Von Willebrand Factor Activity Levels Research Articles

Analytical characterization and reference interval of an enzyme-linked immunosorbent assay for active von Willebrand factor.

BackgroundInteraction of von Willebrand factor (VWF) with platelets requires a conformational change that exposes an epitope within the VWF A1 domain, enabling platelet glycoprotein Ibα binding. Quantification of this ‘‘active” conformation of VWF has been shown to provide pathophysiological insight into conditions characterized by excessive VWF-platelet interaction.MethodsWe developed an immunosorbent assay based on a variable heavy chain antibody fragment against the VWF A1 domain as a capture antibody. Assay performance in terms of specificity (binding to active R1306W- and sheared VWF), precision, accuracy, linearity, limits of detection and stability were determined. Active VWF, VWF antigen, VWF ristocetin cofactor activity, VWF:GP1bM and VWF propeptide were measured in citrated plasma and platelet-VWF binding in whole blood from 120 healthy individuals to establish a reference interval for active VWF and to assess associations with other VWF parameters.ResultsIntra- and inter-assay CVs were between 2.4–7.2% and 4.1–9.4%, depending on the level. Mean recovery of spiked recombinant R1306W VWF was 103±3%. The assay was linear in the range of 90.1–424.5% and had a limit of quantification of 101%. The reference interval for active VWF was 91.6–154.8% of NPP. Significant, positive correlations between active VWF and all other VWF parameters were found, with the strongest correlation with VWF:GP1bM binding.ConclusionsWe developed and validated an immunosorbent assay for the accurate detection of active VWF levels in plasma. The assay fulfilled all analytical criteria in this study and a reference interval was established, allowing its use to quantify active VWF in pathological conditions for future research.

Coagulation factor VIII is vital for increasing global coagulation after physical exercise.

In a previous smaller study, we found evidence of a diminished global coagulation capacity after maximal exercise in patients with severe haemophilia A (HA). To validate these results, we repeated the study in a larger cohort. We also examined if the exercise-induced increased levels of von Willebrand factor (VWF) might prolong the effect of factor concentrate administered just before exercise. We studied individual and global coagulation parameters after maximal physical exercise in 10 persons with severe HA and 10 healthy matched control subjects. Blood samples were taken before, 10minutes, 60minutes and 4hours after exercise. Rotational thromboelastometry (ROTEM) and thrombin generation assay-calibrated automated thrombogram (TGA-CAT) showed significantly increased coagulation capacity after maximal exercise in healthy controls but not in patients with severe HA. VWF antigen and activity levels increased significantly in both groups, whereas FVIII:C only showed a significant increase in the control group. No statistically significant differences were seen between FVIII pharmacokinetic results obtained with and without exercise. Our findings do not support the presence of a FVIII-independent mechanism that increases global coagulation, but rather underscores the importance of FVIII in mediating the increased coagulation capacity seen after exercise. Our results could not support the hypothesis that exercise-induced increased levels of VWF for patients with severe HA lead to a prolonged effect of factor concentrate administered just before exercise.

A New Flow-Based Assay to Assess Treatment Response in Congenital Thrombotic Thrombocytopenic Purpura

IntroductionCongenital TTP (cTTP) is an ultra-rare disorder in which deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) results in circulating ultra large Von Willebrand Factor (VWF) multimers and subsequent microthrombi formation. Regular prophylactic therapy aims to improve outcomes from long-term complications, but also ongoing symptoms, including lethargy, headaches and abdominal pain, despite normal blood counts. Existing methods of quantifying ADAMTS13 activity lack the sensitivity to enable their use for evaluating treatment response in patients with cTTP. We present a novel flow-based assay with the aim of assessing treatment response, novel therapeutic options and analyzing the impact of different mutations on disease severity.MethodA VenaFlux semi-automated microfluidic system was used to provide shear flow to mimic in vivo flow rates. Using whole blood, we analyzed platelet adhesion, aggregation and thrombi formation on microchannels coated with type I collagen and mounted onto the stage of an inverted epifluorescence microscope. Fresh, citrated whole blood was treated with DiOC6 to achieve platelet fluorescence and a macro on Image-Pro Premier was designed for automated calculation of total surface coverage. Surface coverage represented increasing thrombus formation with total coverage by thrombus within 180 seconds quantified as 100% coverage.Results were compared to a normal range developed using 43 normal controls (26=female, 17=male) with normal hemoglobin, platelet count and hematocrit. The surface coverage normal range was 6-39%.cTTP samples were analyzed for complete blood count, ADAMTS13 activity, VWF antigen, VWF activity and percentage surface coverage. Samples were taken 30 minutes before and after prophylactic treatment, either plasma infusion or BPL-8Y. Recombinant ADAMTS13 was added in-vitro on all pre-treatment samples with 15 minutes incubation time. Further re-measurement was undertaken after initiation of aspirin for at least ten days.ResultsEighteen patients with cTTP confirmed by genetic analysis and ADAMTS13 levels <5 IU/dl were included (16 = female, 2 = male) with a median age of 33 (range: 15-69 years). Median VWF antigen levels: 114% (range: 54% - 276%, NR: 50-160%) and median VWF activity levels: 173% (range: 83% - 338%, NR: 50-187%).The median pre-treatment surface coverage was 90% (range 47% - 100%). There was no significant difference in surface coverage considering genetic mutation type (median coverage for homozygous patients 88%, heterozygous 67%, p=0.99), mutation location (pre-spacer mutation surface coverage 67%, post spacer mutation surface coverage 84%, p=0.84), or age of first symptom onset (childhood onset surface coverage 59%, adult onset 86%, p=0.19).Plasma infusion improved surface coverage results with pre treatment coverage of 90% compared to 44% post plasma infusion (p=0.0003). In vivo recombinant ADAMTS13 administration on pre prophylaxis samples, resulted in normalization of surface coverage in all patients (p<0.0001)(median post rADAMTS13 coverage 28%, range 3-39%). In patients initiated on aspirin, surface coverage had improved both pre and post prophylaxis. The median pre treatment surface coverage for patients on aspirin was 51% (vs. 90% pre treatment and no aspirin, p=0.004). This improvement persisted after treatment with post treatment surface coverage of 18% (vs. 44% post treatment but not on aspirin, p=0.003). 100% of patients who received aspirin saw surface coverage return to the normal range post treatment compared to 82% with plasma infusion alone (p=0.0195).ConclusionPlasma infusion and aspirin synergistically reduce surface coverage by thrombus in patients with cTTP, demonstrated on peak and trough samples. Furthermore, in vitro addition of recombinant ADAMTS13 completely normalized thrombus formation. There were no major differences in surface coverage by genetic mutation. The newly developed flow-based assay presented can be used to assess treatment options and efficacy in cTTP in addition to demonstrating cTTP disease pathophysiology that has not previously been identified. In combination with clinical symptoms it offers potential to improve and personalize treatment for patients with cTTP. [Display omitted] DisclosuresLiesner:Bayer: Consultancy, Research Funding; Sobi: Speakers Bureau; Roche: Research Funding; Baxalta: Consultancy, Research Funding; Novo Nordisk: Research Funding, Speakers Bureau; Octapharma: Consultancy, Other: Clinical study investigator for NuProtect Study (Octapharma sponsored), Research Funding, Speakers Bureau. Scully:Novartis: Honoraria, Other: Member of Advisory Board, Speakers Bureau.

