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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 in vitro 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.

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN-protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1' residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence-function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4' region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4' region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4' RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.

This article assessed whether plasma collected from patients with COVID-19 at different disease stages could trigger endothelial damage in vitro by using cultured human pulmonary microvascular endothelial cells (HPMVEC) This study also further investigated the association of plasma-induced cytotoxicity with levels of circulating biomarkers related to organ dysfunction (Pao2 [partial pressure of oxygen in arterial blood]/Fio2 [fraction of inspired oxygen], widely used as an indicator of oxygenation requirements, lactate dehydrogenase, creatinine, and aspartate transaminase), endothelial damage (von Willebrand factor antigen;ADAMTS13;plasminogen activator inhibitor-1;syndecan-1), tissue injury (cell-free DNA, a damage-associated molecular patterns marker), and levels of circulating cytokines related to the activation of innate (interleukin [IL]-6 and tumor necrosis factor-a) and adaptative immune cell responses (soluble IL-2 receptor) Inclusion criteria were individuals aged 18 years or older with a positive SARS-CoV-2 real-time reverse-transcriptase polymerase chain reaction on nasal or tracheal samples This data shed new light on the pathophysiology of COVID-19 by demonstrating the direct and rapid cytotoxic effect of plasma collected from critically ill patients on vascular endothelial cells This rapid effect (1 hour after plasma exposure) excludes a direct cytopathic effect of SARS-CoV-2 infection, as the progression of viral infection and visible cytopathogenic effects are in general only apparent 12 to 24 hours after infection 5 A higher cytotoxic effect of plasma on endothelial cells was associated with a more pronounced hypoxemia and organ dysfunction as reflected by the correlation with Pao2/FiO2, lactate dehydrogenase, creatinine, and aspartate transaminase This cytotoxic effect also correlated with circulating markers of endothelial damage, indicating that this in vitro functional assay reflects microvascular endothelial damage in vivo Different pathways could be involved in endothelial cell injury during the course of COVID-19, i e , complement activation, cellular hypoxia, platelets, and direct cytotoxicity of cytokines such as IL-6, IL-1beta, and tumor necrosis factor-a The study observed a relationship between this cytotoxic effect and the level of proinflammatory cytokines, suggesting that cytotoxicity could be related to overproduction of proinflammatory cytokines

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN-protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1' residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence-function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4' region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4' region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4' RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.

We thank Bhogal and colleagues for their positive comments on our manuscript and their perspective on VWF being a potential driver of COVID-19 microvascular thrombosis rather than a mere biomarker of endotheliopathy. It is well-known that VWF promotes neutrophil recruitment to endothelial surfaces under low-shear conditions and supports neutrophil extravasation towards inflamed tissues1 . This pro-inflammatory role of VWF involves two distinct pathways: (1) a direct interaction of neutrophil receptors (PSGL1 and s2-integrins) with VWF, and (2) an indirect interaction in which neutrophils adhere to VWF-bound platelets.

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 in vivo. 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. In vitro, 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 vivo. In conclusion, in type 2B vWD, platelet desialylation has a minor role and is not sufficient to mediate thrombocytopenia.

International audience; 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. Pre-treatment of PSGL1 with sialidase reduced Siglec-5 binding by 79 ± 4%. In confocal immune-fluorescence assays, we observed that 50% of Peripheral Blood Mononuclear Cells (PBMCs) simultaneously express PSGL1 and Siglec-5. Duolink-proximity ligation analysis demonstrated that PSGL1 and Siglec-5 are in close proximity (

Thrombocytopenia and increased platelet clearance observed in von Willebrand disease-type 2B (VWD-2B) may be explained by platelet apoptosis triggered by the constitutive binding of VWF to its receptor, glycoprotein Ib (GPIb). Apoptosis was assessed in platelets from two patients with a severe VWD-2B mutation VWF/p.V1316M and from mice transiently expressing VWF/p.V1316M. We now report that the VWD-2B mutation VWF/p.V1316M which binds spontaneously to its receptor GPIbα does not induce apoptosis. In 2 unrelated patients (P1 and P2) exhibiting different VWF plasma levels (70% and 36%, respectively, compared with normal pooled human plasma given as 100%), inner transmembrane depolarization of mitochondria, characteristic of apoptotic events was undetectable in platelets, whether washed or in whole blood. No or a moderate phosphatidyl serine (PS) exposure as measured by annexin-V staining was observed for P1 and P2, respectively. Expression of pro-apoptotic proteins Bak and Bax, and caspase-3 activity were similar to control platelets. In the VWD-2B mouse model expressing high levels of mVWF/p.V1316M (423%), similar to what is found in inflammatory pathologies, no significant difference was observed between mice expressing mVWF/WT and mVWF/p.V1316M. These results strongly argue against apoptosis as a mechanism for the thrombocytopenia of severe VWD-2B exhibiting the VWF/p.V1316M mutation.

Up until recently, von Willebrand Factor (VWF) structure-function relationships have only been studied through in vitro approaches. A powerful technique known as hydrodynamic gene transfer, which allows transient expression of a transgene by mouse hepatocytes, has led to an important shift in VWF research. Indeed this approach has now enabled us to transiently express a number of VWF mutants in VWF-deficient mice in order to test the relative importance of specific residues in different aspects of VWF biology and functions in an in vivo setting. As a result, mice reproducing various types of von Willebrand disease have been generated, models that will be useful to test new therapies. This approach also allowed a more precise identification of the importance of VWF interaction with subendothelial collagens and with platelets receptors in hemostasis and thrombosis. The recent advances gathered from these studies as well as the pros and cons of the technique will be reviewed here.

