Tissue factor pathway inhibitor is required for cerebrovascular development in mice.

Tissue factor pathway inhibitor (TFPI) inhibits proteases in the blood coagulation cascade that lead to the production of thrombin, including prothrombinase (factor Xa [FXa]/FVa), the catalytic complex that directly generates thrombin. Thus, TFPI and FV are directly linked in regulating the procoagulant response. Studies using knockout mice indicate that TFPI and FV are necessary for embryogenesis, but their contributions to vascular development are unclear. We performed extensive histological analyses of Tfpi-/- and Tfpi-/-F5-/- mouse embryos to investigate the importance of the interplay between TFPI and FV in regulating hemostasis and vascular development during embryogenesis. We observed normal tissue development throughout Tfpi-/- embryos, except in the central nervous system (CNS). The CNS displayed stunted brain growth, delayed development of the meninges, and severe vascular pathology characterized by the formation of glomeruloid bodies surrounding areas of cellular death, fibrin deposition, and hemorrhage. Removing FV from Tfpi-/- embryos completely ameliorated their brain pathology, suggesting that TFPI dampens FV-dependent procoagulant activity in a manner that modulates cerebrovascular development. Thus, we have identified a previously unrecognized role for TFPI activity within the CNS. This TFPI activity likely diminishes an effect of excess thrombin activity on signaling pathways that control cerebral vascular development.

Major Reservoir for Heparin-Releasable TFPIα (Tissue Factor Pathway Inhibitor α) Is Extracellular Matrix.

[Figure: see text].

Measurement of plasma and platelet tissue factor pathway inhibitor, factor V and Protein S in people with haemophilia.

INTRODUCTION: Tissue factor pathway inhibitor (TFPI) is a naturally occurring anticoagulant found in plasma, where it circulates bound to lipoproteins, factor V (FV) or Protein S (PS), and in platelets. Therapeutic agents targeting TFPI are under development for the treatment of haemophilia A and haemophilia B. AIM: To begin to understand how TFPI, FV and PS interact to modulate haemophilia bleeding. METHODS: Plasma and platelet antigen concentrations of these factors were determined in 73 people with haemophilia A and 18 with haemophilia B. Using multiple regression models, these were compared to the same analytes measured in 224 male blood donors. RESULTS: There were no differences in plasma or platelet TFPI, FV or PS concentrations between haemophilia types or severities. However, compared to blood donors, people with haemophilia had approximately one-third lower plasma PS, 9% lower plasma TFPIα, 50% higher platelet FV and 26% lower platelet Protein S. CONCLUSION: Together, the presented data suggest that individuals with haemophilia may have a compensatory procoagulant response of both plasma and platelet proteins to the decreased concentrations of FVIII or FIX.

A balance between TFPI and thrombin-mediated platelet activation is required for murine embryonic development.

Tissue factor pathway inhibitor (TFPI) is a critical anticoagulant protein present in endothelium and platelets. Mice lacking TFPI (Tfpi(-/-)) die in utero from disseminated intravascular coagulation. They are rescued by concomitant tissue factor (TF) deficiency, demonstrating that TFPI modulates TF function in vivo. Recent studies have found TFPI inhibits prothrombinase activity during the initiation of coagulation and limits platelet accumulation during thrombus formation, implicating TFPI in modulating platelet procoagulant activity. To examine whether altered platelet function would compensate for the lack of TFPI and rescue TFPI-null embryonic lethality, Tfpi(+/-) mice lacking the platelet thrombin receptor, protease activated receptor 4 (PAR4; Par4(-/-)), or its coreceptor, PAR3, were mated. PAR3 deficiency did not rescue Tfpi(-/-) embryos, but >40% of expected Tfpi(-/-):Par4(-/-) offspring survived to adulthood. Adult Tfpi(-/-):Par4(-/-) mice did not exhibit overt thrombosis. However, they had focal sterile inflammation with fibrin(ogen) deposition in the liver and elevated plasma thrombin-antithrombin complexes, indicating activation of coagulation at baseline. Tfpi(-/-):Par4(-/-) mice have platelet and fibrin accumulation similar to Par4(-/-) mice following venous electrolytic injury but were more susceptible than Par4(-/-) mice to TF-induced pulmonary embolism. In addition, ∼30% of the Tfpi(-/-):Par4(-/-) mice were born with short tails. Tfpi(-/-):Par4(-/-) mice are the first adult mice described that lack TFPI with unaltered TF. They demonstrate that TFPI physiologically modulates thrombin-dependent platelet activation in a manner that is required for successful embryonic development and identify a role for TFPI in dampening intravascular procoagulant stimuli that lead to thrombin generation, even in the absence of thrombin-mediated platelet activation.