High Molecular Weight von Willebrand Factor Multimer Loss and Bleeding in Patients with Short-Term Mechanical Circulatory Support Devices: A Case Series

Acquired von Willebrand syndrome (VWS) due to loss of high-molecular-weight multimers (HMWMs) has been reported with longer term mechanical devices and is associated with mucosal bleeding, a primary hemostasis type of bleeding. However, little is known whether a similar defect occurs in patients with short-term mechanical circulatory support (STMCS) devices. We reviewed von Willebrand factor (VWF) profiles in patients with STMCS devices who underwent VWS workup from December 2015 to March 2017 at an academic quaternary care hospital. There were a total of 18 patients (57.0 ± 12.7 years old; 83.3% male) including nine with mucosal bleeding and nine with decreasing hemoglobin. The STMCS devices included Impella (n = 11), Impella and right ventricular assist device (n = 2), and an extracorporeal membrane oxygenator (n = 5). The mean HMWM by quantitative VWF multimer analysis was 3.6% ± 1.3% (normal cutoff: 18–34%). In all 10 cases in which VWF activity, fibrinogen, factor VIII, or VWF antigen level were obtained, they were either normal or elevated. All cases demonstrated high normal or elevated levels of low molecular weight multimers (LMWMs). These findings are consistent with type 2 VWS (qualitative defect). This is the first study that quantitatively describes STMCS device–associated HMWM loss, which may contribute to mucosal bleeding. This finding may have implications for intraoperative management during implantation of longer term devices or heart transplantation or other surgery while on STMCS.

Coagulation and inflammation in long‐term cancer survivors: results from the adult population

Background The advances in cancer treatment and detection of early cancer have resulted in a steady increase in the number of of cancer survivors over the years. However, because of the long-term toxic effects of chemotherapy and radiotherapy, the incidence of cardiovascular disease (CVD) is increasing in survivors. Objectives To investigate traditional cardiovascular risk factors (CVRFs), inflammation and the coagulation profile in long-term cancer survivors (cancer diagnosis ≥ 5 years) from a large adult population-based study sample. Methods The presence of cardiovascular risk factors (CVRFs) and laboratory markers were compared in individuals with (n = 723) and without (n = 13626) a long-term history of cancer from the Gutenberg Health Study. Data on coagulation factors, D-dimer and von Willebrand factor (VWF) activity were available for 4974 individuals (n = 244 cancer survivors). Results In multivariable regression models, a history of cancer was, independently of CVRFs and CVD, associated with higher fibrinogen levels (β 6.99, 95% confidence interval [CI] 1.16-12.8), VWF activity (β 5.08, 95% CI 0.02-10.1), and antithrombin activity (β 1.85, 95% CI 0.44-3.27). Cancer survivors with CVD showed notably higher VWF activity than individuals with CVD without a history of cancer, with a difference in the means of 23.0 (7.9-38.1). Multivariate Cox regression analysis, adjusted for CVRFs, confirmed that a long-term history of cancer is associated with a 72% higher mortality. Increased mortality in cancer survivors was dependent on fibrinogen level and VWF activity level. Conclusion Cancer survivors showed a worse inflammation and coagulation profile than individuals without a history of cancer. Overall mortality in long-term cancer survivors was increased independently of traditional CVRFs. These results underline the need to further investigate plasma biomarkers as complementary cardiovascular risk predictors in cancer survivors.

Pregnancy loss in women with von Willebrand disease: a single-center pilot study.

