Von Willebrand Factor Ristocetin Cofactor Research Articles

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

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

Quantitative impact of using different criteria for the laboratory diagnosis of type 1 von Willebrand disease

Only ± 50% of patients with type 1 von Willebrand disease (VWD) have recognized molecular defects and diagnosis still rests on demonstrating low plasma von Willebrand factor (VWF) protein/function. However, no generalized consensus exists regarding the type and number of VWF variables that should be considered for diagnosis. To compare the quantitative impact of four different criteria to diagnose type 1 VWD. We tested four laboratory criteria on 4298 laboratory studies during a 5-year period. The first was the National Heart, Lung, and Blood Institute recommendation, which diagnoses type 1 VWD with plasma VWF antigen (VWF:Ag) and VWF ristocetin cofactor (VWF:RCo) < 30 IU dL(-1) and possible VWD/'low VWF' with values between 30 and 50 IU dL(-1) . Second, diagnosis was established when two of three variables, VWF:Ag, VWF:RCo, VWF collagen binding assay (VWF:CB), were ≤ 2.5th percentile. Diagnostic criterion for possible VWD/'low VWF' using percentiles was also described. The third criterion (European Group on von Willebrand Disease, EUVWD), uses a plasma level of VWF:RCo (or VWF:CB) ≤ 40 IU dL(-1) for diagnosis. Finally, the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCBVWD) diagnoses VWD if VWF:Ag or VWF:RCo are ≤ 40 IU dL(-1) . The three assays had high correlation and excellent agreement at levels < 120 IU dL(-1) . The National Heart, Lung, and Blood Institute recommendation was followed to diagnose 122 (2.8%) patients with type 1 VWD and 704 (16.4%) with possible VWD/'low VWF.' Using percentiles, the diagnosis of type 1 VWD increased to 280 (6.5%) patients; 169 (3.9%) patients had possible VWD and 180 (4.2%) patients had 'low VWF.' Diagnoses using EUVWD and ZPMCBVWD criteria increased to 339 (7.9%) and 357 (8.3%) patients, respectively. Identical data, analyzed using different criteria, led to almost three-fold difference (2.8-8.3%) in diagnostic rate. This increase is mostly explained by increasing the cut-off values of VWF measurements from < 30 to ≈ 40 IU dL(-1) . Further refinement of the laboratory diagnosis of type 1 VWD is a priority.

Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF:RCo

IntroductionRistocetin cofactor activity of Von Willebrand factor (VWF:RCo) and the ratio VWF:RCo to its antigen VWF:Ag are used as routine screening to estimate VWF function and to detect types of Von Willebrand disease (VWD) caused by loss of high molecular weight multimers. However, the VWF:RCo test is prone to analytic imprecisions due to various reasons. We compared an assay for VWF activity (VWF:Ac) with VWF:RCo putting emphasis on the ratios to VWF:Ag. Materials and MethodsWe evaluated 942 samples from 432 patients and evaluated three groups in detail: normal patients (normal multimers, VWF:Ag and VWF:RCo >0.5 U/ml, VWD type 1 excluded; n=258), VWD type 1 (n=76) and acquired Von Willebrand syndrome (AVWS, n=326). In addition, 30 healthy subjects were analysed. ResultsVWF:Ac and VWF:RCo correlated well (Pearson´s r=0.96, p<0.01), so did their ratios to VWF:Ag (Pearson´s r=0.82, p<0.01). We calculated the normal range of VWF:Ac/VWF:Ag for healthy subjects as 0.8-1.16. In comparison, the reference range (mean±2std) derived from normal patient samples was 0.73-1.14. The corresponding ranges for VWF:RCo/VWF:Ag came to 0.74-1.23 (healthy) and 0.62-1.25 (normal patients). The ratios showed similar results regarding VWD type 1. The sensitivity for AVWS was higher with VWF:Ac/VWF:Ag than with VWF:RCo/VWF:Ag (97.5% versus 84.7%). ConclusionsThe data suggest that the results obtained with the VWF:Ac assay are comparable to that of the VWF:RCo assay. An AVWS was more reliably detected by VWF:Ac/VWF:Ag. We assume that the VWF:Ac assay could replace VWF:RCo for routine screening for AVWS, especially when prompt evaluation is required.

Laboratory aspects of von Willebrand disease: test repertoire and options for activity assays and genetic analysis.

The deficiency or abnormal function of von Willebrand factor (VWF) causes von Willebrand disease (VWD), the most frequent inherited bleeding disorder. The laboratory diagnosis of VWD can be difficult as the disease is heterogeneous and an array of assays is required to describe the phenotype. Basic classification of quantitative (type 1 and 3) and qualitative (type 2) VWD variants requires determination of VWF antigenic (VWF:Ag) levels and assaying of VWF ristocetin cofactor (VWF:RCo) activity, determining the capacity of VWF to interact with the platelet GPIb-receptor. Knowing the VWF:RCo activity is essential for identifying, subtyping and monitoring VWD, but the assay is poorly standardized and many protocols do not fulfil the clinical need in all situations. This has led to the development of novel activity assays, independent of ristocetin, with enhanced assay characteristics. Results from the first independent clinical evaluations are promising, showing that they are reliable and suitable for VWD diagnosis. The qualitative type 2 VWF deficiency can be further divided into four different subtypes (A, B, M and N) using specific assays that explore other activities or the size distribution of VWF multimers. These methods are discussed herein. However, in a number of patients it may be difficult to correctly classify the VWD phenotype and genetic analysis may provide the best option to clarify the disorder, through mutation identification.

