Calcium-binding site beta 2, adjacent to the "b" polymerization site, modulates lateral aggregation of protofibrils during fibrin polymerization.

Structural analysis of recombinant fibrinogen fragment D revealed that the calcium-binding site (β2-site) composed of residues BβAsp261, BβAsp398, BβGly263, and yGlul32 is modulated by the "B:b" interaction. To determine the β2-site's role in polymerization, we engineered variant fibrinogen yE132A in which calcium binding to the β2-site was disrupted by replacing glutamic acid at γ132 with alanine. We compared polymerization of yE132A to normal fibrinogen as a function of calcium concentration. Polymerization of γE132A at concentrations of calcium ≤ 1 mM exhibited an uncharacteristic 2-3-fold increase in lateral aggregation and fiber thickness compared to normal fibrinogen, while polymerization of variant and normal were indistinguishable at 10 mM calcium. These results suggest that the β2-site controls the extent of lateral aggregation. That is, when the calcium anchor (β2-site) is eliminated before "B:b" interactions occur then lateral aggregation is enhanced. We solved structures of fragment D of yE132A fibrinogen (rfD-yE132A) with and without Gly-His-Arg-Pro-amide (GHRPam) and found no change to the global structure. X-ray diffraction data showed GHRPam binding in the "a" and "b" polymerization sites and that calcium could still bind to the β2-site of yE132A fibrinogen at 70 mM calcium. We found that the y2 calcium-binding site (in loop γ294-301) did not have calcium bound in the structure of fragment D of γE132A fibrinogen with GHRPam bound (rfD-γE132A+GH). Analysis of structures rfD-γE132A+GH and rfD-BβD398A+GH indicated that differences in calcium occupation of the y2-site resulted from minor conformational changes provoked by crystal packing and GHRPam binding to the "a" site did not directly modulate calcium binding to this site.