Melinda L. Ramsby

Binding of Latent Matrix Metalloproteinase 9 to Fibrin: Activation via a Plasmin-Dependent Pathway

The binding of two matrix metalloproteinases (MMP) to fibrin was evaluated. MMP-2 (72-kDa) and MMP-9 (92-, 130-, and 225-kDa) were selected since both contain a fibronectin-like region and fibronectin binds fibrin. Gelatin zymography indicated selective and dose dependent binding of MMP-9 to fibrin. No MMP-2 binding to fibrin occurred. Densitometry revealed that the 130- and 225-kDa forms demonstrated similar sigmoidal binding profiles whereas 92-kDa uptake was hyperbolic. Fibronectin and TIMP-1 competition studies indicated that the fibronectin and C-terminal MMP-9 domains, respectively, were not involved with fibrin binding. The MMP-9 collagen-like region may be of regulatory significance since type I and II fibrillar and type IV basement membrane collagens demonstrated fibrin binding. During fibrinolysis, latent fibrin-bound MMP-9 was processed to lower molecular weight forms consistent with proteolytic activation. This process was inhibited by ∈-aminocaproic acid, indicating a plasmin-dependent pathway. The significance of these findings to procoagulant activity and MMP-mediated extracellular matrix destruction during inflammation and tumor invasion and metastasis is discussed.

Identification and partial characterization of three calcium- and zinc-independent gelatinases constitutively present in human circulation

Three constitutive gelatinases in human plasma were identified and characterized relative to known matrix metalloproteinase (MMP) gelatinases: MMP‐2 (fibroblast 72‐kDa) and MMP‐9 (neutrophil 92‐, 130‐, and 225‐kDa). Substrate gel electrophoresis (gelatin zymography) revealed an apparent Mw of 78‐, 82‐, and 89‐ kDa for these gelatinases. Densitometry revealed that MMP‐9 and MMP‐2 were highly calcium sensitive requiring 50‐150 μM and 500 μM calcium for half‐maximal activity, respectively. Of the new gelatinases, only the 89‐kDa form demonstrated slight calcium activation. The three gelatinases were unaffected by known MMP inhibitors: EDTA (5 mM), 1,10‐phenanthroline (2 mM), and pepstatin (18 μM). Serine and thiol protease inhibitors (leupeptin, aprotinin, PMSF, TLCK, TPCK, antichymostatin, antipain) were also ineffective. Solution‐phase IEF revealed that the 78‐ and 82‐kDa forms focused at neutral pl 6.72‐7.95 whereas the 89‐kDa focused at an acidic pI 4.89‐5.18 (similar to neutrophil and fibroblast forms). The data indicate that these gelatinases are not MMPs or partially activated MMPs. Their role in normal and pathological conditions is not known.

Amorphous Calcium Phosphate-Mediated Binding of Matrix Metalloproteinase-9 to Fibrin is Inhibited by Pyrophosphate and Bisphosphonate

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 μM) inhibited both ACP formation and subsequent fibrin binding of MMP-9 at orthophosphate concentrations of 250 μM. PCP exhibited a similar inhibitory effect. With both substances, inhibition was slightly overcome (>2.5 μM) by higher phosphate (500 μM). In contrast, supraphysiologic concentrations of either POP or PCP (>50 μM) were required to inhibit MMP-9 binding to preformed ACP or to induce dissociation of preformed ACP:MMP-9 complexes (50–100 μM). 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.

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 μM) (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.

Interaction of amorphous calcium phosphate with fibrin in vitro causes decreased fibrinolysis and altered protease profiles: implications for atherosclerotic disease.

