Tissue Factor Pathway Inhibitor Antigen Research Articles

Quantitative TFPI Antigen Release and Functionality After Intravenous Administration of Heparins Sourced From Various Species in Non‐human Primates

IntroductionTissue factor pathway inhibitor (TFPI) is a multi‐domain Kunitz‐type inhibitor that plays an important role in the regulation of the tissue factor pathway through its release from endothelial sites upon heparin administration. Currently, heparin is primarily sourced from porcine tissue, however, recent advancements have allowed for it to be re‐sources from bovine tissue. In this context, we elected to compare the relative abilities of porcine, bovine, and ovine heparins to release endogenous TFPI following intravenous administrations of gravimetric and USP potency‐adjusted heparin dosages.MaterialsActive pharmaceutical ingredient (API) forms of bovine, ovine, and porcine heparins of mucosal origin were obtained from Kinmaster, Brazil, Ronssi, China, and Medifill, USA, respectively. The active pharmaceutical ingredients were dissolved to obtain 10mg/mL solutions of each type. Individual groups of non‐human primates (n=6) were intravenously injected with 0.5mg/kg and 100U/kg dosages of each heparin type. Blood samples were subsequently drawn at 0, 15, 30, 60, and 120 minutes post‐administration. Citrated blood samples were centrifuged to obtain platelet‐poor plasma and stored at ‐ 80C until analysis. A sandwich ELISA method for TFPI antigen and a chromogenic substrate‐based functional method were used to quantify TFPI antigen release and functionality, respectively. All results were calculated in terms of group means ±SD. Applicable statistical methods were used to compare the heparins for their relative effects on TFPI release.ResultsGravimetric dosages of ovine and porcine heparins produced comparable TFPI release in both the functional and immunologic assays while bovine heparin demonstrated slightly lower peak TFPI antigen release and slightly higher peak function, as shown in Figure 1a and 1b, respectively. When compared following administration of USP‐adjusted 100U/kg dosages, all three heparins consistently demonstrated essentially identical TFPI antigen release and functional profiles, as shown in Figure 1c and 1d, respectively. Overall, any potential differences in the gravimetric dosing regimens were adequately corrected following USP potency adjustment in both TFPI antigen release and functionality, as seen in Figure 1a and 1b and Figure 1c and 1d, respectively. Furthermore, all three heparins demonstrated comparable pharmacokinetic profiles in both TFPI antigen release and function levels, as shown in Table 1.ConclusionThese results demonstrate that heparins from various sources are capable of comparatively releasing TFPI antigen. While the gravimetrically‐dosed studies demonstrated less consistent trajectories, potency‐based adjustments of the heparin dosages allowed for improved pharmacokinetic homogeneity. These studies underscore the potential for the development of alternative heparin sources as viable, pharmacokinetic equivalents.

Severe thrombophilia in a factor V‐deficient patient homozygous for the Ala2086Asp mutation (FV Besançon)

BackgroundCoagulation factor V (FV), present in plasma and platelets, has both pro‐ and anticoagulant functions. ObjectiveWe investigated an FV‐deficient patient (FV:C 3%, FV:Ag 4%) paradoxically presenting with recurrent venous thrombosis (11 events) instead of bleeding. Methods/ResultsThrombophilia screening revealed only heterozygosity for the F2 20210G>A mutation. Although thrombin generation in the patient's platelet‐poor plasma was suggestive of a hypocoagulable state, thrombin generation in the patient's platelet‐rich plasma (PRP) was higher than in control PRP and extremely resistant to activated protein C (APC). This was partially attributable to the complete abolition of the APC‐cofactor activity of FV and a marked reduction of plasma tissue factor pathway inhibitor antigen and activity. The patient was homozygous for a novel missense mutation (Ala2086Asp, FVBesançon) that favors a “closed conformation” of the C2 domain, predicting impaired binding of FV(a) to phospholipids. Recombinant FVBesançon was hardly secreted, indicating that this mutation is responsible for the patient's FV deficiency. Model system experiments performed using highly diluted plasma as a source of FV showed that, compared with normal FVa, FVaBesançon has slightly (≤1.5‐fold) unfavorable kinetic parameters (Km, Vmax) of prothrombin activation, but also a lower rate of APC‐catalyzed inactivation in the presence of protein S. ConclusionsFVBesançon induces a hypercoagulable state via quantitative (markedly decreased FV level) and qualitative (phospholipid‐binding defect) effects that affect anticoagulant pathways (anticoagulant activities of FV, FVa inactivation, tissue factor pathway inhibitor α level) more strongly than the prothrombinase activity of FVa. A possible specific role of platelet FV cannot be excluded.

Overexpression of Platelet Tfpiα Rescues TFPI Deficient Embryos from Mid-Gestational, but Not Late Gestational Lethality

