Tissue Factor Pathway Inhibitor Deficiency Research Articles

Loss of Arl6ip5 Gene Function Suppresses Thrombosis in Mice

Background: The genetic contributors to thrombosis have only been partly described. Heterozygous tissue factor pathway inhibitor deficiency ( Tfpi+/-) is a powerful negative genetic modifier of the homozygous FVL ( F5L/L) phenotype, demonstrating a nearly complete perinatal lethal genetic interaction in F5L/LTfpi+/- mice. To identifymutations suppressing lethal thrombosis, we used this F5L/LTfpi+/- phenotype for a sensitized genome wide ENU mutagenesis screen. We screened over 12,000 mice and generated a total of 31 thrombosis suppressor variant lines. We were able to identify ENU-induced mutations in 10 of these lines, each potentially representing an independent pathway for reducing thrombosis severity. One of the mutations that suppressed thrombosis in F5L/LTfpi+/- mice was a dominant loss of function mutation (i.e. mutation in a single allele) in the ADP Ribosylation Factor Like GTPase 6 Interacting Protein-5 ( Arl6ip5) gene. Interestingly, no direct role(s) of Arl6ip5 gene or protein in hemostasis have been reported. Therefore, the aim of our studies was to investigate the hypothesis that Arl6ip5 deficiency suppresses thrombosis by reducing blood coagulation and platelet reactivity in mice. Methods: We previously generated a mouse model of Arl6ip5 ( Arl6ip5 -) by genome editing, creating a 151 base pair deletion removing an exon 1 splice site. After backcrossing mice carrying the Arl6ip5 - allele to the wildtype C57BL/6J (B6) background, we performed Arl6ip5 +/-intercrosses to determine a potential difference in genotype frequency at weaning. To investigate whether the Arl6ip5 - allele was able to produce a stable mRNA transcript we performed RT-qPCR on whole liver, lung, heart, kidney, and brain samples derived from Arl6ip5 +/+ Arl6ip5 +/- and Arl6ip5 -/- mice. Furthermore, complete blood cell counts were analyzed using the Advia 2120 (N ≥ 10 for each group), and to investigate potential blood coagulation defects, Prothrombin Time (PT) (N ≥ 5 for each group) and activated Partial Thromboplastin Time (aPTT) (N ≥ 8 for each group) assays were performed on plasma from Arl6ip5 +/+ Arl6ip5 +/- and Arl6ip5 -/- mice with the operator blinded to the genotype. Similarly, we performed platelet function analyses on whole blood from Arl6ip5 +/+ Arl6ip5 +/- and Arl6ip5 -/- mice using the Roche Multiplate, with ADP and collagen as agonists (N ≥ 6 for each group). Results: Arl6ip5 +/- x Arl6ip5+/- crosses resulted in normal Mendelian proportions of progeny observed at weaning (N > 400). No Arl6ip5transcripts were present in any of the Arl6ip5 -/- tissue samples analyzed, whereas heterozygous mice had the expected 50% Arl6ip5 expression levels across all organs as compared to wildtype mice. A significant prolongation was observed in aPTT between Arl6ip5+/+ and Arl6ip5-/- animals (p<0.01). Although no differences in platelet counts between genotypes were observed, platelet aggregometry showed significant differences between wildtype Arl6ip5 +/+and Arl6ip5 -/-littermates for platelet aggregation, velocity, and area under the curve in response to ADP agonist (normalized to platelet counts, p<0.01). In addition, collagen-induced aggregometry also yielded significant differences in aggregation velocity. Conclusions: Our results support the hypothesis that Arl6ip5 deficiency contributes to thrombosis suppression by increasing the aPTT and reducing platelet reactivity. Since the PT in Arl6ip5 -/- mice is normal, we speculate that Arl6ip5 -/- is either directly or indirectly affecting one or more of the levels or activity of coagulation factors VIII, IX, XI and XII. Significant differences in ADP- and collagen-induced platelet aggregation between Arl6ip5 +/+wildtype littermates and Arl6ip5 -/- suggests that the lack of Arl6ip5affects an aggregation process common to both pathways. Additional analyses are being performed to refine these mechanisms. Understanding the mechanism by which Arl6ip5 deficiency suppresses thrombosis could provide insights into human thrombosis regulation.

An Intergenic Noncoding Chromosome 18 Variant Suppresses Lethal Thrombosis in Mice By Normalizing Blood Coagulation and Reducing Platelet Reactivity

