Von Willebrand Factor Multimeric Pattern Research Articles

Haemostaseological Changes of VWF and FVIII during Pregnancy and the Oestrus Cycle in a Porcine Model of Von Willebrand Disease

Pregnancy and the oestrus cycle are challenging for female patients suffering from von Willebrand disease (VWD). Therefore, our study aimed to investigate the changes in von Willebrand factor (VWF) and factor VIII (FVIII) during pregnancy and the oestrus cycle in our porcine model of von Willebrand disease compared with the wild-type. Plasma analyses regarding primary hemostasis, secondary hemostasis, and VWF multimers, as well as immunohistochemistry analyses of VWF in the uterus and ovary, were performed. For levels of VWF and FVIII activities, significant elevations were seen in the last trimester. Primary hemostasis improved towards the end of pregnancy. In the oestrus cycle, significantly lower VWF values can be seen in the immunohistochemistry of the ovaries during the oestrus, while values were highest in the metoestrus. VWF multimer patterns in pigs were similar to the ones in human VWD patients. In summary, the course of VWF and FVIII during pregnancy and the oestrus cycle in porcine VWD were investigated for the first time. The porcine model seems to be suitable for haemostaseological studies on VWD. This provides an advantage for investigating reproduction-related bleeding and understanding the underlying mechanisms of post-partum hemorrhage or miscarriage in women with VWD.

Differences and similarities in endothelial and angiogenic profiles of preeclampsia and COVID-19 in pregnancy

BackgroundCOVID-19 presents a spectrum of signs and symptoms in pregnant women that might resemble preeclampsia. Differentiation between severe COVID-19 and preeclampsia is difficult in some cases.ObjectiveTo study biomarkers of endothelial damage, coagulation, innate immune response, and angiogenesis in preeclampsia and COVID-19 in pregnancy in addition to in vitro alterations in endothelial cells exposed to sera from pregnant women with preeclampsia and COVID-19.Study DesignPlasma and sera samples were obtained from pregnant women with COVID-19 infection classified into mild (n=10) or severe (n=9) and from women with normotensive pregnancies as controls (n=10) and patients with preeclampsia (n=13). A panel of plasmatic biomarkers was assessed, including vascular cell adhesion molecule-1, soluble tumor necrosis factor-receptor I, heparan sulfate, von Willebrand factor antigen (activity and multimeric pattern), α2-antiplasmin, C5b9, neutrophil extracellular traps, placental growth factor, soluble fms-like tyrosine kinase-1, and angiopoietin 2. In addition, microvascular endothelial cells were exposed to patients’ sera, and changes in the cell expression of intercellular adhesion molecule 1 on cell membranes and von Willebrand factor release to the extracellular matrix were evaluated through immunofluorescence. Changes in inflammation cell signaling pathways were also assessed by of p38 mitogen-activated protein kinase phosphorylation. Statistical analysis included univariate and multivariate methods.ResultsBiomarker profiles of patients with mild COVID-19 were similar to those of controls. Both preeclampsia and severe COVID-19 showed significant alterations in most circulating biomarkers with distinctive profiles. Whereas severe COVID-19 exhibited higher concentrations of vascular cell adhesion molecule-1, soluble tumor necrosis factor-α receptor I, heparan sulfate, von Willebrand factor antigen, and neutrophil extracellular traps, with a significant reduction of placental growth factor compared with controls, preeclampsia presented a marked increase in vascular cell adhesion molecule-1 and soluble tumor necrosis factor-α receptor I (significantly increased compared with controls and patients with severe COVID-19), with a striking reduction in von Willebrand factor antigen, von Willebrand factor activity, and α2-antiplasmin. As expected, reduced placental growth factor, increased soluble fms-like tyrosine kinase-1 and angiopoietin 2, and a very high soluble fms-like tyrosine kinase-1 to placental growth factor ratio were also observed in preeclampsia. In addition, a significant increase in C5b9 and neutrophil extracellular traps was also detected in preeclampsia compared with controls. Principal component analysis demonstrated a clear separation between patients with preeclampsia and the other groups (first and second components explained 42.2% and 13.5% of the variance), mainly differentiated by variables related to von Willebrand factor, soluble tumor necrosis factor-receptor I, heparan sulfate, and soluble fms-like tyrosine kinase-1. Von Willebrand factor multimeric analysis revealed the absence of von Willebrand factor high-molecular-weight multimers in preeclampsia (similar profile to von Willebrand disease type 2A), whereas in healthy pregnancies and COVID-19 patients, von Willebrand factor multimeric pattern was normal.Sera from both preeclampsia and severe COVID-19 patients induced an overexpression of intercellular adhesion molecule 1 and von Willebrand factor in endothelial cells in culture compared with controls. However, the effect of preeclampsia was less pronounced than the that of severe COVID-19. Immunoblots of lysates from endothelial cells exposed to mild and severe COVID-19 and preeclampsia sera showed an increase in p38 mitogen-activated protein kinase phosphorylation. Patients with severe COVID-19 and preeclampsia were statistically different from controls, suggesting that both severe COVID-19 and preeclampsia sera can activate inflammatory signaling pathways.ConclusionAlthough similar in in vitro endothelial dysfunction, preeclampsia and severe COVID-19 exhibit distinctive profiles of circulating biomarkers related to endothelial damage, coagulopathy, and angiogenic imbalance that could aid in the differential diagnosis of these entities.

Von Willebrand Factor Multimer Patterns in People Living with Human Immunodeficiency Virus Infection.

Endothelial dysfunction contributes to hypercoagulability in people with HIV (PWH). A surrogate marker of this is elevated von Willebrand factor (VWF) which has been documented in PWH. This study compared VWF multimer patterns in PWH who were anti-retroviral therapy (ART) naive and immune reconstituted on ART. VWF multimer analysis was performed with a semi-quantitative electrophoresis assay on plasma from 79 PWH (39 ART-naive and 40 virally suppressed on ART) and 25 normal control samples. The total optical density mean VWF level was significantly increased in PWH and higher in ART-naive versus ART-exposed, virally suppressed patients (p < 0.0001). No quantitative difference was demonstrated in the multimer distribution pattern between the groups. Our findings suggest that there is no difference in multimer distribution between ART-naive and virally suppressed PWH. Due to a small sample size, confirmation in a larger study is required.

