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Activated protein C up-regulates procoagulant tissue factor activity on endothelial cells by shedding the TFPI Kunitz 1 domain.

ABSTRACT: Thrombin and activated protein C (APC) signaling can mediate opposite biologic responses in endothelial cells. Given that thrombin induces procoagulant tissue factor (TF), we examined how TF activity is affected by APC. Exogenous or endogenously generated APC led to increased TF-dependent factor Xa activity. Induction required APC's proteolytic activity and binding to endothelial cell protein C receptor but not protease activated receptors. APC did not affect total TF antigen expression or the availability of anionic phospholipids on the apical cell membrane. Western blotting and cell surface immunoassays demonstrated that APC sheds the Kunitz 1 domain from tissue factor pathway inhibitor (TFPI). A TFPI Lys86Ala mutation between the Kunitz 1 and 2 domains eliminated both cleavage and the enhanced TF activity in response to APC in overexpression studies, indicating that APC up-regulates TF activity by endothelial cell protein C receptor-dependent shedding of the Kunitz 1 domain from membrane-associated TFPI. Our results demonstrate an unexpected procoagulant role of the protein C pathway that may have important implications for the regulation of TF- and TFPI-dependent biologic responses and for fine tuning of the hemostatic balance in the vascular system.

SUBMITTER: Schuepbach RA

PROVIDER: S-EPMC3122952 | biostudies-literature |

REPOSITORIES: biostudies-literature

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TMEM16E regulates endothelial cell procoagulant activity and thrombosis.

Project description:Endothelial cells (ECs) normally form an anticoagulant surface under physiological conditions, but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid "scramblases," such as TMEM16F. TMEM16F-dependent PS externalization is well characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified 2 TMEM16 family members, TMEM16F and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall-dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.

| S-EPMC10231993 | biostudies-literature

Recombinant pig TFPI efficiently regulates human tissue factor pathways.

Project description:Rejected pig-to-primate organ xenografts almost invariably exhibit significant microvascular thrombosis, believed to be due in part to several molecular incompatibilities affecting the regulation of coagulation. In this study, we tested one such proposed incompatibility: whether there is, at least in part, a functional incompatibility in pig tissue factor pathway inhibitor (TFPI) that impedes binding of human factor Xa and regulation of human tissue factor-initiated coagulation. TFPIalpha cDNA was cloned from pig aortic endothelial cells and found to encode a 279-residue mature protein with 79% overall identity to human TFPIalpha, increasing to 88 to 90% in the functional Kunitz-1 and Kunitz-2 domains. Transfected primate cells expressing equivalent levels of GPI-linked pig or human TFPIalpha were assayed for binding of human factor Xa and inhibition of the human factor VIIa/tissue factor complex. The activity of the expressed pig anticoagulant was equivalent to that of the human protein in both measures of TFPI function in these systems. These data indicate that there are no apparent incompatibilities between recombinant pig TFPI and the human tissue factor pathway. Other factors must account for the thromboregulatory failure of pig endothelium and aberrant tissue factor activity in xenograft rejection.

| S-EPMC2517609 | biostudies-literature

Histones Induce the Procoagulant Phenotype of Endothelial Cells through Tissue Factor Up-Regulation and Thrombomodulin Down-Regulation.

Project description:The high circulating levels of histones found in various thrombotic diseases may compromise the anticoagulant barrier of endothelial cells. We determined how histones affect endothelial procoagulant tissue factor (TF) and anticoagulant thrombomodulin (TM). Surface antigens, soluble forms, and mRNA levels of TF and TM were measured by flow cytometry, ELISA, and real-time RT-PCR, respectively. TF and TM activity were measured using procoagulant activity, thrombin generation, or chromogenic assays. Involvement of the toll-like receptor (TLR) was assessed using the neutralizing antibodies. Histones dose-dependently induced surface antigens, activity and mRNA levels of endothelial TF. Histone-treated endothelial cells significantly shortened the lag time and enhanced the endogenous thrombin potential of normal plasma, which was normalized by a TF neutralizing antibody. Histones induced phosphatidylserine and protein-disulfide isomerase expression in endothelial cells. Histones also reduced the surface antigen, activity, and mRNA levels of endothelial TM. Polysialic acid and heparin reversed the histone-induced TF up-regulation and TM down-regulation. Activated protein C did not affect the TF up-regulation, but interrupted TM down-regulation. TLR2, and TLR4 inhibitors partially blocked the TF up-regulation. Histones induced the endothelial procoagulant phenotype through TF up-regulation and TM down-regulation. The effects of histones were partly mediated by TLR2, TLR4. Strategies to inhibit the harmful effects of histones in endothelial cells may be required in order to prevent a thrombotic environment.

| S-EPMC4892514 | biostudies-literature

LIM Domain Only 2 Regulates Endothelial Proliferation, Angiogenesis, and Tissue Regeneration.

