ALBERT

Description: Abstract 2210 Tissue factor pathway inhibitor (TFPI) is a key regulator of factor X (FX) activation in the extrinsic pathway of blood coagulation. TFPI inhibits FXa generation by formation of a quaternary complex consisting of factor VIIa (FVIIa), tissue factor (TF), FXa and TFPI. The main portion (∼80%) of TFPI in humans is reportedly associated with endothelial cells. We used human umbilical vein endothelial cells (HUVECs) as a model to obtain further insight into the function of TFPIα and the glycosylphosphatidylinositol (GPI) anchored TFPI form, which represents TFPIα bound to GPI-anchored surface proteins and/or TFPIβ. In contrast to TFPIα, which consists of 3 Kunitz domains (KD) and a basic C-terminal part, GPI-anchored TFPIβ lacks the third Kunitz domain (KD3) and the basic C–terminal region due to alternative splicing. In TFPIβ these two domains are replaced by a sequence that adds a GPI anchor to the protein linking it to the cell membrane. Treatment of HUVECs with phosphatidylinositol phospholipase C (PI-PLC) that cleaves GPI-anchors and subsequent fluorescence activated cell sorting (FACS) on living cells showed that GPI-anchored TFPI represents about 70–80% of cell surface TFPI. Staining of TFPI on and in fixed and permeabilized cells (total TFPI) demonstrated that GPI-anchored cell surface TFPI contributes to ∼20% of total cellular TFPI. Enzyme-linked immunosorbent assay (ELISA) showed that PI-PLC treatment released a TFPI protein lacking the KD3 and basic C-terminus. These findings strongly suggest that TFPIβ is the predominant GPI-anchored form of TFPI on HUVECs. FX activation assays performed on the cell surface of PI-PLC treated living HUVECs showed the importance of GPI-anchored TFPI on extrinsic Xase complex activity. PI-PLC treatment resulted in increased FX activation. Although GPI-anchored TFPI displays ∼70–80% of cell surface TFPI, overall FXa generation was increased only by ∼50%. In conclusion, HUVEC surface TFPI is predominantly TFPIβ, and GPI-anchored TFPI is the main but not sole regulator of FX activation on the surface of HUVECs. Disclosures: No relevant conflicts of interest to declare.

Description: Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits both FXa and TF-FVIIa and is an important physiological inhibitor of the extrinsic coagulation pathway. The main portion (~80%) of TFPI in humans is reportedly associated with endothelial cells (ECs). At least 2 different isoforms of TFPI exist in humans, namely, TFPI alpha (α) and TFPI beta (β). In contrast to TFPIα, which consists of 3 Kunitz domains (KD) and a basic C-terminal part, TFPIβ lacks the third KD (KD3) and the basic C–terminal region. In TFPIβ, these 2 domains are replaced, due to alternative splicing, by a sequence that adds a glycosylphosphatidylinositol (GPI) anchor to the protein, linking it to the cell membrane. The present study aimed to identify possible differences in the TFPI level of macro- and microvascular ECs of various tissues to obtain further insight into the relevance of TFPI isoforms α and β at the cellular level. Primary macro- and microvascular ECs of various tissues were treated with phosphatidylinositol phospholipase C (PI-PLC) to remove the GPI-anchored TFPI isoform β from the cell surface. After detachment with an enzyme-free cell dissociation solution, living cells were washed and stained with a polyclonal anti human TFPI antibody and subsequently used for fluorescence activated cell sorting (FACS). TFPIα, the non-GPI anchored TFPI isoform, is not affected by PI-PLC treatment and therefore remains on the cell surface. Measuring the fluorescence intensities of these cells by FACS allowed us to determine the relative amount of cell surface TFPIβ. The supernatants and similarly treated cells were used in two different ELISA assays to quantify TFPI antigen. The first ELISA quantifies total TFPI, consisting of both TFPIα and β, whereas the second ELISA specifically determines the TFPIα content using another capture antibody. Subtraction of TFPIα from the total TFPI amount enabled us to determine the concentration of TFPIβ. Three macrovascular (HUVEC, HAoEC and HPAEC) and four microvascular (HDMEC, HDBEC, HCMEC and HPMEC) cell types were used for TFPI analysis. Based on availability, ECs from more than one donor were analyzed to explore individual differences in TFPI expression. FACS results of living cells revealed that ~85% of the cell surface TFPI of all analyzed ECs represents TFPIβ. ELISA measurements of supernatants and cell lysates showed that TFPIα was not affected by PI-PLC treatment, whereas TFPIβ increased in the supernatant and decreased in the total cell lysate after PI-PLC treatment. These results were obtained with all tested cell types. Differences in the absolute TFPI level of individual donors were also detected and microvascular cells were shown to exhibit more total TFPI than macrovascular cells. In conclusion, both TFPIα and β appear to be present on micro- and macrovascular ECs and ~85% of cell surface TFPI to represent TFPIβ. Furthermore, TFPI levels in the various vasculatures were shown to be dependent on the size of the blood vessel and on the individual donor. Disclosures Dockal: Baxter Innovations GmbH, Vienna, Austria: Employment. Pachlinger:Baxter Innovations GmbH, Vienna, Austria: Employment. Baldin-Stoyanova:Baxter Innovations GmbH, Vienna, Austria: Employment. Knofl:Baxter Innovations GmbH, Vienna, Austria: Employment. Ullrich:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.