Reclassification of Pre-Diagnosed Von-Willebrand Disease in the Eldery: A Hospital-Based Cohort Study

Background: von Willebrand disease (VWD) is the commonest inherited bleeding disorder caused by reduced levels or reduced function of von Willebrand factor (VWF). With increasing age, VWD patients undergo changes with regard to VWF levels, FVIII levels and bleeding symptoms. As the number of elderly VWD patients is increasing, the pathophysiology of aging in VWD has become clinically relevant. In this two-center cohort study we re-evaluated patients previously diagnosed with VWD with respect to their bleeding phenotype and VWF levels based on age-and blood group (BG)-dependent reference values.1Methods: Since February 2012 in the study center Lübeck and Kiel 969 consecutively admitted patients presenting with hemorrhagic diathesis (HD) one year before laboratory work-up/re-classification were enrolled. Reclassification was performed on the basis of age- and BG-dependent reference values with respect to the presence or absence of VWD (1). 174 of 504 patients aged 1 to 86 years (median 35) suffering from non-acquired and non-VWD-associated clinically relevant HD (Rodeghiero ISTH bleeding score) were diagnosed with VWD type 1 (n=154), type 2 (n=17) or 3 (n=3) within the last two decades prior re-classification based on VWF levels, multimeric pattern and/or genotyping. Laboratory cut-off values for re-classification (VWF:ac & VWF:ag; < 10th percentiles) derived from healthy population-based blood donors (n=1010) were as follows: < 60% (BG non-0: 72%) until the age of 49, < 100% (BG non-0: 120%) patients aged 50 to 59 years, and < 113% (BG non-0: 135%) in subjects > 60 years of age.Results: In the present patient follow-up visits in patients aged 20 to < 49 years 42 of 230 individuals (18,3%) compared to 55 of 131 older patients aged 50 to 86 years (median 66: 42%) the formerly classification of VWD type 1 would have been missed (p<0,001) without using age- and BG-dependent cut-off values. In patients < 20 years of age previous diagnosis were confirmed in all cases (n=39). Of note, inclusion of the variable blood group “0” versus “non-0” in our model has improved the detection rate of previously diagnosed VWD by 5%. Interestingly, although the bleeding phenotype in women with VWD type 1 switched over the aging process from epistaxis (prior puberty) to menorrhagia back to epistaxis and non-vaginal mucosal bleeding the severity of bleeding documented via ISTH score did not mitigate during the aging process.Conclusion: Based on the model of “developmental hemostasis” including not only the youngest but also elderly patients classification of inherited HD especially of VWD by using age- and BG-dependent reference values is suggested in order not to miss patients prone to possible bleeding episodes during emergency or elective interventions DisclosuresNowak Goettl:Octapharma: Research Funding. Kenet:Alnylam: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Opko biologics: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

Platelet activation and aggregation in different centrifugal-flow left ventricular assist devices

Left-ventricular assist devices (LVADs) improve outcomes in end-stage heart failure patients. Two centrifugal-flow LVAD systems are currently approved, HeartMate 3 (HM3) and Medtronic/Heartware HVAD (HVAD). Clinical findings suggest differences in thrombogenicity between both systems. We compared markers of platelet activation and aggregation between HM3 and HVAD. We prospectively included 59 LVAD patients (40 HM3, 19 HVAD). Platelet P-selectin expression, activated glycoprotein (GP) IIb/IIIa and monocyte-platelet aggregates (MPA) were assessed by flow-cytometry. Platelet aggregation was measured by light-transmission aggregometry (LTA) and multiple-electrode aggregometry (MEA). Von-Willebrand factor (VWF) antigen (VWF:Ag), VWF activity (VWF:Ac), and VWF multimer pattern analysis were determined. Soluble P-selectin (sP-selectin) was measured with an enzyme-linked immunoassay. P-selectin, GPIIb/IIIa and MPA levels in vivo and in response to arachidonic acid, adenosine diphosphate, and thrombin receptor activating peptide were similar between HM3 and HVAD (all p > .05). Likewise, agonist-inducible platelet aggregation by LTA and MEA did not differ between HM3 and HVAD (all p > .05). VWF:Ag levels and FVIII:C were similar between both systems (both p > .05), but patients with HVAD had significantly lower VWF:Ac (p = .011) and reduced large VWF multimers (p = .013). Finally, sP-selectin levels were similar in patients with HVAD and HM3 (p = .845). In conclusion, on-treatment platelet activation and aggregation are similar in HM3 and HVAD patients. Potential clinical implications of observed differences in VWF profiles between both LVAD systems need to be addressed in future clinical trials.

High-molecular-weight von Willebrand Factor multimer ratio differentiates true-severe from pseudo-severe classical low-flow, low-gradient aortic stenosis.

Upon high wall shear stress, high-molecular-weight (HMW) von Willebrand Factor (VWF) multimers are degraded, thus, HMW VWF multimer deficiency mirrors haemodynamics at the site of aortic stenosis (AS). The aim of the present study was to analyse the role of HMW VWF multimer ratio for subcategorization of classical low-flow, low-gradient (LF/LG) AS. Eighty-three patients with classical LF/LG AS were prospectively recruited and HMW VWF multimer pattern was analysed using a densitometric quantification of western blot bands. Patients were subclassified into true-severe (TS) and pseudo-severe (PS) classical LF/LG AS based on dobutamine stress echocardiography (DSE). Positive and negative predictive values (PPV/NPV) of HMW VWF multimer ratio for diagnosis of the TS subtype were calculated. HMW VWF multimer ratio in TS classical LF/LG AS was significantly decreased compared to PS classical LF/LG AS (0.86 ± 0.27 vs. 1.06 ± 0.09, P < 0.001). HMW VWF multimer deficiency occurred exclusively in the TS subtype with an optimal PPV of 1.000 and NPV of 0.379. HMW VWF multimer ratio showed a strong correlation with mean transvalvular pressure gradients during DSE (r = -0.616; P < 0.001). HMW VWF multimer ratio measured at baseline was higher compared to levels measured after DSE (0.87 ± 0.27 vs. 0.84 ± 0.31; P = 0.031) indicating DSE-induced increased proteolysis. HMW VWF multimer ratio represents a valuable biomarker for classical LF/LG AS subclassification and mirrors haemodynamics during DSE. HMW VWF multimer ratio identifies the TS subtype without the use of other imaging techniques.