Background: Factor V Leiden (FVL) is a common thrombosis susceptibility variant in humans. It is incompletely penetrant; this indicates that there are modifiers of FVL that alter thrombosis susceptibility. We used mouse models of FVL (F5L) and heterozygous tissue factor pathway inhibitor deficiency (Tfpi+/-), to identify a perinatal lethal genetic interaction when mice inherited F5L/L Tfpi+/-. This phenotype was used as the basis for a sensitized genome wide ENU mutagenesis screen to identify mutations suppressing lethal thrombosis in F5L/L Tfpi+/- mice. From this screen, we generated multiple independent lines of thrombosuppressed mice, called MF5L, for Modifier of F5L. MF5L16 was a large, highly penetrant (77.2%), multigenerational pedigree containing 136 viable F5L/L Tfpi+/- mice.Aims: In the present study, we aimed to identify and functionally characterize the thrombosuppressor mutation present in MF5L16.Methods: Genomic analyses: We performed whole genome sequencing (WGS) on four MF5L16 F5L/L Tfpi+/- mice. We used comparative bioinformatic analyses to identify variants inherited by all four mice and compiled these variants into candidate variant list. PCR and Sanger sequencing were used to analyze the 136 F5L/L Tfpi+/- mice for inheritance of each of the candidate variants. Functional analyses: We performed biochemical blood coagulation and platelet assays of blood from the Chr18 A mice . Complete blood counts were measured using the Advia 2120 with settings optimized for C57BL/6 mouse blood. Platelet aggregation studies were performed using the Roche Multiplate Aggregometer with ADP and type 1 collagen as the aggregating agents.Results: We analyzed four MF5L16 mice by WGS and identified seven spontaneous mutations that arose in our F5L/L breeding colony that were introduced into MF5L16. Importantly, no coding variants were linked to these variants. Analysis of these seven mutations in all 136 MF5L16 F5L/L Tfpi+/- mice revealed a significant association between a Chromosome 18 intergenic variant (Chr18 G to A, Chr18 A) and F5L/L Tfpi+/- mouse survival (p=0.003). To re-create the suppression of the lethal F5L/L Tfpi+/- phenotype, we bred F5+/L Tfpi+/- Chr18 +/A triple heterozygous mice to F5L/L Chr18 A/A mice to observe the effects of Chr18 A on F5L/L Tfpi+/- mouse survival. Out of 109 mice from this cross, two F5L/L Tfpi+/- Chr18 +/A mice were produced (expected ratio ~1:8). This suggests that the Chr18 A variant suppresses F5L/L Tfpi+/- lethal thrombosis at ~15% penetrance. Complete blood count analysis on Chr18 +/+,Chr18 +/A, and Chr18 A/A mice determined that Chr18 A/A mice had reduced platelet count and distribution width and increased variability in red blood cell (RBC) mean corpuscular volume (n≥4; p<0.05). The Chr18 A/A mice did not display differences in PT or aPTT assays, but had significantly reduced platelet aggregation velocity when stimulated by both ADP and collagen agonists (n≥4; p=0.0002). Additionally, blood smears revealed the presence of poikilocytic RBCs in the Chr18 A/A mice.Conclusions and future directions: Our results establish that a noncoding intergenic Chr18 variant at nucleotide position 62,970,011 (G>A, Chr18 A) contributes to thrombosuppression by reducing platelet reactivity. The observed platelet and RBC phenotypes suggest that a major mechanism of Chr18 A thrombosuppression could be through regulation of gene expression in cells of the myeloid lineage. We are performing additional platelet and blood coagulation analyses to refine the phenotypic differences due to the Chr18 A variant. Comparative transcriptomic analyses are also being performed to identify the genetic pathways involved. Understanding the mechanism in which this intergenic mutation suppresses thrombosis could provide insights into human thrombosis regulation. DisclosuresNo relevant conflicts of interest to declare.

Maintaining extraembryonic expression allows generation of mice with severe tissue factor pathway inhibitor deficiency

AbstractTissue factor pathway inhibitor (TFPI) is a serine protease with multiple anticoagulant activities. The Kunitz1 (K1) domain of TFPI binds the active site of factor VIIa and is required for inhibition of tissue factor (TF)/factor VIIa catalytic activity. Mice lacking TFPI K1 domain die in utero. TFPI is highly expressed on trophoblast cells of the placenta. We used genetic strategies to selectively ablate exon 4 encoding TFPI K1 domain in the embryo, while maintaining expression in trophoblast cells. This approach resulted in expected Mendelian frequency of TFPI K1 domain–deficient mice. Real-time polymerase chain reaction confirmed 95% to 99% genetic deletion and a similar reduction in transcript expression. Western blotting confirmed the presence of a truncated protein instead of full-length TFPI. Mice with severe TFPI K1 deficiency exhibited elevated thrombin-antithrombin (TAT) levels, frequent fibrin deposition in renal medulla, and increased susceptibility to TF-induced pulmonary embolism. They were fertile, and most lived normal life spans without any overt thrombotic events. Of 43 mice observed, 2 displayed extensive brain ischemia and infarction. We conclude that in contrast to complete absence of TFPI K1 domain, severe deficiency is compatible with in utero development, adult survival, and reproductive functions in mice. Inhibition of TFPI activity is being evaluated as a means of boosting thrombin generation in hemophilia patients. Our results show that in mice severe reduction of TFPI K1 activity is associated with a prothrombotic state without overt developmental outcomes. We note fibrin deposits in the kidney and rare cases of brain ischemia.

Knockdown of TFPI-Anchored Endothelial Cells Exacerbates Lipopolysaccharide-Induced Acute Lung Injury Via NF-κB Signaling Pathway.

ABSTRACTAs activation of the coagulation system is both a consequence and contributor to acute lung injury (ALI), pulmonary coagulopathy has become a potential target for therapeutic intervention in ALI patients. We investigated the effects and possible mechanisms of endothelial cell (EC)-anchored tissue factor pathway inhibitor (TFPI) on lipopolysaccharide (LPS)-induced ALI in mice. To assess the effect of EC-anchored TFPI deletion on ALI indices, TFPI knockout (cKO) mice were generated. Mice were instilled by direct intratracheal injection LPS for the preparation of an ALI model. Evans blue dye (EBD) was injected intravenously 2 h prior to animal sacrifice (48 h post-LPS). Lungs were fixed for histopathology and the prepared tissue was homogenized or used to extract bronchoalveolar lavage fluid (BALF) or detect EBD concentration. TFPI knockdown mice with ALI were compared to wild-type (WT) mice with ALI to assess the effect of TFPI on endothelial barrier function and inflammation. TFPI deletion markedly exacerbated LPS histopathological changes in lung, and the LPS changes in protein, EBD extravasation, proinflammatory cytokines TNF-α, IL-1β, and IL-6 in BALF in lung. The number and infiltration of white blood cells (WBCs) from BALF and lung tissue of TFPI cKO mice with LPS-challenged ALI was increased compared to WT mice with LPS-challenged ALI. We also found further increased toll-like receptor 4 and nuclear factor kappa-light-chain-enhancer of activated B cells activation and additional expression of vascular cell adhesion molecule 1 and reduction of angiotensin converting enzyme 2 expression in TFPI cKO+LPS mice compared with WT+LPS mice. Endothelial-specific TFPI deficiency promoted LPS-induced pulmonary inflammation and endothelial barrier permeability possibly via toll-like receptor 4-mediated nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathway activation.

