Mast, Alan E.

Abstract TFPI is an endothelium associated anticoagulant protein that directly inhibits tissue factor (TF) pro-coagulant activity and prevents disease produced by intravascular thrombosis. TFPI is produced in three alternatively spliced isoforms in mice, α, β and γ, that differ in domain structure and mechanism for association with endothelium. TFPIα has three Kunitz-type serine protease inhibitory domains and a basic C-terminal region. It produces anticoagulant activity by direct inhibition of factor Xa (fXa) via the second Kunitz domain (K2) and, in a fXa dependent manner, inhibition of TF-fVIIa via the first Kunitz domain (K1). The third Kunitz domain (K3) and C-terminal region do not directly inhibit proteolysis. However, alignment of sequences from various vertebrate species demonstrates that K3 has maintained a high degree of sequence conservation for over 430 million years suggesting that K3 maintains a physiological function. TFPIα indirectly associates with endothelium by binding to a glycosylphosphatidyl inositol (GPI) anchored protein and binds non-specifically to glycosaminoglycans. TFPIβ has K1 and K2 but lacks K3 and has a distinct C-terminal region that encodes a GPI-anchor attachment sequence. TFPIγ has K1 and K2 but lacks K3 and has a distinct C-terminal region that results in production of a secreted protein. It is not known if these structurally diverse forms of TFPI with different mechanisms for cell surface association have variable efficacy in their ability to inhibit TF activity in vivo. We characterized TFPI isoform expression in mouse tissues in order to define specific physiological functions for alternative splicing of TFPI and how it may hinder diseases mediated by intravascular TF activity. Message for TFPIα is ~16-fold more abundant than message for TFPIβ or TFPIγ in mouse tissues. In situ hybridization studies of mouse heart and lung found that the isoforms are expressed in the same pattern, primarily in endothelial cells. No cell or vascular bed exclusively producing a single isoform was identified. Western blot analyses revealed the somewhat surprising finding that TFPIβ is the predominant isoform produced by adult mouse tissues. Thus, protein synthesis of TFPI isoforms in mice is regulated at least in part at the level of protein translation. Mouse plasma TFPI levels increase only 10–20% following heparin infusion. These data contrast with the 2- to 4-fold increase in plasma TFPI observed following heparin infusion in humans. The presence of only small amounts of heparin releasable TFPI in mouse plasma is consistent with TFPIβ as the major isoform because TFPIβ is directly GPI-anchored to endothelium and lacks the highly basic C-terminal region that allows TFPIα to associate non-specifically with endothelium glycosaminoglycans. To our knowledge, these are the first data demonstrating TFPIβ production in vivo. In contrast to the adult tissues, western blot analysis of mouse placenta revealed that it produces primarily TFPIα. This finding led us to examine mouse fetal tissue to determine if there is developmental regulation of the production of different TFPI isoforms. These studies demonstrated that TFPIα and TFPIβ are produced in approximately equal amounts in E14.5 embryos. The relative proportion of TFPIα in mouse tissues then decreases during development and very little is produced at 12 weeks of age. The evolutionary conservation of TFPIα and its production in mouse placenta and embryonic tissues suggests that unique features of TFPIα, such as K3, the basic C-terminal region and/or its binding to the endothelial surface through a GPI-anchored co-receptor, perform specific functions in the regulation of intravascular TF activity during vasculogenesis/angiogenesis that are not performed by TFPIβ or TFPIγ.