: The risk of pregnancy loss in von Willebrand disease (VWD) has been inconsistently reported. Von Willebrand factor (VWF) is a known regulator of angiogenesis, so has the potential to affect placental function. We sought to determine the risk of pregnancy loss and placenta-mediated pregnancy complications in women with VWD, compared with women without VWD. Women with VWD followed in the Southern Alberta Rare Blood and Bleeding Disorders Clinic were invited to participate in a questionnaire (February-June 2014). The same questionnaire was sent to women without VWD identified in a low-risk obstetrical clinic in Calgary, Alberta, Canada. The primary outcome was the proportion of pregnancies that ended in pregnancy loss. Secondary outcomes were preeclampsia, fetal growth restriction, placental abruption, and preterm labor less than 37 weeks gestation. Of the 30 (31.6%) VWD participants that responded, 26 (86.7%) were diagnosed with Type 1 VWD, of which 11 (42.3%) had VWF antigen or activity levels less than 30%. In women with VWD, there were 20 pregnancy losses out of 80 pregnancies [25.0%, 95% confidence interval (CI), 16.81-35.48], compared with eight losses out of 50 pregnancies in the control group (16.0%, 95% CI, 8.34-28.51; P = 0.28). There was no difference in the risk of preeclampsia (1.7 versus 0%, P = 1.00) or preterm labor (16.7 versus 7.1%, P = 0.23) among VWD and control groups. There were no other placenta-mediated pregnancy complications reported. There is no significant difference in pregnancy loss between women with and without VWD; however, a large multicenter study is needed to clarify the risk of pregnancy loss in women with VWD.

The Involvement of Copper Transport in Von Willebrand Factor Multimer Assembly

In the event of vascular injury, the underlying prothrombotic extracellular matrix becomes exposed to circulating blood. Endothelial cells at the site of injury secrete multimeric von Willebrand factor (VWF) that will bind to the exposed extracellular matrix and create a scaffold to which platelets can adhere, forming a platelet plug. These critical steps after vascular injury are essential for creating a stable clot. Intracellular VWF multimer assembly is required for its extracellular function, and defects in VWF assembly cause the bleeding diathesis, von Willebrand disease. In a cell-free in vitro system, we and others find that EDTA or EGTA inhibits multimer formation, and calcium ions have been proposed to be required for this process. However, we find that the binding affinity of calcium ions for VWF D domains is very weak (Kdof 300-400 μM) and that buffers containing less than 2 μM calcium ions support multimer assembly even though few calcium binding sites could be occupied. These results suggest a role for cations other than calcium in VWF multimer assembly. Here we show that copper ions support VWF multimer assembly in a cell-free system and that the intracellular copper transporter, ATP7A, is required for VWF multimer assembly in cultured cells.Some calcium binding proteins undergo a conformational change upon binding calcium ions and exhibit a decrease in the apparent molecular weight (an electrophoretic mobility shift) as assessed by SDS-PAGE and Western blot. Although the D domains of VWF bind calcium ions, they do not exhibit this behavior. We tested a panel of other divalent cations including: iron, magnesium, manganese, zinc, and copper. We found that the VWF N-terminal D’D3 dimer bound copper ions as demonstrated by a shift in electrophoretic mobility. We therefore assessed the effect of copper ions on VWF multimer assembly in vitro. Briefly, addition of monensin (1.5 μM) to human umbilical vein endothelial cells (HUVEC) caused secretion of C-terminal dimers of proVWF into the media. Pretreatment of the conditioned media with the divalent cation chelator, EGTA, or the copper chelator, tetraethylenepentamine (TEPA), preceded immunopurification of C-terminal dimers of proVWF using a rabbit polyclonal anti-hVWF antibody (DAKO A0082) bound to protein A magnetic beads. Beads were incubated at pH 7.4 or pH 5.8, and in the presence or absence of divalent cations for 3 hours at 37°C before analyzing the VWF by gel electrophoresis. Addition of calcium ions (100 μM) to proVWF dimers pretreated with only EGTA allowed some VWF multimer assembly; however, copper ions (100 μM) induced the assembly of higher molecular weight multimers from proVWF dimers pretreated with either EGTA or TEPA. With either divalent cation, multimers assembled at low pH, but not at neutral pH. These findings support a role for copper ions in VWF synthesis.In vivo, free copper ions are toxic; therefore, they are sequestered by a system of chaperones and transporters. We examined the role of the intracellular copper transporter, ATP7A, in VWF multimerization because it is expressed in the trans Golgi network of endothelial cells, the site of VWF multimer assembly. Mick Petris (University of Missouri-Columbia) provided both wildtype and ATP7A knockout mouse embryonic fibroblasts (MEFs). We stably expressed full length human VWF and analyzed the secreted multimers by agarose gel electrophoresis. Wildtype MEFs produced multimeric VWF; however, culturing the MEFs in the presence of the copper chelator, TEPA, markedly impaired VWF multimer assembly. Moreover, ATP7A-null MEFs secreted only VWF dimers. These results indicate that copper ions and ATP7A contribute to VWF multimer assembly in cells.The dependence of VWF assembly on ATP7A in cultured cells suggests that humans with congenital ATP7A deficiency (Menkes disease) could have detectable abnormalities in VWF multimer structure, although dietary copper and parenteral copper replacement therapy could affect this phenotype. Our preliminary analyses have identified a Menkes disease patient with low plasma levels of VWF antigen, multimers and activity. Further study will be required to determine the prevalence and significance of altered VWF multimer assembly in Menkes disease. DisclosuresSadler:BioMarin: Consultancy; 23andMe: Consultancy; Ablynx: Consultancy.

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

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

Strenuous exercise induces a hyperreactive rebalanced haemostatic state that is more pronounced in men.

