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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 FeCl 3 -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., (© 2022 International Society on Thrombosis and Haemostasis.)

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 αIIbβ3 integrin compared to wild-type-rVWF., Conclusions: We report two mutations of the D4 domains that induced combined qualitative and quantitative defects., (© 2022 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

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 αIIbβ3 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-β3 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-β3 proximity in cells adherent to immobilized fibrinogen were assessed before and after αIIbβ3 activation by the protein kinase C agonist phorbol 12-myristate 13-acetate., Results: Our results show that FLNa-actin and FLNa-β3 interactions negatively regulate αIIbβ3 activation. Moreover, FLNa-actin interaction represses Rac activation, contributing to the negative regulation of αIIbβ3 activation. In contrast, the FLNa dimerization domain, which maintains Rho inactive, was found to negatively regulate αIIbβ3 outside-in signaling., Conclusion: We conclude that FLNa negatively controls αIIbβ3 activation by regulating actin polymerization and restraining activation of Rac, as well as outside-in signaling by repressing Rho., (© 2022 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

The multimeric plasma glycoprotein (GP) von Willebrand factor (VWF) is best known for recruiting platelets to sites of injury during primary hemostasis. Generally, mutations in the VWF gene lead to loss of hemostatic activity and thus the bleeding disorder von Willebrand disease. By employing cone and platelet aggregometry and microfluidic assays, we uncovered a platelet GPIIb/IIIa-dependent prothrombotic gain of function (GOF) for variant p.Pro2555Arg, located in the C4 domain, leading to an increase in platelet aggregate size. We performed complementary biophysical and structural investigations using circular dichroism spectra, small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, molecular dynamics simulations on the single C4 domain, and dimeric wild-type and p.Pro2555Arg constructs. C4-p.Pro2555Arg retained the overall structural conformation with minor populations of alternative conformations exhibiting increased hinge flexibility and slow conformational exchange. The dimeric protein becomes disordered and more flexible. Our data suggest that the GOF does not affect the binding affinity of the C4 domain for GPIIb/IIIa. Instead, the increased VWF dimer flexibility enhances temporal accessibility of platelet-binding sites. Using an interdisciplinary approach, we revealed that p.Pro2555Arg is the first VWF variant, which increases platelet aggregate size and shows a shear-dependent function of the VWF stem region, which can become hyperactive through mutations. Prothrombotic GOF variants of VWF are a novel concept of a VWF-associated pathomechanism of thromboembolic events, which is of general interest to vascular health but not yet considered in diagnostics. Thus, awareness should be raised for the risk they pose. Furthermore, our data implicate the C4 domain as a novel antithrombotic drug target., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Aims: Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary haemostasis, while also having additional roles beyond haemostasis 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 signalling 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 significantly reduced 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., Conclusion: VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvβ3 signalling. 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., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

The protein Z (PZ)-dependent plasma protease inhibitor (ZPI) is a glycoprotein that inhibits factor XIa and, in the presence of PZ, FXa. Recently, ZPI has been shown to be an acute-phase protein (APP). As usually APPs downregulate the harmful effects of inflammation, we tested whether ZPI could modulate the increase of cytokines observed in inflammatory states. We observed that recombinant human ZPI (rhZPI) significantly decreases the levels of interleukin (IL)-1, IL-6, and tumor necrosis factor- α (TNF-α) induced by lipopolysaccharide (LPS) in a whole blood model. This inhibitory effect was unaffected by the presence of PZ or heparin. A ZPI mutant within the reactive loop center ZPI (Y387A), lacking anticoagulant activity, still had an anti-inflammatory activity. Surprisingly, rhZPI did not inhibit the synthesis of IL-6 or TNF-α when purified monocytes were stimulated by LPS, whereas the inhibitory effect was evidenced when lymphocytes were added to monocytes. The requirement of lymphocytes could be due to the synthesis of CCL5 (RANTES), a chemokine mainly produced by activated lymphocytes which is induced by rhZPI, and which can reduce the production of proinflammatory cytokines in whole blood. Lastly, we observed that the intraperitoneal injection of rhZPI significantly decreased LPS-induced IL-6 and TNF-α production in mouse plasma., Competing Interests: Conflict of Interest No author has conflicts of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. ( https://creativecommons.org/licenses/by/4.0/ ).)

