vWF Antigen Levels Research Articles

The severe von Willebrand Disease variant p.M771V leads to impaired anterograde trafficking of Von Willebrand factor in patient-derived and base-edited ECFCs

BackgroundVon Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects of VWF. The p.M771V VWF variant leads to a severe bleeding phenotype in homozygous patients. However, the exact molecular mechanism remains unclear, which prevents personalized treatment of those VWD patients. ObjectiveThis study aims to characterize the underlying molecular mechanisms of the p.M771V variant in multiple representative ex-vivo cell models. MethodsECFCs were isolated from venous blood of VWD patients from the Willebrand in the Netherlands (WiN) cohort carrying homozygous and heterozygous p.M771V VWF variants. The p.M771V variant was also introduced in cord-blood derived ECFCs (CB-ECFCs) through adenine base editing and was overexpressed in HEK293 cells. Biosynthesis, storage, and secretion of VWF was studied using biochemical methods and confocal microscopy. ResultsTwo unrelated homozygous p.M771V patients presented with very low VWF activity and antigen levels in plasma. Patient ECFCs showed impaired proVWF processing into mature VWF with secreted VWF being severely reduced when compared to ECFCs of healthy donors. Multimer analysis of p.M771V ECFCs showed a deficiency of high molecular weight VWF multimers. Immunofluorescent staining revealed VWF retention in the endoplasmic reticulum (ER); this was confirmed in various populations of base edited CB-ECFCs harboring the p.M771V variant. ConclusionThe severe endothelial phenotype observed in patient-derived p.M771V ECFCs, HEK293 cells, and an original base-edited CB-ECFC modelling system, show that ER retention of VWF and failure to undergo subsequent proteolytic processing underpins the severe bleeding phenotype of patients with homozygous variants at M771.

Clinical Utility of Recently Food and Drug Administration-Approved IntelliSep Test (Sepsis Biomarker) for Early Diagnosis of Sepsis: Comparison with Other Biomarkers.

Context: IntelliSep by Cytovale has received United States (U.S.) Food and Drug Administration (FDA) approval as a sepsis biomarker test. However, the clinical utility of this new test is not assessed in emergency departments. Objective: We investigated the clinical utility of this test using 44 patients visiting the emergency department at The University of Kansas Medical Center by comparing it with the monocyte distribution width (MDW) and other biomarkers including the von Willebrand factor (vWF) and ADAMTS13. Design and Methods: IntelliSep assesses the cellular host response via deformability cytometry of biophysical leukocyte properties and produces a score (IntelliSep Index; ISI: from 0.1 (lowest risk) to 10 (highest risk). We measured the ISI in 44 patients (19 high probability and 25 low probability of sepsis groups) using EDTA-anticoagulated blood. Left over plasma was used for measuring the plasma von Willebrand factor (vWF) and ADAMTS13 antigen by ELISA assays. The MDW was obtained during routine CBC analysis using a Beckman hematology analyzer. The lactate and high-sensitivity troponin I levels were measured using a Beckman analyzer. Procalcitonin was measured using a Cobas e801 analyzer. Results: The median ISI was twofold higher in the high-probability group than in the low-probability group (p < 0.01) while the median MDW was 34.5% higher in the high-probability group than in the low-probability group (p < 0.01). However, the correlation between the ISI and MDW was only modest (r = 0.66). In addition, significantly higher levels of plasma vWF antigen but lower levels of plasma ADAMTS13 antigen in the high-probability group were found, resulting in significantly higher vWF/ADAMTS13 ratios in the high-probability group than in the low-probability group. Conclusions: The new IntelliSep test along with vWF/ADAMTS13 ratios may be useful for the early diagnosis of sepsis in patients visiting the emergency department, which appears to be superior to the traditional marker, MDW.

ROTEM and von Willebrand Factor in COVID patients presenting with acute ischemic stroke: A case series: ROTEM and von Willebrand Factor in COVID-19 Related Stroke

