Publications by authors named "Peter J Lenting"

120 Publications

Von Willebrand disease: What does the future hold?

Blood 2021 03 4. Epub 2021 Mar 4.

INSERM, Le Kremlin-Bicetre, France.

Von Willebrand disease (VWD) is characterized by its heterogeneous clinical manifestation, which complicates its diagnosis and management. The clinical management of VWD has remained essentially unchanged over the last 30 years or so, using von Willebrand factor (VWF) concentrates, desmopressin and anti-fibrinolytic agents as main tools to control bleeding. This is in contrast to hemophilia A, for which a continuous innovative path has led to novel treatment modalities. Despite current VWD management being considered effective, quality-of-life studies consistently reveal a higher than anticipated burden of VWD on patients, which is particularly true for women. Apparently, despite our perceived notion of current therapeutic efficiency, there is space for innovation with the goal to reach superior efficacy. Developing innovative treatments for VWD is complex, especially given the heterogeneity of the disease and the multifunctional nature of VWF. In this perspective article, we describe several potential strategies that could provide the basis for future VWD treatments. These include genetic approaches such as gene therapy using dual-vector adeno-associated virus and transcriptional silencing of mutant alleles. Furthermore, protein-based approaches to increase FVIII levels in VWD-type 3 or 2N patients will be discussed. Finally, antibody-based options to interfere with VWF degradation (for congenital VWD-type 2A or acquired Von Willebrand syndrome-type 2A) or increase endogenous VWF levels (for VWD-type 1) will be presented. By highlighting these potential strategies, we hope to initiate an innovative path, which ultimately would allow us to better serve VWD patients and their specific needs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2020008501DOI Listing
March 2021

The VWF/LRP4/αVβ3-axis represents a novel pathway regulating proliferation of human vascular smooth muscle cells.

Cardiovasc Res 2021 Feb 12. Epub 2021 Feb 12.

INSERM, UMR_S 1116, Vandœuvre-lès-Nancy, France; Université de Lorraine, DCAC, Nancy, France.

Aims: Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary hemostasis, while also having additional roles beyond hemostasis namely in cancer, inflammation, angiogenesis and potentially in vascular smooth muscle cell (VSMC) proliferation. Here, we addressed how VWF modulates VSMC proliferation and investigated the underlying molecular pathways and the in vivo pathophysiological relevance.

Methods And Results: VWF induced proliferation of human aortic VSMCs and also promoted VSMC migration. Treatment of cells with a siRNA against αv integrin or the RGT-peptide blocking αvβ3 signaling abolished proliferation. However, VWF did not bind to αvβ3 on VSMCs through its RGD-motif. Rather, we identified the VWF A2 domain as the region mediating binding to the cells. We hypothesized the involvement of a member of the LDL-related receptor protein (LRP) family due to their known ability to act as co-receptors. Using the universal LRP-inhibitor receptor-associated protein, we confirmed LRP-mediated VSMC proliferation. siRNA experiments and confocal fluorescence microscopy identified LRP4 as the VWF-counterreceptor on VSMCs. Also co-localization between αvβ3 and LRP4 was observed via proximity ligation analysis and immuno-precipitation experiments. The pathophysiological relevance of our data was supported by VWF-deficient mice having significant reduced, if any, hyperplasia in carotid artery ligation and artery femoral denudation models. In wild-type mice, infiltration of VWF in intimal regions enriched in proliferating VSMCs was found. Interestingly, also analysis of human atherosclerotic lesions showed abundant VWF accumulation in VSMC-proliferating rich intimal areas.

Conclusions: VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvβ3 signaling. Our findings provide new insights into the mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall. In addition, the VWF/LRP4-axis may represent a novel therapeutic target to modulate VSMC proliferation.

Translational Perspective: The mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall are complex and only partially understood. Specifically, the role of subendothelial-matrix proteins remains unclear. Here, we show that the hemostatic protein von Willebrand factor (VWF) accumulates in the vascular wall of atherosclerotic lesions and localizes to areas of vascular smooth muscle cell (VSMC) proliferation. VWF was found to use its A2-domain for binding to the VSMC-receptor LRP4, which in turn triggered outside-in signaling via integrin αVβ3, thereby inducing VSMC proliferation. Interfering with A2-domain/LRP4 interactions might offer innovative and additional therapeutical approaches to limit pathological hyperplasia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cvr/cvab042DOI Listing
February 2021

Impaired adhesion of neutrophils expressing Slc44a2/HNA-3b to VWF protects against NETosis under venous shear rate.

Blood 2021 02 8. Epub 2021 Feb 8.

Aix-Marseille Univ, INSERM, INRAE, C2VN, Marseille, France.

