Von Willebrand Factor Multimer Size Research Articles

First 24-Hour-Long Intensive Care Unit Testing of a Clinical-Scale Microfluidic Oxygenator in Swine: A Safety and Feasibility Study.

Microfluidic membrane oxygenators are designed to mimic branching vasculature of the native lung during extracorporeal lung support. To date, scaling of such devices to achieve clinically relevant blood flow and lung support has been a limitation. We evaluated a novel multilayer microfluidic blood oxygenator (BLOx) capable of supporting 750-800 ml/min blood flow versus a standard hollow fiber membrane oxygenator (HFMO) in vivo during veno-venous extracorporeal life support for 24 hours in anesthetized, mechanically ventilated uninjured swine (n = 3/group). The objective was to assess feasibility, safety, and biocompatibility. Circuits remained patent and operated with stable pressures throughout 24 hours. No group differences in vital signs or evidence of end-organ damage occurred. No change in plasma free hemoglobin and von Willebrand factor multimer size distribution were observed. Platelet count decreased in BLOx at 6 hours (37% dec, P = 0.03), but not in HFMO; however, thrombin generation potential was elevated in HFMO (596 ± 81 nM·min) versus BLOx (323 ± 39 nM·min) at 24 hours ( P = 0.04). Other coagulation and inflammatory mediator results were unremarkable. BLOx required higher mechanical ventilator settings and showed lower gas transfer efficiency versus HFMO, but the stable device performance indicates that this technology is ready for further performance scaling and testing in lung injury models and during longer use conditions.

Safety and Pharmacokinetics of Recombinant ADAMTS13 in Patients with Sickle Cell Disease: A Phase 1 Randomized, Double-Blind, Placebo-Controlled Study

Background Sickle cell disease (SCD) is an autosomal recessive hemoglobinopathy associated with chronic hemolysis and vaso-occlusive crises (VOCs). Despite the use of treatments such as hydroxyurea, many patients continue to experience VOCs. There is emerging evidence implicating the dysregulation of the von Willebrand factor (VWF)-ADAMTS13 axis in the pathophysiology of VOCs. The size and platelet-binding activity of ultra-large VWF multimers is regulated by the VWF-cleaving metalloprotease ADAMTS13; therefore, the administration of recombinant ADAMTS13 (rADAMTS13), particularly during VOCs, may be therapeutically beneficial in patients with SCD. Here, we present findings from a phase 1 study (NCT03997760) investigating the safety and pharmacokinetics (PK) of three doses of rADAMTS13 (TAK-755; Takeda Development Center Americas, Inc., Lexington, MA, USA) in patients with SCD. Aims To assess the safety, tolerability, and PK of 40 IU/kg, 80 IU/kg, and 160 IU/kg rADAMTS13 in patients with SCD at baseline health. Methods This phase 1, randomized, double-blind, placebo-controlled, multicenter, ascending single dose study (NCT03997760) enrolled patients aged between 18 and 65 years with a documented history of HbSS or HbSβ o thalassemia. Concurrent treatment with a stable dose of hydroxyurea was permitted. Patients were at baseline health: those diagnosed with acute VOC in the 21 days prior to dose administration were excluded. Patients who had received a blood transfusion within 30 days of screening were also excluded. Patients were randomized 3:1 to receive a single intravenous infusion of either rADAMTS13 or placebo in three sequential dose cohorts: cohort 1, 40 IU/kg; cohort 2, 80 IU/kg; cohort 3, 160 IU/kg. Patients were followed for 28 days post-infusion. Each dose cohort was opened sequentially following a review of safety data by a dose escalation committee. Primary safety endpoints included treatment-emergent adverse events (TEAEs), serious TEAEs, and the incidence of neutralizing antibodies against ADAMTS13. Secondary endpoints included an assessment of PK, comprising the maximum concentration (C max) and the area under the concentration-time curve (AUC) from zero (pre-dose) to 72 hours post-dose (AUC 0-72) estimated from ADAMTS13 antigen and activity levels, among other parameters. Results Five patients received placebo and 14 patients received a single dose of rADAMTS13 (cohort 1, n=4; cohort 2, n=6; cohort 3, n=4). The median age (range) of patients who received placebo was 36.0 (20-43) years and 2 (40.0%) were male. Median age (range) of patients who received rADAMTS13 was 38.5 (23-50) years and 9 (64.3%) were male. TEAEs were reported in 2 (40.0%) patients who received placebo and 8 (57.1%) patients who received rADAMTS13. TEAEs considered related to rADAMTS13 were reported in 4 patients (28.6%; Table). One serious TEAE (sickle cell anemia with crisis) not considered related to rADAMTS13 was reported in a patient who received 40 IU/kg rADAMTS13. No bleeding-related TEAEs were reported during the study and no TEAEs led to study discontinuation or death. No anti-ADAMTS13 neutralizing antibodies were detected within 28 days following rADAMTS13 administration. For the PK assessment, mean (standard deviation [SD]) baseline ADAMTS13 antigen was 0.68 (0.16) μg/mL, and rADAMTS13 demonstrated a dose-proportional increase in ADAMTS13 antigen level ( Figure). For cohorts 1, 2, and 3, respectively, baseline-adjusted ADAMTS13 antigen mean C max was 0.51 μg/mL (n=4), 1.07 μg/mL (n=5), and 2.08 μg/mL (n=4), and mean AUC 0-72 was 18.21 h*μg/mL (n=3), 43.01 h*μg/mL (n=5), and 74.51 h*μg/mL (n=4). Mean (SD) baseline ADAMTS13 activity was 1.06 (0.31) IU/mL. For cohorts 1, 2 and 3, respectively, mean C max for baseline-adjusted ADAMTS13 activity was 0.94 IU/mL (n=4), 2.09 IU/mL (n=5), and 3.08 IU/mL (n=4). Conclusions No safety signals or dose-limiting toxicities, such as bleeding events, were observed for the three evaluated doses of rADAMTS13 up to 160 IU/kg, and no rADAMTS13-related serious TEAEs were reported. No anti-ADAMTS13 neutralizing antibodies were detected during the study. rADAMTS13 demonstrated a dose-proportional increase in ADAMTS13 antigen levels, which is consistent with linear PK. Findings from this study support the further investigation of rADAMTS13 as a potential therapeutic option for patients with SCD.