ObjectivesSARS-CoV-2 (COVID) induces systemic thrombotic complications including acute ischemic stroke. In this case series, we report markers of inflammation, coagulation factors including von Willebrand factor antigen, and rotational thromboelastometry (ROTEM) data. Materials and methodsRetrospective case series of COVID patients seen at a single comprehensive stroke center between 2020–2022. For patients undergoing mechanical thrombectomy (MT), ROTEM data was collected during the procedure and analyzed on ROTEM delta system. ResultsFifteen patients (33.3% female) median age 65-years-old presented with COVID and acute ischemic stroke. Thirteen had LVO. The mean NIHSS was 15 (range 0–35) on admission and 18 (0–42) at discharge. Most were cryptogenic (N=7, 46.7%), followed by cardioembolic (N=6, 40%) and large artery-to-artery embolization (N=2, 13.3%). mRS was < 3 in 8 (53%) patients at discharge. None of the patients were on anticoagulation, and five were on antiplatelet therapy pre-hospitalization. Seven received thrombolytics with alteplase (tPA), and 10 had MT.Baseline platelet count was 102 K/uL (range 102–291 K/uL). vWF was measured in 12 patients, all elevated, with seven having levels >400 (180%). ROTEM data was collected in six patients. Three who received tPA had abnormal EXTEM and FIBTEM data (CT extem > 85secs, A10 EXTEM < 45mm, and A10 FIBTEM < 10mm). Notably, INTEM (CT INTEM >208secs) was abnormal in five of the six patients, two of whom did not receive tPA. ConclusionsElevated vWF antigen levels with abnormal ROTEM data suggests that COVID induces changes in the clotting cascade. More robust research is needed to investigate these findings. Thrombolytics, MT, and antiplatelet agents should be utilized to treat COVID-related ischemic stroke based on current clinical guidelines.

Molecular mechanisms and clinical manifestations of hereditary hemorrhagic telangiectasia

IntroductionHereditary Hemorrhagic Telangiectasia (HHT) is charactered by telangiectasia and arteriovenous malformations (AVMs). Recurrent visceral and mucocutaneous bleeding is frequently reported among HHT patients, while data on the prevalence of thrombosis remains limited. This study aims to describe the clinical manifestations and molecular biological characteristics of HHT patients. MethodsWe conducted a retrospective study at Ruijin Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. A total of 24 HHT patients, observed between January 2019 and December 2023, were included. We recorded the biological, clinical, and therapeutic events, with particular attention to bleeding and thrombotic events. Gene mutation analysis and blood constituent measurements were performed. ResultsThe prevalence of bleeding among all HHT patients was 100 %, while thrombotic events were noted in 41.70 % of cases. Hepatic arteriovenous malformations (HAVMs) were identified in six patients, pulmonary arteriovenous malformations (PAVMs) in five patients, and cerebral arteriovenous malformations (CAVMs) in one patient. For patients with thrombosis, the discontinuation rates were 23.08 % for antiplatelet therapy and 33.33 % for anticoagulant therapy due to the increased risk of bleeding. Genetic mutations related to HHT were present in 16 patients, with ACVRL1 (activin A receptor-like type 1) mutations being the most frequent at 41.67 %, followed by ENG (endoglin) mutations at 20.83 %, and GDF2 (growth differentiation factor 2) mutations at 4.17 %. The incidence of PAVMs was 75.00 % in HHT1 patients with ENG mutations and 20 % in HHT2 patients with ACVRL1 mutations, while HAVMs occurred in 0 % and 40.00 % of these groups, respectively. Patients were divided into non-AVMs and AVMs groups. Compared to normal controls, von Willebrand factor (vWF) activity was significantly increased in all HHT patients (149.10 % vs. 90.65 %, P < 0.001). In the non-AVMs group, the median level of stromal cell-derived factor-1 (SDF-1) was significantly elevated (124.31 pg/mL vs. 2413.57 pg/mL, P < 0.05), while vWF antigen levels were markedly higher in the AVMs group (165.30 % vs. 130.60 %, P = 0.021). Further grouping of HHT patients based on bleeding and thrombosis phenotypes revealed that those with thrombosis had significantly higher median percentages of schistocytes (3.50 % vs. 0 %, P = 0.002), ferritin concentrations (318.50 μg/L vs. 115.50 μg/L, P = 0.001), and lactate dehydrogenase (LDH) levels (437 U/L vs. 105 U/L, P < 0.001). There were no significant differences in the activity of vWF, protein C (PC), protein S (PS), and factor VIII (FVIII) between the two groups. ConclusionThis study highlighted the complex relationship between arteriovenous malformations and genetic mutations in HHT patients. A comprehensive assessment of bleeding and thrombosis risks should be conducted for each HHT patient, additionally, further clinical studies are needed to explore the risk factors for thrombosis and anticoagulant-related bleeding in HHT.

