vWF Antigen Levels Research Articles

Elevated Von Willebrand Factor Antigen Serum Levels Are Associated With Poor Prognosis In Patients With Symptomatic Waldenstrom's Macroglobulinemia

Waldenstrom's Macroglobulinemia (WM) is an uncommon malignancy which is characterized by the infiltration of the bone marrow by lymphoplasmacytic cells, which produce a monoclonal IgM. The manifestations of the disease are related to the infiltration of the bone marrow and the quantity, physical and immunological properties of the monoclonal IgM. The interactions of lymphoplasmatic cells with other cells in their microenvironment, including mast cells and endothelial cells, support their survival and proliferation and may induce resistance to therapy. Von Willebrand factor (vWF), a glycoprotein produced by the endothelial cells and megakaryocytes, plays a key role in primary hemostasis but is also a marker of endothelial “stimulation”. Recently, it was shown that high levels of vWF, measured in citrated plasma, are associated with adverse prognosis in patients with symptomatic WM and it was suggested that vWF levels may reflect interactions between lymphoplasmacytic cells and other cells of their microenvironment such as mast cells and endothelial cells (Hivert et al; Blood 2012;120:3214-21). Thus, we evaluated the prognostic importance of vWF antigen levels in the serum of patients with symptomatic previously untreated WM, in order to validate vWF as a possible prognostic marker for progression free (PFS) and overall (OS) survival and to validate the measurement of vWF in the serum instead of citrated plasma.The vWF antigen (vWF Ag) levels were measured in serum collected before initiation of therapy by means of a latex particle-enhanced immunoturbidimetric assay (HemosIL vWF antigen) with an automated coagulometer (ACL Top 3G, Instrumentation Laboratory, Lexington, MA, USA). The inter-assay and intra-assay coefficients of variation were 2% and 3% at a concentration of 123.5 U/dL, respectively, and the lower limit of detection was 2.2 U/dL.The analysis included 42 patients with symptomatic WM, treated at the Department of Clinical Therapeutics, University of Athens (Greece), from 1999 to 2012, and 19 healthy controls of matched gender and age. The median age of patients with symptomatic WM was 65 years (range: 37-83 years) and 54% of them were males. Anemia (<11.5 g/dL) was present in 78% of patients, lower platelet counts (<100x109/L) in 17%, beta2-microglobulin >3 mg/dl in 56%, while 7.5% had serum LDH ≥250 U/L and 58% had serum albumin <3.5 g/dL. Median serum IgM was 3340 mg/L (range 246-9563 mg/dL). According to IPSSWM, 22% had low, 43% intermediate and 35% high risk WM, respectively. Reasons to initiate therapy included cytopenias in 42% of patients, B-symptoms in 15%, hyperviscosity in 12%, neuropathy in 10% and other reasons in 21%. Primary therapy based on rituximab was given in 93% of the patients and 54% achieved at least 50% reduction of IgM.Median serum level of vWF Ag was 101 U/dL (mean 132.5 U/dL, range 19.9-399 U/dL) and were slightly higher compared to the serum levels of healthy controls (median 85 U/L, mean 85 U/L, range 48-124 U/L). However, 6/42 (14%) had vWF Ag levels <40 U/L; which could be compatible with acquired vWF syndrome but no further investigation was performed.There was an inverse correlation of platelet counts with levels of vWF Ag (R=-0.336, p=0.032) and vWF Ag ≥median was more frequent in patients with beta2-microglobulin >3 mg/L (p=0.006) and less frequent in patients with low (11%) vs. patients with intermediate (59%) or high (62%) risk IPSS (p=0.036). There was no correlation of vWF Ag levels with IgM levels or with the extent of bone marrow infiltration or with other manifestations of the disease.Median follow up of symptomatic patients was 4 years. Patients with vWF Ag levels within the upper quartile (i.e. vWF Ag ≥200 U/dL) had a median progression free survival of 12 months vs. 63 months of patients with vWF Ag < 200 U/L (p<0.001), while the median survival for patients with vWF Ag ≥200 U/dL was 37 months (4-year survival 29%) vs. 4-year survival of 97% for patients with vWF Ag <200 U/L (p<0.001; Figure).In conclusion, the serum levels of vWF antigen provide significant prognostic information in patients with symptomatic WM and patients with levels ≥200 IU/dL have a very poor prognosis compared to patients with lower levels. vWF measured in the serum, may become an important prognostic marker in WM and needs further investigation. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Increased Platelet Thrombus Formation Under Flow Condition In Myeloproliferative Neoplasms (MPN)

