Decreased lateral aggregation of a variant recombinant fibrinogen providesinsight into the polymerization mechanism

We analyzed the polymerization of B beta A68T fibrinogen, the recombinant c ounterpart of fibrinogen Naples, a variant known to have decreased thrombin binding. When polymerized with equal thrombin concentrations, B beta A68T fibrinogen had a longer lag time and lower rate of lateral aggregation, V-m ax, than normal recombinant fibrinogen, but a similar final turbidity. At t hrombin concentrations that equalized the rates of fibrinopeptide A release , B beta A68T fibrinogen polymerized with a lag time and V-max similar to n ormal, but reached a significantly lower final turbidity. Similar results w ere produced when B beta A68T was polymerized with Ancrod, which cleaves fi brinopeptide A at the same rate from either fibrinogen, and when B beta A68 T desA monomers were polymerized. The polymerization of desAB fibrin monome rs, which circumvents fibrinopeptide release, was the same for both fibrino gens. We confirmed that turbidity was indicative of fiber thickness by scan ning electron microscopy of fibrin clots. Here, we present the first experi mental evidence of fibrin polymerization with a normal period of protofibri l formation and rate of lateral aggregation, but with a significantly decre ased extent of lateral aggregation. We conclude that the decreased lateral aggregation seen in B beta A68T fibrinogen is due to an altered step in the enzymatic phase of its polymerization process. We propose that during norm al polymerization a subtle conformational change in the E domain occurs, be tween the release of FpA and FpB, and that this change modulates the mechan ism of lateral aggregation. Without this change, the lateral aggregation of B beta A68T fibrinogen is impaired such that variant clots have thinner fi bers than normal clots.