ECMO produces very rapid changes in primary hemostasis detected by PFA-200 during lung transplantation: An observational study

BackgroundThe pathology of primary hemostasis is a common complication of extracorporeal membrane oxygenation (ECMO) support. Scientific data describing its changes in patients on short-term ECMO support and the ability and speed of the restoration of its functions are limited. AimsThe aim of this study was to describe the pathology of primary hemostasis induced by short-term ECMO support and its development over time using PFA-200®, ROTEM® platelet and von Willebrand factor (vWF) analyses. MethodsIn patients undergoing lung transplantation surgery using intra-operative veno-arterial ECMO support, blood samples were analyzed using the following tests: PFA-200®, ROTEM® platelet tests, vWF antigen, ristocetin cofactor (RCo) and collagen binding protein (CB) before, during and after ECMO support. ResultsBlood samples from 32 patients were analyzed. All three PFA-200® tests (COL/EPI, COL/ADP and COL/P2Y) showed significant deterioration during ECMO support with rapid restoration after ECMO cessation (p<0.05), suggesting an ECMO-induced primary hemostasis disorder. A significant increase of vWF antigen after ECMO cessation (p<0.05) was found with an increase of ristocetin cofactor and collagen binding protein levels, although it was not significant (p>0.05). ConclusionsShort-term ECMO support induces primary hemostasis pathology. It occurs immediately after initiation but is rapidly restored after ECMO cessation, which is detectable by PFA-200®. Despite there being persistent platelet dysfunction after ECMO cessation as seen with the ROTEM® platelet results, the increased levels of vWF antigen might explain the normal results of primary hemostasis detected by PFA-200®.

ADAMTS13-Mediated Proteolytic Cleavage of Unusually Large von Willebrand Factor Polymers on Endothelial Cells in the Absence of Fluid Shear Stress

To investigate the molecular mechanism of proteolytic cleavage of unusually large von Willebrand Factor(ULVWF) on endothelial cells by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) in the absence of fluid shear stress, so as to provide a theoretical basis for the pathogenesis of thrombotic thrombocytopenic purpura (TTP) and other thrombotic disorders. The ADAMTS13-mediated proteolysis of ULVWF on the surface of endothelial cells in the absence of fluid shear stress was observed through immunofluorescence microscopy. The variation in VWF antigen levels in the conditioned media were determined by ELISA assay. The levels of VWF and the proteolytic fragments released into the conditioned media were determined by ELISA assay and Western blot in the absence and presence of fluid shear stress or FVIII. The effect of ADAMTS13-mediated ULVWF cleavage on the normal distribution of plasma VWF multimers was evaluated by multimer analysis. Histamine stimulated human umbilical vein endothelial cells (HUVECs) were incubated with ADAMTS13 and various N- and C-terminally truncated mutants. Then the ULVWF that maintained binding to the cells were observed through immunofluorescence microscopy and the soluble ULVWF released from endothelial cells was determined by ELISA, so as to demonstrate the domains of ADAMTS13 required for proteolysis of ULVWF on endothelial cells. The ULVWF strings on the endothelial cell surface were rapidly proteolyzed by recombinant and plasma ADAMTS13 in the absence of fluid shear stress. This proteolytic processing of ULVWF depended on incubation time and ADAMTS13 concentration, but not shear stress and FVIII. The distribution of VWF releaseded by ADAMTS13-mediated proteolysis was quite similar to that secreted by endothelial cells under histamine stimulation, suggesting the ULVWF cleavage occured at the cell surface. The proteolysis of the ULVWF on endothelial cells required the Cys-rich(CysR) and spacer domains, but not the TSP1 2-8 and CUB domains of ADAMTS13. The ULVWF polymers on endothelial cells are sensitive to ADAMTS13-mediated cleavage even in the absence of fluid shear stress. The findings provide novel insight into the molecular mechanism of ADAMTS13-mediated ULVWF cleavage at the cellular level and may contribute to understanding of the pathogenesis of TTP and other thrombotic disorders.

Bleeding Disorder of Unknown Cause: A Diagnosis of Exclusion.

Patients with an unexplained mild to moderate bleeding tendency are diagnosed with bleeding disorder of unknown cause (BDUC), a classification reached after ruling out other mild to moderate bleeding disorders (MBD) including von Willebrand disease (VWD), platelet function defects (PFDs), coagulation factor deficiencies (CFDs), and non-hemostatic causes for bleeding. This review outlines our diagnostic approach to BDUC, a diagnosis of exclusion, drawing on current guidelines and insights from the Vienna Bleeding Biobank (VIBB). According to guidelines, we diagnose VWD based on VWF antigen and/or activity levels ≤50 IU/dL, with repeated VWF testing if VWF levels are <80 IU/dL. This has been introduced in our clinical routine after our findings of diagnostically relevant fluctuations of VWF levels in a high proportion of MBD patients. PFDs are identified through repeated abnormalities in light transmission aggregometry (LTA), flow cytometric mepacrine fluorescence, and glycoprotein expression analysis. Nevertheless, we experience diagnostic challenges with regard to reproducibility and unspecific alterations of LTA. For factor (F) VIII and FIX deficiency, a cutoff of 50% is utilized to ensure detection of mild hemophilia A or B. We apply established cutoffs for other rare CFD being aware that these do not clearly reflect the causal role of the bleeding tendency. Investigations into very rare bleeding disorders due to hyperfibrinolysis or increase in natural anticoagulants are limited to cases with a notable family history or distinct bleeding phenotypes considering cost-effectiveness. While the pathogenesis of BDUC remains unknown, further explorations of this intriguing area may reveal new mechanisms and therapeutic targets.

