ALBERT

Description: In recent years, the traditional view of the hemostatic system as being regulated by a coagulation factor cascade coupled with platelet activation has been increasingly challenged by new evidence that activation of the immune system strongly influences blood coagulation and pathological thrombus formation. Leukocytes can be induced to express tissue factor and release proinflammatory and procoagulant molecules such as granular enzymes, cytokines, and damage-associated molecular patterns. These mediators can influence all aspects of thrombus formation, including platelet activation and adhesion, and activation of the intrinsic and extrinsic coagulation pathways. Leukocyte-released procoagulant mediators increase systemic thrombogenicity, and leukocytes are actively recruited to the site of thrombus formation through interactions with platelets and endothelial cell adhesion molecules. Additionally, phagocytic leukocytes are involved in fibrinolysis and thrombus resolution, and can regulate clearance of platelets and coagulation factors. Dysregulated activation of leukocyte innate immune functions thus plays a role in pathological thrombus formation. Modulation of the interactions between leukocytes or leukocyte-derived procoagulant materials and the traditional hemostatic system is an attractive target for the development of novel antithrombotic strategies.

Description: Key Points CLEC4M plays a role in the clearance of VWF. CLEC4M polymorphisms contribute to the genetic variability of VWF plasma levels.

Description: Thrombosis is the leading cause of mortality worldwide and can result from uncontrolled immune activation in inflammatory conditions. Von Willebrand factor (VWF) is an acute phase glycoprotein that mediates primary hemostasis by binding to platelets. It is stored in platelets and in Weibel-Palade bodies (WPB) in endothelial cells. Cell-free DNA (CF-DNA) and histones are released during cell damage and have been shown to stimulate innate immune and pro-thrombotic responses including, in the case of histones, thrombocytopenia in mice and VWF release due to platelet activation/aggregation. We recently demonstrated the correlation between VWF and CF-DNA plasma levels in human and mouse models of chronic inflammation associated with aging. In these studies we investigate the influence of CF-DNA and histones on VWF release and platelet-binding in murine and endothelial cell models. C57BL6 mice received a retro-orbital injection of unfractionated (UFH), lysine-rich (H1) or arginine-rich (H3/H4) calf thymus histone (20 mg/kg), calf thymus DNA (4 mg/kg) or saline and blood was collected 1 hr post-injection from the inferior vena cava into buffered citrate. Platelet count was quantified from whole blood via a complete blood count. Blood-outgrowth endothelial cells (BOEC) from normal individuals were stimulated with UFH, H1, H3/H4, DNA and DNA/histone combinations (25 µg/mL) for 75 min. VWF and ANG-2 (stored exclusively in WPB) was quantified from plasma and BOEC media by ELISA and results expressed as fold-change ± SD. To visualize the influence of histone on platelet binding to BOEC surface associated VWF, BOEC were seeded onto collagen coated flow chamber slides. BOEC were stimulated for 20 min. with UFH (50 µg/mL) or histamine (25µM). Washed platelets were labelled with DiOC6 and flowed at a shear rate of 500s-1 over BOEC for 10 min. VWF-platelet strings (≥ 3 consecutive platelets) were quantified from still images obtained post-flow. In C57BL6 mice, VWF:Ag levels were significantly increased by UFH infusion (1.31±0.17, p=0.0015, n=8) but not DNA (1.09±0.19, p=0.20, n=10) relative to saline controls (1.00±0.16, n=14). H1 (1.64±0.20, n=5) mediated a greater VWF release than H3/H4 (1.21±0.20, p=0.01, n=6) and UFH (p=0.006). As previously described, we observed that UFH (0.74±0.08, p=0.0005), H1 (0.73±0.09, p=0.0011) and H3/H4 (0.87±0.14, p=0.08) induced thrombocytopenia in normal mice. ANG-2 levels were also significantly higher in H3/H4 (1.38±0.20, p=0.004) than saline (1.00±0.20) treated mice. Mice receiving UFH (1.25±0.31, p=0.13) and H1 (1.36±0.33, p=0.07) also had elevated ANG-2 levels although not statistically significant. Taken together this data suggests that both platelet and endothelial cells may contribute to VWF release upon histone-treatment. In vitro treatment of BOEC with H1 induced greater VWF (2.13±0.61, p=0.004) and ANG-2 (2.66±0.60, p=0.004) release as compared to the unstimulated condition (1.00±0.36). Interestingly, when