Physical exercise is recommended for a healthy lifestyle. Strenuous exercise, however, may trigger the haemostatic system, increasing the risk of vascular thrombotic events and the incidence of primary cardiac arrest. Our goal was to study the effects of strenuous exercise on risk factors of cardiovascular disease. Blood was collected from 92 healthy volunteers who participated in the amateur version of the pro-tour Amstel Gold cycling race, before and directly after the race. Thrombin generation showed a shortening of the lag time and time to peak and an increase of the velocity index. Interestingly, the endogenous thrombin potential measured in plasma decreased due to reduced prothrombin conversion. Platelet reactivity increased and this effect was stronger in men than in women. Lower fibrinogen and higher D-dimer levels after exercise indicated higher fibrin formation. On the other hand, fibrinolysis was also elevated as indicated by a shortening of the clot lysis time. Exercise activated the endothelium (von Willebrand factor (VWF) and active VWF levels were elevated) and the immune system (concentrations IL-6, IL-8, MCP-1, RANTES and PDGF increased). Additionally, an increased cardiac troponin T level was measured post-exercise. Strenuous exercise induces a temporary hyperreactive state in the body with enhanced pro- and anticoagulant responses. As strenuous exercise has a more pronounced effect on platelet function in male subjects, this gives a possible explanation for the higher incidence of sudden cardiac death during exercise compared to women. This trial is registered at www.clinicaltrials.gov as NCT02048462.

Von Willebrand Disease Lab Diagnosis.

C), VWF binding to immobilized collagen (VWF:CB), ristocetin-induced platelet aggregation (RIPA), VWF multimers patterns, factor VIII binding of immobilized VWF (VWF:FVIIIB), among others. From the moment the diagnosis is confirmed, the appropriate treatment for each patient is sought, with the purpose of increasing plasma concentrations of the deficient protein, both in bleeding episodes, as for invasive procedures. Although diagnosis facilitates treatment other approach in the present scenario is prenatal diagnosis which, is the need of the hour.

Management of Perioperative Anticoagulation in Patients with VWD

▪Objectives: von Willebrand disease (VWD) is the most common hereditary bleeding disorder. This study reviews the management of perioperative anticoagulation in patients with VWD undergoing surgical procedures. Risk factors for VTE with von Willebrand factor (VWF) concentrate use are older age, previous VTE, obesity, surgery, hormone replacement therapy use, antifibrinolytic therapy use and high post infusion FVIII levels. Currently, there are few data from randomized clinical trials assessing efficacy and possible complications of perioperative VTE prophylaxis in VWD patients.Methods: A total number of 116 surgeries were performed (minor: n=64 and major: n=52) in this retrospective, single-centre study. They were divided into groups of perioperative non-anticoagulation (n=54) and perioperative anticoagulation (n=62), who all received coagulation factor concentrate (CFC). Sub-analyses were done according to the type of concentrate used. Anticoagulation was performed using different low molecular weight heparins (LMWH) according to standard protocols or body-weight adapted doses in patients with either elevated body-mass-index or additional thrombosis risk factors. Blood samples had been collected pre- and post-surgery (up to 21 days) to analyse PT, aPTT, PFA, and trough levels of FVIII coagulant activity (FVIII:C), VWF activity (VWF:GPIbM) and antigen (VWF:Ag), respectively. Furthermore, the median doses of CFC/kg and the median total number of infusions were calculated. The rates of clinically overt thrombosis as well as bleeding were assessed during the post-operative phase.Results: The majority of patients suffered from VWD type 1 (104), 9 patients with type 2A, 2 with type 2M and 1 with type 3 VWD. Humate-P (H) was used in 55 patients and 61 patients received Wilate (W). Using W, we found parallel curves for FVIII:C, VWF-antigen and VWF:GPIbM, respectively. Using H, less concordance between VWF:Ag and VWF:GPIbM was visible and FVIII:C tends to increase between D3 to D10 in spite of decreasing VWF:Ag and VWF:GPIbM. This observation was visible in minor as well as major surgical procedures. LMWH (Enoxaparin, Nadroparin and Certaparin) were used in doses between 30 and 100 mg/injection (mean 46.0 ± 18.5 mg/injection) and a mean of 32.2 ± 24.6 injections in total (range: 8-112). In one patient a significant haematoma occurred (1/116; 0.9%), also one thrombotic event was documented in a different patient (1/116; 0.9%).Conclusion: Using standard dose LMWH in patients with no overt increased thrombosis risk as well as body-weight-adapted LMWH in high risk patients seem to be safe and effective in VWD patients receiving coagulation factor concentrate perioperatively. However, prospective randomized comparative studies are required to determine the optimal indication as well as type of anticoagulation according to the CFC treatment regimen in this setting. DisclosuresNo relevant conflicts of interest to declare.

Kinetics of coagulation in ST-elevation myocardial infarction following successful primary percutaneous coronary intervention