Von Willebrand factor (VWF) is a large multimeric protein, the function of which has been demonstrated to be pivotal to the haemostatic system. Indeed, quantitative and/or qualitative abnormalities of VWF are associated with the bleeding disorder Von Willebrand disease (VWD). Moreover, increased plasma concentrations of VWF have been linked to an increased risk for thrombotic complications. In the previous decades, many studies have contributed to our understanding of how VWF is connected to the haemostatic system, particularly with regard to structure-function relationships. Interactive sites for important ligands of VWF (such as factor VIII, collagen, glycoprotein Iba, integrin aIIbb3 and protease ADAMTS13) have been identified, and mutagenesis studies have confirmed the physiological relevance of the interactions between VWF and these ligands. However, we have also become aware that VWF has a more versatile character than previously thought, given its potential role in various non-hemostatic processes, like intimal thickening, tumor cell apoptosis and inflammatory processes. In the presence review, a summary of our knowledge on VWF structure-function relationships is provided in the context of the "classical" haemostatic task of VWF and in perspective of pathological processes beyond haemostasis.

A major problem for diagnosing the antiphospholipid syndrome (APS) is the high variability between commercial anti-beta(2)glycoprotein I (beta(2)GPI) assays. Predominantly antibodies reactive against cryptic epitope Glycine40-Arginine43 (G40-R43) in domain I are associated with an increased risk for thrombosis. Upon interaction with anionic surfaces beta(2)GPI opens up, thereby exposing G40-R43. Objectives: To examine whether suboptimal exposure of epitope G40-R43 explains the variations in results observed between commercial assays. Methods: Two patient-derived monoclonal antibodies were tested on neutral versus anionic plates. Antibody P1-117 reacts with G40-R43 in the open conformation while P2-6 recognizes beta(2)GPI irrespective of its conformation. These antibodies were tested in commercial anti-beta(2)GPI assays (A-E). Results: In assay A, both antibodies showed equal reactivity towards beta(2)GPI, indicating that all the beta(2)GPI exposes G40-R43. In other assays P1-117 displayed lower reactivity than P2-6, demonstrating reduced G40-R43 availability. To exclude influences of other assay features, reactivity was re-examined on plates of assay A and B using the protocol/reagents from each assay. In all combinations, reactivity of both antibodies on a plate was comparable to results obtained with its own protocol/reagents, suggesting that the coating, rather than other assay components, accounts for the observed differences. In two patient cohorts we demonstrated that a number of domain I-reactive samples are missed in assays characterized by a decreased exposure of epitope G40-R43. Conclusions: Exposure of epitope G40-R43 on beta(2)GPI is highly variable between commercial anti-beta(2)GPI assays. As a consequence, patients can be falsely assigned negative in assays characterized by a reduced exposure of G40-R43.

Leukocyte-associated Ig-like receptor (LAIR)-1 is a collagen-receptor that inhibits immune cell function upon collagen binding. Next to LAIR-1, the human genome encodes LAIR-2, a putative soluble homolog. In this study we show, for the first time, that the LAIR-2 gene is broadly transcribed in human PBMC, mirroring the expression profile of LAIR-1. LAIR-2 protein is expressed as a soluble receptor exhibiting high affinity for various collagen molecules to which it binds in a hydroxyproline-dependent manner. In vitro stimulation of PBMC induces secretion of LAIR-2. We detect high amounts of LAIR-2 in urine of pregnant women, indicating that the soluble receptor is indeed produced in vivo and can be cleared from the body via urine. Furthermore, LAIR-2 levels are increased in synovial fluid of patients with rheumatoid arthritis as compared with osteoarthritis patients. We hypothesize that soluble LAIR-2 may function as a natural competitor for LAIR-1, thereby regulating its inhibitory potential. Indeed, LAIR-2 prevents binding of human LAIR-1 to collagens and LAIR-1 cross-linking in vitro, suggesting that the protein has an immunoregulatory function in vivo. Hence, we reveal a novel mechanism of immune regulation by a soluble LAIR receptor regulating the inhibitory potential of the membrane-bound LAIR-1 via competition for ligands.

Background and Objectives. Patients with severe hemophilia A have considerably different factor VIII half-lives. Whether this is associated with their clinical characteristics has not been reported. The aim of this study was to describe the association of factor VIII half-lives with treatment and clinical characteristics of patients with severe hemophilia A, who have been treated with individually tailored prophylaxis. Design and Methods. Patients were selected from a single-center cohort of 214 patients with severe hemophilia, born between 1944 and 1995. To improve efficiency we measured factor VIII half-life in 42 patients selected from the extremes of the distribution of phenotypes of severe hemophilia. We assessed information on life-long joint bleeds and clotting factor consumption. Orthopedic outcome was assessed by the Pettersson score. Results. Among these patients with severe hemophilia, factor VIII half-life ranged from 7.4-20.4 hours (median 11.8 hours). A one-hour increase in factor VIII half-life was associated with 96 (95% confidence interval (Cl) 2-190) IU less clotting factor use per kg per year. Age was an important determinant of factor VIII half-life, and explained a large part of the association between factor VIII half-life and clotting factor consumption. The median number of joint bleeds per year and arthropathy were similar for patients with different half-lives. Interpretations and Conclusions. Among patients with severe hemophilia, treated prophylactically with clotting factor, those with a shorter factor VIII half-life required slightly more clotting factor to prevent joint bleeds and subsequent arthropathy than similar patients with a longer factor VIII half-life.