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 of reaching 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 adenoassociated virus and transcriptional silencing of mutant alleles. Furthermore, protein-based approaches to increase factor FVIII levels in VWD-type 3 or 2N patients are 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) are 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., (© 2021 by The American Society of Hematology.)

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.

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., (© 2020 International Society on Thrombosis and Haemostasis.)

Blood flow profoundly varies throughout the vascular tree due to its pulsatile nature and to the complex vessel geometry. While thrombus formation has been extensively studied in vitro under steady flow, and in vivo under normal blood flow conditions, the impact of complex hemodynamics such as flow acceleration found in stenosed arteries has gained increased appreciation. We investigated the effect of flow acceleration, characterized by shear rate gradients, on the function of platelets adhering to fibrinogen, a plasma protein which plays a key role in hemostais and thrombosis. While we confirmed that under steady flow, fibrinogen only supports single platelet adhesion, we observed that under shear rate gradients, this surface becomes highly thrombogenic, supporting efficient platelet aggregation leading to occlusive thrombus formation. This shear rate gradient-driven thrombosis is biphasic with an initial step of slow platelet recruitment supported by direct plasma VWF adsorption to immobilized fibrinogen and followed by a second phase of explosive thrombosis initiated by VWF fiber formation on platelet monolayers. In vivo experiments confirmed that shear rate gradients accelerate thrombosis in a VWF-dependent manner. Together, this study characterizes a process of plasma VWF-dependent accelerated thrombosis on immobilized fibrinogen in the presence of shear rate gradients.

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 V H H 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., (© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

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., (© 2020 International Society on Thrombosis and Haemostasis.)

Background: Correct diagnosis of the cause of thrombocytopenia is crucial for the appropriate management of patients. Hyposialylation/desialylation (characterized by abnormally high β-galactose exposure) accelerates platelet clearance and can lead to thrombocytopenia. However, the reference range for β-galactose exposure in healthy individuals has not been defined previously., Objective: The objective of the present study was to develop a standardized assay of platelet β-galactose exposure for implementation in a clinical laboratory., Methods: β-Galactose exposure was measured in platelet-rich plasma by using flow cytometry and Ricinus communis agglutinin (RCA). A population of 120 healthy adults was recruited to study variability., Results: We determined an optimal RCA concentration of 12.5 μg/mL. The measure was stable for up to 4 hours (mean fluorescence intensity [MFI]-RCA: 1233 ± 329 at 0 hour and 1480 ± 410 at 4 hours). The platelet count did not induce a variation of RCA and the measure of RCA was stable when tested up to 24 hours after blood collection (MFI-RCA: 1252 ± 434 at day 0 and 1140 ± 297 24 hours after blood sampling). To take into account the platelet size, results should be expressed as RCA/forward scatter ratio. We used the assay to study variability in 120 healthy adults, and we found that the ratio is independent of sex and blood group., Conclusion: We defined a normal range in a healthy population and several preanalytical and analytical variables were evaluated, together with positive and negative controls. This assay may assist in the diagnosis of thrombocytopenic diseases linked to changes in β-galactose exposure., (© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

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., (Copyright© 2020 Ferrata Storti Foundation.)

The development of an immune response against therapeutic factor VIII is the major complication in hemophilia A patients. Oligomannose carbohydrates at N239 and/or N2118 on factor VIII allow its binding to the macrophage mannose receptor expressed on human dendritic cells, thereby leading to factor VIII endocytosis and presentation to CD4+ T lymphocytes. Here, we investigated whether altering the interaction of factor VIII with mannose-sensitive receptors on antigen-presenting cells may be a strategy to reduce factor VIII immunogenicity. Gene transfer experiments in factor VIII-deficient mice indicated that N239Q and/or N2118Q factor VIII mutants have similar specific activities as compared to non-mutated factor VIII; N239Q/N2118Q mutant corrected blood loss upon tail clip. Production of the corresponding recombinant FVIII mutants or light chains indicated that removal of the N-linked glycosylation site at N2118 is sufficient to abrogate in vitro the activation of FVIII-specific CD4+ T cells by human monocyte-derived dendritic cells. However, removal of mannose-ending glycans at N2118 did not alter factor VIII endocytosis and presentation to CD4+ T cells by mouse antigen-presenting cells. In agreement with this, the N2118Q mutation did not reduce factor VIII immunogenicity in factor VIII-deficient mice. Our results highlight differences in the endocytic pathways between human and mouse dendritic cell subsets, and dissimilarities in tissue distribution and function of endocytic receptors such as CD206 in both species. Further investigations in preclinical models of hemophilia A closer to humans are needed to decipher the exact role of mannose-ending glycans in factor VIII immunogenicity., (Copyright © 2020 Delignat, Rayes, Dasgupta, Gangadharan, Denis, Christophe, Bayry, Kaveri and Lacroix-Desmazes.)