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.

Determination of vWF, ADAMTS-13 and Thrombospondin-1 in Venous Thromboembolism and Relating Them to the Presence of Factor V Leiden Mutation.

Thrombophilia in venous thromboembolism (VTE) is multifactorial. Von Willebrand factor (vWF) plays a major role in primary hemostasis. While elevated vWF levels are well documented in VTE, findings related to its cleaving protease (ADAMTS-13) are contradicting. The aim of this study was to determine vWF, ADAMTS-13, and the multifactorial Thrombospondin-1 (TSP-1) protein levels in patients after 3-6 months following an unprovoked VTE episode. We also explored a possible association with factor V Leiden (FVL) mutation. vWF, ADAMTS-13 and TSP-1 were analyzed using ELISA kits in 60 VTE patients and 60 controls. Patients had higher levels of vWF antigen (P = .021), vWF collagen-binding activity (P = .008), and TSP-1 protein (P < .001) compared to controls. ADAMTS-13 antigen was lower in patients (P = .046) compared to controls but ADAMTS-13 activity was comparable between the two groups (P = .172). TSP-1 showed positive correlation with vWF antigen (rho = 0.303, P = .021) and negative correlation with ADAMTS-13 activity (rho = -0.244, P = .033) and ADAMTS-13 activity/vWF antigen ratio (rho = -0.348, P = .007). A significant association was found between the presence of FVL mutation and VTE (odds ratio (OR): 9.672 (95% confidence interval (CI) 2.074-45.091- P = .004), but no association was found between the mutation and the studied proteins (P > .05). There appears to be an imbalance between vWF and ADAMTS-13 in VTE patients even after 3-6 months following the onset of VTE. We report that the odds of developing VTE in carriers of FVL mutation are 9.672 times those without the mutation, but the presence of this mutation is not associated with the studied proteins.

The effects of ambient particulate matter air pollution on platelets and hemostasis.

Elevated ambient pollution exposure is potentially linked to thromboembolism. However, the mechanisms by which particulate matter (PM) interferes with the balance of hemostatic system remain unclear. This study investigates PM-mediated hemostatic changes in individuals across unique seasonal variations of ambient pollution. This prospective study was conducted between February and July 2020 during alterations in ambient pollution in Chiang Mai, Thailand. Blood tests from 30 healthy subjects were assessed at four-week intervals, four times in total. Various coagulation tests, including prothrombin time (PT), activated partial thromboplastin time (aPTT), von Willebrand factor (vWF), platelet count, and platelet functions, were evaluated. A mixed-effects model was used to analyze the impact of high PM2.5 and PM10 on hemostatic parameters. Thirty male subjects with mean age of 38.9 ± 8.2 years, were included. High levels of PM2.5 and PM10 were significantly associated with PT shortening, with no such effect observed in aPTT. PM2.5 and PM10 values also positively correlated with vWF function, while vWF antigen levels remained unchanged. Soluble P-selectin showed a strong positive association with PM2.5 and PM10 levels. Platelet function analysis revealed no correlation with PM values. Short-term exposure to elevated PM2.5 and PM10 concentrations was linked to shortened PT and enhanced vWF function in healthy individuals. Exploring the impact of these changes on clinically relevant thrombosis is crucial. Additional studies on the pathogenesis of pollution-related thrombosis are warranted for maintaining good health.

Searching for New Biomarkers to Assess COVID-19 Patients: A Pilot Study.

