An Inhibitory Single-Domain Antibody against Protein Z-Dependent Protease Inhibitor Promotes Thrombin Generation in Severe Hemophilia A and FXI Deficiency.

Background:  Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin that targets factor Xa (FXa) in the presence of protein Z (PZ), and factor XIa (FXIa). In factor-VIII-deficient mice, PZ or ZPI gene knock-out mitigates the bleeding phenotype, and pharmacological inhibition of PZ enhances thrombin generation in plasma from patients with hemophilia.

Aims:  To develop a single-domain antibody (sdAb) directed against ZPI to inhibit its anticoagulant activity.

A small-molecule hemostatic agent for the reversal of direct oral anticoagulant-induced bleeding.

Background: The bleeding risk associated with direct oral anticoagulants (DOACs) remains a major concern, and rapid reversal of anticoagulant activity may be required. Although specific and nonspecific hemostatic biotherapies are available, there is a need for small-molecule DOAC reversal agents that are simple and cost-effective to produce, store, and administer.

Objectives: To identify and characterize a small molecule with procoagulant activity as a DOAC reversal agent.

N-Glycosylation Deficiency Reduces the Activation of Protein C and Disrupts Endothelial Barrier Integrity.

Phosphomannomutase 2 (PMM2) deficiency is the most prevalent congenital disorder of glycosylation. It is associated with coagulopathy, including protein C deficiency. Since all components of the anticoagulant and cytoprotective protein C system are glycosylated, we sought to investigate the impact of an -glycosylation deficiency on this system as a whole.

Antithrombotic potential of a single-domain antibody enhancing the activated protein C-cofactor activity of protein S.

Background: Protein S (PS) is a natural anticoagulant acting as a cofactor for activated protein C (APC) in the proteolytic inactivation of activated factors V (FVa) and VIII (FVIIIa), but also for tissue factor pathway inhibitor α (TFPIα) in the inhibition of activated factor X (FXa).

Objective: For therapeutic purposes, we aimed at generating single-domain antibodies (sdAbs) that could specifically modulate the APC-cofactor activity of PS in vivo.

Methods: A llama-derived immune library of sdAbs was generated and screened on recombinant human PS by phage display.

Anti-inflammatory Activity of the Protein Z-Dependent Protease Inhibitor.

The protein Z (PZ)-dependent plasma protease inhibitor (ZPI) is a glycoprotein that inhibits factor XIa and, in the presence of PZ, FXa. Recently, ZPI has been shown to be an acute-phase protein (APP). As usually APPs downregulate the harmful effects of inflammation, we tested whether ZPI could modulate the increase of cytokines observed in inflammatory states.

The Proteolytic Inactivation of Protein Z-Dependent Protease Inhibitor by Neutrophil Elastase Might Promote the Procoagulant Activity of Neutrophil Extracellular Traps in Sepsis.

Septic shock is the archetypal clinical setting in which extensive crosstalk between inflammation and coagulation dysregulates the latter. The main anticoagulant systems are systematically impaired, depleted, and/or downregulated. Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin that not only targets coagulation factors Xa and XIa but also acts as an acute phase reactant whose plasma concentration rises in inflammatory settings.

Serpins in Hemostasis as Therapeutic Targets for Bleeding or Thrombotic Disorders.

Bleeding and thrombotic disorders result from imbalances in coagulation or fibrinolysis, respectively. Inhibitors from the serine protease inhibitor (serpin) family have a key role in regulating these physiological events, and thus stand out as potential therapeutic targets for modulating fibrin clot formation or dismantling. Here, we review the diversity of serpin-targeting strategies in the area of hemostasis, and detail the suggested use of modified serpins and serpin inhibitors (ranging from small-molecule drugs to antibodies) to treat or prevent bleeding or thrombosis.

Inflammation in deep vein thrombosis: a therapeutic target?

Deep vein thrombosis is a common disease associated with a variety of complications including post-thrombotic syndrome as a late complication. It is now clear that in addition to classical deep vein thrombosis triggers such as blood flow disturbance, hypercoagulability, and vessel wall changes, inflammation has a key role in the pathophysiology of deep vein thrombosis, and there is a close relationship between inflammation and coagulation. As attested by changes in several plasma biomarkers, inflammation may have a significant role in the development of post-thrombotic syndrome.

Elevated thrombin generation in patients with congenital disorder of glycosylation and combined coagulation factor deficiencies.

Background: Congenital disorders of glycosylation are rare inherited diseases affecting many different proteins. The lack of glycosylation notably affects the hemostatic system and leads to deficiencies of both procoagulant and anticoagulant factors.

Objective: To assess the hemostatic balance in patients with multiple coagulation disorders by using a thrombin generation assay.

An ADAM-10 dependent EPCR shedding links meningococcal interaction with endothelial cells to purpura fulminans.

