Von Willebrand Factor Parameters Research Articles

Mitral regurgitation is associated with similar loss of von Willebrand factor large multimers but lower frequency of anemia compared with aortic stenosis

BackgroundVarious cardiovascular diseases cause acquired von Willebrand syndrome (AVWS), which is characterized by a decrease in high-molecular-weight (large) von Willebrand factor (VWF) multimers. Mitral regurgitation (MR) has been reported as a cause of AVWS. However, much remains unclear about AVWS associated with MR. ObjectivesTo evaluate VWF multimers in MR patients and examine their impact on clinical characteristics. MethodsModerate or severe MR patients (n = 84) were enrolled. VWF parameters such as the VWF large multimer index (VWF-LMI), a quantitative value that represents the amount of VWF large multimers, and clinical data were prospectively analyzed. ResultsAt baseline, the mean hemoglobin level was 12.9 ± 1.9 g/dL and 58 patients (69.0%) showed loss of VWF large multimers defined as VWF-LMI < 80%. VWF-LMI in patients with degenerative MR was lower than in those with functional MR. VWF-LMI appeared to be restored the day after mitral valve intervention, and the improvement was maintained 1 month after the intervention. Seven patients (8.3%) had a history of bleeding, 6 (7.1%) of whom had gastrointestinal bleeding. Gastrointestinal endoscopy was performed in 23 patients (27.4%) to investigate overt gastrointestinal bleeding, anemia, etc. Angiodysplasia was detected in 2 of the 23 patients (8.7%). ConclusionModerate or severe MR is frequently associated with loss of VWF large multimers, and degenerative MR may cause more severe loss compared with functional MR. Mitral valve intervention corrects the loss of VWF large multimers. Gastrointestinal bleeding may be relatively less frequent and hemoglobin level remains stable in MR patients.

Use of Vonicog Alpha and Acquired von Willebrand Syndrome, a New Approach: A Case Report.

Therapeutic management of acquired von Willebrand syndrome (AVWS) can be challenging, particularly in cases of AVWS associated with monoclonal IgM such as Waldenström macroglobulinemia (WM) where several therapeutic options may be ineffective. Here, we describe the case of an 88-year-old patient who developed AVWS during follow-up for WM. The presence of a severe bleeding symptomatology not controlled by several therapies (plasma-derived von Willebrand factor, plasmapheresis) led us to introduce a supplementation with recombinant von Willebrand factor, vonicog α (Veyvondi, Takeda, Japan), starting at a dose of 50 IU/kg/d. This supplementation allowed clinical (no further bleeding) and biological (hemoglobin level, von Willebrand factor parameters) improvements. Because of the persistence of bleeding risk factors, the treatment was maintained at a prophylactic dose (20 UI/kg three times a week), without recurrence of bleeding events for a period of 9 months.

VWF–ADAMTS13 axis dysfunction in children with sickle cell disease treated with hydroxycarbamide vs blood transfusion

AbstractPrevious studies have reported elevated von Willebrand factor (VWF) levels in patients with sickle cell disease (SCD) and demonstrated a key role for the VWF-ADAMTS13 axis in the pathobiology of SCD vaso-occlusion. Although blood transfusion is the gold standard for stroke prevention in SCD, the biological mechanisms underpinning its improved efficacy compared with hydroxycarbamide are not fully understood. We hypothesized that the improved efficacy of blood transfusion might relate to differences in VWF–ADAMTS13 axis dysfunction. In total, 180 children with a confirmed diagnosis of SCD (hemoglobin SS) on hydroxycarbamide (n = 96) or blood transfusion (n = 84) were included. Despite disease-modifying treatment, plasma VWF and VWF propeptide were elevated in a significant proportion of children with SCD (33% and 47%, respectively). Crucially, all VWF parameters were significantly higher in the hydroxycarbamide compared with the blood transfusion cohort (P < .05). Additionally, increased levels of other Weibel-Palade body–stored proteins, including factor VIII (FVIII), angiopoietin-2, and osteoprotegerin were observed, indicated ongoing endothelial cell activation. Children treated with hydroxycarbamide also had higher FVIII activity and enhanced thrombin generation compared with those in the blood transfusion cohort (P < .001). Finally, hemolysis markers strongly correlated with VWF levels (P < .001) and were significantly reduced in the blood transfusion cohort (P < .001). Cumulatively, to our knowledge, our findings demonstrate for the first time that despite treatment, ongoing dysfunction of the VWF–ADAMTS13 axis is present in a significant subgroup of pediatric patients with SCD, especially those treated with hydroxycarbamide.

