Fitusiran reduces bleeding in Factor X-deficient mice.

Factor X (FX)-deficiency is a rare bleeding disorder manifesting a bleeding tendency caused by low FX activity levels. We aimed to explore the use of fitusiran (an investigational siRNA that silences antithrombin expression) to increase thrombin generation and the in vivo hemostatic potential under conditions of FX-deficiency. We therefore developed a novel model of inducible FX-deficiency, generating mice expressing <1% FX activity and antigen (f10low-mice). Compared to control f10WT-mice, f10low-mice had 6- and 4-fold prolonged clotting times in Prothrombin Time- and activated Partial Prothrombin Time-assays, respectively (p<0.001). Thrombin generation was severely reduced, irrespective whether tissue factor or factor XIa was used as initiator. In vivo analysis revealed near-absent thrombus formation in a laser-induced vessel injury-model. Furthermore, in two distinct bleeding models, f10low-mice displayed an increased bleeding tendency compared to f10WT-mice. In the tail-clip assay blood loss was increased from 12±16 microliter to 590±335 microliter (p<0.0001). In the saphenous vein puncture (SVP)-model, the number of clots generated was reduced from 19±5 clots/30 min for f10WT-mice to 2±2 clots/30 min (p<0.0001) for f10low-mice. In both models, bleeding was corrected upon infusion of purified FX. Treatment of f10low-mice with fitusiran (2x10 mg/kg at one-week interval) resulted in 17±6% residual antithrombin activity and increased thrombin generation (4-fold and 2-3-fold increase in endogenous thrombin potential and thrombin peak, respectively). In the SVP-model, the number of clots was increased to 8±6 clots/30 min (p=0.0029). Altogether, we demonstrate that reduction of antithrombin levels is associated with improved hemostatic activity under conditions of FX-deficiency.

Impact of allele-selective silencing of von Willebrand factor in mice based on a single nucleotide allelic difference in von Willebrand factor.

INTRODUCTION: Von Willebrand factor (VWF) plays a pathophysiological role in hemostatic disorders. Partial inhibition of the VWF gene through small interfering RNA (siRNA)-mediated allele-selective silencing could be a promising therapeutic strategy. For von Willebrand disease, allele-selectively inhibiting dominant-negative VWF-alleles might ameliorate the phenotype. For thrombotic disorders, partial VWF reduction can lower thrombotic risk, while avoiding bleeding. Previously, we demonstrated the feasibility of Vwf-silencing in homozygous C57BL/6J (B6) or 129S1/SvImJ (129S) mice. The present study investigated allele-selective Vwf-silencing in a complex heterozygous setting of crossed B6 and 129S mice and its subsequent hemostatic impact. MATERIALS AND METHODS: Heterozygous B6.129S mice were treated with siRNAs targeting Vwf expressed from either B6- (siVwf.B6) or 129S-alleles (siVwf.129S). Plasma VWF and lung Vwf mRNA were determined. siVwf.B6-treated B6.129S mice were subjected to ferric chloride-induced mesenteric vessel thrombosis and tail-bleeding. RESULTS: In B6.129S mice, siVwf.B6 reduced Vwf mRNA of the targeted B6-allele by 72% vs. only 12% of the non-targeted 129S-allele (41% total mRNA reduction), lowering plasma VWF by 46%. Oppositely, siVwf.129S reduced Vwf mRNA by 45%, now selectively inhibiting the 129S-allele over the B6-allele (58% vs. 9%), decreasing plasma VWF by 43%. The allele-selective VWF reduction by siVwf.B6 coincided with decreased thrombus formation in mesenteric arterioles, without prolonging tail-bleeding times. CONCLUSIONS: This study demonstrates the feasibility of allele-selective Vwf-silencing in a heterozygous setting, achieving a controlled close to 50% reduction of plasma VWF. The observed thromboprotection and absence of prolonged bleeding times underline the potential of allele-selective Vwf-silencing as a therapeutic strategy in hemostatic disorders.