Evaluation of an automated method for measuring von Willebrand factor activity in clinical samples without ristocetin.

The development of an automated, von Willebrand factor (VWF) activity assay, Innovance(®) VWF Ac (VWF:Ac), which measures VWF binding to the platelet receptor glycoprotein Ibα without ristocetin, led us to evaluate the assay for diagnosing von Willebrand disease (VWD) and monitoring therapy. After validating that the assay could be performed on an instrument from a different manufacturer, we compared VWF:Ac to VWF ristocetin cofactor activity (VWF:RCo) findings, including ratios of activity/antigen, for 100 healthy controls and 262 consecutive clinical samples from 217 patients (197 adults, 64 children, n = 1 age unknown) referred for VWF testing. There was excellent correlation (R(2) = 0.96) between VWF:Ac results run at two different sites on two different instruments. VWF:Ac had greater precision and sensitivity to low levels of VWF than the VWF:RCo method. Although there was good correlation between VWF:Ac and VWF:RCo results among healthy controls and patient subjects, VWF:Ac results were undetectable and/or significantly lower than VWF:RCo among patients who had types 2A, 2B, or 2M VWD. Additionally, a higher proportion of patient samples were classified as showing qualitative defects using the VWF:Ac compared with VWF:RCo method. While most samples drawn on VWD therapy had similar VWF levels by VWF:Ac and VWF:RCo, a type 2B VWD subject on replacement had much lower activity estimated by VWF:Ac. We conclude that Innovance(®) VWF Ac is suitable for the diagnosis, classification, and monitoring of VWD, and that it has a number of advantages over VWF:RCo method.

Platelet-Type von Willebrand Disease: Toward an Improved Understanding of the “Sticky Situation”

We are pleased to highlight in this issue of Seminars in Thrombosis & Hemostasis, the study by Wood et al,1 wherein the authors describe a novel mutation in the platelet GP1BA gene creating a hyperresponsive GPIbα protein—a receptor for von Willebrand factor (VWF)—and causing platelet-type von Willebrand disease (PT-VWD). This is a new naturally occurring mutation in a 23-year-old male patient and is considered the sixth reported mutation thus far in patients described with this disease worldwide. Despite being a rare bleeding disorder, PT-VWD represents a significant challenging clinical problem as it may cause life-threatening bleeding, if not appropriately treated, particularly in situations related to surgeries and childbirth. The clinical diagnosis challenge stems from the close similarity of PT-VWD to the more common bleeding disorder, type 2BVWD.2,3 The discrimination and correct diagnosis can only be made after carefully assessing less commonly performed laboratory tests,3–8 and confirmed only after DNA analysis of the binding regions in the two genes VWF and GP1BA9 has been performed. The report by Woods et al1 highlights some important issues that add to our understanding of this rare but potentially life-threatening bleeding disorder. First, the patient presented with severe bleeding symptoms (rather than mild/moderate bleeding symptoms, and unlike previously reported cases) while also showing other typical laboratory phenotypic data known in this disease such as macrothrombocytopenia, mild spontaneous platelet aggregation, absence from plasma of high-molecular-weight VWF multimers, positive ristocetin-induced platelet aggregation (RIPA) at 0.3 and 0.4 mg/mL, VWF ristocetin cofactor (VWF:RCo) T GP1BA gene mutation that predicted the protein change Try246Leu. This mutation was absent in the unaffected mother and also in 100 healthy control subjects. Second, in that report,1 an attempt was made to quantify the bleeding symptoms in a patient with PT-VWD (bleeding score of 13). The knowledge about variation in bleeding symptoms and the ability to objectively assess these symptoms have proven to be important inmanagement of bleeding disorders and can help predict disease outcome and also aid in treatment.10 The only other such attempt was in a small case series reported recently. This series showed considerable variability in bleeding severity among PT-VWD that is independent of age or gender. The phenotypic variability in type 2B VWD has been reviewed in relation to various mutations and different patients’ cohorts.12 However, a systematic analysis of PT-VWD phenotype and the genotype–phenotype relationship remains to be investigated. Third, for the first time, the level of VWF propeptide (VWFpp) and VWFpp/VWF:Ag ratio was reported in a patient with PT-VWD.1 VWFpp is a 741 amino acid portion that gets cleaved from mature VWF by proteolysis. After cleavage, the VWFpp remains in noncovalent association with the VWF multimers, and both are stored together in the α-granules (megakaryocytes/platelets) or Weibel–Palade bodies (endothelial cells). Upon release and under physiologic pH, the VWF multimers and VWFpp dissociate and are secreted in 1:1 stoichiometric amounts.13 Studies have shown a regulatory role for VWFpp as an intramolecular chaperone for the mature VWF protein and an aid to its storage and multimerization.14,15 The VWFpp circulates in the plasma for a short time, with a half-life of approximately 2 to 3 hours and plasma levels of approximately 1 μg/mL, whereas multimeric VWF circulates with a half-life of approximately 8 to 12 hours and plasma levels of approximately 10 μg/mL.16,17 Recent

