TFPIß is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes.

Tissue factor pathway inhibitor (TFPI) produces factor Xa-dependent feedback inhibition of factor VIIa/tissue factor-induced coagulation. Messages for 2 isoforms of TFPI have been identified. TFPIα mRNA encodes a protein with an acidic N-terminus, 3 Kunitz-type protease inhibitor domains and a basic C-terminus that has been purified from plasma and culture media. TFPIß mRNA encodes a form in which the Kunitz-3 and C-terminal domains of TFPIα are replaced with an alternative C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPIß protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment is TFPIα, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPIß based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and (3) it contains a GPI anchor. Immunoassays demonstrate that, although endothelial cells secrete TFPIα, greater than 95% of the TFPI released by PIPLC treatment from the surface of endothelial cells and from placental microsomes is TFPIß.

Deletion of tissue factor pathway inhibitor isoform beta or gamma, but not alpha, improves clotting in hemophilic mice.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) regulates tissue factor-triggered coagulation. Humans and mice express transcripts encoding for multidistributed (endothelial, platelet, and plasma) 3-Kunitz domain TFPIα and endothelial membrane-anchored 2-Kunitz TFPIß. Mice express a third transcript, γ, that encodes plasma lipoprotein-associated 2-Kunitz TFPI. In humans, proteolysis of α and/or ß produces plasma lipoprotein-associated 2-Kunitz TFPI at lower levels. In clinical trials, monoclonal antibodies that target all TFPI isoforms extend coagulation and correct bleeding in hemophilic patients but with some thrombosis risks. OBJECTIVES: To determine the impact of TFPI isoform-specific deletions on promoting clotting in hemophilic mice. METHODS: Engineered TFPI isoform-specific, hemophilic (factor VIII-null) mice were evaluated for clotting. RESULTS: Mice expressing any single TFPI isoform were healthy. Thrombin generation assays identified TFPIγ as the dominant anticoagulation isoform in mouse plasma. Hemostasis was assessed by serial bleeding times from a tail vein laceration. Repeatedly, after a clot forms, it was manually disrupted; the number of clots/disruptions occurring over a 15-minute period were reported. C57BL/6 and hemophilic mice clot on average 25.6 vs 5.4 times, respectively. On a hemophilia background, TFPIß or TFPIγ-specific deletion improved clotting to 14.6 and 15.2 times, respectively (P < .0001). TFPIα-specific deletion was without impact, clotting 5.1 times. Heterozygous deletion of TFPIß was effective, clotting 11.8 times (P < .0001). Heterozygous deletion of TFPIα or TFPIγ alone was ineffective, clotting 3.0 and 6.1 times, respectively, but heterozygous TFPIαγ deletion improved clotting to 11.2 times (P < .001). CONCLUSION: In hemophilic mice, endothelial TFPIß and plasma γ-derived 2-Kunitz TFPI individually contribute more to bleeding than total TFPIα.

Peripheral blood mononuclear cell tissue factor (F3 gene) transcript levels and circulating extracellular vesicles are elevated in severe coronavirus 2019 (COVID-19) disease.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with excessive coagulation, thrombosis, and mortality. OBJECTIVE: To provide insight into mechanisms that contribute to excessive coagulation in coronavirus 2019 (COVID-19) disease. PATIENTS/METHODS: Blood from COVID-19 patients was investigated for coagulation-related gene expression and functional activities. RESULTS: Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from severe COVID-19 patients revealed a 5.2-fold increase in tissue factor (TF [F3 gene]) transcript expression levels (P < .05), the trigger of extrinsic coagulation; a 7.7-fold increase in C1-inhibitor (SERPING1 gene; P < .01) transcript expression levels, an inhibitor of intrinsic coagulation; and a 4.4-fold increase in anticoagulant thrombomodulin (TM [THBD gene]) transcript expression levels (P < .001). Bulk RNA-seq analysis of sorted CD14+ monocytes on an independent cohort of COVID-19 patients confirmed these findings (P < .05). Indicative of excessive coagulation, 41% of COVID-19 patients' plasma samples contained high D-dimer levels (P < .0001); of these, 19% demonstrated extracellular vesicle TF activity (P = .109). COVID-19 patients' ex vivo plasma-based thrombin generation correlated positively with D-dimer levels (P < .01). Plasma procoagulant extracellular vesicles were elevated ∼9-fold in COVID-19 patients (P < .01). Public scRNA-seq data sets from bronchoalveolar lung fluid and our peripheral blood mononuclear cell scRNA-seq data show CD14+ monocytes/macrophages TF transcript expression levels are elevated in severe but not mild or moderate COVID-19 patients. CONCLUSIONS: Beyond local lung injury, SARS-CoV-2 infection increases systemic TF (F3) transcript levels and elevates circulating extracellular vesicles that likely contribute to disease-associated coagulation, thrombosis, and related mortality.

Part II: piperazinyl-glutamate-pyridines as potent orally bioavailable P2Y12 antagonists for inhibition of platelet aggregation.