PB0825 Effect of von Willbrand Factor Concentrate on von Willebrand Factor Parameters during ECMO

PB0825 Effect of von Willbrand Factor Concentrate on von Willebrand Factor Parameters during ECMO

Impact of transcatheter aortic valve implantation on circulating von Willebrand factor in patients with severe aortic stenosis.

Severe aortic stenosis (AS) can lead to degradation of high molecular weight (HMW) von Willebrand factor (VWF) which can result in haemostatic abnormalities. While studies have explored changes in VWF profiles before and after surgical aortic valve replacement (SAVR), the longer-term changes in VWF profiles pre- and post-transcatheter aortic valve implantation (TAVI) are less understood. Our primary objective was to identify differences in VWF multimer profiles and VWF function pre-TAVI and 1-month post-TAVI. Our secondary objective was to correlate VWF markers with measures of AS severity. Adult patients with severe AS referred for TAVI at our institution were prospectively enrolled in this cohort study. Blood samples were collected for plasma analysis at three time points for all patients: 1day pre-TAVI, 3days post-TAVI, and 1-month post-TAVI. VWF antigen, activity, propeptide, collagen binding, multimers, and factor VIII coagulant activity were determined at each time point. Correlations between VWF parameters and severity of AS were assessed. Twenty participants (15 males, five females) with severe AS were recruited for the study. There was a significant increase in HMW VWF between pre-procedure and 1-month post-TAVI (p<.05). There was a transient increase in VWF antigen levels and activity at 3-days post TAVI that decreased to pre-TAVI levels at 1-month. There were no statistically significant correlations between VWF markers and AS severity. This is the first study to elucidate longer-term (>1week) improvements in HMW VWF after a TAVI procedure in severe AS patients.

Comprehensive Analyses of Coagulation Parameters in Patients with Vascular Anomalies

Vascular anomalies comprise a diverse group of rare diseases with altered blood flow and are often associated with coagulation disorders. The most common example is a localized intravascular coagulopathy in venous malformations leading to elevated D-dimers. In severe cases, this may progress to a disseminated intravascular coagulopathy with subsequent consumption of fibrinogen and thrombocytes predisposing to serious bleeding. A separate coagulopathy is the Kasabach-Merritt phenomenon in kaposiform hemangioendothelioma characterized by platelet trapping leading to thrombocytopenia and eventually consumptive coagulopathy. Our previous work showed impaired von Willebrand factor and platelet aggregometry due to abnormal blood flow, i.e., in ventricular assist devices or extracorporeal membrane oxygenation. With altered blood flow also present in vascular anomalies, we hypothesized that, in particular, the von Willebrand factor parameters and the platelet function may be similarly impacted. We prospectively recruited 73 patients with different vascular anomaly entities and analyzed their coagulation parameters. Acquired von Willebrand syndrome was observed in both of our patients with Kasabach-Merritt phenomenon. In six out of nine patients with complex lymphatic anomalies, both the vWF antigen and activity were upregulated. Platelet aggregometry was impaired in both patients with Kasabach-Merritt phenomenon and in seven out of eight patients with an arteriovenous malformation. The analysis of coagulation parameters in our patients with vascular anomalies advanced our understanding of the underlying pathophysiologies of the observed coagulopathies. This may lead to new treatment options for the, in part, life-threatening bleeding risks in these patients in the future.