BackgroundEssential Thrombocythemia (ET) and Polycythemia Vera (PV) are MPNs characterized by a high incidence of both arterial and venous thrombosis, and microcirculatory disturbances. Platelet abnormalities have been variably involved in the thrombotic diathesis of these patients, without providing conclusive evidence. Remarkably, no studies have explored so far the MPN platelet thrombus formation capacity underflow conditions. AimIn order to evaluate whether and to what extent the MPN platelet membrane abnormalities may influence their interactions with the vessel wall components in a dynamic system, we wanted to characterize the ET and PV platelet adhesion capacity under flow conditions, by using an in vitro system based on a parallel flow chamber connected to the EVOS fluorescence microscope. The effects of the V617F JAK2 mutational status, cytoreductive therapy, and circulating von Willebrand Factor (vWF) on platelet adhesion in this system were also explored. MethodsNine ET (3M/6F; age 63 years, range 60-81) and 6 PV (3M/4F; age 74 years, range 58-82) patients were enrolled into the study upon informed consent. Eleven healthy subjects (5M/6F; age 44 years, range 35-55) acted as a control group (CTR). Peripheral venous whole blood was withdrawn in sodium citrate, recalcified in the presence of heparin, and perfused over a collagen-coated surface for 4 min at a shear rate of 1000 s-1. Platelets were then stained with an anti-CD62P (P-selectin)-FITC antibody as a platelet activation index, and annexin V-AlexaFluor647 as a measure of the procoagulant phosphatidylserine (PS) expression. After staining, images of adherent platelets in random fields were taken using phase contrast and fluorescence imaging with the EVOS fluorescence microscope system. Results were the mean±SD of the percentage of area covered by all platelets, or as the % of adherent platelets positive to either P-selectin or annexin V. In parallel, plasma vWF antigen and activity levels were measured by ELISA. Statistical analysis was performed by SPSS software package. ResultsPlatelet adhesion was significantly greater (p<0.05) in both ET (49.3±14.5%) and PV patients (55.3±12.7%) compared to controls (31.6±7.3%). Among patients, platelet adhesion was significantly (p<0.05) increased in those positive for the V617F JAK2 mutation compared to the negative ones, with the highest values in the homozygous subjects. Patients on hydroxyurea (HU) therapy (n=7) had significantly lower platelet adhesion (45.2±13.0%) compared to non-HU-treated patients (56.9±10.4%; p<0.05).The % platelet adhesion directly related to either platelet (r=0.623, p=0.001) and leukocyte (r=0.506, p<0.01) counts, but not to plasma vWF levels. Multivariate regression analysis adjusted for age, sex, and HU therapy, confirmed platelet count and V617F JAK2 mutation as significant determinants of platelet adhesion. Although adhesion was increased, the platelet surface expression of P-selectin and PS was reduced in ET (P-sel: 70±11% pos. platelets; PS: 11±6% pos. platelets) and PV (P-sel: 66±13%; PS: 9±5%) patients compared to controls (P-sel: 74±8%; PS: 20±8%). ConclusionsThe data show for the first time, in MPN patients, an increased platelet adhesion capacity to collagen under flow conditions, indicating a greater thrombus formation potential. This phenomenon is likely not related to the expression of membrane P-selectin and phosphatidylserine, but is significantly influenced by the V617F JAK2 mutation burden and is sensitive to the cytoreductive treatment with HU. Prospective studies are worth to establish the role of the EVOS assay in assessing the levels of thrombotic risk in MPN patients. Disclosures:No relevant conflicts of interest to declare.

Elevated Serum Levels Of Von Willebrand Factor (vWF) Predict For Early Death and Shorter Survival In Patients With Primary Systemic Light Chain (AL) Amyloidosis Independently Of Cardiac Biomarkers

Organ dysfunction in systemic light chain (AL) amyloidosis is caused by the deposition of amyloid fibrils composed of immunoglobulin light chains in tissue microcirculation. vWF is mainly produced, stored and secreted by endothelial cells (ECs) and it is critical for thrombus formation. The secretion of vWF by ECs is triggered by several conditions and it has been proposed that changes in the levels of circulating vWF may reflect a state of “stimulation” of the endothelium. Cardiac involvement is the main determinant of prognosis in AL amyloidosis, but the role of endothelium in this disease has not been extensively studied and no marker of endothelial dysfunction has been evaluated, as yet. Thus, we studied the prognostic role of vWF in patients with AL amyloidosis who were treated with novel agents.The vWF antigen (vWFag) levels were measured using a latex particle-enhanced immunoturbidimetric assay (HemosIL vWF antigen) with an automated coagulometer (ACL Top 3G, Instrumentation Laboratory, Lexington, MA, USA). The inter- and intra-assay CVs were 2% and 3% at a concentration of 123.5 U/dL, respectively, and the lower limit of detection was 2.2 U/dL.The analysis included 81 consecutive patients with newly diagnosed AL amyloidosis who were treated in a single center (Department of Clinical Therapeutics, University of Athens, Greece) from 2005 to 2012. Median age of the patients was 68 years (range 42-82 years) and the median number of involved organs was 2; heart was involved in 62%, kidneys in 74%, peripheral nerve in 24%, liver in 9% and soft tissue in 21% of patients. Median NTproBNP level was 2,318 pg/mL (range 33-75,000 pg/mL); 36% had NTproBNP levels ≥4,000 pg/mL and 28%, 38% and 34% of patients had Mayo stage -1, -2 and -3, respectively. Primary therapy based on bortezomib was given in 52% of patients, on lenalidomide in 44%, while 4% received MDex. Median survival of the cohort was 47 months; 3- and 6-month mortality was 12% and 20%, respectively.The median serum level of vWFag in patients with AL amyloidosis was 181 (range 20-557) U/dL and was significantly higher than that measured in healthy controls (median: 84 U/dL, range 48-124; p <0.001). There was no significant association of vWFag levels with renal, cardiac, nerve or liver involvement. There was no significant correlation of vWFag levels with the levels of NTproBNP (or BNP) or Mayo stage and there was no significant correlation with the degree of renal dysfunction, serum albumin levels or proteinuria. Levels of involved free light chains did not have any correlation with vWFag levels either.We then examined the prognostic significance of vWFag and we found that levels of vWFag within the top quartile (≥230 U/dL) were associated with a very poor outcome (median survival 4 months vs. 47 months, p=0.001). Because the most important predictor of early death in patients with AL amyloidosis is cardiac involvement, we performed a multivariate analysis which included NTproBNP levels: vWFag levels ≥230 U/dL were independently associated with survival (HR: 2.64, 95% CI 1.2-5.8, p=0.01), along with NTproBNP levels ≥4,000 pg/ml (HR: 4.17, 95% CI 1.98- 8.8, p<0.001). Because the survival curves indicated that patients with vWFag ≥230 U/dL had a significant probability of early death, we performed an analysis to identify whether vWFag is a significant predictor of early death, adjusting for the levels of NTproBNP ≥4000 pg/mL. Thus, in multivariate analysis, vWFag ≥230 U/dL was independent predictor of death within 6 months from initiation of therapy (HR: 15, 95% CI 2.6-84, p=0.002) together with NTproBNP ≥4,000 pg/mL (HR: 16.8, 95% CI 3- 94, p=0.001). A combination of the two risk factors was able to identify patients at a very high risk of early death: 75% of patients with both risk factors present died within 3 months from initiation of therapy (figure).In conclusion, vWFag levels are elevated in patients with AL amyloidosis but are not correlated with other features of the disease, such as pattern of organ involvement and cardiac biomarkers. For the first time, we found that high levels of vWFag are associated with a high risk of early death and shorter survival in patients with AL amyloidosis, independently of cardiac biomarkers. In addition, wWFag levels improve the prognostic ability of cardiac biomarkers in patients with AL amyloidosis. Our data also justify the investigation of the role of endothelial dysfunction in AL amyloidosis. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Quantitative and qualitative changes of von Willebrand factor and their impact on mortality in patients with end-stage kidney disease