Tissue Factor Pathway Inhibitor (TFPI) is an alternatively spliced Kunitz-type protease inhibitor required for murine embryonic development. On a mixed 129-C57BL/6 background, deletion of the first Kunitz (K1) domain of TFPI (Tfpitm1Gjb; Tfpi-/-) results in complete intrauterine lethality that occurs in about 60% of Tfpi-/- embryos during mid-gestation with the remaining 40% dying during late gestation. We recently discovered that this late gestation lethality is associated with severe cerebrovascular defects that are completely absent in Tfpi-/- embryos harboring genetic deletion of Factor V (FV), indicating that regulation of FV-dependent procoagulant activity by TFPI is necessary for proper cerebral vascular development. In mice, TFPIα is the only isoform present in platelets, TFPIβ is the major isoform on endothelium, and TFPIγ is the major isoform in plasma. All isoforms inhibit the tissue factor-Factor VIIa (TF-FVIIa) catalytic complex and Factor Xa (FXa) via the K1 and K2 domains, respectively. TFPIα is distinct from other isoforms as it contains a basic C-terminus that enables inhibition of early forms of prothrombinase (FXa-FVa). To better understand the biological activities of platelet TFPIα (pTFPIα), a transgenic mouse model expressing TFPIα under control of the platelet specific murine Gp1ba promoter was generated on the C57BL/6NCrl background. The transgene (Tg) was bred onto the Tfpitm1Gjb congenic C57BL/6J (B6J) background to examine how overexpression of pTFPIα alters embryonic survival of Tfpi-/- mice. Out of 407 offspring derived by crossing Tfpi+/- females to Tfpi+/- Tg+ males, the expected 50% Tg transmission ratio was observed in Tfpi+/+ and Tfpi+/- mice at wean, yet no Tfpi-/- Tg+ or Tfpi-/- Tg0 mice were produced. When examining Tg function in assays measuring TF-FVIIa mediated generation of FXa, it was found that Tfpi+/+ and Tfpi+/- Tg+ mice had 4- to 5-fold increased platelet TFPI inhibitory activity over Tg0 littermates, while plasma TFPI activity and antigen were unaltered indicating functional expression and storage of transgenic TFPIα within platelets. To study the impact of pTFPIα overexpression on Tfpi-/- survival, 120 late gestation embryos derived from Tfpi+/- x Tfpi+/- Tg+ backcrosses were harvested between E14.5-18. In comparison to the 5% frequency of Tfpi-/- 129-C57BL/6 embryos observed at this time interval, the Tfpi alleles were normally distributed with 28% Tfpi+/+, 49% Tfpi+/- and 23% Tfpi-/-. Of the Tfpi-/- embryos, 86% were Tg+ and 14% were Tg0. However, only 21% of Tfpi+/+ embryos were Tg+ (50% expected) suggesting that the Tg caused lethality in Tfpi+/+ embryos, a finding not observed in the weaning age cohort. Given this, we hypothesized that underlying genetic factors were complicating survival outcomes. PCR-based DNA walking uncovered that non-independent assortment played a major role in the disproportionate embryo frequencies, as the Tg integrated into an intergenic region on chromosome 2, ~36.4 megabases proximal to, and in linkage with, the endogenous Tfpi locus. Additionally, by intercrossing Tfpi+/- mice on the congenic B6J background, we found that 14% of 359 embryos harvested between E14.5-16.5 were Tfpi-/- , which was higher than the 5% observed in 129-C57BL/6 embryos, indicating that strain specific genetic interactions modulate Tfpi-/- mid-gestational lethality. After accounting for recombination rates, male breeder haplotypes, and expected frequencies at late gestation, pTFPIα overexpression completely suppressed mid-gestational lethality of Tfpi-/- embryos. Histological analyses revealed that the Tg had no effect on development of cerebrovascular defects in Tfpi-/- embryos during late gestation consistent with their lack of survival to wean. In conclusion, underlying genetic factors complicated our analyses illustrating the importance of meticulously characterizing transgenic mouse models to avoid spurious interpretation of results. After accounting for these unexpected genetic effects, our analyses indicated that pTFPIα overexpression totally rescued Tfpi-/- embryos from mid-gestational lethality. However, late gestational lethality persisted likely because overexpression of pTFPIα did not prevent the severe cerebrovascular developmental defects occurring at this stage of development in Tfpi-/- embryos. Disclosures Mast: Novo Nordisk: Honoraria, Research Funding.

Tissue Factor Pathway Inhibitor (TFPI) release by bovine, ovine and porcine unfractionated heparins in primates. Comparative studies at mass and potency adjusted intravenous dosing

BackgroundUnfractionated heparin (UFH) is a highly sulfated glycosaminoglycan (GAG) that consists of repeating disaccharide units, containing iduronic acid (or glucuronic acid) and glucosamine, exhibiting variable degrees of sulfation. UFHs release tissue factor pathway inhibitor (TFPI) which inhibits extrinsic pathway of coagulation by rapidly inactivating of Factor Xa and the Factor VIIa/TF complex. Bovine mucosal heparins (BMH) and ovine mucosal heparin (OMH) are currently being developed for re‐introduction for both medical and surgical indications. In contrast to the porcine mucosal heparins (PMH), the active pharmaceutical ingredient (API) BMH exhibits a somewhat weaker anti‐coagulant activity as cross‐referenced against PMH. In this study, we determined the TFPI antigen release level by various dosages of UFHs using ELISA method. Also, we projected that potency adjusted BMH at equivalent dosage has the same TFPI release profile compared to OMH and PMH.Materials & MethodsBMH, OMH and PMH were obtained from various commercial sources. Potencies of each heparin were determined by an amidolytic anti‐Xa assay in relation to the USP heparin standard. Individual groups of primates (n=4) were administered 0.5 mg/kg or (50 or 100) U/kg dosage of bovine, ovine or porcine UFH via intravenous route. Blood samples were collected at baseline, 15, 30, 60 and 120 minutes post‐administration. Tissue Factor Pathway Inhibitor (TFPI) antigen levels were measured using a commercially available sandwich ELISA methods. All results were compiled as mean ± SD.ResultsAt a gravimetric level (0.5 mg/kg), BMH exhibited a lower TFPI level than OMH & PMH at all time points with a maximum value of (200 ng/ml) at 15 mins post drug administration. OMH and PMH showed comparable TFPI levels at all time points with maximum values of (250 ng/ml) and (265 ng/ml) respectively at 15 mins post drug administration. However, USP cross‐referenced anti‐Xa potency adjusted based dosing showed comparable TFPI antigen levels for all three UFHs at all time points with a maximum value of 250 ng/ml at 15 mins post drug administration. At 60 mins post drug administration, potency adjusted BMH showed slightly higher TFPI level of (200 ng/ml) compared to both OMH & PMH.ConclusionTissue factor pathway inhibitor (TFPI) levels at 15 minutes after IV administration of 0.5 mg/kg heparins to non‐human primates showed that both PMH and OMH release comparable TFPI antigen levels which were significantly higher than BMH. Potency equated dosing resulted in comparable TFPI release profiles for all three heparins. Therefore such dosing may provide uniform levels of anticoagulation for the parenteral indications for UFHs. These observations suggest that potency equated bovine heparin has comparable release of TFPI and therefore is biosimilar to the OMH and PMH.