Increased von Willebrand factor levels in polycythemia vera and phenotypic differences with essential thrombocythemia

BackgroundAcquired von Willebrand factor (VWF) deficiency was described in Philadelphia‐negative myeloproliferative neoplasms, especially in essential thrombocythemia (ET). VWF phenotype in contemporary patients with polycythemia vera (PV) remains less explored. ObjectivesTo characterize the VWF phenotype in PV and to compare VWF phenotype in PV with matched healthy subjects and ET patients. Patients/MethodsWe studied 48 PV patients, treated according to current recommendations (hematocrit ≤ 45%, on low‐dose aspirin prophylaxis); 48 healthy and 41 subjects with ET, all sex, age, and blood group matched. We measured VWF antigen, activity, multimeric pattern, ADAMTS‐13, and factor VIII (FVIII) antigen. ResultsIn patients with PV, VWF antigen and activity were significantly higher than in healthy subjects (antigen: 119[96‐137] vs 93[79‐107] IU/dL; activity: 114[95‐128] vs 90[79‐107] IU/dL, respectively, medians and interquartile, P < 0.01), with normal multimeric distribution. ADAMTS‐13 levels were similar between patients with PV and healthy subjects. FVIII levels were higher in PV than in healthy subjects (141[119‐169] versus 98[88‐123] IU/dL, respectively, P < 0.01). By multivariable analysis, JAK2‐p.V617F allelic burden, erythrocyte count, and male sex significantly predicted VWF antigen and activity levels. As compared to patients with ET, patients with PV showed similar VWF antigen levels but approximately 40% higher activity (79[49‐104] vs 112[93‐125] IU/dL, respectively, P < 0.01). ConclusionsPatients with PV show increased VWF and FVIII levels, predicted by JAK2‐p.V617F burden and erythrocyte count. At variance with ET, acquired VWF defect was not observed in PV. High VWF/FVIII levels may sustain the thrombotic diathesis of PV and may be investigated as biomarkers for risk stratification.

Plasma Level of von Willebrand Factor Propeptide at Diagnosis: A Marker of Subsequent Renal Dysfunction in Autoimmune Rheumatic Diseases.

Introduction:Patients with systemic autoimmune rheumatic diseases (SARDs) such as rheumatoid arthritis, systemic lupus erythematosus (SLE), Sjögren syndrome, and systemic sclerosis, which are chronic inflammatory diseases, are prone to develop renal dysfunction, which is related to vascular endothelial cell damage.Material and Methods:We evaluated plasma levels of von Willebrand factor (VWF), VWF propeptide (VWF-pp), disintegrin-like and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), and VWF multimer pattern in patients with SARDs at diagnosis and investigated whether they may serve as markers to identify patients destined to develop renal dysfunction within 1 year. Renal dysfunction was defined as subsequent reduced estimated glomerular filtration rate (eGFR) by >25% or the new appearance of abnormal urine findings such as proteinuria (protein > 30 mg/dL) or hematuria (red blood cells >20/HPF in urine sediments). Overall, 63 patients with SARDs were studied.Results and Conclusions:We observed a significant increase of VWF-pp and a significant decrease of ADAMTS13 in patients with SARDs compared with normal healthy controls. The highest level of VWF-pp was observed in patients with SLE among the groups. The levels of VWF and multimer pattern of VWF were not different compared with normal healthy controls. Von Willebrand factor propeptide predicted a subsequent decrease in eGFR at a cutoff point of 210% (sensitivity, 78.6%; specificity, 73.5%) and new urinary abnormal findings at a cutoff point of 232% (sensitivity, 77.8%; specificity, 77.8%) Using these cutoff points, multivariable analysis revealed that VWF-pp was a significant risk factor for renal dysfunction at an odds ratio of 8.78 and 22.8, respectively, and may lead to a new therapeutic approach to prevent vasculitis and renal dysfunction.

Cryptic non-canonical splice site activation is part of the mechanism that abolishes multimer organization in the c.2269_2270del von Willebrand factor

We report a new pathogenic mechanism in von Willebrand disease involving the use of a non-canonical splicing site. The proband, carrying the homozygous c.2269_2270del mutation previously classified as a type 3 mutation, showed severely reduced plasma and platelet von Willebrand factor antigen levels and functions, and no factor VIII binding capacity. A particular von Willebrand factor multimer pattern emerged in plasma, characterized by the presence of only two oligomers: the dimer and an unusually large band, with no intermediate components. There were von Willebrand factor multimers in platelets, but each band ran more slowly than the normal counterpart. No anti-von Willebrand factor antibodies were detectable. The proband was classified as having severe type 1 von Willebrand disease. Seeking the relationship between phenotype and genotype, we found the c.2269_2270del mutation associated with three different RNA: r.2269_2270del (RNAI), giving a truncated von Willebrand factor protein; r.[2269_2270del;2282_2288del] (RNAII), resulting from activation of a cryptic “AG” splicing site; and r.[2269_2270del;2281_2282insAG] (RNAIII), where the wild-type “AG” acceptor of exon 18 was retained due to the non-canonical 2279-2280 “CG” acceptor splicing site being used. The aberrant RNAII and RNAIII caused the alteration of the furin cleavage and binding sites, respectively, both resulting in a von Willebrand factor protein characterized by the persistence of von Willebrand factor propeptide, as confirmed by western blot analysis of the recombinant mutated von Willebrand factor molecules produced in vitro. Taken together, these findings explain the residual von Willebrand factor synthesis, slower-running multimers, and absent factor VIII binding capacity. The apparently pure gene null mutation c.2269_2270del profoundly alters von Willebrand factor gene splicing, inducing a complex RNA expression pattern.