Previously, we demonstrated that amorphous calcium phosphate (ACP), chemical precursor to apatite, strongly interacted with fibrin and facilitated binding of matrix metalloproteinase (MMP)-9, a type IV collagenase. Plasmin-dependent fibrinolysis resulted in coordinate MMP-9 activation. Here we report on the effect(s) of ACP on fibrin degradation and binding of endogenous plasma proteases. Electrophoresis (8.5% SDS-PAGE) revealed that fibrin formed in the presence of ACP demonstrated characteristic γ-γ dimers (90-kDa) and β-monomers (55-kDa), but resisted spontaneous fibrinolysis (72 h, 37°C) or degradation by plasminogen activators (uPA, tPA). Casein zymography revealed an ACP-dependent decrease in fibrin binding of a low molecular weight (Mw) protease triplet (47-, 43-, 42-kDa) and increased fibrin binding of two high Mw proteases (94- and 84-kDa). The low Mw triplet also possessed gelatinolytic activity, but was not an MMP since 1,10-phenanthroline was ineffective as an inhibitor. Fibrin-binding proteases were inhibited to some degree by the serine protease inhibitor aprotinin. Competition/dissociation experiments with ∈-aminocaproic acid revealed that the low Mw triplet lacked kringle regions whereas the 94- and 84-kDa proteases were tentatively identified and glu-/lys-plasmin(ogen)s. The triplet may, however, represent one or more kringle deficient mini-plasminogen(s), since electrophoretic mobility and substrate specificity was similar to elastase-generated mini-plasminogen. To explore these findings in a clinically relevant setting, a series of plasma samples was collected from a patient with unstable angina prior to, during, and post coronary artery bypass graft (CABG) surgery. Fibrin formed from plasma collected during and immediately post CABG was associated with increased fibrinolytic capacity and enhanced binding of a) MMP-9, b) the low Mw protease triplet (described above), and c) PA (as putative 110-kDa tPA:PAI-1 complex). The relevance of these findings to pathologic calcification of atherosclerotic plaques is discussed.

Zymographic Evaluation of Plasminogen Activators and Plasminogen Activator Inhibitors

Publisher Summary The plasminogen activator/plasminogen activator inhibitor (PA/PAI) system is of considerable fundamental importance in elucidating the biochemical mechanisms of many physiological and patho-physiological processes. Although many techniques are available to assess the PA/PAI system, including ELISA and molecular assays, fibrin overlay zymography provides a highly versatile tool for the detection and semiquantitative investigation of PAs, PAIs, and PA/PAI complexes. The chapter discusses monitoring the PA/PAI system such as enzyme-linked immunosorbent assay, molecular methods, substrate gel electrophoresis, fibrin overlay zymography, and advantages of overlay zymography. The chapter also discusses the usefulness of fibrin overlay zymography for the detection of PAs and PAIs in complex biological systems. Several semiquantitative results such as fibrin zymography of PAs, densitometric analysis of PA activity, fibrin zymography of PA induction in cell culture, reverse fibrin zymography for PAIs, and fibrin zymography of PAs from CABG patients are discussed in this chapter. Unique to the overlay method is the opportunity to incorporate into the indicator gel multiple components including activators, inhibitors, antibodies, zymogens, cofactors, and allosteric modifiers. This enables highly sensitive and specific assays to be developed, and thus affords extensive biochemical investigation of diverse proteolytic components including those of the PA/PAI system. The chapter describes the materials and methods required to perform fibrin overlay zymography such as material used, sample preparation, SDS-PAGE, preparation of fibrin indicator gel, fibrin zymography, and reverse zymography.

Gelatinolytic and fibrinolytic activity in fresh-frozen plasma

Fresh-frozen plasma (FFP) was evaluated for gelatinolytic and fibrinolytic activity. Gelatin zymography revealed that gelatinase A (MMP-2) was constitutively present in FFP whereas gelatinase B (MMP-9) was present at variable levels. The presence of MMP-9 likely represents differential release from neutrophils during FFP collection or processing. Although fibrin matrices generated from FFP or freshly prepared plasma contained characteristic crosslinked gamma-gamma dimers and beta-monomers, matrices generated from FFP were resistant to spontaneous plasmin-dependent fibrinolysis. This observation likely stems from the plasminogen activator instability and could potentially lead to a hypofibrinolytic state. The impact of these in vitro findings to protease balance in patients receiving multiple FFP doses remains to be determined.