Thrombin generation on vascular cells in the presence of factor VIII and/or emicizumab.

BACKGROUND: The effect of factor VIII (FVIII) or emicizumab on thrombin generation is usually assessed in assays using synthetic phospholipids. Here, we assessed thrombin generation at the surface of human arterial cells (aortic endothelial cells [hAECs] and aortic vascular smooth muscle cells [hVSMCs]). OBJECTIVES: To explore the capacity of hAECs (resting or stimulated) and hVSMCs to support thrombin generation by FVIII or emicizumab. METHODS: Primary hVSMCs and hAECs were analyzed for tissue factor (TF)-activity and antigen, phosphatidylserine (PS)-exposure, tissue factor pathway inhibitor (TFPI)-content and thrombomodulin expression. Cells were incubated with FVIII-deficient plasma spiked with FVIII, emicizumab, activated prothrombin complex concentrate (APCC) or combinations thereof. RESULTS: TF activity and PS-exposure were present on both hVSMCs and hAECs. In contrast, thrombomodulin and TFPI were expressed on hAECs, while virtually lacking on hVSMCs, confirming the procoagulant nature of hVSMCs. Tumor necrosis factor α-mediated stimulation of hAECs increased not only TF antigen, TF activity, and PS-exposure but also TFPI and thrombomodulin expression. As expected, FVIII and emicizumab promoted thrombin generation on nonstimulated hAECs and hVSMCs, with more thrombin being generated on hVSMCs. Unexpectedly, FVIII and emicizumab increased thrombin generation to a lesser extent on stimulated hAECs compared with nonstimulated hAECs. Finally, adding emicizumab to FVIII did not further increase thrombin generation, whereas the addition of emicizumab to APCC resulted in exaggerated thrombin generation. CONCLUSION: Tumor necrosis factor stimulation of hAECs increases both pro- and anticoagulant activity. Unexpectedly, the increased anticoagulant activity is sufficient to limit both FVIII- and emicizumab-induced thrombin generation. This protective effect disappears when emicizumab is combined with APCC.

Differences in venous clot structures between hemophilic mice treated with emicizumab versus factor VIII or factor VIIIFc.

Recombinant factor VIII (rFVIII), rFVIIIFc and emicizumab are established treatment options in the management of hemophilia A. Each has its unique mode of action, which can influence thrombin generation kinetics and therefore also the kinetics of thrombin substrates. Such differences may potentially result in clots with different structural and physical properties. A starting observation of incomplete wound closure in a patient on emicizumab-prophylaxis led us employ a relevant mouse model in which we noticed that emicizumab-induced clots appeared less stable compared to FVIII-induced clots. We thus analyzed fibrin formation in vitro and in vivo. In vitro fibrin formation was faster and more abundant in the presence of emicizumab compared to rFVIII/rFVIIIFc. Furthermore, the time-interval between the initiation of fibrin formation and factor XIII activation was twice as long for emicizumab compared to rFVIII/rFVIIIFc. Scanning-electron microscopy and immunofluorescent spinning-disk confocal-microscopy of in vivo generated clots confirmed increased fibrin formation in the presence of emicizumab. Unexpectedly, we also detected a different morphology between rFVIII/rFVIIIFc- and emicizumab-induced clots. Contrary to the regular fibrin-mesh obtained with rFVIII/rFVIIIFc, fibrin-fibers appeared to be fused into large patches upon emicizumabtreatment. Moreover, fewer red blood cells were detected in regions where these fibrin patches were present. The presence of highly-dense fibrin-structures associated with a diffuse fiber-structure in emicizumab-induced clots was also observed when using superresolution imaging. We hypothesize that the modified kinetics of thrombin, fibrin and factor XIIIa generation contribute to differences in structural and physical properties between clots formed in the presence of FVIII or emicizumab.

The role of platelets and von Willebrand factor in the procoagulant phenotype of inflammatory bowel disease.

