Abstract
Abstract 1154
Fibrinogen, a 340-kDa glycoprotein has essential roles in blood coagulation and platelet aggregation. Fibrinogen is a complex molecule consisting of 3 pairs of Aα, Bβ, and γ chains intertwined to form a tri-nodular molecule with 2 terminal D regions and a central E region. The fibrinogen γ-nodule, a part of D region, has several important sites relating to fibrinogen function, which are the high affinity calcium binding site, hole ‘a’ that binds with knob ‘A’, and the D:D interface. Residue γAla341, which is located in the vicinity of those sites and conserved between all available species, is altered in two variant fibrinogens: fibrinogen Seoul (γAla341Asp) (Song et al., Clin Appl Thromb Hemost 2006) and fibrinogen Tolaga Bay (γAla341Val) (Davis et al., Thromb Haemost 2007). Fibrinogen Seoul showed hypodysfibrinogenemia, and fibrinogen Tolaga Bay hypofibrinogenemia. We have expressed these two variant fibrinogens in CHO cells, purified them from the culture media and performed biochemical tests to elucidate their function. Thrombin-catalyzed kinetics of FpA release was not different (p-value, 0.3, n=3) from normal fibrinogen. Average specificity constant, kcat/Km for FpA was 7.4±1.8, 5.7±1.8, and 8.1±1.2 (mean±SD, 106M-1s-1) for normal, γAla341Val, and γAla341Asp, respectively. However, FpB release from both variants was slower than that of normal (p-value, 0.005 in One Way ANOVA; p-value, 0.006 and 0.002 for normal vs γAla341Val, and normal vs γAla341Asp, respectively in multiple comparison). Average specificity constant for FpB was 3.6±1.1, 1.7±0.4, and 1.4±0.3 (mean±SD, 106M-1s-1) for normal, γAla341Val, and γAla341Asp, respectively. We measured fibrin polymerization by turbidity with the final thrombin and fibrinogen concentration being 0.2mg/mL and 0.1U/mL, respectively. At 10mM calcium we saw no turbidity rising with either variant. Both variants showed impaired polymerization with a longer lag period and a slower Vmax than normal fibrinogen at physiologic 1mM calcium. Lag period, which reflects protofibril formation, for normal, γAla341Val, and γAla341Asp was 110±30, 2,500±150, and 1,200±40, respectively (mean±SD, sec). Vmax, which reflects lateral aggregation, was 81±26, 15±3, and 3±1 (mean±SD, 10-5s-1) for normal, γAla341Val, and γAla341Asp. With the FXIIIa cross-linking, measured by SDS-PAGE, we found that γ and α chain cross-linking was delayed in both variants. We tested the calcium binding and the functionality of ‘a’ polymerization site with the plasmin protection assay. Both variants were not protected from plasminolysis in the presence of 1mM calcium or 0.55mM GPRP, indicative of impaired binding of calcium and knob ‘A’. Given these results, both fibrinogen Seoul and Tolaga Bay likely have a conformational change in their calcium and GPRP binding sites resulting in the impaired fibrin polymerization. In conclusion, we think fibrinogen residue γAla341 is important for calcium binding, ‘A-a’ interactions and the conformation of the γ-nodule.
Disclosures:
No relevant conflicts of interest to declare.
Characterization of the Novel Ene Reductase Ppo-Er1 from Paenibacillus Polymyxa