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., (Copyright© 2019 Ferrata Storti Foundation.)

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 i  = 28-45 nM) in a competitive manner. KB-FVIIa-004 also inhibited FVIIa-mediated FX activation (K i  = 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., (© 2019 International Society on Thrombosis and Haemostasis.)

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., (© 2019 by The American Society of Hematology.)

The frequent von Willebrand factor (VWF) variant p.Phe2561Tyr is located within the C4 domain, which also harbors the platelet GPIIb/IIIa-binding RGD sequence. To investigate its potential effect on hemostasis, we genotyped 865 patients with coronary artery disease (CAD), 915 with myocardial infarction (MI), and 417 control patients (Ludwigshafen Risk and Cardiovascular Health Study) and performed functional studies of this variant. A univariate analysis of male and female carriers of the Tyr2561 allele aged 55 years or younger revealed an elevated risk for repeated MI (odds ratio, 2.53; 95% confidence interval [CI], 1.07-5.98). The odds ratio was even higher in females aged 55 years or younger, at a value of 5.93 (95% CI, 1.12-31.24). Cone and plate aggregometry showed that compared with Phe2561, Tyr2561 was associated with increased platelet aggregate size both in probands' blood and with the recombinant variants. Microfluidic assays revealed that the critical shear rate for inducing aggregate formation was decreased to 50% by Tyr2561 compared with Phe2561. Differences in C-domain circular dichroism spectra resulting from Tyr2561 suggest an increased shear sensitivity of VWF as a result of altered association of the C domains that disrupts the normal dimer interface. In summary, our data emphasize the functional effect of the VWF C4 domain for VWF-mediated platelet aggregation in a shear-dependent manner and provide the first evidence that a functional variant of VWF plays a role in arterial thromboembolism., (© 2019 by The American Society of Hematology.)

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., (© 2019 by The American Society of Hematology.)

The ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first EPHB2 variant affecting platelets in 2 siblings (P1 and P2) from a consanguineous family with recurrent bleeding and normal platelet counts. Whole-exome sequencing identified a c.2233C>T variant (missense p.R745C) of the EPHB2 gene. P1 and P2 were homozygous for this variant, while their asymptomatic parents were heterozygous. The p.R745C variant within the tyrosine kinase domain was associated with defects in platelet aggregation, αIIbβ3 activation, and granule secretion induced by G-protein-coupled receptor (GPCR) agonists and convulxin, as well as in thrombus formation on collagen under flow. In contrast, clot retraction, flow-dependent platelet adhesion, and spreading on fibrinogen were only mildly affected, indicating limited effects on αIIbβ3 outside-in signaling. Most importantly, Lyn, Syk, and FcRγ phosphorylation, the initial steps in glycoprotein VI (GPVI) platelet signaling were drastically impaired in the absence of platelet-platelet contact, indicating a positive role for EPHB2 in GPVI activation. Likewise platelet activation by PAR4-AP showed defective Src activation, as opposed to normal protein kinase C activity and Ca 2+ mobilization. Overexpression of wild-type and R745C EPHB2 variant in RBL-2H3 (rat basophilic leukemia) cells stably expressing human GPVI confirmed that EPHB2 R745C mutation impaired EPHB2 autophosphorylation but had no effect on ephrin ligand-induced EPHB2 clustering, suggesting it did not interfere with EPHB2-ephrin-mediated cell-to-cell contact. In conclusion, this novel inherited platelet disorder affecting EPHB2 demonstrates this tyrosine kinase receptor plays an important role in platelet function through crosstalk with GPVI and GPCR signaling., (© 2018 by The American Society of Hematology.)