OBJECTIVE: Although the risk for thrombosis is well documented for inflammatory bowel disease (IBD) patients, the underlying pathological mechanism seems to be different from other thrombotic conditions. Deciphering the actors responsible for the increased risk of thrombosis in IBD would help to improve management of this frequent complication. DESIGN: We studied the interplay between platelets, coagulation, and von Willebrand factor (VWF) in 193 IBD patients and in experimental models (acute and chronic) of colitis in wild-type and VWF-deficient mice. RESULTS: We found a platelet-dependent increase in thrombin generation in IBD patients and in our mouse model of colitis. Agglutinated platelets were present in the blood of patients and mice. Interestingly, we observed not only a significant increase in total VWF antigen, but we were able to detect the presence of active VWF (VWF in its platelet-binding conformation; 3.2±2.7µg/ml) in the plasma of 30% of all IBD patients. In healthy controls, active VWF levels were below 0.3µg/ml. This led us to further explore experimental colitis in VWF-deficient mice and we observed that these mice were protected against the procoagulant state triggered by the colitis. Unexpectedly, these mice also manifested a significant worsening of colitis severity both in acute and chronic models. CONCLUSION: Platelets and VWF (including its active form) appear to be central players in the procoagulant phenotype in IBD. We observed that the role of VWF in hemostasis differs from its role in colic tissue healing, potentially opening new therapeutic avenues for a life-threatening complication in IBD patients.

Shear Forces Induced Platelet Clearance Is a New Mechanism of Thrombocytopenia.

BACKGROUND: Thrombocytopenia has been consistently described in patients with extracorporeal membrane oxygenation (ECMO) and associated with poor outcome. However, the prevalence and underlying mechanisms remain largely unknown, and a device-related role of ECMO in thrombocytopenia has been hypothesized. This study aims to investigate the mechanisms underlying thrombocytopenia in ECMO patients. METHODS: In a prospective cohort of 107 ECMO patients, we investigated platelet count, functions, and glycoprotein shedding. In an ex vivo mock circulatory ECMO loop, we assessed platelet responses and VWF (von Willebrand factor)-GP Ibα (glycoprotein Ibα) interactions at low- and high-flow rates, in the presence or absence of red blood cells. The clearance of human platelets subjected or not to ex vivo perfusion was studied using an in vivo transfusion model in NOD/SCID (nonobese diabetic/severe combined Immunodeficient) mice. RESULTS: In ECMO patients, we observed a time-dependent decrease in platelet count starting 1 hour after device onset, with a mean drop of 7%, 35%, and 41% at 1, 24, and 48 hours post-ECMO initiation (P=0.00013, P<0.0001, and P<0.0001, respectively), regardless of the type of ECMO. This drop in platelet count was associated with a decrease in platelet GP Ibα expression (before: 47.8±9.1 versus 24 hours post-ECMO: 42.3±8.9 mean fluorescence intensity; P=0.002) and an increase in soluble GP Ibα plasma levels (before: 5.6±3.3 versus 24 hours post-ECMO: 10.8±4.1 µg/mL; P<0.0001). GP Ibα shedding was also observed ex vivo and was unaffected by (1) red blood cells, (2) the coagulation potential, (3) an antibody blocking VWF-GP Ibα interaction, (4) an antibody limiting VWF degradation, and (5) supraphysiological VWF plasma concentrations. In contrast, GP Ibα shedding was dependent on rheological conditions, with a 2.8-fold increase at high- versus low-flow rates. Platelets perfused at high-flow rates before being transfused to immunodeficient mice were eliminated faster in vivo with an accelerated clearance of GP Ibα-negative versus GP Ibα-positive platelets. CONCLUSIONS: ECMO-associated shear forces induce GP Ibα shedding and thrombocytopenia due to faster clearance of GP Ibα-negative platelets. Inhibiting GP Ibα shedding could represent an approach to reduce thrombocytopenia during ECMO.

Angiopoietin-2 binds to multiple interactive sites within von Willebrand factor.

