Amorphous calcium phosphate-mediated binding of matrix metalloproteinase-9 to fibrin is inhibited by pyrophosphate and bisphosphonate.

Abstract

Coordinate regulation of fibrinolytic and collagenolytic systems is essential for normal tissue remodeling and wound healing. To define the molecular mechanisms which link these two proteolytic systems, we have investigated the role of fibrin in matrix metalloproteinase (MMP) function. Both active and latent forms of MMP-9 (gelatinase B) bind to fibrin in a selective, dose-dependent manner; latent enzyme is activated by plasmin during fibrinolysis. Fibrin binding of MMP-9 is mediated by amorphous calcium phosphate (ACP), and proceeds in a step-wise fashion with formation of ACP as the first and rate-limiting step. MMP-9 rapidly binds preformed ACP to yield a transient ACP: MMP-9 complex that avidly binds fibrin. Here we report the effect(s) on fibrin: ACP: MMP-9 formation/dissociation of pyrophosphate (POP), an endogenous calcification inhibitor, and its bisphosphonate analog, alendronate (PCP). MMP-9 was obtained from neutrophil lysate and ACP formation was monitored turbidimetrically. Free MMP-9, ACP: MMP-9 and fibrin: ACP: MMP-9 complexes were analyzed by gelatin zymography. POP at physiologic concentrations (0.5-2.5 microM) inhibited both ACP formation and subsequent fibrin binding of MMP-9 at orthophosphate concentrations of 250 microM. PCP exhibited a similar inhibitory effect. With both substances, inhibition was slightly overcome (>2.5 microM) by higher phosphate (500 microM). In contrast, supraphysiologic concentrations of either POP or PCP (>50 microM) were required to inhibit MMP-9 binding to preformed ACP or to induce dissociation of preformed ACP: MMP-9 complexes (50-100 microM). Neither POP nor PCP had any effect on preformed fibrin: ACP: MMP-9 at concentrations up 1 mM. POP is an endogenous by-product of numerous metabolic pathways and may regulate bone turnover, soft tissue calcification, and contribute to the pathogenesis of calcium pyrophosphate crystal disease (CPPD). These studies support another role for POP and fibrin: ACP: MMP-9 complexes in physiologic and pathologic processes, including tumorigenesis and cancer metastasis.