Using design thinking to create and implement a 3D digital library of anatomical specimens.

Design thinking (DT) is a five-stage process (empathize, define, ideate, prototype, and test) that guides the creation of user-centered solutions to complex problems. DT is in common use outside of science but has rarely been applied to anatomical education. The use of DT in this study identified the need for flexible access to anatomical specimens outside of the anatomy laboratory and guided the creation of a digital library of three-dimensional (3D) anatomical specimens (3D Anatomy Viewer). To test whether the resource was fit for purpose, a mixed-methods student evaluation was undertaken. Student surveys (n = 46) were employed using the system usability scale (SUS) and an unvalidated acceptability questionnaire. These verified that 3D Anatomy Viewer was usable (SUS of 72%) and acceptable (agreement range of 77%-93% on all Likert-type survey statements, Cronbach's alpha = 0.929). Supplementary interviews (n = 5) were analyzed through content analysis and revealed three main themes: (1) a credible online supplementary learning resource; (2) learning anatomy with 3D realism and interactivity; (3) user recommendations for expanding the number of anatomical models, test questions, and gamification elements. These data demonstrate that a DT framework can be successfully applied to anatomical education for creation of a practical learning resource. Anatomy educators should consider employing a DT framework where student-centered solutions to learner needs are required.

A workflow for the creation of photorealistic 3D cadaveric models using photogrammetry.

Three-dimensional (3D) representations of anatomical specimens are increasingly used as learning resources. Photogrammetry is a well-established technique that can be used to generate 3D models and has only been recently applied to produce visualisations of cadaveric specimens. This study has developed a semi-standardised photogrammetry workflow to produce photorealistic models of human specimens. Eight specimens, each with unique anatomical characteristics, were successfully digitised into interactive 3D models using the described workflow and the strengths and limitations of the technique are described. Various tissue types were reconstructed with apparent preservation of geometry and texture which visually resembled the original specimen. Using this workflow, an institution could digitise their existing cadaveric resources, facilitating the delivery of novel educational experiences.

Does decreasing the incubation period used in the antibody screen affect its sensitivity?

BACKGROUND: Pre-transfusion testing (PTT) encompasses a set of mandatory laboratory tests performed before red blood cell transfusion. The antibody screen, one component of PTT, commonly includes a 10-20 min incubation. The primary aim of this study was to determine if this period can be reduced when using current immunohematology methodologies. METHODS AND MATERIALS: Antibody screens were performed on reagent samples using Glass or Gel-based column agglutination technologies (CAT) and a solid phase red cell adherence (SPRCA) assay, with incubation periods of 1, 5, 10 and 15 min, and 20 min (SPRCA assay only). For each method, the shortest period producing a minimum of a 1+ reaction with all reagent samples was considered optimal. The sensitivity of each assay using the optimal period was calculated after performing antibody screens on 100 patient samples. RESULTS AND DISCUSSION: It was demonstrated that the incubation period in the SPRCA and Glass CAT systems can be reduced to 5 and 10 min, respectively, while achieving high assay sensitivity (98.9% in both). The incubation period in the Gel CAT system cannot be reduced from 15 min. Significant association between titre and reaction strength was observed for all three screening methods (p < 0.001 for both CAT methods, p = 0.041 for SPRCA). This study demonstrates that the incubation period used in the antibody screen can be reduced when using systems employing the Glass CAT and SPRCA methods, without affecting assay sensitivity. If confirmed, it could result in faster completion of PTT.

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.

Elevated Plasma Factor IXa Activity in Premenopausal Women on Hormonal Contraception.

OBJECTIVE: Combined oral contraceptives induce a reversible hypercoagulable state with an enhanced risk of venous thromboembolism, but the underlying mechanism(s) remain unclear. Subjects on combined oral contraceptives also demonstrate a characteristic resistance to APC (activated protein C) in the thrombin generation assay. Here, we report the potential role of plasma factor IXa (FIXa) as a mechanism for hormone-induced systemic hypercoagulability. APPROACH AND RESULTS: A novel assay was used to determine FIXa activity in plasma samples from volunteer blood donors. Plasma from 36 premenopausal females on hormonal contraception and 35 not on hormonal contraception, 35 postmenopausal females, and 10 males were analyzed for FIXa activity, total PS (protein S), total tissue factor pathway inhibitor (TFPI), and TFPI-α antigen. Premenopausal females on hormonal contraception demonstrated significantly increased FIXa activity and decreased TFPI-α compared with the other groups. Remarkably, FIXa values were not normally distributed in the hormonal contraception group, but skewed toward the high end. Plasma FIXa activity inversely correlated with both TFPI-α and total PS antigen. Ex vivo determination of TF-dependent FIX activation in FV-deficient plasma demonstrated that inhibitory anti-TFPI antibodies enhanced FIXa generation by 2- to 3-fold, whereas addition of 75 nmol/L PS reduced FIXa generation by ≈2-fold. Further, increasing FIXa concentration enhanced APC resistance during TF-triggered plasma thrombin generation. CONCLUSIONS: Elevation of plasma FIXa activity in association with reductions in TFPI-α and PS is a potential mechanism for systemic hypercoagulability and resistance to APC in premenopausal females on hormonal contraception.

