on MCF/MCE were evaluated at normal to supraclinical levels because it is plausible that the catalytic ability of thrombin might be saturated at extremely high levels of fibrinogen. Whole blood samples (n = 8), prepared as above, were aliquoted and mixed with fibrinogen solutions (nine parts whole blood to one part fibrinogen) at varied concentrations to increase the fibrinogen level of each aliquot by 250, 500, 740, 1000, 1230, 1480, 1730, 1975, 2220, and 2470 mg/dL. One aliquot was mixed with isotonic saline as a control. EXTEM and FIBTEM were performed for each sample, and the fibrinogen level was measured by the Clauss method.
Thromboelastometric Data from Thrombocytopenic Patients
ROTEM results from perioperative patients who presented with low platelet count (<50 × 103 mm−3) were analyzed retrospectively to evaluate whether there was a correlation between clot strength and fibrinogen at low platelet counts. In total, 904 ROTEM measurements were available for retrospective analysis. The data were categorized into Group A: severe thrombocytopenia (platelet count <20 × 103 mm−3, n = 107) and Group B: moderate thrombocytopenia (platelet count 20–50 × 103 mm−3, n = 797), and were further subdivided into five subgroups based on the fibrinogen concentration (<100 mg/dL, 100–199 mg/dL, 200–399 mg/dL, 400–599 mg/dL, >599 mg/dL).
Based on previous studies of thrombin generation, the sample size of 5 was needed to detect a 30% change from the controls in peak thrombin generation with a β ≥0.8 and an α <0.05,5 therefore all of our experiments consisted of a sample size of 8. Thromboelastometric variables were analyzed using Kruskal–Wallis H-test followed by Mann–Whitney U-test with Bonferroni correction or using a simple regression analysis. All data are expressed as mean ± sd. A P value equal to or <0.05 was considered significant.
Effects of Platelet Count on Clot Strength
ROTEM analysis of varying platelet counts in PRP demonstrated a positive correlation between changes in the clot strength, expressed as MCE, and increasing platelet counts (r2 = 0.88, P < 0.001). The MCE values decreased with platelet counts below 100 × 103 mm−3, whereas at platelet counts within and above the normal range, the increase in MCE tended to reach a plateau (Fig. 2).
Effects of Fibrinogen Levels on Clot Strength
The baseline fibrinogen level was 268 ± 42 mg/dL. After adding fibrinogen concentrate, 550 mg/dL and 780 mg/dL, to PRP samples, final plasma fibrinogen levels were increased to 823 ± 42 mg/dL and 1045 ± 42 mg/dL, respectively, from 268 ± 42 mg/dL at baseline. At all three platelet levels, the clot strength increased with higher fibrinogen concentrations (Fig. 3). In particular, the MCE doubled from baseline levels even with the lower dose of fibrinogen.
Whole Blood Clotting with Incremental Fibrinogen Levels
The addition of fibrinogen to whole blood led to an exponential increase in clot strength in both EXTEM and FIBTEM (Fig. 4A). Furthermore, the platelet component of clot strength (MCEplatelet) was increased in a fibrinogen concentration-dependent manner (r2 = 0.79, P < 0.001; Fig. 4B).
Thromboelastometric Data from Thrombocytopenic Patients
When data from included surgical patients (n = 904) were subgrouped based on fibrinogen levels, the mean platelet count among subgroups was not significantly different between Groups A and B (Table 1).
There was a trend toward increases in clot strength in a fibrinogen concentration-dependent manner in both groups. There was a moderate correlation between clot strength and fibrinogen concentration in both Group A (r2 = 0.45, P < 0.01) and Group B (r2 = 0.53, P < 0.01).
In moderate thrombocytopenia (Group B), near-normal values of MCF (reference range 54–72 mm) were achieved with fibrinogen at 400–600 mg/dL, whereas in severe thrombocytopenia (Group A) near-normal values of MCF were only reached with fibrinogen above 600 mg/dL.
PI clearly increased in a fibrinogen concentration-dependent manner in both groups (Table 1). Although MCF values were higher in Group B, PI values were larger in Group A. This latter finding most likely indicates that more fibrin molecules are bound to a lower number of platelets because of the presence of abundant GPIIb/IIIa receptors on a single platelet (Fig. 5).
