CD40 ligand shedding is regulated by interaction between matrix metalloproteinase-2 and platelet integrin alpha(IIb)beta(3).
BACKGROUND
CD40 ligand (CD40L, CD154) in the circulatory system is mainly contained in platelets, and surface-expressed CD40L on activated platelets is subsequently cleaved by proteolytic activity to generate soluble CD40L (sCD40L). However, the enzyme responsible for the shedding of CD40L in activated platelets has not been clearly identified yet. We have recently found that molecular interaction of matrix metalloproteinase-2 (MMP-2) with integrin alpha(IIb)beta(3) is required for the enhancement of platelet activation.


OBJECTIVE
To elucidate the biochemical mechanism of MMP-2-associated sCD40L release.


METHODS
Localization of MMP-2 and CD40L in platelets was analyzed by flow cytometry and fluorescence microscopy. The release of sCD40L from activated platelets was measured by enzyme-linked immunosorbent assay. MMP-2 binding to alpha(IIb)beta(3) was analyzed by immunoprecipitation and western blotting. Recombinant hemopexin-like domain and MMP-2-specific inhibitor were used to characterize the nature of MMP-2 binding and catalytic activity.


RESULTS
It was revealed that interaction of MMP-2 with alpha(IIb)beta(3) is required for effective production of sCD40L in activated human platelets. Platelet activation and release of sCD40L were significantly affected by inhibition of platelet-derived MMP-2 activity or by inhibition of binding between the enzyme and the integrin. It was also found in platelet-rich plasma that MMP-2 activity is responsible for generating sCD40L.


CONCLUSIONS
The results presented here strongly suggest that MMP-2 interacts with alpha(IIb)beta(3) to regulate the shedding of CD40L exposed on the surfaces of activated human platelets.
National Research Laboratory, Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea.
W-S
WS
Choi
O-H
OH
Jeon
D-S
DS
Kim
D055162:Biocatalysis; D015153:Blotting, Western; D023201:CD40 Ligand / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D047468:Immunoprecipitation; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D015539:Platelet Activation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011485:Protein Binding
D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D023201:CD40 Ligand; D020778:Matrix Metalloproteinase 2