Background: Biosynthesis of von Willebrand factor (VWF) in endothelial cells drives the formation of storage-organelles known as Weibel-Palade bodies (WPBs). WPBs also contain several other proteins, including angiopoietin-2 (Ang-2). Objectives: At present, the molecular basis of the VWF-Ang-2 interaction is poorly understood. Here, we used immunosorbent-binding assays and specific recombinant VWF fragments to analyze VWF-Ang-2 interactions. Results: We found that VWF bound to immobilized Ang-2 most efficiently (half-maximal binding at 0.5 ± 0.1 µg/mL) under conditions of high CaCl2 (10 mM) and slightly acidic pH (6.4-7.0). Interestingly, several isolated recombinant VWF domains (A1/Fc, A2/Fc, D4/Fc, and D'D3-HPC4) displayed dose-dependent binding to immobilized Ang-2. Binding appeared specific, as antibodies against D'D3, A1, and A2 significantly reduced the binding of these domains to Ang-2. Complexes between VWF and Ang-2 in plasma could be detected by immunoprecipitation- and immunosorbent assays. Unexpectedly, control experiments also revealed complexes between VWF and angiopoietin-1 (Ang-1), a protein structurally homologous to Ang-2. Furthermore, direct binding studies showed dose-dependent binding of VWF to immobilized Ang-1 (half-maximal binding at 1.8 ± 1.0 µg/mL). Interestingly, rather than competing for Ang-1 binding, Ang-2 enhanced the binding of VWF to Ang-1 about 3-fold. Competition experiments further revealed that binding to VWF does not prevent Ang-1 and Ang-2 from binding to Tie-2. Conclusion: Our data show that both Ang-1 and Ang-2 bind to VWF, seemingly using different interactive sites. Ang-2 modulates the binding of VWF to Ang-1, the (patho)-physiological consequences of which remain to be investigated.

Imlifidase, a new option to optimize the management of patients with hemophilia A on emicizumab.

BACKGROUND: Emicizumab is a bispecific, chimeric, humanized immunoglobulin G (IgG)4 that mimics the procoagulant activity of factor (F) VIII (FVIII). Its long half-life and subcutaneous route of administration have been life-changing in treating patients with hemophilia A (HA) with or without FVIII inhibitors. However, emicizumab only partially mimics FVIII activity; it prevents but does not treat acute bleeds. Emergency management is particularly complicated in patients with FVIII inhibitors receiving emicizumab prophylaxis in whom exogenous FVIII is inefficient. We have shown recently that Imlifidase (IdeS), a bacterial IgG-degrading enzyme, efficiently eliminates human anti-FVIII IgG in a mouse model of severe HA with inhibitors and opens a therapeutic window for the administration of exogenous FVIII. OBJECTIVES: To investigate the impact of IdeS treatment in inhibitor-positive HA mice injected with emicizumab. METHODS: IdeS was injected to HA mice reconstituted with human neutralizing anti-FVIII IgG and treated with emicizumab. RESULTS: IdeS hydrolyzed emicizumab in vitro and in vivo, albeit, at slower rates than another recombinant human monoclonal IgG4. While F(ab')2 fragments were rapidly cleared from the circulation, thus leading to a rapid loss of emicizumab procoagulant activity, low amounts of single-cleaved intermediate IgG persisted for several days. Moreover, the IdeS-mediated elimination of the neutralizing anti-FVIII IgG and restoration of the hemostatic efficacy of exogenous FVIII were not impaired by the presence of emicizumab and polyclonal human IgG in inhibitor-positive HA mice. CONCLUSION: Our results suggest that IdeS could be administered to inhibitor-positive patients with HA receiving emicizumab prophylaxis to improve and ease the management of breakthrough bleeds or programmed major surgeries.

Transplacental delivery of therapeutic proteins by engineered immunoglobulin G: a step toward perinatal replacement therapy.