BOEC were stimulated with UFH and DNA together we observed an increase in ANG-2 release (1.39±0.23, n=4, p=0.02) compared to unstimulated BOEC, but not in VWF:Ag when the cells were treated with each stimulus alone. As histones have been shown to bind to VWF, we hypothesize that these results could be explained by an impaired detection of VWF that is partially mitigated by the addition of DNA to the system. Using the in vitro flow chamber model, we observed significantly more VWF-platelet strings formed when cells were pre-incubated with UFH (5.51±0.97 strings, p=0.002) than when not stimulated (0.72±0.77 strings) (Figure 1). The ability of histones to activate platelets may also contribute to VWF-platelet string formation. Histones, but not DNA, stimulate the release of VWF from endothelial cells and platelets and facilitate platelet capture by endothelial VWF in a flow chamber model. As CF-DNA may serve as a surrogate marker for plasma histone levels, the correlation between CF-DNA and VWF:Ag we have previously observed in chronic inflammatory conditions, may be related in part to histone-induced VWF release from platelet and endothelial cell stores. These experiments demonstrate that histones induce VWF release in vivo and suggest a novel connection between the innate immune and hemostatic systems. Figure 1 Representative images taken after flow experiment. Arrows indicate VWF-platelet strings. Graph is expressed in mean ± SD (**p=0.0025). Figure 1. Representative images taken after flow experiment. Arrows indicate VWF-platelet strings. Graph is expressed in mean ± SD (**p=0.0025). Disclosures No relevant conflicts of interest to declare.

Description: Background The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion and aggregation at the site of vessel injury. The adhesive property of VWF is regulated by its multimer length, such that ultra large VWF (ULVWF) multimers, newly released from the endothelium, have greater hemostatic activity. multimer size is regulated by the metalloprotease ADAMTS13, which cleaves the A2 domain to reduce VWF multimer size and functional activity. static conditions, VWF maintains a globular conformation and the ADAMTS13 cleavage site is inaccessible. However, the exposure of endothelial-anchored VWF to tensile forces mediated by platelets and hydrodynamic shear enhance the cleavage of VWF by ADAMTS13. releases VWF of optimal hemostatic length from the endothelium into the plasma. We have previously reported using a flow chamber model which demonstrates that in addition to regulating VWF length and activity at the site of release, ADAMTS13 also associates with VWF at the site of thrombus formation. observed that under conditions of high and very high shear, ADAMTS13 reduced the size of thrombus volume., multi-coloured immunostaining revealed that ADAMTS13 co-localized with VWF and platelets at the top and middle layers of the thrombus, in the presence of very high shear. Aim To better understand the mechanism by which ADAMTS13 regulates thrombus size in our flow chamber model, we assessed the contribution of platelet tensile force to the localization of ADAMTS13 at the site of the thrombus. this model, the contributions of platelet GPIb, GPIIbIIIa, and P-selectin to ADAMTS13 localization were observed. Method Full length mouse VWF and ADAMTS13 cDNA were cloned into pCIneo and pcDNA3.1 plasmid, respectively. The gain of platelet GPIb binding mutation V1316M, and loss of GPIIbIIIa binding mutation (RGD to RGG) were introduced by site-directed-mutagenesis. mCherry was cloned at the C terminus of ADAMTS13 with a 12AA linker. Recombinant mVWF and mADAMTS13-mCherry proteins were produced via HEK293T cells by calcium phosphate transient transfection. mADAMTS13-mCherry (2 U/mL) and wild type or mutant mVWF (4 U/mL) was added to whole blood obtained from VWF-/-/ADAMTS13-/- double knockout mice. Whole blood containing DiOC6-labeled platelets was perfused over a collagen coated flow chamber at very high shear (7500s-1). The role of P-selectin was also analyzed by adding a P-selectin blocking antibody to blood obtained from ADAMTS13-/-knockout mice prior to the flow chamber experiment. After the perfusion, thrombi were fixed and immunostaining was performed to further analyze the distribution of platelets, VWF and ADAMTS13. Result As previously reported, ADAMTS13 localization was observed in the top and middle layers of the thrombus in the presence of wild type mVWF. The GPIb gain-of-function mutation V1316M increased both platelet (126%, p