Device-induced platelet dysfunction in mechanically assisted circulation increases the risks of thrombosis and bleeding.

Thrombotic and bleeding complications are the major obstacles for expanding mechanical circulatory support (MCS) beyond the current use. While providing the needed hemodynamic support, those devices can induce damage to blood, particularly to platelets. In this study, we investigated device-induced alteration of three major platelet surface receptors, von Willebrand factor (VWF) and associated hemostatic functions relevant to thrombosis and bleeding. Fresh human whole blood was circulated in an extracorporeal circuit with a clinical rotary blood pump (CentriMag, Abbott, Chicago, IL, USA) under the clinically relevant operating condition for 4hours. Blood samples were examined every hour for glycoprotein (GP) IIb/IIIa activation and receptor loss of GPVI and GPIbα on the platelet surface with flow cytometry. Soluble P-selectin in hourly collected blood samples was measured by enzyme linked immunosorbent assay to characterize platelet activation. Adhesion of device-injured platelets to fibrinogen, collagen, and VWF was quantified with fluorescent microscopy. Device-induced damage to VWF was characterized with western blotting. The CentriMag blood pump induced progressive platelet activation with blood circulating time. Particularly, GPIIb/IIIa activation increased from 1.1% (Base) to 11% (4hours) and soluble P-selectin concentration increased from 14.1ng/mL (Base) to 26.5ng/mL (4hours). Those device-activated platelets exhibited increased adhesion capacity to fibrinogen. Concurrently, the CentriMag blood pump caused progressive platelet receptor loss (GPVI and GPIbα) with blood circulating time. Specifically, MFI of the GPVI and GPIbα receptors decreased by 17.2% and 16.1% for the 4-hours sample compared to the baseline samples, respectively. The device-injured platelets exhibited reduced adhesion capacities to collagen and VWF. The high molecular weight multimers (HMWM) of VWF in the blood disappeared within the first hour of the circulation. Thereafter the multimeric patterns of VWF were stable. The change in the VWF multimeric pattern was different from the progressive structural and functional changes of platelets with the circulation time. This study suggested that the CentriMag blood pump could induce two opposite effects on platelets and associated hemostatic functions under a clinically relevant operating condition. The device-altered hemostatic function may contribute to thrombosis and bleeding simultaneously as occurring in patients supported by a rotary blood pump. Device-induced damage of platelets may be an important cause for bleeding in patients supported with rotary blood pump MCS systems relative to device-induced loss of HMWM-VWF.

Type 2B von Willebrand disease with or without large multimers: A distinction of the two sides of the disorder is long overdue.

Most, but not all patients with type 2B von Willebrand disease (VWD)—which features gain-of-function mutations in the A1 domain of von Willebrand factor (VWF)—have no circulating large VWF multimers. Similarities and differences were analysed in 33 type 2B patients, 12 with a normal and 21 with an abnormal multimer pattern, to see whether they should be considered separately. The minimum aggregating dose of ristocetin was similarly reduced in both patient groups, and modulated by their underlying VWF mutations. Platelet VWF content was normal in all patients lacking in large multimers, but sometimes reduced in those with a normal multimer pattern. All the former patients and none of the latter had persistent or transient thrombocytopenia. A short VWF half-life (affecting plasma VWF levels) was seen in both groups, but more pronounced in patients without large multimers. Bleeding scores were also high in all patients, but more so in those without large multimers, apparently regardless of their platelet count. The marked phenotypic heterogeneity of type 2B VWD concerns not only patients’ VWF multimer pattern, but also their bleeding risk, and consequently their appropriate treatment too. Hence the need to clearly distinguish between type 2B VWD with normal or abnormal VWF multimers.

Acquired Von Willebrand Syndrome (AVWS) Type 2A as the Presenting Feature of JAK2 Wild type Thrombocythemia in a Child: Effectiveness of Platelet Reduction by Anagrelide

A 9-year-old Caucasian boy presented in 1994 with severe headache, attacks of migraine, aggressive behavior and minor bleeding symptoms. Initial abnormal laboratory data were a platelet count of 1596 × 109/L and slight splenomegaly on echogram. Low-dose aspirin 100 mg/day relieved the cerebral symptoms, but a pronounced spontaneous bleeding tendency became evident. Severe epistaxis, bruises, hematomas and gum bleedings resulted in an iron deficiency state (hemoglobin 5.7 mmol/L,hematocrit 0.30, MCV 77 fl, ferritine 6 ug/l) in November 1995. The combination of mucocutaneous bleeding, high platelet counts (1946 × 109/L), and increase of enlarged megakaryocytes in a bone marrow smear was consistent with the diagnosis of hemorrhagic thrombocythemia. Coagulation studies revealed the presence of an acquired von Willebrand syndrome type 2A featured by a prolonged Ivy bleeding time, near normal factor VIII coagulant activity (0.53 u/ml) and von Willebrand factor (VWF) antigen (0.55 u/ml), low VWF ristocetine cofactor activity (0.29), very low VWF collagen binding activity (0.16 u/ml) and absence of large VWF multimers. A severe epistaxis lasting for several hours stopped immediately after correction of the VWF parameters by substitution of 3000 units Bone marrow histopathology showed a pronounced increase of large immature megakaryocytes with hyperlobulated nuclei and maturation defects of nuclei and cytoplasm, and absence of reticuline fibrosis consistent with the diagnosis of JAK2 wild type thrombocythemia in primary megakaryocytic granulocytic myeloproliferation (PMGM). Initial treatment with hydroxyurea (500 mg daily) followed by anagrelide resulted in correction of platelet count from 2000 × 109/L to near normal (400-600 × 109/L) by), which was associated with relief of bleeding symptoms, and correction of plasma VWF values and VWF multimeric pattern to normal. The subsequent natural history was featured by spontaneous reduction of platelet count to near normal and no progression of JAK2 wild type myeloproliferative neoplasms for more than 13 years follow-up.

