Von Willebrand Factor Activity Research Articles

Evaluation of coagulation and platelet activation state and function in heartworm-infected dogs.

Enhanced platelet responses have been demonstrated in heartworm-infected (HWI) dogs; however, the cause and clinical implications of altered platelet function have not been fully elucidated. This study evaluated platelet function in HWI dogs. Anticoagulated whole blood collected from eight HWI and eight uninfected dogs was evaluated using turbidometric platelet aggregometry, a platelet function analyzer (PFA-100), a total thrombus analysis system (T-TAS), tissue factor-activated and tissue plasminogen activator modified thromboelastography (TF- and tPA-TEG), CBC, von Willebrand Factor activity, and fibrinogen concentrations. Platelet activation state and the presence of reticulated platelets were assessed via flow cytometric expression of P-selection (CD-62P) and thiazole orange staining. Platelet aggregation responses to adenosine diphosphate (ADP, 10 μM) or collagen (20 μg/mL), PFA-100 closure times, and T-TAS occlusion times did not differ between groups. TEG values TF-R, tPA-R, TF-K, and TF-LY60 were decreased (P = .025, P = .047, P = .038, P = .025) and TF-MA, tPA-MA, TF-G, tPA-G and TF-alpha angle were increased (P < .04) in HWI dogs. HWI dogs had higher fibrinogen concentrations (465.6 ± 161 mg/dL vs 284.5 ± 38 mg/dL, P = .008) and eosinophil counts (0.686 ± 0.27 × 103/μL vs 0.267 ± 0.20 × 103/μL, P = .003). There was no difference in hematocrit, activation state, or percent of reticulated platelets. Non-activated reticulated platelets exhibited higher CD62P expression compared with mature platelets. Chronic canine heartworm disease was accompanied by hypercoagulability, hyperfibrinogenemia, and decreased fibrinolysis. Enhanced platelet activation was not identified in this group of HWI dogs.

In vitro study on device-induced damage to blood cellular components and degradation of von Willebrand factor in a CentriMag pump-assisted circulation.

High mechanical shear stress (HMSS) generated by blood pumps during mechanical circulatory support induces blood damage (or function alteration) not only of blood cell components but also of plasma proteins. In the present study, fresh, healthy human blood was used to prime a blood circuit assisted by a CentriMag centrifugal pump at a flow rate of 4.5 L/min under three pump pressure heads (75, 150, and 350 mm Hg) for 4 h. Blood samples were collected for analyses of plasma-free hemoglobin (PFH), von Willebrand factor (VWF) degradation and platelet glycoprotein (GP) IIb/IIIa receptor shedding. The extent of all investigated aspects of blood damage increased with increasing cross-pump pressure and duration. Loss of high-molecular-weight multimers (HMWM)-VWF in Loop 2 and Loop 3 significantly increased after 2 h. PFH, loss of HMWM-VWF, and platelet GPIIb/IIIa receptor shedding showed a good linear correlation with mean shear stress corresponding to the three pump pressure heads. HMSS could damage red blood cells, cause pathological VWF degradation, and induce platelet activation and platelet receptor shedding. Different blood components can be damaged to different degrees by HMSS; VWF and VWF-enhanced platelet activation may be more susceptible to HMSS.

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. MethodsAn enzyme-linked immunosorbent assay–based assay (VWF 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.

Asymmetric dimethylarginine correlates with indicators of prethrombotic state in patients with nonvalvular atrial fibrillation.

The mechanism of asymmetric dimethylarginine (ADMA) in thrombosis in patients with nonvalvular atrial fibrillation (NVAF) is still unclear. Our aim was to investigate the relationship between ADMA and indicators of prethrombotic state in NVAF patients and to analyze the predictive role of ADMA in NVAF thrombosis. A total of 192 NVAF patients were continuously selected from January 2023 to October 2023. Plasma ADMA levels were measured by high-performance liquid chromatography. P-selectin (P-sel), von Willebrand factor (vWF), D-dimer (D-D), and plasminogen activator inhibitor-1 (PAI-1) levels were measured by enzyme-linked immunosorbent assay (ELISA). Nitric oxide (NO) levels were measured by the nitrate reductase assay for plasma nitrite/nitrate, then the Griess method (Shanghai Hailian Biotechnology Co., Shanghai, China) was used to calculate plasma NO levels. In our study, ADMA levels were significantly elevated and positively correlated with P-sel, vWF, D-D, and PAI-1, whereas NO levels were significantly negatively correlated with these prethrombotic factors in NVAF. Furthermore, multifactorial logistic regression analysis showed that ADMA and LA diameter were independent predictors of high thrombosis risk (CHA2DS2-VASc ≥2 score) in patients with NVAF. Our findings suggested that ADMA correlated with the prethrombotic state in NVAF and that reduction of ADMA levels in NVAF patients may be a novel therapeutic strategy for thrombosis risk reduction.