Biology of tissue factor pathway inhibitor.

Recent studies of the anticoagulant activities of the tissue factor (TF) pathway inhibitor (TFPI) isoforms, TFPIα and TFPIß, have provided new insight into the biochemical and physiological mechanisms that underlie bleeding and clotting disorders. TFPIα and TFPIß have tissue-specific expression patterns and anticoagulant activities. An alternative splicing event in the 5' untranslated region allows for translational regulation of TFPIß expression. TFPIα has 3 Kunitz-type inhibitor domains (K1, K2, K3) and a basic C terminus, whereas TFPIß has the K1 and K2 domains attached to a glycosylphosphatidyl inositol-anchored C terminus. TFPIα is the only isoform present in platelets, whereas endothelial cells produce both isoforms, secreting TFPIα and expressing TFPIß on the cell surface. TFPIα and TFPIß inhibit both TF-factor VIIa-dependent factor Xa (FXa) generation and free FXa. Protein S enhances FXa inhibition by TFPIα. TFPIα produces isoform-specific inhibition of prothrombinase during the initiation of coagulation, an anticoagulant activity that requires an exosite interaction between its basic C terminus and an acidic region in the factor Va B domain. Platelet TFPIα may be optimally localized to dampen initial thrombin generation. Similarly, endothelial TFPIß may be optimally localized to inhibit processes that occur when endothelial TF is present, such as during the inflammatory response.

Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation.

Tissue factor (TF) pathway inhibitor (TFPI) is a well-characterized activated factor X (FXa)-dependent inhibitor of TF-initiated coagulation produced in two alternatively spliced isoforms, TFPIα and TFPIß. The TFPIα C terminus has a basic sequence nearly identical to a portion of the factor V (FV) B domain necessary for maintaining FV in an inactive conformation via interaction with an acidic region of the B domain. We demonstrate rapid inhibition of prothrombinase by TFPIα mediated through a high-affinity exosite interaction between the basic region of TFPIα and the FV acidic region, which is retained in FXa-activated FVa and platelet FVa. This inhibitory activity is not mediated by TFPIß and is lost upon removal of the acidic region of FVa by thrombin. The data identify a previously undescribed, isoform-specific anticoagulant function for TFPIα and are a unique description of physiologically relevant inhibition of prothrombinase. These findings, combined with previous descriptions of differential expression patterns of TFPIα and TFPIß in platelets and endothelial cells, suggest that the TFPI isoforms may act through distinct mechanisms to inhibit the initial stages of intravascular coagulation, with TFPIß acting to dampen TF expressed on the surface of vascular cells, whereas TFPIα dampens the initial prothrombinase formed on the activated platelet surface.

Protein S is a cofactor for platelet and endothelial tissue factor pathway inhibitor-α but not for cell surface-associated tissue factor pathway inhibitor.

