Ristocetin Cofactor Research Articles

Trend in Von Willebrand Factor Profile in Women With Von Willebrand Disease During Pregnancy and Postpartum

Introduction: Pregnancy is considered a hypercoagulable state, with levels of multiple coagulation factors including Factor VIII (FVIII) and von Willebrand Factor (VWF), progressively increasing throughout, reaching their peak levels during the third trimester. However, in women with von Willebrand disease (VWD), these changes are variable and could be blunted or absent due to the wide heterogeneity of phenotypes and pathophysiological mechanisms linked to this disorder, posing a significant clinical challenge. This study aims to describe the trend in the VWF profile in women with VWD during each trimester of pregnancy and in the postpartum period. Methods: This is a retrospective single-center study that included adult pregnant women diagnosed with VWD, who were followed at Henry Ford Health between 03/2012 and 11/2022. Patients with other bleeding disorders were excluded from the study. Data on baseline characteristics, including age, race, age at childbirth, and parity were collected. Additional data collected included VWF antigen (VWF:Ag) level, FVIII activity level, and VWF ristocetin cofactor (VWF:Rco) activity during the first, second, and third trimesters, as well as in the postpartum period. Results: A total of 33 cases were included in our study. At baseline, the mean VWF:Ag level in our patients was 50.5% (reference range 50%-150%). Compared to baseline, the VWF:Ag level increased by 48.71% in the first trimester, 80.59% in the second trimester, and 168.71% in the third trimester. Postpartum, the VWF:Ag level decreased by 51.29% compared to the third trimester; however, it remained 30.89% higher than baseline levels. While the FVIII level (reference range 50%-150%) was an average of 64.8% at baseline, it increased by 21.91% in the first trimester, 74.23% in the second trimester, and 143.67% in the third trimester, compared to baseline. Postpartum, the FVIII level decreased by 48.45% from the third trimester but remained 25.62% higher than baseline. Additionally, the VWF:Rco level was an average of 44.3% (reference range 51%-215%) at baseline. Compared to baseline, the VWF:Rco level increased by 61.85% in the first trimester, 90.52% in the second trimester, and 142.66% in the third trimester. It decreased by 49.12% postpartum compared to the third trimester but remained 23.48% higher than baseline. Conclusion: In women with VWD, VWF:Ag level, FVIII level, and VWF:Rco levels steadily increase during pregnancy, reaching their maximum levels during the third trimester. All three levels decreased in the postpartum period; however, they remain higher compared to baseline at a median follow up of 105 days after delivery.

In vitro field study and worldwide survey assessing how clinical haemostasis laboratories analyse recombinant and plasma-derived von Willebrand factor products.

Several well-established clinical laboratory methods are available to measure von Willebrand factor (VWF) in plasma samples, but few data are available on their use for analysing recombinant VWF (rVWF). To evaluate how clinical diagnostic laboratories analyse rVWF and plasma-derived VWF (pdVWF) spiked in vitro into VWF-deficient plasma using quantitative protein and functional assays of VWF. Human VWF-deficient plasma samples were spiked with rVWF (vonicog alfa; Takeda) or pdVWF/factor VIII (pdVWF/FVIII; antihemophilic factor/VWF complex [human], CSL Behring), each at final concentrations of 1.0, 0.6, 0.2, 0.1 IU/mL VWF:ristocetin cofactor activity (VWF:RCo) according to labelled VWF activity. The ISTH SSC secondary coagulation standard was used as a control. Participating laboratories received three sets of these blinded aliquots. Mean results per assay were compared with the expected potency based on the labelled VWF:RCo activity. Among 39 laboratories, the most commonly established assay was VWF:RCo; 22 laboratories reported data from 2214 tests. Despite a trend to lower values, VWF:RCo activities for rVWF were in agreement with target concentrations (71%-109%), whereas VWF:platelet glycoprotein Ib (VWF:GpIb) and VWF collagen-binding activity (VWF:CB) assays gave high recoveries (up to 132% and 127%, respectively). In contrast, pdVWF/FVIII was substantially underestimated by VWF:GpIb and VWF:CB assays (56%-86% recoveries), whereas the VWF:RCo assay gave recoveries of 47%-112% for pdVWF/FVIII. The results of VWF assays used in clinical laboratories differ between rVWF and pdVWF, particularly for VWF:GpIb and VWF:CB assays. These differences may arise from the higher multimeric structure of rVWF compared to pdVWF.

Impact of Anticoagulant Function By the Addition of Heparinoid in Vitro in the Plasmas from Patients with Hemophilia and Von Willebrand Disease