Description: Regulation of plasma levels of the coagulation factors von Willebrand factor (VWF) and factor VIII (FVIII) involves a dynamic balance between biosynthesis, secretion, and clearance. Clearance of VWF and FVIII occurs through receptor-mediated endocytosis, with both LDL receptor gene family (eg. LRP1), and lectin receptors (eg. asialoglycoprotein receptor, siglec-5) contributing to this process. We have previously characterized the endothelial lectin CLEC4M as an endocytic receptor for FVIII and VWF. These proteins represent the first endogenous glycoprotein ligands indentified for CLEC4M. Previous studies have characterized CLEC4M as an adhesive receptor capable of facilitating viral infection in trans. Thus, the mechanism by which CLEC4M internalizes VWF and FVIII, and the subsequent fate of these ligands, is uncharacterized. We hypothesize that CLEC4M is able to endocytose VWF and FVIII via a clathrin-coated pit-dependent mechanism. We further hypothesize that endocytosis of FVIII and VWF by CLEC4M-expressing cells targets FVIII and VWF to lysosomes for degradation. As CLEC4M is expressed exclusively on the endothelial cells of the hepatic sinusoids and lymph nodes, and commercially prepared liver sinusoidal endothelial cells do not retain CLEC4M expression, we generated a HEK 293 cell line that stably expresses CLEC4M (〉90% positive by flow cytometry). We first inhibited the CLEC4M-mediated endocytic pathways by preincubating cells with methyl-β-cyclodextrin to deplete cell membrane cholesterol, dynasore hydrate to inhibit dynamin GTPase activity, and pitstop-2 to block the N-terminus of clathrin. Cells were then incubated with recombinant human FVIII, or plasma derived VWF-FVIII complex for 1 hour and internalization was visualized by immunofluorescence. A quantitative analysis of VWF or FVIII-positive objects was performed using Image Pro software. CLEC4M-expressing 293 cells internalized both FVIII as well as the VWF-FVIII complex. Binding and internalization of VWF was reduced by methyl-β-cyclodextran (65%, p=0.067), by dynasore hydrate (92%, p=0.055), and by pitstop-2 (83%, p=0.032). Binding and internalization of FVIII was similarly reduced by methyl-β-cyclodextran (50%, p=0.0050), by dynasore hydrate (60%, p=0.0225), and by pitstop-2 (90%, p=0.0086). This suggests that CLEC4M mediates endocytosis of VWF and FVIII via a clathrin-coated pit-dependent mechanism that involves lipid rafts. To visualize the endocytic pathway of VWF and FVIII, CLEC4M-expressing 293 cells were incubated with FVIII or the VWF-FVIII complex for 15, 30, or 60 minutes. Colocalization of FVIII, VWF and/or CLEC4M with markers for early endosomes (early endosomal antigen-1), late endosomes (Rab9), and lysosomes (LAMP1) was observed using immunofluorescence. For all conditions, CLEC4M-negative and isotype controls were performed. We have previously confirmed that FVIII and VWF are internalized by CLEC4M expressing cells, and that VWF colocalizes with a marker for early endosomes. When CLEC4M-expressing cells were exposed to the VWF-FVIII complex, we observed a partial colocalization of VWF and FVIII, confirming that CLEC4M is able to internalize the VWF-FVIII complex. When CLEC4M-expressing cells were exposed to FVIII, we observed colocalization between FVIII and early endosomal antigen 1 within 15 minutes, confirming that upon internalization by CLEC4M, VWF and FVIII are targeted to early endosomes. We next observed the transport of VWF and FVIII to late endosomes and lysosomes. When CLEC4M-expressing cells were incubated with VWF and FVIII for 30 minutes, we observed colocalization of both proteins with Rab9, a marker for late endosomes. When CLEC4M-expressing cells were incubated with FVIII for 1 hour, we observed colocalization of FVIII with LAMP1, a lysosomal marker, suggesting that the internalization of FVIII by CLEC4M leads to lysosome-mediated degradation of FVIII. In contrast, incubation of CLEC4M-expressing cells with VWF for up to 2 hours did not result in co-localization with LAMP1. These studies confirm that, in the context of the stable cell system used in these experiments, the cell surface lectin receptor CLEC4M mediates endocytosis of FVIII and VWF through a clathrin-coated pit-dependent pathway. Internalization of VWF and FVIII by CLEC4M targets these proteins to early and late endosomes; FVIII is subsequently targeted to lysosomes for degradation. Disclosures: James: CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.