TFPIα Has Reduced Ability to Inhibit FV Leiden

Introduction: Tissue factor pathway inhibitor (TFPI)α dampens the initiation phase of coagulation by inhibiting the tissue factor/factor VIIa complex and early thrombin generation. Activated protein C (APC) dampens the propagation phase of coagulation by degrading factor Va (FVa) and factor VIIIa. Kinetic assays using purified TFPIα, protein C, and thrombomodulin have shown that TFPIα and APC act synergistically to inhibit thrombin production. In these assays, thrombin generation is markedly altered by factor V Leiden (FVL), a prothrombotic mutation that reduces the ability of APC to degrade FVa. A 50% reduction in TFPIα (2.5nM to 1.25nM) completely abolishes the anticoagulant effect of APC in reactions performed with FVL, but has no effect in reactions performed with FV. The physiological importance of these in vitro results was subsequently supported by the severe perinatal thrombosis observed in mice with homozygous FVL and heterozygous TFPI deficiency. TFPIα inhibits thrombin generation via a high affinity exosite interaction between its basic C-terminus and an acidic region in early forms of FVa. We hypothesized that the Leiden mutation alters this interaction in a manner that reduces TFPIα inhibitory activity during the initiation of coagulation.Methods: Plasma and platelet-rich plasma (PRP) were collected from homozygous FVL, heterozygous FVL, and control donors. Thrombin generation assays were performed in the presence or absence of recombinant TFPIα, a peptide mimicking the TFPIα basic C-terminus (LIKTKRKRKK), or anti-TFPI antibodies. Purified prothrombinase assays were performed using FV810, a B-domainless form of FV(a) that binds and is inhibited by TFPIα, and FVL810.Results: Thrombin generation assays were initiated with limiting FXa to determine an activation threshold. The threshold for thrombin generation was between 10 and 20 pM FXa in all 10 control plasmas, but was reduced in homozygous FVL plasmas, with 4 of 10 responding to only 5 pM FXa. Additional thrombin generation assays were performed using 100 pM FXa to determine the effect of FVL on TFPI activity, as there were no baseline differences between control and FVL plasmas in these conditions. When substances that alter TFPI function were added, the lag time for initiation of thrombin generation was primarily affected. Addition of 1 nM recombinant TFPIα and 1 μM LIKTKRKRKK prolonged the lag time in control plasma by 58% and 28%, respectively, but had progressively less effect in FVL heterozygous (45% and 19%) and homozygous (39% and 9.6%) plasmas (p<0.03 for all comparisons except addition of TFPIα to heterozygous plasma). Conversely, anti-TFPI antibodies directed against the second (anti-K2) and third (anti-K3) Kunitz domains decreased the lag time 32% and 23% in control plasmas, respectively, but only 25% and 17% in FVL homozygous plasma (p=0.03 for both). Thrombin generation assays were repeated using PRP. There were minimal and equivalent effects of recombinant TFPIα or LIKTKRKRKK in control and FVL PRP, possibly due to the presence of additional exogenous TFPIα in platelets. However, the anti-TFPI antibodies decreased the lag time more in control plasma than in FVL homozygous plasma, with anti-K2 decreasing the lag time of control PRP by 28% and FVL homozygous PRP by 20% (p=0.009). In purified prothrombinase assays, TFPIα was a weaker inhibitor of prothrombinase containing FV810 (IC50 = 4.8nM; C.I.= 3.8-6.1nM) compared to FVL810 (IC50 = 7.3nM; C.I. = 6.9-7.9nM). A similar effect of FVL was observed when inhibiting with LIKTKRKRKK.Conclusion: The biochemical mechanism underlying the procoagulant phenotype of FVL is well-described as a resistance to APC, which dampens the propagation phase of clot formation. The data described here suggest a potential second biochemical mechanism underlying this procoagulant phenotype, a reduced ability of TFPIα to inhibit FVL. This effect is evidenced by alterations in the threshold for initiation of thrombin generation in FVL homozygous plasma and PRP, as well as the reduced IC50 for inhibition of thrombin production by prothrombinase assembled with FVL. This effect is enhanced by reducing TFPIα concentration, offering an explanation for the dramatic procoagulant effect of 50% reduced TFPI in assays using human proteins and mice with FVL. The findings could have clinical relevance in patients with FVL who use oral contraceptives that can reduce the plasma TFPIα concentration. DisclosuresCamire:Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Research Funding; Pfizer: Consultancy, Patents & Royalties, Research Funding. Mast:Novo Nordisk: Honoraria, Research Funding.

Hyperactivatable Protein C Expression Partially Rescues the Embryonic Lethality of TFPI Null Mice but Surviving Mice Develop Severe Hydrocephalus

Introduction: Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) are the two anticoagulant proteins directly bound to endothelium. TFPI inhibits the initiation of coagulation by inhibiting TF-FVIIa and early forms of prothrombinase. TM slows the propagation of coagulation by promoting the activation of protein C (APC), which inactivates FVa and FVIIIa. An in vitro model system of purified coagulation proteins has shown that TFPI and thrombomodulin act synergistically to quench tissue factor mediated thrombin generation via neutralization of prothrombinase activity. However, it is unclear how these two proteins cooperate within different vascular beds in vivo, particularly in the brain that has large amounts of tissue factor procoagulant activity. We have previously shown that mice with decreased thrombomodulin function (TMpro) do not have cerebral fibrin deposition even following LPS injection, while partial TFPI deficiency does induce intravascular fibrin deposition in the brain of the TMpro mice, suggesting that these two anticoagulant proteins have distinct functions within the brain vasculature. TFPI null mice die in utero with brain fibrin deposition. A hyperactivatable murine protein C (hMPC) can be produced by altering two acidic amino acids near the thrombin cleavage site. hMPC is activated 30-fold more efficiently by thrombin than wild type protein C and its activation does not require TM. We have used murine model systems to investigate how hMPC expression alters the (patho)physiology of mice with TFPI deficiency.Methods: Transgenic mice producing hMPC under control of the transthyretin promoter were produced. hMPC was bred into TFPI+/- mice, which were then characterized to define the anticoagulant activity of the transgene. Offspring from these initial matings were interbred to determine if hMPC expression would rescue the embryonic lethality of TFPI null mice.Results: hMPC expression elevated plasma protein C 2-fold and plasma APC 3-fold producing a potent anticoagulant effect. In thrombin generation assays, plasma from TFPI+/-/hMPC+ mice had peak thrombin generation of 44.9±7.5 nM vs. 63.0±3.5 nM in TFPI+/-/hMPC- mice (p<0.001). In a TF-induced pulmonary embolism model 5 of 14 TFPI+/-/hMPC+ mice survived over 5 minutes, while only 1 of 10 TFPI+/-/hMPC- did (p=0.02). When TFPI+/-/hMPC+ mice were mated with TFPI+/-/hMPC- mice, TFPI-/-/hMPC+ pups were born at approximately 30% of the expected frequency regardless of whether the transgene was expressed by the mother or the father. About 35% of the surviving TFPI-/-/hMPC+ mice developed a domed shaped head and succumbed to severe hydrocephalus by 8 weeks of age. Those surviving beyond 8 weeks did not develop severe hydrocephalus and were fertile. One TFPI-/-/hMPC+ mouse with a domed shaped head survived long enough to be examined by MRI, which documented severe hydrocephalus. In addition, two lesions (1mm and 0.5 mm diameter) were present in remaining brain tissue. These lesions contained iron suggesting they were areas of blood clot or hemorrhage. MRI exam of 12 week old TFPI-/-/hMPC+ mice identified subclinical hydrocephalus in 1 of 4 mice. Hydrocephalus did not occur in TFPI+/+/hMPC+ or TFPI+/-/hMPC+ mice. Histological examination of brain tissue from TFPI-/-/hMPC+ mice confirmed hydrocephalus with little remaining normal brain tissue. Mice with hydrocephalus had hemorrhage in the ventricles and brain parenchyma that was associated with areas of fibrin(ogen) deposition. There was also congestion of pial vessels and hemorrhage within the subarachnoid space. These findings were not observed in TFPI+/-/hMPC+ mice.Conclusions: Expression of the hMPC transgene produces a potent anticoagulant effect that partially rescues TFPI null embryonic lethality. Surviving TFPI-/-/hMPC+ pups are susceptible to death from severe hydrocephalus associated with hemorrhage and vascular abnormalities in the brain as they mature. These findings demonstrate that TFPI produces physiologically important anticoagulant activity within murine brain vasculature that is not fully compensated by over-expression of activated protein C. DisclosuresMast:Novo Nordisk: Honoraria, Research Funding.