Identification of p.W246L As a Novel Mutation in the GP1BA Gene Responsible for Platelet-Type von Willebrand Disease

Platelet-type von Willebrand disease (PT-VWD) and type 2B von Willebrand disease (2B-VWD) are rare bleeding disorders characterized by increased ristocetin-induced platelet aggregation (RIPA) at low concentrations of ristocetin. Diagnosis of either condition is not easy and the differential diagnosis between the two entities is especially challenging as evidenced by high levels of misdiagnosis of both conditions, but particularly PT-VWD. Five mutations in the GP1BA gene related to PT-VWD and less than 50 patients are currently reported worldwide. We herein describe a patient with severe bleeding symptoms, macrothrombocytopenia, mild spontaneous platelet aggregation, positive RIPA at 0.3 and 0.4 mg/mL, von Willebrand factor ristocetin cofactor (VWF:RCo) to antigen (VWF:Ag) < 0.2, normal VWF propeptide/VWF:Ag ratio, and RIPA mixing tests and cryoprecipitate challenge positive for PT-VWD. GP1BA gene was studied in the patient, in his mother, and in 100 healthy control subjects. We identified a heterozygous substitution G > T located at nucleotide 3805 in the g.DNA of the patient's GP1BA gene, resulting in a Trp to Leu amino acid change at residue 246 (p.W246L). This mutation was absent in his unaffected mother and also in the 100 controls, and was predicted as damaging by in silico analysis. The residue W246 is located within the VWF-binding region and exists in a strongly conserved position in the phylogenetic tree, which is expected to be unable to tolerate substitutions without changing its functional characteristics. These findings argue strongly in favor of the view that this substitution does not represent a polymorphism and is therefore responsible for the PT-VWD phenotype of the patient.

Decreased von Willebrand factor ristocetin cofactor activity and increased ADAMTS13 antigen increase postoperative drainage after coronary artery bypass grafting

Routine coagulation tests and bleed-scores fail to identify patients at risk of excessive postoperative drainage following coronary artery bypass grafting (CABG). We sought to investigate whether lower von Willebrand factor (VWF) and higher ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) are associated with a high postoperative drainage after CABG. In the prospective cohort study, VWF antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCO), VWF collagen-binding (VWF:CB), ADAMTS13 antigen (ADAMTS13:Ag) and ADAMTS13 activity were measured on the day of elective on-pump CABG in 232 consecutive patients without a prior history of haemorrhagic diathesis, including von Willebrand disease (95% discontinued aspirin preoperatively). Postoperative drainage and blood product use were recorded. A comparison of extreme drainage quartiles (n=56) showed that individuals with the highest drainage volumes have mean VWF:RCO lower by 19% (P<0.0001), median VWF:Ag lower by 19% (P<0.0001), ADAMTS13:Ag higher by 8% (P=0.0002), ADAMTS13 activity higher by 9% (P=0.01) and fibrinogen lower by 14% (P=0.03) than those with the lowest drainage. Linear regression analysis showed that preoperative VWF:RCO (b=-4.83, P=0.002) and fibrinogen (b=-61.52, P=0.04) are the only independent predictors of postoperative drainage. Multivariate logistic regression demonstrated that preoperative VWF:RCO in the lowest quartile and ADAMTS13:Ag levels in the highest quartile increased the risk of high (≥1000 ml) drainage (odds ratio [95% confidence interval]=4.88 [1.83-13.02], P=0.001 and 3.77 [1.49-9.52], P=0.005; respectively). Patients undergoing elective CABG with lower preoperative VWF:RCO are at risk of having larger postoperative drainage, which suggests a novel contributor to increased perioperative bleeding in cardiac surgery.

An approach to outreach patients with von Willebrand disease in Egypt by targeting women with heavy menstrual bleeding and/or bleeding symptoms

von Willebrand disease (VWD) is frequently ignored as a cause of menorrhagia. We investigated Egyptian women complaining of heavy menstrual bleeding (HMB) and/or other bleeding symptoms to detect potential VWD cases. Seventy-five female patients complaining of HMB and/or bleeding symptoms and 38 age-matched healthy female controls went through a family history questionnaire, a physical examination and were evaluated for bleeding score, pictorial blood assessment chart (PBAC), complete blood count, serum ferritin, blood group, prothrombin time, activated partial thromboplastin time, factor VIII (FVIII) activity, von Willebrand factor (VWF) ristocetin cofactor (RCo) activity, antigen (Ag), and RCo/Ag ratio. Sixty-eight of 75 patients presented with HMB, out of which 46 had no organic pathology and 7 presented other bleeding symptoms. Six patients were diagnosed with VWD, three with HMB, two with other bleeding symptoms and one with family history of VWD. Two related VWD patients were diagnosed in the control group. There were significant differences in bleeding and PBAC scores, ferritin level, FVIII activity, VWF:RCo and VWF:Ag between VWD patients and controls. This study indicated a high prevalence of VWD among patients with HMB without organic pathology (6.5%) and demonstrated the sensitivity of diagnostic parameters of VWD patients in an outreach campaign. The inexpensive bleeding and PBAC scoring systems are valuable to exclude cases without objective bleeding symptoms. Raising gynaecologists awareness about hereditary bleeding disorders is important to ensure a proper diagnosis and possible referral of these patients. Management of these patients with comprehensive medical care services under a multidisciplinary team would be ideal.

