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Concept: Von Willebrand disease

186

Here, for the first time, we test a novel hypothesis that systemic treatment of stroke with exosomes derived from multipotent mesenchymal stromal cells (MSCs) promote neurovascular remodeling and functional recovery after stroke in rats. Adult male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion (MCAo) followed by tail vein injection of 100 μg protein from MSC exosome precipitates or an equal volume of vehicle phosphate-buffered saline (PBS) (n=6/group) 24 hours later. Animals were killed at 28 days after stroke and histopathology and immunohistochemistry were employed to identify neurite remodeling, neurogenesis, and angiogenesis. Systemic administration of MSC-generated exosomes significantly improved functional recovery in stroke rats compared with PBS-treated controls. Axonal density and synaptophysin-positive areas were significantly increased along the ischemic boundary zone of the cortex and striatum in MCAo rats treated with exosomes compared with PBS control. Exosome treatment significantly increased the number of newly formed doublecortin (a marker of neuroblasts) and von Willebrand factor (a marker of endothelial cells) cells. Our results suggest that intravenous administration of cell-free MSC-generated exosomes post stroke improves functional recovery and enhances neurite remodeling, neurogenesis, and angiogenesis and represents a novel treatment for stroke.Journal of Cerebral Blood Flow & Metabolism advance online publication, 21 August 2013; doi:10.1038/jcbfm.2013.152.

Concepts: Von Willebrand disease, Endothelium, Middle cerebral artery, Cardiovascular system, Atherosclerosis, Blood vessel, Von Willebrand factor, Mesenchymal stem cell

175

Type 2 diabetes is known to cause endothelial activation resulting in the secretion of von Willebrand factor (VWF). We have shown that levels of VWF in a glycoprotein Ib-binding conformation are increased in specific clinical settings. The aim of the current study is to investigate whether active VWF levels increase during aging and the development of diabetes within the population of patients suffering from type 2 diabetes. Patients and controls were divided into two groups based on age: older and younger than 60 years of age. VWF antigen, VWF propeptide, VWF activation factor and total active VWF were measured. Patients older than 60 years of age had increased levels of total active VWF, VWF activation factor and VWF propeptide compared to younger patients and controls. All measured VWF parameters were associated with age in diabetic patients. Total active VWF and VWF propeptide correlated with the period of being diagnosed with diabetes. Regression analyses showed that especially the VWF activation factor was strongly associated with diabetes in patients older than 60 years of age. In conclusion, we found that the conformation of VWF could be involved in the disease process of diabetes and that the VWF in a glycoprotein Ib-binding conformation could play a role as risk marker during the development of diabetes in combination with an increase in age. Our study shows that the active quality of VWF was more important than the quantity.

Concepts: Heyde's syndrome, Diabetes mellitus, Weibel-Palade body, Insulin, Diabetes mellitus type 2, Platelet, Von Willebrand disease, Von Willebrand factor

168

Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII(a), which does not bind VWF. Nano-LC-mass spectrometry analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or FVIIIa. However, Lys773 of peptide Ser766-Leu774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser764-Arg782, which comprises the first 18 amino acid residues of mature VWF, showed a differential modification of both Lys773 and the alpha amino group of Ser764. To verify the role of Lys773 and the N-terminal Ser764 for FVIII binding, we employed VWF variants in which either Lys773 or Ser764 was replaced by an alanine. Surface plasmon resonance analysis and competition studies revealed that VWF-K773A exhibits reduced binding to FVIII and FVIII light chain, which harbors the VWF binding site. In contrast, VWF-S764A revealed more effective binding to FVIII and FVIII light chain as compared to WT-VWF. The results of our study show that the N-terminus of VWF is critical for the interaction with FVIII, and that the residues Ser764 and Lys773 have an opposite role in the binding mechanism.

Concepts: Platelet, Lysine, Surface plasmon resonance, Factor VIII, Von Willebrand disease, Protein, Von Willebrand factor, Amino acid

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Clinically significant portal hypertension (CSPH), defined as hepatic venous pressure gradient (HVPG) ≥10 mmHg, causes major complications. HVPG is not always available, so a non-invasive tool to diagnose CSPH would be useful. VWF-Ag can be used to diagnose. Using the VITRO score (the VWF-Ag/platelet ratio) instead of VWF-Ag itself improves the diagnostic accuracy of detecting cirrhosis/ fibrosis in HCV patients.