In the present study, we demonstrated the difference between platelets and fibrinogen with regard to their ability to increase clot strength on ROTEM. At normal fibrinogen levels (150–350 mg/dL), the clot strength expressed as MCE increased when platelet count changed from <10 × 103 mm−3 to 50–100 × 103 mm−3. The increase in MCE tended to reach a plateau at normal platelet counts. The clot strength increased in a fibrinogen concentration-dependent manner in both EXTEM and FIBTEM. Surprisingly, the contribution of platelets to clot strength, reflected by the platelet component in MCE (MCEEXTEM-MCEFIBTEM), also increased with fibrinogen concentration at constant platelet count. Our present findings at various fibrinogen and platelet levels confirm the previous report by Nielsen et al.,19 which demonstrated the concentration-dependent effects of fibrinogen in plasma below 350 mg/dL.
Fibrinogen is a major procoagulant substrate of thrombin, and fibrin formation is a critical component of normal coagulation. Furthermore, the interaction of platelets with both fibrinogen and fibrin20 is necessary to achieve hemostasis in arterial vasculature. Even in the presence of normal platelet count or function, low fibrinogen level (e.g., afibrinogenemia) or slow fibrin formation (e.g., hemophilia) can be associated with clinical bleeding diathesis.21,22 Because there is an abundance of fibrinogen receptors (GPIIb/IIIa) on a single platelet,23 we investigated whether increasing fibrinogen levels improved clot strength in thrombocytopenic patients.
The contribution of platelets to hemostasis is well known, and a prophylactic transfusion of platelets to avoid hemorrhage is a common practice. However, the review of four randomized, prospective trials comparing the trigger count of 10 × 103 mm−3 vs 20 × 103 mm−3 showed no differences in hemorrhagic complications.24 One mechanism which might contribute to hemostasis is that large amounts of fibrinogen are captured by a few platelets via the abundant GPIIb/IIIa receptors (40,000–50,000 copies per platelet).23 On the surface of an activated platelet, as many as 1680 fibrinogen molecules are efficiently cleaved by a single thrombin molecule.22 Even in the presence of reduced platelet count and thrombin generation (e.g., after cardiopulmonary bypass), fibrinogen addition increases clot strength.14 In the present study, the use of cytochalasin D in FIBTEM largely abolished GPIIb/IIIa assembly,5 thus the contribution of factor XIII and other platelet-derived factors to the clot strength is presumably small.25 Increased PI according to the concentration of fibrinogen suggests that each platelet increasingly engaged with fibrin(ogen) via GPIIb/IIIa receptors (note: PI is the MCEplatelet adjusted to the platelet count). Even when the platelet count was below 20 × 103 mm−3 (i.e., common trigger level for platelet transfusion), fibrinogen addition improved the clot strength (Table 1, Fig. 3).
It is important to note that ROTEM is conducted under a low-shear condition (0.1 s−1), which may not fully represent in vivo conditions.2,3 In arterial vasculature, functional platelets generate a procoagulant milieu against the shear of blood flow. The initial interaction (or primary plug) between the damaged vasculature and platelets is mediated by subendothelial collagen, von Willebrand factor, platelet collagen, and GPIb receptors.2,3 Subsequently, fibrinogen binds to platelet GPIIb/IIIa receptors upon platelet activation.26 Fibrin is formed by cleavage of fibrinogen by thrombin generated on the activated platelet surface.27 The importance of the interactions between fibrin(ogen) and platelet GPIIb/IIIa receptors is clinically evident from bleeding episodes in the congenital GPIIb/IIIa deficiency Glanzmann thromboasthenia,28 pharmacological GPIIb/IIIa blockade (e.g., abciximab29), and congenital afibrinogenemia.21,30
There are experimental and clinical observations that support the compensatory hemostatic function of fibrinogen in thrombocytopenia. In obstetric patients, increased fibrinogen levels of 50%–250% during pregnancy, relative to decreased platelet count (10%–20%), may have a role in limiting blood loss during delivery.12,18 Moreover, it has recently been shown that a plasma fibrinogen level <200 mg/dL is an important predictor of postpartum hemorrhage.31 In a porcine model of traumatic bleeding (liver laceration), animals that received fibrinogen 250 mg/kg experienced less bleeding than control animals that received isotonic saline.13 More recently, the same model has been applied in a thrombocytopenic condition (platelet count <30 × 103 mm−3), and fibrinogen repletion at 250 mg/kg was found superior to the apheresis platelet transfusion according to ROTEM variables and blood loss.32
The optimal plasma fibrinogen level for hemostasis is not yet known, and there have been controversies about a potential association between elevated fibrinogen and myocardial infarction.33,34 It is plausible that fibrinogen merely reflects a proinflammatory state, rather than directly triggering atherosclerosis.35 The use of bedside coagulation monitors, such as ROTEM/TEG, may be useful for dosing fibrinogen concentrate or fibrinogen-rich cryoprecipitate.25 In particular, the Clauss (turbidometric) method may give falsely high levels in the presence of dextran and hydroxyethyl starch,36 and FIBTEM may be a preferred monitor in evaluating the contribution of functional fibrinogen in perioperative settings. Further studies are required to evaluate the safety and efficacy of fibrinogen supplementation.