Recombinant Factor VIII Combined With Recombinant Von Willebrand Factor In Patients With Severe Hemophilia A: A Prospective Clinical Study Of Safety and Pharmacokinetics

Hemophilia A is an X-linked recessive, congenital bleeding disorder caused by a deficiency of circulating coagulation FVIII. Treatment regimens include on-demand or prophylactic FVIII substitution. An important challenge for patient compliance and treatment success is the need for frequent infusions due to the short circulating terminal half-life (T1/2) of FVIII (8 to 14 h). In vivo, FVIII binds to, and is stabilized by, von Willebrand factor (VWF). Studies have shown that the T1/2 of FVIII is positively correlated to baseline levels of VWF. The present study hypothesizes that co-administration of recombinant FVIII (rFVIII) with recombinant VWF can improve the pharmacokinetic (PK) properties of rFVIII.The PK and safety of a rFVIII (ADVATE, Baxter) co-administered with an investigational rVWF were investigated in a prospective clinical trial of 12 previously-treated patients with severe hemophilia A (FVIII:C <1%), aged 18 to 60 years. Subjects were administered 3 infusions 8 to 14 days apart with: 1) rFVIII alone, 2) rFVIII combined with rVWF (low VWF ristocetin co-factor activity [VWF:RCo] dose), and 3) rFVIII combined with rVWF (high VWF:RCo dose). rFVIII was administered at the same dose for each PK infusion. FVIII activity was assessed using a one-stage clotting assay.rFVIII co-administered with rVWF up to the highest investigated dose was well tolerated and safe in hemophilia A patients. No serious adverse events (AEs) and no treatment related AEs occurred, including no signs or symptoms of thrombosis, development of neutralizing antibodies to rVWF or rFVIII, or hypersensitivity reactions were observed.A trend suggesting a prolongation of FVIII activity by the addition of rVWF was observed as shown by an increased area under the concentration curve (AUC), mean residence time (MRT) and T1/2 after infusion of rFVIII combined with rVWF as compared with rFVIII alone, with a greater improvement associated with the higher of the 2 rVWF doses investigated (Table 1). An association between VWF antigen (VWF:Ag) level and rFVIII T1/2 was observed. Subjects with lower VWF:Ag levels at baseline tended to have a more pronounced increase in rFVIII T1/2 after infusion with rFVIII combined with rVWF compared with rFVIII alone (Table 2).Table 2Baseline VWF:Ag levels and geometric mean FVIII:C in individual patients after infusionSubjectBaseline VWF:AGFVIII T ½ after rFVIII aloneFVIII T½ after rFVIII + rVWF (high dose)Ratio of T½ after rFVIII + rVWF (high dose) over T½ after rFVIII alone110616.6515.220.91229826.6323.970.903909.1210.341.13412113.2613.781.04512610.6312.241.156908.2412.171.487795.818.751.5181089.3612.131.30913914.6815.931.091018126.6018.160.681116111.8813.911.17In summary, rVWF was safe and well tolerated when co-administered with rFVIII in hemophilia A patients and slightly sustained FVIII activity, with the highest improvement in circulating FVIII T1/2 associated with lower baseline VWF:Ag levels. These findings provide insight into the FVIII/VWF interaction and its potential impact on therapy. Disclosures:Windyga:Baxter, Bayer, Behring, Novo Nordisk, Octapharma, Pfizer: Honoraria, Research Funding. Draxler:Baxter: Employment. Chapman:Baxter: Employment. Wong:Baxter: Employment. Sørensen:Baxter: Employment. Ewenstein:Baxter: Employment.

Von Willebrand Factor Activity Increases Following Automated But Not Simple Red Blood Cell Exchange Transfusion In Sickle Cell Disease Patients

