Remodeling of Clots without Proteolytic Digestion

Abstract 1184

Fibrin polymerization is a necessary part of hemostasis but thrombi can obstruct blood vessels and cause heart attacks and strokes, so the possibility of reversing clot formation has significant clinical implications. It is generally assumed that fibrin polymerization is an irreversible process and that clots and thrombi are stable structures until proteolytic digestion, but this supposition has not been critically tested. Using the technique of fluorescence recovery after photobleaching (FRAP) of individual fluorescently labeled fibrin fibers in a clot, we demonstrate here for the first time that there is turnover of fibrin in an uncrosslinked clot. These conditions occur in the vasculature during early stages of clotting or thrombosis before extensive Factor XIIIa-induced crosslinking. Increase of fluorescence in the bleached area was observed as soon as 1 second after photobleaching and the intensity increased rapidly. The association/dissociation of fibrin was characterized quantitatively, with the mobile fraction representing an average of 14.1% ± 5.1% of the total fibrin. Analyses of recovery curves showed that there was a linear relationship between both the percentage of fluorescence recovery and the rate of recovery and the surface to volume ratio of the fibers. Thus, it appears that fibrin monomers or oligomers located on or near the surface of the fibers are more likely to dissociate and re-associate. The mechanism of fibrin turnover was further investigated by using the peptide GPRP, mimicking the knobs ‘A’ involved in polymerization. Surprisingly, only 0.1 mM of GPRP was necessary to dissolve completely a preformed fibrin clot, and perfusion of 0.01 mM of GPRP through a clot decreased the mobile fraction of fibrin in FRAP experiments to 6.5% ± 1.7%. The kinetic parameters of fibrin turnover characterized from FRAP recovery curves revealed that the off rate was not affected by the GPRP concentration, but the equilibrium concentration of the bound complex decreased with increasing peptide concentration, likely because the free GPRP competes with fibrin knobs ‘A’ for holes ‘a.’ As a result of the enhanced dissociation induced by this peptide, striking rearrangements in clot structure were visualized by confocal light microscopy and scanning electron microscopy. Transmission electron microscopy of structures released from the clot revealed both monomers and larger aggregates. The implications of this research are that clots and thrombi appear to be much more dynamic structures than has been previously believed. The observed modulation of clot structure in vitro suggests that in vivo clots and thrombi may be dissolved or remodeled as a result of changing the environmental conditions surrounding them. These findings may also be relevant to embolization, which could in part be a consequence of the reversibility of polymerization. Furthermore, such modulation of clot structure could be a target for potential therapeutic intervention.