Objective- PI3Kα (phosphoinositide 3-kinase alpha) is a therapeutic target in oncology, but its role in platelets and thrombosis remains ill characterized. In this study, we have analyzed the role of PI3Kα in vitro, ex vivo, and in vivo in 2 models of arterial thrombosis. Approach and Results- Using mice selectively deficient in p110α in the megakaryocyte lineage and isoform-selective inhibitors, we confirm that PI3Kα is not mandatory but participates to thrombus growth over a collagen matrix at arterial shear rate. Our data uncover a role for PI3Kα in low-level activation of the GP (glycoprotein) VI-collagen receptor by contributing to ADP secretion and in turn full activation of PI3Kβ and Akt/PKB (protein kinase B). This effect was no longer observed at high level of GP VI agonist concentration. Our study also reveals that over a vWF (von Willebrand factor) matrix, PI3Kα regulates platelet stationary adhesion contacts under arterial flow through its involvement in the outside-in signaling of vWF-engaged α IIb β 3 integrin. In vivo, absence or inhibition of PI3Kα resulted in a modest but significant decrease in thrombus size after superficial injuries of mouse mesenteric arteries and an increased time to arterial occlusion after carotid lesion, without modification in the tail bleeding time. Considering the more discrete and nonredundant role of PI3Kα compared with PI3Kβ, selective PI3Kα inhibitors are unlikely to increase the bleeding risk at least in the absence of combination with antiplatelet drugs or thrombopenia. Conclusions- This study provides mechanistic insight into the role of PI3Kα in platelet activation and arterial thrombosis.

This 9th Symposium on Hemostasis is an international scientific meeting held biannually in Chapel Hill, North Carolina. The meeting is in large measure the result of the close friendship between the late Dr. Harold R. Roberts of UNC Chapel Hill and Dr. Ulla Hedner of Novo Nordisk. When Novo Nordisk was developing the hemophilia therapy that would become NovoSeven, they sponsored a series of meetings to understand the basic biology and clinical applications of factor VIIa. The first meeting in Chapel Hill was held April 4-6, 2002 with Dr. Roberts as the organizer. Over the years, the conference emphasis has expanded from discussions of factor VIIa and tissue factor to additional topics in hemostasis and thrombosis. This year's meeting includes presentations by internationally renowned speakers that discuss the state-of-the-art on an array of important topics, including von Willebrand factor, engineering advances, coagulation and disease, tissue factor biology, therapeutic advances, and basic clotting factor biology. Included in this review article are illustrated abstracts provided by our speakers, which highlight the main conclusions of each invited talk. This will be the first meeting without Dr. Roberts in attendance, yet his commitment to excellent science and his focus on turning science to patient care are pervasively reflected in the presentations by our speakers.

During the last decade, the development of improved and novel approaches for the treatment of hemophilia A has expanded tremendously. These approaches include factor VIII (FVIII) with extended half-life (eg, FVIII-Fc and PEGylated FVIII), monoclonal antibodies targeting tissue factor pathway inhibitor, small interfering RNA to reduce antithrombin expression and the bispecific antibody ACE910/emicizumab. Emicizumab is a bispecific antibody recognizing both the enzyme factor IXa and the substrate factor X. By simultaneously binding enzyme and substrate, emicizumab mimics some part of the function exerted by the original cofactor, FVIII, in that it promotes colocalization of the enzyme-substrate complex. However, FVIII and the bispecific antibody are fundamentally different proteins and subject to different modes of regulation. Here, we will provide an overview of the similarities and dissimilarities between FVIII and emicizumab from a biochemical and mechanistical perspective. Such insight might be useful in the clinical decision making for those who apply emicizumab in their practice now or in the future, particularly in view of the thrombotic complications that have been reported when emicizumab is used in combination with FVIII-bypassing agents., (© 2017 by The American Society of Hematology.)

Objective: von Willebrand factor (VWF) is crucial to hemostasis, but also plays a role in inflammatory processes. Unfortunately, no proper monoclonal antibodies to study VWF function in mice are currently available. We therefore aimed to generate single-domain antibodies (sdAbs) recognizing murine VWF and blocking its function in vivo., Approach and Results: Llama-derived sdAbs recognizing both human and murine VWF were isolated via phage display technology. One of them (designated KB-VWF-006) recognized the VWF A1 domain with picomolar affinity. This sdAb avidity was strongly enhanced via dimerization using a triple Ala linker (KB-VWF-006bi). When administered in vivo to wild-type mice, KB-VWF-006bi dose dependently induced bleeding in a tail clip model. In 2 distinct models of inflammation, KB-VWF-006bi efficiently interfered with leukocyte recruitment and vascular leakage., Conclusions: KB-VWF-006bi is an sdAb recognizing the A1 domain of human VWF and murine VWF that interferes with VWF-platelet interactions in vivo. By using this sdAb, we now also show that the A1 domain is pertinent to the participation of VWF in the inflammatory response., (© 2017 American Heart Association, Inc.)