Potential Mechanisms for Enhanced Activity of Von Willebrand Factor in Patients with Sickle Cell Disease

Sickle cell disease (SCD) is a hemoglobinopathy characterized by vaso-occlusion and hemolysis. Even at disease baseline, von Willebrand factor (VWF) quantity and activity is markedly increased and correlates with the extent of hemolysis [Chen et al. Blood, 2011, 117:3680].VWF is an adhesive plasma protein capable of binding platelets, and secondarily, erythrocytes, and leukocytes. Its adhesive activity is regulated at several levels: being decreased by proteolysis by the metalloprotease ADAMTS13 [Dong et al. Blood, 2002, 100:4033] or by inhibition of self-association into more adhesive forms by high density lipoprotein (HDL) [Chung et al. Blood, 2016, 127:637], or enhanced by oxidative modification [Chen et al. Blood, 2010, 115:706; Fu et al. Blood, 2011, 118:5283]. Here, we explored several potential mechanisms accounting for the increased total active VWF in SCD patients: endothelial activation, enhanced VWF self-association, and defective cleavage of endogenous VWF because of ADAMTS13 dysfunction and/or VWF resistance to cleavage due to oxidation.Methods: We analyzed plasma collected from 12 adult SCD patients at disease baseline; for 3 of these we also have samples collected during vaso-occlusive crisis. We also analyzed multiple crisis samples from one additional patient with no baseline sample. To determine if SCD plasma could activate endothelial cells, we incubated plasma with human umbilical vein endothelial cells (HUVECs) cultured in parallel-plate flow chambers for 20 min at 37˚C then washed the plasma away before perfusing fixed platelets through the chamber. The number and length of platelet-decorated VWF strings were then quantified as a measure of endothelial activation. The plasma levels of VWF, apolipoprotein A-I (ApoA-I, the major apolipoprotein in HDL), myeloperoxidase (MPO), and ADAMTS13 were measured by sandwich ELISA. The activity of ADAMTS13 was evaluated by 1) cleavage of a small VWF A2 peptide substrate; 2) cleavage of multimeric VWF in patient plasma in 1.5 M urea and examining the VWF multimer pattern over time; 3) cleavage of preformed VWF-platelet strings on activated HUVECs; and 4) examining the extent of VWF cleavage in vivo using mass spectrometry (MS). The oxidation of specific methionine residues in VWF and ADAMTS13 from patient plasma was measured by MS as described previously [Chen et al. Blood, 2010, 115:706; Wang et al. JBC, 2015, 290:1422].Results: 1) HUVECs incubated with SCD plasma were activated and produced 2 to 25 fold more VWF strings than cells treated with pooled normal plasma (PNP). Activation was inhibited if the plasma was pre-incubated with hemopexin (0.5 mg/ml), implicating heme as an activator. However, purified heme alone even at high concentration did not activate HUVECs to secrete VWF strings, indicating that heme is required but not sufficient. 2) The ratio of VWF to ApoA-I was markedly elevated in SCD patients, especially during crisis (2.3 ± 0.7 fold of PNP), indicating predisposition to VWF self-association. 3) ADAMTS13 from SCD plasma was defective in cleaving VWF strings (11% to 57% of PNP) and multimeric VWF, but not the A2 peptide substrate. 4) Almost all of the SCD plasma had elevated MPO (3.8 ± 2.5 and 12.8 ± 7.6 fold of PNP in baseline and crisis samples, respectively). 5) Our previous studies showed that HOCl oxidation of VWF Met1606 at the ADAMTS13 cleavage site rendered the VWF noncleavable, and oxidation at other sites increased its platelet-binding activity. We also found that HOCl can inactivate ADAMTS13 by oxidizing Met249 within the metalloprotease domain. We therefore evaluated the extent of VWF Met1606 and ADAMTS13 Met249 oxidation, and VWF cleavage by ADAMTS13 in vivo in patient plasma using tandem MS. We found that ADAMTS13 in SCD plasma had elevated oxidation at Met249 (1.6 ± 0.2 fold of normal controls); VWF cleavage by ADAMTS13 in vivo was decreased by as much as 40% in several of these patients, and the extent of cleavage correlated negatively with the extent of VWF Met1606 oxidation.Summary: Multiple mechanisms contribute to enhanced VWF activity in SCD patients. These include increased secretion by activated endothelial cells, enhanced VWF self-association, and defective cleavage of VWF by endogenous ADAMTS13, partially due to oxidative modification of both VWF and ADAMTS13. DisclosuresNo relevant conflicts of interest to declare.

Endothelial activation, haemostasis and thrombosis biomarkers in Ugandan children with severe malaria participating in a clinical trial.

BackgroundMalaria is a major cause of morbidity and mortality in sub-Saharan Africa, and poor outcomes have been associated with endothelial activation. In this study, biomarkers of endothelial activation, haemostasis, and thrombosis were measured in Ugandan children with severe malaria who participated in a clinical trial, in order to investigate associations between these processes.MethodsSerum and plasma were collected from participants at baseline (day 1), and on days 2, 3, 4, and 14. Von Willebrand factor (VWF) antigen was measured in stored plasma samples from all trial participants, and its association with mortality and changes over time were analysed. VWF multimer patterns were evaluated in baseline serum samples by gel electrophoresis followed by Western blotting. Levels of angiopoietins 1 and 2, VWF antigen, total active VWF, ADAMTS13, platelet counts, apolipoprotein A1, and syndecan-1 were measured in stored serum samples from 12 survivors at baseline and day 4.ResultsVWF antigen levels were associated with mortality, and decreased over time in survivors. Baseline VWF antigen and total active VWF levels were elevated, and very large multimers were present in the baseline serum of several patients. Higher platelet counts were associated with higher angiopoietin-1 and apolipoprotein A1 levels, while lower platelet counts were associated with higher syndecan-1, a marker of endothelial damage. Higher angiopoietin-2 to angiopoietin-1 ratio and higher syndecan-1 levels were correlated with lower apolipoprotein A1 levels. There were no correlations between total active VWF, VWF antigen, or ADAMTS13 levels and the other biomarkers at baseline. Changes in biomarker levels between baseline and day 4 were not correlated.ConclusionsThese results confirm that severe malaria is associated with endothelial activation, and suggest that endothelial activation contributes to microvascular thrombosis and endothelial damage.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1106-z) contains supplementary material, which is available to authorized users.