Background: Heparinoid (Hirudoid ®) is a local anticoagulant preparation as skin application that relieves pain and inflammation with addition to promoting healing in superficial thrombophlebitis and bruising. Heparinoid is mainly composed of chondroitin sulfate and has high moisturizing ability due to the large number of hydrophilic group. Thus, it is used as topical treatments for sebum deficiency in Japan. Since heparinoid has a structure similar to heparin, the package insert states the contraindication for hemorrhagic diseases such as hemophilia and von Willebrand disease (VWD) in Japan. Hemophilia A and B (HA/HB) are inherited bleeding disorders caused by a defect in factor (F) VIII and FIX coagulant protein, respectively. Based on its activity levels, the clinical severity is classified as severe (<1 IU/dL), moderate (1-5 IU/dL), and mild (>5-40 IU/dL). VWD is the common congenital bleeding disorders and is caused by a deficiency of von Willebrand factor (VWF). VWD is classified into partial deficiency (type 1), qualitative deficiency (type 2), and complete quantitative deficiency (type 3) of VWF, based on the VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo) level. In the clinical practice, there are cases of hemorrhagic patients using heparinoid at the discretion of their families. However, the influence of heparinoid on coagulant function in human subjects remains to be investigated, and it is unclear if the patients with hemophilia and VWD can use heparinoid safely. Aim: To examine the impact of anticoagulant function of heparinoid in vitro in the plasmas from patients with hemophilia and VWD. Methods: The maximum blood concentration from absorbed heparinoid was estimated to be 2.5 µg/mL, based on the previous report, when heparinoid was applied to the skin as stated in package insert. Heparinoid (0-6.5 µg/mL) or unfractionated heparin (0-0.6 IU/mL) were added to normal plasma (NP), followed by measuring activated partial thromboplastin time (aPTT) by one-stage clotting assay. A modified clot waveform analysis (CWA) using an aPTT reagent were performed on the CS-2400™ (Sysmex), followed by measuring clotting time (CT) and adjusted maximum coagulation velocity (Ad|min1|). Heparinoid was also added to the patients' plasmas with HA, HB, or VWD, followed by CWA assessment. The CT and Ad|min1| of 20 healthy individuals were 31.1±0.1 sec and 6.90±0.05 (median±SEM). Results: aPTT and CT values in NP by the spike of heparinoid were prolonged and Ad|min1| was decreased dose-dependently. The addition of 2.5 µg/mL heparinoid showed that aPTT (34.3 sec; 1.2-fold of control (no heparinoid)), CT (36.3 sec; 1.2-fold of control), and Ad|min1| (9.5; 0.96-fold of control) were comparable to the effect of ~0.2 IU/mL heparin, suggesting that heparinoid exhibited slight anticoagulant effect for NP. To examine the effect of heparinoid on HA/HB plasmas, recombinant (r) FVIII or rFIX (0-50 IU/dL) were added to FVIII- or FIX-deficient plasmas (3 lots each) together with heparinoid (0-6.3 µg/mL), respectively. The addition of heparinoid had prolonged the CT and decreased the Ad|min1| at each concentration of FVIII and FIX dose-dependently. The addition of 2.5 µg/mL heparinoid showed modest anticoagulant effect for HA and HB at each concentration of FVIII and FIX (CT; 1.3- and 1.5-fold of control, Ad|min1|; 0.82- and 0.87-fold of control). Next, the effect of heparinoid on the plasma from one VWD patient each with type 1 (VWF:Ag/VWF:RCo 28 IU/dL/26 IU/dL), type 2 (20 IU/dL/9 IU/dL), and type 3 (<2 IU/dL/<3 IU/dL) was assessed. The addition of 2.5 µg/mL heparinoid showed slight anticoagulant effect for each type of VWD (CT; ~1.3-fold of control, Ad|min1|; ~0.91-fold of control). Conclusion: The presence of heparinoid demonstrated mild anticoagulant effect in vitro in the plasmas of patients with hemophilia and VWD. It might be possible to apply the heparinoid-contained product as skin treatment for these patients.

Characterization of copy-number variants in a large cohort of patients with von Willebrand disease reveals a relationship between disrupted regions and disease type

BackgroundGenetic analysis for von Willebrand disease (VWD) commonly utilizes DNA sequencing to identify variants in the von Willebrand factor (VWF) gene; however, this technique cannot always detect copy-number variants (CNVs). Additional mapping of CNVs in patients with VWD is needed. ObjectivesThis study aimed to characterize CNVs in a large sample of VWF mutation-negative VWD patients. MethodsTo determine the role of CNVs in VWD, a VWF high-resolution comparative genomic hybridization array was custom-designed to avoid multiple sequence variations, repeated sequences, and the VWF pseudogene. This was performed on 204 mutation-negative subjects for whom clinical variables were also available. ResultsAmong the 204 patients, 7 unique CNVs were found, with a total of 24 CNVs (12%). Of the 7 unique CNVs, 1 was novel, 1 was found in a VWF database, and 5 were previously reported. All patients with type 1C VWD and a CNV had the same exon 33 and 34 in-frame deletion. Certain clinical variables were also significantly different between those with and without CNVs. ConclusionThe in-frame deletion in patients with type 1C VWD exactly matches the D4N module of the D4 domain, a region where mutations and deletions are known to affect clearance. We observed significantly higher VWF–to–ristocetin cofactor levels in patients with type 1C VWD and a CNV than in patients without a CNV, suggesting a relationship between CNVs and the increased clearance observed in patients with type 1C VWD. Glycoprotein IbM activity was significantly lower in patients with type 1 VWD and a CNV than in patients without a CNV, suggesting that platelet binding is more affected by CNVs than single base pair mutations. This work elucidates some of the underlying genetic mechanisms of CNVs in these patients.

Comparison of coagulation parameters associated with fibrinogen concentrate and cryoprecipitate for treatment of bleeding in patients undergoing cytoreductive surgery for pseudomyxoma peritonei: Subanalysis from a randomized, controlled phase 2 study.