Endothelial cell-anchored tissue factor pathway inhibitor regulates tumor metastasis to the lung in mice.

Tissue factor pathway inhibitor (TFPI) is a physiological inhibitor of the tissue factor (TF)-initiated coagulation pathway. Both circulating and tumor cell-associated TFPI significantly reduce tumor cell-induced coagulation activation and lung metastasis. However, the significance of endothelial cell-anchored TFPI in cancer biology remains largely unexplored. We generated mice with full-length disruption of TFPI (including TFPIα and TFPIβ isoforms) in endothelial cells, using a Cre-LoxP system and gene inactivation (GI) strategy. Experimental pulmonary tumor metastasis models were used with TFPI-deficient mice to evaluate the role of endothelial cell-anchored TFPI in cancer progression. Finally, lung microvascular permeability and microenvironment were investigated. TFPI-deficient mice were viable and fertile, and showed decreased plasma TFPI levels and lung TFPI levels as compared with their control littermates. TFPI deficiency in endothelial cells promoted pulmonary tumor metastasis with an increased vascular permeability and altered lung microenvironment. Our observations suggest that endothelial cell-anchored TFPI controls lung tumor metastasis, and does so largely through the inhibition of local TF-induced thrombin generation and the regulation of the lung microenvironment in mice.

Tissue Factor Pathway Inhibitor Modulates Fibrin Deposition In The Brain In a Manner That Is Independent Of Thrombomodulin and Activated Protein C

BackgroundFibrin deposition in cerebral vessels contributes to the pathogenesis of stroke and vascular dementia. Under quiescent conditions fibrin deposition is prevented or limited by natural anticoagulants of the endothelium. Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) are the two major endothelial anticoagulants and act on different portions of the clotting cascade. TFPI inhibits the initiation of coagulation by inhibiting TF-FVIIa and FXa. TM slows the propagation of coagulation by promoting the activation of protein C (APC), which inactivates FVa and FVIIIa. Both proteins are reported to have low expression in the brain and their function in prevention of cerebral vascular fibrin deposition is unclear. This is emphasized by findings from murine models of decreased TFPI and TM (TMpro/pro) activity. TFPI null embryos have fibrin deposition in the brain and liver, while TMpro/pro mice have fibrin deposition in the lungs, heart, spleen and liver, but the brain is protected from fibrin deposition even following lethal LPS doses. Interestingly, TFPI heterozygosity combined with TMpro/pro results in cerebral vascular fibrin. Here, we report a detailed analysis of TFPI expression in the brain vasculature, the consequences of vascular TFPI deficiency in adult mice, and the capacity of APC to compensate for TFPI deficiency. Methods and ResultsReal time PCR studies demonstrated that TFPI expression in total brain is lower than in other tissues in comparison to the RPL-19 housekeeping gene. However, when the endothelial-specific genes, VE-cadherin or CD31 were used as housekeeping genes, brain TFPI expression was higher than that of lung endothelium (TFPIa: ∼9 fold higher; TFPIb: ∼5-fold higher). In situ hybridization studies revealed that TFPI and TM are expressed on endothelium throughout the brain parenchyma. In addition, TFPI was expressed within the granule layer of the cerebellum. TFPI null embryos succumb to embryonic lethality with necrotic lesions and fibrin deposition in the brain. To determine if increased TM-APC anticoagulant activity would rescue TFPI null embryos, a transgene over expressing human activated protein C (hHPC) was bred into heterozygous TFPI mice, which were then bred to determine if TFPI null mice expressing hHPC survive embryogenesis. However, the hHPC transgene did not rescue TFPI null embryos. Furthermore, TFPI null embryos had similar brain lesions and brain fibrin deposition regardless of the presence of the hHPC transgene. A hematopoietic cell transplant model system was used to examine the consequences of TFPI deficiency in the adult mouse brain. Mice transplanted with hematopoietic cells lacking TFPI had cerebral vascular fibrin deposition that was not observed in mice transplanted with hematopoietic cells producing normal amounts of TFPI. Interestingly, the fibrin was often found at sites of endothelial injury as demonstrated by co-localization with anti-C1qA, which binds to phosphatidylserine on damaged endothelium. ConclusionsTFPI prevents necrotic brain lesions and cerebral fibrin deposition in developing mouse embryos and over expression of hHPC is not sufficient to compensate for the absence of TFPI. Adult mice lacking TFPI in hematopoietic cells have increased amounts of fibrin deposited at sites of cerebral vascular injury despite having normal endothelial TM. These data indicate that the distinct function of TFPI in the cerebral vasculature is not compensated by the anticoagulant activity of TM in regulating cerebral vascular thrombosis. Disclosures:Mast:Novo Nordisk: Honoraria, Research Funding.

