Fragment X (frag X), a clottable degradation product of fibrinogen, accumulates in patients given tissue-type plasminogen activator (tPA). Previously, we demonstrated that frag X incorporation into fibrin (Fn) clots renders them more susceptible to lysis, raising the possibility that tPA-induced frag X formation contributes to bleeding. To explore the mechanism by which frag X incorporation renders clots more susceptible to lysis, we first examined the effects of clotted frag X on rates of plasminogen (Pg) activation by tPA. Samples containing a total of 3 UM fibrinogen (Fg) and frag X in varying proportions, 0.5 uM Glu-Pg, 0.4 mM S-2251, and 0.1 nM tPA were clotted with 5 nM thrombin, and absorbance was measured at 405 and 490 nm to monitor plasmin (Pn) formation and clot formation/lysis, respectively. Frag X produces a concentration-dependent decrease in lysis times. Rates of Pn formation were 3-fold higher with frag X clots than with Fn clots, suggesting that frag X promotes Pn formation. To explore this possibility, we compared the effect of frag X and Fn on the kinetics of Glu-Pg activation by tPA. Compared with Fn clots, the kci| for Glu-Pg activation by tPA is approximately 2-fold higher with frag X clc ts, whereas the Km is 4-fold lower. Consequently, the catalytic efficiency (kJ is 8-fold hig icr with frag X clots than with Fn clots. Although carboxypeptidase B (CPB) prolongs ihe lysis times of both frag X and Fn clots, the rates of Pn formation with frag X clots remain 3fold higher than those with Fn clots in the presence of CPB, suggesting that increased 'n formation in the presence of frag X clots is not dependent on C-terminal lysine residues. In addition to enhancing Pn formation, frag X also alters clot structure. Frag X produce ; a concentration-dependent increase in turbidity, suggesting that thicker fibres are form :d. Furthermore, frag X-containing clots not only promote Pg activation to a greater extent, ut also are more readily degraded by Pn, suggesting that frag X clots are more susceptible to lysis than Fn clots. To explore the possibility that frag X is preferentially released from he clot, we monitored labelled fibrin degradation products (FDP) released from clots contain ng 1251-labelled Fg or frag X. FDP release from Fn clots and frag X clots is similar, makin ; it unlikely that frag X is preferentially released because it already is partially degraded. Th :se studies reveal that frag X clots are more susceptible to lysis than Fn clots because (a) f'ag X enhances Pn formation by tPA, and (b) frag X clots are more sensitive to plasmin-mediated degradation. !