Studies on Tissue Factor Pathway Inhibitor Antigen Release by Bovine, Ovine and Porcine Heparins Following Intravenous Administration to Non-Human Primates.

Unfractionated heparin (UFH) is a sulfated glycosaminoglycan that consists of repeating disaccharides, containing iduronic acid (or glucuronic acid) and glucosamine, exhibiting variable degrees of sulfation. UFHs release tissue factor pathway inhibitor (TFPI) which inhibits the extrinsic pathway of coagulation by inactivating factor Xa and the factor VIIa/TF complex. Most heparins used clinically are derived from porcine intestinal mucosa however, heparins can also be derived from tissues of bovine and ovine origin. Currently there are some concerns about the shortage of the porcine heparins as they are widely used in the manufacturing of the low molecular weight heparins (LMWHs). Moreover, due to cultural and religious reasons in some countries, alternative sources of heparins are needed. Bovine mucosal heparins (BMH) are currently being developed for re-introduction to the US market for both medical and surgical indications. Compared to porcine mucosal heparin (PMH), BMH exhibits a somewhat weaker anti-coagulant activity. In this study, we determined the TFPI antigen level following administration of various dosages of UFHs from different origins. These studies demonstrated that IV administration of equigravemetric dosages of PMH and ovine mucosal heparin (OMH) to non-human primates resulted in comparable TFPI antigen release from endothelial cells. In addition, the levels of TFPI were significantly higher than TFPI antigen levels observed after BMH administration. Potency adjusted dosing resulted in comparable TFPI release profiles for all 3 heparins. Therefore, such dosing may provide uniform levels of anticoagulation for the parenteral indications for UFHs. These observations warrant further clinical validation in specific indications.

Ovine Mucosal Enoxaparin Exhibit Comparable Pharmacokinetic Profiles to Porcine Mucosal Enoxaparin

BackgroundLow Molecular Weight Heparins (LMWH) such as Enoxaparin are currently derived from porcine mucosal heparin (PMH), the same depolymerization methods can be used to obtain enoxaparin from unfractionated heparin from other sources. The purpose of this study is to compare the pharmacokinetics of ovine mucosal enoxaparin (OME) with that of porcine mucosal enoxaparin (PME).Materials and MethodsBranded Enoxaparin (Lovenox, Sanofi‐Aventis, Bridgewater, NJ) and three batches of OME (Suzhou Ronnsi Pharma, Suzhou, China) were used. Following anesthesia, a baseline blood sample was collected from primates (Macaca mulatta; n=4). LMWH was administered subcutaneously (SC) at a dose of 1 mg/kg. Additional blood samples were collected at various times (2,4,6 hours). Anti‐Xa and anti‐thrombin assays were used to calculate pharmacokinetic parameters. Tissue factor pathway inhibitor antigen levels were also measured using an ELISA method.ResultsIn the samples collected at 2, 4, and 6 hours, comparable levels of branded PME and the three batches of OME were observed using both anti‐Xa and anti‐thrombin assays. The pharmacokinetic parameters such as biologic half‐life, Cmax, volume of distribution, and clearance were comparable for both the ovine and porcine enoxaparin. Tissue Factor Pathway Inhibitor levels returned to baseline levels by 6 hours in OME, but remained slightly elevated with PME.DiscussionAt a 1 mg/kg SC dosage, three OME preparations produced comparable anti‐Xa and anti‐IIa effects which were similar to the ones observed with PME. The PK parameters calculated based on this data were also comparable for both groups. It is concluded that the SC pharmacokinetics of the three batches of OME were not only comparable between batches, but were also similar to the branded product.Support or Funding InformationNone.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lipid-Mediated Relation between Tissue Factor Pathway Inhibitor Activity and Cardiovascular Risk Factors and Diseases in a Large Population Sample