BackgroundRed blood cell (RBC) exchange transfusion is a commonly used therapy for acute and chronic complications of sickle cell disease (SCD). However, the therapeutic benefit of RBC exchange in many clinical situations is not well established. RBC exchange can be performed in two ways: 1) manually, involving repeated cycles of whole blood phlebotomy followed by simple transfusion of donor RBC, or 2) automated exchange utilizing apheresis to rapidly remove patient RBC while returning patient plasma, platelets, and leukocytes along with donor RBC to the patient. Unlike manual exchange transfusion, automated RBC exchange exposes patient blood to high shear stress as blood transits the apheresis machine, reaching wall shear stresses of 30-50 dyne/cm2 (within the range of arterial shear) with regional peak wall shear stresses of over 500 dyne/cm2 (supraphysiologic shear). Increased wall shear stress increases the reactivity of plasma von Willebrand Factor (VWF), in part by enhancing exposure of the VWF A1 domain which is directs binding to the platelet glycoprotein (GP) Ib-IX-V. Increased VWF reactivity has been implicated in SCD pathogenesis, likely by promotion of adhesive interactions between VWF and sickle erythrocytes, platelets, leukocytes, and the vessel wall. HypothesisWe hypothesized that automated RBC exchange would increase the reactivity of VWF as compared to manual RBC exchange in SCD patients. MethodsWe assessed VWF parameters in the plasma of SCD patients before and after manual exchange transfusion and/or automated RBC exchange transfusion performed for non-acute clinical indications. The VWF parameters assessed were VWF antigen level (VWF:Ag), VWF activity (VWF:Act) as assessed by ELISA (DiaPharma) detecting the spontaneous Gp Ib-IX-V binding conformation in the VWF A1 domain, total active VWF (VWF:TA) calculated by VWF:TA = (VWF:Ag) x (VWF:Act), VWF ristocetin cofactor activity (VWF:RCo), and VWF multimer composition. ResultsSix SCD patients on chronic, monthly exchange transfusion support were enrolled in the study. Blood samples were obtained prior to and immediately following multiple separate exchange transfusion episodes for each patient. Three patients underwent only automated RBC exchange transfusion, one underwent only manual RBC exchange transfusion, and two patients underwent both manual and automated RBC exchange transfusion. Compared to normal pooled plasma (NPP), SCD patients prior to RBC exchange transfusion had increased VWF:Ag (1.3-2.1 fold), VWF:Act (mean 124% of normal), and VWF:TA (0.6-2.2, mean 1.52; VWF:TA of NPP= 1.02). There was no statistically significant change in VWF:Ag following manual or automated exchange. There was a statistically significant increase in VWF:Act in patients following automated RBC exchange (mean 25% increase, p<0.05 with range 12%- 53%) but not manual exchange (mean 3.4% increase, range 1.4%-6.3%). In addition, we found an increased VWF:TA following automated (mean 31% increase, p<0.05) but not manual exchange transfusion. Patients exhibiting the highest VWF:TA post-exchange transfusion also had increased VWF:Rco in response to low dose and high dose ristocetin. The patient plasma with the highest VWF:TA post-RBC exchange (3.59) was noted to initiate spontaneous platelet agglutination in the absence of ristocetin. The observed increases in VWF:TA or VWF:RCo did not correlate with differences in VWF multimer composition on non- reducing agarose gels. ConclusionsVWF activity is increased after automated RBC exchange but not manual RBC exchange in adult sickle cell patients on chronic exchange transfusion. These increases are likely a result of shear induced activation of VWF in the apharesis machine circuit. We speculate that the increased VWF reactivity observed following automated RBC exchange transfusion could contribute to VWF-mediated adhesive interactions and aggravate SCD vascular complications, potentially conferring a microangiopathic effect, in certain clinical situations. Maneuvers to decrease automated RBC exchange transfusion-induced VWF activation may lead to improved outcomes for SCD patients requiring exchange transfusion. Disclosures:No relevant conflicts of interest to declare.

Binding Of VWF To Type IV Collagen: An Additional Collagen Binding Mechanism Beyond Types I, III, and VI Collagen?

Von Willebrand disease (VWD) is a common bleeding disorder characterized by defects in the interaction of von Willebrand factor (VWF) with its ligands factor VIII, platelet glycoprotein Ib, and/or collagen. Nearly all assessment of collagen binding is performed with types I or III collagen. Type VI collagen, which is formed as a tight triple helix, has also been reported to interact with VWF. In contrast, type IV collagen lacks this tight helical structure and is located in the basal lamina. Type IV collagen may therefore serve as another important ligand for VWF. We examined this interaction in samples from the Zimmerman Program for the Molecular and Clinical Biology of VWD, a large US study of subjects with all types of VWD.Subjects were enrolled based on a pre-existing diagnosis of VWD and compared to a cohort of healthy controls. VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and type III collagen binding using 1 mcg/mL human placental type III collagen (VWF:CB3) were performed in the clinical laboratory at the BloodCenter of Wisconsin. Type IV and type VI collagen binding (VWF:CB4 and VWF:CB6) were performed in the research laboratory by ELISA using 1 mcg/mL human placental type IV or type VI collagen captured on amine binding plates. DNA sequencing was performed on all subjects to identify VWF sequence variations. Bleeding scores were calculated according to the scoring system used in the MCMDM-1VWD study.For this analysis, 251 healthy controls, 535 type 1, 49 type 2A, 16 type 2B, 18 type 2M, 11 type 2N, and 18 type 3 VWD were included. The mean VWF:CB4/VWF:Ag ratio for the healthy controls was 0.96, with a standard deviation of 0.24. This was similar to the mean VWF:CB6/VWF:Ag ratio (mean 0.95, p=NS), but slightly lower than the mean VWF:CB3/VWF:Ag ratio (mean 1.05, p<0.01). The mean VWF:CB4/VWF:Ag ratio was 0.87 for the type 1 VWD subjects. The mean VWF:CB4/VWF:Ag ratio was significantly lower in type 1 subjects as compared to healthy controls (p<0.01). This was attributed in part to the presence of several A1 domain sequence variations, including p.R1399H, p.F1369I, and p.L1382P. While the latter sequence variations were isolated findings in single subjects, p.R1399H was present in 1% of the healthy controls, 1% of the type 1, and 6% of the type 2M VWD subjects.Mean VWF:CB4/VWF:Ag ratios were low for both type 2A and type 2B VWD subjects, at 0.54 and 0.52 respectively. This was not significantly different from the VWF:CB3/VWF:Ag data (p=NS for both type 2A and 2B VWD), consistent with the loss of high molecular weight multimers in these subjects and the concomitant reduction in collagen binding. As expected, VWF:CB4/VWF:Ag ratios were normal in the type 2N subjects, with a mean of 1.04. The type 2M subjects, however, displayed a significant reduction in type IV collagen binding, with a mean VWF:CB4/VWF:Ag ratio of 0.79. One subject with an 11 amino acid deletion (from p.R1392 through p.Q1402) had absent binding to type IV collagen yet normal binding to type III collagen with a VWF:CB3/VWF:Ag ratio of 0.82. Another subject with p.I1425F and p.R1399H had a VWF:CB4/VWF:Ag ratio of 0.48, and VWF:CB3/VWF:Ag ratio of 1.10. Type IV collagen binding did not correlate with VWF:RCo (R squared less than 0.1). The type 2M subjects with a mean VWF:CB4/VWF:Ag ratio less than 0.7 had higher bleeding scores, with a mean bleeding score of 9 compared to a mean of 6 for type 2M subjects with a mean VWF:CB4/VWF:Ag ratio greater than 0.7. Not surprisingly, the subjects with type 3 VWD and undetectable VWF:Ag also had undetectable VWF:CB4.These data suggest that type IV collagen binding reflects an independent function of VWF apart from platelet binding, and distinct from binding to type III collagen. In our study, more variation was seen between type IV and type III collagen than between type IV and type VI collagen. Neither VWF:CB4 nor VWF:CB6 are currently tested as part of the clinical workup of VWD. Measurement of this function may assist in identification of variant VWD, particularly in the subgroup of patients with type 2M VWD. Disclosures:No relevant conflicts of interest to declare.