OBJECTIVE: Tissue factor pathway inhibitor (TFPI) is produced in 2 isoforms: TFPIα, a soluble protein in plasma, platelets, and endothelial cells, and TFPIß, a glycosylphosphatidylinositol-anchored protein on endothelium. Protein S (PS) functions as a cofactor for TFPIα, enhancing the inhibition of factor Xa. However, PS does not alter the inhibition of prothrombinase by TFPIα, and PS interactions with TFPIß are undescribed. Thus, the physiological role and scope of the PS-TFPI system remain unclear. APPROACH AND RESULTS: Here, the cofactor activity of PS toward platelet and endothelial TFPIα and endothelial TFPIß was quantified. PS enhanced the inhibition of factor Xa by TFPIα from platelets and endothelial cells and stabilized the TFPIα/factor Xa inhibitory complex, delaying thrombin generation by prothrombinase. By contrast, PS did not enhance the inhibitory activity of TFPIß or a membrane-anchored form of TFPI containing the PS-binding third Kunitz domain (K1K2K3) although PS did function as a cofactor for K1K2K3 enzymatically released from the cell surface. CONCLUSIONS: The PS-TFPI anticoagulant system is limited to plasma TFPIα and TFPIα released from platelets and endothelial cells. PS likely functions to localize solution-phase TFPIα to the cell surface, where factor Xa is bound. PS does not alter the activity of membrane-associated TFPI. Because activated platelets release TFPIα and PS, the PS-TFPIα anticoagulant system may act physiologically to dampen thrombin generation at the platelet surface.

Translation of human tissue factor pathway inhibitor-ß mRNA is controlled by alternative splicing within the 5' untranslated region.

OBJECTIVE: Tissue factor pathway inhibitor (TFPI) blocks the initiation of coagulation by inhibiting TF-activated factor VII, activated factor X, and early prothrombinase. Humans produce two 3' splice variants, TFPIα and TFPIß, which are differentially expressed in endothelial cells and platelets and possess distinct structural features affecting their inhibitory function. TFPI also undergoes alternative splicing of exon 2 within its 5' untranslated region. The role of exon 2 splicing in translational regulation of human TFPI isoform expression is investigated. APPROACH AND RESULTS: Exon 2 splicing occurs in TFPIα and TFPIß transcripts. Human tissue mRNA analysis uncovered a wide variability of exon 2 expression. Polysome analysis revealed a repressive effect of exon 2 on TFPIß translation but not on TFPIα. Luciferase reporter assays further exposed strong translational repression of TFPIß (90%) but not TFPIα. Use of a Morpholino to remove exon 2 from TFPI mRNA increased cell surface expression of endogenous TFPIß. Exon 2 also repressed luciferase production (80% to 90%) when paired with the ß-actin 3' untranslated region, suggesting that it is a general translational negative element whose effects are overcome by the TFPIα 3' untranslated region. CONCLUSIONS: Exon 2 is a molecular switch that prevents translation of TFPIß. This is the first demonstration of a 5' untranslated region alternative splicing event that alters translation of isoforms produced via independent 3' splicing events within the same gene. Therefore, it represents a previously unrecognized mechanism for translational control of protein expression. Differential expression of exon 2 denotes a mechanism to provide temporal and tissue-specific regulation of TFPIß-mediated anticoagulant activity.

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.

Cellular expression and biological activities of alternatively spliced forms of tissue factor pathway inhibitor.

PURPOSE OF REVIEW: Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits tissue factor-factor VIIa (TF-fVIIa) and factor Xa (fXa). Recent studies revealed distinct cellular expression patterns for TFPIα and TFPIß and spurred additional experiments to define unique functions for these alternatively spliced TFPI isoforms. RECENT FINDINGS: TFPIα is produced by endothelial cells, localizes to an intracellular granule, and is released following cellular stimulation with thrombin or heparin. TFPIα also is produced by megakaryocytes and released from activated platelets. Platelet TFPIα limits clot growth following vessel injury and alters bleeding in hemophilia, suggesting that its primary physiological role is modulation of clot development. TFPIß is made by endothelial cells, localizes to the endothelium surface, and is not in platelets. TFPIß is an effective inhibitor of TF-mediated cellular migration and may act to dampen the adverse effects of intravascular TF expressed during inflammation. SUMMARY: Knowledge of TFPI isoform expression and activity provides new insights into the biochemical regulation of TF-mediated thrombotic and inflammatory disease. Recent findings have therapeutic implications for use of recombinant TFPI to treat severe sepsis in community-acquired pneumonia or to achieve improved engraftment of hematopoietic stem cells, and for development of TFPI-blocking pharmaceuticals to treat hemophilia.

Caveolae optimize tissue factor-Factor VIIa inhibitory activity of cell-surface-associated tissue factor pathway inhibitor.

