Identification, regulation and role of tissue inhibitor of metalloproteinases-4 (TIMP-4) in human platelets

1 Matrix metalloproteinase-2 (MMP-2) released during activation of human platelets by aggregating agents and cancer cells is known to stimulate platelet aggregation. 2 The expression, activity and role of tissue inhibitors of metalloproteinases (TIMPs), natural inhibitors of MMPs, in isolated human platelets were investigated. 3 Western blot, reverse zymography, immunogold electron microscopy, aggregometry (collagen-, thrombin and HT-1080 human fibrosarcoma cells-induced aggregation), flow cytometry and the release of C-14-serotonin from labelled platelets recruited to the aggregate were used to characterize the presence and function of platelet TIMPs. 4 TIMP-4 (23 kDa) has been identified as the major MMP inhibitor (12- 16 ng per 10(8) platelets) in human platelets. Platelets expressed lower (< 1 ng per 10(8) platelets) amounts of TIMP-1. No other TIMPs were detected using Western blot analysis. 5 TIMP-4 co-localized with MMP-2 in resting platelets and was released upon platelet aggregation induced by collagen and thrombin. 6 Collagen resulted also in the release of higher molecular weight (60 kDa) complexes of TIMP-4. 7 The release of TIMP-4 was reduced by prostacyclin and S-nitroso-glutathione (GSNO), an NO donor. 8 Human recombinant TIMP-4 (rTIMP-4), but not human rTIMP-1, inhibited partially both platelet aggregation and recruitment. 9 The recombinant TIMP-4 potentiated the recruitment inhibitor effects of GSNO. 10 TIMP-4 was not released during platelet aggregation induced by HT-1080 cells. 11 Human rTIMP-4 exerted a biphasic effect on HT-1080 cells-induced aggregation. 12 Thus, TIMP-4 is the major intraplatelet MMP inhibitor and it is involved in regulation of platelet aggregation and recruitment.