A two‐centre comparative evaluation of new automated assays for von Willebrand factor ristocetin cofactor activity and antigen

von Willebrand disease (VWD) is caused by a quantitative and/or qualitative deficiency of the von Willebrand factor (VWF). The laboratory diagnosis of VWD is dependent on the measurement of VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo). The aim of this study was to undertake a two-centre evaluation of two new automated VWF:Ag and VWF:RCo assays systems from Instrumentation Laboratory (Bedford, USA). Using the two new analytical systems that operated with different detection principles: immunoturbidimetric (TOP500 analyser) and chemiluminescent (AcuStar analyser), VWF:Ag and VWF:RCo levels were determined in samples from 171 healthy normal subjects, 80 VWD patients (16 type 1, 58 type 2 and 6 type 3) and 7 acquired von Willebrand syndrome patients. With commercial lyophilized normal and pathological plasmas VWF: Ag and VWF:RCo assays performed on both analysers exhibited low levels of inter-assay imprecision (AcuStar: CV% range 3.3-6.9; TOP500: CV% range 2.6-6.3). Samples from normal healthy subjects (range: VWF:Ag 44.6-173.9 IU dL(-1) ; VWF:RCo 43.1-191.5 IU dL(-1)) and patients (range: VWF:Ag <0.3-115.1 IU dL(-1) ; VWF:RCo <0.5-57.2 IU dL(-1)) showed a good correlation between the two VWF:Ag and VWF:RCo methods (rs = 0.92 and 0.82 respectively), with only a few inconsistent cases among the patients' samples evaluated. The chemiluminescent assays had a lower limit of detection for both VWF:Ag and VWF:RCo compared to immunoturbidimetric tests (0.3 IU dL(-1) vs. 2.2 IU dL(-1) and 0.5 IU dL(-1) vs. 4.4 IU dL(-1) respectively). The TOP500 and AcuStar VWF:Ag and VWF:RCo assays were precise and compare well between centres, making these systems suitable for the diagnosis of VWD in non-specialized and reference laboratories.

An Unbalance Between von Willebrand Factor and ADAMTS13 in Acute Liver Failure: Implications for Hemostasis and Clinical Outcome

Emerging evidence supports the concept of a rebalanced hemostatic state in liver disease as a result of a commensurate decline in prohemostatic and antihemostatic drivers. In the present study, we assessed levels and functionality of the platelet-adhesive protein von Willebrand factor (VWF) and its cleaving protease ADAMTS13 in the plasma of patients with acute liver injury and acute liver failure (ALI/ALF). Furthermore, we explored possible associations between VWF, ADAMTS13, and disease outcome. We analyzed the plasma of 50 patients taken on the day of admission for ALI/ALF. The plasma of 40 healthy volunteers served as controls. VWF antigen levels were highly elevated in patients with ALI/ALF. In contrast, the collagen-binding activity and the ratio of the VWF ristocetin cofactor activity and VWF antigen was significantly decreased when compared with healthy controls. Also, the proportion of high molecular weight VWF multimers was reduced, despite severely decreased ADAMTS13 levels. In spite of these functional defects, platelet adhesion and aggregation were better supported by plasma of patients with ALI/ALF when compared with control plasma. Low ADAMTS13 activity, but not high VWF antigen, was associated with poor outcome in patients with ALI/ALF as evidenced by higher grades of encephalopathy, higher transplantation rates, and lower survival. VWF or ADAMTS13 levels were not associated with bleeding or thrombotic complications. Highly elevated levels of VWF in plasma of patients with ALI/ALF support platelet adhesion, despite a relative loss of function of the molecule. Furthermore, low ADAMTS13 activity is associated with progressive liver failure in the patient cohort, which might be attributed to platelet-induced microthrombus formation in the diseased liver resulting from a substantially unbalanced VWF/ADAMTS13 ratio.