ADAMTS13 inhibition to treat acquired von Willebrand syndrome during mechanical circulatory support device implantation

BackgroundAcquired von Willebrand syndrome (aVWS) is common in patients with mechanical circulatory support (MCS) devices. In these patients, the high shear stress in the device leads to increased shear‐induced proteolysis of von Willebrand factor (VWF) by A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, number 13 (ADAMTS13). As a result, the high molecular weight (HMW) VWF multimers are lost, leading to a decreased VWF function and impaired hemostasis that could explain the bleeding complications that are frequently observed in these patients. To counteract this abnormal VWF degradation by ADAMTS13, we developed a novel targeted therapy, using an anti‐ADAMTS13 monoclonal antibody (mAb) that inhibits the shear‐induced proteolysis of VWF by ADAMTS13. MethodsHuman or bovine blood was circulated through in vitro MCS device systems with either inhibitory anti‐ADAMTS13 mAb 3H9 or 17C7 (20 μg/ml) or control anti‐ADAMTS13 mAb 5C11 or phosphate buffered saline (PBS). VWF multimers and function (collagen binding activity) were determined at different time points. Next, Impella pumps were implanted in calves and the calves were injected with PBS and subsequently treated with mAb 17C7. VWF, ADAMTS13, and blood parameters were determined. ResultsWe demonstrated that blocking ADAMTS13 could prevent the loss of HMW VWF multimers in in vitro MCS device systems. Importantly, our antibody could reverse aVWS in a preclinical Impella‐induced aVWS calf model. ConclusionHence, inhibition of ADAMTS13 could become a novel therapeutic strategy to manage aVWS in MCS device patients.

Von Willebrand factor assays in patients with acquired immune thrombotic thrombocytopenia purpura treated with caplacizumab.

Acquired immune thrombotic thrombocytopenic purpura (iTTP) is a rare disease with a poor prognosis if undiagnosed. It is caused by autoantibody production to the von Willebrand factor (VWF) cleaving protease, A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13). Caplacizumab, an immunoglobulin directed to the platelet glycoprotein Ibα receptor of VWF, has been reported to induce quicker resolution of iTTP compared to placebo. The laboratory measurement of VWF activity was significantly reduced in clinical trials of caplacizumab. Several VWF assays are available in the UK and this study investigated whether differences in VWF parameters were present in 11 patients diagnosed with iTTP and treated with daily caplacizumab. Chromogenic factor VIII activity, VWF antigen, collagen binding activity, VWF multimers and six VWF activity assays were measured prior to caplacizumab therapy and on several occasions during treatment. VWF antigen and collagen binding activity levels were normal or borderline normal in all patients. Ultra-large molecular weight multimers were present in all patients following treatment. VWF activity assays were normal or reduced during treatment, but this was reagent and patient dependant. In the unusual scenario of a caplacizumab-treated patient requiring measurement of VWF activity, it is important that laboratories understand how their local reagents perform as results cannot be predicted.

The Impact of Age and BMI on the VWF/ADAMTS13 Axis and Simultaneous Thrombin and Plasmin Generation in Hospitalized COVID-19 Patients.

Aging and obesity independently contribute toward an endothelial dysfunction that results in an imbalanced VWF to ADAMTS13 ratio. In addition, plasma thrombin and plasmin generation are elevated and reduced, respectively, with increasing age and also with increasing body mass index (BMI). The severity risk of Corona Virus Disease 2019 (COVID-19) increases in adults older than 65 and in individuals with certain pre-existing health conditions, including obesity (>30 kg/m2). The present cross-sectional study focused on an analysis of the VWF/ADAMTS13 axis, including measurements of von Willebrand factor (VWF) antigen (VWF:AG), VWF collagen binding activity (VWF:CBA), Factor VIII antigen, ADAMTS13 antigen, and ADAMTS13 activity, in addition to thrombin and plasmin generation potential, in a demographically diverse population of COVID-19 negative (−) (n = 288) and COVID-19 positive (+) (n = 543) patient plasmas collected at the time of hospital presentation. Data were analyzed as a whole, and then after dividing patients by age (<65 and ≥65) and independently by BMI [<18.5, 18.5–24.9, 25–29.9, >30 (kg/m2)]. These analyses suggest that VWF parameters (i.e., the VWF/ADAMTS13 activity ratio) and thrombin and plasmin generation differed in COVID-19 (+), as compared to COVID-19 (−) patient plasma. Further, age (≥65) more than BMI contributed to aberrant plasma indicators of endothelial coagulopathy. Based on these findings, evaluating both the VWF/ADAMTS13 axis, along with thrombin and plasmin generation, could provide insight into the extent of endothelial dysfunction as well as the plasmatic imbalance in coagulation and fibrinolysis potential, particularly for at-risk patient populations.