Coagulation factor VIII regulates von Willebrand factor homeostasis invivo

BackgroundCoagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex, but each has its distinct functions. Binding of FVIII to VWF results in a prolongation of FVIII’s half-life in circulation and modulates FVIII’s immunogenicity during hemophilia therapy. However, the biological effect of FVIII and VWF interaction on VWF homeostasis is not fully understood. ObjectivesTo determine the effect of FVIII in VWF proteolysis and homeostasis in vivo. MethodsMouse models, recombinant FVIII infusion, and patients with hemophilia A on a high dose FVIII for immune tolerance induction therapy or emicizumab for bleeding symptoms were included to address this question. ResultsAn intravenous infusion of a recombinant B-domain less FVIII (BDD-FVIII) (40 and 160 μg/kg) into wild-type mice significantly reduced plasma VWF multimer sizes and its antigen levels; an infusion of a high but not low dose of BDD-FVIII into Adamts13+/- and Adamts13-/- mice also significantly reduced the size of VWF multimers. However, plasma levels of VWF antigen remained unchanged following administration of any dose BDD-FVIII into Adamts13-/- mice, suggesting partial ADAMTS-13 dependency in FVIII-augmented VWF degradation. Moreover, persistent expression of BDD-FVIII at ∼50 to 250 U/dL via AAV8 vector in hemophilia A mice also resulted in a significant reduction of plasma VWF multimer sizes and antigen levels. Finally, the sizes of plasma VWF multimers were significantly reduced in patients with hemophilia A who received a dose of recombinant or plasma-derived FVIII for immune tolerance induction therapy. ConclusionOur results demonstrate the pivotal role of FVIII as a cofactor regulating VWF proteolysis and homeostasis under various (patho)physiological conditions.

Proximity proteomics identifies septins and PAK2 as decisive regulators of actomyosin-mediated expulsion of von Willebrand factor

AbstractIn response to tissue injury, within seconds the ultra-large glycoprotein von Willebrand factor (VWF) is released from endothelial storage organelles (Weibel−Palade bodies) into the lumen of the blood vasculature, where it leads to the recruitment of platelets. The marked size of VWF multimers represents an unprecedented burden on the secretory machinery of endothelial cells (ECs). ECs have evolved mechanisms to overcome this, most notably an actomyosin ring that forms, contracts, and squeezes out its unwieldy cargo. Inhibiting the formation or function of these structures represents a novel therapeutic target for thrombotic pathologies, although characterizing proteins associated with such a dynamic process has been challenging. We have combined APEX2 proximity labeling with an innovative dual loss-of-function screen to identify proteins associated with actomyosin ring function. We show that p21 activated kinase 2 (PAK2) recruits septin hetero-oligomers, a molecular interaction that forms a ring around exocytic sites. This cascade of events controls actomyosin ring function, aiding efficient exocytic release. Genetic or pharmacological inhibition of PAK2 or septins led to inefficient release of VWF and a failure to form platelet-catching strings. This new molecular mechanism offers additional therapeutic targets for the control of thrombotic disease and is highly relevant to other secretory systems that employ exocytic actomyosin machinery.

Patient specific approach to analysis of shear-induced platelet activation in haemodialysis arteriovenous fistula.

Shear-induced platelet activation (SIPAct) is an important mechanism of thrombosis initiation under high blood flow. This mechanism relies on the interaction of platelets with the von Willebrand factor (VWF) capable of unfolding under high shear stress. High shear stress occurs in the arteriovenous fistula (AVF) commonly used for haemodialysis. A novel patient-specific approach for the modelling of SIPAct in the AVF was proposed. This enabled us to estimate the SIPAct level via computational fluid dynamics. The suggested approach was applied for the SIPAct analysis in AVF geometries reconstructed from medical images. The approach facilitates the determination of the SIPAct level dependence on both biomechanical (AVF flow rate) and biochemical factors (VWF multimer size). It was found that the dependence of the SIPAct level on the AVF flow rate can be approximated by a power law. The critical flow rate was a decreasing function of the VWF multimer size. Moreover, the critical AVF flow rate highly depended on patient-specific factors, e.g., the vessel geometry. This indicates that the approach may be adopted to elucidate patient-specific thrombosis risk factors in haemodialysis patients.