BACKGROUND: Transplacental delivery of maternal immunoglobulin G (IgG) provides humoral protection during the first months of life until the newborn's immune system reaches maturity. The maternofetal interface has been exploited therapeutically to replace missing enzymes in the fetus, as shown in experimental mucopolysaccharidoses, or to shape adaptive immune repertoires during fetal development and induce tolerance to self-antigens or immunogenic therapeutic molecules. OBJECTIVES: To investigate whether proteins that are administered to pregnant mice or endogenously present in their circulation may be delivered through the placenta. METHODS: We engineered monovalent immunoglobulin G (FabFc) specific for different domains of human factor VIII (FVIII), a therapeutically relevant model antigen. FabFc was injected with exogenous FVIII into pregnant severe hemophilia A mice or pregnant mice expressing human FVIII following AAV8-mediated gene therapy. FabFc and FVIII were detected in the pregnant mice and/or fetuses by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: Administration of FabFc to pregnant mice allowed the maternofetal delivery of FVIII in a FcRn-dependent manner. FVIII antigen levels achieved in the fetuses represented 10% of normal plasma levels in the human. We identified antigen/FabFc complex stability, antigen size, and shielding of promiscuous protein patches as key parameters to foster optimal antigen delivery. CONCLUSION: Our results pave the way toward the development of novel strategies for the in utero delivery of endogenous maternal proteins to replace genetically deficient fetal proteins or to educate the immune system and favor active immune tolerance upon protein encounter later in life.

MAGT1 deficiency in XMEN disease is associated with severe platelet dysfunction and impaired platelet glycoprotein N-glycosylation.

BACKGROUND: X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection, and neoplasia (XMEN) disease is a primary immunodeficiency due to loss-of-function mutations in the gene encoding for magnesium transporter 1 (MAGT1). Furthermore, as MAGT1 is involved in the N-glycosylation process, XMEN disease is classified as a congenital disorder of glycosylation. Although XMEN-associated immunodeficiency is well described, the mechanisms underlying platelet dysfunction and those responsible for life-threatening bleeding events have never been investigated. OBJECTIVES: To assess platelet functions in patients with XMEN disease. METHODS: Two unrelated young boys, including one before and after hematopoietic stem cell transplantation, were investigated for their platelet functions, glycoprotein expression, and serum and platelet-derived N-glycans. RESULTS: Platelet analysis highlighted abnormal elongated cells and unusual barbell-shaped proplatelets. Platelet aggregation, integrin αIIbß3 activation, calcium mobilization, and protein kinase C activity were impaired between both patients. Strikingly, platelet responses to protease-activated receptor 1 activating peptide were absent at both low and high concentrations. These defects were also associated with decreased molecular weights of glycoprotein Ibα, glycoprotein VI, and integrin αIIb due to partial impairment of N-glycosylation. All these defects were corrected after hematopoietic stem cell transplantation. CONCLUSION: Our results highlight prominent platelet dysfunction related to MAGT1 deficiency and defective N-glycosylation in several platelet proteins that could explain the hemorrhages reported in patients with XMEN disease.

Efficacy of platelet-inspired hemostatic nanoparticles on bleeding in von Willebrand disease murine models.

The lack of innovation in von Willebrand disease (VWD) originates from many factors including the complexity and heterogeneity of the disease but also from a lack of recognition of the impact of the bleeding symptoms experienced by patients with VWD. Recently, a few research initiatives aiming to move past replacement therapies using plasma-derived or recombinant von Willebrand factor (VWF) concentrates have started to emerge. Here, we report an original approach using synthetic platelet (SP) nanoparticles for the treatment of VWD type 2B (VWD-2B) and severe VWD (type 3 VWD). SP are liposomal nanoparticles decorated with peptides enabling them to concomitantly bind to collagen, VWF, and activated platelets. In vitro, using various microfluidic assays, we show the efficacy of SPs to improve thrombus formation in VWF-deficient condition (with human platelets) or using blood from mice with VWD-2B and deficient VWF (VWF-KO, ie, type 3 VWD). In vivo, using a tail-clip assay, SP treatment reduced blood loss by 35% in mice with VWD-2B and 68% in mice with VWF-KO. Additional studies using nanoparticles decorated with various combinations of peptides demonstrated that the collagen-binding peptide, although not sufficient by itself, was crucial for SP efficacy in VWD-2B; whereas all 3 peptides appeared necessary for mice with VWF-KO. Clot imaging by immunofluorescence and scanning electron microscopy revealed that SP treatment of mice with VWF-KO led to a strong clot, similar to those obtained in wild-type mice. Altogether, our results show that SP could represent an attractive therapeutic alternative for VWD, especially considering their long half-life and stability.