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.

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.

Tissue factor pathway inhibitor: then and now.

Tissue factor pathway inhibitor (TFPI) is the major physiological regulator of tissue factor (TF)-induced blood coagulation. TFPI inhibits the TF-activated factor VII (FVIIa) complex in an activated factor X (FXa)-dependent manner, helping to control thrombin generation and ultimately fibrin formation. The importance of TFPI is demonstrated in models of hemophilia where lower levels of FVIII or FIX are insufficient to overcome its inhibitory effect, resulting in a bleeding phenotype. There are two major isoforms in vivo; TFPIα contains three Kunitz-type inhibitory domains (designated K1, K2, and K3), is secreted by endothelial cells and requires protein S to enhance its anticoagulant activity. In contrast, TFPIß contains only the K1 and K2 domains, but it is attached to the endothelial surface via a glycosylphosphatidylinositol anchor. This review will initially provide a brief history of the major discoveries related to TFPI, and then discuss new insights into the physiology of TFPI, including updates on its association with protein S and FV, as well as the current understanding of its association with disease.

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.

Further insight into the heparin-releasable and glycosylphosphatidylinositol-lipid--anchored forms of tissue factor pathway inhibitor.

The release of tissue factor pathway inhibitor (TFPI) from human umbilical vein endothelial cells (HUVECs) was investigated using heparin and phospholipase C. The experiment included incubating HUVECs with 0, 1, or 10 U/mL heparin diluted in Dulbecco Modified Eagle's Medium plus 5% fetal calf serum for 1 or 24 hours. A statistically significant increase in TFPI activity levels was seen at 1 hour, but not at 24 hours. A 20-fold increase in the release of TFPI after phospholipase C treatment of HUVECs was demonstrated, confirming that it is glycosylphosphatidylinositol-lipid (GPI) anchored. Sequential treatment of HUVECs with phospholipase C and heparin was performed, and a trend was observed where GPI-anchored TFPI levels were increased after 1 hour of pretreatment with heparin but were decreased after 24 hours. Serum is a requirement for the heparin-dependent release of TFPI from HUVECs. Heparin pretreatment of HUVECs may affect levels of GPI anchored TFPI in a time and dose-dependent manner.

Correlates of plasma and platelet tissue factor pathway inhibitor, factor V, and Protein S.

BACKGROUND: Plasma Tissue Factor Pathway Inhibitor (TFPI) circulates bound to factor V (fV) and Protein S (PS). Estrogen therapy decreases plasma TFPI and PS. TFPI, fV, and PS circulate within platelets, and are released upon activation to modulate thrombus formation. OBJECTIVE: Identify factors affecting the concentrations of plasma and platelet TFPI, fV, and PS. METHODS: Blood samples were obtained from 435 healthy individuals. Plasma total TFPI, TFPIɑ, fV, and PS, and platelet TFPI, fV, and PS were quantified. Correlations between these protein concentrations and age, gender, race, and estrogen use were established. RESULTS: In males, only plasma fV increased with age, while in females, all plasma analytes increased with age. Males had higher plasma total TFPI, TFPIα, and PS than females. The platelet proteins in either sex remained relatively stable with increasing age. Platelet TFPI and PS were comparable in both sexes, while platelet fV was higher in females. Estrogen use was associated with decreased plasma total TFPI and TFPIα, and platelet PS, but not with platelet TFPI concentration. Racial differences in plasma and platelet proteins were observed, some of which were larger than inter-individual differences observed within racial groups. TFPI, fV and PS concentrations correlated in plasma, while only fV and PS correlated in platelets. CONCLUSIONS: Plasma and platelet TFPI, fV and PS differ in their: (i) in vivo association; (ii) demographic correlates; and (iii) alteration by estrogen therapies. Therefore, the plasma and platelet pools of these proteins may modulate hemostasis and thrombosis via different biochemical pathways.