Comparison of a new chemiluminescent immunoassay for von Willebrand factor activity with the ristocetin cofactor-induced platelet agglutination method

Measuring von Willebrand factor (VWF) activity is essential for the diagnosis of von Willebrand disease (VWD). The VWF activity is usually assessed based on measurement of the ristocetin cofactor (VWF:RCo). However, that test is technically challenging and has high intra- and inter-assay variabilities. A new automated chemiluminescent immunoassay VWF activity has recently become commercially available (HemosIL AcuStar von Willebrand Factor Ristocetin Cofactor Activity). The main objective of this study was to evaluate this new method and to compare it with the VWF:RCo assay as the reference method. We studied 91 samples, 18 healthy volunteers samples and 73 samples from patients (VWF:RCo level <50IUdL(-1) ): 29 type 1 VWD, 13 type 2A, 5 type 2B, 5 type 2M, 3 type 2N, 5 type 3, 4 type 3 under treatment, 5 type 3 carriers and 4 samples with other pathologies. The HemosIL AcuStar VWF:RCo assay was 96% sensitive and 100% specific for detecting VWF abnormalities. The good analytical performance, and the sensitivity and specificity of HemosIL AcuStar VWF:RCo to detect VWF deficiency renders it a suitable method for VWD screening.

Time course of acquired von Willebrand disease associated with two types of continuous-flow left ventricular assist devices: HeartMate II and CircuLite Synergy Pocket Micro-pump

Bleeding complications are frequent adverse events in patients supported with axial continuous-flow pumps. Previous retrospective studies demonstrated that bleeding events in patients with the HeartMate II (Thoratec Corp, Pleasanton, CA) were attributed to acquired von Willebrand syndrome. We sought to analyze the von Willebrand factor (VWF) profile in patients receiving a HeartMate II or a CircuLite (Saddle Brook, NJ,) device (Synergy Pocket Micro-pump) prospectively. Prospectively analyzed were 34 patients supported with left ventricular assist device (LVAD; 26 with HeartMate II and 8 with CircuLite). The control group comprised 20 patients who underwent heart transplantation (HTx). Blood samples were taken pre-operatively and at 14 days and 3, 6, 9, and 12 months post-operatively. Patients with LVADs had a high incidence of bleeding complications. From the immediate post-operative phase throughout the entire observation, the VWF ristocetin cofactor activity (Rco)/antigen (Ag) ratio of patients with HeartMate II and CircuLite devices was consistently lower compared with HTx patients. No correlation was found between the individual VWF:Rco/Ag ratio and bleeding events or transfusion requirements. The VWF:Rco/Ag ratio normalized immediately in patients who received HTx. Acquired von Willebrand syndrome was confirmed to occur immediately after the implantation of both types of LVAD and persisted up to 12 months. A lower VWF:Rco/Ag ratio was associated with larger transfusion requirements. Acquired von Willebrand syndrome resolves after LVAD explantation.

Quality in Hemostasis and Thrombosis, Part II

bleeding symptoms, macrothrombocytopenia, mild spontaneous platelet aggregation, positive RIPA at 0.3 and 0.4 mg/mL, von Willebrand factor (VWF) ristocetin cofactor (VWF:RCo) to antigen (VWF:Ag) ratio of T located at nucleotide 3805 in the g.DNA of the patient’s GP1BA gene, resulting in a Trp to Leu amino acid change at residue 246 (p.W246L). This mutation was absent in his unaffected mother and also in the 100 controls and was predicted as damaging by in silico analysis. The residue W246 is located within the VWF binding region and exists in a strongly conserved position in the phylogenetic tree, which is expected to be unable to tolerate substitutions without changing its functional characteristics. These findings argue strongly in favor of the view that this substitution does not represent a polymorphism, and therefore, is responsible for the PT-VWD phenotype of the patient. The accompanying editorial by Othman and Emsley 5 highlights the importance of this new work, and it alsoextends the analysis of the proposed mutation by molecular modeling. The second editorial, 7 along with the accompanying highlighted articles, 8,9 deal with recent initiatives in the standardization and harmonization of antiphospholipid antibody (aPL) testing, essential for the appropriate diagnosis and management of patients with antiphospholipid syndrome (APS) and other hypercoagulable states. 14 The leading issues in aPL testing include high vulnerability to preanalytical, analytical as well as postanalytical problems, the heterogeneous sensitivity of tests and reagents, high intermethod and interlaboratory variability, the clinically meaningful rate of false-negative and false-positive results, an absence of

Variability in platelet‐ and collagen‐binding defects in type 2M von Willebrand disease