N-Acetylcysteine Treatment in Two Patients with Relapsed Thrombotic Thrombocytopenic Purpura Increased ADAMTS13 Activity, Free Thiol Concentration in Plasma, and Inhibited Platelet Activation

▪Introduction: Thrombotic thrombocytopenic purpura (TTP) is a catastrophic and potentially fatal disorder caused by systemic microvascular thrombosis due to von Willebrand factor (VWF)-platelet thrombi. TTP is caused by congenital or acquired deficiency of the plasma metalloprotease ADAMTS13. Based on an earlier study (Chen J et al., J Clin Invest 2011, 121:593-603), we proposed N-acetylcysteine (NAC) as an adjunct treatment for TTP. This study showed that NAC reduced the size and activity of VWF in vitro in human plasma and in vivo in a TTP mouse model. In 2013 and 2014, two case reports described treatment of refractory TTP patients with NAC, one receiving a low dose of NAC [300 mg/kg (total 15 g) for the 1st 24 hrs, followed by 2.5 g/day for two weeks concurrently with plasma exchange] (Shortt J et al., N Engl J Med 2013, 368: 90-92; Shortt J et al., Transfusion 2014, 54:2362-2363) and the other receiving high-dose NAC [300 mg/kg/day (11 g/day) for 10 days between plasma exchanges] (Li GW et al. Transfusion 2014, 54: 1221-1224). The patient treated with high-dose NAC improved rapidly (the patient woke up from coma 18 hr after NAC treatment was initiated), but the patient treated with lower dose NAC did not appear to respond. Thus, it is as yet unclear whether NAC is an effective treatment for TTP. Therefore, more clinical studies and detailed analyses are required to examine the effects of NAC in TTP patients. Here we report the results of clinical and biochemical studies on two patients with relapsed TTP treated with NAC. Before, during, and after NAC treatment, we determined the concentrations of NAC, cysteine, and glutathione in plasma; VWF concentration, multimer structure, and functions; ADAMTS13 concentration and activity; and platelet counts and activation status (P-selectin expression and phosphatidylserine exposure).Methods: Two females with a history of prior episodes of TTP presented with acute TTP [ADAMTS13 < 10%, positive for ADAMTS13 inhibitors, platelet count ≤ 10,000/uL, lactate dehydrogenase (LDH) > 600 IU/L] and both were treated with NAC per IRB-approved protocol [150 mg/kg bolus over 1 hr and 150 mg/kg as continuous infusion until the next therapeutic plasma exchange (TPE)]. They received daily TPE until their platelet counts normalized, and intravenous NAC during days 2-5. Blood was collected daily for 8 days for research assays. ADAMTS13 concentrations in patient plasma were measured by ELISA. ADAMTS13 activity was measured using HRP-conjugated A2 peptide substrate (Wu J-J et al. J Thromb Haemost 2006, 4:129-136). Concentrations of NAC, total cysteine, and total free thiols (free thiol cysteine and free thiol NAC) in plasma were determined by mass spectrometry. Plasma VWF multimer patterns were analyzed by 1.5% agarose gel electrophoresis followed by western blotting with an HRP-conjugated polyclonal VWF antibody. Platelets in whole blood were labeled for platelet markers (CD41a or CD42b) together with one of the activation markers, P-selectin or phosphatidylserine (lactadherin). The labeled platelets were analyzed by flow cytometry.Results: Platelet counts in both patients started to increase 1 day after NAC infusion and continued to increase after discontinuation of NAC and TPE. After NAC infusion, the free thiol concentration (NAC and cysteine) in plasma increased 4 and 59 fold in patients 1 and 2, respectively. This was accompanied by increasing ADAMTS13 specific activity (ADAMTS13 activity/ADAMTS13 antigen). In patient 1, the specific activity increased from 127% (prior to NAC infusion but after TPE) to 270% during NAC infusion; in patient 2, the specific activity increased from 56% to 86%. In patient 1, the VWF multimer size decreased during NAC treatment and the VWF multimers migrated slightly faster. NAC also appeared to inhibit platelet activation. Before NAC infusion, the platelets in both patients were positive for phosphatidylserine (PS, > 30%) and P-selectin (> 15%), compared to 2% and 5%, respectively, in a normal control. The percentages of PS- and P-selectin-positive platelets decreased to less than 18% and 10% respectively, during NAC treatment.Summary: NAC treatment of two patients with TTP in conjunction with TPE was well tolerated and associated with recovery of platelet count and LDH, increased ADAMTS13 specific activity and total free thiol concentration in plasma, reduced platelet activation, and decreased VWF multimer size in one patient. DisclosuresKonkle:CSL Behring: Consultancy; Pfizer: Consultancy; Baxalta: Consultancy, Research Funding; Biogen: Consultancy, Research Funding; Octapharma: Research Funding; Novo Nordisk: Consultancy.

Variable content of von Willebrand factor mutant monomer drives the phenotypic variability in a family with von Willebrand disease

AbstractVon Willebrand disease (VWD) is an inherited bleeding disorder characterized by incomplete penetrance and variable expressivity. We evaluated a 24-member pedigree with VWD type 2 caused by a T>G mutation at position 3911 that predicts a methionine to arginine (M1304R) change in the platelet-binding A1 domain of von Willebrand factor (VWF). This mutation manifests as an autosomal-dominant trait, with clinical and biochemical phenotypic variability among affected individuals, including differences in bleeding tendency and VWF quantity, activity, and multimer pattern. Sequencing of all VWF coding regions in 3 affected individuals did not identify additional mutations. When expressed in heterologous cells, M1304R was secreted in lower quantities, failed to drive formation of storage granules, and was defective in multimerization and platelet binding. When cotransfected in equal quantities with the wild-type complementary DNA, the mutant complementary DNA depressed VWF secretion, although multimerization was only mildly affected. A llama nanobody (AU/VWFa-11) that detects the mutant A1 domain demonstrated highly variable binding to VWF from different affected members, indicating that the VWF contained different percentages of mutant monomers in different individuals. Thus, the observed variability in VWD phenotypes could in part be determined by the extent of mutant monomer incorporation in the final multimer structure of plasma VWF.