Decreased tissue factor pathway inhibitor (TFPI)‐dependent anticoagulant capacity in patients with cirrhosis who have decreased protein S but normal TFPI plasma levels

Protein S acts as a cofactor for tissue factor pathway inhibitor (TFPI) in the down regulation of thrombin generation, and acquired and congenital protein S deficiencies are associated with a concomitant TFPI deficiency. In contrast, in patients with liver diseases, decreased protein S, but normal or increased levels of TFPI have been reported. We compared TFPI and protein S plasma levels between 26 patients with cirrhosis and 20 healthy controls and found that TFPI levels were comparable between patients (111 ± 38%) and controls (108 ± 27%), despite reduced protein S levels (74 ± 23% in patients vs. 98 ± 10% in controls). Subsequently, we quantified the activity of the TFPI-protein S system by measuring thrombin generation in the absence and presence of neutralizing antibodies to protein S or TFPI. Ratios of peak thrombin generation in the absence and presence of these antibodies were calculated. Both the protein S and the TFPI ratios were increased in patients with cirrhosis compared to controls. Protein S ratios were (0·62 [0·08-0·93] in patients vs. 0·32 [0·20-0·54] in controls; TFPI ratios were 0·50 [0·05-0·90] in patients vs. 0·18 [0·11-0·49] in controls). Thus, although the acquired protein S deficiency in patients with cirrhosis is not associated with decreased TFPI levels, the TFPI/protein S anticoagulant system is functionally impaired.

A Sensitized Whole Genome ENU Mutagenesis Screen Identifies an Arp2 Missense Mutation As a Novel Suppressor of Lethal Thrombosis in the Factor V Leiden Mouse

Abstract 493Only ∼10% of individuals carrying the common risk factor, Factor V Leiden (FVL), will develop venous thrombosis. In order to identify potential FVL modifier genes, we performed a sensitized dominant ENU mutagenesis screen based on the perinatal synthetic lethal thrombosis observed in mice homozygous for FVL (FVQ/Q) and hemizygous for tissue factor pathway inhibitor deficiency (Tfpi+/−). The screen was performed by crossing ENU-treated male FVQ/Q mice with FVQ/+ Tfpi+/− females. Surviving G1 offspring were analyzed to identify survivors with the lethal FVQ/Q Tfpi+/− genotype. Analysis of 7,128 G1 offspring (∼2X genome coverage) identified 98 FVQ/Q Tfpi+/− mice that survived to weaning. Fourteen FVQ/Q Tfpi+/− G1 mice exhibited successful transmission of a putative suppressor mutation to two or more FVQ/Q Tfpi+/− G2 offspring. Whole exome sequencing was performed on a progeny tested member of 8 of the 14 lines using the Agilent SureSelect mouse whole exome capture kit resulting in ∼100 fold coverage. Variant analysis revealed a small number of high confidence novel heterozygous (dominant) single nucleotide variants (SNVs) in each sample. Sanger re-sequencing of all 11 SNVs in a cohort of mice from line 1 confirmed that the G to C mutation (chromosome 11 base 19,977,300) in the Actr2 gene (present in 15/16 re-sequenced progeny p<0.0001) is the dominant FVL modifier in this line. This mutation resulted in an R286G substitution in a highly conserved amino acid in the Arp2 protein, which is essential for the function of the Arp2/3 complex. Arp2/3 is responsible for intracellular actin branching and polymerization, which is critical for the regulation of cell shape. Analysis of 31 progeny from an Arp2 R/G × Arp2 R/G cross revealed only one live Arp2 G/G mouse (p<0.05), suggesting that R286G is a loss of function mutation and that homozygous deficiency is lethal. Complete blood counts (Advia 2120) performed on 14 Actr2 heterozygous and 10 wildtype littermates revealed no significant differences in platelet count, red and white blood cell counts, hematocrit or hemoglobin. However, measurements of platelet size and size distribution including mean platelet dry mass, platelet volume distribution width and the platelet component distribution width were significantly altered in Actr2 heterozygous mutant mice (p<0.05 for each measure). Thus, partial deficiency of Arp2 appears to alter platelet structure/function resulting in a shift in hemostatic balance facilitating survival of the otherwise lethal FVQ/Q Tfpi+/− phenotype. These results suggest that variation in Arp2 or related genes could potentially modify thrombosis risk in humans, and might also identify novel therapeutic targets for the treatment of this class of disorders. Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Absence of hematopoietic tissue factor pathway inhibitor mitigates bleeding in mice with hemophilia

Tissue factor pathway inhibitor (TFPI) blocks thrombin generation via the extrinsic blood coagulation pathway. Because the severe bleeding in patients with hemophilia occurs from deficiency of intrinsic blood coagulation pathway factor VIII or IX, pharmacological agents that inactivate TFPI and, therefore, restore thrombin generation via the extrinsic pathway, are being developed for treatment of hemophilia. Murine models of combined TFPI and factor VIII deficiency were used to examine the impact of TFPI deficiency on bleeding and clotting in hemophilia. In breeding studies, Factor VIII null (F8(-/-)) did not rescue the embryonic death of TFPI null (Tfpi(-/-)) mice. Tfpi(+/-) did not alter the bleeding phenotype of F8(-/-) mice. However, total inhibition of intravascular TFPI through injection of anti-TFPI antibody mitigated tail vein bleeding. Interestingly, tail blood loss progressively decreased at doses greater than needed to totally inhibit plasma TFPI, suggesting that inhibition of a sequestered pool of TFPI released at the injury site mitigates bleeding. Because TFPI is sequestered within platelets and released following their activation, the function of platelet TFPI was examined in F8(-/-) mice lacking hematopoietic cell TFPI that was generated by fetal liver transplantation. Blood loss after tail transection significantly decreased in Tfpi(+/-);F8(-/-) mice with hematopoietic Tfpi(-/-) cells compared with Tfpi(+/-);F8(-/-) mice with Tfpi(+/+) hematopoietic cells. Additionally, following femoral vein injury, Tfpi(+/-);F8(-/-) mice with Tfpi(-/-) hematopoietic cells had increased fibrin deposition compared with identical-genotype mice with Tfpi(+/+) hematopoietic cells. These findings implicate platelet TFPI as a primary physiological regulator of bleeding in hemophilia.