von Willebrand factor (vWF) antigen levels are elevated in patients with end-stage kidney disease (ESKD). We determined the quantitative and qualitative abnormalities of vWF and factors influencing vWF proteolysis in participants with ESKD compared with age-matched controls and determined the association between abnormalities in vWF and mortality over 4 years of follow-up. vWF : Ag and von Willebrand factor propeptide (vWFpp) levels, vWF functional activity (vWF :RCo), vWF multimer profiles, ADAMTS-13, thrombospondin 1 (TSP-1), and interleukin 6 (IL-6) were evaluated before and after a single hemodialysis treatment in 55 individuals with vascular disease and an age-matched group of controls (n = 21). vWF : Ag and vWF activity were significantly higher in the ESKD patients and levels increased further following the dialysis procedure. The percentage of high molecular weight multimers (%HMWMs) was significantly elevated in the ESKD patients compared with controls. TSP-1 was lower and IL-6 was higher providing possible explanation for the increase in %HMWM in ESKD. The %HMWM dropped significantly in the postdialysis sample. Mortality at 4 years was significantly associated with vWF : Ag. There are higher plasma vWF : Ag levels and a small increase in HMWMs in the ESKD milieu. The acute drop in the %HMWM of vWF postdialysis appears to be due to shear forces encountered during the dialysis procedure. The contribution of these abnormalities to either a pro-thrombotic and/or pro-bleeding phenotype in this population requires further study.

Time course of soluble P-selectin and von Willebrand factor levels in trauma patients: a prospective observational study

BackgroundCoagulopathy often develops in patients with serious trauma and is correlated with the clinical outcome. The contribution of platelet activity and endothelial dysfunction to trauma-induced coagulopathy remain to be defined. The purpose of this study was to investigate the time courses of soluble P-selectin (sPsel, an index of platelet activation) and von Willebrand factor (VWF, an index of endothelial dysfunction) in trauma patients and elucidate their relationship to coagulation parameter levels, the presence of coagulopathy, and patient outcome.MethodsThis prospective observational study, which took place in a university hospital intensive care unit (ICU), included 82 severely injured trauma patients. The sPsel, VWF antigen, protein C, and factor VII levels were measured and routine coagulation tests were performed upon admission to ICU and daily within the first week. The 30-day mortality rate was also determined.ResultsThirty-seven (45.1%) patients developed coagulopathy upon admission to the ICU, and the 30-day mortality rate was 20.7% (n = 17). Both the admission sPsel and VWF levels were lower in patients with coagulopathy than in those without (p < 0.05) and were significantly correlated with the protein C and factor VII levels, respectively (all p < 0.05). The VWF levels were lower during the first 3 days and higher on day 7 after admission in nonsurvivors than in survivors (all p < 0.05). No significant differences in sPsel levels were found between nonsurvivors and survivors on each day during the first week.ConclusionIn severely injured trauma patients in the ICU, lower levels of sPsel and VWF on admission were associated with the presence of coagulopathy and might not predict a better outcome. An increase in the VWF level at the end of the first week after admission to ICU was associated with increased 30-day mortality.

Deep intronic ‘mutations’ cause hemophilia A: application of next generation sequencing in patients without detectable mutation in F8 cDNA

In a small group of typical hemophilia A (HA) patients no mutations in the F8 coding sequence (cDNA) could be found. In the current study, we performed a systematic screening of genetic and non-genetic parameters associated with reduced FVIII:C levels in a group of mostly mild HA (only one moderate) patients with no detectable mutations in F8 cDNA. We determined FVIII and VWF activity and antigen levels and performed VWF-FVIII binding (VWF:FVIIIB) and VWF-collagen binding assays (VWF:CB) as well as VWF multimer analysis. VWF was completely sequenced to exclude mutations. The F8 locus, including the introns, was sequenced using overlapping long-range PCRs (LR-PCRs) combined with a next generation sequencing (NGS) approach. Moreover, the F8 mRNA was analyzed quantitatively and qualitatively by real-time PCR (qRT) and overlapping reverse transcription (RT) PCRs, respectively. All VWF tests were normal. The LR-PCRs demonstrated the integrity of the F8 locus. Eight unique polymorphisms were found in the patients, with two being recurrent. Furthermore, RT-PCRs analysis confirmed that two of the unique variants create detectable new cryptic splice sites in the patients that result in the introduction of intronic DNA sequences into the mRNA and create premature stop codons. By systematically excluding all possible causes of HA, we could with great certainty conclude that deep intronic mutations in F8, although rare, cause abnormal mRNA splicing, leading to mild HA.

Retrospective Survey of Hemostatic Complications in 680 Patients Undergoing Hematopoietic Stem-Cell Transplantation (HSCT)

s / Biol Blood Marrow Transplant 19 (2013) S279eS312 S292 thrombotic complications had decreased plasma ADAMTS13 activity (P < .01) and increased VWF antigen level after pretreatment (P< .01) as comparedwith the non-thrombotic patients; three out of 8 (37.5%) showed more than 60% decrease in plasma ADAMTS13 activity. The level of ADAMTS13 activity dropped in the 49 patients with aGVHD as compared with healthy controls (P< .01), but therewas no significant difference between patients with and without aGVHD. Twenty-five patients showed decreased plasma ADAMTS13 activities only at the onset of aGVHD occurrence (P < .01), in which two of them decreased more than 60% (6%). Logistic regression analysis showed that the ADAMTS13 activity declined by more than 60% was the risk of thrombosis (P < .01). Conclusions: We observed decreased plasma ADAMTS13 activity and increased plasma level of VWF antigen in patients following HSCT after pretreatment, especially in the patients with thrombotic complications. A decrease more than 60% in plasma ADAMTS13 activity is the risk factor of thrombotic complications. Therefore, the plasma ADAMTS13 activity could be an important parameter for the development of vascular disorder, which has a potential role for the early diagnosis of thrombotic complications.