During the initial diagnosis of urgent medical conditions, which include acute infectious diseases, it is important to assess the severity of the patient's clinical state as quickly as possible. Unlike individual biochemical or physiological indicators, derived indices make it possible to better characterize a complex syndrome as a set of symptoms, and therefore quickly take a set of adequate measures. Recently, we reported on novel diagnostic indices containing butyrylcholinesterase (BChE) activity, which is decreased in COVID-19 patients. Also, in these patients, the secretion of von Willebrand factor (vWF) increases, which leads to thrombosis in the microvascular bed. The objective of this study was the determination of the concentration and activity of vWF in patients with COVID-19, and the search for new diagnostic indices. One of the main objectives was to compare the prognostic values of some individual and newly derived indices. Patients with COVID-19 were retrospectively divided into two groups: survivors (n = 77) and deceased (n = 24). According to clinical symptoms and computed tomography (CT) results, the course of disease was predominantly moderate in severity. The first blood sample (first point) was taken upon admission to the hospital, the second sample (second point)-within 4-6 days after admission. Along with the standard spectrum of biochemical indicators, BChE activity (BChEa or BChEb for acetylthiocholin or butyrylthiocholin, respectively), malondialdehyde (MDA), and vWF analysis (its antigen level, AGFW, and its activity, ActWF) were determined and new diagnostic indices were derived. The pooled sensitivity, specificity, and area under the receiver operating curve (AUC), as well as Likelihood ratio (LR) and Odds ratio (OR) were calculated. The level of vWF antigen in the deceased group was 1.5-fold higher than the level in the group of survivors. Indices that include vWF antigen levels are superior to indices using vWF activity. It was found that the index [Urea] × [AGWF] × 1000/(BChEb × [ALB]) had the best discriminatory power to predict COVID-19 mortality (AUC = 0.91 [0.83, 1.00], p < 0.0001; OR = 72.0 [7.5, 689], p = 0.0002). In addition, [Urea] × 1000/(BChEb × [ALB]) was a good predictor of mortality (AUC = 0.95 [0.89, 1.00], p < 0.0001; OR = 31.5 [3.4, 293], p = 0.0024). The index [Urea] × [AGWF] × 1000/(BChEb × [ALB]) was the best predictor of mortality associated with COVID-19 infection, followed by [Urea] × 1000/(BChEb × [ALB]). After validation in a subsequent cohort, these two indices could be recommended for diagnostic laboratories.

The Utility of a Coagulation Laboratory Profile in Addition to Clinical Risk Factors for Thrombosis Risk Evaluation in Patients with Myeloproliferative Neoplasms: A Pilot Study

Background: Patients (pts) with myeloproliferative neoplasms (MPN) have a significantly increased risk of arterial and venous thrombotic events (TEs), while some pts may paradoxically suffer from bleeding manifestations. Thrombosis risk evaluation in polycythemia vera (PV) and essential thrombocythemia (ET) has important implications for patient management, however, the currently used risk stratification models are extremely limited. Methods: We designed a pilot study to assess the utility of an expanded coagulation laboratory profile in addition to other clinical variables for thrombosis and bleeding risk evaluation in a heterogenous MPN patient population. Our laboratory profile included PT, aPTT, fibrinogen, D-dimer, von-Willebrand Factor (vWF) antigen and activity testing, platelet (PLT) aggregation studies and thrombin generation assay (TGA, ST-Genesia, Stago, France), along with CBC and comprehensive biochemistry studies. Blood was drawn from pts visiting the hematology clinics or day care at Hadassah Medical Center. Clinical and demographic data were collected retrospectively from electronic medical records. The study was approved by the local IRB committee. Results:74 pts were included in the study; the mean age was 59.7 years and 51% were women. Sixty percent had a diagnosis of PV, 20% had ET, 17.6% had myelofibrosis (MF) and 2.7% had MPN unclassifiable. 78% of the pts harbored a JAK2V617F mutation, 8% and 2.7% harbored CALR and MPL mutations, respectively, and 11% were negative to all three MPN driver mutations. 63% of the pts had at least one cardiovascular risk factor, most commonly hypertension (HTN) and dyslipidemia. Most of the pts were treated with antiplatelet agents (62%), anticoagulants (16%) and/or cytoreductive agents (58%). Of 74 pts, 31% had a history of TEs (20% arterial, 11% venous), 22% reported suffering from microvascular symptoms, and 20% had experienced bleeding. The high rate of bleeding manifestations in our study population suggests an underestimation of the bleeding burden in MPN, as proposed by our group previously [Rottenstreich, Eur J Intern Med 2017]. Other patient characteristics were overall comparable to MPN patient populations in other studies (Table 1). In multivariate analyses, history of TEs was significantly associated with older age, type 2 diabetes mellitus (DM2), HTN and dyslipidemia, but not with gender, MPN subtype, driver mutation status or smoking. Pts with history of TEs had significantly lower hematocrit and prolonged PT/INR, associated with a higher rate of treatment with cytoreduction and anticoagulation, respectively, in these pts. Interestingly, pts with TEs had significantly higher vWF antigen levels (p=0.007), which were independent of age, blood counts and treatments, a finding not previously reported in MPN. No association was observed between TEs and PLT or WBC counts, D-dimer levels and vWF activity (Table 2), however, vWF activity was decreased in pts with higher PLT counts. TGA was of key interest in this study. In 62 pts not receiving anticoagulation, we observed an association between prolonged lag time (suggesting decreased coagulability) and older age, male gender and (near significant trend) JAK2V617 negative status, but did not find a correlation to TEs, blood counts and other variables. PLT aggregation indices were not associated with TEs, bleeding history or blood counts. In multivariate logistic regression analyses, history of DM2 and HTN, and vWF antigen levels, showed the strongest association to TEs, which was independent of age, blood counts and all other variables. A risk-model incorporating these variables showed a sensitivity of 77% and specificity of 92% for history of TEs in our study population. Conclusions: Despite being limited by a small and heterogenous study population and retrospective data collection; these findings support the utility of a coagulation laboratory profile in the evaluation of thrombosis and bleeding risk in MPN. The potential of TGA in risk evaluation in MPN is intriguing but merits further research. The multivariate risk-model incorporating history of DM2 and HTN along with vWF antigen levels merits validation in larger studies. Based on this pilot, a prospective study following over 250 pts with PV and ET (excluding overt MF) was initiated. This study will include NGS mutational profiling and JAK2V617F allele burden testing and omit the use of PLT aggregation studies.