Genome wide association studies linked expression of the human neutrophil antigen 3b (HNA-3b) epitope on the Slc44a2 protein with a 30% decreased risk of venous thrombosis (VT) in humans. Slc44a2 is a ubiquitous transmembrane protein identified as a receptor for Von Willebrand factor (VWF). To explain the link between Slc44a2 and VT we wanted to determine how Slc44a2 expressing either HNA-3a or HNA-3b on neutrophils could modulate their adhesion and activation on VWF under flow. Transfected HEK293T cells or neutrophils homozygous for the HNA-3a- or the HNA-3b-coding allele were purified from healthy donors and perfused in flow chambers coated with VWF at venous shear rates (100s-1). HNA-3a expression was required for Slc44a2-mediated neutrophil adhesion to VWF at 100s-1. This adhesion could occur independently of β2 integrin and was enhanced when neutrophils are preactivated with lipopolysaccharide (LPS). Moreover, specific shear conditions with high neutrophil concentration could act as a "second hit", inducing the formation of neutrophil extracellular traps. Neutrophil mobilization was also measured by intravital microscopy in venules from SLC44A2-knockout and wild-type mice after histamine-induced endothelial degranulation. Mice lacking Slc44a2 showed a massive reduction in neutrophil recruitment in inflamed mesenteric venules. Our results show that Slc44a2/HNA-3a is important for the adhesion and activation of neutrophils in veins under inflammation and when submitted to specific shears. Neutrophils expressing Slc44a2/HNA-3b not being associated with these observations, these results could thus explain the association between HNA-3b and a reduced risk for VT in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2020008345DOI Listing
February 2021

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

Gene Ther 2021 Jan 17. Epub 2021 Jan 17.

Généthon, 91000, Evry, France.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41434-020-00218-6DOI Listing
January 2021

Camelid-derived single-chain antibodies in hemostasis: Mechanistic, diagnostic, and therapeutic applications.

Res Pract Thromb Haemost 2020 Oct 9;4(7):1087-1110. Epub 2020 Sep 9.

HITh UMR_S1176 INSERM Université Paris-Saclay Le Kremlin-Bicêtre France.

Hemostasis is a complex process involving the concerted action of molecular and vascular components. Its basic understanding as well as diagnostic and therapeutic aspects have greatly benefited from the use of monoclonal antibodies. Interestingly, camelid-derived single-domain antibodies (sdAbs), also known as VH or nanobodies, have become available during the previous 2 decades as alternative tools in this regard. Compared to classic antibodies, sdAbs are easier to produce and their small size facilitates their engineering and functionalization. It is not surprising, therefore, that sdAbs are increasingly used in hemostasis-related research. In addition, they have the capacity to recognize unique epitopes unavailable to full monoclonal antibodies. This property can be used to develop novel diagnostic tests identifying conformational variants of hemostatic proteins. Examples include sdAbs that bind active but not globular von Willebrand factor or free factor VIIa but not tissue factor-bound factor VIIa. Finally, sdAbs have a high therapeutic potential, exemplified by caplacizumab, a homodimeric sdAb targeting von Willebrand factor that is approved for the treatment of thrombotic thrombocytopenic purpura. In this review, the various applications of sdAbs in thrombosis and hemostasis-related research, diagnostics, and therapeutic strategies will be discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/rth2.12420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590285PMC
October 2020

In vivo modulation of a dominant-negative variant in mouse models of von Willebrand disease type 2A.

J Thromb Haemost 2021 01 3;19(1):139-146. Epub 2020 Nov 3.

Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche Scientifique, Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France.

Essentials Treatment options for von Willebrand disease (VWD) patients are limited. The p.P1127_C1948delinsR deletion/variant is a useful model to study VWD in vitro and in vivo. Counteracting dominant-negative effects restores von Willebrand factor multimerization in mice. This is the first siRNA-based treatment applied to a mouse model of VWD-type 2A. ABSTRACT: Background Treatment options for patients suffering from von Willebrand disease (VWD) are limited. Von Willebrand factor (VWF) is a polymeric protein that undergoes regulated dimerization and subsequent multimerization during its biosynthesis. Numerous heterozygous variants within the VWF gene display a dominant-negative effect and result in severe VWD. Previous studies have suggested that preventing the assembly of wild-type and mutant heteropolymers using siRNAs may have beneficial effects on VWF phenotypes in vitro. Objectives To study heterozygous dominant-negative variants in vivo, we developed a mouse model of VWD-type 2A and tested two independent strategies to modulate its detrimental effect. Methods The p.P1127_C1948delinsR deletion/variant, causing defective VWF multimerization, was expressed in mice as a model of VWD-type 2A variant. Two corrective strategies were applied. For the first time in a mouse model of VWD, we applied siRNAs selectively inhibiting translation of the mutant transcripts and we combined the VWD-type 2A deletion with the Cys to Arg substitution at position 2773, which is known to prevent dimerization. Results The RNA silencing approach induced a modest but consistent improvement of the VWF multimer profile. However, due to incomplete efficiency, the dominant-negative effect of the original variant could not be completely prevented. In contrast, the DNA approach resulted in increased antigen levels and restoration of a normal multimer profile. Conclusions Our data showed that preventing the detrimental impact of dominant-negative VWF variants by independent molecular mechanisms has beneficial consequences in vivo, in mouse models of dominant VWD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jth.15131DOI Listing
January 2021

Coagulation markers are independent predictors of increased oxygen requirements and thrombosis in COVID-19: Response from Original Authors Susen, et al.

