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Regulatory mechanism of matrix metalloprotease-2 enzymatic activity by factor Xa and thrombin.

ABSTRACT: Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and serine proteases. The mechanism of pro-MMP-2 activation by metalloproteases is well established, and the most studied activation mechanism involves cleavage of the propeptide by membrane type 1-MMP (MT1-MMP). In contrast, serine protease activation has not been thoroughly studied, although studies suggest that MT1-MMP may be involved in activation by thrombin and plasmin. Here, we demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 in vascular smooth muscle cells and endothelial cells. Factor Xa and thrombin directly cleaved the propeptide on the carboxyl terminal sides of the Arg(98) and Arg(101) residues, whereas plasmin only cleaved the propeptide downstream of Arg(101). Moreover, processed MMP-2 showed enzymatic activity that was enhanced by intermolecular autoproteolytic processing at the Asn(109)-Tyr peptide bond. In addition to its role in activation, factor Xa rapidly degraded MMP-2, thereby restricting excessive MMP-2 activity. Thrombin also degraded MMP-2, but the degradation was reduced greatly under cell-associated conditions, resulting in an increase in processed MMP-2. Overall, factor Xa and thrombin regulate MMP-2 enzymatic activity through its activation and degradation. Thus, the net enzymatic activity results from a balance between MMP-2 activation and degradation.

SUBMITTER: Koo BH

PROVIDER: S-EPMC2749111 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Regulatory mechanism of matrix metalloprotease-2 enzymatic activity by factor Xa and thrombin.

Koo Bon-Hun BH Park Michael Y MY Jeon Ok-Hee OH Kim Doo-Sik DS

The Journal of biological chemistry 20090706 35

Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and serine proteases. The mechanism of pro-MMP-2 activation by metalloproteases is well established, and the most studied activation mechan ...[more]

PMID: 19581306

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Project description:BackgroundThe dry root and rhizome of Ligusticum chuanxiong Hort., or Chuanxiong, has been used as a blood-activating and stasis-removing traditional Chinese medicine for 1000 years. Our previous studies have shown the inhibitory activity on platelet and thrombin (THR) of Chuanxiong. THR and factor Xa (FXa) play significant roles in the coagulation cascade and their inhibitors are of valuable in the treatment of thromboembolic diseases. The aim of the present study is to screen THR and FXa inhibitors from Chuanxiong.MethodsFour extracts [ethyl acetate (EA), butanol (BA) and remained extract (RE) from 75% ethanol extract, and water extract (WE)] of Chuanxiong were prepared, and their THR/FXa inhibitory activities were assessed in vitro. Following silica-gel column chromatography (SC), the active EA extract and BA extract was further partitioned, respectively. Their active fractions (EA-SC1 to EA-SC5; BA-SC1 to BA-SC5) were obtained and analyzed by LC-MS. After modeling by the principal component analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLS-DA), the specific marker compounds were predicted and identified. Their enzyme inhibitory was assessed in vitro and interactions with THR/FXa were investigated by molecular docking analysis.ResultsChuanxiong EA extract showed strong activity against THR and BA extract was more effective in inhibiting FXa activity, and their fractions exhibited obvious difference in enzyme inhibitory activity. Furthermore, marker compounds a-h were predicted by PCA and OPLS-DA, and their chemical structures were identified. Among them, senkyunolide A, Z-ligustilide, ferulic acid and senkyunolide I (IC50 was determined as 0.77 mM) with potential THR inhibitory activity, as well as isochlorogenic acid A with FXa inhibitory activity were screened out. It was found that the four components could interact with the active site of THR, and the binding energy was lower than - 5 kcal/mol. Isochlorogenic acid A were bound to the active site of FXa, and the binding energy was - 9.39 kcal/mol. The IC50 was determined as 0.56 mM.ConclusionsTHR/FXa inhibitory components in different extracts of Chuanxiong were successfully characterized by the method of enzyme inhibition activity assays with ultra performance liquid chromatography-quadrupole time of flight mass spectrometry-based multivariate statistical analysis.

Dataset Information

Regulatory mechanism of matrix metalloprotease-2 enzymatic activity by factor Xa and thrombin.

Publications

Regulatory mechanism of matrix metalloprotease-2 enzymatic activity by factor Xa and thrombin.

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