Laboratory characterization of obligate carriers of type 3 von Willebrand disease with a potential role for Platelet Function Analyzer (PFA-200).

Type 3 von Willebrand disease (VWD) is a rare autosomal recessive disorder characterized by undetectable von Willebrand Antigen (VWF:Ag). Carriers of type 3 VWD carry one null allele and have von Willebrand factor (VWF) at about 50% of normal. The aim of this study was to characterize type 3 VWD carriers and to study the role of Platelet Function Analyzer (PFA-200) in this cohort. This was a cross-sectional study where data were collected from carriers (parents/offspring) of type 3 VWD patients and evaluated with activated partial thromboplastin time, factor VIII, blood group, ristocetin cofactor assay (VWF:RCo), VWF:Ag, and closure time on PFA-200 with collagen/epinephrine (COL/EPI), and collagen/ADP (COL/ADP). One hundred carriers were included in the study of which 85 were included for PFA-200 analysis. The mean (SD) of VWF:Ag (IU/ml) and VWF:RCo (IU/ml) was 0.63 (0.24) and 0.61 (0.26), respectively. Among the 100 carriers, based on VWF levels (VWF:Ag and/or VWF:RCo) and bleeding history, there were 7 type 1 VWD, 10 type 2 VWD, 25 borderline VWF (0.30-0.50IU/ml and no bleeding), and 58 normal VWF (>0.50IU/ml). PFA-200 was prolonged in 71% of the carriers, all carriers with type 1 and type 2 VWD phenotype, 80% carriers with borderline VWF, and 59% with normal VWF. COL/EPI was more sensitive than COL/ADP and showed better correlation with VWF parameters than COL/ADP. Carriers of type 3 VWD can have a variable laboratory phenotype. PFA-200 showed good sensitivity among the carriers at VWF levels <0.50IU/ml.

Thrombin generation in children using ThromboScreen reagent kit with ST Genesia—A pilot study

Thrombin generation assays assess overall coagulation system and are widely used in research; however, they still need standardization and clinical validation. The new ST Genesia is a benchtop, automated analyzer that normalizes each thrombin generation parameter using a reference plasma. The ThromboScreen reagent kit has two triggers, one of which contains thrombomodulin to assess the effect of the protein C pathway. This study aimed to make a pilot approach to the ThromboScreen reference range in children and evaluate the impact of sex, age, and pro- and anticoagulant plasma proteins on thrombin generation parameters. This study included 55 healthy children from the following age groups: 1-6years (n=14), 7-11years (n=15), and 12-17years (n=26). Children younger than 1year were excluded from the study. We measured thrombin generation using ThromboScreen, coagulation routine and test, pro- and anticoagulant proteins. Age did not influence ThromboScreen results. Males showed significantly lower endogenous thrombin potential and peak height values than females. The strongest determinants of endogenous thrombin potential were von Willebrand factor parameters, whereas for endogenous thrombin potential inhibition, the strongest determinants were protein C and protein S. No statistically significant differences were found betweengroups on temporal parameters. For the ThromboScreen reagent kit, it may not be necessary to subdivide reference ranges according to age for children (>1year).

