Structural analysis of recombinant fibrinogen fragment D revealed that the calcium-binding site (beta2-site) composed of residues BbetaAsp261, BbetaAsp398, BbetaGly263, and gammaGlu132 is modulated by the "B:b" interaction. To determine the beta2-site's role in polymerization, we engineered variant fibrinogen gammaE132A in which calcium binding to the beta2-site was disrupted by replacing glutamic acid at gamma132 with alanine. We compared polymerization of gammaE132A to normal fibrinogen as a function of calcium concentration. Polymerization of gammaE132A at concentrations of calcium <or=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 beta2-site controls the extent of lateral aggregation. That is, when the calcium anchor (beta2-site) is eliminated before "B:b" interactions occur then lateral aggregation is enhanced. We solved structures of fragment D of gammaE132A fibrinogen (rfD-gammaE132A) 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 beta2-site of gammaE132A fibrinogen at 70 mM calcium. We found that the gamma2 calcium-binding site (in loop gamma294-301) did not have calcium bound in the structure of fragment D of gammaE132A fibrinogen with GHRPam bound (rfD-gammaE132A+GH). Analysis of structures rfD-gammaE132A+GH and rfD-BbetaD398A+GH indicated that differences in calcium occupation of the gamma2-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.