Von Willebrand Factor Antigen Levels Research Articles

Von Willebrand factor antigen, von Willebrand factor propeptide and ADAMTS13 activity in TIA or ischaemic stroke patients changing antiplatelet therapy

Data are limited on the impact of commencing antiplatelet therapy on von Willebrand Factor Antigen (VWF:Ag) or von Willebrand Factor propeptide (VWFpp) levels and ADAMTS13 activity, and their relationship with platelet reactivity following TIA/ischaemic stroke.In this pilot, observational study, VWF:Ag and VWFpp levels and ADAMTS13 activity were quantified in 48 patients ≤4 weeks of TIA/ischaemic stroke (baseline), and 14 days (14d) and 90 days (90d) after commencing aspirin, clopidogrel or aspirin+dipyridamole. Platelet reactivity was assessed at moderately-high shear stress (PFA-100® Collagen-Epinephrine / Collagen-ADP / INNOVANCE PFA P2Y assays), and low shear stress (VerifyNow® Aspirin / P2Y12, and Multiplate® Aspirin / ADP assays).VWF:Ag levels decreased and VWFpp/VWF:Ag ratio increased between baseline and 14d and 90d in the overall population (P ≤ 0.03). In the clopidogrel subgroup, VWF:Ag levels decreased and VWFpp/VWF:Ag ratio increased between baseline and 14d and 90d (P ≤ 0.01), with an increase in ADAMTS13 activity between baseline vs. 90d (P ≤ 0.03). In the aspirin+dipyridamole subgroup, there was an inverse relationship between VWF:Ag and VWFpp levels with both PFA-100 C-ADP and INNOVANCE PFA P2Y closure times (CTs) at baseline (P ≤ 0.02), with PFA-100 C-ADP, INNOVANCE PFA P2Y and C-EPI CTs at 14d (P ≤ 0.05), and between VWF:Ag levels and PFA-100 INNOVANCE PFA P2Y CTs at 90d (P = 0.03). There was a positive relationship between ADAMTS13 activity and PFA-100 C-ADP CTs at baseline (R2 = 0.254; P = 0.04).Commencing/altering antiplatelet therapy, mainly attributed to commencing clopidogrel in this study, was associated with decreasing endothelial activation following TIA/ischaemic stroke. These data enhance our understanding of the impact of VWF:Ag and VWFpp especially on ex-vivo platelet reactivity status at high shear stress after TIA/ischaemic stroke.

Ratio of von Willebrand factor to ADAMTS13 is a useful predictor of esophagogastric varices progression after sustained virologic response in patients with hepatitis C virus-related liver cirrhosis.

Esophagogastric varices (EGV) are a serious complication of hepatitis C virus (HCV)-related liver cirrhosis (HCV-LC). In most cases, portal hypertension improves after a sustained virologic response (SVR) is achieved with direct-acting antiviral (DAA) treatment; however, in some cases, EGV exacerbation occurs after HCV elimination. We investigated whether von Willebrand factor (VWF) and a disintegrin-like metalloproteinase with thrombospondin type-1 motif 13 (ADAMTS13) can predict EGV progression with HCV-LC after SVR achievement. This retrospective study enrolled 47 patients with HCV-LC who achieved an SVR after DAA treatment. Eighteen patients experienced EGV progression after the SVR was achieved (EGV progression group). Twenty-nine patients did not experience EGV progression after the SVR was achieved (non-EGV progression group). Plasma VWF antigen levels and ADAMTS13 activity were measured the day before DAA treatment. The EGV progression group had significantly higher plasma VWF antigen levels (p=0.00331) and VWF-to-ADAMTS13 ratios (p=0.000249) than the non-EGV progression group. Multivariate logistic regression models found that a VWF-to-ADAMTS13 ratio >2.3 was the only risk factor for EGV progression after the SVR was achieved (hazard ratio [HR], 18.4; 95% confidence interval [CI], 3.08-109; p=0.00138). During the observation period, patients with a VWF-to-ADAMTS13 ratio >2.3 had a significantly higher cumulative incidence of EGV progression after SVR achievement than patients with a VWF-to-ADAMTS13 ratio ≤2.3 (HR, 6.4; 95% CI, 1.78-22.96; p=0.0044). The VWF-to-ADAMTS13 ratio before DAA treatment for HCV could predict EGV progression after SVR achievement.