Hemostasis and pathological thrombus formation are dynamic processes that require multiple adhesive receptor-ligand interactions, with blood platelets at the heart of such events. Many studies have contributed to shed light on the importance of von Willebrand factor (VWF) interaction with its platelet receptors, glycoprotein (GP) Ib-IX-V and αIIbβ3 integrin, in promoting primary platelet adhesion and aggregation following vessel injury. This review will recapitulate our current knowledge on the subject from the rheological aspect to the spatio-temporal development of thrombus formation. We will also discuss the signaling events generated by VWF/GPIb-IX-V interaction, leading to platelet activation. Additionally, we will review the growing body of evidence gathered from the recent development of pathological mouse models suggesting that VWF binding to GPIb-IX-V is a promising target in arterial and venous pathological thrombosis. Finally, the pathological aspects of VWF and its impact on platelets will be addressed.

Von Willebrand disease (VWD) type 2B is characterized by mutations causing enhanced binding of von Willebrand factor (VWF) to platelets. Bleeding tendency is associated with heterogeneous clinical manifestations, including moderate to severe thrombocytopenia. The underlying mechanism of the thrombocytopenia has remained unclear. Here, a mouse model of VWD type 2B was used to investigate pathways contributing to thrombocytopenia. Immunohistochemical analysis of blood smears revealed that mutant VWF was exclusively detected on platelets of thrombocytopenic VWD type 2B mice, suggesting that thrombocytopenic VWD type 2B mice were elevated two- to threefold upon chemical macrophage depletion. Colocalization of platelets with CD68-positive Kupffer cells and CD168-positive marginal macrophages in liver and spleen, respectively, confirmed the involvement of macrophages in the removal of VWF/platelet complexes. Significantly more platelets were found in liver and spleen of VWD type 2B mice compared with control mice. Finally, platelet survival was significantly shorter in VWD type 2B mice compared with control mice, providing a rationale for lower platelet counts in VWD type 2B mice. In conclusion, our data indicate that VWF type 2B binds to platelets and that this is a signal for clearance by macrophages, which could contribute to the thrombocytopenia in patients with VWD type 2B.

Thrombosis is the pathological face of the hemostatic process, and is still the major cause of death in the Western society. Many different components have been identified that contribute to the thrombotic occlusion of the vasculature and several therapeutic approaches have been developed to treat this severe clinical complication. In the last several years, a number of new agents have been under (pre)clinical investigation that are targeting von Willebrand factor (VWF), a protein that nicely exemplifies the thin line between the normal hemostatic process and an overly active system that gives rise to thrombotic events. Indeed, several epidemiological studies have found that increased plasma levels of VWF are associated with an increased risk for cardiovascular complications. VWF is a multimeric protein that is pertinent to the recruitment of platelets to the growing thrombus. VWF and platelets circulate together without interacting under normal conditions, and should combine into a thrombus selectively when necessary. This delicate process is highly regulated by various endothelial- and plasma proteins as well as by changes in shear stress. In the present review, an update is provided about our current knowledge on VWF as a risk factor, mediator and pharmacological target in association with thrombosis.

Two unrelated families were recruited in the French Reference Center for von Willebrand Disease with moderate bleeding symptoms associated with low von Willebrand factor (VWF) antigen levels, decreased collagen binding assay, and no or partial response to desmopressin. Genetic analysis showed the presence of heterozygous mutations in the A3 domain away from the collagen-binding surface: 1 never reported previously (p.L1696R) and another (p.P1824H) described in a Spanish family. The mutations were reproduced by site-directed mutagenesis and mutant VWF was expressed in different expression systems, COS-7 cells, baby hamster kidney cells, and in VWF-deficient mice through hydrodynamic injection. p.L1696R and p.P1824H were associated with very low expression levels both in vitro and in vivo, with intracellular retention for p.P1824H. Both homozygous mutants displayed decreased binding to collagen types I and III but also decreased binding to platelet glycoproteins Ib and IIbIIIa. Co-transfections with wild-type VWF partially corrected these defects, except that collagen binding remained abnormal. The in vivo thrombosis response was severely reduced for both heterozygous mutants. In conclusion, we report 2 VWF A3 domain mutations that induce a combined qualitative and quantitative defect.

