Polymerized fibrin strongly enhances tissue plasminogen activator (tPA)-mediated plasminogen activation, concomitant with exposure of 'fibrin-specific' epitopes at 'Aα148-160' and 'γ312-324'. To investigate which aspects of polymerization are involved in these activities, we explored the fibrin polymerization process by evaluating the ability of factor XIIla-crosslinked fibrinogen polymers to expose 'fibrin-specific' epitopes and enhance plasminogen activation. Crosslinked normal fibrinogen, fibrinogen with deficient [des Bβ 1-42] or defective [Birmingham (AαR16H)] fibrin 'D:E' assembly sites ('E(A)'), or with defective end-to-end self-association sites ('D:D') [Cedar Rapids (γR275C)], exposed both 'fibrin-specific' epitopes and enhanced tPA-dependent plasminogen activation, whereas non-crosslinked fibrinogens showed minimal or no such activities. Epitope expression in crosslinked fibrinogen was retained in the presence of the fibrin E(A) site peptide homolog, gly-pro-arg-pro (GPRP), which inhibits fibnin D:E association, except for the Aα148-160 epitope in des Bβ1-42 fibrinogen, which was nor expressed. Fibrin prepared from crosslinked normal or abnormal fibrinogen, except for the des Bβ1-42 fibrin epitopes, which were reduced or absent, expressed 'fibrin-specific' epitopes even in the presence of GPRP, which otherwise impairs such expression in non-crosslinked fibrin. Epitope exposure in fibrin prepared from non-crosslinked fibrinogen was nearly normal in Cedar Rapids fibrin (heterozygous D:D defect), but reduced in Birmingham fibrin (heterozygous E(A) defect), nil in des Bβ1-42 fibrin (E(A) deficient), and absent in all cases in the presence of GPRP. In contrast, plasminogen activation stimulatory activity that had been exposed in crosslinked normal fibrinogen or in crosslinked des Bβ1-42 or Cedar Rapids fibrin, was preserved to a large extent in the presence of GPRP, suggesting that once enhanced stimulatory activity and epitopes are exposed, they are not completely reversible. The findings indicate that end-to-end intermolecular associations (D:D) are not critical for 'fibrin-specific' epitope exposure, but that polymerization brought about in fibrinogen through factor XIIIa crosslinking, or in fibrin through 'D:E' interactions, is necessary for 'fibrin-specific' (more correctly, 'polymerization-specific') epitope exposure and enhancement of plasminogen activation.