J Thromb Haemost 2020 12;18(12):3385-3386

Inserm, UMR_1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jth.15101DOI Listing
December 2020

Coagulation biomarkers are independent predictors of increased oxygen requirements in COVID-19.

J Thromb Haemost 2020 11 18;18(11):2942-2953. Epub 2020 Sep 18.

Department of Hematology and Transfusion, Pôle de Biologie Pathologie Génétique, Institut Pasteur de Lille, UMR1011-EGID, Univ. Lille, Inserm, CHU Lille, Lille, France.

Background: Hypercoagulability seems to contribute to SARS-CoV-2 pneumonia pathogenesis. However, age and metabolic syndrome are potential confounders when assessing the value of coagulation biomarkers' prediction of COVID-19 outcomes. We assessed whether coagulation biomarkers, including factor VIII (FVIII) and von Willebrand factor (VWF) levels, measured at time of admission, were predictive of COVID-19 adverse outcomes irrespective of age and major comorbidities associated with metabolic syndrome.

Methods: Blood was sampled at admission in 243 adult COVID-19 patients for analysis of coagulation biomarkers including FVIII and VWF on platelet-poor plasma. The association between baseline C-reactive protein (CRP), activated partial thromboplastin time ratio, prothrombin time ratio, D-dimers, fibrinogen, FVIII, VWF antigen (VWF:Ag), and FVIII/VWF:Ag ratio levels and adverse outcomes (increased oxygen requirements, thrombosis, and death at day 30) was assessed by regression analysis after adjustment on age, sex, body mass index (BMI), diabetes, and hypertension.

Results: In univariable regression analysis increased CRP (subdistribution hazard ratio [SHR], 1.68; 95% confidence interval [CI], 1.26-2.23), increased fibrinogen (SHR, 1.32; 95% CI, 1.04-1.68), and decreased FVIII/VWF:Ag ratio (SHR, 0.70; 95% CI, 0.52-0.96) levels at admission were significantly associated with the risk of increased oxygen requirement during follow-up. Leucocytes (SHR, 1.36; 95% CI, 1.04-1.76), platelets (SHR,1.71; 95% CI, 1.11-2.62), D-dimers (SHR, 2.48; 95% CI, 1.66-3.78), and FVIII (SHR, 1.78; 95% CI, 1.17-2.68) were associated with early onset of thrombosis after admission. After adjustment for age, sex, BMI, hypertension, and diabetes, these associations were not modified.

Conclusion: Coagulation biomarkers are early and independent predictors of increased oxygen requirement in COVID-19 patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jth.15067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461161PMC
November 2020

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins.

Nat Commun 2020 07 29;11(1):3778. Epub 2020 Jul 29.

Genethon, 91000, Evry, France.

Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients' cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-17552-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391635PMC
July 2020

Development and characterization of single-domain antibodies neutralizing protease nexin-1 as tools to increase thrombin generation.

J Thromb Haemost 2020 09;18(9):2155-2168

Institut National de la Santé et de la Recherche Médicale, UMR_S 1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France.

Background: Protease nexin-1 (PN-1) is a member of the serine protease inhibitor (Serpin)-family, with thrombin as its main target. Current polyclonal and monoclonal antibodies against PN-1 frequently cross-react with plasminogen activator inhibitor-1 (PAI-1), a structurally and functionally homologous Serpin.

Objectives: Here, we aimed to develop inhibitory single-domain antibodies (VHHs) that show specific binding to both human (hPN-1) and murine (mPN-1) PN-1.

Methods: PN-1-binding VHHs were isolated via phage-display using llama-derived or synthetic VHH-libraries. Following bacterial expression, purified VHHs were analyzed in binding and activity assays.

Results And Conclusions: By using a llama-derived library, 2 PN-1 specific VHHs were obtained (KB-PN1-01 and KB-PN1-02). Despite their specificity, none displayed inhibitory activity toward hPN-1 or mPN-1. From the synthetic library, 4 VHHs (H12, B11, F06, A08) could be isolated that combined efficient binding to both hPN-1 and mPN-1 with negligible binding to PAI-1. Of these, B11, F06, and A08 were able to fully restore thrombin activity by blocking PN-1. As monovalent VHH, half-maximal inhibitory concentration values for hPN-1 were 50 ± 10, 290 ± 30, and 960 ± 390 nmol/L, for B11, F06, and A08, respectively, and 1580 ± 240, 560 ± 130, and 2880 ± 770 nmol/L for mPN-1. The inhibitory potential was improved 4- to 7-fold when bivalent VHHs were engineered. Importantly, all VHHs could block PN-1 activity in plasma as well as PN-1 released from activated platelets, one of the main sources of PN-1 during hemostasis. In conclusion, we report the generation of inhibitory anti-PN-1 antibodies using a specific approach to avoid cross-reactivity with the homologous Serpin PAI-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jth.14940DOI Listing
September 2020

Laboratory monitoring of hemophilia A treatments: new challenges.