Reduced Prothrombinase Inhibition by Tissue Factor Pathway Inhibitor Contributes to the Factor V Leiden Hypercoagulable State.

Activated factor V (FVa) and factor X (FXa) form prothrombinase, which converts prothrombin to thrombin. The α isoform of tissue factor (TF) pathway inhibitor (TFPI) dampens early procoagulant events, partly by interacting with FV. FV Leiden (FVL) is the most common genetic thrombophilia in Caucasians. Thrombosis risk is particularly elevated in women with FVL taking oral contraceptives, which produce acquired TFPIα deficiency. In mice, FVL combined with 50% reduction in TFPI causes severe thrombosis and perinatal lethality. However, a possible interaction between FVL and TFPIα has not been defined in humans. Here, we examined this interaction using samples from patients with FVL in thrombin generation and fibrin formation assays. In dilute TF- or FXa-initiated reactions, these studies exposed a TFPI-dependent activation threshold for coagulation initiation that was greatly reduced by FVL. The reduced threshold was progressively overcome with higher concentrations of TF or FXa. Plasma assays using anti-TFPI antibodies or a TFPI peptide that binds and inhibits FVa demonstrated that the decreased activation threshold resulted from reduced TFPIα inhibition of prothrombinase. In assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL than with FV. Thus, FVL reduces the threshold for initiating coagulation, and this threshold is further reduced in situations of low TFPIα concentration. Individuals with FVL are likely prone to thrombosis in response to weak procoagulant stimuli that would not initiate blood clot formation in individuals with FV.

The effects of tissue factor pathway inhibitor and anti-beta-2-glycoprotein-I IgG on thrombin generation.

BACKGROUND AND OBJECTIVES: Recent evidence suggests that autoantibodies to tissue factor pathway inhibitor (TFPI) and/or antiphospholipid antibodies (aPL) may contribute to upregulation of the tissue factor (TF) pathway of blood coagulation and the development of thrombotic complications in the antiphospholipid syndrome (aPS). The aim of this study was to determine the influence of aPL e.g. anti-beta-2-glycoprotein-I (anti-beta2GPI) and anti-prothrombin, on in vitro TF-induced thrombin generation in the presence and absence of TFPI. DESIGN AND METHODS: IgG fractions were collected from subjects with aPL (n=21) and normal controls (n=36). Anti-TFPI activity was determined after incubation of IgG isolated from control or subject plasma with pooled normal plasma using an amidolytic assay for TFPI. The influence of IgG fractions and purified aPL (anti-beta2GPI and anti-prothrombin) on TF-induced in vitro thrombin generation was determined using a chromogenic assay of thrombin activity. RESULTS: Patients with aPL had significantly elevated thrombin generation (median [interquartile range]) compared to normal controls (112.0 [104.0-124.0]% vs 89.9 [85.7-100.9]%, respectively; p<0.001). Thrombin generation was significantly correlated with anti-TFPI activity in patients with aPL (r(s)=0.452; p=0.039). It was also demonstrated that anti-beta2GPI, but not anti-prothrombin IgG antibodies, significantly enhanced TF-induced thrombin generation in the presence of TFPI, using both purified and patients' samples. INTERPRETATION AND CONCLUSIONS: Our findings support the hypothesis that anti-beta2GPI IgG antibodies accelerate thrombin generation in the presence of TFPI and may contribute to hypercoagulability in patients with aPS.

Alternatively spliced isoforms of tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) is the major regulator of tissue factor (TF)-induced coagulation. It down regulates coagulation by binding to the TF/fVIIa complex in a fXa dependent manner. It is predominantly produced by microvascular endothelial cells, though it is also found in platelets, monocytes, smooth muscle cells, and plasma. Its physiological importance is demonstrated by the embryonic lethality observed in TFPI knockout mice and by the increase in thrombotic burden that occurs when heterozygous TFPI mice are bred with mice carrying genetic risk factors for thrombotic disease, such as factor V Leiden. Multiple TFPI isoforms, termed TFPIalpha, TFPIbeta, and TFPIdelta in humans and TFPIalpha, TFPIbeta, and TFPIgamma in mice, have been described, which differ in their domain structure and method for cell surface attachment. A significant functional difference between these isoforms has yet to be described in vivo. Both human and mouse tissues produce, on average, approximately 10 times more TFPIalpha message when compared to that of TFPIbeta. Consistent with this finding, several lines of evidence suggest that TFPIalpha is the predominant protein isoform in humans. In contrast, recent work from our laboratory demonstrates that TFPIbeta is the major protein isoform produced in adult mice, suggesting that TFPI isoform production is translationally regulated.