ADP receptor P2Y12 is the capstone of the cross-talk between Ca2+ mobilization pathways dependent on Ca2+ ATPases sarcoplasmic/endoplasmic reticulum type 3 and type 2b in platelets.

Background: Blood platelet Ca2+ stores are regulated by 2 Ca2+-ATPases (SERCA2b and SERCA3). On thrombin stimulation, nicotinic acid adenosine dinucleotide phosphate mobilizes SERCA3-dependent stores, inducing early adenosine 5'-diphosphate (ADP) secretion, potentiating later SERCA2b-dependent secretion. Objectives: The aim of this study was to identify which ADP P2 purinergic receptor (P2Y1 and/or P2Y12) is(are) involved in the amplification of platelet secretion dependent on the SERCA3-dependent Ca2+ mobilization pathway (SERCA3 stores mobilization) as triggered by low concentration of thrombin. Methods: The study used the pharmacologic antagonists MRS2719 and AR-C69931MX, of the P2Y1 and P2Y12, respectively, as well as Serca3 -/- mice and mice exhibiting platelet lineage-specific inactivation of the P2Y1 or P2Y12 genes. Results: We found that in mouse platelets, pharmacological blockade or gene inactivation of P2Y12 but not of P2Y1 led to a marked inhibition of ADP secretion after platelet stimulation with low concentration of thrombin. Likewise, in human platelets, pharmacological inhibition of P2Y12 but not of P2Y1 alters amplification of thrombin-elicited secretion through SERCA2b stores mobilization. Finally, we show that early SERCA3 stores secretion of ADP is a dense granule secretion, based on parallel adenosine triphosphate and serotonin early secretion. Furthermore, early secretion involves a single granule, based on the amount of adenosine triphosphate released. Conclusion: Altogether, these results show that at low concentrations of thrombin, SERCA3- and SERCA2b-dependent Ca2+ mobilization pathways cross-talk via ADP and activation of the P2Y12, and not the P2Y1 ADP receptor. The relevance in hemostasis of the coupling of the SERCA3 and the SERCA2b pathways is reviewed.

Development of a dual hybrid AAV vector for endothelial-targeted expression of von Willebrand factor.

Von Willebrand disease (VWD), the most common inherited bleeding disorder in humans, is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWD represents a potential target for gene therapy applications, as a single treatment could potentially result in a long-term correction of the disease. In recent years, several liver-directed gene therapy approaches have been exploited for VWD, but their efficacy was generally limited by the large size of the VWF transgene and the reduced hemostatic activity of the protein produced from hepatocytes. In this context, we aimed at developing a gene therapy strategy for gene delivery into endothelial cells, the natural site of biosynthesis of VWF. We optimized an endothelial-specific dual hybrid AAV vector, in which the large VWF cDNA was put under the control of an endothelial promoter and correctly reconstituted upon cell transduction by a combination of trans-splicing and homologous recombination mechanisms. In addition, we modified the AAV vector capsid by introducing an endothelial-targeting peptide to improve the efficiency for endothelial-directed gene transfer. This vector platform allowed the reconstitution of full-length VWF transgene both in vitro in human umbilical vein endothelial cells and in vivo in VWD mice, resulting in long-term expression of VWF.