In conclusion, these data suggest that clot strength, measured by ROTEM, increases in a fibrinogen concentration-dependent manner. Even with the low platelet counts associated with severe thrombocytopenia, thrombin-activated platelets bind to large amounts of fibrin(ogen) via GPIIb/IIIa receptors, resulting in increased clot strength. Further clinical investigations are necessary to verify whether bleeding complications associated with thrombocytopenia could be safely compensated for by the supplementation of fibrinogen in perioperative settings.
The authors thank Mr. Gerald Hochleitner for his skilled technical assistance.
1.Hoffman M, Monroe DM 3rd. A cell-based model of hemostasis. Thromb Haemost 2001;85:958–65
2.Ruggeri ZM. The role of von Willebrand factor and fibrinogen in the initiation of platelet adhesion to thrombogenic surfaces. Thromb Haemost 1995;74:460–3
3.Ruggeri ZM. Mechanisms initiating platelet thrombus formation. Thromb Haemost 1997;78:611–6
4.Katori N, Tanaka KA, Szlam F, Levy JH. The effects of platelet count on clot retraction and tissue plasminogen activator-induced fibrinolysis on thrombelastography. Anesth Analg 2005;100:1781–5
5.Lang T, Toller W, Gutl M, Mahla E, Metzler H, Rehak P, Marz W, Halwachs-Baumann G. Different effects of abciximab and cytochalasin D on clot strength in thrombelastography. J Thromb Haemost 2004;2:147–53
6.Ray MJ, Hawson GA, Just SJ, McLachlan G, O’Brien M. Relationship of platelet aggregation to bleeding after cardiopulmonary bypass. Ann Thorac Surg 1994;57:981–6
7.Sarode R, Rawal A, Lee R, Shen YM, Frenkel EP. Poor correlation of supratherapeutic international normalised ratio and vitamin K-dependent procoagulant factor levels during warfarin therapy. Br J Haematol 2006;132:604–7
8.Spiess BD, Royston D, Levy JH, Fitch J, Dietrich W, Body S, Murkin J, Nadel A. Platelet transfusions during coronary artery bypass graft surgery are associated with serious adverse outcomes. Transfusion 2004;44:1143–8
9.Gajic O, Rana R, Winters JL, Yilmaz M, Mendez JL, Rickman OB, O’Byrne MM, Evenson LK, Malinchoc M, DeGoey SR, Afessa B, Hubmayr RD, Moore SB. Transfusion related acute lung injury in the critically ill: prospective nested case-control study. Am J Respir Crit Care Med 2007;176:886–9
10.Nuttall GA, Oliver WC, Santrach PJ, Bryant S, Dearani JA, Schaff HV, Ereth MH. Efficacy of a simple intraoperative transfusion algorithm for nonerythrocyte component utilization after cardiopulmonary bypass. Anesthesiology 2001;94:773–81
11.Shore-Lesserson L, Manspeizer HE, DePerio M, Francis S, Vela-Cantos F, Ergin MA. Thromboelastography-guided transfusion algorithm reduces transfusions in complex cardiac surgery. Anesth Analg 1999;88:312–9
12.Kettner SC, Panzer OP, Kozek SA, Seibt FA, Stoiser B, Kofler J, Locker GJ, Zimpfer M. Use of abciximab-modified thrombelastography in patients undergoing cardiac surgery. Anesth Analg 1999;89:580–4
13.Fries D, Krismer A, Klingler A, Streif W, Klima G, Wenzel V, Haas T, Innerhofer P. Effect of fibrinogen on reversal of dilutional coagulopathy: a porcine model. Br J Anaesth 2005;95:172–7
14.Tanaka KA, Taketomi T, Szlam F, Calatzis A, Levy JH. Improved clot formation by combined administration of activated factor VII (NovoSeven®) and fibrinogen (Haemocomplettan® P). Anesth Analg 2008;106:732–8
15.Khurana S, Mattson JC, Westley S, O’Neill WW, Timmis GC, Safian RD. Monitoring platelet glycoprotein IIb/IIIa-fibrin interaction with tissue factor-activated thromboelastography. J Lab Clin Med 1997;130:401–11
16.Nielsen VG, Geary BT, Baird MS. Evaluation of the contribution of platelets to clot strength by thromboelastography in rabbits: the role of tissue factor and cytochalasin D. Anesth Analg 2000;91:35–9