Efforts to refine the SAR of the piperazinyl-glutamate-pyridines for more potent analogs with improved pharmacokinetic profiles are described. Exploring substituted piperidines and other ring systems at the 4-pyridyl position led to compounds with improved potency and pharmacokinetic properties over candidate I. In particular, compounds 4t and 5t were discovered with a 10-fold improvement over potency and improved pharmacokinetic profiles in both the rat and dog.

Piperazinyl-glutamate-pyrimidines as potent P2Y12 antagonists for inhibition of platelet aggregation.

Piperazinyl-glutamate-pyrimidines were prepared with oxygen, nitrogen, and sulfur substitution at the 4-position of the pyrimidine leading to highly potent P2Y12 antagonists. In particular, 4-substituted piperidine-4-pyrimidines provided compounds with exceptional potency. Pharmacokinetic and physicochemical properties were fine-tuned through modifications at the 4-position of the piperidine ring leading to compounds with good human PRP potency, selectivity, clearance and oral bioavailability.

Piperazinyl-glutamate-pyridines as potent orally bioavailable P2Y12 antagonists for inhibition of platelet aggregation.

Polymer-assisted solution-phase (PASP) parallel library synthesis was used to discover a piperazinyl-glutamate-pyridine as a P2Y(12) antagonist. Exploitation of this lead provided compounds with excellent inhibition of platelet aggregation as measured in a human platelet rich plasma (PRP) assay. Pharmacokinetic and physiochemical properties were optimized leading to compound (4S)-4-[({4-[4-(methoxymethyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic acid 22J with good human PRP potency, selectivity, in vivo efficacy and oral bioavailability.

Polymer-assisted solution-phase library synthesis and crystal structure of alpha-ketothiazoles as tissue factor VIIa inhibitors.

A solution-phase synthesis of an alpha-ketothiazole library of the general form D-Phe-L-AA-Arg-alpha-ketothiazole is described. The five-step synthesis is accomplished using a combination of polymeric reagents and polymer-assisted solution-phase purification concepts, including reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multistep synthesis affords desired alpha-ketothiazole products in excellent purities and yields. A variety of L-amino acid inputs were used to probe the S2 pocket of tissue Factor VIIa enzyme to influence both potency and selectivity. An X-ray crystal structure of compound 10k bound to the TF/VIIa complex was obtained that explains the observed selectivity. The alpha-ketothiazoles were found to be potent, reversible-covalent inhibitors of tissue Factor VIIa, with some analogues demonstrating selectivity over thrombin.

Design, parallel synthesis, and crystal structures of pyrazinone antithrombotics as selective inhibitors of the tissue factor VIIa complex.

Structure-based drug design (SBDD) and polymer-assisted solution-phase (PASP) library synthesis were used to develop a series of pyrazinone inhibitors of the Tissue Factor/Factor VIIa (TF/VIIa) complex. The crystal structure of a tripeptide-alpha-ketothiazole complexed with TF/VIIa was utilized in a docking experiment to identify the pyrazinone core as a starting scaffold. The pyrazinone core could orient the substituents in the correct spatial arrangement to probe the S1, S2, and S3 pockets of the enzyme. A multistep PASP library synthesis was designed to prepare the substituted pyrazinones varying the P1, P2, and P3 moieties. Hundreds of pyrazinone TF/VIIa inhibitors were prepared and tested in several serine protease enzyme assays involved in the coagulation cascade. The inhibitors exhibited modest activity on TF/VIIa with excellent selectivity over thrombin (IIa) and Factor Xa. The structure-activity relationship of the pyrazinone inhibitors will be discussed and X-ray crystal structures of selected compounds complexed with the TF/VIIa enzyme will be described. This study ultimately led to the synthesis of compound 34, which exhibited 16 nM (IC50) activity on TF/VIIa with >6250 x selectivity vs Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for preclinical, intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side effects in a nonhuman primate model of electrolytic-induced arterial thrombosis.

Factor V, tissue factor pathway inhibitor, and east Texas bleeding disorder.

In a report reading like a fascinating detective story, Vincent and colleagues crack the mysterious case of east Texas bleeding disorder. They show that affected individuals have a mutation in exon 13 of the coagulation F5 gene that causes increased expression of an alternatively spliced transcript, which encodes a previously unrecognized factor V (FV) isoform they call FV-short. This FV isoform lacks a large portion of the B domain of FV, which is normally released upon the proteolytic activation of FV by thrombin and binds tightly to the coagulation regulator tissue factor pathway inhibitor-α (TFPIα). This interaction leads to an approximately 10-fold increase in the level of TFPIα circulating in plasma and a resultant anticoagulant effect that produces a hemorrhagic diathesis.

Tissue factor pathway inhibitor: structure-function.