The FORMA-05 study compared the efficacy and safety of human fibrinogen concentrate (HFC) versus cryoprecipitate for hemostasis in bleeding patients undergoing cytoreductive surgery for pseudomyxoma peritonei (PMP). This subanalysis explores coagulation parameters in the FORMA-05 patients, with a focus on the seven patients who developed thromboembolic events (TEEs). FORMA-05 was a prospective, randomized, controlled phase 2 study in which patients with predicted blood loss ≥2 L received HFC (4 g) or cryoprecipitate (two pools of five units), repeated as needed. Plasma fibrinogen, platelet count, factor (F) XIII, FVIII, von Willebrand Factor (VWF) antigen and ristocetin cofactor activity levels, EXTEM A20, FIBTEM A20, and endogenous thrombin potential (ETP) were measured perioperatively. Fibrinogen, platelet count, EXTEM and FIBTEM A20, FXIII, FVIII, VWF levels, and ETP were maintained throughout surgery in both the HFC group (N = 21) and the cryoprecipitate group (N = 23). Seven TEEs were observed in the cryoprecipitate group. The two patients developing deep vein thromboses (DVT) appeared to have a procoagulant status preoperatively, with distinctively higher fibrinogen level, FIBTEM A20, and platelet levels, all of which persisted perioperatively. The five patients developing pulmonary embolism (PE) had slightly higher VWF levels preoperatively, with a disproportionate increase intraoperatively (postcryoprecipitate administration) and postoperatively. Patients treated with HFC versus cryoprecipitate showed broad overlaps in coagulation parameters. Patients with PE experienced a disproportionate VWF rise following cryoprecipitate administration, whereas patients developing DVT displayed a procoagulant status before and following surgery. Preoperative testing may allow these patients to be identified.

Dynamic and functional linkage between von Willebrand factor and ADAMTS-13 with aging: an Atherosclerosis Risk in Community study

Backgroundvon Willebrand factor (VWF) is a multimeric glycoprotein critically involved in hemostasis, thrombosis, and inflammation. VWF function is regulated by its antigen levels, multimeric structures, and the state of enzymatic cleavage. Population studies in the past have focused almost exclusively on VWF antigen levels in cross-sectional study designs. ObjectiveTo identify subjects in the Atherosclerosis Risk in Community study who had persistently low and high VWF antigen over 10 years and to quantify longitudinal changes in the biological activities and cleavage of VWF in these subjects. MethodsWe measured VWF antigen, propeptide, adhesive activities, and cleavage by ADAMTS-13 quantified using a mass spectrometry method that detected the cleaved VWF peptide EQAPNLVY, as well as coagulation factor VIII activity. ResultsWe determined the mean subject-specific increase in VWF to be 22.0 International Units (IU)/dL over 10 years, with 95% between -0.3 and 59.7 IU/dL. This aging-related increase was also detected in VWF propeptide levels, ristocetin cofactor activity, and VWF binding to collagen. We identified 4.1% and 25.0% of subjects as having persistently low (<50 IU/dL) and high (>200 IU/dL) VWF antigen, respectively. Subjects with persistently low VWF had enhanced ristocetin cofactor activity, whereas those with persistently high VWF had elevated levels of ADAMTS-13, resulting in a comparable rate of VWF cleavage between the 2 groups. ConclusionsThese results provide new information about the effects of aging on VWF antigens and adhesive activity and identify a functional coordination between VWF and the rate of its cleavage by ADAMTS-13.

Effect of 10-Day Treatment with 50 mg Prednisolone Once-Daily on Haemostasis in Healthy Men-A Randomised Placebo-Controlled Trial.

Synthetic corticosteroids are widely used due to their anti-inflammatory and immunosuppressant effects. Their use has been associated with venous thromboembolism, but it is unknown whether thromboembolism has a causal relationship with corticosteroid treatment. In a randomised, double-blind, placebo-controlled trial in normal to overweight healthy men, the effect of the corticosteroid prednisolone on haemostasis using either 50 mg prednisolone or matching placebo once daily for ten days was investigated. The primary outcome was a change from baseline in the viscoelastic measurement maximal amplitude of clot in kaolin-activated thromboelastography (TEG). Changes from baseline in other TEG measurements, D-dimer, von Willebrand factor (VWF) antigen, and ristocetin cofactor activity (RCo), antithrombin, protein C, prothrombin, fibrinogen, INR, APTT, and platelet count were secondary outcomes. Thirty-four men participated in this study. Compared to placebo, prednisolone treatment did not affect maximal amplitude of clot (difference -0.77 (95% confidence interval (CI) -2.48, 0.94) mm, p = 0.37, missing: n = 2), but it altered VWF antigen (28%, p = 0.0004), VWF:RCo (19%, p = 0.0006), prothrombin (5%, p = 0.05), protein C (31%, p < 0.0001), antithrombin (5%, p = 0.013), and fibrinogen (-15%, p = 0.004). Thus, prednisolone treatment did not alter TEG-assessed maximal amplitude of clot, despite that it affected prothrombotic markers (increased prothrombin, VWF antigen, VWF:RCo, prothrombin, and decreased fibrinogen) and increased antithrombotic markers (protein C and antithrombin).

Challenges in the classification of von Willebrand disease with a limited test panel: An experience from an upcoming hemostasis laboratory in southern India

Objectives: The diagnosis and classification of von Willebrand disease (VWD) is an intricate process requiring multiple hemostatic tests. Cutoff values of the tests vary with the subtype. Sometimes, all the tests are not available. This study was done to analyze the clinical and coagulation profile of VWD diagnosed and broadly classified based on a simplified algorithm. Material and Methods: This was a cross-sectional study done over 6 years. After screening tests, a simplified algorithm taking cutoff for various tests based on updated guidelines using the primary panel of von Willebrand factor antigen assay (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and Factor VIII:C was used. Multimer assay was done in a few cases. Data were compiled using summary statistics. Results: Forty patients fitted the diagnosis of VWD: Type 3 in 13 (32.5%), Type 2N (likely) in 8 (20%), Type 2 (not further categorized) in 9 (22.5%), and VWD, not further categorized in 10 (25%). The mean age was in the second or third decade with female predominance. Diagnosis of Type 3 was relatively straightforward due to markedly deranged parameters. Type 2N was provisionally diagnosed based on bleeding pattern and markedly reduced Factor VIII:C; further, subtyping of Type 2 and categorization of some cases was not possible due to non-availability of some tests. Conclusion: VWF:Ag assay, VWF:RCo, and Factor VIII:C form the cornerstone for diagnosing major VWD types. The under-representation of milder phenotypes and a greater proportion of severe VWD subtypes observed is likely due to hospital referral bias and may not represent population prevalence.

