Background: Human endothelial cells produce two alternatively spliced tissue factor pathway inhibitor (TFPI) isoforms that maintain anticoagulant properties of the vasculature. TFPIβ attaches to the cell surface via a glycosylphosphatidylinositol anchor. TFPIα has a basic C-terminus sharing homology with vascular endothelial growth factor (VEGF) and is a heparin-releasable protein. The reservoir for heparin-releasable TFPIα is thought to be endothelial cell surface glycosaminoglycans (GAGs).

Aims: Further define the source of heparin-releasable TFPIα.

Methods: Biochemical and imaging methods including ELISA, flow cytometry, mass spectrometry, immunohistochemistry and immunofluorescence were used to determine the location of heparin-releasable TFPIα in cultured and primary human endothelial cells, umbilical veins and kidney.

Results: TFPIα was not detectable on the surface of Ea.hy926 cells examined by flow cytometry, while TFPIβ was abundant. In contrast, heparin-releasable TFPIα was abundant in ECM produced by cultured Ea.hy926 or by umbilical vein endothelial cells examined by ELISA, while TFPIβ was not detectable. Immunofluorescent imaging of Ea.hy926 cells localized TFPIα to internal granules within the cytoplasm and to the ECM located directly below individual cells. The localization of heparin-releasable TFPIα to ECM was confirmed with ELISA and immunohistochemistry studies of umbilical cord veins. The TFPIα C-terminus interacted with Ea.hy926 ECM GAGs in competitive binding experiments, and a homologous VEGF basic peptide competed for this binding, suggesting these interactions may modulate VEGF responses. An immobilized TFPIα C-terminal peptide bound to several ECM proteoglycans in Ea.hy926 conditioned media. Immunofluorescence studies of human kidney colocalized TFPIα with four of these proteoglycans surrounding the microvasculature: glypican-1, syndecan-4, thrombospondin, and laminin-5. The absence of TFPIα on the surface of endothelial cells and its co-localization with specific ECM proteins suggests TFPIα binds to unique proteoglycan structures.

Conclusions: ECM contained the primary vascular pool of heparin-releasable TFPIα. TFPIα within ECM is optimally positioned to inhibit the procoagulant activity of tissue factor surrounding the vasculature.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/a-major-reservoir-for-heparin-releasable-tfpi%ce%b1-is-extracellular-matrix/