A Mutation in Complement Factor H (W1183R) Enhances VWF Proteolysis By ADAMTS13 Under Shear

Introduction: Complement factor H (CFH) is the primary negative regulator of complement activation in the alternative pathway in both fluidic phase and on cell surface. Loss of function of CFH such as W1183R is associated with atypical hemolytic uremic syndrome (aHUS). Homozygous mutation W1206R in the murine CFH, corresponding to W1183R in human CFH, results in acute aHUS. Interestingly, in these mice, while plasma VWF antigen levels are significantly increased, plasma VWF multimer size is markedly reduced in ADAMTS13-depedent manner. However, the molecular mechanism underlying such a dramatic effect of the mutant CFH (W1206R) on VWF multimer distribution is not known. The goal of this study is to determine how wild type CFH and mutant CFH W1183R affects VWF proteolysis under shear, and the mechanism of action. Methods: Recombinant WT CFH, CFH W1183R, ADAMTS13, and VWF fragments were expressed in mammalian cells and purified to homogeneity. The effects of WT CFH or CFH W1183R on proteolytic cleavage of VWF73 peptide and full-length multimeric VWF were determined. Additionally, the binding and unfolding events were determined by micro scale thermophoresis (MST) and optical tweezer, respectively. Results : WT CFH showed no effect on the cleavage of peptide VWF73 substrate, but CFH W1183R enhanced the proteolytic cleavage of VWF73 by ADAMTS13 in a dose-dependent manner ( Fig. 1). Counterintuitively, WT CFH appeared to inhibit the proteolytic cleavage of multimeric VWF by ADAMTS13 under arterial shear, but CFH W1183R exhibited an opposite effect, modestly enhancing the proteolytic cleavage of multimeric VWF by ADAMTS13 under the same conditions. The MST binding assay demonstrated that both WT CFH and CFH W1183R bind to a short A1, a long AIM-A1, and A1-A2-A3 fragments with a dissociation constant ( K D) of 6.4 x 10 -7, 3.0 x 10 -9 M, and 3.3 x10 -9 mol/L, respectively. These results suggest that CFH may bind to the AIM, A1-A2 junction, and A2 domain. To further assess the impact of CFH binding to A2, we performed optical tweezer experiments. The results showed that both WT CFH (Lc: from 27.4 nm at 12 nM to 40 nm at 1.0 mM) and CFH W1183R (Lc: from 26 nm at 12 nM to 31.8 nm at 1.0 mM) resulted in significantly extended contour length (Lc) of VWF-A2 domain in a concentration-dependent manner ( Fig. 2), suggesting a dramatic conformational change in the A2 domain upon binding by WT CFH or CFH W1183R. Conclusions : Our results demonstrate that WT CFH at physiological concentrations may exhibit a modest inhibitory activity towards cleavage of VWF by ADAMTS13 under shear, but such an inhibitory activity disappears as the result of a point mutation in the C-terminal end of CFH (e.g., W1183R). Instead, the mutant CFH W1183R appears to enhance the VWF cleavage by ADAMTS13, likely through direct binding to the AIM, A1-A2 junction, and A2 domain of VWF, resulting in conformational changes in the central A2 domain.

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.