IntroductionST-elevated myocardial infarction (STEMI) is most frequently caused by coronary occlusion due to formation of an intracoronary thrombus in reaction to rupture of atherosclerotic plaques. Little is known about kinetics of coagulation markers after STEMI in patients treated according to current guidelines. We aimed to investigate kinetics of important coagulation markers in percutaneous coronary intervention (PCI)-treated STEMI patients. Materials and methods60 consecutive PCI-treated STEMI patients were prospectively included. Blood samples were collected immediately after as well as 1, 4 and 7days following PCI. Samples collected 90days after PCI served as baseline values. ADAMTS13 activity, VWF (von Willebrand factor) activity, VWF antigen, VWF propeptide, fibrinogen antigen, D-dimer, alpha2-antiplasmin (α2AP), plasmin-alpha2-antiplasmin complex (PAP), prothrombin fragment F1+2 (F1+2), prothrombin time (PT), activated partial thromboplastin time (aPTT), and anti-factor Xa (anti-Xa) were measured. Cardiac magnetic resonance (CMR) was performed at 4–6 and 90days after PCI in 49 patients and left ventricular ejection fraction (LVEF), infarct size and microvascular injury (MVI) were determined. ResultsImmediately after PCI, ADAMTS13 activity, fibrinogen antigen and α2AP levels were significantly decreased and VWF activity, VWF antigen and VWF propeptide levels were significantly elevated, compared to baseline. Individual coagulation markers and different combinations thereof were not related to LVEF or infarct size at 90days, or the occurrence of MVI at 4–6days after PCI. ConclusionCoagulation parameters show a very dynamic profile in the early days after STEMI. However, individual coagulation parameters or combinations thereof do not predict CMR-defined LVEF, infarct size or MVI.

Higher and lower active circulating VWF levels: different facets of von Willebrand disease.

Most circulating von Willebrand factor (VWF) is normally inactive and incapable of binding platelets, but numerous disorders may modify the proportion of active VWF. We explored active VWF levels in patients with von Willebrand disease (VWD) whose VWF had a higher affinity for platelet glycoprotein (GP)Ib, but different susceptibilities to ADAMTS13 and multimer patterns (9 patients lacking large multimers, 10 with a normal pattern); 12 patients with VWF C2362F and R1819_C1948delinsS mutations, which make VWF resistant to ADAMTS13 were also studied. Type 2B patients with abnormal or normal multimers had significantly more active VWF (3·33 ± 1·6 and 3·74 ± 0·74, respectively; normal 0·99 ± 0·23). The type of VWF mutation influenced VWF activation: V1316M was associated with the highest levels in patients with abnormal multimers, and R1341W in those with normal multimers. Pregnancy induced gradually rising active VWF levels and declining platelet counts in one type 2B VWD patient without large multimers. Active VWF levels dropped significantly in patients homozygous for the C2362F mutation or heterozygous for R1819_C1948delinsS mutations (0·2 ± 0·03 and 0·23 ± 0·1, respectively), and less in cases heterozygous for the VWF C2362F mutation (0·55 ± 0·17). We demonstrate that VWF may be more or less activated, with or without any direct involvement of the A1 domain, and regardless of ADAMTS13.

Epidural Analgesia Use in Women with Von Willebrand Disease

▪There is little safety data available on the use of epidural analgesia during pregnancy in women with von Willebrand disease (VWD), a common disorder comprising up to 0.1% of the population. Despite physiological increases in von Willebrand factor antigen and activity levels to normal or near levels during third trimester in Type 1 VWD patients, epidural analgesia use is varied and often withheld. We conducted a cross-sectional questionnaire in order to further characterize the local practice and provide additional safety data on epidural use in the VWD population.We invited all women with VWD followed by the Southern Alberta Rare Blood and Bleeding Disorders Comprehensive Care Program to participate in an online or paper questionnaire. The questionnaire was part of a larger study identifying pregnancy complications in VWD, with additional questions collected on the diagnosis and severity of VWD, epidural use and associated outcomes, as well as the perceived contraindications of epidural analgesia. All questions were pre-tested by hematologists, obstetricians and laypeople. With patient consent, supplemental data was collected from clinic and hospital records. We included patients who were 18 years of age or older with a diagnosis of VWD, defined as von Willebrand factor (VWF) antigen and activity levels less than 50 percent or a historical diagnosis, in combination with a bleeding score of 4 or more (condensed MCMDM-1 bleeding questionnaire). Patients were excluded if they had no past pregnancies, no mailing address, or had an alternative bleeding disorder. Confidence intervals for proportions were calculated using the Wilson’s score method.Of the 98 women with VWD who met the study inclusion criteria, 31 (32%) completed the questionnaire. There were 25 (81%) diagnosed with Type 1, 5 (16%) with Type 2, and 1 (3%) with Type 3 VWD. The mean bleeding score was 10.3 (SD 3.6). There were a total of 83 pregnancies (mean 2.7, range 1-6), with 60 deliveries assessed because of 20 pregnancy losses, 2 elective terminations, and 1 patient currently pregnancy. Of the 60 deliveries, the rate of epidural use was 28.3% (95% confidence interval 18.5-40.8, n=17). All epidural analgesia use was reported in women with Type 1 VWD. Of the 43 pregnancies without an epidural, the stated reasons were as follows: 10 because of concern for bleeding (23.3%), 25 because of personal preference (58.1%), 7 'other’ (16.3%), and 1 participant without a response (2.3%). In the 10 pregnancies where an epidural was not used due to bleeding concerns, 6 patients had Type 1 VWD and 1 patient had Type 2N VWD.The outcome of epidural use in the VWD population was reassuring, 2 women reported their epidural analgesia to be ineffective, and 4 women reported neurological symptoms such as numbness, tingling or weakness. All neurological symptoms were temporary with no persistent deficits or documented complication of bleeding.Due to the retrospective nature of this study, VWF antigen and activity levels at the time of delivery were not available, limiting the conclusions that can be made on the safety of epidural use based on VWF levels. It is our local practice to proceed with epidural analgesia if a patient’s VWF antigen and activity levels measured in third trimester are greater than 50%. For VWF levels less than 50%, epidural analgesia would only be considered with appropriate DDAVP or factor concentrate replacement.In summary, our local rate of epidural use was close to 30% in the VWD population, which was primarily composed of Type 1 VWD patients. Outcome data is reassuring, but a larger cohort study is needed to further quantify rare risks such as neuraxial bleeding. DisclosuresNo relevant conflicts of interest to declare.

