Characterization of Stromelysin 1 (MMP-3), Matrilysin (MMP-7), and Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Derived Fibrin(ogen) Fragments D-Dimer and D-like Monomer: NH2-Terminal Sequences of Late-Stage Digest Fragments

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Abstract
Matrix metalloproteinases (MMPs) participate in physiological remodeling of the extracellular matrix. Recently we determined that both fibrinogen (Fg) and cross-linked fibrin (XL-Fb) are substrates for selected MMPs. Specifically, XL-Fb clots were solubilized by MMP-3 (stromelysin 1) by cleavage at γ Gly 404−Ala 405, resulting in a D-like monomer fragment. Similarly, MMP-7 (matrilysin) and MT1-MMP (membrane type 1 matrix metalloproteinase) solubilized XL-Fb clots. However, the molecular mass of fragment D-dimer, obtained after MMP-7 and MT1-MMP degradation of XL-Fb, is similar to that of fragment D-dimer from plasmin degradation (∼186 kDa). In contrast, fragment D-like monomer, from MMP-3 degradation of both fibrinogen (Fg) and XL-Fb, is similar to fragment D from plasmin degradation of Fg (∼94 kDa). Reduced chains from MMP-3, MMP-7, and MT1-MMP digests of Fg and XL-Fb were subjected to direct sequence analyses and D/D-dimer α-chain showed cleavage at both α Asp 97−Phe 98 and α Asn 102−Asn 103. Degradation of the β-chain resulted in microheterogeneity of cleavage sites at β Asp 123−Leu 124, β Asn 137−Val 138, and β Glu 141−Tyr 142, whereas all three enzymes cleaved the γ-chain at γ Thr 83−Leu 84. In both Fg and XL-Fb, several cleavage sites obtained by proteolysis with MMP-3, MMP-7, and MT1-MMP were found to be in very close proximity to those obtained by plasmin on these same substrates. That does not occur with other MMPs such as MMP-1, -2, and -9 and MT2-MMP. The degradation of XL-Fb by MMPs suggests both plasmin-dependent and independent mechanisms of fibrinolysis that might be relevant in inflammation, angiogenesis, arthritis, and atherosclerosis.