Characterization of Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Human Plasma

Abstract

In this study, we have identified and characterized metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) in human plasma. Treatment of plasma with trypsin or aminophenylmercuric acetate resulted in activation of latent gelatinolytic activity. Fractionation of plasma by gelatin Sepharose chromatography resulted in the isolation of 72 kDa and 92 kDa gelatinases/type IV collagenases. The 72 kDa gelatinase was purified by gel filtration chromatography. Stromelysin-1 was isolated from plasma by Matrex green A affinity chromatography. Immunoblotting of plasma fractions with antibodies to unique peptide regions of human gelatinases differentiated the 72 kDa gelatinase from the 92 kDa gelatinase. Antibodies to the amino terminal peptides of each enzyme were used to determine that plasma gelatinases circulate as latent proenzymes. Immunoblotting with antibodies directed against human stromelysin identified a 57 kDa stromelysin. TIMP-1 (28 kDa) and TIMP-2 (21 kDa) were also identified by immunoblotting of gelatin Sepharose bound plasma proteins using non-crossreacting antibodies to each protein.

Oncostatin M Differentially Regulates Tissue Inhibitors of Metalloproteinases TIMP‐1 and TIMP‐3 Gene Expression in Human Synovial Lining Cells

“…TIMP-I, on the other hand, is a secretory protein and can be determined in body fluids such as plasma and synovial fluids [42][43][44][45][46]. Since oncostatin M induces TIMP-1 but suppresses TIMP-3, its physiological role in inflammatory processes might be to destabilize the inatrix in the local environment but to stabilize it systemically.…”

Thrombin Induces the Activation of Progelatinase A in Vascular Endothelial Cells

“…Western blotting was performed using affinity-purified rabbit polyclonal antibodies directed against purified native proteins (TIMP-1, TIMP-2) and a synthesized peptide corresponding to the sequence of gelatinase A (amino acids 67-89) as described previously (35). Recombinant proteins were used as positive controls in immunoblotting.…”

Serum or Plasma Samples?

“…13 Approximately 20 years ago, it was first described that peripheral blood contains several forms of MMPs 47 (ie, soluble constitutive form in plasma, and intracellular zymogens in platelets and leukocytes). 48,49 The discrepancy in MMP concentrations between serum and plasma was reported for the first time Ϸ15 years ago, 14 but only beginning in 1996 17 has the influence of the blood sampling process on MMP concentrations and zymographic profiles been carefully evaluated. 18 -41 In particular, although MMP-2 and TIMP-2 (activity and concentration) show no significant difference between plasma and serum, 15,19,23,24,26,30 -32,34,36,50 the levels and zymographic separation of the MMP-1, -8, and -9 forms and TIMP-1 are strongly affected by anticoagulants.…”

“…23,64,65 The major differences between plasma and serum involve TIMP-1 and MMP-1, -8, and -9 forms, and to a much lesser extent MMP-2, which is considered the constitutive gelatinase circulating in blood. [47][48][49] The presence of MMPs in higher amounts in serum than in plasma is related not only to disease status but also to coagulation/fibrinolytic pathways. 38 In fact, the rapid separation of serum (minimizing the time between blood drawing and centrifugation) does not prevent the artificially high MMP-9 content in serum.…”