Recombinant von Willebrand factor and tranexamic acid for heavy menstrual bleeding in patients with mild and moderate von Willebrand disease in the USA (VWDMin): a phase 3, open-label, randomised, crossover trial

Heavy menstrual bleeding occurs in 80% of women with von Willebrand disease and is associated with iron deficiency and poor response to current therapies. International guidelines indicate low certainty regarding effectiveness of hormonal therapy and tranexamic acid. Although von Willebrand factor (VWF) concentrate is approved for bleeds, no prospective trials guide its use in heavy menstrual bleeding. We aimed to compare recombinant VWF with tranexamic acid for reducing heavy menstrual bleeding in patients with von Willebrand disease. VWDMin, a phase 3, open-label, randomised crossover trial, was done in 13 haemophilia treatment centres in the USA. Female patients aged 13-45 years with mild or moderate von Willebrand disease, defined as VWF ristocetin cofactor less than 0·50 IU/mL, and heavy menstrual bleeding, defined as a pictorial blood assessment chart (PBAC) score more than 100 in one of the past two cycles were eligible for enrolment. Participants were randomly assigned (1:1) to two consecutive cycles each of intravenous recombinant VWF, 40 IU/kg over 5-10 min on day 1, and oral tranexamic acid 1300 mg three times daily on days 1-5, the order determined by randomisation. The primary outcome was a 40-point reduction in PBAC score by day 5 after two cycles of treatment. Efficacy and safety were analysed in all patients with any post-baseline PBAC scores. The trial was stopped early due to slow recruitment on Feb 15, 2022, by a data safety monitoring board request, and was registered at ClinicalTrials.gov, NCT02606045. Between Feb 12, 2019, and Nov 16, 2021, 39 patients were enrolled, 36 of whom completed the trial (17 received recombinant VWF then tranexamic acid and 19 received tranexamic acid then recombinant VWF). At the time of this unplanned interim analysis (data cutoff Jan 27, 2022), median follow-up was 23·97 weeks (IQR 21·81-28·14). The primary endpoint was not met, neither treatment corrected PBAC score to the normal range. Median PBAC score was significantly lower after two cycles with tranexamic acid than with recombinant VWF (146 [95% CI 117-199] vs 213 [152-298]; adjusted mean treatment difference 46 [95% CI 2-90]; p=0·039). There were no serious adverse events or treatment-related deaths and no grade 3-4 adverse events. The most common grade 1-2 adverse events were mucosal bleeding (four [6%] patients during tranexamic acid treatment vs zero during recombinant VWF treatment) and other bleeding (four [6%] vs two [3%]). These interim data suggest that recombinant VWF is not superior to tranexamic acid in reducing heavy menstrual bleeding in patients with mild or moderate von Willebrand disease. These findings support discussion of treatment options for heavy menstrual bleeding with patients based on their preferences and lived experience. National Heart Lung Blood Institute (National Institutes of Health).

Pharmacokinetic-Pharmacodynamic Comparison of Recombinant and Plasma-Derived von Willebrand Factor in Patients with von Willebrand Disease Type 3.

Recombinant von Willebrand factor (rVWF, vonicog alfa, Vonvendi/Veyvondi, Takeda Pharmaceuticals USA, Lexington, MA) and several plasma-derived VWF/factor VIII (pdVWF/FVIII) concentrates are available for treating bleeding episodes in patients with von Willebrand disease (VWD). To develop population pharmacokinetic (PK)/pharmacodynamic (PD) models that describe VWF:ristocetin cofactor (VWF:RCo) activity and its relationship with FVIII activity (FVIII:C) over time following intravenous administration of either rVWF or a pdVWF/FVIII concentrate (VWF:RCo/FVIII:C 2.4:1) in patients with VWD; to use the final PK/PD models for an in silico comparison of rVWF and pdVWF/FVIII. The population PK model for rVWF was based on data from four clinical studies in which rVWF was administered to adult patients with VWD type 1, 2 or 3 (phase 1: NCT00816660; phase 3: NCT01410227 and NCT02283268) or severe hemophilia A (phase 1: EudraCT 2011-004314-42). The PK and PK/PD models for pdVWF/FVIII were based on data from the phase 1 study (NCT00816660) in patients with type 3 VWD who received either rVWF plus recombinant FVIII (rFVIII, octocog alfa, ADVATE®, Takeda Pharmaceuticals USA, Lexington, MA, USA) or pdVWF/FVIII. There was a marked difference in clearance following rVWF administration compared with pdVWF/FVIII in type 3 VWD, leading to a ~1.75 longer mean residence time (ie, persistence of VWF:RCo activity in the body) and half-life for rVWF versus pdVWF/FVIII. Simulations showed that following repeated administration of rVWF (50 IU/kg), a FVIII:C activity of >40 IU/dL can be maintained for the full 72 h dosing interval. The slower elimination of VWF:RCo following rVWF administration results in a prolonged effect on FVIII turnover compared with pdVWF/FVIII administration.

Effects of Hypobaric Hypoxia on Coagulation in Healthy Subjects Exposed to 3,500 m Altitude.