Description: Abstract 16 Type 1 von Willebrand's Disease (VWD) can result from decreased synthesis or accelerated clearance of von Willebrand Factor (VWF), resulting in partial quantitative deficiency. Approximately 35% of individuals with Type 1 VWD do not have a putative mutation in their VWF gene, suggesting that genes other than VWF may contribute to the pathophysiology of this disease. Recently, the CHARGE GWAS meta-analysis identified single nucleotide polymorphisms in the gene encoding the C-type lectin domain family 4 member M (CLEC4M) as being significantly associated with plasma VWF levels in normal individuals. CLEC4M is a pathogen recognition receptor with a polymorphic extracellular neck region consisting of a variable number of tandem repeats (VNTR) (3 – 9 repeats). We hypothesize that CLEC4M binds to and clears VWF from the circulation, and that different CLEC4M VNTR alleles may contribute to differences in plasma levels of VWF in normal subjects and patients with Type 1 VWD. Previously, genotyping of 555 subjects (196 cases with type 1 VWD, and 362 family members) for the CLEC4M VNTR number showed that the most frequently documented alleles were VNTR 5 (29%), 6 (15%), and 7 (53%). Family-based association analysis on kindreds with type I VWD has demonstrated a significant excess transmission of VNTR 6 to the type I VWD phenotype (p=0.005) and an association of this VNTR allele with VWF:RCo (p=0.037). In the present studies, we have complemented this genetic association data with experiments to directly evaluate the ability of CLEC4M to bind and internalize VWF. Further, we characterized the ability of different CLEC4M VNTR alleles to facilitate VWF clearance. Binding of VWF to CLEC4M was assessed with a modified ELISA using a recombinant CLEC4M-Fc chimera. CLEC4M-Fc bound to Humate P (plasma-derived VWF-FVIII) in a dose-dependent manner. CLEC4M-Fc also bound to recombinant human VWF, and factor VIII-free plasma-derived VWF. CLEC4M-Fc demonstrated a 70% increase in binding to de-O-glycosylated Humate P (p=0.041), and a 75% decrease in binding to de-N-glycosylated Humate P (p=0.046) relative to controls. Additionally, pre-incubation of CLEC4M-Fc with the polysaccharide mannan attenuated binding to all VWF preparations by approximately 50%. Binding and internalization of VWF by HEK 293 cells stably expressing CLEC4M (VNTR allele 7) was assessed with immunofluorescence and ELISA. Binding of VWF co-localized with CLEC4M expression on HEK 293 cells. CLEC4M and VWF co-localized with early endosomal antigen-1, suggesting that CLEC4M participates in receptor-mediated endocytosis of VWF. CLEC4M-expressing cells bound and internalized VWF in a dose- and time-dependent manner relative to controls. Preincubation of CLEC4M expressing cells with mannan inhibited VWF binding and internalization by 50% (p=0.0088). The contribution of CLEC4M genetic variability to VWF binding and internalization was measured using HEK 293 cells expressing CLEC4M with 4, 6, 7, and 9 tandem repeats. Decreased binding and internalization of VWF was observed with cells expressing CLEC4M 4 and 9 tandem repeat constructs as compared to CLEC4M with 7 tandem repeats (CLEC4M 4 – 60% reduction, p 〈 0.001; CLEC4M 9 – 45% reduction, p=0.006). Cells expressing the CLEC4M VNTR combination 4 and 9, had a 55% decrease in binding and internalization of VWF relative to cells expressing CLEC4M with 7 VNTRs (p 〈 0.001). These VNTR associated differences in VWF binding/internalization were not accounted for by variances in the CLEC4M expression levels in the transfected HEK 293 cells. These studies demonstrate that the C-type lectin CLEC4M binds to and internalizes VWF through an N-glycan-dependent mechanism. Additionally, it provides further evidence that polymorphisms in the CLEC4M gene contribute to quantitative VWF deficiency. Disclosures: Montgomery: Gen-Probe/GTI Diagnostics: Consultancy; CSL Behring: Consultancy; Octapharma: Consultancy. James:CSL-Behring, Baxter, Bayer: Honoraria, Research Funding.

