A coagulation defect arising from heterozygous premature termination of tissue factor.

Tissue factor (TF) is the primary initiator of blood coagulation in vivo and the only blood coagulation factor for which a human genetic defect has not been described. As there are no routine clinical assays that capture the contribution of endogenous TF to coagulation initiation, the extent to which reduced TF activity contributes to unexplained bleeding is unknown. Using whole genome sequencing, we identified a heterozygous frameshift variant (p.

Next-generation sequencing for the diagnosis of MYH9-RD: Predicting pathogenic variants.

The heterogeneous manifestations of MYH9-related disorder (MYH9-RD), characterized by macrothrombocytopenia, Döhle-like inclusion bodies in leukocytes, bleeding of variable severity with, in some cases, ear, eye, kidney, and liver involvement, make the diagnosis for these patients still challenging in clinical practice. We collected phenotypic data and analyzed the genetic variants in more than 3,000 patients with a bleeding or platelet disorder. Patients were enrolled in the BRIDGE-BPD and ThromboGenomics Projects and their samples processed by high throughput sequencing (HTS).

Identification of PDI Substrates by Mechanism-Based Kinetic Trapping.

Protein disulphide isomerase (PDI) is secreted by activated platelets and endothelial cells and is required for thrombus formation upon vascular injury. PDI catalyzes the reduction, oxidation, or isomerization of disulphide bonds in its substrate proteins. The specific substrates of PDI during thrombus formation have largely remained elusive, in part due to the transient nature of the PDI-substrate interaction.

Thiol isomerase ERp57 targets and modulates the lectin pathway of complement activation.

ER protein 57 (ERp57), a thiol isomerase secreted from vascular cells, is essential for complete thrombus formation , but other extracellular ERp57 functions remain unexplored. Here, we employed a kinetic substrate-trapping approach to identify extracellular protein substrates of ERp57 in platelet-rich plasma. MS-based identification with immunochemical confirmation combined with gene ontology enrichment analysis revealed that ERp57 targets, among other substrates, components of the lectin pathway of complement activation: mannose-binding lectin, ficolin-2, ficolin-3, collectin-10, collectin-11, mannose-binding lectin-associated serine protease-1, and mannose-binding lectin-associated serine protease-2.

Targeting protein disulfide isomerase with the flavonoid isoquercetin to improve hypercoagulability in advanced cancer.

Background: Protein disulfide isomerase (PDI) is a thiol isomerase secreted by vascular cells that is required for thrombus formation. Quercetin flavonoids inhibit PDI activity and block platelet accumulation and fibrin generation at the site of a vascular injury in mouse models, but the clinical effect of targeting extracellular PDI in humans has not been studied.

Methods: We conducted a multicenter phase II trial of sequential dosing cohorts to evaluate the efficacy of targeting PDI with isoquercetin to reduce hypercoagulability in cancer patients at high risk for thrombosis.

Regulatory role of thiol isomerases in thrombus formation.

The protein disulfide isomerase (PDI) family of thiol isomerases are intracellular enzymes known to catalyze the oxidation, reduction and isomerization of disulfide bonds during protein synthesis in the endoplasmic reticulum. PDI and related members of the thiol isomerase family are known to localize extracellularly where they possess various functions. Among these, the role of PDI in the initiation of thrombus formation is best characterized.

A long-acting PAI-1 inhibitor reduces thrombus formation.

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type and urokinase-type plasminogen activators (t/uPA) and plays an important role in fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis, indicating that PAI-1 inhibitors may be used as effective antithrombotic agents. We previously designed a PAI-1 inhibitor (PAItrap) which is a variant of inactivated urokinase protease domain.

Kinetic-based trapping by intervening sequence variants of the active sites of protein-disulfide isomerase identifies platelet protein substrates.

Thiol isomerases such as protein-disulfide isomerase (PDI) direct disulfide rearrangements required for proper folding of nascent proteins synthesized in the endoplasmic reticulum. Identifying PDI substrates is challenging because PDI catalyzes conformational changes that cannot be easily monitored ( compared with proteolytic cleavage or amino acid phosphorylation); PDI has multiple substrates; and it can catalyze either oxidation, reduction, or isomerization of substrates. Kinetic-based substrate trapping wherein the active site motif CGHC is modified to CGHA to stabilize a PDI-substrate intermediate is effective in identifying some substrates.

Protein disulfide isomerase secretion following vascular injury initiates a regulatory pathway for thrombus formation.

Protein disulfide isomerase (PDI), secreted by platelets and endothelial cells on vascular injury, is required for thrombus formation. Using PDI variants that form mixed disulfide complexes with their substrates, we identify by kinetic trapping multiple substrate proteins, including vitronectin. Plasma vitronectin does not bind to αvβ3 or αIIbβ3 integrins on endothelial cells and platelets.

Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation.

: Protein disulfide isomerase (PDI) is required for thrombus formation. We previously demonstrated that glycosylated quercetin flavonoids such as isoquercetin inhibit PDI activity and thrombus formation in animal models, but whether extracellular PDI represents a viable anticoagulant target in humans and how its inhibition affects blood coagulation remain unknown. : We evaluated effects of oral administration of isoquercetin on platelet-dependent thrombin generation in healthy subjects and patients with persistently elevated anti-phospholipid antibodies.

A substrate-driven allosteric switch that enhances PDI catalytic activity.

Protein disulfide isomerase (PDI) is an oxidoreductase essential for folding proteins in the endoplasmic reticulum. The domain structure of PDI is a-b-b'-x-a', wherein the thioredoxin-like a and a' domains mediate disulfide bond shuffling and b and b' domains are substrate binding. The b' and a' domains are connected via the x-linker, a 19-amino-acid flexible peptide.

