Ratio Of Von Willebrand Factor Research Articles

The Ratio of von Willebrand Factor Antigen to ADAMTS13 Activity: Usefulness as a Prognostic Biomarker in Acute-on-Chronic Liver Failure

Acute-on-chronic liver failure (ACLF) has a high risk of short-term mortality. A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) is a metalloproteinase that specifically cleaves multimeric von Willebrand factor (VWF). Imbalance between ADAMTS13 and VWF is associated with portal hypertension, which induces ACLF development. A previous study reported that ADAMTS13 activity (ADAMTS13:AC) and VWF antigen (VWF:Ag) are predictive biomarkers of ACLF development in patients with cirrhosis. This study investigated the changes in ADAMTS13:AC and VWF:Ag levels from before to after the development of ACLF to determine their usefulness as a prognostic biomarker in patients with ACLF. In total, 101 patients with cirrhosis were enrolled in this study. The level of ADAMTS13:AC and VWF:Ag was determined by an enzyme-linked immunosorbent assay. Cox proportional hazard regression analysis was conducted to determine independent prognostic factors for patients with liver cirrhosis in the post-ACLF group. ADAMTS13:AC levels gradually decreased in the order of non-ACLF group, pre-ACLF group, and finally post-ACLF group. VWF:Ag and the ratio of VWF:Ag to ADAMTS13:AC (VWF:Ag/ADAMTS13:AC) levels gradually increased in the order of non-ACLF group, pre-ACLF group, followed by post-ACLF group. VWF:Ag/ADAMTS13:AC and CLIF-C ACLF scores were associated with prognosis in the post-ACLF group in multivariate analysis. The cumulative survival of the post-ACLF group was significantly lower for patients with high VWF:Ag/ADAMTS13:AC (>9) compared with those with low VWF:Ag/ADAMTS13:AC (≤9) (HR: 10.72, 95% confidence interval: 1.39-82.78, p < 0.05). The VWF:Ag/ADAMTS13:AC increased according to the progression of ACLF in patients with cirrhosis and predicted prognosis in patients with cirrhosis with ACLF.

Temporal Changes in Extracellular Vesicle Hemostatic Protein Composition Predict Favourable Left Ventricular Remodeling after Acute Myocardial Infarction.

The subset of plasma extracellular vesicles (EVs) that coprecipitate with low-density lipoprotein (LDL-EVs) carry coagulation and fibrinolysis pathway proteins as cargo. We investigated the association between LDL-EV hemostatic/fibrinolysis protein ratios and post-acute myocardial infarction (post-AMI) left ventricular (LV) remodeling which precedes heart failure. Protein concentrations of von Willebrand factor (VWF), SerpinC1 and plasminogen were determined in LDL-EVs extracted from plasma samples obtained at baseline (within 72 h post-AMI), 1 month and 6 months post-AMI from 198 patients. Patients were categorized as exhibiting adverse (n = 98) or reverse (n = 100) LV remodeling based on changes in LV end-systolic volume (increased or decreased ≥15) over a 6-month period. Multiple level longitudinal data analysis with structural equation (ML-SEM) model was used to assess predictive value for LV remodeling independent of baseline differences. At baseline, protein levels of VWF, SerpinC1 and plasminogen in LDL-EVs did not differ between patients with adverse versus reverse LV remodeling. At 1 month post-AMI, protein levels of VWF and SerpinC1 decreased whilst plasminogen increased in patients with adverse LV remodeling. In contrast, VWF and plasminogen decreased whilst SerpinC1 remained unchanged in patients with reverse LV remodeling. Overall, compared with patients with adverse LV remodeling, higher levels of SerpinC1 and VWF but lower levels of plasminogen resulted in higher ratios of VWF:Plasminogen and SerpinC1:Plasminogen at both 1 month and 6 months post-AMI in patients with reverse LV remodeling. More importantly, ratios VWF:Plasminogen (AUC = 0.674) and SerpinC1:Plasminogen (AUC = 0.712) displayed markedly better prognostic power than NT-proBNP (AUC = 0.384), troponin-I (AUC = 0.467) or troponin-T (AUC = 0.389) (p < 0.001) to predict reverse LV remodeling post-AMI. Temporal changes in the ratios of coagulation to fibrinolysis pathway proteins in LDL-EVs outperform current standard plasma biomarkers in predicting post-AMI reverse LV remodeling. Our findings may provide clinical cues to uncover the cellular mechanisms underpinning post-AMI reverse LV remodeling.

ADAMTS-13/von Willebrand factor ratio: A prognostic biomarker for portal vein thrombosis in compensated cirrhosis. A prospective observational study

Background and aimsIn cirrhosis, decreased portal flow velocity, thrombophilia factors, and portal hypertension are considered risk factors for portal vein thrombosis (PVT). In cirrhosis, the transformation of the stellate cells causes a progressive decrease of ADAMTS-13, while VWF multimers secretion by endothelial cells is strongly enhanced. This imbalance leads to an accumulation of ultra-large VWF multimers that in sinusoidal circulation could favor PVT both in intra- and extra-hepatic branches, mostly in decompensated cirrhosis. This prospective study was aimed at identifying possible clinical, biochemical, and hemostatic factors predictive for non-tumoral PVT in a cohort of patients with compensated cirrhosis. MethodsSeventynine compensated cirrhosis patients were prospectively followed for 48 months, receiving a periodic Doppler-ultrasound liver examination associated with an extensive evaluation of clinical, biochemical, and hemostatic profile. ResultsFive patients developed PVT (cumulative prevalence = 6.3%), occurring 4–36 months after enrollment. In logistic regression analysis, the ADAMTS-13/VWF:GpIbR ratio < 0.4 was the only independent variable significantly associated with PVT (OR 14.6, 95% C.I.:1.36–157.2, p = 0.027). A Cox-regression-analysis confirmed this finding (HR = 7.7, p = 0.027). ConclusionsThe ADAMTS-13/VWF ratio < 0.4 measured in compensated cirrhosis could be a reliable predictive biomarker for PVT development, paving the way to novel therapeutic strategies to prevent and treat PVT in this clinical setting.