Authors:
Peter J Lenting

Blood Adv 2020 05;4(9):2111-2118

Hémostase Inflammation Thrombose, INSERM, Unité Mixte de Recherche Scientifique 1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France.

Monitoring factor VIII (FVIII) activity has traditionally been complicated by discrepancies between assays for the various sorts of FVIII molecules. The advent of novel nonfactor therapies (emicizumab, fitusiran, and anti-tissue factor pathway inhibitor antibodies) in hemophilia A poses a new level of difficulty on the laboratory monitoring of these patients. To use the correct assays and for a proper interpretation of their results, it is pertinent to understand the mode of action of these nonfactor agents. Furthermore, the biochemical consequences for the different types of activity assays (whether it be specific FVIII activity assays or global coagulation assays) should be taken into account as well. In this review, these aspects will be discussed. In addition, the use of various animal models to estimate FVIII-equivalence of the nonfactor therapies will be presented.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2019000849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218434PMC
May 2020

A hemophilia A mouse model for the in vivo assessment of emicizumab function.

Blood 2020 08;136(6):740-748

Unité Mixte de Recherche Scientifique (UMR_S) 1176, INSERM/Université Paris-Sud/Université Paris-Saclay, Le Kremlin-Bicêtre, France.

The bispecific antibody emicizumab is increasingly used for hemophilia A treatment. However, its specificity for human factors IX and X (FIX and FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here, we describe a novel mouse model that allows emicizumab function to be examined. Briefly, FVIII-deficient mice received IV emicizumab 24 hours before tail-clip bleeding was performed. A second infusion with human FIX and FX, administered 5 minutes before bleeding, generated consistent levels of emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg doses) and of both FIX and FX (85 and 101 U/dL, respectively, after dosing at 100 U/kg). Plasma from these mice display FVIII-like activity in assays (diluted activated partial thromboplastin time and thrombin generation), similar to human samples containing emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail-clip-bleeding model using FVIII-deficient mice. However, reduction was incomplete compared with mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted FVIII-like activity from emicizumab that corresponded to a dose of 4.5 U of FVIII per kilogram (ie, 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), emicizumab provided enough additive activity to allow complete bleeding arrest. This model could be useful for further in vivo analysis of emicizumab.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2019004334DOI Listing
August 2020

Single-domain antibodies targeting antithrombin reduce bleeding in hemophilic mice with or without inhibitors.

EMBO Mol Med 2020 04 11;12(4):e11298. Epub 2020 Mar 11.

HITh, UMR_S1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre, France.

Novel therapies for hemophilia, including non-factor replacement and in vivo gene therapy, are showing promising results in the clinic, including for patients having a history of inhibitor development. Here, we propose a novel therapeutic approach for hemophilia based on llama-derived single-domain antibody fragments (sdAbs) able to restore hemostasis by inhibiting the antithrombin (AT) anticoagulant pathway. We demonstrated that sdAbs engineered in multivalent conformations were able to block efficiently AT activity in vitro, restoring the thrombin generation potential in FVIII-deficient plasma. When delivered as a protein to hemophilia A mice, a selected bi-paratopic sdAb significantly reduced the blood loss in a model of acute bleeding injury. We then packaged this sdAb in a hepatotropic AAV8 vector and tested its safety and efficacy profile in hemophilic mouse models. We show that the long-term expression of the bi-paratopic sdAb in the liver is safe and poorly immunogenic, and results in sustained correction of the bleeding phenotype in hemophilia A and B mice, even in the presence of inhibitory antibodies to the therapeutic clotting factor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.201911298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136963PMC
April 2020

The von Willebrand Factor A1 domain mediates thromboinflammation, aggravating ischemic stroke outcome in mice.

Haematologica 2021 Mar 1;106(3):819-828. Epub 2021 Mar 1.

Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

von Willebrand factor (VWF) plays an important role in ischemic stroke. However, the exact mechanism by which VWF mediates progression of ischemic stroke brain damage is not completely understood. Using flow cytometric analysis of single cell suspensions prepared from brain tissue and immunohistochemistry, we investigated the potential inflammatory mechanisms by which VWF contributes to ischemic stroke brain damage in a mouse model of cerebral ischemia/reperfusion injury. Twenty-four hours after stroke, flow cytometric analysis of brain tissue revealed that overall white blood cell recruitment in the ipsilesional brain hemisphere of VWF KO mice was 2 times lower than WT mice. More detailed analysis showed a specific reduction of proinflammatory monocytes, neutrophils and T-cells in the ischemic brain of VWF KO mice compared to WT mice. Interestingly, histological analysis revealed a substantial number of neutrophils and T-cells still within the microcirculation of the stroke brain, potentially contributing to the no-reflow phenomenon. Specific therapeutic targeting of the VWF A1 domain in WT mice resulted in reduced immune cell numbers in the affected brain and protected mice from ischemic stroke brain damage. More specifically, recruitment of proinflammatory monocytes was reduced two-fold, neutrophil recruitment was reduced five-fold and T-cell recruitment was reduced two-fold in mice treated with a VWF A1-targeting nanobody compared to mice receiving a control nanobody. In conclusion, our data identify a potential role for VWF in the recruitment of proinflammatory monocytes, neutrophils and T-cells to the ischemic brain via a mechanism that is mediated by its A1 domain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2019.241042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927893PMC
March 2021

A Thrombin-Activatable Factor X Variant Corrects Hemostasis in a Mouse Model for Hemophilia A.

Thromb Haemost 2019 Dec 22;119(12):1981-1993. Epub 2019 Oct 22.

Institut National de la Santé et de la Recherche Médicale, UMR_S 1176, Univ. Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France.

Engineered recombinant factor X (FX) variants represent a promising strategy to bypass the tenase complex and restore hemostasis in hemophilia patients. Previously, a thrombin-activatable FX variant with fibrinopeptide-A replacing the activation peptide (FX-delAP/FpA) has been described in this regard. Here we show that FX-delAP/FpA is characterized by a sixfold shorter circulatory half-life compared with wild-type FX, limiting its therapeutical applicability. We therefore designed a variant in which the FpA sequence is inserted C-terminal to the FX activation peptide (FX/FpA). FX/FpA displayed a similar survival to wt-FX in clearance experiments and could be converted into FX by thrombin and other activating agents. In in vitro assays, FX/FpA efficiently restored thrombin generation in hemophilia A and hemophilia B plasmas, even in the presence of inhibitory antibodies. Expression following hydrodynamic gene transfer of FX/FpA restored thrombus formation in FVIII-deficient mice in a laser-induced injury model as well as hemostasis in a tail-clip bleeding model. Hemostasis after tail transection in FVIII-deficient mice was also corrected at 5 and 90 minutes after injection of purified FX/FpA. Our data indicate that FX/FpA represents a potential tenase-bypassing agent for the treatment of hemophilia patients with or without inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/s-0039-1697662DOI Listing
December 2019

A single-domain antibody that blocks factor VIIa activity in the absence but not presence of tissue factor.

J Thromb Haemost 2019 12 3;17(12):2035-2046. Epub 2019 Sep 3.

Laboratory of Hemostasis-Inflammation-Thrombosis, Institut National de la Santé et de la Recherche Médicale, UMR_S 1176, Univ. Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France.

Background: Activated factor VII (FVIIa) is pertinent to the initiation of blood coagulation. Proteolytic and amidolytic activity of FVIIa are greatly enhanced by its cofactor, tissue factor (TF).

Objective: We aimed to generate a single-domain antibody (sdAb) that recognizes free FVIIa rather than TF-bound FVIIa.

Methods: A llama-derived phage library was used to screen for anti-FVIIa sdAbs.

Results: One sdAb, KB-FVIIa-004, bound to FVIIa, but not to its precursor FVII or to homologous proteins (prothrombin, factor X, or their activated derivatives). FVIIa amidolytic activity was inhibited by KB-FVIIa-004 (K  = 28-45 nM) in a competitive manner. KB-FVIIa-004 also inhibited FVIIa-mediated FX activation (K  = 26 nM). In contrast, KB-FVIIa-004 was inefficient in prolonging the clotting time of the prothrombin time-test, which was prolonged by a maximum of 10 s at high sdAb concentrations (10 μM). Furthermore, FVIIa/TF amidolytic activity or FVIIa/TF-mediated FX activation remained unaffected up to a 50-fold to 1000-fold molar excess of KB-FVIIa-004. These data suggest that KB-FVIIa-004 loses its inhibitory activity in the presence of TF. A KB-FVIIa-004/albumin fusion-protein (004-HSA) was generated for in vivo testing. By using 004-HSA, we observed that this sdAb blocked the therapeutic capacity of FVIIa to correct bleeding in FVIII-deficient mice.

Discussion: This observation is compatible with the view that FVIIa functions independently of TF under these conditions. In conclusion, we have generated a sdAb that specifically blocks TF-independent activity of FVIIa. This antibody can be used to gain insight into the roles of TF-bound and TF-free FVIIa.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jth.14615DOI Listing
December 2019

Targeting protease nexin-1, a natural anticoagulant serpin, to control bleeding and improve hemostasis in hemophilia.

Blood 2019 11;134(19):1632-1644

INSERM, UMR_S 1148-Laboratory for Vascular Translational Science, Université de Paris, Paris, France.