Improving our understanding on the clinical role of plasmin-mediated von Willebrand factor degradation.

Von Willebrand factor (VWF) plays a pivotal role in primary hemostasis. A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13 (ADAMTS13) is primarily responsible for cleaving ultra-large VWF multimers into smaller, less adhesive forms. However, plasmin has also been shown to cleave VWF multimers. This proteolytic cleavage of VWF results in a decreased multimer size and, hence, a lower VWF activity. This review aims to present a comprehensive overview of the involvement of plasmin-mediated VWF proteolysis in (micro)thrombosis. Plasmin-mediated VWF proteolysis has been suggested to play a role in various pathologies involving microthrombosis in combination with an imbalance in VWF antigen levels and ADAMTS13 activity, as well as activation of the fibrinolytic system, but quantitative assays to demonstrate this were lacking. Recently, a VHH-based bioassay was developed designed specifically to quantify plasmin-cleaved VWF (cVWF). The novel ELISA assay holds significant promise for gaining further insights into the clinical relevance of plasmin-mediated VWF proteolysis in several pathologies. Furthermore, local plasmin activation at the site of microthrombosis has been shown to be a promising treatment strategy by degrading VWF-rich microthrombi. Plasmin-mediated proteolysis of VWF is observed during microthrombosis; however, it remains unclear whether it impacts disease severity. A novel ELISA method to detect cVWF will improve our understanding of the clinical role of plasmin-mediated VWF degradation.

Nanobody activator improves sensitivity of the von Willebrand factor activity assay to multimer size

BackgroundThe activity of von Willebrand factor (VWF) in facilitating platelet adhesion and aggregation correlates with its multimer size. Traditional ristocetin-dependent functional assays lack sensitivity to multimer sizes. Recently, nanobodies targeting the autoinhibitory module and activating VWF were identified. ObjectivesTo develop an assay that can differentiate the platelet-binding activity of VWF multimers. MethodsA novel enzyme-linked immunosorbent assay (nanobody-triggered glycoprotein Ib binding assay [VWF:GPIbNab]) utilizing a VWF-activating nanobody was developed. Recombinant VWF, plasma-derived VWF (pdVWF), and selected gel-filtrated fractions of pdVWF were evaluated for VWF antigen and activity levels. A linear regression model was developed to estimate the specific activity of VWF multimers. ResultsOf the 3 activating nanobodies tested, 6C11 with the lowest activation effect exhibited the highest sensitivity for high-molecular-weight multimers (HMWMs) of VWF. VWF:GPIbNab utilizing 6C11 (VWF:GPIbNab6C11) produced significantly higher activity/antigen ratios for recombinant VWF (>2.0) and HMWM-enriched pdVWF fractions (>2.0) than for pdVWF (∼1.0) or fractions enriched with shorter multimers (<1.0). The differences were much larger than those produced by VWF:GPIbNab utilizing other nanobodies, VWF:GPIbM, VWF:GPIbR, or VWF:CB assays. Linear regression analysis of 5 pdVWF fractions of various multimer sizes produced an estimated specific activity of 2.7 for HMWMs. The analysis attributed >90% of the VWF activity measured by VWF:GPIbNab6C11 to that of HMWMs, which is significantly higher than all other activity assays tested. ConclusionThe VWF:GPIbNab6C11 assay exhibits higher sensitivity to HMWMs than ristocetin-based and collagen-binding assays. Future studies examining the application of this assay in clinical settings and any associated therapeutic benefit of doing so are warranted.