Tonsillectomy in a Patient with Type 3 Von Willebrand Disease

Introduction: Type 3 Von Willebrand Disease (VWD) is a rare autosomal recessive bleeding disorder characterized by a complete deficiency of von Willebrand factor (VWF) and markedly reduced levels of FVIII, which confers a severe bleeding phenotype. Conventional management is with plasma-derived von Willebrand factor / FVIII concentrates. These products do not fully correct the bleeding diathesis, for several possible reasons: (a) platelets remain deficient in VWF; (b) subendothelial matrix remains deficient in VWF; and (c) available products lack the highest molecular weight VWF multimers. Because of the low prevalence of type 3 VWD, reports on surgical procedures are scarce. We reviewed the literature and identified one case report of a patient with type 3 VWD who underwent tonsillectomy (Alusi et al. 1995). That case was complicated by major intra-operative bleeding as well as prolonged hemorrhage in the two weeks following surgery, despite factor replacement with concentrate, cryoprecipitate, platelet transfusion and tranexamic acid. We report on the management of a patient with type 3 VWD undergoing a tonsillectomy, which demonstrates the importance of local measures in hemostatic control.Case: A 29 year old woman with Type 3 VWD (baseline VWF:Ag 4 IU/dL, VWF:RCoF <5 IU/dL, FVIII 1 IU/dL) had a 2 year history of recurrent tonsillar hemorrhage despite regular prophylaxis with plasma-derived VWF concentrate (Humate-P®). Each episode occurred in the absence of trauma or infection, and bleeding was difficult to control despite Humate-P and tranexamic acid. Lee et al. (2010) have previously reported that VWD is a cause of spontaneous tonsillar hemorrhage. Due to the recurrent nature of the bleeding episodes, a tonsillectomy was recommended.In preparation for the surgical procedure, we performed pharmacokinetic studies, which revealed a yield of VWF activity of 2.35IU/dL per U/kg and a half-life of VWF activity of 8.8 hours. We administered an initial loading dose of 63IU/kg (RCoF) of Humate-P preoperatively, achieving a VWF activity level (by latex agglutination: HemosIL vWF activity, Instrumentation Laboratories) of 126%. The tonsillectomy was uneventful, and the estimated blood loss intra-operatively was <50 mL. Surgical hemostasis was augmented by administration of tranexamic acid 1000mg intravenously pre-operatively and by continuous infusion intra-operatively, as well as 5 mL of fibrin sealant (Tisseel) administered to each tonsillar bed. A dose of Humate-P of 22.6 IU/kg was given post-operatively, and subsequent doses of 11.3 IU/kg were given q 4 hourly to maintain trough VWF activity of approximately 100IU/dL. Post-operative doses of tranexamic acid were omitted due to a transcribing error until day #3. On day #4 the Humate P dosing was reduced to 11.5U/kg q6h, which maintained trough VWF activity levels of approximately 50%. No bleeding occurred until post-operative day #9, when the eschar at the wound side detached. Bleeding continued despite re-bolus of Humate-P (23IU/kg) followed by resumption of q4h dosing, administration of tranexamic acid topically in the form of a 5% solution in addition to systemic administration, and application of topical silver nitrate. Hypotension transiently developed and the hemoglobin fell from 106 to 66g/L, requiring a return to the operating room. Further application of fibrin sealant promptly secured hemostasis.Conclusion: Surgery in a patient with type 3 VWD requires careful planning and collaboration between hematologists with expertise in bleeding disorders, the bleeding disorders nursing staff and surgical team. This case highlights the importance of monitoring for an extended period post-operatively, and demonstrates that bleeding can occur despite maintaining adequate VWF levels. Topical measures such as fibrin sealant play a key role in controlling bleeding in this context. DisclosuresNo relevant conflicts of interest to declare.