Description: Introduction: Von Willebrand factor (VWF) is a multimeric glycoprotein that serves as the carrier for the essential coagulation cofactor, factor VIII (FVIII). Both plasma levels of VWF and its FVIII-binding ability can influence plasma levels of FVIII. Type 2N von Willebrand disease (VWD) is associated with a reduced binding affinity of VWF for FVIII, resulting in accelerated proteolysis and clearance of FVIII (plasma levels 5 – 30% of normal). Type 2N VWD is a recessive trait and patients are either homozygous or compound heterozygous for 2N alleles. We hypothesize that type 2N VWD mutations can alter the expression and FVIII-binding ability of VWF. In these studies, we characterize three type 2N VWD mutations in vitro and in a murine model. R854Q (20-30% FVIII) is the most common 2N allele and is associated with a mild phenotype, while R816W (

Description: Background: Although Factor VIII (FVIII) concentrates are now routinely used for the prophylactic treatment of hemophilia A (HA), the optimal doses and intervals between administrations are difficult to predict because of variable pharmacokinetics of FVIII (FVIII-PK) between patients. Previous studies in HA have revealed a close relationship between FVIII-PK and the FVIII carrier protein, von Willebrand factor (VWF). A large genome-wide association study from the CHARGE consortium highlighted several novel loci associated with plasma levels of VWF and FVIII in normal subjects, and the five genetic loci associated with FVIII levels coincided with those influencing VWF levels (Smith, 2010). Objective: To investigate the effects of VWF synthesis, clearance and genetic variability on FVIII-PK in young HA patients. We hypothesized that 1) plasma VWF:Ag levels (VWF secretion and clearance), 2) polymorphic variants within the FVIII binding region of VWF, and 3) the glycosylation pattern of VWF (N-linked and ABO blood group antigen) would influence FVIII-PK. Methods: HA males recruited at two large academic pediatric hemophilia centers (The Hospital for Sick Children in Toronto and the Medical University of Vienna) were enrolled. Blood was collected at 5 time points (pre, post FVIII-infusion: 1, 9, 24, and 48 h), and FVIII-PK parameters, clearance (CL), volume of distribution (VD) and half-life (HL), were calculated based on a Bayesian model. Plasma levels of VWF (VWF:Ag), VWF propeptide (VWFpp) and FVIII binding ability of VWF (VWF:FVIIIB) were also evaluated. Genetic analysis of the FVIII-binding region and glycosylation sites of VWF was performed. Results: Samples from 33 boys [median age 10.9 years (range 6.5-17.9)] with severe HA were evaluated. Median values of FVIII-CL, VD and HL were 0.032 dl/h/kg (range 0.018-0.062), 0.47 dl/kg (0.29-0.78), and 10.2 h (6.7-16.8), respectively. VWF:Ag, VWFpp, VWFpp/VWF:Ag ratio and VWF:FVIIIB were 86.6 IU/dl (39.9-141.6), 88.2 U/dl (43.5-156.6), 1.09 (0.33-1.71) and 70.3% (41.2-101.9), respectively. FVIII-CL (r=-0.41, p