Evaluation of an automated von Willebrand factor glycoprotein IbM activity assay compared with 3 alternative von Willebrand factor activity assays

BackgroundTo overcome deficiencies of the traditional von Willebrand factor (VWF) ristocetin cofactor activity assay (VWF:RCo), several automated assays for VWF platelet-binding activity have been developed. Information on the performance of these assays and their diagnostic utility remains limited. ObjectivesTo validate the VWF:glycoprotein IbM assay INNOVANCE VWF Ac and compare it with an automated VWF:RCo assay as well as with an automated assay and a manual VWF:Ab assay and to generate reference ranges and analyze reproducibility of the VWF:glycoprotein IbM assay. MethodsClinical sites enrolled healthy subjects and patients representing the intended use population; VWF activity assays were performed, and results were analyzed. The performance of the INNOVANCE VWF Ac assay was also compared between the BCS XP System and the CS-2500 and CS-5100 analyzers. ResultsThe INNOVANCE VWF Ac assay correlated well with the VWF:RCo assay and the automated HemosIL VWF:Ab assay, with Pearson coefficients of >.9 and a predicted bias of ≤5.0 IU/dL at VWF levels of 30 IU/dL and ≤5.8 IU/dL at the levels of 50 IU/dL, but correlation and bias were not as good when compared with the REAADS manual VWF:Ab assay. Reference ranges observed for healthy subjects correlated well with previously published findings. Reproducibility of the INNOVANCE VWF Ac assay on the BCS XP System and the CS analyzers was excellent, as was correlation among devices. ConclusionThe characteristics of the INNOVANCE VWF Ac assay regarding comparability with other VWF activity assays, reference ranges, and precision support the use of this assay for evaluation of patients with concern for von Willebrand disease.

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.

Platelet volume indices and von Willebrand factor levels in blood exposed to polymer- or heparin-coated membrane oxygenators.

To understand the behavior of platelet volume indices and the von Willebrand factor (VWF), in vitro experiments using whole human blood were performed with extracorporeal circulation (ECC) circuits, including membrane oxygenators coated with acrylate copolymer (ACP) or immobilized heparin (IHP). Heparinized blood was circulated through two distinct experimental circuits: an ACP-coated reservoir and tubes, as well as membranes coated with either ACP or IHP (comprising five pieces of each type). The platelet distribution width, mean platelet volume (MPV), platelet large cell ratio (P-LCR), VWF quantity (VWFQ), and VWF activity (VWFA) were measured at 0, 8, 16, 24, and 32 h in each experiment. A two-way analysis of variance (ANOVA) was performed to determine whether the coating type or circulation duration affected the transition of each measurement. Two-way ANOVA indicated that the transitions of MPV, P-LCR, and VWFA were significantly affected by the circulation duration (p = 0.030, 0.001, and <0.001, respectively) and that the transitions of VWFQ and VWFA were significantly affected by the coating type (p = 0.022 and 0.006, respectively). Factor interactions between the coating type and circulation duration were not observed for each transition (p > 0.05). Our findings suggest that P-LCR is a good index for platelet activation in blood-circulating ECC and that VWFA and VWFQ are significantly attenuated in blood-circulating ECC with ACP-coated membranes, indicating the advantage of IHP coating regarding platelet activation.

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.

Conformational activation and inhibition of von Willebrand factor by targeting its autoinhibitory module

AbstractActivation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.