The Rate of Pregnancy Loss in Von Willebrand Disease: A Cross-Sectional Study

Pregnancy and delivery represent a major hemostatic challenge in von Willebrand disease (VWD). In addition to maintaining adequate hemostasis, von Willebrand factor (VWF) is an important regulator of angiogenesis, with angiodysplasia contributing to morbidity in patients with VWD. Placental development is complex, with vascular development important for fetal growth and viability. It has been hypothesized that antepartum bleeding or abnormal placental angiogenesis may lead to pregnancy loss in women with VWD. Large studies have reported an increased risk of postpartum bleeding in patients with VWD, however, little information is available for rates of pregnancy loss. We conducted a cross-sectional study to determine the rate of pregnancy loss in women with VWD, when compared to a control population. We invited all women with VWD followed by the Southern Alberta Rare Blood and Bleeding Disorders Comprehensive Care Program to participate in an online or paper questionnaire, along with a control group of women without VWD identified through a low-risk obstetrical clinic in Calgary, Alberta. We included patients who were 18 years of age or older with a diagnosis of VWD, defined as VWF antigen and activity levels less than 50 percent or a historical diagnosis, in combination with a bleeding score of 4 or more (condensed MCMDM-1 bleeding questionnaire). Patients were excluded if they had no past pregnancies, no mailing address, or had an alternative bleeding disorder. Our 20-item questionnaire was reviewed and pre-tested by hematologists, obstetricians and laypeople. With patient consent, data was supplemented from clinical and hospital records to verify and expand on the information collected in the questionnaire. The primary outcome was the incidence of spontaneous abortion (fetal loss &lt;20 weeks) or late pregnancy loss (≥20 weeks) in women with VWD. The secondary outcomes were antepartum and postpartum bleeding. Data on co-morbidities including placental abnormalities were also collected. Confidence intervals for proportions were calculated using the Wilson’s score method. Groups were compared using χ2 testing, with p values &lt; 0.05 considered significant. Of the 98 women with VWD who met the study inclusion criteria, 31 (32%) completed the questionnaire. The mean bleeding score was 10.3 in VWD patients (SD 3.6). The majority (81%) of participants were diagnosed with Type 1 VWD, with 16% and 3% diagnosed as Type 2 and 3 VWD, respectively. Twenty-two women in the control group completed the questionnaire. The mean age at recruitment was 46.1 (SD 13.7) and 32.6 (SD 4.2) in the VWD and control group, respectively. There were 83 and 48 pregnancies in the VWD and control group, respectively. Two women in the VWD group and 1 woman in the control group had an elective pregnancy termination. In women with VWD, the rate of pregnancy loss was 24.7% out of 81 pregnancies (95% confidence interval 16.6-35.1), with 17 (85%) losses occurring less than 20 weeks, and 3 (15%) with unknown timing. In contrast, the rate of pregnancy loss in the control group was 12.8% (95% CI 5.96-25.17) (p=0.106), all occurring less than 20 weeks. In 83 pregnancies, the number of antepartum bleeding episodes in VWD women was 45 (29 first trimester, 9 second trimester, 7 third trimester), compared to the control group (9 first trimester, 3 second trimester, 1 third trimester) in 48 pregnancies. More women with VWD reported excessive postpartum bleeding compared to controls (56.7% versus 13.6%, p=0.002), with more delayed (&gt;24 hour) bleeding post-delivery in women with VWD. In VWD women with and without pregnancy loss, there was no difference in mean bleeding score (9.7 vs. 10.9). There was also no difference in cases of pre-eclampsia, placental abruption or gestational hypertension in VWD women with and without pregnancy loss. Two VWD patients with previous pregnancy loss developed gastrointestinal angiodysplasia later in life. There was no significant increase in the rate of pregnancy loss in women with VWD, however, a larger cohort study is needed to confirm the rate of pregnancy loss before further conclusions can be made. Disclosures No relevant conflicts of interest to declare.

Laboratory Assessment of Anti-Coagulant Properties of a Von Willebrand Factor Targeted Aptamer

Introduction: Many cardiovascular procedures including percutaneous coronary intervention, coronary bypass surgery, and peripheral vascular surgery require antithrombotic therapy during the procedure. Heparin is frequently used, due to low cost, ease of monitoring and reversibility with protamine. Antiplatelet agents such as aspirin and clopidogrel are also frequently used but these agents are not reversible. Alternative reversible antithrombotic strategies that interfere with platelet-mediated thrombosis are needed. We are investigating the use of a reversible agent that is specific for von Willebrand factor (VWF) for use during cardiovascular procedures.We previously developed an antidote-controlled 80 oligonucleotide RNA aptamer Ch-9.14-T10 (T10). (Nimjee, S.M., Lohrmann, Wang, H, et al. Molecular Therapy 2012; 2:391-397). T10 bound to VWF with high affinity and inhibited thrombosis in a murine carotid artery damage model. The aptamer also greatly increased bleeding in mice that were surgically challenged by tail transection and blood loss decreased when complementary antidote oligonucleotides were administered. In addition, T10 inhibited primary hemostasis in the PFA-100 assay and this inhibition was completely reversed by the antidote. This study further characterizes the T10 aptamer, as well as a truncated version of the T10 aptamer, using clinical laboratory assays that would be used to monitor antithrombotic therapies during cardiovascular procedures.Methods: We initiated our study with T10 and then developed a truncated version, T25, a 60 oligonucleotide with which we performed additional investigations. We recruited normal donors for analysis of aptamer and antidotes. All participants had normal platelet counts, VWF activity and antigen levels, and were not taking antiplatelet agents. Laboratory tests included: ristocetin induced platelet agglutination (RIPA), VWF activity, ristocetin cofactor activity, cone and plate(let) analyzer, thromboelastography (TEG) and thromboelastometry (ROTEM).Results: We first determined the effects of the VWF aptamers on RIPA, VWF activity, and ristocetin cofactor activity to confirm functional inhibition of VWF. We found that T10 (500-750 nM) and T25 (250-750nM) completely inhibited RIPA depending on the donor. T25 also inhibited VWF activity with a Ki of 160-320 nM and ristocetin cofactor activity with an IC50 of 3000 nM. Employing a cone and plate(let) analyzer we found that T25 (750 nm) reduced platelet adhesion by 33-49% on a polystyrene surface under arterial flow conditions (1800 s-1, for 2 minutes). The aggregate size was also reduced by 30%.As ACT and TEG are frequently used during cardiovascular procedures, we performed several measures with our VWF aptamer. T10 had no effect on the ACT (730-2000nM), TEG (1600 nM), or ROTEM (750 nM). Standard methods were used for the TEG (Kaolin activation followed by CaCl2) and ROTEM (EXTEM assay) Topf et al. reported a modified ROTEM protocol where they found that incubating citrated whole blood with ristocetin decreased the clot strength as measured by the maximum amplitude (Thromb.Haemost 2011; 105:1091). We reasoned that an aptamer that bound to VWF would inhibit the effect of ristocetin on the maximum amplitude. We found that T25 did inhibit the effect of ristocetin and titration studies showed that 200-400 nM T25 was needed to restore the maximum amplitude to at least 80% of its original value. An example of the ROTEM tracings is shown in figure 1.Conclusion: The VWF-specific aptamer T25 can be detected by several clinical assays, including the RIPA, VWF activity assays, and the cone and plate(let) analyzer. It is not detectable by an ACT or standard TEG or ROTEM, however. We are able to detect the aptamer using a modified ROTEM and are further defining the adaptability of this assay to facilitate monitoring during cardiovascular procedures.Citrated whole blood from was incubated with buffer or ristocetin (0.7 mg/ml) or ristocetin and T25 (300 nM) as indicated. Clotting was initiated with EXTEM (tissue factor pathway activation) and CaCl2. [Display omitted] DisclosuresBecker:Regado Biosciences: Membership on an entity’s Board of Directors or advisory committees.