Novel Splice-Site Mutation in RASGRP2 Leading to Caldag-GEFI Deficiency

Introduction: CalDAG-GEFI (Calcium and diacyglycerol-regulated guanine exchange factor I) deficiency or platelet-type bleeding disorder-18 (BDPLT18) is a relatively new, rare hemorrhagic disorder with autosomal recessive inheritance (OMIM #615888). CalDAG-GEFI activates Rap1 and is essential for αIIbβ3 (GP IIb/IIIa) integrin activation in platelets, a key step in platelet aggregation. The deficiency is due to mutations of the encoding gene, RASGRP2 (HGNC ID 9879), which is located on chromosome 11q13.1. To date, 23 pathogenic variants in the RASGRP2 gene are listed in the Human Gene Mutation Database (HGMD®). Two other RASGRP2 splice-site mutations (c.74-1G&amp;gt;C and 372-3C&amp;gt;G) are described in the literature in patients suffering from CalDAG-GEFI loss of function. Patients and Methods: We investigated a 3 yr-old index patient who was referred to our outpatient clinic because of Glanzmann thrombasthenia (GT)-like symptoms and GT-typical pattern in platelet aggregometry analyses. The boy presented with hematomas and recurrent infections, postnatally, he had shown petechiae. After a tongue bite at the age of 1 yr, he needed tranexamic acid to stop bleeding. So far, no surgery has been performed. His family members including mother, father, elder sisters and brother had no history of bleeding. Informed consent for our study of biochemical and molecular genetic characterization for inherited platelet disorders using NGS was given by all family members. Platelet functions were assessed by light transmission aggregometry using citrated PRP. Membrane glycoprotein quantification (CD41, CD42a and CD42b), fibrinogen binding, von Willebrand factor (VWF) binding and the investigation of alpha- and delta- granule secretion were performed by flow cytometry. For the index patient and his parents we performed NGS panel sequencing (Nextera Rapid Custom Enrichment, Illumina). 80 genes known to be associated with platelet disorders were screened. The variants detected were classified based on the 2015 American College of Medical Genetics (ACMG) guidelines. ClinVar, Human Gene Mutation Database, and in silico pathogenicity prediction were used to assess the detected variants. The segregation of the putative mutation was confirmed by Sanger sequencing of three unaffected siblings. Results: The index patient presented with normal values for platelet count, aPTT, INR, FXIII and VWF parameters. ADP (4, 10 and 20µM/L), epinephrine (8, 16 and 40µM/L) and low concentration collagen (2µM/mL) and TRAP6 (5µM/mL) induced aggregation was markedly reduced. However, normal values after stimulation with 10µM/mL collagen and TRAP6 respectively were achieved. Agglutination after stimulation with ristocetin (1,2mg/mL) was normal. Flow cytometry revealed severely decreased fibrinogen binding after stimulation with ADP (0.25-5.0 μmol/l) compared to control platelets. For CD41 (GP IIb/IIIa-complex), CD42a/b (GP Ib/IX-complex), ristocetin-induced VWF-binding, CD62- and CD63-expression, normal values were measured. Molecular genetic analysis revealed a novel homozygous splice-site mutation of the RASGRP2 gene in the index patient. The mutation was heterozygous in the non- consanguineous parents (Tab.1). Conclusion: We have identified a novel recessive mutation of the RASGRP2 gene in a boy affected by a Glanzmann thrombasthenia-like disorder. Platelet aggregometry analysis showed typical failure of aggregation with low dose collagen and TRAP6, whereas aggregation after stimulation with higher concentration reached normal values. Flow cytometric measured severely reduced fibrinogen binding indicated a functional defect of the αIIbβ3 fibrinogen receptor with normal expression. Comprehensive platelet function analysis and panel sequencing allowed early and precise diagnosis and therapy of this ultra-rare platelet signaling defect. Disclosures Zieger: Biotest: Research Funding; DFG: Research Funding; CSL Behring: Research Funding; Takeda: Research Funding.

ADAMTS‐13 and bleeding phenotype in von Willebrand disease

BackgroundThe bleeding phenotype of von Willebrand disease (VWD) varies highly between patients and can only partly be explained by von Willebrand factor (VWF) parameters. By cleaving large VWF multimers into smaller, less active multimers, ADAMTS‐13 is an important regulator of VWF activity. However, it is unknown what the role of ADAMTS‐13 is in individuals with VWD. ObjectivesWe therefore studied how ADAMTS‐13 activity is associated with the laboratory and bleeding phenotype in individuals with VWD. MethodsWe measured ADAMTS‐13 activity using the fluorescence resonance energy transfer substrate VWF 73 assay in 638 individuals with VWD in the nationwide cross‐sectional Willebrand in the Netherlands study and in 36 healthy controls. The bleeding phenotype was assessed using the Tosetto bleeding score. ResultsADAMTS‐13 activity was similar in individuals with VWD (109% ± 20.6%) and controls (110% ± 19.7%). ADAMTS‐13 activity was higher in individuals with VWD with type 3 than those with type 1 (mean difference, 11.8%; 95% confidence interval [CI], 2.9%‐20.8%) or type 2 (mean difference, 16.1%; 95% CI, 7.1%‐25.1%). ADAMTS‐13 activity was not associated with the Tosetto bleeding score (0.1 Tosetto bleeding score increase per 10% ADAMTS‐13 increase, 95% CI, −0.2 to 0.3). Furthermore, ADAMTS‐13 activity did not differ between individuals with and without a bleeding event during the year preceding blood sampling (mean difference, 1.4%; 95% CI, −2.1% to 4.9%). ConclusionADAMTS‐13 activity was highest in individuals with type 3 VWD, but it had only minor associations with VWF parameters. ADAMTS‐13 activity does not influence the bleeding phenotype in individuals with VWD.