Abstract 19: Von Willebrand Factor Deficiency Improves Long Term Stroke Outcomes in Mice With Respiratory Infections

Background: Preceding respiratory tract infections (RTI) due to bacteria or virus has been linked to an increasing risk of stroke likely by potentiating an inflammatory immune response, endothelial dysfunction, platelet activation, and coagulopathy. Von Willebrand factor (VWF) plays a crucial role in hemostasis and thrombosis by promoting platelet adhesion to the subendothelial matrix and factor VIII stabilization. VWF activity is regulated by the protease ADAMTS13. Prior population-based studies suggest increased VWF levels and reduced ADAMTS13 activity as independent risk factors for stroke; however, a causal role of the VWF/ADAMTS13 axis in RTI-mediated stroke exacerbation is not investigated yet. Objective: To determine the causal role of the VWF/ADAMTS13 axis in a mouse model of preceding RTI and ischemic stroke. Methods: Wild-type (WT) mice were infected intranasally with sub-lethal doses of S. aureus or vehicle on days 0, 2, and 5. On day 6, infection was measured in the lungs (MRI), brain, and blood. VWF levels and ADTMTS13 activity were quantified by ELISA in the plasma on day 6 post-infection. In another set of experiments, on day 6 post-infection S. aureus -infected WT, Vwf -/- , Adamts13 -/- mice were subjected to transient (30 min) middle cerebral artery ischemia using a filament model. Functional outcomes (modified neurological severity score, rota-rod, corner, novel object recognition, and wire-hanging test) were measured in the same cohort of mice up to 4 weeks. Controls were mock-infected WT mice. Result: We demonstrate that a sustained S. aureus infection localized to lungs in WT mice, increases plasma VWF levels (~2-fold) and reduces ADAMTS13 activity that were associated with larger infarcts and worsened long-term functional outcomes (P&lt;0.05 vs. mock-infected mice). S. aureus infected VWF-deficient mice exhibited reduced infarcts and better long-term functional outcomes (P &lt;0.05 vs. S. aureus infected WT). In contrast, S. aureus infected ADAMTS13-deficient mice exhibited larger infarcts and worse long-term functional outcomes (P &lt;0.05 vs. S. aureus infected WT). Conclusion: In a model of respiratory tract infection during the week preceding stroke, VWF has a causal role in worsening stroke outcomes while ADAMTS13 is protective.

Haemostaseological Changes of VWF and FVIII during Pregnancy and the Oestrus Cycle in a Porcine Model of Von Willebrand Disease

Pregnancy and the oestrus cycle are challenging for female patients suffering from von Willebrand disease (VWD). Therefore, our study aimed to investigate the changes in von Willebrand factor (VWF) and factor VIII (FVIII) during pregnancy and the oestrus cycle in our porcine model of von Willebrand disease compared with the wild-type. Plasma analyses regarding primary hemostasis, secondary hemostasis, and VWF multimers, as well as immunohistochemistry analyses of VWF in the uterus and ovary, were performed. For levels of VWF and FVIII activities, significant elevations were seen in the last trimester. Primary hemostasis improved towards the end of pregnancy. In the oestrus cycle, significantly lower VWF values can be seen in the immunohistochemistry of the ovaries during the oestrus, while values were highest in the metoestrus. VWF multimer patterns in pigs were similar to the ones in human VWD patients. In summary, the course of VWF and FVIII during pregnancy and the oestrus cycle in porcine VWD were investigated for the first time. The porcine model seems to be suitable for haemostaseological studies on VWD. This provides an advantage for investigating reproduction-related bleeding and understanding the underlying mechanisms of post-partum hemorrhage or miscarriage in women with VWD.

Acquired von Willebrand syndrome and post-operative drainage: a comparison of patients with aortic stenosis versus coronary artery disease.

Degenerative aortic stenosis and coronary artery disease are considered to be the most prevalent cardiovascular diseases in industrialized countries. This study aims to determine the change over time in von Willebrand factor antigen, von Willebrand factor activity, and factor VIII and where there is a correlation with total post-operative drainage. The single-center retrospective study included 203 consecutive patients (64.5% male), undergoing coronary artery bypass surgery between March 1, 2019 and June 30, 2020 at the University Clinical Center of Serbia in the Clinic for Cardiac Surgery in Belgrade, Serbia. All patients 18years or older who presented with isolated, hemodynamically significant aortic stenosis were included. The control group consisted of patients who presented with only coronary artery disease. Between patients with only coronary artery disease and patients with coronary artery diseases and aortic stenosis, there was a statistically significant difference between pre-op and 1-month post-op fibrinogen, factor VIII, von Willebrand factor antigen, and von Willebrand factor (p < 0.001), post-op drainage, with overall lower drainage in coronary artery disease patients, and consistent increase in von Willebrand factor antigen, von Willebrand factor activity, and Factor VIII post-operatively in patients with coronary artery diseases and aortic stenosis. This study has shown that there is a correlation between von Willebrand factor antigen, von Willebrand factor activity and total drainage to the level of statistical significance in aortic stenosis patients and in the overall study population.