A nanobody against the VWF A3 domain detects ADAMTS13-induced proteolysis in congenital and acquired VWD.

von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2 domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3 domain, the epitope of which overlaps the collagen-binding site. Although KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2 domain modulates exposure of its epitope in the A3 domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact VWF could be detected using this nanobody. By comparing plasma from volunteers to that from congenital von Willebrand disease (VWD) patients, intact-VWF levels were significantly reduced for all VWD types, and most severely in VWD type 2A-group 2, in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD type 1 and VWD type 2M. A significant correlation (r = 0.51, P < .0001) between the relative amount of high-molecular weight multimers and levels of intact VWF was observed. Reduced levels of intact VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3 domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.

A gain-of-function filamin A mutation in mouse platelets induces thrombus instability.

BACKGROUND: Filaminopathies A are rare disorders affecting the brain, intestine, or skeleton, characterized by dominant X-linked filamin A (FLNA) gene mutations. Macrothrombocytopenia with functionally defective platelets is frequent. We have described a filaminopathy A male patient, exhibiting a C-terminal frame-shift FLNa mutation (Berrou et al., Arterioscler Thromb Vasc Biol. 2017;37:1087-1097). Contrasting with female patients, this male patient exhibited gain of platelet functions, including increased platelet aggregation, integrin αIIbß3 activation, and secretion at low agonist concentration, raising the issue of thrombosis risk. OBJECTIVES: Our goal is to assess the thrombotic potential of the patient FLNa mutation in an in vivo model. METHODS: We have established a mutant FlnA knock-in mouse model. RESULTS: The mutant FlnA mouse platelets phenocopied patient platelets, showing normal platelet count, lower expression level of mutant FlnA, and gain of platelet functions: increased platelet aggregation, secretion, and αIIbß3 activation, as well as increased spreading and clot retraction. Surprisingly, mutant FlnA mice exhibited a normal bleeding time, but with increased re-bleeding (77%) compared to wild type (WT) FlnA mice (27%), reflecting hemostatic plug instability. Again, in an in vivo thrombosis model, the occlusion time was not altered by the FlnA mutation, but arteriolar embolies were increased (7-fold more frequent in mutant FlnA mice versus WT mice), confirming thrombus instability. CONCLUSIONS: This study shows that the FlnA mutation found in the male patient induced gain of platelet functions in vitro, but thrombus instability in vivo. Implications for the role of FLNa in physiology of thrombus formation are discussed.

Identification of von Willebrand factor D4 domain mutations in patients of Afro-Caribbean descent: In vitro characterization.

Background: Von Willebrand disease was diagnosed in two Afro-Caribbean patients and sequencing of the VWF gene (VWF) revealed the presence of multiple variants located throughout the gene, including variants located in the D4 domain of VWF: p.(Pro2145Thrfs*5) in one patient and p.(Cys2216Phefs*9) in the other patient. Interestingly, D4 variants have not been studied often. Objectives: Our goal was to characterize how the D4 variants p.(Pro2145Thrfs*5) and p.(Cys2216Phefs*9) influenced VWF biosynthesis/secretion and functions using in vitro assays. Methods: Recombinant VWF (rVWF), mutant or wild-type, was produced via transient transfection of the human embryonic kidney cell line 293T. The use of different tags for the wild-type and the mutant allele allowed us to distinguish between the two forms when measuring VWF antigen in medium and cell lysates. Binding of rVWF to its ligands, collagen, factor VIII, ADAMTS13, and platelet receptors was also investigated. Results: Homozygous expression of the p.(Cys2216Phefs*9)-rVWF mutation resulted in an almost complete intracellular retention of the protein. Heterozygous expression led to secretion of almost exclusively wild-type-rVWF, logically capable of normal interaction with the different ligands. In contrast, the p.(Pro2145Thrfs*5)-rVWF exhibited reduced binding to type III collagen and αIIbß3 integrin compared to wild-type-rVWF. Conclusions: We report two mutations of the D4 domains that induced combined qualitative and quantitative defects.

