BINDING OF MATRIX METALLOPROTEINASE-9 TO FIBRIN IS MEDIATED BY AMORPHOUS CALCIUM-PHOSPHATE

In our previous study we demonstrated selective, dose-dependent bindin g of matrix metalloproteinase-9 (MMP-9), a neutrophil collagenase, to fibrin. Here we investigated the mechanism of this interaction. We fou nd that MMP-9 to fibrin was dependent on formation of a calcium-phosph ate intermediate. The intermediate was precipitable by centrifugation and contained a Ca/P ratio of 1.52-1.54, consistent with amorphous cal cium-phosphate (ACP). ACP formation exhibited a temperature optimum at 37 degrees C. Gelatin zymography revealed that interaction of ACP wit h MMP-9 resulted in formation of a high molecular weight ACP:MMP-9 com plex which was required for MMP-9 binding to fibrin. Complex formation was dependent on the generation of viable ACP that required both calc ium (7.5-10 mM) and phosphate (225-250 mu M) (Ca x P product range, 1. 7-2.5 mM(2)). Carbonate (CO3) and sulfate (SO4) were ineffective as ca lcium counteranions. Preformed ACP rapidly complexed MMP-9. Thus ACP f ormation was rate-limiting for MMP-9 fibrin binding activity. No MMP-9 fibrin binding activity was noted at 25 degrees C, an observation con sistent with lack of ACP production. The significance of these finding s is discussed with respect to normal and pathologic wound healing.