The Von Willebrand factor-ADAMTS-13 axis: a two-faced Janus in bleeding and thrombosis

Von Willebrand factor (VWF), a blood multimeric protein with a very high molecular weight, plays a crucial role in the primary hemostasis, the physiological process characterized by the adhesion of blood platelets to the injured vessel wall. Hydrodynamic forces are responsible for the VWF multimers conformational transitions from a globular to a stretched linear conformation. These characteristics render this protein a valuable object to be investigated by mechanochemistry, the biophysical chemistry branch that studies the effects of shear forces on protein conformation. This review will focus on the structural elements of the VWF molecule involved in the biochemical response to shear forces. The stretched VWF conformation favors the interaction with the platelet GpIb and at the same time with ADAMTS-13, the zinc-protease that cleaves VWF in the A2 domain, limiting its prothrombotic capacity. It is important to consider the level or the function of VWF or ADAMTS-13 always in relation each other, keeping in mind that in many thrombotic forms of microangiopathies the reduction of the ratio between the ADAMTS-13 activity and the VWF level (lower than 0.5) can be a valuable parameter to predict a real thrombotic risk. Hence, a significant increase in VWF level alone, even without any reduction of ADAMTS-13 concentration, would still be responsible for a significant reduction of the ADAMTS-13/VWF ratio, which ultimately could reflect or predict a prothrombotic risk. Future studies will have to validate the concept whether ADAMTS-13/VWF ratio could a valuable and reliable biomarker to predict or confirm the presence of thrombotic risk in several morbid conditions.

Ratio of von Willebrand factor antigen to ADAMTS13 activity is a useful biomarker for acute-on-chronic liver failure development and prognosis in patients with liver cirrhosis.

Acute-on-chronic liver failure (ACLF) is associated with a high risk of short-term mortality after progression to multiple organ failure. A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) is a metalloproteinase that specifically cleaves multimeric von Willebrand factor (VWF). An imbalance between ADAMTS13 enzyme and VWF substrate is associated with liver cirrhosis progression that induces ACLF. This study examined the relationship between ADAMTS13 and VWF and ACLF development to determine whether ADAMTS13 and VWF are useful predictive biomarkers for ACLF development and prognosis of patients with liver cirrhosis. The study enrolled 67 patients with Child-Pugh class A and B liver cirrhosis. ADAMTS13 activity (ADAMTS13:AC) and VWF antigen (VWF:Ag) were measured using enzyme-linked immunosorbent assays. The ratio of VWF:Ag to ADAMTS13:AC (VWF:Ag/ADAMTS13:AC) was used to divide patients into two groups according to the classification and regression tree based on Gray model survival analysis. Compared with patients with Child-Pugh class A liver cirrhosis, class B patients had a higher VWF:Ag/ADAMTS13:AC and a higher risk of ACLF development. Cumulative incidence of ACLF was significantly higher in patients with high (>7.9) versus low (≤7.9) VWF:Ag/ADAMTS13:AC (hazard ratio [HR], 6.50; 95% CI, 2.31-18.29; p<0.001). Cumulative survival was significantly lower in cirrhotic patients with high versus low VWF:Ag/ADAMTS13:AC (HR 5.11; 95% CI, 1.85-14.14; p=0.002). For patients with liver cirrhosis, VWF:Ag/ADAMTS13:AC is associated with functional liver reserve and predicts the development of ACLF and the prognosis.

VWF/ADAMTS13 Ratios Are Potential Markers of Immunothrombotic Complications in Patients with COVID-19: A Cross-Sectional Study