Whole-Exome Sequencing to Identify Mutations in Thrombosis Modifier Genes Isolated From a Factor V Leiden-Dependent Sensitized ENU Suppressor Screen in the Mouse

Abstract 1183Only ∼10% of individuals carrying the common venous thrombosis risk factor, Factor V Leiden (FVL) will develop venous thrombosis in their lifetime. In order to identify potential FVL modifier genes, we performed a sensitized dominant ENU mutagenesis screen, based on the perinatal synthetic lethal thrombus previously observed in mice homozygous for FVL (FVQ/Q) and hemizygous for tissue factor pathway inhibitor deficiency (Tfpi+/−). The genome-wide ENU mutagenesis screen was performed by crossing ENU-treated male FVQ/Q mice with FVQ/+ Tfpi+/− females. Surviving G1 offspring were analyzed to identify survivors with the otherwise lethal FVQ/Q Tfpi+/− genotype. As proof of concept, we demonstrated that reduced tissue factor (Tf+/−) suppresses the lethal FVQ/Q Tfpi+/− phenotype, suggesting that mutations at Tf should be among the suppressor genes identified by our screen. Analysis of 7,128 G1 offspring (∼2X genome coverage) identified 98 FVQ/Q Tfpi+/− mice that survived to weaning. Fourteen FVQ/Q Tfpi+/− G1 mice exhibited successful transmission of a putative suppressor mutation to two or more FVQ/Q Tfpi+/− G2 offspring. Extensive genotyping of mice from an expanded genetic cross from one of these lines mapped a candidate suppressor locus to a chromosome 3 region encompassing the TF gene (LOD=4.93). With continued improvements in next generation sequencing technologies, we have now applied whole exome sequencing to analysis of 8 of the remaining 13 lines. The entire DNA coding region (the “exome”, totaling 49.6 Mb of DNA sequence) from a progeny-tested member of each line was captured using the Agilent SureSelect mouse exome capture system. Whole-exome sequencing using the Illumina HiSeq high-throughput sequencer yielded 12–15 gigabases of sequence data per sample, corresponding to an average of ∼200 fold sequencing coverage for each nucleotide position. Variant analysis using the Gene Analysis Toolkit revealed the presence of a small number of high confidence novel heterozygous (dominant) variants in each sample. Each of these heterozygous variants is a candidate suppressor mutation and these are presently being tested in remaining FVQ/Q Tfpi+/− mice from each respective line. Based on previous studies where ENU-induced mutations in mice have been identified, we anticipate the identification of putative exomic mutations in approximately 80% of tested suppressor lines. Identification of these mutations should provide candidate modifier genes for FVL and other human hemostatic disorders. Disclosures:Ginsburg:Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Catalyst Biosciences: Membership on an entity's Board of Directors or advisory committees; Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Murine Hematopoietic Cell Tissue Factor Pathway Inhibitor Limits Thrombus Growth

Tissue factor (TF)-factor VIIa initiates blood coagulation and is found on microparticles that accumulate within intravascular thrombi. Tissue factor pathway inhibitor (TFPI), a factor Xa (fXa)-dependent inhibitor of TF-factor VIIa, is produced by megakaryocytes and is present in platelets. We sought to determine the role of platelet TFPI in regulation of thrombus growth. Western blot analyses demonstrated that murine platelets produce TFPIα, the most evolutionarily conserved alternatively spliced isoform of TFPI. A mouse model of hematopoietic cell TFPI deficiency was developed by transplanting irradiated TFPI(+/-) mice with TFPI(-/-) fetal liver cells. Platelets from transplanted mice totally lack TFPI inhibitory activity. An electrolytic vascular injury model was used to assess thrombus growth in the femoral vein and carotid artery. Mice lacking hematopoietic TFPI developed larger femoral vein and carotid artery thrombi than TFPI(+/-) mice transplanted with TFPI(+/+) hematopoietic cells, as evidenced by increased platelet accumulation. Hematopoietic TFPI limits thrombus growth following vascular injury. Because platelets are the primary hematopoietic cell accumulating within a growing thrombus, these findings suggest that TFPI present within platelets functions to limit intravascular thrombus growth, likely through inhibition of the procoagulant activity of blood borne TF.

Hereditary and acquired protein S deficiencies are associated with low TFPI levels in plasma.

Protein S and tissue factor pathway inhibitor (TFPI) act together in down-regulating coagulation. To investigate the TFPI/protein S system in hereditary and acquired protein S deficiency. Plasma antigen levels of protein S and full-length TFPI were determined in heterozygous type I protein S-deficient individuals (n=35), patients on oral anticoagulant treatment (OAT) (n=29), oral contraceptive (OC) users (n=10) and matched controls. Thrombin generation was determined using calibrated automated thrombography. Full-length TFPI levels were lower in type I protein S-deficient individuals (76.8+/-33.8%) than in age- and sex-matched controls (128.0+/-59.4%, P<0.001). Among protein S-deficient individuals with thrombosis, those on OAT had not only lower total protein S levels (25.7+/-8.2% vs. 54.7+/-8.2%, P<0.001), but also lower full-length TFPI levels (52.6+/-15.0% vs. 75.4+/-22.9%, P=0.009) than those not on OAT. Similarly, OC users had lower protein S (73.8+/-11.5% vs. 87.9+/-10.8%, P=0.005) and full-length TFPI levels (73.7+/-27.7% vs. 106.4+/-29.2%, P=0.007) than non-users. When triggered with tissue factor, plasma from protein S-deficient individuals generated 3-5-fold more thrombin than control plasma. The difference was only partially corrected by normalization of the protein S level, full correction requiring additional normalization of the TFPI level. Protein S-immunodepletion experiments indicated that free protein S and full-length TFPI form a complex in plasma, and the protein S/TFPI interaction was confirmed by surface plasmon resonance analysis. Full-length TFPI binds to protein S in plasma and is reduced in genetic and acquired protein S deficiency. The concomitant TFPI deficiency substantially contributes to the hypercoagulable state associated with protein S deficiency.