Kammerer, Tobias, Anna Walzl, Thomas Müller, Philipp Groene, Giulia Roveri, Rachel Turner, Johanna Roche, Hannes Gatterer, Christoph Siebenmann, and Simon T. Schäfer. Effects of hypobaric hypoxia on coagulation in healthy subjects exposed to 3,500 m altitude. High Alt Med Biol. 24:94-103, 2023. Background: Hypoxia is discussed as a trigger for prothrombotic changes both in intensive care and high altitude medicine. This research study aimed to evaluate the effect of isolated hypobaric hypoxia (HH) on coagulation in females in a highly standardized setting. Methods: Twelve healthy female subjects were studied under HH (equivalent to 3,500 m) and normoxia (NX) during two 4-day sojourns, in a strictly controlled crossover design. Nutrition, fluid intake, hormonal status (i.e., menstrual cycle variation), and physical stress were standardized. Functional coagulation and blood lysis were measured by viscoelastometry and compared between HH and NX. In addition, plasma-based coagulation tests (PBCTs), namely prothrombin time, activated partial thromboplastin time, fibrinogen, factor VIII coagulation activity (FVIII:C), von Willebrand factor antigen (vWF:Ag), and von Willebrand factor ristocetin cofactor activity (vWF:RCo) were measured. Results: Neither for Viscoelastic Haemostatic Assays nor for PBCTs significant changes were found for HH compared with NX (all p > 0.05). Specifically, the lysis ability, as well as clotting time, clot formation, clot amplitude, and maximum clot firmness unchanged were similar between HH and NX. This also applied to all other variables. Conclusion: We demonstrate that moderate HH per se has no influence on blood coagulation in healthy females.

Constructing and evaluating the effect of micron-scale structures on von Willebrand factor damage

The incidence of clinical complication gastrointestinal bleeding has been proved as consequence of von Willebrand factor (VWF) damage after mechanical circulatory support in clinic. Many studies have been conducted to evaluate VWF damage, of which the most studied influencing factors are mechanical factors such as shear stress. However, in addition to mechanical factors, VWF damage may also be affected by interface factors. To address this issue, a roller pump circulation platform was established to investigate the effect of material surface micron-scale structures distribution on VWF damage in flow state. A composite micro-structure combining microngrating and micronpost was designed and constructed on the surface of Si wafer by lithography and reactive ion etching, and detailed characterization of material surfaces was also performed. Then the changes of VWF antigen, VWF ristocetin cofactor activity, and the degradation of high molecular weight VWF on these surfaces were investigated and compared. The results showed that, with the encryption of surface micro-structures arrangement, the material surface tends to be more hydrophobic, which is beneficial to reduce VWF damage. Therefore, in the design of material surface inside the mechanical circulatory support devices, it can be considered to add some surface micro-structures with a certain distribution density to change the hydrophilicity and hydrophobicity, so as to minimize the VWF damage. These results can provide important references for the evaluation of VWF damage caused by interface factors, and aid in designing material surface inside the mechanical circulatory support devices.

Novel Antiplatelet Activity of Ginsenoside Re Through the Inhibition of High Shear Stress-Induced Platelet Aggregation.

Bleeding is one of the most serious side effects of antiplatelet drugs. Efforts have been made to find new antiplatelet agents without bleeding complications. Shear-induced platelet aggregation (SIPA) occurs only under pathological conditions and is a promising target for overcoming bleeding problems. This work demonstrates that the ginsenoside Re selectively inhibits platelet aggregation induced by high shear stress. Human platelets were exposed to high shear stress using microfluidic chip technology, and aggregation, activation, and phosphatidylserine (PS) exposure were measured. The Von Willebrand Ristocetin Cofactor (vWF:RCo) assay and western blot were used to evaluate the effect of the vWF-GPⅠb/PI3K/Akt signal pathway. The coagulation and bleeding risk were evaluated by measuring the coagulation parameters PT, APTT, TT, and thromboelastography. The 3-dimensional morphology of platelet aggregates was observed by a microscopic 3-dimensional imaging. Re was a potent inhibitor of SIPA, with an IC 50 of 0.071 mg/mL. It effectively blocked shear stress-induced platelet activation without any significant toxicity. It was highly selective against SIPA, effectively inhibiting vWF-GPIb and the downstream PI3K/Akt signaling pathway. Most importantly, Re did not affect normal blood coagulation and did not increase the risk of bleeding. In conclusion, Re inhibits platelet activation through the inhibition of the vWF-GPIb/PI3K/Akt pathway. Thus, it might be considered as a new antiplatelet drug in the prevention of thrombosis without increasing the risk of bleeding.

A comparative study in patients with type 2 von Willebrand disease using 4 different platelet-dependent von Willebrand factor assays

BackgroundSeveral assays are now available to evaluate platelet-dependent von Willebrand factor (VWF) activity. ObjectiveTo report the results obtained using 4 different assays in patients with von Willebrand disease (VWD) carrying variants mainly in the A1 domain, which is critical for VWF binding to glycoprotein Ib (GPIb) and ristocetin. MethodsWe evaluated 4 different assays, 2 gain-of-function mutant GPIb binding (VWF:GPIbM) and 2 ristocetin cofactor (VWF:RCo) assays, in 76 patients with type 2 VWD. Patients and healthy controls were tested using VWF:GPIbM enzyme-linked immunosorbent assay (ELISA), VWF:GPIbM automated, VWF:RCo aggregometric, and VWF:RCo automated assays. ResultsThere was a good correlation (Pearson’s r>0.82) and agreement (Bland-Altman plots assessment) between the 4 assays, although several outliers existed among the type 2B without high-molecular-weight multimers (HMWM). The VWF activity/VWF:antigen ratios, calculated for each assay, were used to establish the percentage of a correct diagnosis of type 2 (ratio<0.60) in these patients: VWF:RCo aggregometric, 2A(100%), 2M(78%), 2M/2A(100%), 2B(68%); VWF:RCo automated, 2A(88%), 2M(89%), 2M/2A(100%), 2B(63%); VWF:GPIbM ELISA, 2A(96%), 2M(67%), 2M/2A(67%), 2B(0%); VWF:GPIbM automated, 2A(73%), 2M(44%), 2M/2A(75%), 2B(84%). In type 2B patients with HMWM, all assays gave a ratio ≥0.60. ConclusionThe VWF:GPIbM-automated assay is the most effective to diagnose as type 2 the 2B variants, whereas the VWF:RCo assays are the most effective in detecting 2M and 2M/2A variants. The VWF:GPIbM ELISA greatly overestimates the activity of the type 2B patients lacking HMWM. In this study, the use of a VWF activity/VWF:antigen ratio cut-off of 0.70 halved the number of misdiagnosed patients.