Associations of multiple genetic variations with plasma levels of Von Willebrand Factor and clinical phenotype in Iranian patients with Von Willebrand disease type 1

BackgroundGenetic variations influence the Von Willebrand Factor plasma level and function. This study aims to evaluate the frequency and clinical phenotype effects of eight single nucleotide polymorphism candidates in four genes (VWF, STXBP5, CLEC4M, and ABO) in Iranian patients with VWD type 1. MethodThe study recruited 50 patients with VWD type 1 and 100 healthy individuals. The demographic data from all participants were collected, and the High-Resolution Melting technique was used to determine the frequency of specific single nucleotide polymorphisms. Bleeding scores were also obtained from all patients to assess how these genetic variations might affect the severity of their bleeding symptoms. ResultsThe study found notable variations in the occurrence of certain SNPs (rs7853989 and rs8176743 for ABO gene and rs1063856 and rs1063857 for VWF gene) between the control group and the patients. Additionally, the study discovered that two SNPs (rs868875 for CLEC4M gene and rs9390459 for STXBP5 gene) were significantly linked to the severity of bleeding, and two others (rs868875 for CLEC4M gene and rs8176746 for ABO gene) were associated with reduced levels of VWF antigen in the patients. ConclusionAccording to this study, the above-selected SNPs can cause variations in VWF plasma levels in patients with VWD type 1. Furthermore, the effects of SNPs on bleeding phenotype prove the role of these SNPs in the severity of bleeding manifestations in patients.

Evaluating von Willebrand factor and ADAMTS13 levels in thalassemia major patients and assessing a possible association with Thrombospondin-1.

Alterations in the endothelium and endothelial adhesion proteins such as von Willebrand factor (vWF) play major roles in hypercoagulability in thalassemia. vWF protein release leads to platelet aggregation and thrombi formation at the site of vascular injury. It is then degraded by the proteolytic enzyme ADAMTS13. Thrombospondin-1 is a multifactorial glycoprotein, which was reported to compete with ADAMTS13 for sites of vWF proteolysis. In this study, levels of vWF, ADAMTS13, and TSP-1 proteins were determined in β-thalassemia major patients. A possible association between TSP-1 and vWF and ADAMTS-13 was also evaluated. The study was conducted on 80 β-thalassemia major patients and 80 age and sex matched healthy controls. The 80 patients were sub-divided into two groups; splenectomised and non-splenectomised. vWF, ADAMTS13 and TSP-1 plasma level were measured using ELISA technique. There was no significant difference in vWF and TSP-1 levels between patients and controls (p > 0.05). However, ADAMTS13 levels and ADAMTS13 activity/vWF antigen ratio were significantly higher in patients compared to controls (p < 0.05). VWF antigen and TSP-1 level were significantly higher in splenectomised patients (p = 0.025 and p < 0.001, respectively). We also observed a significant decrease in ADAMTS13 activity/vWF antigen ratio among splenectomised compared to non- splenectomised patients (p = 0.019). Correlation analysis showed a significant negative correlation between TSP-1 and vWF Collagen Binding Activity (r = -0.394, p = 0.021) and a positive correlation with ADAMTS13 activity/vWF antigen ratio (r = 0.356, p = 0.039) in splenectomised compared to non- splenectomised patients. Our findings highlight the adequacy of patient management protocols for β-TM in Kuwait as patients presented with comparable levels of platelets, vWF and TSP-1 compared to normal controls. The reported increase in ADAMTS13 in patients may be required to maintain normal levels of vWF. Although no active thrombotic episodes were reported at the time of the study, the significant rise in platelets, vWF:Ag and TSP-1 levels in splenectomised patients may indicate a tendency towards hypercoagulability. Monitoring of splenectomised patients is recommended.

Clinical significance of ADAMTS-13/vWF axis in pregnant women at different trimesters of gestation