ADAMTS13 activity and genetic mutations in Japan

Thrombotic thrombocytopenic purpura (TTP), a life threatening disease, can be induced by congenital or acquired deficiency of plasma metalloprotease ADAMTS13. Since the publication of the first genetic analysis in patients with congenital ADAMTS13 deficiency in 2001, more than 100 genetic defects in the ADAMTS13 gene have been reported worldwide. Genetic analysis in patients with ADAMTS13 deficiency has greatly contributed to the understanding of the etiology of TTP. A rapid and quantitative assay method for the plasma ADAMTS13 activity was developed recently in 2005 and opened a new area of TTP research - namely genetic research using a general population to evaluate age and gender differences of ADAMTS13 activity as well as phenotype - genotype correlations of genetic polymorphisms and estimation of a homozygote or a compound heterozygote ADAMTS13 deficiencies. The Japanese general population study included 3616 individuals with an age between 30 - 80 years confirming other studies that while ADAMTS13 activity decreased with age, VWF antigen increased and VWF antigen levels are lowest in blood group O indviduals, whereas ADAMTS13 activity levels were not associated with the AB0 blood group. 25 polymorphisms with a minor allele frequency of more than 0.01 were found, among them 6 missense mutations and 19 synonymous mutations, except P475S missense polymorphisms that was only idenitified in an East Asian population, characterized by reduced ADAMTS13 activity. Prevalence of congenital ADAMTS13 deficiency in the Japanese population was estimated about one individual in 1.1 x 106 to be homozygote or compound heterozygote for ADAMTS13 deficiency. So far more than 40 mutations in Japanese congenital TTP patients were found, but R193W, Q449*, C754Afs*24 (c.2259delA) and C908Y were identified in more than four patients suggesting the precipitaion of these mutations in the Japanese population.

Von Willebrand Factor Inhibition Improves Endothelial Function in Patients with Stable Angina

ALX-0081 is a novel nano-antibody inhibiting von Willebrand factor (vWF). We evaluated whether direct inhibition of vWF by ALX-0081 improves endothelial function. Stable patients (pts, n = 55) with single vessel disease undergoing percutaneous coronary intervention (PCI) were randomized to ALX-0081 (n = 38) or placebo (n = 17). vWF inhibition was assessed by vWF antigen level (vWF:Ag) and activity by ristocetin test (vWF:RiCo). Endothelial function was assessed before (BL), 6 h and 24 h after PCI by: (a) endothelial peripheral arterial tonometry (Endoscore); (b) endothelial microparticles (EMPs) by flow cytometry. vWF:Ag and vWF:RiCo decreased within 1 h from ALX-0081. In the placebo group, no significant Endoscore changes occurred from BL to 24 h. In ALX-0081 group, Endoscore increased from BL to 24 h (p = 0.014). A decrease in EMPs was observed after ALX-0081 (p < 0.01), while no changes occurred in placebo pts. An inhibition of vWF with ALX-0081 significantly improves peripheral endothelial function.

Genetic Determinants of Plasma Von Willebrand Factor (VWF) and VWF Propeptide Levels

Abstract 3313Plasma VWF levels vary by approximately five-fold in healthy populations and are influenced by both environmental and inherited factors. Low levels of VWF are associated with bleeding and elevated levels with increased risk for thrombosis. Increased levels of VWF occur with age, may rise acutely due to inflammation or infection, and may serve as a biomarker for endothelial dysfunction and atherosclerosis. Genetic factors are thought to account for 60 – 70% of the variance in VWF antigen levels with ∼30% of this effect due to ABO blood type. VWF multimers are assembled in the Golgi apparatus where the VWF propeptide is cleaved to form mature VWF. Because the half life of plasma VWF propeptide (ppVWF) is ∼5X faster than the mature VWF antigen, the ratio of ppVWF/VWF:Ag in plasma primarily reflects clearance rates of mature VWF while alterations in biosynthesis and/or secretion of VWF affect the level of both ppVWF and VWF:Ag. In order to identify additional quantitative trait loci (QTL) for VWF antigen variation and altered clearance of VWF:Ag, we performed genetic analyses in a healthy young sibling cohort, the Genes and Blood Clotting Study (GABC, n=1,152). The cohort was SNP genotyped with an Illumina Omni-1 array resulting in 763,195 SNPs available for analysis after extensive QC. Plasma VWF antigen (VWF:Ag) and VWF propeptide levels were determined by AlphaLISA (Perkin Elmer, Waltham, MA), using polyclonal anti-human VWF antibodies (DAKO, Glostrup, Denmark) or monoclonal anti-ppVWF antibodies (Blood Center of Wisconsin, Milwaukee, WI). The median levels were 108.1 IU/dL, 99.97 IU/dL and 1.0 for VWF:Ag, ppVWF and ppVWF/VWF:Ag respectively. The estimated heritability (h2) was 64.5%, 55.72%, and 64.26% for VWF:Ag, ppVWF, and ppVWF/VWF:Ag respectively. A genome-wide association study (GWAS) for VWF:Ag in this cohort revealed significant signals at the ABO and VWF loci, consistent with previously published reports. However, GWAS for ppVWF levels demonstrated a signal only at 12p13, overlying the VWF gene itself. The top SNP, rs1800378 (beta 4.297, p-value 1.58E–11), encodes a nonsynonymous substitution (His484Arg) in the VWF propeptide. In contrast, GWAS for ppVWF/VWF:Ag showed a strong signal at 9q34, the ABO locus, (top SNP rs687289, beta −0.2131, p-value 1.88E–62) and no significant signal at 12p13. These results suggest that ABO is the major common determinant of variability in VWF clearance rates and a common SNP, rs1800378, is associated with VWF propeptide levels. We next utilized the sibling structure in this cohort to perform linkage analyses. Linkage analysis for VWF:Ag identified a novel locus at 2p12-2q13 in addition to the expected signal at ABO. The effect size of this chromosome 2 locus on VWF variation (19.2% variance explained) was comparable to the effect of the ABO locus (24.5%). Linkage analysis for ppVWF demonstrated novel loci at 7q21 (LOD score 5.03) and at 11q21 (LOD score 4.12), with no significant signal on chromosome 2. In contrast, linkage analysis for ppVWF/VWF:Ag identified signals at 2p12 (LOD score 3.17), 8p22 (LOD score 3.48) and 11q22 (LOD score 3.41). Taken together, these results identify a number of potential candidate loci for genetic regulation of plasma VWF through alteration of synthesis, storage, secretion or clearance mechanisms. We hypothesize that signals present in linkage but not association are due to VWF regulatory genes with high allelic heterogeneity, harboring many genetic variants that are individually rare, but in aggregate present in a large portion of sibships. Segregation of these variants among sibs could then explain the strong signal by linkage, but lack of signal by association in unrelated individuals. Replication of the strong 2p12-2q13 signal in both VWF:Ag and ppVWF/VWF:Ag linkage studies suggests that the responsible variants at this locus affect VWF levels through altered clearance. The novel QTLs for ppVWF/VWF:Ag levels on chromosome 8p22 and 11q22 are also likely to work through altered clearance mechanisms while the novel signals for ppVWF levels on 7q21 and 11q21, not significant in the VWF:Ag linkage study, may affect efficiency of cleavage or clearance of ppVWF. Identification of the underlying genes at these novel loci and their mechanism of action could lead to improved understanding of the severity, penetrance and treatment for von Willebrand Disease as well as the risk for venous thromboembolic disease associated with elevated VWF. Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Pharmacokinetic Evaluation of a Newly Introduced Von Willebrand Factor Concentrate At Two London Haemophilia Centres