A noncanonical splicing variant c.875-5 T > G in von Willebrand factor causes in-frame exon skipping and type 2A von Willebrand disease

IntroductionA novel variant involving noncanonical splicing acceptor site (c.875-5 T > G) in propeptide coding region of von Willebrand factor (VWF) was identified in a patient with type 2A von Willebrand disease (VWD), who co-inherited with a null variant (p.Tyr271*) and presented characteristic discrepancy of plasma level of VWF antigen and activity, and a selective reduction of both intermediate-molecular-weight (IMWMs) and high-molecular-weight VWF multimers (HMWMs). Materials and methodsVWF mRNA transcripts obtained from peripheral leukocytes and platelets of the patients were investigated to analyze the consequence of c.875-5 T > G on splicing. The impact of the variant on expression and multimer assembly was further analyzed by in vitro expression studies in AtT-20 cells. The intracellular processing of VWF mutant and the Weibel-Palade bodies (WPBs) formation was evaluated by immunofluorescence staining and electron microscopy. ResultsThe mRNA transcript analysis revealed that c.875-5 T > G variant led to exon 8 skipping and an in-frame deletion of 41 amino acids in the D1 domain of VWF (p.Ser292_Glu333delinsLys), yielding a truncated propeptide. Consistent with the patient's laboratory manifestations, the AtT-20 cells transfected with mutant secreted less VWF, with the VWF antigen level in conditioned medium 47 % of wild-type. A slight retention in the endoplasmic reticulum was observed for the mutant. Almost complete loss of IMWMs and HMWMs in the medium and impaired WPBs formation in the cell, indicating truncated VWF propeptide lost its chaperon-like function for VWF multimerization and tubular storage. ConclusionsThe VWF splicing site variant (c.875-5 T > G) causes propeptide truncation, severely compromising VWF multimer assembly and tubular storage.

Von Willebrand Factor Antigen Levels Predict Poor Outcomes in Patients With Stroke and Cancer: Findings From the Multicenter, Prospective, Observational SCAN Study.

Patients with acute ischemic stroke and active cancer have more severe neurological symptoms, elevated risks of stroke recurrence, and death compared with the general population. We examined whether von Willebrand factor (vWF) antigen levels at stroke onset were associated with the poor outcomes of patients with stroke and cancer. Using data from 90 patients with acute ischemic stroke and active cancer who were registered in the SCAN (Ischemic Stroke in Patients With Cancer and Neoplasia) study, a prospective multicenter, observational study in Japan, we divided patients into 2 groups according to their median vWF antigen levels (high, n=46; or low, n=44). The high-vWF group had a significantly higher initial National Institutes of Health Stroke Scale score (median, 7 [interquartile range, 3-11.25] versus 3 [interquartile range, 1-8.5]; P<0.05) and a significantly higher incidence of cryptogenic stroke (32 [70%] versus 16 [36%]; P<0.01) and venous thromboembolism (7 [15%] versus 0 [0%]; P<0.01), as well as multiple lesions (28 [62%] versus 12 [27%]; P<0.001), than the low-vWF group. We observed no significant difference in the rate of stroke recurrence within 1 year between the groups. However, increased vWF levels were an independent predictor of death within 1 year of stroke onset, after adjusting for potential confounders (odds ratio, 6.77 [95% CI, 1.49-30.78]; P<0.05). Elevated vWF antigen levels were associated with adverse outcomes in patients with cancer-associated stroke and may represent a useful biomarker to guide future therapeutic interventions.

Type 1 VWD classification revisited: novel insights from combined analysis of the LoVIC and WiN studies

AbstractThere is significant ongoing debate regarding type 1 von Willebrand disease (VWD) defintion. Previous guidelines recommended patients with von Willebrand factor (VWF) levels <30 IU/dL be diagnosed type 1 VWD, whereas patients with significant bleeding and VWF levels from 30 to 50 IU/dL be diagnosed with low VWF. To elucidate the relationship between type 1 VWD and low VWF in the context of age-induced increases in VWF levels, we combined data sets from 2 national cohort studies: 162 patients with low VWF from the Low VWF in Ireland Cohort (LoVIC) and 403 patients with type 1 VWD from the Willebrand in The Netherlands (WiN) studies. In 47% of type 1 VWD participants, VWF levels remained <30 IU/dL despite increasing age. Conversely, VWF levels increased to the low VWF range (30-50 IU/dL) in 30% and normalized (>50 IU/dL) in 23% of type 1 VWD cases. Crucially, absolute VWF antigen (VWF:Ag) levels and increase of VWF:Ag per year overlapped between low VWF and normalized type 1 VWD participants. Moreover, multiple regression analysis demonstrated that VWF:Ag levels in low VWF and normalized type 1 VWD patients would not have been different had they been diagnosed at the same age (β = 0.00; 95% confidence interval, −0.03 to 0.04). Consistently, no difference was found in the prevalence of VWF sequence variants; factor VIII activity/VWF:Ag or VWF propeptide/VWF:Ag ratios; or desmopressin responses between low VWF and normalized type 1 VWD patients. In conclusion, our findings demonstrate that low VWF does not constitute a discrete clinical or pathological entity. Rather, it is part of an age-dependent type 1 VWD evolving phenotype. Collectively, these data have important implications for future VWD classification criteria.