Changes in von Willebrand factor and ADAMTS-13 in patients following arthroplasty.

The glycoprotein, von Willebrand factor (VWF) is a carrier protein for factor VIII. When bound to platelets and the extracellular matrix, it promotes aggregation or adhesion of platelets to areas of vascular damage. A disintegrin and metalloproteinase with thrombospondin motif, member 13 (ADAMTS13) cleaves between the tyr1605 and met1606 residues in the central A2 domain of VWF decreasing its activity. The levels of ADAMTS13 and VWF are positively correlated with the risk of developing thrombosis and inversely correlated with the risk of bleeding. A total of 93 patients were observed, who underwent total hip arthroplasty or total knee arthroplasty. Blood samples were collected preoperatively and on postoperative days (PODs) 1, 2, 3, 5 and 7. Plasma levels of the ADAMTS13 antigen were determined using western blotting. The proteolytic activity was validated with the FRETS‑VWF73 assay. VWF:Ag and VWF:RCo activity were measured using an enzyme‑linked immunosorbent assay. Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), antithrombin III and plasma fibrinogen levels were measured on a Sysmex® CA500 system with corresponding reagents. D‑dimer levels were measured on a STA‑R fully automated coagulation analyzer. The results demonstrated that, the levels of VWF antigen and activity in the patient increased from postoperative day (POD) 1. By contrast, the level of the ADAMTS13 antigen and its activity in the patients decreased significantly. Starting on POD1, fibrinogen and D‑dimer levels increased. No significant changes were observed in PT, APTT and TT. It was concluded that the ADAMTS13 and VWF levels exhibited a marked association with thrombosis risk. The levels of ADAMTS13 and VWF may be potentially useful as markers for predicting thrombotic complications following arthroplasty and inhibiting the activity of VWF may be a novel prophylaxis to reduce postoperative deep venous thrombosis and pulmonary embolism.

Plasma levels of active Von Willebrand factor are increased in patients with first ST-segment elevation myocardial infarction: a multicenter and multiethnic study.

Von Willebrand factor (VWF), a key player in hemostasis and thrombosis, is released from endothelial cells during inflammation. Upon release, VWF is processed by ADAMTS13 into an inactive conformation. The aim of our study was to investigate whether plasma levels of active VWF, total VWF, ADAMTS13, osteoprotegerin (OPG) and the ratios between VWF and ADAMTS13 are risk factors for first ST-segment elevation myocardial infarction (STEMI). We assessed 1026 patients with confirmed first STEMI and 652 control subjects from China, Italy and Scotland, within six hours after their cardiovascular event. Median plasma levels of total VWF, active VWF, OPG and ratios VWF/ADAMTS13 were increased, while plasma levels of ADAMTS13 were decreased in patients compared to controls. The odds ratio (OR) of STEMI in patients with high plasma levels of active VWF was 2.3 (interquartile range (IQR): 1.8-2.9), total VWF was 1.8 (1.4-2.3), ADAMTS13 was 0.6 (05-0.8), OPG was 1.6 (1.2-2.0) and high VWF/ADAMTS13 ratios was 1.5 (1.2-2.0). The OR for total VWF, active VWF and ratios VWF/ADAMTS13 remained significant after adjustment for established risk factors, medical treatment, C-reactive protein, total VWF, ADAMTS13 and OPG. When we adjusted for levels of active VWF, the significance of the OR for VWF and ratios VWF/ADAMTS13 disappeared while the OR for active VWF remained significant. We found evidence that plasma levels of active VWF are an independent risk factor for first STEMI in patients from three different ethnic groups. Our findings confirm the presence of VWF abnormalities in patients with STEMI and may be used to develop new therapeutic approaches.

Active von Willebrand factor predicts 28-day mortality in patients with systemic inflammatory response syndrome

AbstractEndothelial dysfunction contributes to the pathology of systemic inflammatory response syndrome (SIRS). However, endothelial biomarkers are not routinely evaluated in this setting. Here, 275 patients with SIRS and plasma levels of von Willebrand factor (VWF), thrombospondin-1, myeloperoxidase, ADAMTS-13, and active VWF (aVWF) were studied in relation to 28-day mortality. On admission, aVWF levels were higher in nonsurvivors vs survivors (0.69 vs 0.47 µg/mL, P = .019). Patients in the highest tertile of aVWF levels had a lower cumulative survival (86% vs 75%, P = .017) and twofold increased hazard ratio (HR). When adjusted for the Acute Physiology and Chronic Health Evaluation IV (APACHE-IV) score, this difference remained significant (HR 1.82, 95% confidence interval, 1.03-3.3). On admission, no significant differences were measured for the other proteins. These observations suggest that the stimulated release of VWF is not predictive for mortality in patients with SIRS, opposite of the processing of VWF after release. aVWF could be used with the APACHE-IV score to stratify SIRS patients at high mortality risk.