AbstractAbstract 3384Introducing new factor concentrates in VWD patients is complicated by VWD subtype and PK variability. Wilate, a VWF concentrate with a 1:1 VWF: FVIII ratio and has not yet been widely used in the UK. We conducted PK studies with Wilate, to determine its efficacy, peak VWF activity and FVIII levels, and clearance in patients with VWD. Where feasible, we compared the data with previously used concentrate (Haemate-P) handling. 17 VWD patients (4 type 1, 6 type 2, 7 type 3) from two London Haemophilia centres were evaluated. The median age was 36 yrs (range 12– 67 yrs); sex M:F 6:11; mean body weight 73 kg (range 39–122 kg). Seven were blood group O, 1 group AB, 4 group A and 4 group B. The mean dose of Wilate administered was 44 iu/kg VWF: RiCoF (range 17–61). VWF activity, VWF antigen and FVIII levels were measured pre- and post for up to 8 hrs. The mean dose of Wilate required to reach target VWF:RiCof activity level of >65 iu/dl in the type 3 VWD was 49.6 iu/kg, and 38.7 iu/kg in type 2 VWD. Of the type 1 patients mean dosing was 43.0 iu/kg. The median peak VWF:RiCoF activity was 82 iu/dl in type 3 and 115 iu/dl in type 2, and 92 iu/dl in type 1. VWF Antigen levels were 116 iu/dl in type 3, and 164 iu/dl in type 2 and 135 iu/dl in type 1. VWF handling in some individuals was suggestive of increased VWF clearance with a half-life below the suggested half-life of 12 hours. This observation indicates the importance of prolonged PK studies in individual cases. As expected, peak FVIII levels were generally higher with Wilate compared to previously used concentrate (94 iu/dl in type 3 and 82 iu/dl in type 2) but the PK profiles between the products were similar, with some inter-individual variability. In conclusion, Wilate achieves adequate VWF activity and FVIII levels and has similar PK properties to previously used concentrate. However, based on the results of this study prolonged PK assessment appears important in selected cases. Disclosures:Hegener:Octapharma AG: Employment. Austin:Baxter: Advisory Board Other; Pfizer: Advisory Board, Advisory Board Other.

Severity of the Loss of High Molecular Weight Multimers Is Associated with Heyde Syndrome in Patients with Severe Aortic Stenosis.

AbstractAbstract 2230 Background:Heyde syndrome describes patients with severe aortic stenosis (AS) who have bleeding diathesis, especially gastrointestinal bleeding. Although acquired won Willebrand syndrome (AVWS) due to loss of the highest molecular weight multimers (HMWM) presents in most patients with severe AS, not all such patients develop Heyde syndrome. The goal of this study was to explore whether the severity of HMWM loss was significantly associated with Heyde syndrome. Methods:We first performed chart reviews on patients referred for double balloon enteroscopy (DBE) for GI bleeding or iron-deficiency anemia (GIB/FeDefic) in two time periods, from 2005–2008, and from 2009–2011. Next, patients who could have AVWS due to AS or other cardiovascular or hematologic disorders were identified. Laboratory tests including VWF antigen (VWF:Ag), VWF ristocetin cofactor activity or VWF latex immunoturgidic activity (VWF:Ltx) and/or VWF multimer analysis were performed as requested by physicians. From this population, patients with Heyde syndrome were identified. Their clinical and VWF laboratory testing results were compared to a separated group of patients with severe AS patients who had no clinically significant bleeding, but had matched gender and hemodynamic severity by echocardiogram (Matched AS Controls). These patients all had VWF:Ag, VWF:Ltx, and VWF multimers performed. In both Heyde syndrome patients and Matched AS controls, VWF multimer densitometry ratio of the HMWM (>15 mers) to low to intermediate multimers (2–15 mers) was assessed (VWF:multi ratio). Results:Overall, 437 patients underwent DBE for GIB/FeDef. Between 2005 and 2008, 1% of 318 patients had VWF testing. From 2009 to 2011, 23% of 119 patients were tested. Heyde syndrome was noted in 15 patients. Of the 437 patients, 134 patients (31%) likely had AVWS based on diagnoses of moderate-severe AS (30%), moderate to severe mitral regurgitation (25%), moderate to severe pulmonary hypertension (16%), prosthetic valve regurgitation or stenosis (13%), hypertrophic cardiomyopathy (10%), and hematologic disorders (4%). Among these 134 patients, 29 patients had VWF tests performed, and 22 had VWF multimer analysis. VWF antigen levels were similar in Heyde syndrome and Matched AS Controls (176±89% versus 155±77% respectively). Despite similar hemodynamic severity, more severe HMWM loss as indicated by lower VWF:Ltx/Ag and VWF:multi ratios was present in Heyde syndrome patients than matched AS controls (table, P<0.0007).Age, yearsMean gradient, mm HgVWF: Ltx/VWF:AgVWF:multi ratiosPFA-CADPHeyde (n=15)76±9†47±12†0.78±0.06*0.106±0.033*203±57†Matched AS Controls (n=25)78±1147±150.89±0.050.143±0.015174±121N=10 for VWF activity/antigen and PFA-CAPD in Heyde syndrome patients.*p<0.0007.†p=NS Conclusion:If AVWS is suspected in patients with GIB/FeDef, laboratory testing for VWF indices is important to confirm the diagnosis of AVWS. Despite similar hemodynamic severity, Heyde syndrome patients have more severe VWF HMWM loss indicated by significantly decreased VWF:Ltx/VWF:Ag and VWF:multi ratios. Conversely, though highly sensitive for AVWS, PFA-CADP does not predict bleeding tendency in patients with severe AS. Disclosures:No relevant conflicts of interest to declare.