Abstract 5479: Endothelial Cell TFPI Regulates Murine Arterial Thrombosis but Not Hemostasis or Development

Background: Tissue factor pathway inhibitor (TFPI) is a key regulator of the tissue factor/factor VIIa complex. Systemic genetic disruption of TFPI exon 4 results in intrauterine lethality in mice. As such, the ability to study the biologic and tissue specific effects of TFPI deficiency has been limited. To define the tissue specific roles of TFPI we utilized a Cre-lox strategy and generated mice with flanking loxP sites surrounding exon 4 (encodes for the K1 domain). Homozygous floxed mice (TFPI Flox ) were crossed with the Tie2 Cre + or LysM Cre + mice and subsequently inbred to create homozygously floxed mice expressing Cre (TFPI Tie2 or TFPI LysM ). Methods and Results: The presence and efficiency of the deleted mRNA product was determined by RT-PCR. TFPI Tie2 and TFPI LysM mice were phenotypically similar to TFPI Flox littermates including weight, blood counts, and prothrombin and activated partial thromboplastin times. TFPI Tie2 mice have complete deletion in endothelial cells, blood cells and bone marrow cells. TFPI LysM mice show partial deletion in blood cells and complete deletion in macrophages. TFPI Tie2 mice had a 60% reduction (P &lt; 0.0001) in circulating TFPI activity compared to TFPI Flox littermate controls. TFPI LysM mice did not have a significant reduction in circulating TFPI activity levels compared to their TFPI Flox counterparts. Neither TFPI Tie2 nor TFPI LysM mice had significant changes in hemostasis compared to TFPI Flox mice as determined by a tail bleeding assay. In an ADP-induced model of thromboembolism there were no significant changes in mortality between the TFPI Tie2 or TFPI LysM and their TFPI Flox littermates. In a ferric chloride model of arterial thrombosis, TFPI Tie2 mice had a significant reduction (P &lt; 0.05) in time to occlusion while TFPI LysM mice did not differ compared to TFPI Flox mice. Conclusions: Deletion of TFPI from cells expressing Tie2 or LysM is consistent with normal development and hemostasis. TFPI Tie2 mice have decreased circulating levels of TFPI and an enhanced thrombotic response to ferric chloride. These data support an important regulatory role for EC TFPI in arterial thrombosis.

Expression of Tissue Factor Pathway Inhibitor (TFPI) by Brain Endothelium

TFPI and thrombomodulin are the two primary endothelium associated anticoagulant proteins. Studies using an in vitro model system of purified coagulation proteins have shown that TFPI and thrombomodulin act synergistically to quench tissue factor mediated thrombin generation via neutralization of prothrombinase activity. However, it is unclear how these two proteins cooperate within different vascular beds in vivo, particularly in the brain that has large amounts of tissue factor procoagulant activity. Mice with decreased thrombomodulin function do not have fibrin deposition within the brain suggesting that other anticoagulants, such as TFPI, may be important for prevention of cerebral thrombosis. Consistent with this hypothesis, we have demonstrated that partial TFPI deficiency induces intravascular fibrin deposition in the brain of the mice with decreased thrombomodulin function suggesting that TFPI may be the primary anticoagulant within the brain vasculature. Yet, studies of mouse and human tissues have shown that TFPI is expressed at much lower levels in brain than in other tissues. Therefore, we undertook studies to characterize the expression of two alternatively spliced isoforms of TFPI, TFPIα and TFPIβ, in mouse brain. In situ hybridization studies detected abundant expression of TFPIα and TFPIβ on brain endothelium as well as expression by individual astrocytes within the parenchyma of the cerebral cortex. Using RPL-19 as a housekeeper gene, quantitative real time PCR analysis of TFPIα and TFPIβ mRNA in mouse brain demonstrated that TFPIα message is 9-fold more abundant that TFPIβ message. This analysis also demonstrated that brain expresses far less TFPIα and TFPIβ mRNA than any other tissue (TFPIα: 18-fold less than lung, 19-fold less than heart, 372-fold less than placenta; TFPIβ: 15-fold less than lung, 41-fold less than heart, 68-fold less than placenta). Since TFPI mRNA was readily detected within mouse brain endothelium in the in situ hybridization studies, we hypothesized that the low TFPIα and TFPIβ expression in the brain is due to low relative numbers of endothelial cells in brain when compared to other tissues rather than low expression of TFPI by brain endothelium. Quantitative PCR analysis was repeated with TFPI expression levels normalized to CD-31 and VE-cadherin, two relatively specific markers for endothelial cells. In this analysis, placenta has by far the highest level of TFPIα and TFPIβ expression most likely because both the endothelial cells and trophoblasts of the placenta produce TFPI while trophoblasts produce smaller amounts of CD-31 or VE-cadherin. The relative amount of TFPIα mRNA produced by brain endothelium is 7-fold greater than that of lung endothelial cells and 60% that of heart endothelial cells. The amount of TFPIβ mRNA produced by brain endothelium is 20-fold greater than that of lung endothelial cells and 10% that of heart endothelial cells. Therefore, in contrast to previous reports, these data demonstrate that brain endothelium produces abundant amounts of TFPIα and TFPIβ mRNA. The variable amount of TFPI produced by these vascular beds likely contributes to the tissue specific thrombosis observed in mouse models of tissue factor mediated disease. Finally, western blot analysis demonstrated that TFPIβ is the primary TFPI isoform produced by mouse brain indicating that regulation of TFPI isoform production occurs at least in part at the level of protein translation. Taken together, the data demonstrate that TFPI is an abundant anticoagulant protein on brain endothelium where it acts to prevent fibrin deposition within the cerebral vasculature. In mice, this anticoagulant activity is produced primarily by TFPIβ.

