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Binding of matrix metalloproteinase 9 to fibrin is mediated by amorphous calcium-phosphate
1Department of Laboratory Medicine, University of Connecticut Health Center, Farmington, USA.
Abstract:
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 degrees 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 microM) (Ca x 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 degrees C, an observation consistent with lack of ACP production. The significance of these findings is discussed with respect to normal and pathologic wound healing.
Insights
Matrix metalloproteinase-9 (MMP-9) binds to fibrin via amorphous calcium-phosphate (ACP) complexes. ACP formation at 37°C is crucial for this interaction, impacting wound healing.
Area of Science:
- Biochemistry
- Biomaterials Science
- Cell Biology
Background:
- Matrix metalloproteinase-9 (MMP-9), a neutrophil collagenase, selectively binds to fibrin in a dose-dependent manner.
- The precise mechanism underlying MMP-9's interaction with fibrin remained to be elucidated.
Purpose of the Study:
- To investigate the mechanism by which MMP-9 binds to fibrin.
- To determine the role of calcium-phosphate intermediates in MMP-9-fibrin interactions.
Main Methods:
- Investigated MMP-9 binding to fibrin under varying conditions.
- Utilized centrifugation to isolate calcium-phosphate intermediates.
- Determined Ca/P ratios and characterized the intermediate as amorphous calcium-phosphate (ACP).
- Employed gelatin zymography to analyze MMP-9 complex formation with ACP.
- Assessed the impact of temperature and specific ion concentrations on ACP and MMP-9 complex formation.
Main Results:
- MMP-9 binding to fibrin is dependent on the formation of an amorphous calcium-phosphate (ACP) intermediate.
- ACP formation, with an optimal temperature of 37°C, leads to the creation of a high molecular weight ACP:MMP-9 complex.
- This ACP:MMP-9 complex is essential for MMP-9's subsequent binding to fibrin.
- Specific calcium (7.5-10 mM) and phosphate (225-250 microM) concentrations are required for viable ACP generation, with a Ca x P product range of 1.7-2.5 mM².
- Carbonate and sulfate ions were ineffective as calcium counteranions.
- ACP formation was identified as the rate-limiting step for MMP-9 fibrin binding activity; no binding occurred at 25°C due to lack of ACP production.
Conclusions:
- Amorphous calcium-phosphate formation is a critical prerequisite for matrix metalloproteinase-9 (MMP-9) binding to fibrin.
- The temperature-dependent nature of ACP formation influences MMP-9's fibrin-binding capacity.
- These findings provide mechanistic insights relevant to both normal and pathological wound healing processes.