Purpura fulminans is a deadly complication of Neisseria meningitidis infections due to extensive thrombosis of microvessels. Although a Disseminated Intra-vascular Coagulation syndrome (DIC) is frequently observed during Gram negative sepsis, it is rarely associated with extensive thrombosis like those observed during meningococcemia, suggesting that the meningococcus induces a specific dysregulation of coagulation. Another specific feature of N.

Inactivated antithombin as anticoagulant reversal in a rat model of cardiopulmonary bypass: a potent and potentially safer alternative to protamine.

Heparin anticoagulation followed by protamine reversal is commonly used in cardiopulmonary bypass (CPB). As an alternative to protamine, a recombinant inactive antithrombin (riAT) was designed as an antidote to heparin and was previously shown to be as potent as protamine in-vitro. In the present study, riAT was assessed for its ability to neutralize heparin after CPB in a rat model.

Thrombomodulin favors leukocyte microvesicle fibrinolytic activity, reduces NETosis and prevents septic shock-induced coagulopathy in rats.

Background: Septic shock-induced disseminated intravascular coagulation is responsible for increased occurrence of multiple organ dysfunction and mortality. Immunothrombosis-induced coagulopathy may contribute to hypercoagulability. We aimed at determining whether recombinant human thrombomodulin (rhTM) could control exaggerated immunothrombosis by studying procoagulant responses, fibrinolysis activity borne by microvesicles (MVs) and NETosis in septic shock.

Soluble Siglec-5 associates to PSGL-1 and displays anti-inflammatory activity.

Interactions between endothelial selectins and the leukocyte counter-receptor PSGL1 mediates leukocyte recruitment to inflammation sites. PSGL1 is highly sialylated, making it a potential ligand for Siglec-5, a leukocyte-receptor that recognizes sialic acid structures. Binding assays using soluble Siglec-5 variants (sSiglec-5/C4BP and sSiglec-5/Fc) revealed a dose- and calcium-dependent binding to PSGL1.

Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use.

Inactivated antithrombins as fondaparinux antidotes: a promising alternative to haemostatic agents as assessed in vitro in a thrombin-generation assay.

In the absence of specific antidote to fondaparinux, two modified forms of antithrombin (AT), one recombinant inactive (ri-AT) and the other chemically inactivated (chi-AT), were designed to antagonise AT-mediated anticoagulants, e. g. heparins or fondaparinux.

A fast capillary electrophoresis method to assess the binding affinity of recombinant antithrombin toward heparin directly from cell culture supernatants.

With the aim to determine the binding affinity of a new generation of recombinant antithrombin (AT) toward heparin, we developed a dynamic equilibrium-affinity capillary electrophoresis (DE-ACE) method. This method allows the determination of an AT-heparin binding constant (Kd) directly from the cell culture supernatant used to produce the AT variants. Eight measurements per AT variant are sufficient to determine an accurate Kd (uncertainty ≤ 22%, regression coefficient ≥ 0.

Novel self assembling nanoparticles for the oral administration of fondaparinux: synthesis, characterization and in vivo evaluation.

Fondaparinux (Fpx) is the anticoagulant of choice in the treatment of short- and medium-term thromboembolic disease. To overcome the low oral bioavailability of Fpx, a new nanoparticulate carrier has been developed. The nanoparticles (NPs) contain squalenyl derivatives, known for their excellent oral bioavailability.

Auto-associative heparin nanoassemblies: a biomimetic platform against the heparan sulfate-dependent viruses HSV-1, HSV-2, HPV-16 and RSV.

A new, simple and green method was developed for the manufacturing of heparin nanoassemblies active against the heparan sulfate-dependent viruses HSV-1, HSV-2, HPV-16 and RSV. These nanoassemblies were obtained by the auto-association of O-palmitoyl-heparin and α-cyclodextrin in water. The synthesized O-palmitoyl-heparin derivatives mixed with α-cyclodextrin resulted in the formation of crystalline hexagonal nanoassemblies as observed by transmission electron microscopy.

Development of a recombinant antithrombin variant as a potent antidote to fondaparinux and other heparin derivatives.

Heparin derivative-based therapy has evolved from unfractionated heparin (UFH) to low-molecular-weight heparins (LMWHs) and now fondaparinux, a synthetic pentasaccharide. Contrary to UFH or LMWHs, fondaparinux is not neutralized by protamine sulfate, and no antidote is available to counteract bleeding disorders associated with overdosing. To make the use of fondaparinux safer, we developed an antithrombin (AT) variant as a potent antidote to heparin derivatives.

Thrombin-activable factor X re-establishes an intrinsic amplification in tenase-deficient plasmas.

Classical hemophilia results from a defect of the intrinsic tenase complex, the main factor X (FX) activator. Binding of factor VIIa to tissue factor triggers coagulation, but little amplification of thrombin production occurs. Handling of hemophilia by injection of the deficient or missing (thus foreign) factor often causes immunological complications.