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 N-glycosylation deficiency on this system as a whole. To this end, we developed a PMM2 knockdown model in the brain endothelial cell line hCMEC/D3. The resulting PMM2low cells were less able to generate activated protein C (APC), due to lower surface expression of thrombomodulin and endothelial protein C receptor. The low protein levels were due to downregulated transcription of the corresponding genes (THBD and PROCR, respectively), which itself was related to downregulation of transcription regulators Krüppel-like factors 2 and 4 and forkhead box C2. PMM2 knockdown was also associated with impaired integrity of the endothelial cell monolayer-partly due to an alteration in the structure of VE-cadherin in adherens junctions. The expression of protease-activated receptor 1 (involved in the cytoprotective effects of APC on the endothelium) was not affected by PMM2 knockdown. Thrombin stimulation induced hyperpermeability in PMM2low cells. However, pretreatment of cells with APC before thrombin simulation was still associated with a barrier-protecting effect. Taken as a whole, our results show that the partial loss of PMM2 in hCMEC/D3 cells is associated with impaired activation of protein C and a relative increase in barrier permeability.

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. PS binders were tested in a global activated partial thromboplastin time (APTT)-based APC-cofactor activity assay. RESULTS: A PS-specific sdAb (PS003) was found to enhance the APC-cofactor activity of PS in our APTT-based assay, and this enhancing effect was greater for a bivalent form of PS003 (PS003biv). Further characterization of PS003biv demonstrated that PS003biv also enhanced the APC-cofactor activity of PS in a tissue factor (TF)-induced thrombin generation assay and stimulated APC in the inactivation of FVa, but not FVIIIa, in plasma-based assays. Furthermore, PS003biv was directed against the sex hormone-binding globulin (SHBG)-like domain but did not inhibit the binding of PS to C4b-binding protein (C4BP) and did not interfere with the TFPIα-cofactor activity of PS. In mice, PS003biv exerted an antithrombotic effect in a FeCl3 -induced thrombosis model, while not affecting physiological hemostasis in a tail-clip bleeding model. DISCUSSION: Altogether, these results showed that pharmacological enhancement of the APC-cofactor activity of PS through an original anti-PS sdAb might constitute a promising and safe antithrombotic strategy.

New insights into regulation of αIIbß3 integrin signaling by filamin A.

Background: Filamin (FLN) regulates many cell functions through its scaffolding activity cross-linking cytoskeleton and integrins. FLN was shown to inhibit integrin activity, but the exact mechanism remains unclear. Objectives: The aim of this study was to evaluate the role of filamin A (FLNa) subdomains on the regulation of integrin αIIbß3 signaling. Methods: Three FLNa deletion mutants were overexpressed in the erythro-megakaryocytic leukemic cell line HEL: Del1, which lacks the N-terminal CH1-CH2 domains mediating the FLNa-actin interaction; Del2, lacking the Ig-like repeat 21, which mediates the FLNa-ß3 interaction; and Del3, lacking the C-terminal Ig repeat 24, responsible for FLNa dimerization and interaction with the small Rho guanosine triphosphatase involved in actin cytoskeleton reorganisation. Fibrinogen binding to HEL cells in suspension and talin-ß3 proximity in cells adherent to immobilized fibrinogen were assessed before and after αIIbß3 activation by the protein kinase C agonist phorbol 12-myristate 13-acetate. Results: Our results show that FLNa-actin and FLNa-ß3 interactions negatively regulate αIIbß3 activation. Moreover, FLNa-actin interaction represses Rac activation, contributing to the negative regulation of αIIbß3 activation. In contrast, the FLNa dimerization domain, which maintains Rho inactive, was found to negatively regulate αIIbß3 outside-in signaling. Conclusion: We conclude that FLNa negatively controls αIIbß3 activation by regulating actin polymerization and restraining activation of Rac, as well as outside-in signaling by repressing Rho.