Cellular effects of heparin on the production and release of tissue factorpathway inhibitor in human endothelial cells in culture

Tissue factor pathway inhibitor (TFPI), the major downregulator of procoagu lant activity of the tissue factor-factor VIIa complex (TF . FVIIa), is syn thesized and constitutively secreted by endothelial cells (ECs). Here we de scribe the in vitro effects of heparin on the cellular localization, gene e xpression, and release of TFPI in human ECs in culture. Both unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH; Fragmin) time-depend ently induced a significant enhanced secretion of TFPI, paralleled by a red istribution and increase of TFPI on the cell surface and a decrease of intr acellular TFPI. Immunogold electron microscopy showed the presence of clust ers of TFPI, both on the plasmalemma proper and within cell-surface opened caveolae/enlarged caveolar profiles. Activation of FX by TF FVIIa on ECs tr eated with endotoxin was inhibited by both heparins but to a higher extent by LMWH, Inhibition of protein synthesis by cycloheximide did not reduce th e release of TFPI induced by heparin. Long-term incubation (48 hours) resul ted in a time-dependent enhanced production of TFPI. After the first 4 to 8 hours, depletion of intracellular TFPI was observed, more significantly wi th UFH. Northern blot analysis of TFPI mRNA also showed a decrease of the 1 .4-kb transcript after 4 hours of incubation with UFH, followed by recovery and an increase over the control level after 24 hours. Incubation of ECs w ith phorbol ester (PMA) significantly enhanced the secretion of TFPI and in creased its activity on the cell surface, probably by preventing invaginati on of caveolae, Heparin-stimulated release of TFPI decreased significantly in the presence of PMA to a level that was 2.4 times lower than the expecte d additive value for PMA and UFH separately. Pretreatment of ECs with PMA s uppressed a subsequent response to heparin. Altogether, our results suggest that the heparin-induced release of TFPI might involve a more specific mec hanism(s) than the previously hypothesized simple displacement of TFPI from the cell surface glycocalyx, We assume that the increased secretion and re distribution of cellular TFPI induced by heparins in ECs in culture can pla y an important role in the modulation of the anticoagulant properties of th e endothelium.