TFPI (tissue factor pathway inhibitor) is an anticoagulant protein that prevents intravascular coagulation through inhibition of fXa (Factor Xa) and the TF (tissue factor)-fVIIa (Factor VIIa) complex. Localization of TFPI within caveolae enhances its anticoagulant activity. To define further how caveolae contribute to TFPI anticoagulant activity, CHO (Chinese-hamster ovary) cells were co-transfected with TF and membrane-associated TFPI targeted to either caveolae [TFPI-GPI (TFPI-glycosylphosphatidylinositol anchor chimaera)] or to bulk plasma membrane [TFPI-TM (TFPI-transmembrane anchor chimaera)]. Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI-GPI to detergent-insoluble membrane fractions, whereas TFPI-TM localized to the aqueous phase. TFPI-GPI and TFPI-TM were equally effective direct inhibitors of fXa in amidolytic assays. However, TFPI-GPI was a significantly better inhibitor of TF-fVIIa than TFPI-TM, as measured in both amidolytic and plasma-clotting assays. Disrupting caveolae by removing membrane cholesterol from EA.hy926 cells, which make TFPIα, CHO cells transfected with TFPIß and HUVECs (human umbilical vein endothelial cells) did not affect their fXa inhibition, but significantly decreased their inhibition of TF-fVIIa. These studies confirm and quantify the enhanced anticoagulant activity of TFPI localized within caveolae, demonstrate that caveolae enhance the inhibitory activity of both TFPI isoforms and define the effect of caveolae as specifically enhancing the anti-TF activity of TFPI.

Platelet tissue factor pathway inhibitor-α dampens cardiac thrombosis and associated fibrosis in mice.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is the primary inhibitor of events initiating the blood coagulation pathway. Tfpi-/- mice die during embryonic development. The absence of protease-activated receptor (PAR) 4, the major thrombin receptor on mouse platelets, rescues Tfpi-/-mice to adulthood. Among the 3 TFPI isoforms in mice, TFPIα is the only isoform within platelets (pltTFPIα) and the only isoform that inhibits prothrombinase, the enzymatic complex that converts prothrombin to thrombin. OBJECTIVES: To determine biological functions of pltTFPIα. METHODS: Tfpi-/-/Par4-/- mice were irradiated and transplanted with bone marrow from mice lacking or containing pltTFPIα. Thus, PAR4 expression was restored in the recipient mice, which differed selectively by the presence or absence of pltTFPIα and lacked other forms of TFPI. RESULTS: Recipient mice lacking pltTFPIα had reduced survival over the 200-day posttransplant period. Necropsy revealed radiation injury associated with large intraventricular platelet-rich thrombi, whereas other organs were not affected. Thrombi were associated with fibrotic presentations, including increased collagen deposition, periostin-positive activated fibroblasts, myofibroblasts, and macrophage infiltrates. Recipient mice containing pltTFPIα showed evidence of radiation injury but lacked heart pathology. CONCLUSIONS: Tfpi-/-/Par4-/- mice develop severe cardiac fibrosis following irradiation and transplantation with bone marrow lacking pltTFPIα. This pathology is markedly reduced when the mice are transplanted with bone marrow containing pltTFPIα. Thus, in this model system pltTFPIα has an important physiological role in dampening pathological responses mediated by activated platelets within the heart tissue.

Endothelial-derived tissue factor pathway inhibitor regulates arterial thrombosis but is not required for development or hemostasis.

The antithrombotic surface of endothelium is regulated in a coordinated manner. Tissue factor pathway inhibitor (TFPI) localized at the endothelial cell surface regulates the production of FXa by inhibiting the TF/VIIa complex. Systemic homozygotic deletion of the first Kunitz (K1) domain of TFPI results in intrauterine lethality in mice. Here we define the cellular sources of TFPI and their role in development, hemostasis, and thrombosis using TFPI conditional knockout mice. We used a Cre-lox strategy and generated mice with a floxed exon 4 (TFPI(Flox)) which encodes for the TFPI-K1 domain. Mice bred into Tie2-Cre and LysM-Cre lines to delete TFPI-K1 in endothelial (TFPI(Tie2)) and myelomonocytic (TFPI(LysM)) cells resulted in viable and fertile offspring. Plasma TFPI activity was reduced in the TFPI(Tie2) (71% ± 0.9%, P < .001) and TFPI(LysM) (19% ± 0.6%, P < .001) compared with TFPI(Flox) littermate controls. Tail and cuticle bleeding were unaffected. However, TFPI(Tie2) mice but not TFPI(LysM) mice had increased ferric chloride-induced arterial thrombosis. Taken together, the data reveal distinct roles for endothelial- and myelomonocytic-derived TFPI.

