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Platelet release of trimolecular complex components MT1-MMP/TIMP2/MMP2: involvement in MMP2 activation and platelet
I Kazes1, I Elalamy, J D Sraer
1INSERM U489 and Association Claude Bernard, Tenon Hospital, and Unité de Pharmacologie Cellulaire, Unité Associée, Pasteur Institute INSERM U485, Paris, France. kazes@b3e.jussieu.fr
Abstract:
Matrix metalloproteinase 2 (MMP2) has been reported to be secreted by collagen-stimulated platelets, and active MMP2 has been shown to play a role in platelet aggregation. It has been demonstrated that MMP2 activation is dependent on the complex (membrane type 1 [MT1]-MMP/tissue inhibitor of MMP2 [TIMP2]) receptor and MMP2. We have investigated human platelets as a possible source of MT1-MMP, and we have studied its role in MMP2 activation and in platelet aggregation. Gelatin zymograms showed the existence of MMP2 at proforms (68 kd) and activated-enzyme forms (62-59 kd) in supernatants of resting and activated platelets, respectively. No gelatinolytic activity was associated with the platelet pellet after aggregation, suggesting a total release of MMP2 during cell activation. By Western blot analysis in nonreduced conditions, MT1-MMP was found on resting platelet membranes in 2 forms-the inactive 45-kd form and an apparent 89-kd form, which totally disappeared under reduced conditions. After platelet degranulation, only the 45-kd form was detected. Reverse transcription-polymerase chain reaction experiments showed the expression in platelets of messenger RNA encoding for MMP2, MT1-MMP, and TIMP2. Flow cytometry analysis showed that MT1-MMP, MMP2, and TIMP2 expressions were enhanced at the activated platelet surface. MMP inhibitors, recombinant TIMP2, and synthetic BB94 inhibited collagen-induced platelet aggregation in a concentration-dependent manner, indicating the role of activated MT1-MMP in the modulation of platelet function. In conclusion, our results demonstrate the expression of the trimolecular complex components (MT1-MMP/TIMP2/MMP2) by blood platelets as well as the ability of MMP inhibitors to modulate the aggregating response.
Insights
Platelets express matrix metalloproteinase 2 (MMP2) and membrane type 1-MMP (MT1-MMP), crucial for MMP2 activation and platelet aggregation. Inhibiting MMPs modulates platelet function.
Area of Science:
- Biochemistry
- Hematology
- Molecular Biology
Background:
- Matrix metalloproteinase 2 (MMP2) is secreted by collagen-stimulated platelets and influences platelet aggregation.
- MMP2 activation depends on the membrane type 1 (MT1)-MMP/tissue inhibitor of MMP2 (TIMP2) complex.
- Human platelets are investigated as a source of MT1-MMP and its role in MMP2 activation and platelet aggregation.
Purpose of the Study:
- To investigate human platelets as a source of MT1-MMP.
- To elucidate the role of MT1-MMP in MMP2 activation and platelet aggregation.
- To determine the expression of MT1-MMP, MMP2, and TIMP2 in platelets.
Main Methods:
- Gelatin zymography to detect MMP2 forms.
- Western blot analysis for MT1-MMP detection.
- Reverse transcription-polymerase chain reaction (RT-PCR) for gene expression.
- Flow cytometry to analyze protein expression on platelet surfaces.
- Inhibition assays using MMP inhibitors, TIMP2, and BB94.
Main Results:
- MMP2 proforms and activated forms were detected in platelet supernatants.
- MT1-MMP was found on resting platelet membranes and after degranulation.
- Platelets express mRNA for MMP2, MT1-MMP, and TIMP2.
- MT1-MMP, MMP2, and TIMP2 expression increased on activated platelet surfaces.
- MMP inhibitors, TIMP2, and BB94 inhibited collagen-induced platelet aggregation.
Conclusions:
- Human platelets express the trimolecular complex components (MT1-MMP/TIMP2/MMP2).
- Activated MT1-MMP plays a role in modulating platelet function.
- MMP inhibitors can modulate platelet aggregation response.