Higher procoagulatory potential but lower DIC score in COVID-19 ARDS patients compared to non-COVID-19 ARDS patients

BackgroundCOVID-19 is a novel viral disease. Severe courses may present as ARDS. Several publications report a high incidence of coagulation abnormalities in these patients. We aimed to compare coagulation and inflammation parameters in patients with ARDS due to SARS-CoV-2 infection versus patients with ARDS due to other causes. MethodsThis retrospective study included intubated patients admitted with the diagnosis of ARDS to the ICU at Munich university hospital. 22 patients had confirmed SARS-CoV2-infection (COVID-19 group), 14 patients had bacterial or other viral pneumonia (control group). Demographic, clinical parameters and laboratory tests including coagulation parameters and thromboelastometry were analysed. ResultsNo differences were found in gender ratios, BMI, Horovitz quotients and haemoglobin values. The median SOFA score, serum lactate levels, renal function parameters (creatinine, urea) and all inflammation markers (IL-6, PCT, CRP) were lower in the COVID-19 group (all: p < 0.05).INR (p < 0.001) and antithrombin (p < 0.001) were higher in COVID-19 patients. D-dimer levels (p = 0.004) and consecutively the DIC score (p = 0.003) were lower in this group.In ExTEM®, Time-to-Twenty (TT20) was shorter in the COVID-19 group (p = 0.047), these patients also had higher FibTEM® MCF (p = 0.005). Further, these patients presented with elevated antigen and activity levels of von-Willebrand-Factor (VWF). ConclusionCOVID-19 patients presented with higher coagulatory potential (shortened global clotting tests, increased viscoelastic and VWF parameters), while DIC scores were lower. An intensified anticoagulation regimen based on an individual risk assessment is advisable to avoid thromboembolic complications.

Acquired von Willebrand Syndrome and Platelet Function Defects during Extracorporeal Life Support (Mechanical Circulatory Support).

Patients with ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO) suffer from an increased risk for thromboembolic events as well as for hemorrhages. High shear stress in the mechanical device results in acquired von Willebrand syndrome (AVWS), characterized by a loss of high-molecular-weight multimers of von Willebrand factor (VWF) leading to an increased bleeding risk. Onset of AVWS occurs within hours, persists during the whole period of mechanical support, and subsides rapidly after explantation. Patients with the older HeartMate II exhibit more severe AVWS than those with the newer HeartMate III, thanks to lower shear stress in the latter. All ECMO and VAD patients exhibit thrombocytopathia and often thrombocytopenia which further increases the bleeding risk. Etiological models for AVWS are increased cleavage by the metalloproteinase ADAMSTS13, mechanical destruction of VWF, and shear-induced VWF binding to platelets. Platelet secretion defects may be caused by transient platelet activation leading to degranulation. AVWS can be diagnosed by detection of VWF multimers using gel-electrophoresis and functional assays of varying sensitivity (VWF ristocetin cofactor activity, VWF activity, VWF collagen binding). Platelet dysfunction is monitored using light transmission aggregometry and secretion defects are detectable using flow cytometry. Modest use of anticoagulants and a target-controlled therapy based on VWF parameters and other coagulation and platelet parameters are shown to be beneficial in this patient group. Persistent hemorrhages may be controlled with tranexamic acid and platelet concentrates. Prompt weaning from the device, when indicated, is the best therapeutic option to prevent recurrent bleeding.