Von Willebrand factor Ristocetin co-factor activity to von Willebrand factor antigen level ratio for diagnosis of acquired von Willebrand syndrome caused by aortic stenosis

BackgroundSevere aortic stenosis (AS) causes acquired von Willebrand syndrome by the excessive shear stress–dependent cleavage of high molecular weight multimers of von Willebrand factor (VWF). While the current standard diagnostic method is so-called VWF multimer analysis that is western blotting under nonreducing conditions, it remains unclear whether a ratio of VWF Ristocetin co-factor activity (VWF:RCo) to VWF antigen levels (VWF:Ag) of <0.7, which can be measured with an automated coagulation analyzer in clinical laboratories and is used for the diagnosis of hereditary von Willebrand disease. ObjectivesTo evaluated whether the VWF:RCo/VWF:Ag is useful for the diagnosis of AS-induced acquired von Willebrand syndrome. MethodsVWF:RCo and VWF:Ag were evaluated with the VWF large multimer index as a reference, which represents the percentage of a patient’s VWF high molecular weight multimer ratio to that of standard plasma in the VWF multimer analysis. ResultsWe analyzed 382 patients with AS having transaortic valve maximal pressure gradients of >30 mmHg, 27 patients with peripheral artery disease, and 46 control patients free of cardiovascular disease with osteoarthritis, diabetes, and so on. We assumed a large multimer index of <80% as loss of VWF large multimers since 59.0% of patients with severe AS had the indices of <80%, while no control patients or patients with peripheral artery disease, except for 2 patients, exhibited the indices of <80%. The VWF:RCo/VWF:Ag ratios, measured using an automated blood coagulation analyzer, were correlated with the indices (rs = 0.470, P < .001). When the ratio of <0.7 was used as a cut-off point, the sensitivity and specificity to VWF large multimer indices of <80% were 0.437 and 0.826, respectively. ConclusionVWF:RCo/VWF:Ag ratios of <0.7 may indicate loss of VWF large multimers with high specificity, but low sensitivity. VWF:RCo/VWF:Ag ratios in patients with AS having a ratio of <0.7 may be useful for monitoring the loss of VWF large multimers during their clinical courses.

Steady-State Von Willebrand Factor Antigen Levels Predict Low Platelet Counts and Risk for Complications in Hospitalizations for Painful Sickle Cell Vaso-Occlusive Episodes