Von Willebrand factor as a biological sensor of blood flow to monitor percutaneous aortic valve interventions.

Percutaneous aortic valve procedures are a major breakthrough in the management of patients with aortic stenosis. Residual gradient and residual aortic regurgitation are major predictors of midterm and long-term outcome after percutaneous aortic valve procedures. We hypothesized that (1) induction/recovery of high molecular weight (HMW) multimers of von Willebrand factor defect could be instantaneous after acute changes in blood flow, (2) a bedside point-of-care assay (platelet function analyzer-closure time adenine DI-phosphate [PFA-CADP]), reflecting HMW multimers changes, could be used to monitor in real-time percutaneous aortic valve procedures. To investigate the time course of HMW multimers changes in models and patients with instantaneous induction/reversal of pathological high shear and its related bedside assessment. We investigated the time course of the induction/recovery of HMW multimers defects under instantaneous changes in shear stress in an aortic stenosis rabbit model and in patients undergoing implantation of a continuous flow left ventricular assist device. We further investigated the recovery of HMW multimers and monitored these changes with PFA-CADP in aortic stenosis patients undergoing transcatheter aortic valve implantation or balloon valvuloplasty. Experiments in the aortic stenosis rabbit model and in left ventricular assist device patients demonstrated that induction/recovery of HMW multimers occurs within 5 minutes. Transcatheter aortic valve implantation patients experienced an acute decrease in shear stress and a recovery of HMW multimers within minutes of implantation which was sustained overtime. In patients with residual high shear or with residual aortic regurgitation, no recovery of HMW multimers was observed. PFA-CADP profiles mimicked HMW multimers recovery both in transcatheter aortic valve implantation patients without aortic regurgitation (correction) and transcatheter aortic valve implantation patients with aortic regurgitation or balloon valvuloplasty patients (no correction). These results demonstrate that variations in von Willebrand factor multimeric pattern are highly dynamic, occurring within minutes after changes in blood flow. It also demonstrates that PFA-CADP can evaluate in real time the results of transcatheter aortic valve procedures.

Von Willebrand Factor As a Biological Sensor of Blood Flow in Percutaneous Cardiac Procedures

▪Background:Acquired deficiency of von Willebrand Factor (VWF) characterized by a quantitative loss of high molecular weight (HMW) multimers of VWF is associated with cardiovascular disorders such as aortic stenosis. It has been shown that HMW-multimers defect is usually corrected after surgical aortic valve replacement. However, the initial time course of loss/recovery of VWF HMW-multimers following acute changes in blood flow in-vivo has not yet been studied. We hypothesized that recovery of HMW-multimers could occur within minutes following correction of the underlying “high shear” condition and as such could be used to monitor acute changes in flow induced by cardiac interventions. We further investigated the potential underlying mechanisms.Methods:We investigated the time course of HMW-multimers loss/recovery in an animal model of instantaneous and reversible aortic stenosis (AS) specifically developed for that purpose. This model allowed the evaluation, in the same rabbit, of the dynamic time course of loss and recovery of HMW-multimers. We further investigated the time course of HMW-multimers loss/recovery and its related bedside whole blood assessment (PFA-100 analyzer) in 28 patients included in the WITAVI (Willebrand-TAVI) registry and undergoing 1) implantation of an axial-continuous-flow-LVAD (HeartMate-IIÒ, n=8) and 2) transcatheter aortic valve procedures, either BAV (n=10) or TAVI (n=10). VWF antigen (VWF:Ag) and VWF propeptide (VWFpp) levels were measured by ELISA. VWF multimeric analysis was performed as previously described in patients and newly developed for rabbits. PFA-CADP was assessed by platelet-function analyzer PFA-100® using ADP cartridges.Results:In the rabbit model, induction of aortic stenosis was associated with a HMW-multimers defect (normalized ratio = 0.74±0.07; p<0.01 versus no stenosis). Partial recovery of HMW-multimers (0.89±0.12 versus AS baseline; p<0.01) occurred within 5 minutes of stenosis reversion. Thirty minutes after reversion, a complete recovery of HMW-multimers was observed (0.98±0.10). In patients with AS (n=18) a VWF HMW-multimers defect was observed at baseline (0.50±0.18). Among patients with AS, those treated with TAVI experienced an acute decrease in shear stress (4.47±0.41 m.s-1 to 1.81±0.43 m.s-1, p<0.0001, n=10) after treatment while those undergoing BAV alone experienced a modest improvement in shear stress conditions (4.47±0.25m.s-1 to 3.88±0.65m.s-1; p=0.03, n=8). In patients undergoing TAVI (n=10), similar to what was observed in the rabbit model, recovery of HMW-multimers defect was observed within minutes of valve implantation (p<0.001, Table1). By contrast, in patients undergoing BAV (n=8) no recovery of HMW-multimers defect was observed (p=0.21,Table1). A potential role of the vascular endothelium in the HMW-multimers recovery was also evaluated by measuring the VWFpp levels during TAVI and BAV procedures. In TAVI procedures, VWFppsignificantly increased 5 minutes after valve implantation, and further after 30 and 180 minutes when compared to baseline. In BAV procedures, VWFpp did not increase significantly overtime.The time course of PFA-CADP time mimicked the recovery of HMW-multimers defect both in TAVI in patients, in whom a rapid correction of PFA-CADP was observed (p<0.001, Table1), and BAV patients, in whom no correction was observed (p=0.69;Table1). Results are expressed as mean±SD, p values are for overall time course comparison (repeated Anova).Abstract 474. TableTranscatheter aortic valve implantion(TAVI, n=10)Balloon Aortic Valvuloplasty(BAV, n=8)T0T5T30T180pT0T5T30T180pHMW-multimers(ratio vs NP)0.47(±0.14)0.87(±0.24)0.93(±0.17)0.99(±0.16)<0.0010.52(±0.21)0.58(±0.20)0.66(±0.25)0.63(±0.10)nsVWFpp(UI/dL)168(±71)195(±63)215(±100)429(±195)p<0.01256(±121)234(±106)290(±96)275(±136)nsPFA-CT ADP(sec)252(±68)193(±51)116(±40)94(±26)<0.001201(±46)212(±61)204(±71)219(±76)nsConclusion:These results demonstrate that variations in the multimeric pattern of VWF are highly dynamic, occurring within minutes of changes in shear stress status. They further demonstrate that VWF multimeric pattern changes could be used in clinical practice as a tool to monitor the quality of the result of percutaneous aortic valve procedures procedures using a bedside analyzer. DisclosuresNo relevant conflicts of interest to declare.