TFPI is a multivalent, Kunitz-type proteinase inhibitor, which, due to alternative mRNA splicing, is transcribed in three isoforms: TFPIalpha, TFPIdelta, and glycosyl phosphatidyl inositol (GPI)-anchored TFPIbeta. The microvascular endothelium is thought to be the principal source of TFPI and TFPIalpha is the predominant isoform expressed in humans. TFPIalpha, apparently attached to the surface of the endothelium in an indirect GPI-anchor-dependent fashion, represents the greatest in vivo reservoir of TFPI. The Kunitz-2 domain of TFPI is responsible for factor Xa inhibition and the Kunitz-1 domain is responsible for factor Xa-dependent inhibition of the factor VIIa/tissue factor catalytic complex. The anticoagulant activity of TFPI in one-stage coagulation assays is due mainly to its inhibition of factor Xa through a process that is enhanced by protein S and dependent upon the Kunitz-3 and carboxyterminal domains of full-length TFPIalpha. Carboxyterminal truncated forms of TFPI as well as TFPIalpha in plasma, however, inhibit factor VIIa/tissue factor in two-stage assay systems. Studies in gene-disrupted mice demonstrate the physiological importance of TFPI.

Piperazinyl glutamate pyridines as potent orally bioavailable P2Y12 antagonists for inhibition of platelet aggregation.

Polymer-assisted solution-phase (PASP) parallel library synthesis was used to discover a piperazinyl glutamate pyridine as a P2Y(12) antagonist. Exploitation of this lead provided compounds with excellent inhibition of platelet aggregation as measured in a human platelet rich plasma (PRP) assay. Pharmacokinetic and physiochemical properties were optimized through modifications at the 4-position of the pyridine ring and the terminal nitrogen of the piperazine ring, leading to compound (4S)-4-[({4-[4-(methoxymethyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic acid 47s with good human PRP potency, selectivity, in vivo efficacy, and oral bioavailability. Compound 47s was selected for further preclinical evaluations.

Pharmacological intervention at disparate sites in the coagulation cascade: comparison of anti-thrombotic efficacy vs bleeding propensity in a rat model of acute arterial thrombosis.

The Tissue Factor/Factor VIIa (TF/FVIIa) complex is an attractive target for pharmacological interruption of thrombin generation and hence blood coagulation, as this complex is the initiation point of the extrinsic pathway of coagulation. TF is a cell membrane-associated protein that interacts with soluble FVIIa to activate factors IX and X resulting in a cascade of events that leads to thrombin generation and eventual fibrin deposition. The goal of this non-randomized study was to evaluate XK1, a specific protein inhibitor of TF/FVIIa, and compare antithrombotic efficacy and bleeding propensity to a previously described Factor Xa (FXa) inhibitor (SC-83157/SN429) and a direct-acting thrombin inhibitor (SC-79407/L-374087) in an acute rat model of arterial thrombosis. All saline-treated animals experienced occlusion of the carotid artery due to acute thrombus formation within 20 minutes. Rats treated with XK1 exhibited a dose-dependent inhibition of thrombus formation with full antithrombotic efficacy and no change in bleeding time or total blood loss at a dose of 4.5 mg/kg, i.v. administered over a 60 minute period. FXa inhibition with SC-83157 resulted in complete inhibition of thrombus formation at a dose of 1.2 mg/kg, i.v.; however, this effect was associated with substantial blood loss. Thrombin inhibition with SC-79407 also afforded complete protection from thrombus formation and occlusion at a dose of 2.58 mg/kg, i.v., and like SC-83157, was associated with substantial blood loss. These data imply that TF/FVIIa inhibition confers protection from acute thrombosis without concomitant changes in bleeding, indicating that this target (TF/FVIIa) may provide improved separation of efficacy vs. bleeding side-effects than interruption of coagulation by directly inhibiting either FXa or thrombin.

Polymer-assisted solution-phase (PASP) parallel synthesis of an alpha-ketothiazole library as tissue factor VIIa inhibitors.

A solution-phase synthesis of an alpha-ketothiazole library of the general form D-Phe-L-AA-L-Arg-alpha-ketothiazole is described. The five-step synthesis is accomplished using a combination of polymeric reagents and polymer-assisted solution-phase purification protocols, including reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multi-step synthesis affords the desired alpha-ketothiazole products in excellent purities and yields. A variety of L-amino acid inputs were used to probe the S2 pocket of the tissue factor (TF) VIIa enzyme to influence both potency and selectivity. An X-ray crystal structure of compound 10e bound to the TF/VIIa complex was obtained that explains the observed selectivity. The alpha-ketothiazoles were found to be potent, reversible-covalent inhibitors of tissue factor VIIa, with some analogues demonstrating selectivity versus thrombin.

Structure-based drug design of pyrazinone antithrombotics as selective inhibitors of the tissue factor VIIa complex.

Structure-based drug design coupled with polymer-assisted solution-phase library synthesis was utilized to develop a series of pyrazinone inhibitors of the tissue factor/Factor VIIa complex. The crystal structure of a tri-peptide ketothiazole complexed with TF/VIIa was utilized in a docking experiment that identified a benzyl-substituted pyrazinone as a P(2) surrogate for the tri-peptide. A 5-step PASP library synthesis of these aryl-substituted pyrazinones was developed. The sequence allows for attachment of a variety of P(1) and P(3) moieties, which led to synthesis pyrazinone 23. Compound 23 exhibited 16 nM IC(50) against TF/VIIa with >6250x selectivity versus Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for pre-clinical intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side effects in a primate model of thrombosis.