Thrombosis and Von Willebrand Factor in Chuvash Polycythemia

Background: In Chuvash polycythemia (CP), homozygous germline VHLR200W results in augmented hypoxia-sensing, elevated erythropoietin and hemoglobin, and increased morbidity and mortality from venous and arterial thromboses.1 In CP patients and controls from Russia's Chuvash Republic, the probability of new thrombosis in CP increased with age, smoking, and higher expression of THBS1, the gene encoding thrombospondin-1.2 In mice, hypoxia leads to upregulation of THBS1 through HIF-23 and to upregulation of VWF .4 THBS1 reportedly regulates the size of circulating von Willebrand Factor (VWF) multimers5 through inhibiting proteolytic cleavage of VWF by ADAMTS13.6 We hypothesized that upregulation of THBS1 in CP may be a risk for thrombosis through an effect on ADAMTS13 and VWF.Methods: We enrolled into an observational study 155 patients with CP and 154 controls matched by age, sex and place of residence. Written informed consent was obtained from all subjects according to the Declaration of Helsinki. We genotyped for VHLR200W, measured baseline plasma THBS-1 concentrations2 and followed the subjects for a median of 9.4 years. Here, we report results for determining baseline circulating levels of ADAMTS13 antigen, activity and specific activity and of VWF antigen and differential multimers in up to 57 CP patients and 30 controls.Results: CP subjects had higher hemoglobin and erythropoietin concentrations and lower white blood cell and platelet counts (Table 1). They were also more likely to be current smokers. There was a trend to higher THBS-1 concentrations in the CP patients (p=0.084), which was significant after adjusting for platelet count (P=0.016). ADAMTS13 and VWF antigen levels did not differ between CP patients and controls but ADAMTS13 activity and especially specific activity were lower in CP. Plasma THBS-1 concentration correlated positively with ADAMTS13 antigen concentration (r=0.358, P=0.001) but negatively with specific activity (r=-0.363, P=0.001) (Figure 1). Eight (14.0%) CP patients (aged 27-76) years had a history of thrombosis (Table 1). The mean serum VWF antigen concentration (P=0.001) and differential VWF multimer size (P=0.090) were higher in CP patients with a history of thrombosis (Figure 2). During the prospective follow-up period, 10 CP patients (17.5%) experienced a new thrombosis at a median age of 46 (range 26-70) years. Three patients also experienced an additional thrombosis for a total of 13 new thromboses: myocardial infarction (n=5), stroke (n=3), splanchnic thrombosis (n=2), deep vein thrombosis (n=2), pulmonary embolism (n=1). Baseline serum VWF antigen levels (P=0.019) and VWF multimer size (P=0.099) were higher in subjects who developed a new thrombosis during follow-up (Figure 3), and these relationships persisted after adjusting for smoking history.Discussion: THBS1 is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions and facilitates platelet aggregation.7 In mice, platelet-derived THBS1 modulated arterial thrombosis only in the presence of VWF.8 Our present findings suggest that CP subjects have lower ADAMTS13 specific activity than controls and that this lower activity may be related to higher plasma THBS1 concentrations. Lower ADAMTS13 specific activity suggests the presence of an inhibitor or partial inactivation of the protease. Furthermore, higher VWF antigen and multimer size in CP patients is associated with a past history of thrombosis and a greater risk for new thrombosis in the future. More broadly, our findings suggest a possible implication of VWF in thrombosis of conditions associated with an up-regulated hypoxic response. Further studies may identify new pathways of prevention and treatment with broad applicability.

Effects of N-Acetylcysteine Infusion in Patients with Sickle Cell Disease during Vaso-Occlusive Crises

Background: Our earlier studies showed that patients with sickle cell disease (SCD) have a high von Willebrand factor (VWF) activity index, which correlates with the extent of the hemolysis they experience [Chen et al Blood 2011; 117:3680‐3]. We are evaluating whether N-acetylcysteine (NAC), which dissociates VWF multimers, has a therapeutic role in SCD. Previously, we studied stable outpatients with SCD [Özpolat et al Blood Dec 2016, 128 (22) 1299]. In that study, we found that NAC infusion was safe and decreased red blood cell (RBC) fragments, dense cells, the size of VWF multimers, platelet activation, and the percentage of platelet-monocyte complexes. In addition, NAC infusion increased the total and free-thiol concentrations of cysteine and glutathione in blood, relieving oxidative stress in the patients at disease baseline. Given these findings, we have begun a pilot study of NAC in SCD patients admitted as inpatients with vaso-occlusive crisis (VOC) (NCT01800526) to examine the safety of NAC infusion and its effects on laboratory markers in that setting.Protocol and patients: Patients are approached for the study in the outpatient setting and consent is re-affirmed when they are admitted in VOC. We plan to enroll enough patients to treat 20 VOCs with NAC infusion, with each patient being treated up to two times in the study. Inclusion criteria: SCD patients with history of VOC requiring narcotics treatment. Exclusion criteria: SCD patients on chronic RBC exchange, expected to be treated by simple or exchange transfusion, inpatient with VOC in the previous 30 days, already had NAC infusion twice in our trial during VOC, and/or had oral NAC in the past 7 days. NAC (75 mg/kg/hr) is infused over 1 hr every 6 hr, for a total daily dose of 300 mg/kg. All patients also receive standard care. Blood is collected immediately before NAC infusion on the first day of admission, daily during admission, and at medical follow-ups during the first week after discharge. To date, we have completed NAC infusion in two patients with VOC under this protocol. Patient 1 is a 26-year-old male with Sβ0 thalassemia, a history of recurrent VOC and avascular necrosis (AVN) of both hips and the left shoulder, on hydroxyurea and oral narcotics. Patient 2 is a 58-year-old female with SS genotype, recurrent VOC and history of bilateral AVN of the hips and knees, pulmonary embolism, on hydroxyurea, oral narcotics, and rivaroxaban. We analyzed patient samples for dense cells, sickle cells (by imaging flow cytometry), VWF antigen and multimer distribution, ADAMTS13 activity and antigen. In addition, we will analyze the concentrations of NAC, cysteine and glutathione and their oxidized and mixed disulfide forms by mass spectrometry.Results: NAC infusions in the two SCD patients with VOC were safe and well tolerated. After NAC treatment, the number of dense cells were reduced (before vs after NAC treatment: patient 1; 1826/µl vs 46/µl, patient 2: 667/µl vs 36/µl), The VWF multimers migrated slightly faster during NAC infusion, indicating possible reduction of intra-chain disulfide bonds by NAC within VWF monomers [Chen J et al. JCI 2011, 121: 593]. The VWF concentrations were increased at the end of both NAC treatments. For patient 1, the ADAMTS13 activity was decreased during the NAC treatment, but the antigen remained unchanged. For patient 2, both the ADAMTS13 activity and antigen were increased with NAC treatment. For patient 1, phosphatidylserine positive microparticles (MP) and MP generated from RBCs and platelets were decreased with NAC treatment but there was no apparent trend for patient 2.Summary: In the first two SCD patients treated, NAC infusion during VOC was safe and well tolerated. NAC infusion decreased dense cells, and reduced intra-chain disulfide bonds in VWF. The full study should determine if NAC is useful therapy for SCD patients during VOC, acting through diverse mechanisms by improving RBC parameters and reducing adhesive activity of VWF. DisclosuresNo relevant conflicts of interest to declare.