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| S-EPMC5121509 | biostudies-literature

TFPI? is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes.

Project description:Tissue factor pathway inhibitor (TFPI) produces factor Xa-dependent feedback inhibition of factor VIIa/tissue factor-induced coagulation. Messages for 2 isoforms of TFPI have been identified. TFPI? mRNA encodes a protein with an acidic N-terminus, 3 Kunitz-type protease inhibitor domains and a basic C-terminus that has been purified from plasma and culture media. TFPI? mRNA encodes a form in which the Kunitz-3 and C-terminal domains of TFPI? are replaced with an alternative C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPI? protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment is TFPI?, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPI? based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and (3) it contains a GPI anchor. Immunoassays demonstrate that, although endothelial cells secrete TFPI?, greater than 95% of the TFPI released by PIPLC treatment from the surface of endothelial cells and from placental microsomes is TFPI?.

| S-EPMC3277357 | biostudies-literature

Comparison of the inhibitory activities of human tissue factor pathway inhibitor (TFPI)? and TFPI?.

Project description:Tissue factor pathway inhibitor (TFPI) is an alternatively spliced protein with two isoforms, TFPI? and TFPI?, which differ in their C-terminal structure and cellular localization. Detailed characterization of their inhibitory activity is needed to define potentially unique inhibitory roles in tissue factor (TF)-mediated thrombotic and inflammatory disease, and to understand how pharmaceuticals targeted to different structural regions of the TFPI isoforms alter hemostasis in hemophilia patients.The TF inhibitory activity of TFPI? localized to the surface of CHO cells was compared with that of soluble TFPI? by the use of in vitro and in vivo assays.In TF-factor VIIa-mediated FXa generation assays, TFPI? was a slightly better inhibitor than TFPI?, which was approximately three-fold better than TFPI-160, a soluble, altered form of TFPI similar to TFPI?. In direct FXa inhibitory assays, TFPI? had an IC50 2.5-fold lower than that of TFPI? and 56-fold lower than that of TFPI-160. TFPI? inhibited TF-mediated CHO cell migration though Matrigel, whereas TFPI? and TFPI-160 were poor inhibitors, demonstrating that TFPI? effectively blocks TF-initiated signaling events during cellular migration through matrices that are not permeable to soluble forms of TFPI. Furthermore, TFPI? inhibited TF-dependent CHO cell infiltration into lung tissue following tail vein injection into SCID mice, and blocked the development of consumptive coagulopathy.TFPI? is a slightly better inhibitor of TF procoagulant activity than TFPI?. As a surface-associated protein, TFPI? is a much better inhibitor of TF-mediated cellular migration than soluble TFPI?, and may specifically act in the inhibition of TF-mediated signaling events on inflamed endothelium and/or monocytes.

| S-EPMC3656975 | biostudies-literature

Inflammatory stimuli induce shedding of heparan sulfate from arterial but not venous porcine endothelial cells leading to differential proinflammatory and procoagulant responses.

Project description:Endothelial dysfunction is an early event of vascular injury defined by a proinflammatory and procoagulant endothelial cell (EC) phenotype. Although endothelial glycocalyx disruption is associated with vascular damage, how various inflammatory stimuli affect the glycocalyx and whether arterial and venous cells respond differently is unknown. Using a 3D round-channel microfluidic system we investigated the endothelial glycocalyx, particularly heparan sulfate (HS), on porcine arterial and venous ECs. Heparan sulfate (HS)/glycocalyx expression was observed already under static conditions on venous ECs while it was flow-dependent on arterial cells. Furthermore, analysis of HS/glycocalyx response after stimulation with inflammatory cues revealed that venous, but not arterial ECs, are resistant to HS shedding. This finding was observed also on isolated porcine vessels. Persistence of HS on venous ECs prevented complement deposition and clot formation after stimulation with tumor necrosis factor α or lipopolysaccharide, whereas after xenogeneic activation no glycocalyx-mediated protection was observed. Contrarily, HS shedding on arterial cells, even without an inflammatory insult, was sufficient to induce a proinflammatory and procoagulant phenotype. Our data indicate that the dimorphic response of arterial and venous ECs is partially due to distinct HS/glycocalyx dynamics suggesting that arterial and venous thrombo-inflammatory disorders require targeted therapies.

| S-EPMC10024017 | biostudies-literature

Air pollution upregulates endothelial cell procoagulant activity via ultrafine particle-induced oxidant signaling and tissue factor expression.