Hemophilia A and B, diseases caused by the lack of factor VIII (FVIII) and factor IX (FIX) respectively, lead to insufficient thrombin production, and therefore to bleeding. New therapeutic strategies for hemophilia treatment that do not rely on clotting factor replacement, but imply the neutralization of natural anticoagulant proteins, have recently emerged. We propose an innovative approach consisting of targeting a natural and potent thrombin inhibitor, expressed by platelets, called protease nexin-1 (PN-1). By using the calibrated automated thrombin generation assay, we showed that a PN-1-neutralizing antibody could significantly shorten the thrombin burst in response to tissue factor in platelet-rich plasma (PRP) from patients with mild or moderate hemophilia. In contrast, in PRP from patients with severe hemophilia, PN-1 neutralization did not improve thrombin generation. However, after collagen-induced platelet activation, PN-1 deficiency in F8-/-mice or PN-1 blocking in patients with severe disease led to a significantly improved thrombin production in PRP, underlining the regulatory role of PN-1 released from platelet granules. In various bleeding models, F8-/-/PN-1-/- mice displayed significantly reduced blood loss and bleeding time compared with F8-/-mice. Moreover, platelet recruitment and fibrin(ogen) accumulation were significantly higher in F8-/-/PN-1-/- mice than in F8-/-mice in the ferric chloride-induced mesenteric vessel injury model. Thromboelastometry studies showed enhanced clot stability and lengthened clot lysis time in blood from F8-/-/PN-1-/- and from patients with hemophilia A incubated with a PN-1-neutralizing antibody compared with their respective controls. Our study thus provides proof of concept that PN-1 neutralization can be a novel approach for future clinical care in hemophilia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2019000281DOI Listing
November 2019

Emicizumab and thrombosis: The story so far.

J Thromb Haemost 2019 08;17(8):1269-1272

INSERM, U1176, Universite Paris-Sud, Le Kremlin-Bicetre, France.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jth.14556DOI Listing
August 2019

Correction of bleeding in experimental severe hemophilia A by systemic delivery of factor VIII-encoding mRNA.

Haematologica 2020 04 9;105(4):1129-1137. Epub 2019 Jul 9.

Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, Université de Paris, Paris, France

The treatment or prevention of bleeding in patients with hemophilia A relies on replacement therapy with different factor VIII (FVIII)-containing products or on the use of by-passing agents, i.e., activated prothrombin complex concentrates or recombinant activated factor VII. Emerging approaches include the use of bispecific anti-factor IXa/factor X antibodies, anti-tissue factor pathway inhibitor antibodies, interfering RNA to antithrombin, and activated protein C-specific serpins or gene therapy. The latter strategies are, however, hampered by the short clinical experience and potential adverse effects including the absence of tight temporal and spatial control of coagulation and the risk of uncontrolled insertional mutagenesis. Systemic delivery of mRNA allows endogenous production of the corresponding encoded protein. Thus, injection of erythropoietin-encoding mRNA in a lipid nanoparticle formulation resulted in increased erythropoiesis in mice and macaques. Here, we demonstrate that a single injection of transcribed B domain-deleted FVIII-encoding mRNA to FVIII-deficient mice enables endogenous production of pro-coagulant FVIII. Circulating FVIII:C levels above 5% of normal levels were maintained for up to 72 h, with an estimated half-life of FVIII production of 17.9 h, and corrected the bleeding phenotype in a tail clipping assay. The endogenously produced FVIII did however exhibit low specific activity and induced a potent neutralizing IgG response upon repeated administration of the mRNA. Our results suggest that the administration of mRNA is a plausible strategy for the endogenous production of proteins characterized by poor translational efficacy. The use of alternative mRNA delivery systems and improved FVIII-encoding mRNA should foster the production of functional molecules and reduce their immunogenicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2018.210583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7109737PMC
April 2020

von Willebrand Factor and Management of Heart Valve Disease: JACC Review Topic of the Week.

J Am Coll Cardiol 2019 03;73(9):1078-1088

CHU Lille, Université de Lille, Inserm U1011 - EGID, Institut Pasteur de Lille, Lille, France; CHU Lille, Hematology and Transfusion, Lille, France. Electronic address:

For decades, numerous observations have shown an intimate relationship between von Willebrand factor (VWF) multimer profile and heart valve diseases (HVD). The current knowledge of the unique biophysical properties of VWF helps us to understand the longstanding observations concerning the bleeding complications in patients with severe HVD. Not only does the analysis of the VWF multimer profile provide an excellent evaluation of HVD severity, it is also a strong predictor of clinical events. Also of importance, VWF responds within minutes to any significant change in hemodynamic valve status, making it an accurate marker of the quality of surgical and transcatheter therapeutic interventions. The authors provide in this review a practical, comprehensive, and evidence-based framework of the concept of VWF as a biomarker in HVD, advocating for its implementation into the clinical decision-making process besides usual clinical and imaging evaluation. They also delineate critical knowledge gaps and research priorities to definitely validate this concept.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jacc.2018.12.045DOI Listing
March 2019

Relevance of platelet desialylation and thrombocytopenia in type 2B von Willebrand disease: preclinical and clinical evidence.