17.Lang T, Bauters A, Braun SL, Potzsch B, von Pape KW, Kolde HJ, Lakner M. Multi-centre investigation on reference ranges for ROTEM thromboelastometry. Blood Coagul Fibrinolysis 2005;16:301–10
18.Gottumukkala VN, Sharma SK, Philip J. Assessing platelet and fibrinogen contribution to clot strength using modified thromboelastography in pregnant women. Anesth Analg 1999; 89:1453–5
19.Nielsen VG, Cohen BM, Cohen E. Effects of coagulation factor deficiency on plasma coagulation kinetics determined via thrombelastography: critical roles of fibrinogen and factors II, VII, X and XII. Acta Anaesthesiol Scand 2005;49:222–31
20.Plow EF, Haas TA, Zhang L, Loftus J, Smith JW. Ligand binding to integrins. J Biol Chem 2000;275:21785–8
21.Chun R, Poon MC, Haigh J, Seal D, Donahue B, Royston D. Case 1–2005: cardiac surgery in congenital afibrinogenemia with thrombo-occlusive disease. J Cardiothorac Vasc Anesth 2005;19:109–17
22.Elodi S, Varadi K. Optimization of conditions for the catalytic effect of the factor IXa-factor VIII complex: probable role of the complex in the amplification of blood coagulation. Thromb Res 1979;15:617–29
23.Wagner CL, Mascelli MA, Neblock DS, Weisman HF, Coller BS, Jordan RE. Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets. Blood 1996;88:907–14
24.Slichter SJ. Relationship between platelet count and bleeding risk in thrombocytopenic patients. Transfusion Med Rev 2004;18:153–67
25.Chakroun T, Gerotziafas GT, Seghatchian J, Samama MM, Hatmi M, Elalamy I. The influence of fibrin polymerization and platelet-mediated contractile forces on citrated whole blood thromboelastography profile. Thromb Haemost 2006;95:822–8
26.Phillips DR, Charo IF, Parise LV, Fitzgerald LA. The platelet membrane glycoprotein IIb-IIIa complex. Blood 1988;71:831–43
27.Mann KG, Butenas S, Brummel K. The dynamics of thrombin formation. Arterioscler Thromb Vasc Biol 2003;23:17–25
28.Poon MC, D’Oiron R. Recombinant activated factor VII (NovoSeven) treatment of platelet-related bleeding disorders. Blood Coagul Fibrinolysis 2000;11:S55–S68
29.Lincoff AM, LeNarz LA, Despotis GJ, Smith PK, Booth JE, Raymond RE, Sapp SK, Cabot CF, Tcheng JE, Califf RM, Effron MB, Topol EJ. Abciximab and bleeding during coronary surgery: results from the EPILOG and EPISTENT trials. Improve long-term outcome with abciximab GP IIb/IIIa blockade. Evaluation of platelet IIb/IIIa inhibition in STENTing. Ann Thorac Surg 2000;70:516–26
30.Ni H, Denis CV, Subbarao S, Degen JL, Sato TN, Hynes RO, Wagner DD. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. J Clin Invest 2000;106:385–92
31.Charbit B, Mandelbrot L, Samain E, Baron G, Haddaoui B, Keita H, Sibony O, Mahieu-Caputo D, Hurtaud-Roux MF, Huisse MG, Denninger MH, de Prost D. The decrease of fibrinogen is an early predictor of the severity of postpartum hemorrhage. J Thromb Haemost 2007;5:266–73
32.Velik-Salchner C, Haas T, Innerhofer P, Streif W, Nussbaumer W, Klingler A, Klima G, Martinowitz U, Fries D. The effect of fibrinogen concentrate on thrombocytopenia. J Thromb Haemost 2007;5:1019–25
33.Meade TW. Fibrinogen measurement to assess the risk of arterial thrombosis in individual patients: yes. J Thromb Haemost 2005;3:632–4
34.Lowe GD. Fibrinogen measurement to assess the risk of arterial thrombosis in individual patients: not yet. J Thromb Haemost 2005;3:635–7
35.Reinhart WH. Fibrinogen—marker or mediator of vascular disease? Vasc Med 2003;8:211–6
36.Hiippala ST. Dextran and hydroxyethyl starch interfere with fibrinogen assays. Blood Coagul Fibrinolysis 1995;6:743–6