We previously reported that a &amp;gt;20% decline in platelet counts from steady state to presentation predicted severe complications during hospitalization for vaso-occlusive pain crisis (VOC) in 130 adult sickle cell disease (SCD) patients. 1 Sharp platelet decline in acute chest syndrome was reported to be accompanied by large von Willebrand Factor (VWF) aggregates adherent to pulmonary vascular endothelium in SCD. 2 A prospective study of 24 SCD patients found a significant increase of VWF reactivity in VOC admissions relative to steady state, which correlated with low platelet counts and the development of acute chest syndrome. 3 We aimed to investigate the role of steady-state VWF antigen level and ADAMTS13 activity as predictors of platelet count decline and severe complications in VOC admissions. Plasma levels of VWF antigen and ADAMTS13 activity were measured 4 for 56 patients at steady-state outpatient visits (Baseline). VWF antigen was expressed as fold increase or decrease from the concentration in normal pooled plasma of 12.2 ug/ml; ADAMTS13 activity was expressed as fold of the value from normal pooled plasma which was considered 100% activity. Electronic records of VOC admission that occurred &amp;gt;21 days after Baseline were reviewed. Exclusion criteria included non-VOC admissions defined as elective admission for surgery, admission for accident or injury, admission for delivery of an infant, or admission with a pain score &amp;lt;8/10 at presentation. 5 A total of 98 qualifying admissions were identified. Severe complication during the admission was defined as having any of the following: 1) acute chest syndrome, new infiltrate on chest X-ray, or use of ventilator; 2) acute kidney injury; 6 3) acute liver injury; 7 4) thrombotic or hemorrhagic stroke; 5) venous thromboembolism including deep vein thrombosis, atrial thrombosis, and pulmonary embolism; 6) need for transfer to the intensive care unit; 7) need for exchange blood transfusion; 8) death during hospitalization. Platelet counts at presentation to the emergency room (ER) leading to the admissions under study were recorded, as well platelet counts at Baseline. The 56 patients had a median (range) age of 37 (21-66) years; 52% are female, 89% with hemoglobin SS or Sβ 0-thalassemia, and 57% on hydroxyrea (HU). Severe complication occurred in 35 (36%) of the 98 admissions involving 27 (48%) of the patients. Focusing on the first admissions, higher steady-state VWF antigen levels correlated strongly with lower ER platelet counts (ρ= -0.50, P=0.00011, Figure 1A) and higher steady-state ADAMTS13 activity correlated weakly with ER platelet counts (Spearman's ρ= 0.30, P=0.025). The ratio of VWF antigen to ADAMTS13 activity ratio also correlated strongly with lower ER platelet counts (ρ= -0.52, P=0.000052, Figure 1B). In contrast, steady-state VWF antigen level (ρ= -0.17, P=0.23), ADAMTS13 activity (ρ= 0.19, P=0.17), and VWF antigen to ADAMTS13 activity ratio (ρ= -0.23, P=0.098) showed no significant correlation with steady-state platelet counts. The correlations remained by adjusting for age, gender, Hb genotype severity, HU, and treatment of anticoagulant, aspirin, or non-steroidal anti-inflammatory drugs at VOC visits. In analyses restricted to admissions &amp;gt;1 year after Baseline, the correlation between steady-state VWF antigen level and ER platelet counts appeared to persist. Focusing on the 28 first admissions, steady-state VWF antigen level (ρ= -0.50, P=0.0071) and VWF antigen to ADAMTS13 activity ratio (ρ= -0.44, P=0.018) correlated negatively with ER platelet counts. Steady-state ADAMTS13 activity did not correlate with ER platelet counts in admissions that occurred &amp;gt;1 year after Baseline (ρ= 0.16, P=0.43). We further examined the association of steady-state VWF antigen level, ADAMTS13 activity, and their ratio with risk for severe complication in admission. Focusing on the first 56 admissions, which involved 23 complications, steady-state VWF antigen level in the top quartile (1.63) associated with risk for severe complications (OR=6.5, 95% CI 1.4-31, P=0.018, n=56), adjusting for age, gender, Hb genotype severity, and HU. Our observations suggest that steady-state VWF antigen level and the ratio of VWF antigen level to ADAMTS13 activity predict low platelet counts at presentation to VOC admissions, and that steady-state VWF antigen level in SCD patients may be a relatively stable marker for risk of severe VOC complications.

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.

The Impacts of Genetic Polymorphisms on the Von Willebrand Factor Level in Type 1 Von Willebrand Disease