Introduction. The pandemic of a novel coronavirus infection has demonstrated the importance of assessing the ADAMTS-13/vWF axis in patients with COVID-19, because a decline in this ratio mirrors disease severity. However very few data in the global literature on crosstalk and ADAMTS-13/vWF levels in pregnant women remaining very contradictory are available. Taking into consideration an impact of the ADAMTS-13/vWF axis on prevalence of thrombosis and disorders in the hemostasis system, investigation of this issue is highly demanded.Aim: to assess the functioning of the ADAMTS-13/vWF axis during physiological pregnancy.Materials and Methods. A controlled non-randomized study was conducted: main group included 44 women with physiologically occurring pregnancies at I, II and III trimesters; the control group consisted of 45 healthy non-pregnant women. The plasma level of ADAMTS-13 inhibitor (ADAMTS-13:i), ADAMTS-13 antigen (ADAMTS-13:Ag), vWF antigen (vWF:Ag), and ADAMTS-13 activity (ADAMTS-13:Ac) as well as relevant ratio (ADAMTS-13:Ac/vWF:Ag) were measured.Results. It was shown that in parallel with increasing gestational age, significant changes occurred in the ADAMTS-13:Ac/vWF:Ag ratio. In main group, patients at II trimester were found to have level of ADAMTS-13:Ac/vWF:Ag 0.359 ± 0.121, in III trimester –0.253 ± 0.741, which significantly differed (p &lt; 0.01) compared to control group with non-pregnant women (1,134 ± 0,308).Conclusion. Our study provides new insights into the functioning of the ADAMTS-13:/vWF axis in women with physiologically occurring pregnancy at I, II and III trimesters. Decline in ADAMTS-13:Ac was demonstrated along with increasing vWF:Ag level observed in parallel with increasing gestational age. Apparently, the progressive decrease of ADAMTS-13 concentration during pregnancy is associated with its increased consumption due to high vWF level. However, due to the small single-center patient cohort, further studies with larger-scale studies are needed.

Prognostic value of von Willebrand factor levels in patients with metastatic melanoma treated by immune checkpoint inhibitors

BackgroundAn increased incidence of thrombotic complications associated with an increased mortality rate has been observed under immune checkpoint inhibition (ICI). Recent investigations on the coagulation pathways have highlighted the direct...

Endothelial cell activation, Weibel-Palade body secretion, and enhanced angiogenesis in severe COVID-19

BackgroundSevere COVID-19 is associated with marked endothelial cell (EC) activation that plays a key role in immunothrombosis and pulmonary microvascular occlusion. However, the biological mechanisms through which SARS-CoV-2 causes EC activation and damage remain poorly defined. ObjectivesWe investigated EC activation in patients with acute COVID-19, and specifically focused on how proteins stored within Weibel-Palade bodies may impact key aspects of disease pathogenesis. MethodsThirty-nine patients with confirmed COVID-19 were recruited. Weibel-Palade body biomarkers (von Willebrand factor [VWF], angiopoietin-2 [Angpt-2], and osteoprotegerin) and soluble thrombomodulin (sTM) levels were determined. In addition, EC activation and angiogenesis were assessed in the presence or absence of COVID-19 plasma incubation. ResultsMarkedly elevated plasma VWF antigen, Angpt-2, osteoprotegerin, and sTM levels were observed in patients with acute COVID-19. The increased levels of both sTM and Weibel-Palade body components (VWF, osteoprotegerin, and Angpt-2) correlated with COVID-19 severity. Incubation of COVID-19 plasma with ECs triggered enhanced VWF secretion and increased Angpt-2 expression, as well as significantly enhanced in vitro EC tube formation and angiogenesis. ConclusionWe propose that acute SARS-CoV-2 infection leads to a complex and multifactorial EC activation, progressive loss of thrombomodulin, and increased Angpt-2 expression, which collectively serve to promote a local proangiogenic state.

Von Willebrand factor levels in healthy blood donors and their association with blood group: A tertiary care hospital-based study.

von Willebrand disease is a common inherited bleeding disorder caused by the deficiency of von Willebrand factor (vWF).[1] The levels of vWF depend on several factors, including exercise, hormones, and ABO blood type.[2] This study was planned to evaluate plasma vWF levels and factor VIII (fVIII) levels in healthy blood donors and its association with the ABO blood group. The aim of this study was to evaluate plasma vWF levels and fVIII levels in healthy donors and its association with the ABO blood group. This study was done in 2016 healthy adult blood donors. Complete history and relevant examination were done along with ABO and Rh (D) blood group typing, complete blood count, prothrombin time, activated partial thromboplastin time, vWF antigen (Ag) level, fVIII coagulant assay, and other tests for hemostasis. Data were expressed in proportions and mean, median, and standard deviation, respectively. An appropriate test of significance was applied. P < 0.05 was considered statistically significant. vWF level of donors ranged from 24 to 186 IU/dL with a mean of 96.31 IU/dL. Low vWF Ag level (below 50 IU/dl) was found in 2.5% of donors while 0.1% (2/2016) had level <30 IU/dL. O Rh (D)-positive blood group donors had the lowest vWF level (87.85 IU/dL), while ARh (D)-negative donors had the highest vWF level (117.27 IU/dL). fVIII level of the donor population ranged from 22% to 174%, with a mean of 98.82%. About 2.48% of donors had fVIII levels below 50%. There was a statistically significant correlation between fVIII level and vWF level (P < 0.001).