Background: Factor VIII (FVIII) and von Willebrand factor (VWF) circulate in plasma in a tight non-covalent complex, being critical to hemostasis. Although structurally unrelated, both share the presence of sialylated glycan-structures, making them potential ligands for sialic-acid-binding-immunoglobulin-like-lectins (Siglecs)., Design and Methods: We explored the potential interaction between FVIII/VWF and Siglec-5, a receptor expressed in macrophages using various experimental approaches, including binding experiments with purified proteins and cell-binding studies with Siglec-5 expressing cells. Finally, Siglec-5 was overexpressed in mice via hydrodynamic gene transfer., Results: In different systems using purified proteins, saturable, dose-dependent and reversible interactions between a soluble Siglec-5 fragment and both hemostatic proteins were found. Sialidase treatment of VWF resulted in a complete lack of Siglec-5 binding. In contrast, sialidase treatment left interactions between FVIII and Siglec-5 unaffected. FVIII and VWF also bound to cellsurface exposed Siglec-5, as was visualized by classical immunostaining as well as by Duolinkproximity ligation assays. Co-localization of FVIII and VWF with early endosomal markers further suggested that binding to Siglec-5 is followed by endocytosis of the proteins. Finally, overexpression of human Siglec-5 in murine hepatocytes following hydrodynamic gene transfer resulted in a significant decrease in plasma levels of FVIII and VWF in these mice., Conclusions: Our data indicate that FVIII and VWF may act as a ligand for Siglec-5, and that Siglec-5 may contribute to the regulation of plasma levels of the FVIII/VWF complex.

Hemostasis and thrombosis represent two sides of the same coin. Hemostasis maintains blood fluidity in the vascular system while allowing for rapid thrombus formation to prevent excessive hemorrhage after blood vessel injury. Thrombosis is a pathologic extension of the normal hemostatic mechanism, occurring when unwanted clot formation develops in certain pathological situations. The molecular mechanisms underlying both phenomena are fundamentally identical. One of the key players in both processes is the plasma glycoprotein von Willebrand factor, which perfectly illustrates this duality between hemostatic and thrombotic mechanisms. The purpose of this review is to discuss novel findings on the role of von Willebrand factor at this interface, and how some of these findings may help develop new therapeutic strategies.

Von Willebrand disease (VWD)-type 2B originates from a gain-of-function mutation in von Willebrand factor (VWF), resulting in enhanced platelet binding. Clinical manifestations include increased bleeding tendency, loss of large multimers, thrombocytopenia, and circulating platelet aggregates. We developed a mouse model to study phenotypic consequences of VWD-type 2B mutations in murine VWF: mVWF/R1306Q and mVWF/V1316M. Both mutations allow normal multimerization but are associated with enhanced ristocetin-induced platelet aggregation, typical for VWD-type 2B. In vivo expression resulted in thrombocytopenia and circulating aggregates, both of which were more pronounced for mVWF/V1316M. Furthermore, both mutants did not support correction of bleeding time or arterial vessel occlusion in a thrombosis model. They further displayed a 2- to 3-fold reduced half-life and induced a 3- to 6-fold increase in number of giant platelets compared with wild-type VWF. Loss of large multimers was observed in 50% of the mice. The role of ADAMTS13 was investigated by expressing both mutants in VWF/ADAMTS13 double-deficient mice. ADAMTS13 deficiency resulted in more and larger circulating platelet aggregates for both mutants, whereas the full multimer range remained present in all mice. Thus, we established a mouse model for VWD-type 2B and found that phenotype depends on mutation and ADAMTS13.

CD40L, a member of the tumor necrosis factor family of ligands, plays a major role in immune responses via its receptor, CD40. Recently, CD40L has been detected on the surfaces of activated platelets and shown to activate endothelium. Here we further addressed the function of platelet CD40L. We show that absence of CD40L affects the stability of arterial thrombi and delays arterial occlusion in vivo. Infusion of recombinant soluble (rs)CD40L restored normal thrombosis, whereas rsCD40L lacking the KGD integrin-recognition sequence did not. CD40-deficient mice exhibited normal thrombogenesis. rsCD40L specifically bound to purified integrin alphaIIbbeta3 and to activated platelets in a beta3-dependent manner and induced platelet spreading. In addition, rsCD40L promoted the aggregation of either human or mouse platelets under high shear rates. Thus, CD40L appears to be an alphaIIbbeta3 ligand, a platelet agonist, and necessary for stability of arterial thrombi.