Background: von Willebrand disease (VWD) is the most common inherited bleeding disease, which results from deficiency or defect of von Willebrand factor (VWF). In type 1 VWD, there are only 53-81% patient whose mutations of VWF gene can be identified. Some studies reported that variants outside the VWF gene may also contribute to the VWF level. There are 7 genetic variants of single nucleotide polymorphism ( SNP ) reported to be associated with the variation of VWF levels, including CLEC4M, STXBP5, SCARA5, ABO, VWF, STAB2 and UFM1. The aims of this study were to explore the frequency of SNPs of the 7 genes among type 1 VWD patients and the effects of the 7 genetic variants on the VWF levels in type 1 VWD patients. Patients and methods: There were 62 patients ( the ratio of M/F: 1 ) with type 1 VWD enrolled in this study, including 35 (56.5%) patients with found mutations of VWF gene. Each patient's bleeding symptoms were assessed by the MCMDM-1 VWD bleeding score. The results of VWF levels: VWF:Ag and VWF activity ( VWF:ACL or VWF:RCo ) and FVIII: C at the VWD diagnosis were retrospectively collected, as shown in Table 1. Each enrolled patient's genomic DNA was extracted and the SNPs of the 7 gene were targeted by PCR amplification followed by direct sequencing. Results: There was high correlations between VWF:Ag and VWF activity ( r = 0.73, p &amp;lt; 0.0001 ) and between VWF:Ag and FVIII:C ( r = 0.60, p &amp;lt; 0.0001 ), respectively. The patients without variant ( A/A) of UFM1 has significantly lower median VWF:Ag of 33% than 44.7% of patients with heterozygous variant ( A/C ) type ( p = 0.033 ). Patients without variant (A/A ) of STXBP gene had both significantly lower median value of VWF:Ag ( 31.9% vs 46.1%, p = 0.017 ) and VWF activity (26.55% vs 30.4%, p = 0.0467 ) than those patients with heterozygous variant ( A/G ). Four alleles substitution of SNP of UFM1( C&amp;gt;A ) and STXBP genes ( G&amp;gt;A ) variant has remarkably lowest median VWF antigen level of 27.4%, which was significantly lower than 43.05% of the three alleles substitution ( p = 0.0014 ) and the 48.6% of the two alleles substitution ( p = 0.0279 ), as shown in Figure 1. Conclusions: Our study demonstrates that both C to A variant of UFM1 gene and G to A variant of STXBP5 gene were associated with lower VWF: Ag level than those without variant, respectively. The combination of four allelic variant of UFM1 and STXBP5 genes had the most reducing effect on VWF: Ag level.

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 (&amp;lt;1 IU/dL), moderate (1-5 IU/dL), and mild (&amp;gt;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 (&amp;lt;2 IU/dL/&amp;lt;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.

Coagulation factor VIII regulates von Willebrand factor homeostasis invivo

BackgroundCoagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex, but each has its distinct functions. Binding of FVIII to VWF results in a prolongation of FVIII’s half-life in circulation and modulates FVIII’s immunogenicity during hemophilia therapy. However, the biological effect of FVIII and VWF interaction on VWF homeostasis is not fully understood. ObjectivesTo determine the effect of FVIII in VWF proteolysis and homeostasis in vivo. MethodsMouse models, recombinant FVIII infusion, and patients with hemophilia A on a high dose FVIII for immune tolerance induction therapy or emicizumab for bleeding symptoms were included to address this question. ResultsAn intravenous infusion of a recombinant B-domain less FVIII (BDD-FVIII) (40 and 160 μg/kg) into wild-type mice significantly reduced plasma VWF multimer sizes and its antigen levels; an infusion of a high but not low dose of BDD-FVIII into Adamts13+/- and Adamts13-/- mice also significantly reduced the size of VWF multimers. However, plasma levels of VWF antigen remained unchanged following administration of any dose BDD-FVIII into Adamts13-/- mice, suggesting partial ADAMTS-13 dependency in FVIII-augmented VWF degradation. Moreover, persistent expression of BDD-FVIII at ∼50 to 250 U/dL via AAV8 vector in hemophilia A mice also resulted in a significant reduction of plasma VWF multimer sizes and antigen levels. Finally, the sizes of plasma VWF multimers were significantly reduced in patients with hemophilia A who received a dose of recombinant or plasma-derived FVIII for immune tolerance induction therapy. ConclusionOur results demonstrate the pivotal role of FVIII as a cofactor regulating VWF proteolysis and homeostasis under various (patho)physiological conditions.

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.