Subcellular Localization of Von Willebrand Factor Mutants Correlates with Particular VWF Multimer Patterns

Abstract 98Von Willebrand disease (VWD) is a bleeding disorder caused by mutations of von Willebrand factor (VWF), a huge multimeric glycoprotein that is essential for platelet-dependent primary hemostasis. VWF undergoes a highly complex biosynthesis that starts with the production of the 250 kD monomers that are further post-translationally modified within the ER. Here, N-linked glycosylation occurs and dimerization is facilitated by the formation of interchain disulfide bonds. These dimers are being released from the ER to the Golgi apparatus where the high mannose glycans are processed to the complex form and O-linked oligosaccharides are added. The pH-dependent assembly of VWF High Molecular Weight Multimers (HMWM), up to 80 monomers in size, occurs in the trans-Golgi where they become organized in tubules that are subsequently stored in Weibel-Palade bodies (WPB) until stimulated release into the circulation. This process may partly or completely be disrupted, in particular in several VWD type 2A subtypes (IIC, IID, IIE) but also in some VWD type 3 mutants with missense mutations. Mutant VWF in such patients is characterized by lack or a significant decrease of VWF HMWM which can be due to impaired dimerization and multimerization or poor secretion. The phenotypes are ranging from a relative decrease of HMWM (IIE, IID) over a severe reduction (IIC) to their complete absence beyond the size of a dimer in some patients with VWD type 3. To identify the cause of VWF HMWM deficiency in type 2A (subtypes IIC, IID and IIE) and type 3 VWD patients with known or novel missense mutations, we overexpressed representative VWF mutants in HEK293 cells. Confocal immunofluorescence was employed to investigate their intracellular localization by parallel visualization of cellular sub-compartments using antibodies against marker proteins for the ER, Golgi, endosomes and pseudo-WPB.The type IIC mutants (I94N, L526S, and G550R) as well as the IID mutants (C2771R, S2775C and G2752D) did not form any cigar-shaped pseudo-WPB and were almost completely localized within the ER where small granules were formed. The type 2A/IIE mutants W1144G, Y1146C and C1169W in contrast exhibited a massive increase in pseudo-WPB formation with most of their contents remaining intracellularly as a result of poor secretion. VWF Type 3 mutants (W377C, W1120S and C2304Y) are retained in the ER in a similar pattern as type IIC and IID mutants.In conclusion, we show that type 2A (IIC, IID, IIE), and type 3 VWF mutations correspond to a discrete disruption in the VWF intracellular transport, resulting in a characteristic subcellular localization of the particular VWF mutants and correlating with a particular VWF multimer pattern. The respective localization is indicative for the processing and secretion defect of VWF which is ultimately responsible for the manifestation of a specific VWD phenotype. Disclosures:No relevant conflicts of interest to declare.

In Vivo Interrogation of a Human Von Willebrand Type A Domain.

Abstract 2176Proteins containing Von Willebrand A domains play critical roles in hemostasis and arterial thrombosis by promoting cell-cell and cell-ligand interactions. Central to these processes is the formation of an adhesive bond between the A1 domain containing protein Von Willebrand Factor (VWF) and its receptor on platelets known as GPIbα. Although ex-vivo approaches have broadened our understanding of the biophysical properties that govern the interaction between this human receptor–ligand pair, validation of hypotheses based on these intriguing studies will require an appropriate biological model that permits the study of such complex adhesive interactions under appropriate hemodynamic conditions; only then will it be possible to truly ascertain the biological relevance of in vitro observations as they pertain to hemostasis and thrombosis in humans. Moreover, a biological model that permits the study of the function of the human VWF-A1 domain in vivo will be of tremendous value in the development of drugs for use in disorders associated with thrombotic microangiopathy.Given the importance of the interaction between GPIbα and the A1 domain of VWF in hemostasis and thrombus formation in humans, we genetically modified the murine VWF genomic sequence so that it contains the majority of the human A1 domain (VWF HA1) in lieu of its murine counterpart. Mutant mice were viable, born in expected mendelian ratio, and had platelet counts comparable to WT littermates. Importantly, VWF gene transcription, antigen levels, and multimer pattern were also equivalent to WT controls. In contrast, hemostasis was significantly disrupted in homozygous VWF HA1 mice with majority of animals continuing to bleed for >10 minutes after removal of the distal tip of the tail. Evidence that the defect in hemostasis was due to the inability of murine platelets to accumulate at sites of vascular damage is demonstrated by a 4-fold reduction in thrombus size in response to laser-induced arteriolar injury in the microcirculation of the cremaster muscle. To further evaluate the ability of this modified VWF to support interactions with murine platelets, we surfaced immobilized plasma VWF from these animals onto a glass cover slip, which was then incorporated into a parallel plate flow system. WT mouse blood infused over the immobilized substrate was unable to support any significant interactions with plasma VWF HA1 (wall shear rate of 1,600s−1). By contrast, human platelets rapidly accumulated on surface-immobilized VWF HA1 at levels observed for its human plasma counterpart; human platelets administered to VWF HA1 mice were also capable of restoring hemostasis in these animals and formed occlusive thrombi in response to laser-induced arterial injury. Remarkably, the human A1 domain contained within murine VWF was able to support ristocetin-induced human platelet agglutination whereas wild-type murine plasma VWF did not. We are now taking the next important steps in defining the in vivo consequences of altered bond formation between the human VWF-A1 domain and GPIbα as well as evaluating the anti-thrombotic effects of compounds designed to disrupt this interaction in patients with disease-associated thrombotic microangiopathy. Disclosures:No relevant conflicts of interest to declare.