Concepts: Medical terms, Cirrhosis, Blood pressure, Portal venous system, Von Willebrand disease, Hepatic portal vein, Vein, Von Willebrand factor

104

Background Acquired thrombotic thrombocytopenic purpura (TTP) is caused by aggregation of platelets on ultralarge von Willebrand factor multimers. This microvascular thrombosis causes multiorgan ischemia with potentially life-threatening complications. Daily plasma exchange and immunosuppressive therapies induce remission, but mortality and morbidity due to microthrombosis remain high. Methods Caplacizumab, an anti-von Willebrand factor humanized single-variable-domain immunoglobulin (Nanobody), inhibits the interaction between ultralarge von Willebrand factor multimers and platelets. In this phase 2, controlled study, we randomly assigned patients with acquired TTP to subcutaneous caplacizumab (10 mg daily) or placebo during plasma exchange and for 30 days afterward. The primary end point was the time to a response, defined as confirmed normalization of the platelet count. Major secondary end points included exacerbations and relapses. Results Seventy-five patients underwent randomization (36 were assigned to receive caplacizumab, and 39 to receive placebo). The time to a response was significantly reduced with caplacizumab as compared with placebo (39% reduction in median time, P=0.005). Three patients in the caplacizumab group had an exacerbation, as compared with 11 patients in the placebo group. Eight patients in the caplacizumab group had a relapse in the first month after stopping the study drug, of whom 7 had ADAMTS13 activity that remained below 10%, suggesting unresolved autoimmune activity. Bleeding-related adverse events, most of which were mild to moderate in severity, were more common with caplacizumab than with placebo (54% of patients vs. 38%). The frequencies of other adverse events were similar in the two groups. Two patients in the placebo group died, as compared with none in the caplacizumab group. Conclusions Caplacizumab induced a faster resolution of the acute TTP episode than did placebo. The platelet-protective effect of caplacizumab was maintained during the treatment period. Caplacizumab was associated with an increased tendency toward bleeding, as compared with placebo. (Funded by Ablynx; ClinicalTrials.gov number, NCT01151423 .).

Concepts: Clinical trial, Coagulation, Idiopathic thrombocytopenic purpura, Thrombocytopenia, Von Willebrand disease, Von Willebrand factor, Platelet, Thrombotic thrombocytopenic purpura

31

Background The development of neutralizing anti-factor VIII alloantibodies (inhibitors) in patients with severe hemophilia A may depend on the concentrate used for replacement therapy. Methods We conducted a randomized trial to assess the incidence of factor VIII inhibitors among patients treated with plasma-derived factor VIII containing von Willebrand factor or recombinant factor VIII. Patients who met the eligibility criteria (male sex, age <6 years, severe hemophilia A, and no previous treatment with any factor VIII concentrate or only minimal treatment with blood components) were included from 42 sites. Results Of 303 patients screened, 264 underwent randomization and 251 were analyzed. Inhibitors developed in 76 patients, 50 of whom had high-titer inhibitors (≥5 Bethesda units). Inhibitors developed in 29 of the 125 patients treated with plasma-derived factor VIII (20 patients had high-titer inhibitors) and in 47 of the 126 patients treated with recombinant factor VIII (30 patients had high-titer inhibitors). The cumulative incidence of all inhibitors was 26.8% (95% confidence interval [CI], 18.4 to 35.2) with plasma-derived factor VIII and 44.5% (95% CI, 34.7 to 54.3) with recombinant factor VIII; the cumulative incidence of high-titer inhibitors was 18.6% (95% CI, 11.2 to 26.0) and 28.4% (95% CI, 19.6 to 37.2), respectively. In Cox regression models for the primary end point of all inhibitors, recombinant factor VIII was associated with an 87% higher incidence than plasma-derived factor VIII (hazard ratio, 1.87; 95% CI, 1.17 to 2.96). This association did not change in multivariable analysis. For high-titer inhibitors, the hazard ratio was 1.69 (95% CI, 0.96 to 2.98). When the analysis was restricted to recombinant factor VIII products other than second-generation full-length recombinant factor VIII, effect estimates remained similar for all inhibitors (hazard ratio, 1.98; 95% CI, 0.99 to 3.97) and high-titer inhibitors (hazard ratio, 2.59; 95% CI, 1.11 to 6.00). Conclusions Patients treated with plasma-derived factor VIII containing von Willebrand factor had a lower incidence of inhibitors than those treated with recombinant factor VIII. (Funded by the Angelo Bianchi Bonomi Foundation and others; ClinicalTrials.gov number, NCT01064284 ; EudraCT number, 2009-011186-88 .).

Concepts: Regression analysis, Multivariate statistics, Epidemiology, Von Willebrand disease, Von Willebrand factor, Factor VIII

28

von Willebrand factor (VWF) is amongst others synthesized by endothelial cells and stored as ultra-large (UL) VWF multimers in Weibel-Palade bodies. Although UL-VWF is proteolysed by ADAMTS13 (a disintegrin-like and metalloprotease domain with thrombospondin type-1 motif, number 13) on secretion from endothelial cells, in vitro experiments in the absence of ADAMTS13 have demonstrated that a proportion of these UL-VWF multimers remain anchored to the activated endothelium. These multimers unravel, bind platelets, and wave in the direction of the flow. These so-called VWF “strings” have also been visualized in vivo, lining the lumen of activated mesenteric veins of Adamts13(-/-) mice. Various studies have demonstrated the extraordinary length of these VWF strings, the availability of their platelet binding and ADAMTS13 cleavage sites, and the possible nature of their endothelial attachment. VWF strings are also capable of tethering leukocytes and parasite-infected red blood cells. However, the majority of studies have been performed in the absence of ADAMTS13, a condition only experienced in thrombotic thrombocytopenic purpura. A normal functional role of VWF strings in healthy persons or in other disease pathologies remains unclear. In this review, we discuss some of the puzzling characteristics of VWF strings, and we debate whether the properties of VWF strings in the absence of ADAMTS13 might be relevant for understanding (patho)physiologic mechanisms.