Vascular thiol isomerases.

Thiol isomerases are multifunctional enzymes that influence protein structure via their oxidoreductase, isomerase, and chaperone activities. These enzymes localize at high concentrations in the endoplasmic reticulum of all eukaryotic cells where they serve an essential function in folding nascent proteins. However, thiol isomerases can escape endoplasmic retention and be secreted and localized on plasma membranes.

A specific plasminogen activator inhibitor-1 antagonist derived from inactivated urokinase.

Fibrinolysis is a process responsible for the dissolution of formed thrombi to re-establish blood flow after thrombus formation. Plasminogen activator inhibitor-1 (PAI-1) inhibits urokinase-type and tissue-type plasminogen activator (uPA and tPA) and is the major negative regulator of fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis.

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs.

Extracellular Thiol Isomerases and Their Role in Thrombus Formation.

Significance: The mammalian endoplasmic reticulum (ER) houses a large family of twenty thioredoxin-like proteins of which protein disulfide isomerase (PDI) is the archetypal member. Although the PDI family is best known for its role in oxidative protein folding of secretory proteins in the ER, these thioredoxin-like proteins fulfill ever-expanding roles, both within the secretory pathway and beyond.

Recent Advances: Secreted PDI family proteins have now been shown to serve a critical role in platelet thrombus formation and fibrin generation.

Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b'x Domain.

Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b' domain of PDI with a 1:1 stoichiometry.

Human phenotype ontology annotation and cluster analysis to unravel genetic defects in 707 cases with unexplained bleeding and platelet disorders.

Background: Heritable bleeding and platelet disorders (BPD) are heterogeneous and frequently have an unknown genetic basis. The BRIDGE-BPD study aims to discover new causal genes for BPD by high throughput sequencing using cluster analyses based on improved and standardised deep, multi-system phenotyping of cases.

Methods: We report a new approach in which the clinical and laboratory characteristics of BPD cases are annotated with adapted Human Phenotype Ontology (HPO) terms.

Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria.

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors, and thus bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a new target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond-forming enzymes Escherichia coli DsbB (EcDsbB) or Mycobacterium tuberculosis VKOR (MtbVKOR), which can replace EcDsbB, although the two are not homologs.

Both platelet- and endothelial cell-derived ERp5 support thrombus formation in a laser-induced mouse model of thrombosis.

Protein disulfide isomerase (PDI) and endoplasmic reticulum protein 57 (ERp57) are emerging as important regulators of thrombus formation. Another thiol isomerase, endoplasmic reticulum protein 5 (ERp5), is involved in platelet activation. We show here the involvement of ERp5 in thrombus formation using the mouse laser-injury model of thrombosis and a specific antibody raised against recombinant ERp5.

Defective PDI release from platelets and endothelial cells impairs thrombus formation in Hermansky-Pudlak syndrome.

Protein disulfide isomerase (PDI), secreted from platelets and endothelial cells after injury, is required for thrombus formation. The effect of platelet and endothelial cell granule contents on PDI-mediated thrombus formation was studied by intravital microscopy using a mouse model of Hermansky-Pudlak syndrome in which platelet dense granules are absent. Platelet deposition and fibrin generation were nearly absent, and extracellular PDI was significantly reduced in HPS6(-/-) mice after vascular injury.

How I treat poisoning with vitamin K antagonists.

Severe deficiency of vitamin K-dependent proteins in patients not maintained on vitamin K antagonists is most commonly associated with poisoning by or surreptitious ingestion of warfarin, warfarin-like anticoagulants, or potent rodenticides ("superwarfarins"), such as brodifacoum. Serious bleeding manifestations are common. Superwarfarins are 2 orders of magnitude more potent than warfarin and have a half-life measured in weeks.

Therapeutic implications of protein disulfide isomerase inhibition in thrombotic disease.

The study of thrombus formation has increasingly applied in vivo tools such as genetically modified mice and intravital microscopy to the evaluation of molecular and cellular mechanisms of thrombosis. Among several unexpected findings of this approach was the discovery that protein disulfide isomerase serves an essential role in thrombus formation at sites of vascular injury. The observation that the commonly ingested quercetin flavonoid, quercetin-3-rutinoside, inhibits protein disulfide isomerase and blocks thrombus formation in preclinical studies has set the stage for clinical trials using protein disulfide isomerase antagonists as antithrombotics.

Platelets are required for enhanced activation of the endothelium and fibrinogen in a mouse thrombosis model of APS.

Antiphospholipid syndrome (APS) is defined by thrombosis, fetal loss, and the presence of antiphospholipid antibodies, including anti-β2-glycoprotein-1 autoantibodies (anti-β2GP1) that have a direct role in the pathogenesis of thrombosis in vivo. The cellular targets of the anti-β2GP1 autoantibody/β2GP1 complex in vivo were studied using a laser-induced thrombosis model of APS in a live mouse and human anti-β2GP1 autoantibodies affinity-purified from APS patients. Cell binding of fluorescently labeled β2GP1 and anti-β2GP1 autoantibodies revealed their colocalization on the platelet thrombus but not the endothelium.

Thiol isomerases in thrombus formation.

Protein disulfide isomerase (PDI), ERp5, and ERp57, among perhaps other thiol isomerases, are important for the initiation of thrombus formation. Using the laser injury thrombosis model in mice to induce in vivo arterial thrombus formation, it was shown that thrombus formation is associated with PDI secretion by platelets, that inhibition of PDI blocked platelet thrombus formation and fibrin generation, and that endothelial cell activation leads to PDI secretion. Similar results using this and other thrombosis models in mice have demonstrated the importance of ERp5 and ERp57 in the initiation of thrombus formation.