Type 2M von Willebrand disease (VWD) includes qualitative defects in von Willebrand factor (VWF) function, with normal multimer distribution but a defect in VWF activity with respect to platelet or collagen binding. We characterized novel VWF gene mutations found in type 2M VWD subjects enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD. Subjects were enrolled based on a pre-existing diagnosis of type 2M VWD. Testing included full-length gene sequencing, VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), VWF collagen binding and multimer distribution. Recombinant VWF variants were synthesized using site-directed mutagenesis and expressed in HEK293T cells. Platelet binding was measured by flow cytometry with fixed platelets and ELISA with recombinant glycoprotein Ibα (GPIbα). Four novel VWF A1 domain mutations were found in individuals with type 2M VWD: S1358N, S1387I, S1394F and Q1402P. All subjects had a history of bleeding, VWF:RCo < 40 IU dL(-1) , VWF:RCo/VWF:Ag ratios <0.6 and normal multimer distribution. No defect in expression, secretion, or multimerization was found for any of the mutations. All showed decreased binding to intact platelets, and decreased or absent binding to a mutant GPIbα construct with spontaneous VWF binding. 1387I had decreased binding to all collagen types tested. 1402P had reduced binding exclusively to type VI collagen. Type 2M VWD is a heterogeneous category comprised of both collagen- and platelet-binding defects. Understanding the precise defect for each mutation may ultimately lead to better diagnosis and treatment.

P-026 Frequency of thrombophilia in females with stroke in association with oral contraceptive use

s / Thrombosis Research 131, Suppl. 1 (2013) S71–S103 S83 P-025 Adrenomedullin is associated with vonWillebrand factor activity in non-pregnant but not in pregnant women M. Poyhonen-Alho1, R. Lassila 2, L. Joutsi-Korhonen2, G. Nicholls 3, R. Kaaja 4 1Department of Obstetrics and Gynaecology, Helsinki University Central Hospital/Jorvi Hospital, Helsinki, Finland; 2Coagulation Unit, Departments of Hematology and Clinical Chemistry (HUSLAB Laboratory Services), Helsinki University Central Hospital, Helsinki, Finland; 3Christchurch Hospital, University of Otago, Christchurch, New Zealand; 4Turku University and Satakunta Central Hospital, Pori, Finland Introduction: Adrenomedullin has many cardiovascular beneficial effects, such as vasodilation, angiogenesis and tolerance of cells to oxidative stress and hypoxic injury. In addition, adrenomedullin has shown to augment the release and production of tissue factor pathway inhibitor in human aortic endothelial cells suggesting a role in anticoagulation on endothelial surface. Objective: We aimed to study the nocturnal variability of coagulation and plasma adrenomedullin in gestational diabetes, normal pregnancy and in non-pregnant women. Methods:We studied 41 women with gestational diabetes, 22 healthy pregnant and 14 non-pregnant controls. We assayed serial nocturnal changes of plasma adrenomedullin and coagulation variables, including factor VIII activity (FVIII:C), von Willebrand factor ristocetin cofactor activity (vWF:RCo), antigen (vWF:Ag) and collagen binding activity (vWF:CB) at 12 pm, 4 am and 7 am. Results: Adrenomedullin was higher in pregnant compared to non-pregnant women with no difference between the pregnant groups. In gestational diabetes, FVIII:C was lower than in normal pregnancy. AM correlated strongly with vWF:Ag (ρ=0.83, P=0.000), vWF:RCo (ρ=0.80, P=0.001) and vWF:CB (ρ=0.68, P=0.010) in non-pregnant controls. In contrast, these correlations disappeared in both pregnant groups. No correlation of adrenomedullin to FVIII:C existed within the study groups. Conclusions: These findings strengthen the view of adrenomedullin as a relevant factor in human coagulation. Distinct correlations of adrenomedullin with vWF and FVIII, usually working in tandem, could suggest an unravelled mechanism which is involved in the coupling of vWF with FVIII. This finding needs more insight from future studies. P-026 Frequency of thrombophilia in females with stroke in association with oral contraceptive use P. Dulicek1, M. Kost’a 1, P. Sadilek1, M. Beranek2 1IVth Department of Internal – Hematology; 2Department of Clinical Biochemistry, University Hospital and Medical Faculty Hradec Kralove, Czech

Von Willebrand disease: Pathogenesis and management

Recent studies have suggested a unifying pathophysiological concept to explain the underlying defects of von Willebrand factor (VWF) causing von Willebrand disease (VWD) and have highlighted the relevance of simple VWF related activities in producing a useful diagnosis. A standardized bleeding history condensed into a final bleeding score and few widely available laboratory tests, such as VWF ristocetin cofactor activity, VWF antigen and factor VIII (FVIII), in the index case and in his/her relatives are of critical importance. Ristocetininduced platelet aggregation (RIPA) should also be tested. Trial with desmopressin should be carried out in patients, except those with virtual absence of VWF or with increased RIPA. Desmopressin should be used in all responsive patients as first choice. Substitutive treatment with VWF/FVIII containing products should be used in unresponsive patients, in those with heightened response to desmopressin or in those undergoing interventions requiring good hemostasis for more than 3–5 days. Special consideration should be deserved to the treatment of menorrhagia and parturition.