Murine hematopoietic cell tissue factor pathway inhibitor limits thrombus growth.

OBJECTIVE: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Factor V east Texas variant causes bleeding in a three-generation family.

BACKGROUND: The factor V east Texas bleeding disorder (FVETBD) is caused by increased plasma tissue factor pathway inhibitor-α (TFPIα) concentration. The underlying cause is a variant in F5 causing alternative splicing within exon 13 and producing FV-short, which tightly binds the C-terminus of TFPIα, prolonging its circulatory half-life. OBJECTIVES: To diagnose a family presenting with variable bleeding and laboratory phenotypes. PATIENTS/METHODS: Samples were obtained from 17 family members for F5 exon 13 sequencing. Plasma/platelet TFPI and platelet FV were measured by ELISA and/or western blot. Plasma thrombin generation potential was evaluated using calibrated automated thrombography. RESULTS: The FVET variant was identified in all family members with bleeding symptoms and associated with elevated plasma TFPIα (4.5- to 13.4-fold) and total TFPI (2- to 3-fold). However, TFPIα and FV-short were not elevated in platelets. TF-initiated thrombin generation in patient plasma was diminished but was restored by a monoclonal anti-TFPI antibody or factor VIIa. TFPIα localized within vascular extracellular matrix in an oral lesion biopsy from an affected family member. CONCLUSIONS: Factor V east Texas bleeding disorder was diagnosed in an extended family. The variant was autosomal dominant and highly penetrant. Elevated plasma TFPIα, rather than platelet TFPIα, was likely the primary cause of bleeding. Plasma FV-short did not deplete TFPIα from extracellular matrix. In vitro thrombin generation was restored with an anti-TFPI antibody or factor VIIa suggesting effective therapies may be available. Increased awareness of, and testing for, bleeding disorders associated with F5 exon 13 variants and elevated plasma TFPI are needed.

Intrauterine lethality in Tfpi gene disrupted mice is differentially suppressed during mid- and late-gestation by platelet TFPIα overexpression.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein required for murine embryonic development. Intrauterine lethality of Tfpi-/- mice occurs at mid- and late gestation, the latter of which is associated with severe cerebrovascular defects. Megakaryocytes produce only the TFPIα isoform, which is stored within platelets and released upon activation. OBJECTIVES: To examine biological activities of platelet TFPIα (pTFPIα) by characterizing effects of pTFPIα overexpression in Tfpi-/- mice. METHODS: Transgenic mice overexpressing pTFPIα were generated and crossed onto the Tfpi-/- background. Genetic and histological analyses of embryos were performed to investigate the function of pTFPIα during embryogenesis. RESULTS: The transgene (Tg) increased pTFPIα 4- to 5-fold without altering plasma TFPI in adult Tfpi+/+ and Tfpi+/- mice but did not rescue Tfpi-/- mice to wean. Analyses of the impact of pTFPIα overexpression on Tfpi-/- survival, however, were complicated by linkage between the Tg integration site and the endogenous Tfpi locus on chromosome 2. Strain-specific genetic interactions also modulated Tfpi-/- embryonic survival. After accounting for these underlying genetic factors, pTFPIα overexpression completely suppressed mid-gestational lethality of Tfpi-/- embryos but had no effect on development of cerebrovascular defects during late gestation resulting in their lack of survival to wean. CONCLUSIONS: pTFPIα overexpression rescued Tfpi-/- embryos from mid-gestational but not late gestational lethality. The prevalence of underlying genetic factors complicating analyses within our study illustrates the importance of meticulously characterizing transgenic mouse models to avoid spurious interpretation of results.