▪Background: Tissue factor pathway inhibitor (TFPI), a Kunitz-type serine protease, is a potent anticoagulant protein in the extrinsic coagulation pathway and acts by inhibiting both the FXa and the Tissue Factor-FVIIa complex. In contrast to total and free TFPI antigen levels, the reference values and clinical determinants of total TFPI activity have not yet been studied in detail in the general population. In the present study, we aim to identify the cardiovascular determinants for total TFPI activity and investigate its association with cardiovascular disease (CVD) and total mortality, in a population at large.Methods: For this study, the first 4779 subjects of the population-based Gutenberg Health Study were examined in a highly standardized setting. Total TFPI activity was assessed in platelet poor plasma by the Actichrome TFPI activity assay (American Diagnostica, Stamford, CT, USA). Sex-specific nomograms were developed to demonstrate the relation of total TFPI activity with age. Multivariable regression analysis was performed to assess the determinants of total TFPI activity and to evaluate the association with CVD. Cox-regression models, adjusted for age, sex, cardiovascular risk factors (CVRFs) and CVD, were calculated to investigate the association between total TFPI activity and total mortality.Results: The multivariable linear regression analysis identified smoking (β, 0.0952 [0.0541 to 0.136],) as a positive determinant for total TFPI activity, while diabetes (β, -0.0716 [-0.134 to -0.00905],), obesity (β, -0.0627 [-0.101 to -0.024],) and history of coronary artery disease (CAD) were negatively associated with TFPI activity, independent of age, sex and the remaining CVRFs. After adjustment for high-density lipoprotein levels (HDL), low-density lipoproteins (LDL) and triglycerides, the association between TFPI activity levels and obesity and CAD was lost. The analysis additionally reveals a strong positive association between TFPI activity levels and LDL (β, 0.221 [0.204 to 0.237],). The Cox regression models revealed that a higher TFPI activity, above 97.5th percentile of the reference group, was associated with an increased mortality risk (HR = 2.58, [95% CI: 1.49/4.47], p=0.00074) independent of age, sex, CVRFs, CVD, oral contraceptives and hormonal replacement therapy. After additional adjustment for LDL levels, this relation became stronger.Conclusion: To the best of our knowledge, this is the first large, epidemiological study of the general population, revealing an increased mortality risk in individuals with higher TFPI activity, independent of CVRFs and CVD and emphasized by LDL-levels. Additionally, it demonstrates the negative association between total TFPI activity levels and CVD, particularly CAD. Furthermore, an association between TFPI activity and obesity, mediated through lipoprotein particles is explored. Lastly, it describes the age- and sex-related reference range of total TFPI activity levels in a large population-based data set. DisclosuresNo relevant conflicts of interest to declare.

Role of ADTRP (Androgen-Dependent Tissue Factor Pathway Inhibitor Regulating Protein) in Vascular Development and Function.

BackgroundThe physiological function of ADTRP (androgen‐dependent tissue factor pathway inhibitor regulating protein) is unknown. We previously identified ADTRP as coregulating with and supporting the anticoagulant activity of tissue factor pathway inhibitor in endothelial cells in vitro. Here, we studied the role of ADTRP in vivo, specifically related to vascular development, stability, and function.Methods and ResultsGenetic inhibition of Adtrp produced vascular malformations in the low‐pressure vasculature of zebrafish embryos and newborn mice: dilation/tortuosity, perivascular inflammation, extravascular proteolysis, increased permeability, and microhemorrhages, which produced partially penetrant lethality. Vascular leakiness correlated with decreased endothelial cell junction components VE‐cadherin and claudin‐5. Changes in hemostasis in young adults comprised modest decrease of tissue factor pathway inhibitor antigen and activity and increased tail bleeding time and volume. Cell‐based reporter assays revealed that ADTRP negatively regulates canonical Wnt signaling, affecting membrane events downstream of low‐density lipoprotein receptor‐related protein 6 (LRP6) and upstream of glycogen synthase kinase 3 beta. ADTRP deficiency increased aberrant/ectopic Wnt/β‐catenin signaling in vivo in newborn mice and zebrafish embryos, and upregulated matrix metallopeptidase (MMP)‐9 in endothelial cells and mast cells (MCs). Vascular lesions in newborn Adtrp −/− pups displayed accumulation of MCs, decreased extracellular matrix content, and deficient perivascular cell coverage. Wnt‐pathway inhibition reversed the increased mmp9 in zebrafish embryos, demonstrating that mmp9 expression induced by Adtrp deficiency was downstream of canonical Wnt signaling.ConclusionsOur studies demonstrate that ADTRP plays a major role in vascular development and function, most likely through expression in endothelial cells and/or perivascular cells of Wnt‐regulated genes that control vascular stability and integrity.

Role of Androgen Dependent TFPI-Regulating Protein (ADTRP) in Vascular Development and Function

We used in vitro and in vivo models to characterize the physiological role of the novel protein encoded by C6ORF105. This gene’s expression is androgen-responsive, and the encoded protein is predicted to be palmitoylated and membrane multi-spanning. Previously we showed that C6ORF105 expression co-regulates with tissue factor pathway inhibitor (TFPI)in human endothelial cells (EC); hence we named this protein "androgen-dependent TFPI-regulating protein" (ADTRP). Using in vitro cell-based TOP-Flash reporter assay we identified ADTRP as a negative regulator of canonical Wnt signaling in human cells. Overexpressing ADTRP in HEK293T cells inhibited the activity of beta-catenin/TCF-dependent transcriptional reporter, while silencing ADTRP increased the expression of Wnt target genes LEF-1, AXIN-2, IL-8 and DKK-2 in EA.hy926 EC line and HUVEC. Addition of LiCl showed that the effect of ADTRP was upstream of GSK3, therefore we focused the investigations on the Wnt signalosome proteins. ADTRP expression in HEK293T cells led to decreased phosphorylation of Wnt co-receptor LRP6, suggesting that ADTRP can affect this critical membrane-located event of Wnt signaling. Furthermore, ADTRP expression in reporter cells transfected with a constitutively phosphorylated form of LRP6 (LRP6DN mutant) inhibited Wnt3a- induced signaling, which suggests that ADTRP can interfere with events downstream of LRP6 phosphorylation, such as Axin-2 binding. Altogether, these data indicate that the Wnt signaling inhibitory activity of ADTRP takes place at the plasma membrane level. Site directed mutagenesis of the predicted palmitoylation site Cys61 showed that Wnt inhibitory effects of ADTRP require palmitoyl-mediated anchoring, highlighting the importance of proper membrane location of ADTRP for Wnt pathway inhibition. In vivo morpholino-based knockdown of adtrp in zebrafish embryos produced aberrant angiogenesis, defective branching and ruptured vessels, hemorrhage spots, pericardial edema and slow heart-beat, all reminiscent of defects caused by activation of canonical Wnt signaling. Indeed, adtrp knock down increased Wnt mediated lef-1 and pax-2a as well as mmp2 and mmp9 mRNA expression. Co-injection of ADTRP mRNA partially recovered the adtrp morpholino- induced morphologic abnormalities. Also, knock down of adtrp in a Wnt reporter zebrafish showed increased expression of ectopic Wnt signaling. Furthermore, our recently established Adtrp-/- mice also display some typical Wnt-mediated vascular defects, including: (i) abnormal patterning, increased capillary tortuosity, abnormal branching and increased density of the capillary network; (ii) dilated vessels, especially venules and veins; (iii) increased leakeage of permeability tracers (Evans blue and fluorescent dextran) without evident changes in endothelial junctions; (iv) hemorrhage spots in the skin, meningeal layers, heart, bladder and kidneys; (v) intravascular and interstitial fibrin deposition in the lung, liver and kidney. ADTRP deficiency decreased plasma TFPI antigen by ~2-times. Furthermore, TFPI antigen and anticoagulant activity in lung extracts and isolated lung EC were similarly decreased, which confirms our previous in vitro data. We aslo noticed increased tail bleeding time (>500 sec vs. 200 sec in WT littermates) and blood volume loss, which likely was caused by increased dilation of the tail vein. Gene expression analysis of whole organs showed upregulation of Wnt target genes involved in vascular contractility (Nos3), and extracellular matrix remodeling (Mmp2). Similarly, skin fibroblasts and lung EC isolated from Adtrp-/- mice showed increased expression of Wnt target genes (Lef-1, Cyclin D, Dkk2, c-Myc), which indicates constitutive activation of canonical Wnt signaling. In conclusion, we used genetic animal models and cell culture systems to show for the first time that the novel protein ADTRP plays major roles in vascular development and function. Lack of, or low levels of ADTRP associate with activation of coagulation and vascular development defects, which may be due, at least in part, to intrinsic high levels of ectopic canonical Wnt signaling. DisclosuresNo relevant conflicts of interest to declare.

Generation and Initial Characterization of Three Murine Models with Altered Platelet TFPI Expression

Introduction: Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits tissue factor and prothrombinase during early stages of the procoagulant response. It is produced in alternatively spliced isoforms. In humans, TFPIα is within platelets and released upon activation and also is present in plasma, while TFPIβ is bound to the endothelial surface. Mice differ from humans in that they do not have TFPIα in plasma, making them an optimal model system for study of the function of platelet TFPIα. Studies of murine hematopoietic cell TFPI using fetal liver transplantation models have found that platelet TFPI dampens platelet accumulation in a growing thrombus following vascular injury and alters bleeding severity in mice with hemophilia. However, results of these studies are potentially confounded by altered TFPI expression on other hematopoietic cells, such as monocytes. To better understand the function of platelet TFPI, murine models of total TFPIα deletion (TFPIα KO), of platelet-specific TFPIα deletion (pTFPIα KO) and platelet-specific TFPIα over expression (pTFPIα high) were produced and characterized.Methods: A TFPIα deleted mouse was generated by inserting a stop codon within exon 10 of the TFPI gene effectively blocking translation of the highly conserved basic C-terminal region of TFPIα, but not altering TFPIβ. The PF4-Cre transgene was bred into the floxed-TFPI(K1) mouse to produce mice specifically lacking platelet TFPIα. A transgenic mouse over expressing platelet TFPIα under control of the GP1b promoter was produced. Mice were characterized for platelet and plasma TFPI antigen by ELISA, for activity by TF-FVIIa inhibition and whole blood platelet aggregation, and for bleeding by tail clip.Results: All 3 strains were viable and reproduced. Platelet TFPI antigen was decreased approximately 25-fold in the TFPIα KO and pTFPIα KO mice and elevated approximately 5-fold in the pTFPIα high mice with corresponding changes in TF-FVIIa inhibitory activity. Plasma TFPI antigen was no different between pTFPIα KO (20.6±4.4 nM) and wild type (21.5±6.2 nM) littermates. Plasma thrombin-antithrombin complex levels were not elevated in any strain. In tail clip bleeding time assays, there was no difference between pTFPIα KO (0.23±0.16 mg/ml Hb) and wild type (0.23±0.37 mg/ml Hb), but there was a trend towards increased blood loss in pTFPIα high mice (1.25±1.8 mg/ml Hb). The pTFPIα high mice also had altered whole blood platelet aggregation that was evident primarily in a prolonged time for initiation of aggregation with 6.50uM ADP (81±12 sec vs. 6.1±5.6 sec for control) or 0.8ug/ml collagen (35±17 sec vs. 16±21 sec for control). Finally, the pTFPIα high transgene was bred into TFPI heterozygous mice which were then bred to determine if over expression of platelet TFPIα would rescue the TFPI null embryonic lethal phenotype. However, no live TFPI null pups were observed following genotyping of over 50 pups from these matings.Conclusions: Three new mouse models of altered TFPI expression have been made providing valuable tools to study the role of platelet TFPI in bleeding and clotting disorders. Plasma TFPI is unchanged in mice lacking platelet TFPI demonstrating that platelet TFPI does not contribute to plasma TFPI in mice. Platelet TFPIα antigen and activity correlate in all three models. Deletion of the basic C-terminal region in TFPIα in the TFPIα KO mice produces a TFPIα hypomorphic mouse, with only trace amounts of truncated TFPIα present in platelets. This lack of TFPIα does not produce a generalized procoagulant state. The 5-fold excess TFPIα antigen in the pTFPIα high mice substantially delays the onset of platelet aggregation in whole blood assays initiated with collagen or ADP. Results from tail clip bleeding assays suggest that over expression of platelet TFPIα may prolong bleeding in adult mice, but it is not sufficient to rescue the embryonic lethality of TFPI null mice. DisclosuresMast:Novo Nordisk: Honoraria, Research Funding.

Syndecan-3 and TFPI Colocalize on the Surface of Endothelial-, Smooth Muscle-, and Cancer Cells

BackgroundTissue factor (TF) pathway inhibitor (TFPI) exists in two isoforms; TFPIα and TFPIβ. Both isoforms are cell surface attached mainly through glycosylphosphatidylinositol (GPI) anchors. TFPIα has also been proposed to bind other surface molecules, like glycosaminoglycans (GAGs). Cell surface TFPIβ has been shown to exert higher anticoagulant activity than TFPIα, suggesting alternative functions for TFPIα. Further characterization and search for novel TFPI binding partners is crucial to completely understand the biological functions of cell associated TFPI.Methods and ResultsPotential association of TFPI to heparan sulphate (HS) proteoglycans in the syndecan family were evaluated by knock down studies and flow cytometry analysis. Cell surface colocalization was assessed by confocal microscopy, and native PAGE or immunoprecipitation followed by Western blotting was used to test for protein interaction. Heparanase was used to enzymatically degrade cell surface HS GAGs. Anticoagulant potential was evaluated using a factor Xa (FXa) activity assay. Knock down of syndecan-3 in endothelial,- smooth muscle- and breast cancer cells reduced the TFPI surface levels by 20-50%, and an association of TFPIα to syndecan-3 on the cell surface was demonstrated. Western blotting indicated that TFPIα was found in complex with syndecan-3. The TFPI bound to syndecan-3 did not inhibit the FXa generation. Removal of HS GAGs did not release TFPI antigen from the cells.ConclusionsWe demonstrated an association between TFPIα and syndecan-3 in vascular cells and in cancer cells, which did not appear to depend on HS GAGs. No anticoagulant activity was detected for the TFPI associated with syndecan-3, which may indicate coagulation independent functions for this cell associated TFPI pool. This will, however, require further investigation.

Fluvastatin Upregulates the Expression of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are cholesterol-lowering drugs with a variety of pleiotropic effects including antithrombotic properties. Tissue factor pathway inhibitor (TFPI), which is produced predominantly in endothelial cells and platelets, inhibits the initiating phase of clot formation. We investigated the effect of fluvastatin on TFPI expression in cultured endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with fluvastatin (0-10μM). The expression of TFPI mRNA and antigen were detected by RT-PCR and western blotting, respectively. The effects of mevalonate intermediates, small GTP-binding inhibitors, and signal transduction inhibitors were also evaluated to identify which pathway was involved. A luciferase reporter assay was performed to evaluate the effect of fluvastatin on TFPI transcription. The stability of TFPI mRNA was estimated by quantitating its levels after actinomycin D treatment. Fluvastatin increased TFPI mRNA expression and antigen in HUVECs. Fluvastatin-induced TFPI expression was reversed by co-treatment with mevalonate or geranylgeranylpyrophosphate (GGPP). NSC23766 and Y-27632 had no effect on TFPI expression. SB203580, GF109203, and LY294002 reduced fluvastatin-induced TFPI upregulation. Moreover, fluvastatin did not significantly affect TFPI promoter activity. TFPI mRNA degradation in the presence of actinomycin D was delayed by fluvastatin treatment. Fluvastatin increases endothelial TFPI expression through inhibition of mevalonate-, GGPP-, and Cdc42-dependent signaling pathways, and activation of the p38 MAPK, PI3K, and PKC pathways. This study revealed unknown mechanisms of the anticoagulant effect of statins and gave a new insight to its therapeutic potential for the prevention of thrombotic diseases.

Tissue Factor Pathway Inhibitor Isoforms of Macro- and Microvascular Endothelial Cells

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. DisclosuresDockal: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.

Both red and blond orange juice intake decreases the procoagulant activity of whole blood in healthy volunteers

Numerous epidemiological studies suggest that exposure to flavonoid-rich fruits has beneficial influence on risk factors for cardiovascular disease. We investigated whether intake of orange juice (OJ) could affect whole blood (WB) procoagulant activity. 17 healthy subjects (aged 31 ± 1.5 SEM 10 males) were randomized to receive, according to a cross-over design, either red or blond OJ, enriched or free of anthocyanins, respectively. After one week run-in period on a controlled diet, the subjects were randomly allocated to receive either type of OJ for 4 weeks, with a 4-week wash-out period. Venous blood was collected on citrate before and at the end of each treatment period. WB was incubated with or without an inflammatory stimulus (tumor necrosis factor-α or bacterial endotoxin LPS). Procoagulant activity was evaluated by a one-stage clotting assay. Tissue factor (TF) and TF pathway inhibitor (TFPI) were measured in plasma by ELISA. Intake of either type of OJ caused a prolongation of unstimulated and stimulated WB clotting times, without any difference between the two treatments. Intake of OJ did not modify TF levels. On the contrary, an increase in circulating TFPI antigen was detected following either treatment. Orange juice intake by healthy volunteers decreases procoagulant activity, possibly through mechanisms independent of its anthocyanin content.

The role of tissue factor pathway inhibitor in atherosclerosis and arterial thrombosis

Tissue factor pathway inhibitor (TFPI) is the main inhibitor of tissue factor (TF)-mediated coagulation. In atherosclerotic plaques TFPI co-localizes with TF, where it is believed to play an important role in attenuating TF activity. Findings in animal models such as TFPI knockout models and gene transfer models are consistent on the role of TFPI in arterial thrombosis as they reveal an active role for TFPI in attenuating arterial thrombus formation. In addition, ample experimental evidence exists indicating that TFPI has inhibitory effects on both smooth muscle cell migration and proliferation, both which are recognized as important pathological features in atherosclerosis development. Nonetheless, the clinical relevance of these antithrombotic and atheroprotective effects remains unclear. Paradoxically, the majority of clinical studies find increased instead of decreased TFPI antigen and activity levels in atherothrombotic disease, particularly in atherosclerosis and coronary artery disease (CAD). Increased TFPI levels in cardiovascular disease might result from complex interactions with established cardiovascular risk factors, such as hypercholesterolemia, diabetes and smoking. Moreover, it is postulated that increased TFPI levels reflect either the amount of endothelial perturbation and platelet activation, or a compensatory mechanism for the increased procoagulant state observed in cardiovascular disease. In all, the prognostic value of plasma TFPI in cardiovascular disease remains to be established. The current review focuses on TFPI in clinical studies of asymptomatic and symptomatic atherosclerosis, coronary artery disease and ischemic stroke, and discusses potential atheroprotective actions of TFPI.

Circulating monocytes mirror the imbalance in TF and TFPI expression in carotid atherosclerotic plaques with lipid-rich and calcified morphology

BackgroundThrombogenicity of atherosclerotic plaque largely depends on plaque morphology and their content of tissue factor (TF) and tissue factor pathway inhibitor (TFPI). The relationship between morphological composition of plaque (lipid-rich or calcified) and expression of TF and TFPI in circulating blood monocytes and within the plaques is not characterized. ObjectiveTo investigate whether lipid-rich (echolucent) or calcified (echogenic) morphology of carotid atherosclerotic plaques is associated with differences in TF and TFPI expression in circulating blood monocytes and within carotid atherosclerotic plaques. MethodsWe studied levels of monocyte TF and TFPI mRNA and protein expression and association with traditional risk factors for atherosclerosis in asymptomatic subjects with echolucent (n=20) or echogenic (n=20) carotid plaques, or controls without carotid atherosclerosis (n=20) determined by ultrasonography. Sections of calcified or lipid-rich carotid plaques obtained from symptomatic patients were assessed for TF and TFPI antigen expression. ResultsTF and TFPI surface presentation, surface TF/TFPI ratio, and TF activity were higher in monocytes obtained from subjects with echolucent than with echogenic plaques or controls without carotid atherosclerosis. Multiple regression analyses revealed inverse association between serum apoA1 and monocyte surface TF antigen expression (p=0.007), and positive association between serum apoB and monocyte surface TFPI expression (p=0.028). Sections from lipid-rich carotid plaques contained 2.5-fold more TF and 1.5-fold more TFPI antigens relative to calcified lesions, also yielding a higher TF/TFPI ratio. ConclusionsOur findings indicate that circulating monocytes of asymptomatic individuals with echolucent lipid-rich carotid atherosclerosis express an imbalance between TF and TFPI expression cohering with changes found within advanced carotid atherosclerotic plaques obtained from symptomatic patients.

Pharmacodynamic Differences in the Two Generic Brands of Enoxaparin

Abstract 1251Several generic versions of enoxaparin have recently become available. While these generic versions of enoxaparin exhibit similar molecular profiles and comparable anti-Xa activities; product specific differences in global anticoagulant (APTT, Heptest and thrombin generation inhibition) have been reported. The purpose of this study was to compare a generic version of enoxaparin Sandoz from Argentina (Fibrinox lot 002) and from the US (enoxaparin lot 914786) in various in vitro whole blood and plasma based clotting tests. Despite comparable molecular profile and anti-Xa potency, product specific differences were noted between the products and the US generic enoxaparin showed a cumulatively stronger activity in most of the assays. To further test the pharmacodynamic profile of these products, individual groups of monkeys (n=4–8) were administered with each product at a 1 mg/kg SC and blood samples were collected for up to 28 hours. Clot based assays such as the APTT, Heptest, thrombin time, amidolytic anti-Xa and anti-IIa activities were carried out. In addition, tissue factor pathway inhibitor (TFPI) antigen, thrombin activatable fibrinolysis inhibitor (TAFI) activity and thrombin generation assays were also performed. Variable differences were noted in the clot based and amidolytic assays. Interestingly, the US generic product exhibited a lower release in the TFPI antigen whereas in the thrombin generation assays it produced a stronger inhibition of thrombin in terms of the AUC. TAFI activity profile also showed wide variations. These differences were more prevalent during the 1–4 hour time period. No differences were noted at >6 hours. The hysterisis PK/PD plots revealed marked differences between the two products. These results indicate that the products for the same generic suppliers may exhibit variations according to market places. Moreover, these observations underscore the need for a more stringent pharmacodynamic profile to demonstrate product equivalence. Disclosures:No relevant conflicts of interest to declare.

Increased risk of gestational vascular complications in women with low free tissue factor pathway inhibitor plasma levels out of pregnancy

Tissue factor pathway inhibitor (TFPI) plays a crucial role in haemostasis by regulating TF-induced initiation of coagulation. Since it is expressed by endothelial and trophoblastic cells, TFPI is of particular importance at the placental level and might be involved in the occurrence of gestational vascular complications (GVC). In the present study, we investigated plasma free TFPI antigen in four groups of women: healthy non-pregnant women without history of pregnancy complications; women at the beginning (<12 weeks) and women during the third trimester of a normal pregnancy; women with late pregnancy complications (pre-eclampsia / HELLP syndrome, intra-uterine fetal death, fetal growth retardation) at the time of obstetrical event and/or at distance from pregnancy. In normal pregnancy, TFPI increased between first trimester and delivery (median 5.0 ng/ml vs. 7.1 ng/ml; p<0.0001) but remained lower than in non-pregnant controls (median 8.2 ng/ml; p<0.0001). In patients, when measured concomitantly to the obstetrical event, TFPI showed no difference with normal late pregnancy levels. In contrast, at distance from pregnancy, in the absence of any hormonal influence, TFPI was significantly lower than in non-pregnant controls (median 5.9 vs. 8.2ng/ml, p < 0.0001). After categorisation into quartiles, an inverse dose-effect relationship was demonstrated between TFPI categories recorded apart from pregnancy and GVC risk, with a crude odds ratio of 43.5 (95% confidence interval 8.2-230) for patients with TFPI values in the lowest quartile (< 5.7 ng/ml). In conclusion, low free TFPI at distance from pregnancy appears to be a strong indicator of GVC risk.

Tissue Factor Pathway Inhibitor Causes Unresponsiveness to Activated Prothrombin Complex Concentrates for Hemophilia A Patients with Inhibitors.

AbstractAbstract 3663Bypassing agents such as activated prothrombin complex concentrates (APCC, FEIBA®) and recombinant activated factor VII (rFVIIa, NovoSeven®) are effective for most hemophiliac patients with inhibitors. While, some patients exhibit unresponsiveness to the treatment with APCC and/or rFVIIa, but their mechanisms remain unknown. We had a severe hemophilia A patient with inhibitor whose bleeding worsened despite of consecutive infusion of APCC. Switching from APCC to rFVIIa was very effective for his bleeding symptoms, and one-week cessation of bypassing agents had restored good response for APCC. Comprehensive coagulation assay such as thromboelastometry and thrombin generation test (TGT) provided us clear evidence of unresponsiveness to APCC. In particular, tissue factor (TF)-triggered TGT showed two significant features, the prolonged lag time and reduced peak thrombin level even after APCC infusion. Although FII, FVII(a), FIX, FX(a) and protein C contained in APCC were elevated in his plasmas after APCC infusion, increased amounts of these factors did not affect the parameters of TGT described above in vitro. We focused on a natural anticoagulant, tissue factor pathway inhibitor (TFPI), since the prolonged lag time in TF-triggered TGT might result from the impairment of FVII/TF-induced initial reaction of blood coagulation. In vitro experiment on the addition of TFPI to FVIII-deficient plasma with APCC showed similar inhibitory pattern in TGT. TFPI antigen levels (total and free forms) in his plasma actually increased above normal range after APCC infusion, whilst these levels unchanged after rFVIIa infusion and returned to the normal range after one-week cessation, speculating that TFPI might contributes to unresponsiveness to APCC. To confirm this, plasmas from several hemophiliac patients with APCC and/or rFVIIa infusion, including 4 patients with poor response pattern in TGT, were prepared. Among 12 pairs of plasmas (a pair; pre and post bypassing agents), each of 4 pairs were for APCC-poor response (APCC-PR), APCC-good response (APCC-GR), and rFVIIa-good response (FVIIa-GR). Free form TFPI antigen levels (normal; 15–35 ng/ml) increased after infusion in APCC-PR (pre/post; 38±4/51±3 ng/ml, p<0.05) and APCC-GR (28±4/37±4 ng/ml, p<0.05), but not increased in FVIIa-GR (23±3/21±3 ng/ml, p>0.05). Post-infusion levels in APCC-PR were significantly higher than those in APCC-GR (p<0.05). By adding anti-TFPI antibody, plasmas in APCC-PR showed marked increase of peak thrombin levels than those in APCC-GR in TGT, supporting that APCC-PR possessed more TFPI activity. Unexpectedly, ELISAs revealed that total TFPI were contained in APCC at 24±4 ng/unit (corresponded to 25≂f50% of physiological concentration), and 34% of them were free form, speculating that APCC infusion with ≂f90 units/kg appeared to increase free TFPI by ≂f15 ng/ml in plasma. Taken together, our results supported that TFPI contained in APCC attenuated the potentials of thrombin generation in hemophilia A patients with inhibitors, and some patients exhibited APCC-resistance due to TFPI accumulated by the consecutive use of APCC. Disclosures:Ogiwara:Baxter Hemophilia Scientific Research and Education Fund in Japan 2009: Research Funding. Nogami:Bayer Hemophilia Award Program 2009: Research Funding.

Calibrated automated thrombography demonstrates hypercoagulability in patients with idiopathic pulmonary arterial hypertension

Introduction Pathogenesis of idiopathic pulmonary arterial hypertension (iPAH) includes endothelial dysfunction and in situ thrombosis. A hypercoagulable state has also been postulated but never demonstrated. Our objective was to determine whether patients with iPAH had a hypercoagulable state using calibrated automated thrombography (CAT), a new tool to phenotype coagulation in vitro. Patients and methods 16 patients with iPAH and 29 controls were studied. In vitro platelet dependent coagulation phenotyping by CAT monitored the activity of thrombin generation over time. Plasma levels of soluble thrombomodulin, tissue factor pathway inhibitor (TFPI) and von Willebrand factor (VWF) were measured as endothelial biomarkers. Results Endogenous thrombin potential (ETP) in the absence of activated protein C (APC) tended to be increased in patients compared to controls (1769 versus 1656 nM.min; p = 0.053). ETP was higher in the presence of APC 25 nM (ETP-APC) in patients (781 versus 494 nM.min; p = 0.005). Five patients had ETP-APC higher than the 95th centile of controls. Other CAT parameters (lag time, peak thrombin and time to peak) were all consistent with some degree of hypercoagulability in patients. Regarding endothelial plasma biomarkers sTM was lower (28.4 versus 40.6 μg/l, p = 0.0108) in patients; TFPI antigen and activity (respectively: 14.3 versus 10.5 μg/l, p = 0.0167; 1.155 versus 1.070, p = 0.0021) and VWF (1300 versus 976%, p = 0.0108) were higher in patients. Conclusion We have demonstrated that at least some patients with iPAH have a hypercoagulable phenotype.