Von Willebrand factor processing in patients with advanced chronic liver disease and its relation to portal hypertension and clinical outcome

Background and aimsEndothelial dysfunction and portal hypertension (PH) are reflected by increased von Willebrand factor antigen (VWF-Ag) levels in advanced chronic liver disease (ACLD). This study investigated VWF release and cleavage and their association with PH and clinical outcomes.MethodsLevels of VWF-Ag, VWF-N (VWF-propeptide), and VWF-A (VWF processed by the main VWF-cleaving protease ADAMTS13) were assessed in 229 patients with clinically stable ACLD (hepatic venous pressure gradient [HVPG] ≥ 6 mmHg; absence of bacterial infections or acute decompensation) undergoing HVPG-measurement. Liver-healthy individuals served as controls (n = 24).ResultsVWF-Ag and VWF-N were similarly accurate for the identification of clinically significant PH (CSPH; HVPG ≥ 10 mmHg) in compensated ACLD (AUROC: VWF-Ag 0.748; VWF-N 0.728). ADAMTS13 activity was similar between patients with ACLD and controls and did not correlate with PH and disease severity, whereas VWF cleavage decreased in patients with CSPH (i.e., VWF-Ag/-A-ratio increased). In vitro VWF activity strongly reflected VWF-Ag levels (Spearman’s r = 0.874, p < 0.001), but decreased (vs. controls) in patients with CSPH when normalized to VWF-Ag levels (VWF-activity/-Ag-ratio). VWF-Act/-Ag ratio correlated negatively with ADAMTS13 activity (r =– 0.256, p < 0.001). ADAMTS13 activity was independently predictive for (i) portal vein thrombosis (PVT) and (ii) hepatic decompensation or liver-related death.ConclusionsVWF-Ag levels and its propeptide are similarly suitable surrogates of PH in patients with compensated ACLD. ADAMTS13-Act was not linked to disease and PH severity, however, when normalized to VWF-Ag, both VWF cleavage and VWF activity were decreased in patients with CSPH, as compared to liver-healthy individuals. Low ADAMTS13-Act was associated with presumably more procoagulant VWF and adverse outcomes.Clinical trial numberNCT03267615

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

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

A performance evaluation of sthemO 301 coagulation analyzer and associated reagents.

The study objective was to evaluate the performance of sthemO 301 system and to compare it with the analyzer used in our university hospital laboratory (STA R Max® 2), for a selection of hemostasis parameters. Method comparison (according to CLSI EP09-A3), carryover (according to CLSI H57-A), APTT sensitivity to heparin (according to CLSI H47-A2), HIL level assessment, and productivity were performed using leftover samples from our laboratory (n > 1000). Commercial quality control materials were used to evaluate precision (according to CLSI EP15-A3) and accuracy. The assays tested on sthemO 301 were: PT, APTT (silica and kaolin activators), fibrinogen (Fib), thrombin time (TT), chromogenic and clotting protein C (PC) activity, and von Willebrand factor antigen (VWF:Ag) levels. All intra-assay and inter-assay precision CVs were below the maximal precision limit proposed by the French Group for Hemostasis and Thrombosis (GFHT). Accuracy was verified with bias below GFHT criteria and most Z-scores were between -2 and +2. No clinically relevant carryover was detected. Silica APTT reagent sensitivity to unfractionated heparin was moderate, as expected. Productivity results were consistent over the 10 repeats performed. The overall agreement between the two systems was excellent for all assays, with Spearman rank correlation coefficient all above 0.9 and slopes of Passing-Bablok correlation near 1 and intercepts close to 0. For the methods tested, sthemO 301 system met all the criteria to implement a novel coagulation analyzer in the laboratory and result comparability with STA R Max® 2 was good.

Factor XIII and Endothelial Dysfunction in Patients with Systemic Sclerosis.

Systemic sclerosis (SSc, scleroderma) is a severe autoimmune connective tissue disease which affects the skin and internal organs. There has been evidence that coagulation factor XIII (FXIII) has a positive impact on clinical results in patients with SSc. In a single-center cohort study, we investigated the relationship between coagulation FXIII, endothelial dysfunction, and skin infection in SSc. Fifty-six patients could be included and were divided into two groups (with and without scleroderma). Markers of inflammation, coagulation, and endothelial dysfunction like C-reactive protein, leucocytes, fibrinogen, FVIII, VWF-Ag (von Willebrand factor antigen), D-dimers, and vascular endothelial growth factor were analyzed as well as MRSS (modified Rodnan skin scores) data were evaluated. Reduced daily activities were evaluated by the Scleroderma Health Assessment Questionnaire (SHAQ). There were no significant correlations between FXIII activity, MRSS, and SHAQ score. There were correlations between FXIII activity and Raynaud's phenomenon-related symptoms and a weak but not significant positive correlation with the level of pain. A significant correlation between VWF-Ag and lung-associated complaints (n = 56; p = 0.41, p < 0.0001) was found. Moreover, the study showed a correlation between VWF-Ag and MRSS (r [N = 48] = 0.4, p = 0.01), which means that higher VWF-Ag levels come along with more severe skin involvement. A trend toward a negative correlation between FXIII activity and VWF-Ag as marker of endothelial dysfunction was found (r [N = 56] = - 0.20, p = 0.15). In our cohort, there is no FXIII deficiency in patients with SSc. FXIII might have a role in improving cutaneous manifestations indirectly by means of a moderating influence on endothelial dysfunction. Further clinical evaluation is needed.