Haematologica 2019 12 28;104(12):2493-2500. Epub 2019 Feb 28.

HITh, UMR_S 1176, INSERM Université Paris-Sud, Université Paris-Saclay, F-94270 Le Kremlin-Bicêtre

Patients with type 2B von Willebrand disease (vWD) (caused by gain-of-function mutations in the gene coding for von Willebrand factor) display bleeding to a variable extent and, in some cases, thrombocytopenia. There are several underlying causes of thrombocytopenia in type 2B vWD. It was recently suggested that desialylation-mediated platelet clearance leads to thrombocytopenia in this disease. However, this hypothesis has not been tested The relationship between platelet desialylation and the platelet count was probed in 36 patients with type 2B von Willebrand disease (p.R1306Q, p.R1341Q, and p.V1316M mutations) and in a mouse model carrying the severe p.V1316M mutation (the 2B mouse). We observed abnormally high elevated levels of platelet desialylation in both patients with the p.V1316M mutation and the 2B mice. , we demonstrated that 2B p.V1316M/von Willebrand factor induced more desialylation of normal platelets than wild-type von Willebrand factor did. Furthermore, we found that N-glycans were desialylated and we identified αIIb and β3 as desialylation targets. Treatment of 2B mice with sialidase inhibitors (which correct platelet desialylation) was not associated with the recovery of a normal platelet count. Lastly, we demonstrated that a critical platelet desialylation threshold (not achieved in either 2B patients or 2B mice) was required to induce thrombocytopenia In conclusion, in type 2B vWD, platelet desialylation has a minor role and is not sufficient to mediate thrombocytopenia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2018.206250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959185PMC
December 2019

Antibodies in the Treatment of Haemophilia A-A Biochemical Perspective.

Hamostaseologie 2019 Feb 29;39(1):36-41. Epub 2019 Jan 29.

Unité Mixte de Recherche Scientifique 1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France.

Replacement therapy has been proven effective in the management of bleedings in haemophilia A. Nevertheless, this approach comes with several shortcomings, like the need for frequent intravenous infusions and the development of neutralizing antibodies in 20 to 30% of the patients with severe haemophilia A replacement. This has led to the development of novel strategies to expand the spectrum of treatment options, some of which are based on antibody technology. These include a bispecific antibody that bridges enzyme factor IXa and substrate factor X, monoclonal antibodies that block the function of tissue factor pathway inhibitor, and a factor VIII-nanobody fusion protein with strongly enhanced von Willebrand factor binding. In this review, functional and mechanistic considerations on the use of these antibody variants will be discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/s-0038-1677521DOI Listing
February 2019

Structure and dynamics of the platelet integrin-binding C4 domain of von Willebrand factor.

Blood 2019 01 10;133(4):366-376. Epub 2018 Oct 10.

Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

Von Willebrand factor (VWF) is a key player in the regulation of hemostasis by promoting recruitment of platelets to sites of vascular injury. An array of 6 C domains forms the dimeric C-terminal VWF stem. Upon shear force activation, the stem adopts an open conformation allowing the adhesion of VWF to platelets and the vessel wall. To understand the underlying molecular mechanism and associated functional perturbations in disease-related variants, knowledge of high-resolution structures and dynamics of C domains is of paramount interest. Here, we present the solution structure of the VWF C4 domain, which binds to the platelet integrin and is therefore crucial for the VWF function. In the structure, we observed 5 intra- and inter-subdomain disulfide bridges, of which 1 is unique in the C4 domain. The structure further revealed an unusually hinged 2-subdomain arrangement. The hinge is confined to a very short segment around V2547 connecting the 2 subdomains. Together with 2 nearby inter-subdomain disulfide bridges, this hinge induces slow conformational changes and positional alternations of both subdomains with respect to each other. Furthermore, the structure demonstrates that a clinical gain-of-function VWF variant (Y2561) is more likely to have an effect on the arrangement of the C4 domain with neighboring domains rather than impairing platelet integrin binding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2018-04-843615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450055PMC
January 2019

Real-Time Monitoring of von Willebrand Factor in the Catheterization Laboratory: The Seatbelt of Mini-Invasive Transcatheter Aortic Valve Replacement?