Concepts: Red blood cell, Weibel-Palade body, Von Willebrand disease, Thrombotic thrombocytopenic purpura, Blood, Coagulation, Platelet, Von Willebrand factor

28

BACKGROUND: The von Willebrand factor (VWF) gene is highly polymorphic, with variants correlated with VWF antigen levels, adhesion activity, clearance, and factor VIII binding. VWF mutations are detected in patients with von Willebrand disease (VWD), whereas polymorphic variants could be associated with thrombosis. However, information on the ethnic diversity of VWF variants and their association with diseases is limited. OBJECTIVES: To characterize novel VWF variants from different ethnicities in the general population. Patients/Methods: We analyzed samples from 1,092 subjects of 14 ethnicities available in the 1000 Genomes database for VWF variants and their potential functional impacts RESULTS: We identified 2,728 SNPs and 91 insertions and deletions that had a high level of ethnic diversity, with Africans having the highest number of variants. The highest level of diversity was found in the D' and D2 domains. Among 94 non-synonymous variants, 31 were predicted to be deleterious, including 19 that were previously associated with VWD. Most of these “VWD variants” had allele frequencies consistent with disease incidence in European subjects; but some had a significantly higher frequency in other ethnicities. The mutation R2185Q, H817Q and M740I associated with type 1 and type 2N VWD were present in more than 13% of African subjects. CONCLUSIONS: These results highlight the complexity of VWF variations in different ethnic groups and emphasize the importance of interrogating variations on multiple ethnic backgrounds for associations with bleeding and thrombosis. © 2012 International Society on Thrombosis and Haemostasis.

Concepts: Weibel-Palade body, Ristocetin induced platelet agglutination, Heyde's syndrome, Platelet, Von Willebrand disease, Von Willebrand factor

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While von Willebrand factor (vWF) has been reported to be elevated in smokers, there are no reports on the effects of smoking on its cleaving protease ADAMTS-13, particularly in subjects of Arab ethnicity. This study was conducted to determine the effects of smoking on vWF and ADAMTS-13 antigen and activity levels in Arab males. Venous blood samples from 80 smoking (at rest) and 80 non-smoking healthy males were collected after asking subjects to fast and refrain from smoking for 8 hours. Similar sampling was done for 40 smokers (acute smokers), who were asked to smoke one cigarette immediately before blood collection. Plasma was used to measure ADAMTS-13 antigen and activity levels, as well as vWF antigen and collagen binding activity levels using commercial ELISA kits. Compared to non-smokers, ADAMTS-13 and vWF activities were significantly lower in smokers at rest (p < 0.05). Acute smokers had significantly higher levels of vWF activity and ADAMTS-13 antigen and activity levels (p < 0.01), compared to smokers at rest. Our results suggest that high vWF activity is accompanied by an increase in ADAMTS-13 activity as a natural physiological mechanism to degrade the elevated vWF molecules. If not followed by a subsequent smoke, the activities of both proteins subside. It is possible that the repeated increase in vWF and constant degradation by ADAMTS-13 results in lower overall levels of both proteins in smokers (at rest) compared to nonsmokers who do not experience a similar (repeated) injury to the endothelium.

Concepts: Heyde's syndrome, ABO blood group system, Weibel-Palade body, Platelet, Hematology, Von Willebrand disease, Blood, Von Willebrand factor

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Acquired von-Willebrand syndrome (AVWS) is described in patients with Waldenström macroglobulinemia (WM). Assessment of ristocetin cofactor activity (VWF:RCo) and von-Willebrand factor (VWF) antigen (VWF:Ag) in 72 consecutive WM patients demonstrated a negative relationship between VWF levels <130 U/dL and both monoclonal immunoglobulin M concentration (mIgMC) and viscosity. Ten patients with VWF:RCo <50 U/dL (<40 for group O patients) fulfilled the AVWS criteria. They had higher mIgMC and viscosity. Reduction in mIgMC was associated with increase in VWF levels. The low VWF:RCo/VWF:Ag ratio suggested that high viscosity might be associated with increased shear force and cleavage of multimers. Surprisingly, 43 patients (59%) presented with high VWF:Ag (>110 U/dL). They had higher bone marrow (BM) microvessel density and vascular endothelial growth factor expression on BM mast cells. Five-year survival rates of patients with VWF:Ag <110, between 110 and 250 and >250 U/dL were 96%, 71%, and 44% respectively (p<0.0001). High VWF:Ag was also a significant adverse prognostic factor for survival after first-line therapy (p<0.0001), independently of the international scoring system. These results support systematic assessment of VWF in WM patients. The adverse prognostic value of high VWF levels raises issues on interactions between lymphoplasmacytic cells, mast cells and endothelial cells in WM.

Concepts: White blood cell, Shear stress, Weibel-Palade body, Viscosity, Ristocetin, Von Willebrand disease, Von Willebrand factor, Immune system