Utility of the platelet function analyser (PFA-100/200) for exclusion or detection of von Willebrand disease: A study 22 years in the making

Introductionvon Willebrand disease (VWD) is the most common inherited bleeding disorder and may alternatively arise as an acquired condition (AVWS). These represent deficiency and/or defects in von Willebrand factor (VWF). Closure times (CTs) obtained from the platelet function analyser (PFA) are highly sensitive to both VWD/AVWS. The current study has evaluated the utility of the PFA-100/-200 to exclude or detect laboratory identified VWD. Materials and methodsAn evaluation of the success or otherwise of prospective PFA testing to help exclude or detect VWD using data from a locally maintained database. This database contains patient information, results of PFA testing, results of VWF testing, platelet count and hematocrit, and additional information, for a current total of 3678 entries representing over 2 decades of testing. ResultsAccording to selection criteria, a total of 142 samples were identified as derived from patients with VWD. All but one of these were also identified to have abnormal PFA CTs. Additional data sets of patients with ‘low VWF’ (n = 137), or borderline normal VWF (n = 163) were also identified, as well as patients with thrombocytopenia and/or low hematocrit (n = 487). In these cohorts, PFA CTs were often (but not always) abnormal. There was a strong association between VWF test parameter values and PFA CTs. Additional study samples comprised cases with normal VWF parameters but prolonged CTs (n = 594), as well as cases with normal VWF and normal CTs (n = 1292), permitting calculation of sensitivity of abnormal PFA for VWD (99.3%), as well as specificity (68.5%), negative predictive value (99.9%) and positive predictive value (19.2%), for a prevalence of 7.0%. Comparatively, normal PFA CTs were better able to exclude VWD than normal test results for individual VWF parameters. ConclusionThis study reports on an evaluation of PFA CTs for identification or exclusion of VWD. In our dataset, representing over 22 years of experience, normal PFA CTs were able to predict absence of VWD with higher sensitivity than individual VWF test results.

Von Willebrand factor parameters as potential biomarkers for disease activity and coronary artery lesion in patients with Kawasaki disease.

Elevated von Willebrand factor (vWF):Antigen plasma levels have been observed in conjunction with cardiovascular diseases or vasculitis. The association of Kawasaki disease, a vascular inflammatory disease and vWF:Antigen, vWF:Collagen binding activity, and vWF multimers is unknown. We therefore investigated vWF parameters in 28 patients with acute Kawasaki disease in association with disease activity and coronary artery lesions. VWF:Antigen and vWF:Collagen binding activity were assessed via enzyme-linked immunoassay. The ratio of both (vWF:Collagen binding activity and VWF:Antigen) was calculated and vWF multimeric structure analysis performed. We analyzed the association between vWF parameters and our clinical data focusing on coronary artery outcome. VWF:Antigen and vWF:Collagen binding activity levels were significantly higher in the acute than in the disease's convalescence phase, and correlated positively with CRP levels. Neither variable was associated with coronary artery lesions. The vWF:Collagen binding activity/vWF:Antigen ratio, however, was significantly decreased in patients with a coronary artery lesion (z-score > 2; N = 10; mean ratio 0.96 vs. 0.64; p = 0.031) and even more so in those with a coronary artery aneurysm (z-score > 2.5; N = 8; mean ratio 0.94 vs. 0.55; p = 0.02). In a sub-analysis, those patients with a very low ratio in the acute phase presented a persistent coronary artery aneurysm at their 1-year follow-up.Conclusion: This study suggests that comprehensive analysis of vWF parameters may help to both monitor KD inflammation and facilitate the identification of those patients carrying an increased risk for coronary artery lesion.What is Known:• Von Willebrand factor (VWF)-parameters represent surrogate markers for vascular inflammation.• Kawasaki disease is a generalized vasculitis in children, which can be complicated by coronary artery lesions.What is New:• In those Kawasaki disease patients with coronary artery lesions, the vWF:CB/vWF:Ag ratio was significantly decreased.• VWF parameters may help to identify patients at risk for coronary artery lesions.