Abstract Background: Use of direct-acting oral anticoagulants (DOAC) is increasingly common among patients with atrial fibrillation and venous thromboembolic disease. Differences in the mechanisms of action as compared to warfarin could impact transfusion patterns and clinical outcomes in patients, especially for those presenting with major hemorrhage. The management of patients taking these newer medications and corresponding outcomes are relevant to optimizing clinical decision making in situations of major hemorrhage. Methods: We tested the hypothesis that inpatient all-cause mortality among patients presenting with major hemorrhage differs based on the home-administered anticoagulant medication class (DOAC versus warfarin). A cohort of patients presenting to twelve US hospitals from 2013 to 2016 was identified using the Recipient Epidemiology and Donor Evaluation Study (REDS)-III Recipient Database. Primary ICD diagnosis codes, issued blood products, laboratory data, and early mortality events were used in the application of the International Society on Thrombosis and Hemostasis definition of major hemorrhage. Exposure status was defined as a record of home-administered DOAC (apixaban, dabigatran, edoxaban, or rivaroxaban; exposed) or warfarin (non-exposed). Patients with multiple encounters and those transferred into or out of network were excluded from the analysis. Proportional hazards regression was used to compare all-cause mortality and hospital length of stay. We then repeated the analysis using a cohort matched on propensity scores to account for confounding by age, gender, concurrent aspirin and anti-platelet use, liver and renal dysfunction, cancer, CHA2DS2-VASc score, traumatic injury, and hospital. We then repeated the propensity score matched analysis stratified by anatomic location of bleed and traumatic injury. Results: More than 1.5 million hospitalizations were screened for eligibility. Exclusion of minors, outpatients, hospitalizations without a medication of interest, absence of major hemorrhage, multiple hospitalizations, and hospital transfers resulted in 3,731 patients available for the unadjusted analysis. Inpatient all-cause mortality was lower among DOAC users when the entire cohort was considered (HR = 0.60, 95%CI 0.45 - 0.80, p=0.0005). Implementation of propensity score matching to account for confounding abrogated this difference (HR=0.84, 95%CI 0.58 - 1.22, p=0.36). Time to hospital discharge was shorter for DOAC users (HR = 1.17, 95%CI 1.05 - 1.30, p=0.0034). Transfusion patterns were similar by medication, except for plasma transfusion occurring in 42% of warfarin encounters and 11% of DOAC encounters. Vitamin K was administered in 63% of warfarin encounters, whereas specific DOAC reversal agents were largely unavailable during the analysis period [used in 5 (1%) DOAC encounters]. There were no statistically significant differences in inpatient all-cause mortality in the stratified analysis (warfarin as reference): HR = 0.69 (95%CI 0.31 - 1.55) for traumatic head injuries; HR = 1.10 (95%CI 0.62 - 1.95) for non-traumatic head injuries; HR = 0.62 (95%CI 0.20 - 1.94) for traumatic, non-head injuries; and HR = 0.69 (95%CI 0.29 - 1.63) for non-traumatic, non-head injuries. Conclusions: Analysis of a population taking oral anticoagulation and presenting with major hemorrhage showed that transfusion of plasma was more commonly employed to treat major hemorrhage among warfarin users than DOAC users. Inpatient all-cause mortality was lower among DOAC users in the overall cohort; however, accounting for potential confounding factors using propensity score matching abrogated this difference. Hospital length of stay was shorter for DOAC users compared to warfarin users. Stratification by location of bleed and traumatic injury did not alter these findings. Less plasma use and a shorter length of hospitalization in this study, combined with no observable difference in inpatient all-cause mortality, suggests that outcomes following major hemorrhage are at least no different for DOAC users as compared to warfarin users. Disclosures Mast: Novo Nordisk: Research Funding. Kor:NIH: Consultancy; NIH: Research Funding; UpToDate: Patents & Royalties; CSL Behring: Honoraria.

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

AbstractThe 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 (TFPIFlox) which encodes for the TFPI-K1 domain. Mice bred into Tie2-Cre and LysM-Cre lines to delete TFPI-K1 in endothelial (TFPITie2) and myelomonocytic (TFPILysM) cells resulted in viable and fertile offspring. Plasma TFPI activity was reduced in the TFPITie2 (71% ± 0.9%, P < .001) and TFPILysM (19% ± 0.6%, P < .001) compared with TFPIFlox littermate controls. Tail and cuticle bleeding were unaffected. However, TFPITie2 mice but not TFPILysM mice had increased ferric chloride–induced arterial thrombosis. Taken together, the data reveal distinct roles for endothelial- and myelomonocytic-derived TFPI.