The contribution of TFPIα to the hemostatic response to injury in mice.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an essential regulator of coagulation, limiting thrombin generation and preventing thrombosis. In humans and mice, TFPIα is the sole isoform present in platelets. OBJECTIVE: Here, we asked whether TFPIα, because of its release from platelets at sites of injury, has a unique role in limiting the hemostatic response. METHODS: TFPIα-mutant (TfpiΔα/Δα ) mice were generated by introducing a stop codon in the C-terminus. Platelet accumulation, platelet activation, and fibrin accumulation were measured following penetrating injuries in the jugular vein and cremaster muscle arterioles, and imaged by fluorescence and scanning electron microscopy. Time to bleeding cessation was recorded in the jugular vein studies. RESULTS: TfpiΔα/Δα mice were viable and fertile. Plasma TFPI levels were normal in the TfpiΔα/Δα mice, no TFPI protein or activity was present in their platelets and thrombin-antithrombin complex levels were indistinguishable from Tfpi+/+ littermates. There was a small, but statistically significant reduction in the time to bleeding cessation following jugular vein puncture injury in the TfpiΔα/Δα mice, but no measurable changes in platelet or fibrin accumulation or in hemostatic plug architecture following injury of the micro- or macrovasculature. CONCLUSION: Loss of TFPIα expression does not produce a global prothrombotic state in mice. Platelet TFPIα is expected to be released or displayed in a focal manner at the site of injury, potentially accumulating to high concentrations in the narrow gaps between platelets. If so, the data from the vascular injury models studied here indicate this is not essential for a normal hemostatic response in mice.

Modelling the domestic poultry population in the United States: A novel approach leveraging remote sensing and synthetic data methods.

Comprehensive and spatially accurate poultry population demographic data do not currently exist in the United States; however, these data are critically needed to adequately prepare for, and efficiently respond to and manage disease outbreaks. In response to absence of these data, this study developed a national-level poultry population dataset by using a novel combination of remote sensing and probabilistic modelling methodologies. The Farm Location and Agricultural Production Simulator (FLAPS) (Burdett et al., 2015) was used to provide baseline national-scale data depicting the simulated locations and populations of individual poultry operations. Remote sensing methods (identification using aerial imagery) were used to identify actual locations of buildings having the characteristic size and shape of commercial poultry barns. This approach was applied to 594 U.S. counties with > 100,000 birds in 34 states based on the 2012 U.S. Department of Agriculture (USDA), National Agricultural Statistics Service (NASS), Census of Agriculture (CoA). The two methods were integrated in a hybrid approach to develop an automated machine learning process to locate commercial poultry operations and predict the number and type of poultry for each operation across the coterminous United States. Validation illustrated that the hybrid model had higher locational accuracy and more realistic distribution and density patterns when compared to purely simulated data. The resulting national poultry population dataset has significant potential for application in animal disease spread modelling, surveillance, emergency planning and response, economics, and other fields, providing a versatile asset for further agricultural research.

Assessing Blood Clotting and Coagulation Factors in Mice.

The mammalian blood coagulation system was designed to restrict blood loss due to injury as well as keep the blood fluid within the blood vessels of the organism. Blood coagulation activity in inbred mouse strains varies widely among strains, suggesting that many genomic variants affect hemostasis. Some of these molecules have been discovered and characterized; however, many are still unknown. Genetically modified mouse technologies are providing a plethora of new mouse models for investigating the regulation of blood coagulation. Here we provide a protocol for the tail bleeding time as a primary assessment of in vivo blood coagulation, as well as in vitro methods such as the prothrombin time, activated partial thromboplastin time, and thrombin generation assay. We also provide protocols for the assessment of the activities of specific known factors involved in blood coagulation. © 2019 by John Wiley & Sons, Inc.

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, TFPIbeta, is a truncated form of TFPI that is directly attached to a GPI-anchor. However, it is not clear that human endothelial cells produce TFPIbeta. Platelets produce TFPI but not TFPIbeta. 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.