An Evaluation of Fibrin(ogen) Determinants of Thromboelastography

Abstract 2251

Thromboelastography (TEG) was used in ∼200 studies published in 2009, is mainly used for global blood coagulation assessment, and is thrombin and fibrinogen concentration-dependent. Reflecting the time course of fibrin polymerization, TEG measures lag time, rate, and maximum amplitude (MA) of clot stiffness, and is substantially enhanced by platelets. To identify more specific fibrin(ogen) determinants potentially relevant to interpretation we investigated MA under different TEG conditions. The procedure utilizes a polymethylmethacrylate (hydrophobic surface) cup and pin set, the cup oscillating through 4° 45' (6 cycles/minute). The pin (sensor) is suspended via a torsion wire, and transmits a tuning fork-like signal, displayed as a graph, as the clot links pin and cup. Clots cross-linked (by factor XIIIa) were shown insoluble in 6 M urea, and were obtained by adding thrombin to fibrinogen solutions, pH 7.4, containing 8 mM CaCl₂ and either 50 nM factor XIII or 30% afibrinogenemic plasma with or without gel-sieved platelets (190,000/μl). Except where otherwise stated, 6 μM fibrinogen was employed. In three sets of experiments without platelets, increasing thrombin (0.001 to 2 U /ml) progressively increased MA. For example, levels of 0.005, 0.1, 0.3, and 0.5 U/ml the yielded respective MAs of 16.7, 49, 62.8, and 65.3 mm. TEG enhancement by platelets reflects mainly their binding of the fibrin(ogen) C-terminal γ dodecapeptide and ensuing clot retraction. Several sets of experiments at different fibrinogen concentrations disclosed that the lower the concentration the more pronounced the platelet enhancement. For example, at of 0.3, 1, 3, and 6 μM fibrinogen (thrombin 0.4 U/ml), the respective control MAs were 0, 2.9, 15.9, 30.4, and 44.9 mm. The presence of platelets increased these to 10.6, 30.4, 44.9, and 64.8 mm, respectively. To test the role of the fibrin(ogen) αC region, two coagulable fibrinogen isolates (termed des-αC) lacking major C-terminal segments of varying length from their αC were prepared as described (Mosesson et al, J Biol Chem 249:4656). They displayed <5% intact Aα chains by gel scanning densitometry (sodium dodecyl sulfate-polyacrylamide electrophoresis of reduced protein bands). Estimated from the size of their Aα core remnants, most des-αC molecules lack the αC domain (Aα392–610) part of αC. Their clots yielded MA <2% that of fibrin with intact αC, n=5. In the presence of platelets, however, the MA was only moderately decreased (e.g. 53% ± 11%, SD, n=4) of controls. Fibrinogen adsorption to hydrophobic surfaces is known to be characteristically tight and to displace other plasma proteins, requires its D region, reaches near maximum of 2–3 molecule thick coating, ∼600 ng/cm² within 2 minutes, and enhances its coagulability (Koo et al, J Thromb Haemost, 8: 2727–35). Cup and pin (CP) were exposed to intact fibrinogen (100 μg/ml, 2 hours), and washed. In a duplicate experiment, addition and clotting of des-αC fibrinogen again yielded MA <2% of control. Coating with des-αC fibrinogen did not alter the MA of intact fibrin clots. Monoclonal IgG antibodies, anti-Aα518–584 (mAb1), and anti-Aα241–476 (mAb2), of which both moderately decrease clot turbidity but do not impair coagulability, were tested (3 mols/mol fibrin with intact αC, non-coated CP) in the presence of platelets. In two separate sets of experiments, mAb1 decreased MA to 11.6% and 9% and mAb2 to 0.6% and 1.7% of respective non-immune IgG controls. In two other separate experiments, coating of CP with incoagulable intact fibrinogen (Frankfurt XIII, homodimeric AαR16C), followed by addition and clotting normal intact fibrinogen yielded MAs of 31% and 38% of respective normal controls. To evaluate hydrophobic surface effects, the water contact angle was decreased from 68° to 13° by CP exposure to UV ozone plasma (5 minutes, n=2). MA of clots formed in treated CP was 41% and 36% of untreated controls. We conclude that fibrin(ogen) adsorption to the hydrophobic CP surface, coagulability of adsorbed fibrinogen, and the αC domain of adsorbed and non-adsorbed fibrin(ogen) are significant TEG determinants. The results also suggest a significant or auxiliary role by the αC domain in the platelet enhancement of TEG. The pronounced stiffness of clots formed on hydrophobic, relative to hydrophilic, surface is of possible relevance in atherothrombotic lesions and in medical implants with hydrophobic surface.