Binding of Matrix Metalloproteinase 9 to Fibrin is Mediated by Amorphous Calcium-Phosphate

In our previous study we demonstrated selective, dose-dependent binding of matrix metalloproteinase-9 (MMP-9), a neutrophil collagenase, to fibrin. Here we investigated the mechanism of this interaction. We found that MMP-9 to fibrin was dependent on formation of a calcium-phosphate intermediate. The intermediate was precipitable by centrifugation and contained a Ca/P ratio of 1.52–1.54, consistent with amorphous calcium-phosphate (ACP). ACP formation exhibited a temperature optimum at 37°C. Gelatin zymography revealed that interaction of ACP with MMP-9 resulted in formation of a high molecular weight ACP : MMP-9 complex which was required for MMP-9 binding to fibrin. Complex formation was dependent on the generation of viable ACP that required both calcium (7.5–10 mM) and phosphate (225–250 M) (Ca × P product range, 1.7–2.5 mM²). Carbonate (CO₃) and sulfate (SO₄) were ineffective as calcium counteranions. Preformed ACP rapidly complexed MMP-9. Thus ACP formation was rate-limiting for MMP-9 fibrin binding activity. No MMP-9 fibrin binding activity was noted at 25°C, an observation consistent with lack of ACP production. The significance of these findings is discussed with respect to normal and pathologic wound healing.