Von Willebrand factor antigen: a biomarker for severe pregnancy complications in women with hereditary thrombotic thrombocytopenic purpura?

BackgroundHereditary thrombotic thrombocytopenic purpura (hTTP) is associated with severe obstetric morbidity (SOM) during pregnancy. Treatment with fresh frozen plasma (FFP) mitigates the risk in some women, but others respond poorly and continue to suffer obstetric complications. ObjectivesTo determine a possible association between SOM and elevated nonpregnant von Willebrand factor (NPVWF) antigen levels in women with hTTP and whether the latter can predict the response to FFP transfusion. MethodsThis was a cohort-based study of women with hTTP due to homozygous c.3772delA mutation of ADAMTS-13 who had pregnancies both with and without FFP treatment. Occurrences of SOM were determined from medical records. Generalized estimated equation logistic regressions and receiver operating characteristic curve analysis determined the NPVWF antigen levels associated with the development of SOM. ResultsFourteen women with hTTP had 71 pregnancies; of which 17 (24%) culminated in pregnancy loss and 32 (45%) were complicated by SOM. FFP transfusions were administered in 32 (45%) of the pregnancies. Treated women had decreased SOM (28% vs 72%, p < .001) and preterm thrombotic thrombocytopenic purpura exacerbations (18% vs 82%, p < .001) and higher median NPVWF antigen levels than those of women with uncomplicated pregnancies (p = .018). Among the treated women, median NPVWF antigen levels were higher in those with SOM than in those without SOM (225% vs 165%, p = .047). Logistic regression models demonstrated a significant 2-way association between elevated NPVWF antigen levels (for SOM, odds ratio, 1.08; 95% CI, 1.001-1.165; p = .046) and SOM (for elevated NPVWF antigen levels, odds ratio, 1.6; 95% CI, 1.329-1.925; p < .001). The receiver operating characteristic curve analysis demonstrated that an NPVWF antigen level of 195% had 75% sensitivity and 72% specificity for SOM. ConclusionElevated NPVWF antigen levels are associated with SOM in women with hTTP. Women with levels >195% may benefit from increased surveillance and more intensive FFP treatment during pregnancy.

Current Understanding of Inherited Modifiers of FVIII Pharmacokinetic Variation.

The inherited bleeding disorder hemophilia A involves the quantitative deficiency of the coagulation cofactor factor VIII (FVIII). Prophylactic treatment of severe hemophilia A patients with FVIII concentrates aims to reduce the frequency of spontaneous joint bleeding and requires personalized tailoring of dosing regimens to account for the substantial inter-individual variability of FVIII pharmacokinetics. The strong reproducibility of FVIII pharmacokinetic (PK) metrics between repeat analyses in the same individual suggests this trait is genetically regulated. While the influence of plasma von Willebrand factor antigen (VWF:Ag) levels, ABO blood group, and patient age on FVIII PK is well established, estimates suggest these factors account for less than 35% of the overall variability in FVIII PK. More recent studies have identified genetic determinants that modify FVIII clearance or half-life including VWF gene variants that impair VWF-FVIII binding resulting in the accelerated clearance of VWF-free FVIII. Additionally, variants in receptors that regulate the clearance of FVIII or the VWF-FVIII complex have been associated with FVIII PK. The characterization of genetic modifiers of FVIII PK will provide mechanistic insight into a subject of clinical significance and support the development of personalized treatment plans for patients with hemophilia A.