Long-lasting prothrombotic state implied by changes of plasma von Willebrand factor parameters after radical prostatectomy for prostate malignancy

ObjectivesThromboembolic complications are present in 0.8%–16.8% of the cases after radical prostatectomy (RP). Association between elevated plasma von Willebrand factor (VWF) levels—as an endothelial activation marker—and increased risk of thrombotic events has been evidenced.We aimed to elicit new data on the VWF after RP in prostate cancer patients and explore the role of it as a thrombotic risk factor. Upon perioperative plasma VWF levels (VWF:Ag) its collagen-binding (CB) activity (VWF:CB), multimerization, and cleaving enzyme (ADAMTS13 [a disintegrin and metalloprotease with thrombospondin type repeats, motif 1, type 13]) of the VWF multimers were quantitated along with Factor VIII and routine laboratory parameters in this observational pilot study. MethodsPlasma samples of 24 prostate cancer patients were collected before (-1 day; D-1) and after RP (1 hour, 6 days, 1 month, and 10 months; H1, D6, M1, and M10). VWF:Ag, VWF:CB, ADAMTS13:Ag were measured by ELISA, and the multimer distribution by electrophoresis and quantitative densitometry. Factor VIII, fibrinogen, D-dimer, and other routine laboratory parameters were determined as well. Preoperative values served as baselines which were compared to controls (24 healthy individuals). ResultsVWF:Ag and CB elevated by 122% and 143% respectively at H1 after RP then plateaued at D6 compared to baseline values. ADAMTS13/VWF:Ag ratio reduced by 41% at H1, and by 46% at D6, meanwhile the ratio of high molecular weight multimers increased as well. Values returned to baseline at M1 and further reduced to the levels of the controls at M10. All of the 24 patients at H1 and D6 and 14 at M10 were in potential prothombotic state according ROC analysis of the VWF parameters as indicators. ConclusionsProstate malignancy and then surgical stress, and inflammatory reactions induced release of VWF from the endothelial cells, along with an increasing amount of large multimers and relative reduction of ADAMTS13 level. Because these changes mark a prothrombotic state even at M1 after RP, more than 1 month follow-up and prophylactic targeting through the thrombotic and inflammatory activity of the VWF is proposed. Evaluation of VWF parameters provides new information about the long-term disturbances of primary hemostasis after radical pelvic oncologic surgery like RP and might improve the understanding the physiological and pathological recovery.

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.

Increased von Willebrand factor parameters in children with febrile seizures.

IntroductionPrimary blood coagulation and wound sealing are orchestrated by von Willebrand factor (VWF), a large multimeric glycoprotein. Upon release of arginine vasopressin (AVP), VWF containing high molecular weight multimers is secreted. By measuring copeptin, the C-terminal part of the AVP prohormone, we recently found strongly increased AVP levels in children with febrile seizures (FS) as compared to children with fever but without seizures. It is unknown if increased AVP levels in FS are of any biological function. Therefore, our a priori hypothesis was that children with FS have increased VWF parameters in parallel with higher AVP levels.MethodsWe conducted a prospective, cross-sectional study of children aged between 6 months and 5 years. Children that presented at our emergency department with fever or a recent FS (within four hours) were evaluated to be included to the study. We measured serum copeptin and VWF parameters, including analyses of VWF:Antigen (WVF:Ag), VWF:collagen binding activity (VWF:CB) and VWF multimers in children with FS, febrile infections without seizures and additionally, in a non-febrile control group.ResultsWe included 54 children in our study, 30 with FS, 10 in the febrile control group, and 14 in the non-febrile control group. Serum copeptin levels were significantly higher in children with FS (median [IQR] 24.73 pmol/l [13.65–68.65]) compared to the febrile control group (5.66 pmol/l [4.15–8.07], p = 0.002) and the non-febrile control group (4.78 pmol/l [3.33–5.3], p<0.001). VWF:CB levels were also significantly higher in children with FS (VWF:CB 2.29 U/ml [1.88–2.97]) as compared to the febrile (VWF:CB 1.41 U/ml [1.27–1.93], p = 0.048) and the non-febrile control group (VWF:CB 1.15 U/ml [0.98–1.21], p<0.001). VWF:Ag tended to be higher in children with FS compared to both control groups. Multivariate regression analysis revealed FS and copeptin as major determinants of VWF:CB.ConclusionsOur results suggest that increased secretion of AVP in children with FS is associated with higher plasma levels of VWF parameters. Especially VWF:CB may serve as additional biomarker in the diagnosis of FS.