BACKGROUND. COVID-19 is a prothrombotic disease, characterized by both arterial and venous thrombosis. The pathogenesis of coagulopathy in COVID-19 is multifactorial, with activation of endothelial cells, platelets, neutrophils, and other immune system effectors playing crucial roles. High levels of von Willebrand factor (VWF) and reduced levels of ADAMTS13 have been observed in COVID-19, sepsis, and immunothrombotic states, and an elevated VWF/ADAMTS13 ratio has been previously described in ischemic stroke1. In this study, we sought to evaluate potential associations of VWF activity and antigen ratios to ADAMTS13 with clinical outcomes in hospitalized patients with COVID-19 and to compare these ratios with other cytokines and markers of inflammation in critical illness.METHODS. Institutional Review Board approval was obtained for this study. Blood was collected from 49 hospitalized patients with a confirmed diagnosis of COVID-19 between April 13 and April 24, 2020, which included 40 patients in an intensive care unit and 9 on a non-ICU unit. Medical records were reviewed, and patient characteristics including age, sex, comorbidities, pertinent medications, imaging studies, history of arterial or venous thrombosis, and other pertinent patient information were compiled. Blood was collected in sodium citrate tubes and centrifuged at 4000 rpm for 20 minutes, with the resulting plasma supernatant used for further testing. Measurements of VWF antigen and VWF activity were performed on an ACL TOP machine using our institution’s standard clinical laboratory protocol. Biomarker profiling analyses were performed at Eve Technologies (Calgary, Alberta, Canada). Statistical analysis was performed using GraphPad Prism (v8.4.3, GraphPad Software, San Diego, CA) and R (v4, R Core Team, 2020). P values <0.05 were considered statistically significant.RESULTS. The median age was 64 years; 36 (73%) were male, 11 (22.4%), 29 (59.2%), and 9 (18.4%) had BMI<25, between 25 and 40, and >40 kg/m2, respectively. The ratio of VWF activity/ADAMTS13 was significantly higher in patients with higher BMI (median [interquartile range, IQR] VWF activity/ADAMTS13 ratios: 0.66 [0.39-0.88] for BMI<25 kg/m2, 0.865 [0.769-1.102] for BMI 25-40 kg/m2, and 1.041 [0.747-2.032] for BMI>40 kg/m2; p = 0.0408, Kruskal-Wallis test). In patients who required mechanical ventilation, median [IQR] VWF activity/ADAMTS13 was 0.876 [0.75-1.769] vs 0.792 [0.566-0.932] for those who did not (p = 0.0497, Mann-Whitney U-test). The difference for the VWF antigen/ADAMTS13 ratio was more pronounced, 1.32 [0.991-2.456] vs 0.981 [0.628-1.162] (p = 0.0045, Mann-Whitney U-test). There was also a significant correlation between both VWF activity/ADAMTS13 and VWF antigen/ADAMTS13 ratios and neutrophil indices such as absolute neutrophil count (R = 0.57, p <0.0001 and R = 0.54, p = 0.0001, respectively) and neutrophil to lymphocyte count (R = 0.6, p <0.0001 and R = 0.57, p <0.0001, respectively). We also found significant correlations of these ratios with markers of neutrophils activation (HGF, resistin, and lipocalin-2; Figure 1).CONCLUSIONS. Ratios VWF activity/ADAMTS13 and VWF antigen/ADAMTS13 are elevated in patients with more severe COVID-19 disease and correlate with markers of neutrophils activation. These findings and that azurophillic granules in neutrophils contain α-defensins implicated in thrombosis2 suggest possible mechanistic links among VWF, ADAMTS13, contact pathway, and neutrophil function in mediating immunothrombotic complications in COVID-19, which might highlight potential therapeutic targets to attenuate the severity of coagulopathy and merit further investigation with a larger cohort of patients.REFERENCES1. Taylor A, et al. Blood Adv. 2020;4(2):398-4072. Abu-Fanne R, et al. Blood. 2019;133(5):481-493DisclosuresNo relevant conflicts of interest to declare.

The von Willebrand Factor antigen to platelet ratio (VITRO) score predicts hepatic decompensation and mortality in cirrhosis.

The ratio of von Willebrand Factor to platelets (VITRO) reflects the severity of fibrosis and portal hypertension and might thus hold prognostic value. Patients with compensated cirrhosis were recruited. VITRO, Child-Pugh score (CPS) and MELD were determined at study entry. Hepatic decompensation was defined as variceal bleeding, ascites or hepatic encephalopathy. Liver transplantation and death were recorded. One hundred and ninety-four patients with compensated cirrhosis (CPS-A 89%, B 11%; 56% male; median age 56years; 50% with varices) were included. During a median follow-up of 45months (IQR 29-61), decompensation occurred in 35 (18%) patients and 14 (7%) patients deceased. The risk of hepatic decompensation was significantly increased in the n = 88 (45%) patients with a VITRO ≥ 2.5 (p < 0.001). Patients with a VITRO ≥ 2.5 had a higher probability of decompensation at 1-year 9% (95% CI 3-16) vs. 0% (95% CI 0-0) and at 2-years 18% (95% CI 10-27%), vs. 4% (95% CI 0-8%) as compared to patients with VITRO < 2.5. Patients with VITRO ≥ 2.5, the estimated 1-year/2-year survival rates were at 98% (95% CI 95-100%) and 94% (95% CI 88-99%) as compared to 100% (95% CI 100-100%) both in the patients with a VITRO < 2.5 (p < 0.001). After adjusting for age, albumin and MELD, VITRO ≥ 2.5 remained as significant predictor of transplant-free mortality (HR 1.38, CI 1.09-1.76; p = 0.007). Patients with compensated cirrhosis and VITRO > 2.1 after hepatitis C eradication remained at significantly increased risk for decompensation (p = 0.033). VITRO is a valuable prognostic tool for estimating the risk of decompensation and mortality in patients with compensated cirrhosis-including the setting after hepatitis C eradication.

Atherosclerosis, myocardial infarction and primary hemostasis: Impact of platelets, von Willebrand factor and soluble glycoprotein VI

IntroductionLittle is known about peril constellations in primary hemostasis contributing to an acute myocardial infarction (MI) in patients with already manifest atherosclerosis. The study aimed to establish a predicting model based on six biomarkers of primary hemostasis: platelet count, mean platelet volume, hematocrit, soluble glycoprotein VI, fibrinogen and von Willebrand factor ratio. Materials and methodsThe biomarkers were measured in 1.491 patients with manifest atherosclerosis of the Leipzig (LIFE) heart study. Three groups were divided: patients with coronary artery disease (900 patients) and patients with atherosclerosis and either ST-elevated MI (404 patients) or Non-ST-elevated MI (187 patients). Correlations were analyzed by non-linear analysis with Self Organizing Maps. Classification and discriminant analysis was performed using Learning Vector Quantization.Results and conclusions.The combination of hemostatic biomarkers is regarded as valuable tool for identifying patients with atherosclerosis at risk for MI. Nevertheless, our study contradicts this belief. The biomarkers did not allow to establish a predicting model usable in daily patient care. Good specificity and sensitivity for the detection of MI was only reached in models including acute phase parameters (specificity 0,9036, sensitivity 0,7937 in men; 0,8977 and 0,8133 in women). In detail, hematocrit and soluble glycoprotein VI were significantly different between the groups. Significant dissimilarities were also found for fibrinogen (in men) and von Willebrand factor ratio. In contrast, the most promising parameters mean platelet volume and platelet count showed no difference, which is an important contribution to the controversy concerning them as new risk and therapy targets for MI.