A Compound Heterozygosis of Two Novel Mutations in vWF Exacerbates vWD in a Chinese Pedigree.

von Willebrand disease (vWD), caused by mutations in the von Willebrand factor (vWF) coding gene, is a disease characterized by abnormal coagulation activity and a severe tendency for hemorrhage. Therefore, identifying mutations in vWF is important for diagnosing congenital vWD. We studied a 23-year-old male vWD patient and his parents. Clotting methods were used to determine activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen (FIB) levels, FVIII activity. Chromogenic substrate method was used to determine vWF antigen and activity. The platelet count was determined. Mutations were searched using whole-exome sequencing and certified by Sanger sequencing. Clinical data, including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen levels, FX activity, FX antigen levels, and the platelet count were collected. A mixing study was performed to eliminate the presence of coagulation factor inhibitors and lupus anticoagulants. Mutations were screened by using whole-exome sequencing (WES) and were verified by using Sanger sequencing. The proband showed severely decreased vWF antigen, vWF activity, and FVIII activity. RIPA (RISTO-CETIN-induced platelet aggregation) was 0%. Data from WES showed that the proband carried compound heterozygous variants vWF: NM_000552.5 (c.3213C>A p.Cys1071Ter) and vWF: NM_000552.5 (c.6598+2T>C). The proband's mother carried variant vWF: NM_000552.5 (c.3213C>A p.Cys1071Ter) while the proband's father carried variant vWF: NM_000552.5 (c.6598+2T>C). All laboratory test indexes of the proband's parents, including vWF antigen, vWF activity, and FVIII activity, were within the normal ranges. We identified a compound heterozygosis with two novel mutations in vWF (c.3213C>A, c.6598+2T >C) in a family pedigree, and our results demonstrate that the compound heterozygous mutations probably exacerbate vWD.

Analysis of laboratory parameters before the occurrence of hepatic sinusoidal obstruction syndrome in children, adolescents, and young adults after hematopoietic stem cell transplantation

PurposeHepatic sinusoidal obstruction syndrome (SOS) is a serious complication following hematopoietic stem cell transplantation (HSCT) in which early diagnosis improves patient outcome. The aim of our study was to detect laboratory parameters following HSCT that can predict the occurrence of SOS.MethodsThis retrospective study included 182 children, adolescents, and young adults who underwent allogeneic or autologous HSCT for the first time (median age 7.2 years). The diagnosis of SOS was based on the pediatric criteria of European Society for Blood and Marrow Transplantation (EBMT). We investigated 15 laboratory parameters after HSCT before the onset of SOS.ResultsThe overall incidence of SOS was 14.8%. SOS developed in 24 of 126 allogeneic (19.1%) and in 3 of 56 autologous (5.4%) HSCT patients at a median time of 13 days after HSCT. We observed a low SOS mortality rate of 11.1% within 100 days after HSCT. International normalized ratio (INR) ≥ 1.3, activated partial thromboplastin time (aPTT) ≥ 40 s, reptilase time ≥ 18.3 s, factor VIII ≤ 80%, antithrombin III ≤ 75%, protein C ≤ 48%, D-dimer ≥ 315 µg/L, bilirubin ≥ 9 µmol/L, and ferritin ≥ 3100 µg/L showed significant associations with the onset of SOS in the univariate analyses. In the multivariate analysis, INR ≥ 1.3 [odds ratio (OR) = 8.104, p = 0.006], aPTT ≥ 40 s (OR = 10.174, p = 0.001), protein C ≤ 48% (OR = 5.215, p = 0.014), and ferritin ≥ 3100 µg/L (OR = 7.472, p = 0.004) could be confirmed as independent risk factors after HSCT before SOS. If three of the four significant cut-off values were present, the probability of developing SOS was more than 70%. The probability of SOS was 96%, if all four laboratory parameters were changed according to the cut-off values. The values of factor XIII, von Willebrand factor (vWF), von Willebrand factor activity (vWF activity), protein S, fibrinogen, and alanine aminotransferase (ALT) were not relevant for the occurrence of SOS.ConclusionIn summary, the laboratory parameters INR, aPTT, protein C, and ferritin were very useful to predict the occurrence of SOS. In addition, this is the first report on a significant association between SOS and high values of INR and aPTT after HSCT before SOS.