Expression of tissue factor pathway inhibitor by endothelial cells and platelets

Tissue factor pathway inhibitor (TFPI) is a potent anticoagulant protein that abrogates the activity of the tissue factor-factor VIIa catalytic complex that activates blood coagulation in vivo. The importance of TFPI in the regulation of blood coagulation is emphasized by how its activity is modulated in human disease. Decreased TFPI activity contributes to the development of both arterial and venous thrombosis and has been implicated in the thrombotic events occurring in women using oral contraceptives and in patients with paroxysmal nocturnal hemoglobinuria. Both endothelial cells and platelets produce TFPI. Our laboratory is interested in the mechanisms for expression of TFPI on the surface of these cells to better understand how TFPI prevents intravascular thrombosis. Studies of cultured endothelial cells and human placenta have demonstrated that TFPI associates with the cell surface through a glycosyl phosphatidyinositol (GPI)-anchor in a manner that is not dependent on GAGs or altered by heparin. TFPI is not directly bound to the GPI-anchor; instead it appears to bind tightly to a GPI-anchored protein. This GPI-anchored protein appears to be necessary for proper trafficking of TFPI to the cell surface. An alternatively spliced form of TFPI, TFPIβ, is a truncated form of TFPI that is directly attached to a GPI-anchor. However, it is not clear that human endothelial cells produce TFPIβ. Platelets produce TFPI but not TFPIβ. TFPI is expressed on the platelet surface following dual activation with collagen plus thrombin, but not through a GPI-anchor. Studies using mouse models of TFPI deficiency are currently being conducted in our laboratory to determine if distinct physiological functions of endothelial and platelet TFPI exist in vivo.

Combined Tissue Factor Pathway Inhibitor (TFPI) and Thrombomodulin (TM) Deficiency Produces an Augmented Hypercoagulable State with Tissue Specific Fibrin Deposition.

Background: TFPI and TM are endothelial associated anticoagulant proteins thought to control hemostasis in specific vascular beds. Mice lacking either TFPI or TM have an intrauterine lethal phenotype. A prothrombotic phenotype has not previously been described in TFPI+/− mice. TMpro mice have a Glu to Pro mutation between the fourth and fifth epidermal growth factor domains. This mutation reduces TM expression and disrupts TM dependent activation of protein C. TMpro/pro mice are viable and fertile and do not exhibit spontaneous thrombosis on the C57/Bl6 background. TFPI+/−/TMpro/pro mice were generated to examine the consequences of TFPI deficiency in the presence of a compounding procoagulant state caused by reduced TM function. TFPI and TM are located 37 centimorgans apart of chromosome 2 resulting in allele segregation in about 40% of meioses. Therefore, breeding studies were performed without defining haplotypes of individual mice.Methods and Results: TFPI+/−/TMpro/pro mice were generated in three crosses in which the mother had a different prothrombotic phenotype. Embryonic lethality was observed in two of the crosses. TFPI+/−/TMpro/pro mice are born at less than expected frequency in either TFPI+/−/TMpro/+ female with TMpro/pro male crosses (7/151 or 4.6%) or TMpro/pro female with TFPI+/−/TMpro/+ male crosses (11/129 or 8.5%). Timed matings revealed similar reduced numbers at E18.5, but not E13.5, demonstrating that the mice die in utero during embryonic development. TMpro/+ are the least prothrombotic mothers that can produce TFPI+/−/TMpro/pro mice. In contrast to the other crosses, TFPI+/−/TMpro/pro mice are born at near the expected frequency in TMpro/+ mothers crossed with TFPI+/−/TMpro/pro fathers (15/75 or 20%). Our results show that both maternal and fetal genotypes contribute to embryonic survival. Adult TFPI+/−/TMpro/pro mice have elevated thrombin-antithrombin complex in plasma (11.4±8.12 ng/ml for TFPI+/−/TMpro/pro n=16; 4.82±3.05 ng/ml for TMpro/pro n=10, p<0.03; wild type and TFPI+/− had no detectable TAT complex) and increased thrombus volume in an electrical injury model of venous thrombosis (mean thrombus volume of wild type is 0.042±0.03 mm3; of TFPI+/−/TMpro/pro 0.125±0.063 mm3 p<0.005). Interestingly, TFPI+/− mice also have increased thrombus volume in this model (0.092±0.045 p<0.02), demonstrating for the first time a thrombotic phenotype in these mice. A fluorescently labeled cyclic peptide (CGLIIQKNEC) that specifically binds to fibrin-fibronectin complexes was used to detect tissue fibrin deposition (Pilch et al. PNAS 2006; 103:2800). In striking contrast to TFPI+/− and TMpro/pro mice, TFPI+/−/TMpro/pro mice exhibit augmented fibrin deposition in the liver and in the cerebral microvasculature.Conclusions: TFPI+/−/TMpro/pro mice display a prothrombotic phenotype with tissue specific fibrin deposition in embryonic development and as adults. They exhibit partial intrauterine lethality when carried by mothers with an underlying prothrombotic state, demonstrating for the first time that TFPI-dependent control of hemostasis in the vascular bed of the placenta fulfills a critical role for successful pregnancy outcome. In addition to the placenta, partial TFPI deficiency interacts with decreased TM function in an organ selective manner within adult mice to produce fibrin deposition in a second specific vascular bed, the brain microcirculation.

Combined tissue factor pathway inhibitor and thrombomodulin deficiency produces an augmented hypercoagulable state with tissue-specific fibrin deposition.

Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) are endothelial-associated anticoagulant proteins thought to control hemostasis in specific vascular beds. Here, we have examined the consequences of TFPI deficiency in the presence of a compounding procoagulant state caused by reduced TM function. TFPI(+/-)/TM(pro/pro) mice are born at less than expected frequency in either TFPI(+/-)/TM(pro/+) or TM(pro/pro) mothers but are born at near the expected frequency in TM(pro/+) mothers. Adult TFPI(+/-)/TM(pro/pro) mice have elevated thrombin-antithrombin complex and increased thrombus volume in an electrical injury model of venous thrombosis. In striking contrast to mice with single deficiency of TFPI or TM, TFPI(+/-)/TM(pro/pro) mice exhibit augmented fibrin deposition not only in the liver, but also in the cerebral microvasculature. TFPI(+/-)/TM(pro/pro) mice exhibit partial intrauterine lethality when carried by mothers with an underlying prothrombotic state, providing the first experimental evidence in an animal model that TFPI-dependent control of hemostasis in the vascular bed of the placenta fulfills a critical role for successful pregnancy outcome. In addition to the placenta, partial TFPI deficiency interacts with decreased TM function in an organ selective manner to produce fibrin deposition in other specific vascular beds, the liver and brain.

29 Pregnancy morbidity, tissue factor pathway inhibitor deficiency and resistance to activated protein C

29 Pregnancy morbidity, tissue factor pathway inhibitor deficiency and resistance to activated protein C