Project description:Air pollution exposure is associated with cardiovascular events triggered by clot formation. Endothelial activation and initiation of coagulation are pathophysiological mechanisms that could link inhaled air pollutants to vascular events. Here we investigated the underlying mechanisms of increased endothelial cell procoagulant activity following exposure to soluble components of ultrafine particles (soluble UF). Human coronary artery endothelial cells (HCAEC) were exposed to soluble UF and assessed for their ability to trigger procoagulant activity in platelet-free plasma. Exposed HCAEC triggered earlier thrombin generation and faster fibrin clot formation, which was abolished by an anti-tissue factor (TF) antibody, indicating TF-dependent effects. Soluble UF exposure increased TF mRNA expression without compensatory increases in key anticoagulant proteins. To identify early events that regulate TF expression, we measured endothelial H2O2 production following soluble UF exposure and identified the enzymatic source. Soluble UF exposure increased endothelial H2O2 production, and antioxidants attenuated UF-induced upregulation of TF, linking the procoagulant responses to reactive oxygen species (ROS) formation. Chemical inhibitors and RNA silencing showed that NOX-4, an important endothelial source of H2O2, was involved in UF-induced upregulation of TF mRNA. These data indicate that soluble UF exposure induces endothelial cell procoagulant activity, which involves de novo TF synthesis, ROS production, and the NOX-4 enzyme. These findings provide mechanistic insight into the adverse cardiovascular effects associated with air pollution exposure.

| S-EPMC4133589 | biostudies-literature

Peroxisome Proliferator-Activated Receptor <i>?</i> Induces the Expression of Tissue Factor Pathway Inhibitor-1 (TFPI-1) in Human Macrophages.

Project description:Tissue factor (TF) is the initiator of the blood coagulation cascade after interaction with the activated factor VII (FVIIa). Moreover, the TF/FVIIa complex also activates intracellular signalling pathways leading to the production of inflammatory cytokines. The TF/FVIIa complex is inhibited by the tissue factor pathway inhibitor-1 (TFPI-1). Peroxisome proliferator-activated receptor gamma (PPAR?) is a transcription factor that, together with PPAR? and PPAR?/?, controls macrophage functions. However, whether PPAR? activation modulates the expression of TFP1-1 in human macrophages is not known. Here we report that PPAR? activation increases the expression of TFPI-1 in human macrophages in vitro as well as in vivo in circulating peripheral blood mononuclear cells. The induction of TFPI-1 expression by PPAR? ligands, an effect shared by the activation of PPAR? and PPAR?/?, occurs also in proinflammatory M1 and in anti-inflammatory M2 polarized macrophages. As a functional consequence, treatment with PPAR? ligands significantly reduces the inflammatory response induced by FVIIa, as measured by variations in the IL-8, MMP-2, and MCP-1 expression. These data identify a novel role for PPAR? in the control of TF the pathway.

| S-EPMC5223051 | biostudies-literature

Novel protein ADTRP regulates TFPI expression and function in human endothelial cells in normal conditions and in response to androgen.

Project description:Thrombosis and cardiovascular disease (CVD) represent major causes of morbidity and mortality. Low androgen correlates with higher incidence of CVD/thrombosis. Tissue Factor Pathway Inhibitor (TFPI) is the major inhibitor of tissue factor-factor VIIa (TF-FVIIa)-dependent FXa generation. Because endothelial cell (EC) dysfunction leading to vascular disease correlates with low EC-associated TFPI, we sought to identify mechanisms that regulate the natural expression of TFPI. Data mining of NCBI's GEO microarrays revealed strong coexpression between TFPI and the uncharacterized protein encoded by C6ORF105, which is predicted to be multispan, palmitoylated and androgen-responsive. We demonstrate that this protein regulates both the native and androgen-enhanced TFPI expression and activity in cultured ECs, and we named it androgen-dependent TFPI-regulating protein (ADTRP). We confirm ADTRP expression and colocalization with TFPI and caveolin-1 in ECs. ADTRP-shRNA reduces, while over-expression of ADTRP enhances, TFPI mRNA and activity and the colocalization of TF-FVIIa-FXa-TFPI with caveolin-1. Imaging and Triton X-114-extraction confirm TFPI and ADTRP association with lipid rafts/caveolae. Dihydrotestosterone up-regulates TFPI and ADTRP expression, and increases FXa inhibition by TFPI in an ADTRP- and caveolin-1-dependent manner. We conclude that the ADTRP-dependent up-regulation of TFPI expression and activity by androgen represents a novel mechanism of increasing the anticoagulant protection of the endothelium.

| S-EPMC3204913 | biostudies-literature

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