Recombinant Fibrinogen Studies Reveal That Thrombin Specificity Dictates Order of Fibrinopeptide Release

During cleavage of fibrinogen by thrombin, fibrinopeptide A (FpA) release precedes fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A'beta fibrinogen, replacing FpB with a fibrinopeptide A-like peptide, FpA' (G14V). Analyses of fibrinopeptide release from A'beta fibrinogen showed that FpA release and FpA' release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA' were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA' release from A'beta fibrinogen was unaffected. Thus, with A'beta fibrinogen, fibrinopeptide release from the beta chain is similar to fibrinopeptide release from the alpha chain. We conclude that the ordered release of fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A'beta fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A'beta fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the fibrinopeptide from the N terminus of the beta chain does not affect polymerization of fibrinogen

Does Topology Drive Fiber Polymerization?

[Image: see text] We have developed new procedures to examine the early steps in fibrin polymerization. First, we isolated fibrinogen monomers from plasma fibrinogen by gel filtration. Polymerization of fibrinogen monomers differed from that of plasma fibrinogen. The formation of protofibrils was slower and the transformation of protofibrils to fibers faster for the fibrinogen monomers. Second, we used formaldehyde to terminate the polymerization reactions. The formaldehyde-fixed products obtained at each time point were examined by dynamic light scattering and transmission electron microscopy (TEM). The data showed the formaldehyde-fixed products were stable and representative of the reaction intermediates. TEM images showed monomers, short oligomers, protofibrils, and thin fibers. The amount and length of these species varied with time. Short oligomers were less than 5% of the molecules at all times. Third, we developed models that recapitulate the TEM images. Fibrin monomer models were assembled into protofibrils, and protofibrils were assembled into two-strand fibers using Chimera software. Monomers were based on fibrinogen crystal structures, and the end-to-end interactions between monomers were based on D-dimer crystal structures. Protofibrils assembled from S-shaped monomers through asymmetric D:D interactions were ordered helical structures. Fibers were modeled by duplicating a protofibril and rotating the duplicate 120° around its long axis. No specific interactions were presumed. The two protofibrils simply twisted around one another to form a fiber. This model suggests that the conformation of the protofibril per se promotes the assembly into fibers. These findings introduce a novel mechanism for fibrin assembly that may be relevant to other biopolymers