Discrepant low von Willebrand factor activity results on the ACL TOP analyzer are frequent in unselected patients with myeloproliferative neoplasms and show no correlation with high-molecular-weight multimer loss or bleeding phenotype

BackgroundBleeding complications are common in patients with myeloproliferative neoplasms (MPNs), with a subset developing acquired von Willebrand disease. Despite this association, a wide spectrum of von Willebrand factor (VWF) abnormalities are described, and the performance of modern assays remains unclear. ObjectivesTo comprehensively describe the pattern of VWF laboratory abnormalities in the MPN population. MethodsWe collected samples from 74 unselected clinic patients with MPNs to evaluate VWF quantitatively and qualitatively via multiple methods, correlating findings with a retrospective analysis of clinical bleeding data. VWF assays were performed on both ACL TOP (Instrumentation Laboratory) and Acustar (Instrumentation Laboratory) analyzers using HemosIL reagents (Instrumentation Laboratory), along with multimer analysis by gel electrophoresis. ResultsFunctional VWF measurements were not concordant between assays, with a median ACL TOP VWF glycoprotein IbR to antigen ratio (VWF:GPIbR/VWF:Ag) of 0.57 (IQR, 0.43-0.71) compared to a median Acustar VWF:GPIbR/VWF:Ag of 0.91 (IQR: 0.82-1.03;P < .001). The ACL TOP showed disproportionately lower results, with 73% of patients having a ratio <0.7. Despite this, no patient experienced loss of high-molecular-weight multimers by gel electrophoresis. An inverse relationship was observed between platelet count and functional ratios on both ACL TOP (R2 = 0.20; P < .001) and Acustar (R2 = 0.18; P = .0011) analyzers. While clinically significant bleeding events were relatively common (11% patients), there was no association with VWF assay abnormalities, and generally, an alternate cause(s) was identified. ConclusionDiscrepancies in functional VWF assays are common in patients with MPN, particularly by ACL TOP VWF:GPIbR. Based on our limited series, a VWF functional to an antigenic ratio of <0.7 (“type 2 pattern”) alone is poorly predictive of bleeding risk.

The rapid change of shear rate gradient is beneficial to platelet activation

Fluid shear plays a key role in hemostasis and thrombosis, and the purpose of this study was to investigate the effect of shear gradient change rate (SGCR) on platelet reactivity and von Willebrand factor (vWF) activity and its mechanism. In this study, we developed a set of microfluidic chips capable of generating different shear gradients and simulated the shear rate distribution in the flow field by COMSOL Multiphysics software. Molecular markers of platelet activation (P-selectin, activated GPIIb/IIIa, phosphatidylserine exposure, and monocyte-platelet aggregate formation) were analyzed by flow cytometry. Platelet aggregation induced by shear gradient was studied by a microfluidic experimental platform, and plasma vWF ristocetin cofactor (vWF: RCO) activity was investigated by flow cytometry. The expression of p-Akt was studied by Western blotting. The results showed that the faster the SGCR, the higher the expression of platelet p-Akt, and the stronger the platelet reactivity and vWF activity. This indicates that fluid shear stress can activate platelets and vWF in a shear gradient-dependent manner through the PI3K/AKT signal pathway, and the faster the SGCR, the more significant the activation effect.

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.

Key Drivers of Coagulation Factor Use in Von Willebrand Disease During Hospitalization: An Overview of the French BERHLINGO Cohort.

Von Willebrand disease (VWD) is the most common inherited bleeding disorder. However, studies of hospitalisation patterns with replacement treatment are scarce. The aim of this study was to investigate the current therapeutic management of VWD and determine the key drivers of coagulation factor uses in patients during hospitalisation. Hopscotch-WILL was a multi-centric retrospective study conducted over a 48-month period in any patients with VWD. The data were collected from the BERHLINGO Research Database and the French Hospital database. A total of 988 patients were included; 153 patients (15%) were hospitalised during 293 stays requiring treatment with von Willebrand factor (VWF) concentrates-pure or in association with Factor VIII (FVIII). Their median basal concentrations of VWF and FVIII were significantly lower than in untreated patients: VWF antigen < 30 IU/dL, VWF activity < 20 IU/dL and FVIII:C < 40 IU/dL. The median (interquartile range) concentrate consumption was similar between highly purified VWF or VWF combined with FVIII (72 [110] vs 57 [89] IU/kg/stay, p = 0.154). The use of VWF was highly heterogeneous by VWD type; type 3 had a particularly high impact on VWF consumption in non-surgical situations. The main admissions were for ear/nose/throat, hepato-gastroenterology, and trauma/orthopaedic conditions, besides gynaecological-obstetric causes in women. The use of VWF concentrates is mostly influenced by low basal levels of VWF and FVIII, but also by VWD type or the cause for hospitalisation. These results could inform future studies of newly released recombinant VWF.