Description: Factor VIII (FVIII) pharmacokinetic (PK) properties show high interpatient variability in hemophilia A patients. Although previous studies have determined that age, body mass index, von Willebrand factor antigen (VWF:Ag) levels, and ABO blood group status can influence FVIII PK, they do not account for all observed variability. In this study, we aim to describe the genetic determinants that modify the FVIII PK profile in a population of 43 pediatric hemophilia A patients. We observed that VWF:Ag and VWF propeptide (VWFpp)/VWF:Ag, but not VWFpp, were associated with FVIII half-life. VWFpp/VWF:Ag negatively correlated with FVIII half-life in patients with non-O blood type, but no correlation was observed for type O patients, suggesting that von Willebrand factor (VWF) half-life, as modified by the ABO blood group, is a strong regulator of FVIII PK. The FVIII-binding activity of VWF positively correlated with FVIII half-life, and the rare or low-frequency nonsynonymous VWF variants p.(Arg826Lys) and p.(Arg852Glu) were identified in patients with reduced VWF:FVIIIB but not VWF:Ag. Common variants at the VWF, CLEC4M, and STAB2 loci, which have been previously associated with plasma levels of VWF and FVIII, were associated with the FVIII PK profile. Together, these studies characterize the mechanistic basis by which VWF clearance and ABO glycosylation modify FVIII PK in a pediatric population. Moreover, this study is the first to identify non-VWF and non-ABO variants that modify FVIII PK in pediatric hemophilia A patients.

Description: Introduction The formation of factor VIII (FVIII)- neutralizing antibodies is the most critical complication in the treatment of hemophilia A (HA). Recent clinical evidence suggests that recombinant FVIII (rFVIII) produced in baby hamster kidney (BHK-rFVIII) cells is more immunogenic than that produced in Chinese hamster ovary (CHO-rFVIII) cells. This difference in FVIII immunogenicity may be attributed to differences in protein glycosylation, which can impact the removal of FVIII from circulation through mechanisms leading to clearance and antigen presentation. Here, we document significant differences among the 25 potential N-linked glycans between these products, and we provide in vivo animal model-based evidence that supports these clinical observations. Methods Factor VIII lectin binding was assessed by ELISA to detect exposed glycans. Commercially-available rFVIII products were adsorbed at 1 ug/mL and specific glycan moieties were detected using a panel of biotinylated lectins and HRP-conjugated streptavidin. Confirmation of differences and determination of N-linked glycan structures was conducted by LC-MS/MS. Eight to 12 week old transgenic C57BL/6 HA mice were used in these studies. This model contains a murine f8 exon 16 KO and additionally harbors a human F8 transgene with a R593C point mutation. While these mice have undetectable plasma levels of human FVIII antigen and activity, they are tolerant to intravenously infused human rFVIII. Clearance was assessed following a 6 IU (~240 IU/kg) infusion of each rFVIII product, and FVIII activity was measured by chromogenic assay and normalized to a 5-minute time point. The number of interferon (IFN)-γ secreting splenocytes from rFVIII-primed naïve mice was determined by ELISPOT. FVIII immune responses were elicited by subcutaneous infusion (6 IU twice-weekly for 2 weeks) and adjuvant-coupled intravenous infusion (1 ug lipopolysacharride with the first infusion as described above) with either rFVIII product. Week 5 plasma samples were assessed for FVIII-specific IgG by ELISA, and FVIII inhibitors by one-stage clotting assay. Results Lectin binding showed that rFVIII produced in BHK cell lines exhibit lower proportions of high-mannose glycans (p