Acquired von willebrand syndrome in patients with philadelphia-negative myeloproliferative neoplasm.

Acquired von Willebrand syndrome (AVWS) has not been investigated in Korean patients with Philadelphia chromosome-negative myeloproliferative neoplasm. This study analyzed the prevalence at diagnosis and clinical features of AVWS in patients with essential thrombocythemia (ET), polycythemia vera (PV), prefibrotic/early primary myelofibrosis (pre-PMF), or overt PMF (PMF) diagnosed between January 2019 and December 2021 at Chungam National University Hospital, Daejeon, Korea. AVWS was defined as below the lower reference limit (56%) of ristocetin cofactor activity (VWF:RCo). Sixty-four consecutive patients (36 with ET, 17 with PV, 6 with pre-PMF, and 5 with PMF; 30 men and 34 women) with a median age of 67 years (range, 18‒87 yr) were followed for a median of 25.1 months (range, 2.6‒46.4 mo). AVWS was detected in 20 (31.3%) patients at diagnosis and was most frequent in ET patients (41.4%), followed by patients with pre-PMF (33.3%) and PV (17.6%) patients. VWF:RCo was negatively correlated with the platelet count (r=0.937; P=0.002). Only one episode of minor bleeding occurred in a patient with ET and AVWS. Younger age (<50 yr) [odds ratio (OR), 7.08; 95% confidence interval (CI), 1.27‒39.48; P=0.026] and thrombocytosis (>600×109/L) (OR, 13.70; 95% CI, 1.35‒138.17; P=0.026) were independent risk factors for developing AVWS. AVWS based on VWF:RCo was common in patients with ET and pre-PMF, but less common in patients with PV in the Korean population. Clinically significant bleeding is rare in these patients.

Effect of Supplemental Oxygen on von Willebrand Factor Activity and Ristocetin Cofactor Activity in Patients at Risk for Cardiovascular Complications Undergoing Moderate-to High-Risk Major Noncardiac Surgery-A Secondary Analysis of a Randomized Trial.

Increased von Willebrand Factor (vWF) activity mediates platelet adhesion and might be a contributor to the development of thrombotic complications after surgery. Although in vitro studies have shown that hyperoxia induces endovascular damage, the effect of perioperative supplemental oxygen as a possible trigger for increased vWF activity has not been investigated yet. We tested our primary hypothesis that the perioperative administration of 80% oxygen concentration increases postoperative vWF activity as compared to 30% oxygen concentration in patients at risk of cardiovascular complications undergoing major noncardiac surgery. A total of 260 patients were randomly assigned to receive 80% versus 30% oxygen throughout surgery and for two hours postoperatively. We assessed vWF activity and Ristocetin cofactor activity in all patients shortly before the induction of anesthesia, within two hours after surgery and on the first and third postoperative day. Patient characteristics were similar in both groups. We found no significant difference in vWF activity in the overall perioperative time course between both randomization groups. We observed significantly increased vWF activity in the overall study population throughout the postoperative time course. Perioperative supplemental oxygen showed no significant effect on postoperative vWF and Ristocetin cofactor activity in cardiac risk patients undergoing major noncardiac surgery. In conclusion, we found no significant influence of supplemental oxygen in patients undergoing major non-cardiac surgery on postoperative vWF activity and Ristocetin cofactor activity.

Impact of ABO Blood Group on Thromboembolic and Bleeding Complications in Patients with Left Ventricular Assist Devices.

The ABO blood group system is linked to hemostasis via its relationship with von Willebrand factor (VWF) and factor VIII (FVIII). In the current study, we investigated the association of the ABO system with clinical outcomes as well as VWF and platelet function in patients with left ventricular assist devices (LVADs). Bleeding and thromboembolic complications were assessed in 111 patients during 1 year after LVAD implantation. In 67 LVAD patients, VWF antigen, VWF activity, VWF ristocetin cofactor, VWF collagen-binding, and FVIII activity were assessed. Platelet surface P-selectin and activated glycoprotein IIb/IIIa were determined by flow cytometry, and soluble P-selectin was measured with an enzyme-linked immunoassay. Platelet aggregation was assessed by light transmission and impedance aggregometry. Thirty-six patients (32.4%) experienced a bleeding and 22 patients (19.8%) a thromboembolic event. In univariate analyses, patients with blood group O had numerically more bleeding complications and less thromboembolic events as compared to patients with blood group non-O (both p ≥ 0.05). After multivariable adjustment, blood group O was significantly associated with a higher risk of bleeding (hazard ratio 2.42 [95% confidence interval 1.03-5.70], p = 0.044) but not linked to thromboembolic complications. Patients with blood group O had significantly lower levels of VWF and FVIII (all p < 0.05), whereas P-selectin expression in response to thrombin-receptor activating peptide and soluble P-selectin were higher as compared to patients with blood group non-O (both p < 0.05). LVAD patients with blood group O are at an increased bleeding risk, potentially due to lower VWF and FVIII levels.