Ibrutinib (PCI 32765) Rapidly Improves Platelet Counts in Chronic Lymphocytic Leukemia / Small Lymphocytic Lymphoma (CLL/SLL) Patients and Has Minimal Effects On Platelet Aggregation

AbstractAbstract 1789 INTRODUCTION:Ibrutinib (PCI 32765) is an orally administered covalent inhibitor of Bruton's Tyrosine Kinase (BTK). Ibrutinib has significant activity in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and is typically well tolerated (Byrd ASCO 2011, O'Brien ASH 2011). Rarely serious bleeding in patients concurrently on oral anticoagulation has been reported but was not related to thrombocytopenia (O'Brien ASH 2011). However, grade 1 or 2 ecchymosis/contusion is a frequent adverse event in patients on ibrutinib. In addition to being essential for B cell receptor signaling BTK is also involved in the signaling of the glycoprotein (GP)VI and GPIV von Willebrand (vW) receptors (Liu, Blood 2006). Thus, it is possible that ibrutinib could increase the bleeding risk by interfering with thrombus formation. In addition, lymphoproliferative disorders and some drugs have been associated with acquired vW-disease (AvWD). METHODS AND PATIENTS:In an ongoing single center, open label phase II trial we treat CLL/SLL patients with ibrutinib 420 mg daily on 28 day cycles (NCT01500733). We measured platelet (PLT) function on the PFA-100 instrument, vW-factor (vWF) antigen levels and activity (vWF-Ag/vWF-Act), and factor VIII (FVIII) on baseline, days 2 and 28. Here we report on effects of ibrutinib on platelet counts and function in 25 patients who completed >2 cycles. RESULTS:PLT counts prior to treatment ranged from 36 k/μl to 256 k/μl with a median of 102 k/μl. Twelve (48%) patients had a pre-treatment PLT count <100 k/μl. Median PLT counts for days 14, 28, and 56 increased to 140, 137, and 135 k/μl, respectively (P<.01). 76% of patients showed an increase after only 2 weeks on drug (median increase 25 k/μl (range 4–183 k/μl) that was sustained at subsequent timepoints. On day 14, 6 patients (24%) had a decrease in PLT count by a median of 13 k/μl from baseline; of these, 3 had a pre-treatment PLT count of <100 k/ul and 1 developed grade III thrombocytopenia (42 k/μl) that resolved to >100 k/μl by day 56. 20% (5 of 25) of patients reported grade 1 spontaneous ecchymosis with no correlation to platelet count, PFA testing, or vWF measurements. Of note we performed lymph node core biopsies in 35 patients taking ibrutinib with minimal bruising. Only 2 patients had more extensive local bruising/ecchymosis at the biopsy site.Test (normal range)baselineday 2day 28PLT count (161-347 k/μl)102140137PFA-100 EPI (86-154 sec)109110110PFA-100 ADP (73-129 sec)747979vWF-ACT (52-156 IU/dL)182nd99vWF-AG (50-197 IU/dL)158nd101FVIII (41-184 IU/dL)181nd157All values are median, nd = not doneIn 19 patients PFA-100 measurements of epinephrine (EPI) and adenosine diphosphate (ADP) stimulated platelet aggregation times were available (test requires PLT count >100 k/μl). Median changes in closure times with EPI and ADP on treatment were not significantly different from baseline (See table). Four (21%) patients started with abnormally prolonged EPI closure times (one on aspirin, one on ibuprofen; discontinued with the start of ibrutinib) which resolved by day 28 in 3 and decreased in 1. Three (16%) patients had a prolongation of EPI closure times on day 2 that resolved by day 28 in 2 and decreased in 1. All closure times on ADP were low or normal. No patients with abnormal PFA testing demonstrated spontaneous ecchymosis. From baseline to day 28 vWF-Act, vWF-Ag and FVIII decreased (P<0.05; n=24). All 3 values were high normal to elevated prior to treatment and decreased to normal on treatment. CONCLUSION:This preliminary report does not identify any significant ibrutinib effect on platelet function. PLT counts improved rapidly in the majority of patients and when seen transient decreases have been minimal. Three patients (16%) developed an abnormal reading in PLT function tests on treatment but none developed spontaneous echymosis or bleeding. The observed normalization of mildly elevated baseline levels of vWF and FVIII seems most consistent with a reduction in acute phase reactants and there was no evidence for AvWD on ibrutinib. The apparent functional tolerance of BTK inhibition in platelets is likely attributable to redundancy in the affected signaling pathways.This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for participating in these research studies. Disclosures:No relevant conflicts of interest to declare.