JACC Cardiovasc Interv 2018 09;11(17):1775-1778

CHU Lille, Institut Coeur-Poumon, Cardiology, Lille, France; University Lille, Inserm U1011 - EGID, Lille, France; Institut Pasteur de Lille, Lille, France. Electronic address:

Significant paravalvular regurgitation (PVR) remains a relatively frequent (4% to 9%) and deleterious complication of transcatheter aortic valve replacement (TAVR), even with the latest generation of bioprosthesis. Although mini-invasive TAVR without general anesthesia or transesophageal echocardiography (TEE) is progressively becoming the predominant approach, identification and grading of PVR in the catheterization laboratory remain an important and challenging clinical issue. The authors discuss how a recently reported blood biomarker reflecting the von Willebrand factor activity, that is, the closure time with adenosine diphosphate, can be successfully applied during the TAVR procedure to detect and monitor PVR in real time, with an excellent negative predictive value. This point-of-care testing performed directly in the catheterization laboratory may improve the diagnosis of PVR and rationalize the decision of whether or not to perform corrective measures. They further discuss how such a test could be a substitute for the multimodal approach combining TEE, hemodynamics, and cine-angiography, and help to secure the transition to the mini-invasive approach and facilitate the expanding indications of less invasive procedures to lower-risk patients without jeopardizing procedural and clinical outcomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jcin.2018.05.047DOI Listing
September 2018

A factor VIII-nanobody fusion protein forming an ultrastable complex with VWF: effect on clearance and antibody formation.

Blood 2018 09 31;132(11):1193-1197. Epub 2018 Jul 31.

INSERM, Unité Mixte de Recherche Scientifique 1176, Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France; and.

Von Willebrand factor (VWF) modulates factor VIII (FVIII) clearance and the anti-FVIII immune response. Despite the high affinity that defines the FVIII/VWF interaction, association/dissociation kinetics dictates 2% to 5% FVIII being present as free protein. To avoid free FVIII when studying the FVIII-VWF complex in vivo, we designed a FVIII-nanobody fusion protein, with the nanobody part being directed against VWF. This fusion protein, designated FVIII-KB013bv, had a 25-fold higher affinity compared with B-domainless FVIII (BDD-FVIII) for VWF. In vitro analysis revealed full cofactor activity in 1-stage clotting and chromogenic assays (activity/antigen ratio 1.0 ± 0.3 and 1.1 ± 0.3, respectively). In vivo, FVIII-013bv displayed a twofold increased mean residence time compared with BDD-FVIII (3.0 hours vs 1.6 hours). In a tail clip-bleeding assay performed 24 hours after FVIII infusion, blood loss was significantly reduced in mice receiving FVIII-KB013bv vs BDD-FVIII (15 ± 7 μL vs 194 ± 146 μL; = .0043). Unexpectedly, when examining anti-FVIII antibody formation in FVIII-deficient mice, the immune-response toward FVIII-KB013bv was significantly reduced compared with BDD-FVIII (1/8 vs 14/16 mice produced anti-FVIII antibodies after treatment with FVIII-KB013bv and BDD-FVIII, respectively). Our data show that a stabilized interaction between FVIII and VWF is associated with a prolonged survival of FVIII and a reduced immune response against FVIII.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2018-01-829523DOI Listing
September 2018

Protein kinase C signaling dysfunction in von Willebrand disease (p.V1316M) type 2B platelets.

Blood Adv 2018 06;2(12):1417-1428

McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC.

von Willebrand disease (VWD) type 2B is characterized by gain-of-function mutations in von Willebrand factor (VWF), enhancing its binding affinity for the platelet receptor glycoprotein (GP)Ibα. VWD type 2B patients display a bleeding tendency associated with loss of high-molecular-weight VWF multimers and variable thrombocytopenia. We recently demonstrated that a marked defect in agonist-induced activation of the small GTPase, Rap1, and integrin αIIbβ3 in VWD (p.V1316M) type 2B platelets also contributes to the bleeding tendency. Here, we investigated the molecular mechanisms underlying impaired platelet Rap1 signaling in this disease. Two distinct pathways contribute to Rap1 activation in platelets: rapid activation mediated by the calcium-sensing guanine nucleotide exchange factor CalDAG-GEF-I (CDGI) and sustained activation that is dependent on signaling by protein kinase C (PKC) and the adenosine 5'-diphosphate receptor P2Y12. To investigate which Rap1 signaling pathway is affected, we expressed VWF/p.V1316M by hydrodynamic gene transfer in wild-type and mice. Using αIIbβ3 integrin activation as a read-out, we demonstrate that platelet dysfunction in VWD (p.V1316M) type 2B affects PKC-mediated, but not CDGI-mediated, activation of Rap1. Consistently, we observed decreased PKC substrate phosphorylation and impaired granule release in stimulated VWD type 2B platelets. Interestingly, the defect in PKC signaling was caused by a significant increase in baseline PKC substrate phosphorylation in circulating VWD (p.V1316M) type 2B platelets, suggesting that the VWF-GPIbα interaction leads to preactivation and exhaustion of the PKC pathway. Consistent with PKC preactivation, VWD (p.V1316M) type 2B mice also exhibited marked shedding of platelet GPIbα. In summary, our studies identify altered PKC signaling as the underlying cause of platelet hypofunction in p.V1316M-associated VWD type 2B.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2017014290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020816PMC
June 2018