Mechanical Forces Impacting Cleavage of Von Willebrand Factor in Laminar and Turbulent Blood Flow

Von Willebrand factor (VWF) is a large multimeric hemostatic protein. VWF is critical in arresting platelets in regions of high shear stress found in blood circulation. Excessive cleavage of VWF that leads to reduced VWF multimer size in plasma can cause acquired von Willebrand syndrome, which is a bleeding disorder found in some heart valve diseases and in patients receiving mechanical circulatory support. It has been proposed that hemodynamics (blood flow) found in these environments ultimately leads to VWF cleavage. In the context of experiments reported in the literature, scission theory, developed for polymers, is applied here to provide insight into flow that can produce strong extensional forces on VWF that leads to domain unfolding and exposure of a cryptic site for cleavage through a metalloproteinase. Based on theoretical tensile forces, laminar flow only enables VWF cleavage when shear rate is large enough (>2800 s−1) or when VWF is exposed to constant shear stress for nonphysiological exposure times (>20 min). Predicted forces increase in turbulence, increasing the chance for VWF cleavage. These findings can be used when designing blood-contacting medical devices by providing hemodynamic limits to these devices that can otherwise lead to acquired von Willebrand syndrome.

Platelet activation via dynamic conformational changes of von Willebrand factor under shear.

Shear-induced conformational changes of von Willebrand factor (VWF) play an important role in platelet activation. A novel approach describing VWF unfolding on the platelet surface under dynamic shear stress is proposed. Cumulative effect of dynamic shear on platelet activation via conformational changes of VWF is analysed. The critical condition of shear-induced platelet activation is formulated. The explicit expression for the threshold value of cumulative shear stress as a function of VWF multimer size is derived. The results open novel prospects for pharmacological regulation of shear-induced platelet activation through control of VWF multimers size distribution.

Histone-induced thrombotic thrombocytopenic purpura in adamts13 -/- zebrafish depends on von Willebrand factor.

Thrombotic thrombocytopenic purpura (TTP) is caused by severe deficiency of ADAMTS13 (A13), a plasma metalloprotease that cleaves endothelium-derived von Willebrand factor (VWF). However, severe A13 deficiency alone is often not sufficient to cause an acute TTP; additional factors may be required to trigger the disease. Using CRISPR/Cas9, we created and characterized several novel zebrafish lines carrying a null mutation in a13−/−, vwf, and both. We further used these zebrafish lines to test the hypothesis that inflammation that results in neutrophil activation and release of histone/DNA complexes may trigger TTP. As shown, a13−/− zebrafish exhibit increased levels of plasma VWF antigen, multimer size, and ability of thrombocytes to adhere to a fibrillar collagen-coated surface under flow. The a13−/−zebrafish also show an increased rate of occlusive thrombus formation in the caudal venules after FeCl3 injury. More interestingly, a13−/− zebrafish exhibit ~30% reduction in the number of total, immature, and mature thrombocytes with increased fragmentation of erythrocytes. Administration of a lysine-rich histone results in more severe and persistent thrombocytopenia and a significantly increased mortality rate in a13−/−zebrafish than in wildtype (wt) ones. However, both spontaneous and histone-induced TTP in a13−/− zebrafish are rescued by the deletion of vwf. These results demonstrate a potentially mechanistic link between inflammation and the onset of TTP in light of severe A13 deficiency; the novel zebrafish models of TTP may help accelerate our understanding of pathogenic mechanisms and the discoveries of novel therapeutics for TTP and perhaps other arterial thrombotic disorders.

Thrombotic microangiopathy as a cause of cardiovascular toxicity from the BCR-ABL1 tyrosine kinase inhibitor ponatinib

AbstractThe third-generation tyrosine kinase inhibitor (TKI) ponatinib has been associated with high rates of acute ischemic events. The pathophysiology responsible for these events is unknown. We hypothesized that ponatinib produces an endothelial angiopathy involving excessive endothelial-associated von Willebrand factor (VWF) and secondary platelet adhesion. In wild-type mice and ApoE−/− mice on a Western diet, ultrasound molecular imaging of the thoracic aorta for VWF A1-domain and glycoprotein-Ibα was performed to quantify endothelial-associated VWF and platelet adhesion. After treatment of wild-type mice for 7 days, aortic molecular signal for endothelial-associated VWF and platelet adhesion were five- to sixfold higher in ponatinib vs sham therapy (P < .001), whereas dasatinib had no effect. In ApoE−/− mice, aortic VWF and platelet signals were two- to fourfold higher for ponatinib-treated compared with sham-treated mice (P < .05) and were significantly higher than in treated wild-type mice (P < .05). Platelet and VWF signals in ponatinib-treated mice were significantly reduced by N-acetylcysteine and completely eliminated by recombinant ADAMTS13. Ponatinib produced segmental left ventricular wall motion abnormalities in 33% of wild-type and 45% of ApoE−/− mice and corresponding patchy perfusion defects, yet coronary arteries were normal on angiography. Instead, a global microvascular angiopathy was detected by immunohistochemistry and by intravital microscopy observation of platelet aggregates and nets associated with endothelial cells and leukocytes. Our findings reveal a new form of vascular toxicity for the TKI ponatinib that involves VWF-mediated platelet adhesion and a secondary microvascular angiopathy that produces ischemic wall motion abnormalities. These processes can be mitigated by interventions known to reduce VWF multimer size.

Effect of Intravenous and Oral N-Acetylcysteine Treatment in a Patient with Sickle Cell Disease at Disease Baseline

Background: We previously showed that patients with sickle cell disease (SCD) have elevated levels of von Willebrand factor (VWF) with high adhesive activity and the level of total active VWF correlated with hemolysis at disease baseline. These findings suggest that VWF plays a role in the pathophysiology of SCD [Chen et al, Blood. 2011; 117:3680‐3]. We also showed that N-acetylcysteine (NAC), a mucolytic and antioxidant drug, can reduce the level of large VWF multimers and platelet adhesion in human plasma in vitro and in mice in vivo [Chen et al, JCI, 2011, 121:522]. Based on these findings, we hypothesized that administration of NAC in SCD patients might reduce VWF multimers in vivo and reduce oxidative stress by scavenging reactive oxygen species (ROS). We started a pilot study to evaluate the effects of intravenous (IV) and oral NAC administration in SCD patients at disease baseline (NCT01800526). In this report, we administered NAC to one patient by IV and orally and compared the effect on biomarkers by the two routes of NAC administration.Patient and protocol: The subject is a 51-year-old male with SS genotype, a history of leg ulcers and acute chest syndrome, and who was not taking hydroxyurea at the time of the study. NAC was infused at a low dose (150 mg/kg over 8 hours) and infusion was repeated at high dose (300 mg/kg over 8 hrs) after 4 weeks. Blood samples were collected before (0), during (4 hr) and after the NAC infusions (8, 24 and 72 hr). Five months after the high dose IV infusion, during which parameters returned to baseline levels, oral NAC was administered for 4 weeks at 2.4g/day. Weekly blood samples were collected four weeks before the start of oral NAC, during oral NAC, and one week after oral NAC administration. We measured RBC parameters and RBC fragments, dense cells and percentage of platelet-monocyte complexes (PMC) by flow cytometry. We also analyzed VWF antigen, VWF multimers, ADAMTS13 antigen and activity levels in plasma. We also measured total, oxidized and reduced forms of NAC, cysteine and glutathione levels in plasma and whole blood by mass spectrometry.Results: 1) The subject did not experience adverse side effect during and after the treatment period. 2) RBC, hemoglobin and hematocrit levels did not significantly change with either IV or oral NAC treatment. Oral and IV NAC decreased dense cell and RBC fragments. NAC reduced the number of dense cells 13% during low dose IV, 79% during high dose IV and 73% with the oral administration. Compared to baseline, RBC fragments decreased 40% during low dose IV, 28% during high dose IV, and 27% with the oral NAC treatment. 3) NAC reduced formation of PMC from 71% to 24% (low dose IV), from 72% to 35% (high dose IV), and from 62% to 27% (oral). 4) VWF multimer size significantly decreased during low and high dose NAC infusions but did not change with oral NAC administration. VWF antigen and ADAMTS13 activity levels did not change with either routes of NAC administration. 5) The concentration of NAC in whole blood increased during treatments: elevated to 0.5 mM and 1 mM at the end of low- and high-dose IV infusions, respectively. At the conclusion of oral administration, the NAC concentration was 4 µM. Total glutathione concentrations in whole blood did not change significantly during and after NAC treatment at either dose or by either route. Free Cys concentrations in plasma increased compared to respective baseline levels 10-fold for low dose infusion (from 10 µM to 101 µM), 20-fold for high dose infusion (from 9 µM to 200 µM), but the level did not change significantly for oral administration (from 5 µM to 7 µM). Protein-bound Cys levels in plasma as markers of oxidative stress were significantly decreased with low- and high-dose IV infusion. However, the level did not change significantly with oral NAC administration.Summary: Both IV and oral administration of NAC were safe and well tolerated in the patient with SCD. Both IV and oral administration reduced the number of dense cells, RBC fragmentation, and formation of PMCs. IV, but not oral NAC, reduced VWF multimer size. These finding suggest that IV administration resulted in higher concentration and had more biologic effects; however both approaches may have a role in treatment of patients with SCD. DisclosuresKonkle:Sangamo: Research Funding; Gilead: Consultancy; Shire: Research Funding; Genentech: Consultancy; CSL Behring: Consultancy; Bioverativ: Research Funding; BioMarin: Consultancy; Spark: Consultancy, Research Funding; Pfizer: Research Funding.

Abstract 535: Proteomics Identifies Endothelium-derived Clusterin as a Novel Regulator of Von Willebrand Factor Multimerization

Background: Secretion of vascular endothelial cells (ECs) is considered to be the first line of defense against vascular injury. Vascular endothelial growth factor (VEGF) is one of the most important endothelial growth factors, involved in various endothelial functions including the regulatory secretion of ECs. However, the systematic analysis of the VEGF-induced secretory proteins has not yet been carried out. Objectives: To investigate the secretory proteins rapidly released from ECs under VEGF stimulation and their functions on the ECs. Methods: A non-serum culture system was established for analyzing the proteins secreted form human primary ECs in response to VEGF with mass spectrometry. Results: In the proteomic result, 362 proteins were identified and assigned to the VEGF-induced secretome, including the known von Willebrand factor (VWF), a multimeric protein mediating adhesion of platelets at vascular injury sites. Clusterin, a multifunctional protein involved in various diseases including Alzheimer disease and cancers, was selected for further study as a new VEGF-induced secretory protein. Clusterin was co-localized with VWF both in Weibel-Palade bodies (WPBs) and extracellular VWF strings of ECs. We showed that the localization of clusterin in pseudo-WPBs in 293T cells depends on the carboxy-terminal domains of VWF. Functional study showed that deficiency of clusterin in ECs increased the size of VWF multimers. Conclusions: We not only obtained a VEGF-induced secretome using primary ECs, but also determined clusterin as a novel component of WPBs, which is capable of maintaining the normal pattern of VWF multimers, and suggest its function on vascular thrombosis and haemostasis.

Activity of Von Willebrand factor and levels of VWF-cleaving protease (ADAMTS13) in preterm and full term neonates

Von Willebrand Factor (VWF) has a central role in primary hemostasis. Its biological activity is related to the size of VWF multimers, spontaneously binding to platelets and inducing circulating microthrombi formation. This process is down-regulated by the VWF cleaving protease ADAMTS13 (A Disintegrin and Metalloprotease with ThromboSpondin motif). To date, information regarding the levels of ADAMTS13 in neonates and preterm infants is scarce. Our aim was to study ADAMTS13, VWF antigen (Ag) and Ristocetin cofactor (RiCof) activity in neonates and evaluate potential correlations with perinatal complications.Our cohort consisted of 128 (48/128: born preterm) neonates, born in Sheba Medical Center and followed until hospital discharge. Control group consisted of 20 healthy adults. As expected, a significant elevation of VWF:Ag was observed in preterm and term infants compared to adults. VWF:Ag levels were highest in full term infants (Median 129.0 IQR 33.8) and lowest in adults (Median 119.0 IQR 58.5) (p<0.05), and RiCoF levels in neonates were higher than in adults. ADAMTS13 was significantly (p<0.05) higher in preterm babies in comparison to full term and adult controls. Neonates that underwent stressful conditions or experienced vascular complications such as IUGR, ROP, NEC, had lower levels of ADAMTS13 in our study. Further studies are required to validate and asses potential significance of these findings.

A Pilot Study of High-Dose N-Acetylcysteine Infusion in Patients with Sickle Cell Disease

▪We have completed a clinical trial evaluating the safety of N-acetylcysteine (NAC) infusion in adult patients with sickle cell disease (SCD) at disease baseline (NCT01800526). This trial was inspired by the hypothesis that von Willebrand factor (VWF) has an important role in the pathophysiology of vaso-occlusion and hemolysis in SCD and that NAC can reduce signs and symptoms of the disease by reducing the activity of VWF. A secondary hypothesis was that NAC could also act by reversing or preventing some of the oxidative changes associated with SCD.VWF is stored in the Weibel-Palade bodies of endothelial cells and α-granules of platelets and released from endothelial cells upon activation and mediates the attachment of platelets to the vessel wall, and secondarily, of erythrocytes and leukocytes. We previously showed that VWF levels and adhesive activity in SCD plasma were elevated and that total active VWF correlated with the extent of hemolysis (determined by plasma LDH levels), suggesting that VWF participates in the pathophysiology of SCD (Chen et al, Blood. 2011; 117:3680‐3). In another study, we showed that NAC was able to reduce VWF multimer size and platelet-binding activity ex vivo in human plasma and in vivo in mice (Chen et al, JCI, 2011, 121:522).In this clinical trial, we enrolled 5 subjects at disease baseline, 4 with SCD and 1 with sickle trait. All subjects received two high-dose NAC infusions: initially 150 mg/kg and 4 weeks later 300 mg/kg. NAC infusion was administered in subjects 1 and 2 as a bolus infusion of half of the dose in the 1sthour and the remaining half in 7 hr. Subjects 3-5 received NAC as constant infusion for 8 hr. Blood was collected immediately before infusion, during infusion (at 1 and 4 hr), immediately after infusion (8 hr), and 24 and 72 hr after infusion. We examined blood for red blood cell (RBC) fragments, dense cells, platelet activation status, platelet-monocyte complexes and the concentrations of reduced and oxidized glutathione. We also examined plasma for VWF concentration and multimer distribution, ADAMTS13 antigen and activity, and evaluated plasma redox status by measuring the concentrations of NAC and cysteine in their reduced, oxidized, and mixed disulfide forms, as well as the concentration of protein-bound cysteine. The small molecule thiols were measured by mass spectrometry.Results:1) All subjects tolerated NAC infusion well, except that subjects 1 and 2 experienced pruritus during the bolus infusion at the 300 mg/kg dose. We therefore changed the protocol to deliver the drug over 8 hr by continuous infusion for subjects 3 through 5. 2) During NAC infusion, the percentage of dense cells and concentration of RBC fragments decreased rapidly (average decrease: low dose 44%; high dose 31%) and the change persisted up to 72 hr. 3) Platelet activation also decreased, as determined by reduced P-selectin expression (low dose, 7.7 ± 0.7% to 5.0 ± 1.9%; high dose, 17.8 ± 0.07% to 7.3% ± 0.4), PAC-1 binding (evaluates activated αΙΙbβ3), and formation of platelet-monocyte complexes (monocytes with attached platelets: low dose, 52 ± 16% to 27 ± 12%; high dose, 77 ± 12% to 50 ± 17%). 4) The highest molecular weight VWF multimers transiently disappeared during the NAC infusion and recovered by 24 hr. There were no appreciable changes in VWF antigen or ADAMTS13 antigen or activity with NAC infusion. 5) With NAC infusion, the concentrations of Cys and NAC increased in plasma, both total and reduced forms, with a concomitant decrease in protein-bound Cys in the one patient studied (free thiol Cys increased 23 fold with the high dose infusion at 8 hr; protein-bound Cys decreased 87%). Similarly, the concentrations of reduced and oxidized glutathione increased in whole blood.Summary: NAC infusion in SCD patients at disease baseline appears safe and is well tolerated. NAC infusion decreases RBC fragments, dense cells, and the size of large VWF multimers, and inhibits platelet activation and formation of platelet-monocyte complexes. In addition, NAC infusion increases the total and free-thiol concentrations of cysteine and glutathione in blood, relieving oxidative stress in SCD. Our data suggest that NAC may be of benefit for SCD patients during vaso-occlusive crisis through a variety of mechanisms, which include improvements in red cell parameters, decreased platelet adhesion, reduced adhesive activity of VWF, and protection against oxidative damage. DisclosuresNo relevant conflicts of interest to declare.

Platelet activation risk index as a prognostic thrombosis indicator.

Platelet activation in blood flow under high, overcritical shear rates is initiated by Von Willebrand factor. Despite the large amount of experimental data that have been obtained, the value of the critical shear rate, above which von Willebrand factor starts to activate platelets, is still controversial. Here, we recommend a theoretical approach to elucidate how the critical blood shear rate is dependent on von Willebrand factor size. We derived a diagram of platelet activation according to the shear rate and von Willebrand factor multimer size. We succeeded in deriving an explicit formula for the dependence of the critical shear rate on von Willebrand factor molecule size. The platelet activation risk index was introduced. This index is dependent on the flow conditions, number of monomers in von Willebrand factor, and platelet sensitivity. Probable medical applications of the platelet activation risk index as a universal prognostic index are discussed.

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.

Neutrophil Recruitment Is Regulated By Adamts-13 in a Murine Model of Invasive Aspergillosis

▪Introduction: During inflammation von Willebrand factor (VWF) multimers are secreted as an acute phase protein whereupon the size and the prothrombotic activity play an essential role. The size of VWF multimers is regulated by the specific proteolytic activity of ADAMTS-13 (a disintegrin and metalloprotease with ThromboSpondin type 1 repeats-13) which is diminished under several pathological conditions. Employing a murine model of invasive pulmonary aspergillosis (IPA) we aimed to determine the relevance of this regulatory pathway for innate inflammatory responses and polymorphonuclear neutrophil (PMN) recruitment which is crucial for fungal clearance and survival.Methods: IPA was induced by intratracheal application of Aspergillus fumigatus (A. fumigatus) conidia in wildtype (129/Sv/Pas) or Adamts13 deficient (Adamts13-/-) mice. 24 h after infection some mice were sacrificed for analysis of fungal load and histology. To assess fungal load as CFU lung homogenates were plated and cultured on Sabourough agar plates. Paraffin sections of the lungs were prepared and stained with mouse complement component C3d antibody for histological analysis. In addition, degranulation, oxidative burst activity and CD62L shedding were assessed in PMN of wildtype and Adamts13-/- mice. Chemotactic properties of A. fumigatus -activated and control serum from wildtype and knock-out mice was evaluated by migration of purified human PMN, isolated by dextran sedimentation and Histopaque® centrifugation, in a transwell assay (Corning® HTS Transwell®-96 well permeable support; 3µm).Results: Adamts13 deficiency in mice was accompanied by a worse outcome of IPA infections compared to wildtype controls. Adamts13-/- mice displayed more severe signs of disease and a lethal course was observed in about 24% of animals. Compared to Adamts13-/- mice all neutropenic mice developed lethal IPA after infection, but all wild-type mice survived the disease. Besides, examination of the lungs revealed a higher fungal burden along with increased signs of acute lung injury, complement deposition and levels of pro-inflammatory cytokines in Adamts13-/- mice. Furthermore a more pronounced perivascular leukocyte infiltration was observed in histological sections indicating a dysregulated inflammatory response in Adamts13-/- mice. Importantly, the activation of neutrophil effector functions in response to TLR agonist or in PMN-mediated fungal killing of conidia or hyphae revealed no general defect in vitro. Despite enhanced complement deposition in the lungs in Adamts13-/- mice, PMN migration towards complement-activated serum revealed unaltered chemotactic properties comparing A. fumigatus -activated serum of wildtype and knock-out mice.Conclusion: In acute inflammatory processes such as fungal pneumonia the proteolytic regulation of VWF by ADAMTS-13 is an important mechanism to control PMN recruitment and outcome of infections. DisclosuresNo relevant conflicts of interest to declare.