Elevated Plasma Von Willebrand Factor and Decreased ADAMTS13 Antigen Levels in Patients with Immune Thrombocytopenia (ITP)

Abstract 1096 Introduction:ITP is an autoimmune disorder characterized by increased platelet destruction and/or decreased platelet production. In addition to risks of bleeding, an increased risk of thrombosis has been identified, despite low platelet counts. Reasons behind this remain unclear. Elevated levels of VWF and decreased levels of ADAMTS13 have been observed in patients with myocardial infarction and ischaemic stroke. Given the interaction of these plasma proteins with platelets, we hypothesized that changes in VWF/ADAMTS13 levels may contribute to the thrombotic risk in patients with ITP. Methods:Adult patients (>18 years) with a diagnosis of ITP were included. ELISAs were used to measure the concentrations of VWF, ADAMTS13 and IL-6. Platelet count and C-Reactive protein (CRP) levels were recorded. Control samples for VWF and ADAMTS13 were taken from a previous study (n=626) (Andersson et al. Blood 2012; 119(6):1555–60). Published data from a population-based study (Marques-Vidal et al. PLoS One 2011; 6(6): e21002) enabled comparison of IL-6 levels to normal controls. Results:93 samples were taken from 48 patients with ITP between March-June 2012. All samples were analyzed for VWF:Ag levels. Plasma concentrations of ADAMTS13 and IL-6 were analyzed in 58 and 34 plasma samples respectively. Plasma VWF antigen (VWF:Ag) levels were elevated in patients with ITP compared to control subjects (p=0.0369) (Figure 1). Levels of ADAMTS13 in patients with ITP were lower than those for control subjects (p=0.0050) (Figure 2). Plasma VWF levels did not correlate with platelet counts. Rather high VWF levels were specific to certain patients. Two patients with VWF:Ag levels >40 μg/mL had a past history of thrombosis; both had additional co-morbidities. Two further patients with acute disease (one recent acute intracranial bleed and one new relapse of ITP) also had high VWF levels. In these two patients, VWF, ADAMTS13 and platelets change over time (example shown in Figure 3). CRP levels did not correlate with platelet count (p=0.6091) or with VWF:Ag levels (p=0.5170). Median IL-6 levels were raised in patients with ITP (3.785 pg/mL) compared to healthy controls (1.47 pg/ml) (p=<0.0001). IL-6 correlated positively with CRP levels (p=0.0183, r=0.4211) however no association between IL-6 and VWF, ADAMTS13 or platelet count was established. Conclusion:The increased ratio of VWF: ADAMTS13 may be contributory in the pathogenesis of thrombosis, in particular in patients with acute and unstable disease. Increased VWF:ADAMTS13 ratio may also result in an increased VWF activity, even without a rise in total VWF. Future studies comparing VWF:Ag to collagen binding are planned.The mechanism behind fluctuating levels of VWF and ADAMTS13 remains unclear. We investigated whether inflammatory mediators associated with acute disease could play a role. CRP levels were elevated in many patients with ITP, but this did not correlate with VWF levels. Levels of IL-6 were also significantly raised in patients compared to healthy controls and correlated with CRP but did not correlate with VWF or with platelet count.In summary, ITP appears to be a heterogeneous disease. In some patients, autoimmunity may be associated with an inflammatory process, and in other patients, low platelets may interfere with other aspects of the coagulation system. Either may predispose to thrombosis or bleeding. Further investigation of the interactions of platelets, with inflammatory cytokines and the coagulation system may help us to better understand the disease, and to recognize those patients at risk of bleeding, or conversely thrombosis. [Display omitted] [Display omitted] [Display omitted] Disclosures:Cooper:Amgen: Consultancy; GSK: Consultancy; Easai: Consultancy.

Prognostic value of plasma von Willebrand factor and its cleaving protease ADAMTS13 in patients with atrial fibrillation

BackgroundThe von Willebrand factor (vWF) is essential for platelet adhesion and arterial thrombosis. It is degraded into less active multimers by ADAMTS13. Patients with atrial fibrillation (AF) exhibit higher plasma vWF and lower ADAMTS13 antigen levels. The vWF/ADAMTS13-ratio might help to estimate the pro-thrombotic risk of patients with AF. We therefore investigated whether a high ratio of vWF/ADAMTS13, independently of clinical risk scores, predicts major adverse cardiovascular events (MACE) in patients with AF. MethodsThis prospective longitudinal single center study included 269 patients with AF. Blood samples were analyzed for vWF and ADAMTS13-antigen concentration by means of enzyme-linked immunoassay kits. ResultsAfter adjustment for all univariable predictors for MACE (p≤0.1), ADAMTS13≤49.77% (HR 1.833 (95% CI 1.089–3.086); p=0.023) and vWF/ADAMTS13-ratio>27.57 (HR 2.174 (95% CI 1.238–3.817); p=0.007) remained independently associated with outcome. vWF>1434.92mU/ml (HR 1.539 (95% CI 0.883–2.682); p=0.128) alone failed to independently predict MACE. In patients with low and intermediate risk for MACE according to the CHADS2-score the addition of high vWF/ADAMTS13-ratio levels (>27.57) had significant impact on the patients' outcome. ConclusionA high ratio of vWF/ADAMTS13 independently predicts MACE in patients with AF. Therefore, vWF and its cleaving protease ADAMTS13 might play an important role in the development and perpetuation of vascular disease in AF patients. This might be a novel target for future treatment strategies or an additional help for risk stratification in AF patients.

Smoking, von Willebrand factor and ADAMTS-13 in healthy males

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

Von Willebrand factor antigen--a possible biomarker of disease activity in childhood central nervous system vasculitis?