Evaluation of Global Hemostasis Assays in Von Willebrand Disease Type 3 in Comparison with Mild to Moderate Hemophilia a

Introduction: Von Willebrand disease (VWD) is the most common inherited bleeding disorder. Type 3 VWD is the most severe form of this disease with a prevalence of about 0.5 to 1 per million in Western countries but is much more common in communities with high levels of consanguinity like South India. Type 3 VWD is characterised by undetectable Von Willebrand Factor and reduced Factor VIII levels. (1,2) Global hemostasis assays clot waveform analysis (CWA) [3,4] and thrombin generation test (TGT)[5,6] reflect the entire process of thrombin generation, and thus reflect the actual bleeding severity . Rotational Thromboelastogram (ROTEM) demonstrates the dynamics of clot formation over time. Global Hemostasis Assays is extensively studied in Hemophilia A(HA) patients, however there are very few studies which analysed the same in type 3 VWD in account of rarity of disease Our aim was to study ROTEM, TGT and APTT-CWA parameters in cases of vWD 3. We also aimed to compare global hemostasis parameters in vWD3 to those in mild to moderate hemophilia A with similar levels of Factor VIII. Materials and Methods: Retrospective study for a period of 2 years which included 27 cases of vWD3 (with undetectable vWF antigen) and 34 cases of Hemophilia A- 25 moderate and 9 mild deficiency (F VIII-5-10%). Complete coagulation workup was done which included International Society of Thrombosis and Hemostasis based Bleeding Assessment Tool(BAT) Score questionnaire, Bleeding time, Platelet Function Analyser 200, Prothrombin Time(PT), APTT, Ristocetin Cofactor, Von Willebrand Antigen, Factor assays, Ristocetin induced platelet aggregation and Global Hemostasis Assays-ROTEM, TGT and CWA. BAT score was done to assess severity of bleeding. TGT was performed by Calibrated Automated Thrombogram, CWA by photo-optical coagulometer and factor levels by one stage clot-based assays on ACL Top 750. Rotem was performed in whole blood by modified extem. TGT was could be done only on 16/34 Hemophilia A cases and 14/27 vWD3 cases. Statistical Analysis: For continuous data, the descriptive statistics mean, SD, and for non-normally distributed data median, IQR were done. Based on the normality of data, the parametric t-test or non-parametric Mann Whitney test was used to find the difference between groups. All tests were two-sided at alpha () =0.05 level of significance. All the statistical analysis was performed using Statistical Package for Social sciences (SPSS) version 21.0. Results: Results are summarized in Table 1Global hemostasis tests in vWD3 A.1)ROTEM- CT (Clotting Time) was prolonged in 44% cases, 55 % cases it was within normal range and shortened in rest. CFT (Clot Formation Time) and alpha angle were within normal range in 81.1% cases, prolonged in 0.3% cases and shortened in rest. MCF (Maximum Clot Firmness) was reduced in 2 cases, and normal in remaining cases. ML(Maximum Lysis) was within range for all cases. A.2) APTT CWA- Min1 was decreased in 25.9 % cases, rest 74% had within normal range. Min2 was decreased in 96% cases. Max 2 was decreased in all cases. A.3) TGT- Lag time was prolonged in 13/14 (93%) cases. ETP was increased in 7/14 cases (50%). Peak height was decreased in all cases. TTP was prolonged in 13/14 cases (93%). Start tail was prolonged in 12/14 cases.B) Comparison between vWD3 and Mild to Moderate Hemophilia Refer to table -1 APTT was significantly prolonged (P value 0.008) in HA group than vWD3, though Factor VIII was similar in both groups. CWA were comparable to HA cases. Min 2 was statistically lower in vWD3 compared to HA group between the 2 groups (P value- 0.018) ROTEM were comparable between the 2 groups. In TGT, the median lag time (min)was significantly more prolonged in vWD3 with median of 5.50(4.95-6.37) than Hemophilia A patients as seen with APTT. ETP and Peak Thrombin were not significantly different between the 2 groups. Time to peak and start tail were statistically lower among cases of vWD 3 and haemophilia A, p value being <0.05. CONCLUSION: Global hemostasis test in von Willebrand disease showed a hypocoagulable state in Thrombin Generation Tests and APTT CWA. ROTEM didn't show any significant change despite Factor VIII levels being low Tests which measure Factor VIII assay and tests which reflect Factor VIII activity as in global hemostasis assay do not show significant difference in patients who have low Factor VIII level irrespective of the diagnosis being von Willebrand disease or Hemophilia A. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characterization of CNVs in a Large Cohort of VWD Patients Reveals Relationship between Disrupted Regions, Clinical Variables and Disease Type