In Vitro Assessment of von Willebrand Factor in Cryoprecipitate, Antihemophilic Factor/VWF Complex (Human), and Recombinant von Willebrand Factor.

Patients with von Willebrand disease (VWD) often require treatment with supplemental von Willebrand factor (VWF) prior to procedures or to treat bleeding. Commercial VWF concentrates and more recently recombinant human VWF (rVWF) have replaced cryoprecipitate as the mainstay of therapy. In comparison with cryoprecipitate, the VWF content and multimer distribution under current manufacturing processes of these commercial products has not been reported. We measured the factor VIII (FVIII:C), VWF antigen (VWF:Ag), VWF collagen-binding activity (VWF:CB), VWF platelet-binding activity by GPIbM enzyme-linked immunosorbent assay (VWF:GPIbM), and percentage of high-molecular-weight (HMWM) VWF in 3 pools of group A and O cryoprecipitate, 3 vials of VWF concentrate (Humate-P), and 1 lot of rVWF (Vonvendi). We found that both group O and group A cryoprecipitate have significantly higher ratios of VWF:GPIbM activity and FVIII:C activity relative to VWF:Ag and have better preservation of HMWM than Humate-P. Although not compared statistically, rVWF appears to have more HMWM VWF and a higher ratio of VWF:GPIbM to VWF:Ag than Humate-P and cryoprecipitate. The estimated acquisition cost for our hospital for treating one major bleeding episode was more than 4-fold higher with Humate-P and 7- to 10-fold higher with rVWF than with cryoprecipitate.

Superiority of the Rapid Von Willebrand Factor (VWF) VWF:GPIbR and VWF:GPIbM Assays in Type 2A, 2B and 2M Von Willebrand Disease

A complete set of rapid activity and classical von Willebrand factor (VWF) assays for Willebrand disease (VWD) diagnosis was used in the present study to characterize VWD type 1, 2A, 2B and 2M patients due to mutations in the A1, A2 and A3 domains. The VWF:RCo/VWF:Ag, VWF:GPIbM/VWF:Ag and VWF:GPIbR/VWF:Ag ratios at cuttt off value of 0.7 separated VWD type 1 and LowVWF from VWD type 2. The results from the Brno cohort of VWD 2A patients with the G1579R mutation in the A2 domain in sixteen affected member from five families and in one case with the G1609R in the A2 domain revealed that the VWF:GPIbM/VWF:Ag and VWF:GPIbR/VWF: Ag ratios are marked decreased (range 0.03-0.27) to a similar degree as compared to VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios (range 0.03-0.27) due to the proteolytic loss of large and intermediate VWF multimers. The results in VWD 2B patients due to gain of ristocetin induced platelet agglutination (RIPA) function mutations R1306W, R1308C and R1341 in the A1 domain demonstrated that the ratios for VWF:GPIbM/VWF:Ag and VWF:GPIbR/VWF:Ag as compared to VWF:RCo/VWF:Ag ratio were markedly decreased in VWD 2B, whereas the VWF:GPIbM/VWF:Ag ratio was somewhat higher (range 0.32 to 0.36) in VWD 2M. VWD 2M patients due to loss of RIPA function mutation R1359K in the A1 domain are featured by decreased VWF ratios for WVF:RCo/Ag and VWF:GPIbR/Ag, but less decreased for the VWF:GPIbM/Ag ratio and normal VWF ratio for VWF:CB/Ag ratio the need to retain the VWF:CB assay to make a correct diagnosis of VWD 2M for its differentiation from VWD type 1. The G1415D mutation in the A1 domain is featured by decreased RIPA and decreased VWF:RCo/VWF:Ag ratio (VWD 2M) but normal values for VWF:CB/VWF:Ag, VWF:GPIbM/VWF:Ag and VWFGPIbR/VWF:Ag ratios consistent with VWD 2M. Double heterozygous P1266L/V1278I mutation in two patients and heterozygous E1292D/WT mutation in three patients in the A1 domain were diagnosed as VWD 2M or 1M associated with a secretion defect (SD). The Platelet Function Analyzer Closure Times (PFA-CT) are strongly prolonged in VWD 2A, 2B and 2M. and moderately prolonged between the upper limit of normal to 300 seconds in heterozygous mutated VWD type 1 patients.

Comparative analysis of von Willebrand factor profiles after implantation of left ventricular assist device and total artificial heart

Background Bleeding remains a challenge during mechanical circulatory support and underlying mechanisms are incompletely understood. Functional von Willebrand factor (VWF) impairment because of loss of high-molecular-weight multimers (MWMs) produces acquired von Willebrand disease (VWD) after left ventricular assist device (LVAD). Little is known about VWF multimers with total artificial hearts (TAHs). Here, VWF profiles with LVADs and TAHs are compared using a VWD panel. Methods VWD evaluations for patients with LVAD or TAH (2013-14) were retrospectively analyzed and included: VWF activity (ristocetin cofactor, VWF:RCo), VWF antigen (VWF:Ag), ratio of VWF:RCo to VWF:Ag, and quantitative VWF multimeric analysis. Results Twelve patients with LVADs and 12 with TAHs underwent VWD evaluation. All had either normal (47.8%) or elevated (52.2%) VWF:RCo, normal (26.1%) or elevated (73.9%) VWF:Ag and 50.0% were disproportional (ratio ≤ 0.7). Multimeric analysis showed abnormal patterns in all patients with LVADs: seven with high MWM loss; five with highest MWM loss. With TAH, 10/12 patients had abnormal patterns: all with highest MWM loss. High MWM loss correlated with presence of LVAD and highest MWM loss with TAH. Increased low MWMs were detected in 22/24. Conclusion Using VWF multimeric analysis, abnormalities after LVAD or TAH were detected that would be missed with measurements of VWF level alone: loss of high MWM predominantly in LVAD, loss of highest MWM in TAH, and elevated levels of low MWM in both. This is the first study to describe TAH-associated highest MWM loss, which may contribute to bleeding.

Reduced ADAMTS13 levels in patients with acute and chronic cerebrovascular disease.

Von Willebrand Factor (VWF) plays a major role in thrombosis and hemostasis and its thrombogenicity is controlled by ADAMTS13. Whereas increasing evidence shows a clear association between VWF levels and acute ischemic stroke, little is known about a correlation with ADAMTS13. Therefore, the aim of this study was to compare plasma levels of ADAMTS13 between 85 healthy volunteers (HV), 104 patients with acute ischemic stroke and 112 patients with a chronic cerebrovascular disease (CCD). In this case-control study, plasma ADAMTS13 antigen levels were measured by ELISA and plasma VWF levels, measured previously, were next used to calculate VWF:ADAMTS13 ratios. ADAMTS13 levels and VWF:ADAMTS13 ratios were subsequently correlated with key demographic and clinical parameters. ADAMTS13 levels were significantly lower in acute ischemic stroke patients (82.6 ± 21.0%) compared with HV (110.6 ± 26.9%). Also, CCD patients (99.6 ± 24.5%) had significantly lower ADAMTS13 levels compared with HV however these were still higher than in acute stroke patients. Furthermore, when assessing the VWF:ADAMTS13 ratios, an even greater difference was revealed between stroke patients (2.7 ± 1.9), HV (1.1 ± 0.5) and CCD patients (1.7 ± 0.7). The VWF:ADAMTS13 ratio was significantly associated with stroke severity and modality. In conclusion, both in acute and chronic cerebrovascular disease patients, ADAMTS13 levels were significantly decreased, with the lowest ADAMTS13 levels found in acute stroke patients. This difference was even more distinct when the ratio of VWF:ADAMTS13 was considered. These results demonstrate the potentially important involvement of the VWF/ADAMTS13 axis in ischemic stroke.

Acquired Von Willebrand syndrome in patients on long-term support with HeartMate II.

Impaired binding of Von Willebrand factor (VWF) to platelets and to collagen due to acquired Von Willebrand syndrome (AVWS) is associated with support from a ventricular assist device (VAD) and can contribute to bleeding tendencies in patients with VADs. The onset of AVWS has been shown to occur immediately after VAD implantation. Our aim was to determine long-term data on AVWS in VAD patients. We analysed 278 data sets of 74 patients on HeartMate II (HMII) support for 3-80 months after implantation (11.2 ± 12.1, median 6.3 months.). Ristocetin cofactor activity (VWF:RCo), collagen binding capacity (VWF:CB), VWF antigen (VWF:Ag) and the ratios of VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag were determined. Further, the presence of high molecular weight (HMW) multimers of VWF was investigated. Abnormally low values of VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag were found in 69% and 97% of blood samples, respectively. Only ten of 181 multimer analyses showed a normal pattern, and HMW multimers were present in both specimens in only one of the 74 patients. No significant changes in these parameters were observed over time. The VWF:CB/VWF:Ag ratio correlated with the multimer patterns, whereas the VWF:RCo/VWF:Ag ratio seemed to be less sensitive for AVWS. Our data indicate that AVWS is a typical phenomenon in patients with VAD support and that there are no time-dependent changes in these parameters apparent in most patients on long-term support with HMII.

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.

Interim Canadian Data from a Prospective, Observational Study of Routine Clinical Use of a Von Willebrand Factor Concentrate (Wilate®)

Introduction:Von Willebrand disease (VWD) is the most common congenital bleeding disorder with a prevalence of symptomatic cases of 1 in 1,000. VWD is characterized by quantitative or qualitative abnormalities in von Willebrand factor (VWF), resulting in impaired haemostasis. To increase VWF levels, VWF concentrates are the only treatment option for approximately 20% of VWD patients for whom 1-deamino-8-D-arginine vasopressin (DDAVP) is not effective or is contraindicated. Wilate® (Octapharma AG, Lachen, Switzerland) is a high purity, double virus-inactivated, freeze-dried concentrate of VWF and FVIII. The ratio of VWF to FVIII is near the physiological value of 1:1 and is designed to simplify dosing and monitoring. Wilate®, initially approved in 2005 in Germany, is currently broadly approved worldwide for treatment of VWD and Hemophilia A. This non-interventional, prospective study in VWD patients was designed to collect information on the safety, tolerability and efficacy of Wilate® in routine clinical practice. We present the results of an interim analysis conducted in Canadian patients who received Wilate® in a routine clinical setting as prophylaxis, on-demand treatment and/or in a surgical setting. Methods:Ethics committee approval was obtained from all participating centres, and all VWD patients prescribed Wilate® from participating physicians were eligible for inclusion. Patients were excluded for the presence of VWF inhibitors, bleeding disorders other than VWD, a history of non-compliance, prohibitively difficult venous access, or the inability to follow study requirements. Routine clinical use of Wilate® for prophylaxis, on demand treatment of bleeding episodes, and prophylaxis for major and minor surgeries were documented. Parameters of routine clinical use are reported here and include exposure days, dosing, and number of infusions. Adverse drug reactions (ADRs), tolerability assessments, and haemostatic efficacy assessments are also reported. Results:Interim data from 13 patients, with a mean age of 45.6 (range 16 to 78 years), enrolled in participating Canadian centres was available for this analysis. Patients consisted of 3 males and 10 females, nine with type 1, three with type 2, and one with type 3 VWD. Two patients were treated for on-demand bleeding episodes, 2 were treated prophylactically, whereas 11 patients were treated for surgeries. A total of 1 051 000 IU Wilate® was infused in the thirteen patients with an average dose for all indications of 46 ± 9.7 IU/kg. Tolerability was 'excellent' in 284 of 285 (99.6%) assessed infusions. Hemostatic efficacy of Wilate® was assessed as 'excellent' or 'good' for all on-demand treatments of bleeding episodes. All 29 breakthrough bleeds, which were primarily GI bleeding, were treated successfully in one long-term prophylactic patient with severe VWD in 17 months. One other patient was treated for seventeen days following surgery and experienced no bleeding episodes. Eleven patients underwent 12 surgical procedures, one patient underwent 2 minor dental procedures, and 10 patients underwent ten major surgeries. In all 12 surgeries the haemostatic efficacy of Wilate® treatment was assessed as 'excellent.' No thrombotic events or severe ADRs were observed. Conclusion:This study is a part of ongoing pharmacovigilance of Wilate® and these interim observations support its safety and efficacy for VWD patients in routine clinical settings. In our Canadian cohort, under routine clinical conditions, bleed resolution, prevention, and tolerability were high. Disclosures Cosentino: Octapharma: Employment. James:CSL Behring: Research Funding; Octapharma: Research Funding; Biogen: Consultancy; Basalt: Consultancy; Bayer: Research Funding. Sholzberg:Shire (previously Baxter, Baxalta): Honoraria, Research Funding; Novonordisk: Honoraria.

Assessment of the Diagnostic Value of Plasma Levels, Activities, and Their Ratios of von Willebrand Factor and ADAMTS13 in Patients with Cerebral Infarction

Raised levels of von Willebrand factor (VWF) and reduced levels of a disintegrin and a metalloproteinase with a thrombospondin type I motif, member 13 (ADAMTS13) activity are associated with thrombosis. We aimed to investigate the relationships between plasma levels of VWF and ADAMTS13, their ratios, and the occurrence of cerebral infarction and to understand the roles of VWF and ADAMTS13 in cerebral infarction. Ninety-four patients with cerebral infarction and 103 controls were analyzed. Plasma levels of VWF antigen (VWF: Ag), VWF ristocetin cofactor activity (VWF: Rcof), and VWF collagen binding activity (VWF: CB) were measured by enzyme-linked immunosorbent assay (ELISA). The ADAMTS13 activity (ADAMTS13) was measured with FRETS-VWF73. The relationship between plasma levels and ratios of VWF and ADAMTS13 and the occurrence of cerebral infarction were analyzed. Patients with cerebral infarction displayed higher VWF: Ag and VWF: Rcof levels and lower ADAMTS13, VWF: CB/VWF: Ag, ADAMTS13/VWF: Ag, and ADAMTS13/VWF: Rcof levels compared to controls (P < .01). The highest quartiles of VWF: Ag (odds ratio [OR] = 5.11, 95% confidence interval [CI], 1.49-17.50) and VWF: Rcof (OR = 5.04, 95% CI, 1.62-15.66) and the lowest quartiles of VWF: CB/VWF: Ag (OR = 5.91, 95% CI, 1.95-17.93), ADAMTS13/VWF: Ag (OR = 9.11, 95% CI, 2.49-33.33), and ADAMTS13/VWF: Rcof (OR = 3.73, 95% CI, 1.39-10.03) are associated with cerebral infarction. An association was found between reduced levels of VWF: CB/VWF: Ag, ADAMTS13/VWF: Ag, and ADAMTS13/VWF: Rcof ratios and cerebral infarction. Our data suggest that increased levels of VWF and reduced levels of ADAMTS13 activity may contribute to the pathogenesis of cerebral infarction.

Decreased Platelet Thrombus Size, Due to a Heightened Proteolysis of VWF By ADAMTS13, Is Quickly Restored after Valve Replacement in Aortic Stenosis Patients

▪Background: The patients with severe aortic-valve stenosis (AS) are often complicated with bleeding episodes. The association between AS and gastrointestinal bleeding due to angiodysplasia is reported as Heyde’s syndrome, which is categorized as one of the acquired von Willebrand disease (AVWD) in cardiovascular disorders. An international survey has shown that Type 2A is the common subtype of AVWD in the patients with AS. AVWD Type 2A is characterized by impaired platelet-dependent VWF function caused by marked decrease or absence of the most hemostatically active HMW-VWFM. In the patients with AS, a significant correlation between the increased high shear stress and loss of HMW-VWFM in vivo. Further, the absence of HMW-VWFM and bleeding tendency are normalized after valve replacement. These results suggest that enhanced proteolysis of von Willebrand factor (VWF) as it passes through the stenotic valve may induce the loss of HMW-VWFM because high shear stress can induce structure changes in VWF, which is sensitive form to the VWF cleaving protease, termed ADAMTS13. Here, we performed investigation of plasma levels of VWF antigen (VWF:Ag), ADAMTS13 activity (ADAMTS13:AC), and platelet thrombus formation in the patients with AS by valve replacement to confirm the pathophysiological mechanism of this rare disease.Patients and Methods: Ten consecutive patients who underwent aortic valve replacement for AS in Nara Medical University Hospital were enrolled in this study. The severity of AS was judged by the American Heart Association guideline. All patients had no bleeding history and received bovine tissue valves replacement followed by administration of warfarin and/or anti-platelet agents for prevention of thrombosis a week after surgery. We collected a series of blood samples from these patients before and day1, 8, 15, 22 after valve replacement. Excluding one patient who developed critical cardiac failure just after valve replacement, 9 patients were eventually evaluated by analyses of VWF:Ag, VWF multimers, ADAMTS13:AC, and mural thrombus formation using flow chamber system. VWF:Ag was measured by sandwich ELISA using a rabbit anti-human VWF polyclonal antiserum. Analysis of VWF multimers was performed according to the method of Ruggeri and Zimmerman. ADAMTS13:AC was measured by a chromogenic ADAMTS13-act-ELISA. Platelet thrombus formation was evaluated by thrombus generation under a high shear stress in a parallel plate flow chamber system. Briefly, whole blood anti-coagulated with argatroban was incubated with the fluorescent dye DiOC6 (1uM), and these samples containing DiOC6 -labeled platelets were perfused for 7 min over a type I collagen-coated glass surface under a high shear rate (1500 s-1). The DiOC6 fluorescence corresponding to the platelets was examined at an excitation wavelength of 488 nm with a barrier filter at 500 nm. The percentage of the area covered by adhering platelets (surface coverage) and each thrombus volume were evaluated.Results: Plasma levels of VWF:Ag before surgery were 78.1 % (median) and those on day 1, 8, 15, 22 after surgery were 130, 224, 155, and 134 %, respectively (Fig 1). Conversely, these levels of ADAMTS13:AC were 50.5, 35.5, 25.5, 25.1, and 30.3 %, respectively (Fig 2). The ratio of VWF:Ag/ADAMTS13:AC at before and day 1, 8, 15, 22 after surgery were 1.6, 4.5, 8.1, 6.1, and 4.1, respectively. In VWF multimer analysis, we found the obvious defect of HMW-VWFM in 7 of 9 patients before surgery, who were diagnosed with severe AS. The remaining two patients had moderate AS with the slight defect of HMW-VWFM. These defects were improved within 14 days after surgery. In platelet thrombus formation, the amount of thrombus volumes significantly increased at day 8, 15, and 22 after compared with before surgery (Fig 3).Conclusion: The dramatic recovery of platelet thrombus formation was observed in the patients with AS by valve replacement. The rapid increment of VWF and normalization of VWFM pattern, together with reduction of ADAMTS13 after valve replacement suggested heightened proteolysis of VWF by ADAMTS13 under high shear stress would be a major cause of this unique bleeding complication. The highest ratio of VWF:Ag/ADAMTS13:AC at day 8 after surgery might imply the necessity of blockade of heightened VWF function with anti-platelet agents. [Display omitted] DisclosuresMatsumoto:Alfresa Pharma Corporation: Patents & Royalties. Fujimura:Alfresa Pharma Corporation: Patents & Royalties.

The Diagnostic Value of Plasma Level, Activity, Ratios between von Willebrand Factor and ADAMTS13 in Patients with Cerebral Infarction

Introduction: Large population studies have revealed that higher levels of von Willebrand Factor (VWF) and lower levels of ADAMTS13 activity (ADAMTS13) are associated with a risk of thrombosis. In this study, we aimed to investigate the relationship among the plasma levels of VWF, ADAMTS13 and the occurrence of cerebral infarction in patients.Methods: In a case-control study of 94 cerebral infarction patients (CIP) and 103 age-matched healthy controls (Control), the plasma levels of VWF antigen (VWF:Ag ), VWF ristocetin cofactor activity (VWF:Rcof), and VWF collagen binding activity (VWF:CB) were measured using ELISA. ADAMTS13 activity (ADAMTS13) was measured with FREST-VWF73. The study was approved by the institutional Ethics Committee at the First Affiliated Hospital of Soochow University, China. All participants were given write informed consents.Results: The levels of VWF:Ag and VWF:Rcof in cerebral infraction patients (mean±SEM, 223±15% and 256±20%) were significantly higher compared with controls (117±8% and 128±9%). Levels of ADAMTS13 and ratios of VWF:CB/VWF:Ag, ADAMTS13/VWF:Ag and ADAMTS13/VWF:Rcof in patients (107±4%, 0.73±0.08, 0.76±0.07 and 0.86±0.10, respectively) were significantly lower compared with controls (125±3%, 1.37±0.08, 1.69±0.14 and 1.45±0.10, P<0.001, respectively). However, there was no difference between cerebral infarction patients and controls in the levels of VWF:CB, VWF:Rcof/VWF:Ag and ADAMTS13/VWF:CB (P>0.05). Unconditional logistic regression analysis demonstrated the highest quartile of VWF:Ag (odds ratio [OR] = 5.11, 95% confidence interval [CI], 1.49-17.50), VWF:Rcof (OR = 5.04, 95% CI, 1.62-15.66), and the lowest quartile of VWF:CB/VWF:Ag (OR = 5.91, 95% CI, 1.95-17.93), ADAMTS13/VWF:Ag (OR = 9.11, 95% CI, 2.49-33.33), ADAMTS13/VWF:Rcof (OR = 3.73, 95% CI, 1.39-10.03) were associated with cerebral infarction (P<0.01). The receiver operating characteristic (ROC) curves analysis demonstrated the cutoff values of VWF:Ag (137.88%), VWF:Rcof (135.88%) and VWF:CB/VWF:Ag (0.538), ADAMTS13/VWF:Ag (0.974), ADAMTS13/VWF:Rcof (0.946) showed a better diagnostic value of cerebral infarction (AUC > 0.7).Conclusions: High levels of VWF:Ag, VWF:Rcof and lower levels of VWF:CB/VWF:Ag, ADAMTS13/VWF:Ag, ADAMTS13/VWF:Rcof are relative risk factors. The threshold of VWF:Ag ( > 137.88%), VWF:Rcof ( > 135.88%), VWF:CB/VWF:Ag ( ≤ 0.538), ADAMTS13/VWF:Ag ( ≤ 0.974), ADAMTS13/VWF:Rcof ( ≤ 0.946) can be diagnostic indicators of cerebral infarction.Research funding: This work was supported by grants from National Scientific Foundation of China (NSFC) (81270593), Jiangsu Provincial Special Program of Medical Science (BL2012005) and Jiangsu Province’s Key Medical Center (ZX201102), China DisclosuresNo relevant conflicts of interest to declare.

Utility of Various Von Willebrant Factor Laboratory Tests in Assessment of Acquired Von Willebrant Syndrome in Patients with Aortic Stenosis

Background: Aortic stenosis-associated acquired von Willebrand syndrome (AS-AVWS) is caused by an accelerated degradation of the highest molecular weight von Willebrand factor (VWF) multimers (HMWM). We recently reported that the severity of the HMWM loss correlates with the severity of AS and patients’ bleeding tendency (Am J Cardiol, 2013;111:374-81). As for the missing HMWM, it is still uncertain if they are cleared from circulation or redistributed with medium-low MWMs, which can be assessed by VWF propeptide (Pro)/antigen (Ag) ratio. Besides the tedious VWF multimer analysis, it is uncertain if VWF activity by immunoturbidic method (Lx)/ Ag ratio or VWF collagen binding activity (CBA)/Ag ratio can be used as a screening test to predict AS-AVWS. It has been speculated in the literature that VWF:CBA is more sensitive than VWF:Lx for detecting a HMWM loss. Our goal was to exam the laboratory characteristics of various VWF tests in evaluating AS-AVWS and the potential mechanism of HMWM loss.Method: Sixty-six patients (between years 2010-2012, 43% male) with varying degrees of AS (16 mild, 20 moderate, and 30 severe) and separate 21 patients who had aortic valve replacement were assessed with VWF:Ag, VWF:Lx, VWF:CBA, platelet function analyzer collagen plus adenosine diphosphate (PFA-CADP), VWF multimer analysis and multimer ratio by densitometry (Am J Cardiol, 2013;111:374-81) after their echocardiography. The clinical endpoints of this study are AS severity measured by mean gradient (MG) by echocardiography and clinically significant bleeding. Statistical analyses including Spearman rank correlation test, estimation of the area under the receiver operating characteristics (ROC) curve, and Wilcoxon rank sum tests were performed.Results: VWF:Lx and VWF:CBA had strong correlation (Spearman r=0.94, P<0.001), and the correlation of VWF:CBA/Ag and VWF:Ltx/Ag was 0.49, p<0.001). As previously published, in patients with AS, MG correlated with VWF:Lx/Ag (Spearman r = -0.41, p <0.001), PFA-CADP (r = 0.49, p <0.001), and VWF multimer ratio (r = -0.76, p <0.001). MG also correlated with VWF:CBA/Ag (r= -0.38, p=0.002), but not with VWF: Pro/Ag (r=-0.20, p=0.12). Among AS and AVR patients, the area under the ROC curve for detection of the loss of HMWM was 0.88 (95% CI: 0.80-0.95) by VWF multimer ratio, 0.76 (95% CI: 0.65-0.86) by PFA-CADP, 0.70 (95% CI: 0.58-0.82) by VWF:CBA/Ag ratio, 0.67 (95% CI: 0.56-0.70) by VWF:Lx/Ag ratio, and 0.53 (95% CI: 0.41-0.65) by VWF:Pro/Ag . In addition to previously published results, clinically significant bleeding, in 14% of AS patients, was not associated with VWF:CBA/Ag (p=0.25) or VWF:Pro/Ag ratio (p=0.83). VWF: CBA/Ag ratio was significantly lower in patients with severe AS (MG > 40 mmHg) compared to AVR patients (median: 0.84 vs. 1.06, p<0.001), while there was no evidence of a difference in VWF:Pro/Ag (p=0.45).Conclusion: HMWM losses are highly prevalent in patients with moderate to severe AS. Of the VWF tests, PFA-CADP and VWF multimer analysis and ratio had the highest predictive ability for AS-AVWS followed by VWF:CBA/Ag and VWF:Lx/Ag. The VWF:CBA is not overly superior than VWF:Lx for predicting a HMWM loss. The normal VWF:Pro/Ag suggests that AS-AVWS is different from other types of AVWS due to accelerated clearance of HMWM. DisclosuresNo relevant conflicts of interest to declare.

Cost-consequence analysis of long-term prophylaxis in the treatment of von Willebrand disease in the Italian context.

PurposeProphylaxis with von Willebrand factor (VWF)/factor VIII (FVIII) concentrates is a potential approach for patients with severe von Willebrand disease (VWD). As far as we are aware, to date there have been no pharmacoeconomic analyses in order to assess the economic impact of treatments for severe VWD. The analysis presented here estimates the cost–benefit ratio of VWF with a low FVIII content when compared with VWF/FVIII concentrates currently used in Italy for long-term prophylaxis in patients with severe VWD.MethodsA cost–consequence analysis was undertaken to assess the economic impact of the treatment of severe VWD from the perspective both of the Italian National Health Service and society. The analysis was based on four case reports of long-term prophylaxis with VWD with VWF/FVIII concentrates and VWF with a low FVIII content. The costs per patient included direct and indirect costs for each treatment.ResultsConsidering the four case reports, health care costs (without cost of treatment) and indirect costs per patient per year were lower with VWF with a low FVIII content than VWF/FVIII concentrates. The total health care costs (without cost of treatment) and indirect costs avoided with VWF with a low FVIII content per patient per year ranged from €2,295 to €17,530 and from €1,867 to €4,978, respectively.ConclusionVWF with a low FVIII content seems to be a cost-effective treatment option for patients with severe VWD. Although the drug cost per se is higher, the use of VWF with a low FVIII content is associated with decreased consumption of hospital resources and fewer lost working days due to bleedings and consequently with an improvement of the quality of life of the patients.