Von Willebrand Factor Is a Cofactor in Complement Regulation

Complement, besides its involvement in eliminating microbes, participates in such diverse processes as synapse maturation, clearance of immune complexes, angiogenesis and tissue regeneration. Delicate balance between complement activation and regulation contributes to complement’s role in physiology. Any trigger that tips this balance can induce self-attack. Atypical hemolytic uremic syndrome (aHUS) is a systemic disease characterized by non-immune hemolytic anemia, thrombocytopenia, and renal impairment. In over 50% of cases, aHUS is known to be caused by uncontrolled activation of the complements. Von Willebrand factor (VWF) is a large multimeric glycoprotein that plays an important role in stopping the escape of blood from vessels following vascular injury. VWF and VWF cleavage enzyme ADAMTS13 gene defects have been identified in patients with aHUS, raising the possibility that VWF could have contributed to complement regulation. To examine the role of VWF in complement activation, we investigated whether VWF functions as a cofactor for FI-mediated C3b cleavage through in vitro assay. We found that C3b binds to VWF. In the presence of plasma-purified VWF (pVWF), FI cleaves C3b to 68kD and 43kD degradation products (iC3b) (Figure (A)). VWF alone, or FI alone, did not have any effect on C3b cleavage. C3b, not C3 or iC3b, was the mainly substrate for FI/VWF proteolysis. Increasing VWF concentration or prolonging the incubation time with VWF enhanced FI-mediated C3b cleavage. To remove the possibility that another plasma protein co-purified with pVWF that affects our results, we used recombinant VWF dimers purified from human embryonic kidney (HEK) 293 cell expressing VWF-Dpro cDNA, and detected a similar cofactor activity (Figure (B)).To investigate whether the size of VWF multimers have any effect on C3b cleavage, we compared the cofactor activity of pVWF, recombinant VWF dimers, and ULVWF multimers. While pVWF and dimer VWF enhanced C3b cleavage by FI, ULVWF did not have any effect on C3b cleavage (Figure (B)). In plasma, complement proteins Factor B and Factor D coexist with the inhibitory protein FI. To investigate the effect of VWF on complement activity in the presence of both pro-activation and inhibitory complement proteins, we incubated C3, FB, FD, and FI with VWF. In the presence of FB and FD, C3 be activated and resulted in the generation of C3a and C3b. Addition of recombinant VWF put a brake on complement activation and shifted C3 toward the generation of iC3b (Figure (C)). We conclude that normal plasma VWF, function as a cofactor, prevents complement activation through steers the complement pathway toward the generation of inactive iC3b. ULVWF multimers, as are present in patients with thrombotic microangiopathy, lack an inhibitory effect on complement and permit complement activation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The Ability of Von Willebrand Factor (VWF) to Bind Factor VIII (FVIII) Can Decrease during Pregnancy

Background: The coagulation protein von Willebrand Factor (VWF) and its circulating partner protein, Factor VIII, are known to be elevated in pregnancy. However, the nature of changes in VWF which occur during pregnancy are not well understood. We previously presented results from a study of 46 healthy pregnancies in which we measured VWF antigen (VWF:Ag) and Factor VIII (FVIII determined both by activity and antigen measurements). We found that VWF:Ag and FVIII increased during pregnancy, consistent with less FVIII relative to VWF. We have also observed that VWF multimer size can decrease during pregnancy. We hypothesized that pregnancy can induced molecular changes in VWF, and that such changes could impact the interaction of VWF with its ligands, specifically FVIII.Methods: We adapted a Type 2N von Willebrand Disease (VWD) assay to test the ability of plasma VWF to bind recombinant FVIII. In brief, serial dilutions of plasma VWF were captured using a polyclonal anti-VWF antibody (DAKO), endogenous FVIII was stripped using a CaCl2 wash, recombinant FVIII (Recombinate) was applied, and bound FVIII was detected using an anti-FVIII antibody (Cedarlane). In parallel, a VWF:Ag ELISA was performed using the same anti-VWF capture antibody. The VWF-FVIII binding ratio (VWF-FVIIIb) was then determined by dividing the quantity of rFVIII detected by the amount of VWF:Ag immobilized on the plate. The range, reproducibility, and variance of the assay was first determined in a healthy blood donor repository. We then tested VWF-FVIIIb in healthy pregnancies for which 3rd trimester and non-pregnant baseline samples were available. Additional time points during pregnancy were also studied, when available. VWF-FVIIIb results for each trimester and the non-pregnant baseline were analyzed by one-way ANOVA (All Pairwise Multiple Comparison Procedures, Holm-Sidak method).Results: Forty-six healthy pregnancies with non-pregnant baseline samples were studied. Samples tested were drawn in the first (n=6), second (n=38), and third (n=43) trimesters, at 38 weeks gestational age (n=13), and at a non-pregnant time point (baseline). In non-pregnant samples, the VWF-FVIIIb was close to 1.0 (0.988 +/- 0.084), similar to a normal blood donor repository. We identified a significant decrease in VWF-FVIIIb in the second trimester (0.931 +/- 0.82; p = 0.013), which became more pronounced in the third trimester (0.899 +/- 0.75; p < 0.001) and at 38 weeks gestational age (0.88 +/- 0.072; p < 0.001). The means, quartiles, and standard deviations for each pregnancy time point are shown in Figure 1.Conclusions: These data support the hypothesis that VWF can acquire a decreased ability to interact with FVIII during pregnancy. A decreased capacity to carry FVIII could contribute to the increased VWF:FVIII ratio we and others have observed during pregnancy, although other factors, such as increased VWF production relative to FVIII, could also play a role. This work provides further evidence for a new model of acquired VWF changes in the setting of pregnancy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.