BACKGROUND: Copy-number variants (CNVs) have been associated to all VWD types. Although many of the underlying pathological mechanisms have not been investigated, in-frame deletions have been shown to cause defects in VWF biosynthesis and secretion. While there are previous studies describing CNVs in VWD patients, these only discuss a small number of CNVs each, and are in varying populations, making it difficult to compare between populations as the differences could be population specific. METHODS: A VWF high-resolution aCGH array was custom designed using long oligonucleotide probes ~60 bp in length to avoid multiple sequence variations, repeated sequences and the pseudogene. Data were manually inspected and analyzed with CytoSure Interpret Software. A multiplex PCR approach was used to map deletion breakpoints and to confirm the presence and zygosity of the deletions in the index case as well as family members. CNVs were mapped and t-tests performed on clinical laboratory values within each VWD type for those with and without a CNV. aCGH was performed in two datasets on low-VWF and VWD index cases with no known pathogenic VWF sequence variant for a total of 205 patients. RESULTS: Of the 205 patients, CNVs were found in 25 (12%). No CNVs were found in low-VWF patients and the only duplication occurred in a Type 2 (n=1) patient. The remaining CNVs were deletions in VWD Type 1+1S (n=12), 1C (n=5), and 3 (n=7) patients. All Type 1C patients (n=5) had the same exon 33-34 deletion. All but 1 of the Type 1+1S patients (n=11) had the same exon 4-5 deletion, while the Type 3 patients (n=7) had deletions spread across the gene. Further, within the Type 1+1S patients, VWFpp/VWF:Ag ratio was significantly lower in those with a CNV than those without (two-tailed t-test, p=0.02). Lastly, ristocetin cofactor was significantly higher in the Type 1C patients with a CNV versus those without a CNV (two-tailed t-test, p=8.2x10-5). DISCUSSION: Here we have analyzed a large dataset of CNVs defined by aCGH in VWD patients with no known pathogenic VWF sequence variant. The location of the deletion in all Type 1C patients exactly matches the D4N module of the D4 domain of the mature VWF protein, a region where mutations and deletions are known to affect clearance. The D4 domain is the only D domain to also include an N module. Although previous work has shown that deletions of exons 33-34 potentially cause a mild reduction in VWF:Ag levels and only a moderate impact on secretion, we observed significantly higher VWF:RCo levels in those Type 1C patients with a CNV when compared to those without. As expected, Type 1C patients had a significantly higher VWFpp/VWF:Ag ratio than Type 1+1S patients overall (p=2.11x10-15). However, the Type 1+1S patient with a CNV had significantly lower VWFpp/VWF:Ag ratio than those without a CNV. This work elucidates some of the underlying genetic mechanisms of CNVs in these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

To Repeat or Not Repeat, That Is the Question: Von Willebrand Testing in Adolescent Girls with Heavy Menstrual Bleeding

Heavy menstrual bleeding (HMB) is often the initial manifestation of a bleeding disorder in adolescent females. Von Willebrand disease (VWD) is the most common bleeding disorder, yet diagnosing VWD in HMB is challenging given that menstruation, acute illness, and physiologic stress all elevate von Willebrand factor (VWF) laboratory values. Thus, the interpretation of initial VWF results may be challenging, and the need for repeat VWF testing in this population may be unclear. The aims of this study were to understand VWD diagnostic testing patterns in adolescent females evaluated within the Texas Children's Hospital (TCH) network and to develop a prediction model to determine whether repeat testing was indicated based on initial VWF testing results. Female patients aged 9 to 21 years who had any VWF laboratory test over a 5-year period (January 1, 2017 through December 31, 2021) at a TCH facility (greater Houston metroplex) were identified. Data collection included patient demographics, VWF-related testing (antigen and activity [ristocetin cofactor or glycoprotein Ib]), factor VIII activity, hemoglobin concentration and serum ferritin. Both initial and subsequent laboratory evaluations were collected. The site of clinical testing was also identified, including both acute care settings (emergency department, inpatient, and critical care), and outpatient settings (adolescent medicine, gynecology, hematology, otolaryngology, other outpatient TCH primary care clinics). To ensure that subsequent laboratory testing was not mistaken as the initial testing within the cohort, patients who underwent VWF laboratory testing from 2010 to 2016 were excluded. VWF levels obtained after the administration of VWF replacement products were also removed from the data set. Based on the ASH ISTH NHF WFH 2021 guidelines on the diagnosis of VWD, and with the presumption that adolescents with HMB met the criteria for abnormal bleeding, we analyzed the need to repeat VWF testing to identify any patient with a low VWF antigen level consistent with Type 1 VWD, defined as <50%. A Bayesian linear regression model was developed to predict follow-up VWF antigen levels using the initial levels. Prediction intervals were analyzed to identify thresholds for patients who may not require retesting. This study was approved by the Baylor College of Medicine Institutional Review Board. A total of 6,125 adolescent females underwent VWD testing at a TCH facility during the study period; 1,204 (19.7%) had repeat VWF antigen values performed and were included in the analysis (TABLE). Of those, 1,037 subjects (86.1%) had normal VWF values (≥50%) on both initial and repeat testing. Seventy-five subjects (6.2%) had low VWF antigen values on both initial and repeat testing. Ninety-two subjects had discrepant initial and repeat testing. Forty-seven (3.9%) had low initial VWF antigen values that normalized on subsequent testing. Forty-five (3.7%) had normal initial VWF values that subsequently fell below 50%. Based on our prediction model, in subjects with initial VWF antigen values of ≥80% there was ≥97% probability of having normal repeat VWF values. Values of ≥100% carry a 99.4% probability of having normal repeat VWF testing (FIGURE). No association was identified between age and VWF antigen level. Subjects assessed in outpatient adolescent medicine or gynecology clinics were more likely to have low VWF values (on any testing) whereas subjects with normal values were more frequently assessed in the acute care settings (P<0.001). The median serum ferritin and hemoglobin values in the cohort were 13 ng/ml and 12.8 g/dl, respectively, without a significant difference between those with normal versus low VWF antigen values. Repeat testing in adolescent females whose initial VWF antigen testing values are 80% and higher is unlikely to identify more patients with VWF values below 50%. In this cohort, subjects undergoing hemostatic evaluation, presumably for HMB, by adolescent medicine or gynecology providers were more likely to identify abnormal VWD testing compared to the acute care setting. Iron deficiency screening should be considered for all adolescent females with HMB and not just those girls with a confirmed bleeding disorder. Additional studies are needed to determine the effects of iron deficiency and anemia on VWD laboratory testing in adolescent females with HMB and to validate the accuracy of the prediction model. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal