FIBRIN STRUCTURES DURING TISSUE-TYPE PLASMINOGEN ACTIVATOR-MEDIATED FIBRINOLYSIS STUDIED BY LASER-LIGHT SCATTERING - RELATION TO FIBRIN ENHANCEMENT OF PLASMINOGEN ACTIVATION

The aim was to relate fibrin structure and the stimulatory effect of f ibrin on plasminogen activation during t-PA-mediated fibrinolysis usin g Lys(78)-plasminogen as activator substrate. Structural studies were undertaken by static and dynamic laser light scattering, cryo transmis sion electron microscopy and by the measurement of conversion of fibri n to X-, Y- and D-fragments. The kinetics of plasmin formation were mo nitored by measurement of the rate of pNA-release from Val-Leu-Lys-pNA . The process of fibrin formation and degradation comprised three phas es. In the first phase, protofibrils with an average length of about 1 0 times that of fibrinogen were formed. The duration of this phase dec reased with increasing t-PA concentration. The second phase was charac terized by a sudden elongation and lateral aggregation of fibrin fiber s, most pronounced at low levels of t-PA, and by formation of fragment X-polymer. The third phase was dominated by fragmentation of fibers a nd by formation of Y- and D-fragments. Plasmin degraded the fibers fro m within, resulting in the formation of long loose bundles, which subs equently disintegrated into thin filaments with a length of less than 10 and a mass per length close to one relative to fibrinogen. Plasmin generation at high t-PA concentrations sets in just prior to (and at l ow t-PA concentrations shortly after) the onset of the rapid second ph ase of elongation and lateral aggregation of fibrin fibers. The maxima l rate of plasmin formation per mol t-PA was the same at all concentra tions of activator and was achieved close to the time of the peak leve l of fragment X-polymer. Plasmin formation ceased after formation of s ubstantial amounts of Y- and D-fragments. At this stage the length was between 300 and 3 and the mass per length close to 1, both relative t o fibrinogen. In conclusion our results indicate that (1) formation of short fibrin protofibrils is the minimal requirement for the onset of the stimulatory effect of fibrin on plasminogen activation by t-PA, ( 2) formation of fragment X protofibrils is sufficient to induce optima l stimulation of plasminogen activation, and (3) plasmin degrades late rally aggregated fibrin fibers from within, resulting in the conversio n of the fibers into long loose bundles, which later disintegrate into thin filaments.