Neuraxial Analgesia in Pregnant Individuals with Bleeding Disorders: A Comprehensive Examination of Obstetric Anesthesia Practices and Outcomes

Introduction: Neuraxial analgesia in obstetric anesthesia offers multiple advantages when compared with general anesthesia, including better pain relief and patient satisfaction, reduced opioid-related side effects, and decreased maternal morbidity and mortality. However, neuraxial analgesia carries a risk of hematoma, which can lead to permanent neurological complications, and is considered contraindicated in patients with uncorrected bleeding disorders. Hemostatic thresholds for safe neuraxial analgesia remain undetermined. Current evidence for neuraxial analgesia in pregnant individuals with bleeding disorders is limited to a few case reports or small case series, leaving significant gaps in clinical practices based on expert opinions. We sought to examine obstetric analgesia practices and outcomes in pregnant individuals with bleeding disorders. Specifically, we aimed to determine the methods used at the time of labor and delivery, the concordance with antenatal recommendations, and the outcomes in relation to the bleeding disorder, hemostatic thresholds, and hemostatic therapy support. Methods: A retrospective cohort study was conducted on all pregnant individuals with bleeding disorders who delivered between 2010-2020 at our institution, a large tertiary academic care center with high-risk obstetrical care in Ontario, Canada. We collected data on patient and disease characteristics, delivery mode, hemostatic laboratory values, hemostatic support, obstetric analgesia method, antenatal anesthesia and hematology recommendations, and post-procedure outcomes. A descriptive analysis was performed to present the findings. Results: The study cohort comprised of 56 pregnant patients, resulting in 82 deliveries. The median maternal age was 31.5 years (interquartile range (IQR): 28-34), and the median gestational age was 39 weeks (IQR: 38-40). Most patients delivered vaginally (60/82, 73.2%). von Willebrand disease type 1 (VWD-1) (28/82, 34.1%) and hemophilia A (24/82, 29.3%) were the most prevalent bleeding disorders. Neuraxial analgesia was used in most deliveries (59/82, 72.0%), followed by opioids (25/82, 30.5%), and nitrous oxide (15/82, 18.3%). Antenatal neuraxial anesthesia recommendations were documented by anesthesiology in 59.8% (50/82) and by hematology in 82.9% (68/82) of deliveries. The concordance rate between the two specialties was high at 95.7%. In two cases with discordant recommendations, neuraxial anesthesia was performed without hemostatic therapy, and no adverse events were reported. A diagnosis of bleeding disorder was established prior to the pregnancy in 79 deliveries (96.3%). Three patients were diagnosed after pregnancy: one with VWD-1 and baseline von Willebrand factor (VWF) activity at 0.34 IU/mL, one with VWD-1 and baseline VWF activity at 0.44 IU/mL, and one with Factor XIII deficiency and baseline levels at 0.42 IU/mL. They had neuraxial anesthesia administered without bleeding complications. Hemostasis laboratory values in the third trimester were considered adequate for neuraxial analgesia in 68.4% (54/79) of deliveries. Those who did not reach adequate levels received hemostatic therapy support prior to neuraxial analgesia, except for three patients (one with VWD-1 with VWF activity &amp;lt; 0.50 IU/mL, one with VWD type 2 with VWF activity &amp;lt; 0.50 IU/mL, one with factor XI deficiency and levels &amp;lt; 0.40 IU/mL) who fortunately did not develop post-procedural hematomas. We observed no bleeding events following neuraxial analgesia, but there were two adverse events (pain at epidural site) necessitating emergency room visits. Conclusions: Our study represents the largest cohort of pregnant individuals with bleeding disorders receiving neuraxial anesthesia and provides evidence supporting the safe administration of neuraxial analgesia, with high level of adherence to antenatal recommendations. In most cases, hemostatic laboratory values were deemed adequate, obviating the need for additional hemostatic therapy support. These findings contribute to our understanding of obstetric anesthesia practices in this population, thereby facilitating the provision of safe and effective analgesia during labor and delivery. Future prospective and multicenter studies focusing on the obstetrical management of pregnant persons with bleeding disorders, with specific emphasis on neuraxial analgesia, are warranted.