The main objective was to develop a trajectory for von Willebrand factor (vWF) antigen in childhood primary CNS vasculitis (cPACNS) after treatment and compare this with disease activity and other inflammatory markers. A single-centre cohort study of consecutive children diagnosed with cPACNS was performed. Demographic, clinical, laboratory, imaging and histological data were collected at diagnosis and during standardized clinic visits. Outcome measures included disease activity measured by physician global assessment and serial measures of vWF antigen. Analysis included descriptive statistics and parametric methods. The study cohort consisted of 39 children diagnosed with cPACNS: 25 with angiography-negative cPACNS and 14 with angiography-positive disease. Twenty-one patients were female, median age at diagnosis was 9.8 years, and median follow-up was 18 months. All patients presented with neurological deficits. Disease activity and neurological outcome improved significantly during follow-up. vWF antigen levels were increased at diagnosis in 65% of children with cPACNS and were decreased significantly after treatment. In contrast, levels of CRP and ESR fluctuated over time. Higher vWF antigen levels at diagnosis were associated with lower measures of disease activity at 12 months. In our study, all children with cPACNS improved over time. Changes in CRP and ESR, other laboratory tests, and MRI did not consistently reflect altered disease activity. However, vWF antigen may help clinicians to identify changes in disease activity during follow-up and predict treatment response. Controlled studies are necessary to evaluate the sensitivity and specificity of vWF antigen as a biomarker of disease activity.

Donor Age Affects Liver Regeneration during Early Period in the Graft Liver and Late Period in the Remnant Liver after Living Donor Liver Transplantation

The aim of the present study was to evaluate the influence of donor age on liver regeneration and surgical outcomes in recipients and donors. Among 101 cases of adult-to-adult living donor liver transplantation (LDLT) between March 2002 and March 2011, according to donor age: younger (Y) <50 years of age or older (O) ≥ 50 years of age, the donors and recipients using right (R) or left (L) graft were divided into groups Y/R (n = 51) and O/R (n = 17), and groups Y/L (n = 26) and O/L (n = 7), respectively. Remnant liver volume (RemLV) and graft liver volume (GLV) were estimated by computed tomography (CT) volumetry. A disintegrin and metalloprotease with thrombospondin type I domain 13 (ADAMTS13) activities and von Willebrand factor (vWF) antigen levels were measured as factors reflecting thrombotic microangiopathy. Among the donors, RemLV/total liver volume (TLV) was lower in group O/R than in group Y/R, although there were no significant differences by t-test with the Bonferroni correction (rough p value = 0.02 at 6 months and rough p value > 0.05 at 1, 3, and 12 months). Donor age (≥ 50 years) was independently correlated with impaired remnant liver regeneration at 6 months in right lobe LDLT (p = 0.04). Among the recipients, GLV/standard liver volume (SLV) was lower during the first month, although there were no significant differences between the two groups by t-test with the Bonferroni correction (rough p value = 0.03 at 1 week and rough p value >0.05 at 2 weeks and 1 and 3 months). Donor age (≥ 50 years) was independently correlated with impaired graft liver regeneration at 1 week in both right and left lobe LDLT (p < 0.05). ADAMTS13 activities were lower in group O/R than in group Y/R, although there were no significant differences by t-test with the Bonferroni correction (rough p value = 0.049 on postoperative days (POD) 1 and 28 and rough p value >0.05 on POD 7 and 14). vWF/ADAMTW13 ratios were higher on POD 14, although there were no significant differences between the two groups by t-test with the Bonferroni correction (rough p value = 0.044 on POD 14 and rough p value >0.05 on POD 1, 7, 14, and 28). The surgical outcomes using older donor livers for LDLT were comparable to those using younger donor livers. When using older donor livers, however, we should pay attention to the liver volume for recipients as well as donors, because older donor livers might have impaired regenerative ability.

High VWF, low ADAMTS13, and oral contraceptives increase the risk of ischemic stroke and myocardial infarction in young women

AbstractVWF and ADAMTS13 are major determinants of platelet adhesion after vessel injury. In the present study, we aimed to determine whether VWF or ADAMTS13 plasma antigen levels influence the risks of ischemic stroke (IS) or myocardial infarction (MI) in young women and how these risks are affected by oral contraceptive (OC) use. VWF and ADAMTS13 plasma antigen levels were measured in a frequency-matched case-control study of 1018 young (18-49 years) women including 175 IS patients and 205 MI patients. Increasing levels of VWF and decreasing levels of ADAMTS13 were associated with the risk of IS and MI in a dose-dependent manner. Having both high VWF and low ADAMTS13 resulted in an odds ratio (OR) of 6.9 (95% confidence interval [95% CI], 2.0-23.0) for IS and 11.3 (95% CI, 3.6-35.2) for MI. Use of OCs increased the risk of IS and MI associated with high VWF (OR = 12; 95% CI, 5.5-26.2 and OR = 7.5, 95% CI, 3.6-15.7, respectively) and the risk of IS associated with low ADAMTS13 (OR = 5.8, 95% CI, 2.7-12.4). We conclude that high VWF and low ADAMTS13 plasma levels both increase the risk of IS and MI. The risks associated with high VWF or low ADAMTS13 levels are further increased by the use of OCs.

Plasma Von Willebrand factor antigen levels are elevated in the classic phenotypes of polycystic ovary syndrome

We aimed to assess plasma Von Willebrand factor (vWF) levels in women with polycystic ovary syndrome (PCOS) and to compare these levels among the different PCOS phenotypes. We studied 140 women with PCOS and 40 age and body mass index (BMI)- matched healthy women (control group). Plasma vWF antigen levels were higher in women with PCOS than in controls (p=0.017). Plasma vWF antigen levels were also higher in patients with phenotypes 1 [i.e. with anovulation (ANOV), biochemical hyperandrogenemia or clinical manifestations of hyperandrogenemia (HA) and polycystic ovaries (PCO)] and 2 (i.e. with ANOV and HA but without PCO) than in controls (p=0.017). In contrast, plasma vWF antigen levels did not differ between controls and patients with phenotypes 3 (i.e. with HA and PCO but without ANOV) and 4 (i.e. with ANOV and PCO but without HA) or between patients with phenotypes 1 and 2 and patients with phenotypes 3 and 4. When overweight/obese and normal weight subjects were analyzed separately, plasma vWF antigen levels did not differ between patients with PCOS (regardless of phenotype) and controls. Plasma vWF levels are elevated in women with PCOS. This increase appears to be more pronounced in women with phenotypes 1 and 2 of PCOS. Given the association between vWF levels and increased incidence of cardiovascular events, the evaluation of vWF levels in women with PCOS might be helpful for cardiovascular risk stratification, but prospective studies are needed to support this hypothesis.