COVID-19 increases extracorporeal coagulation during hemodialysis associated with upregulation of vWF/FBLN5 signaling in patients with severe/critical symptoms.

BACKGROUND: COVID-19 has been shown to increase the risk of extracorporeal coagulation during hemodialysis in patients, but the underlying mechanism remains unclear. This study aimed to investigate the effect and mechanism of COVID-19 on the risk of extracorporeal coagulation in patients with chronic kidney disease undergoing hemodialysis. METHODS: A retrospective analysis of the extracorporeal coagulation status of 339 hemodialysis patients at our center before and after COVID-19 infection was performed, including subgroup analyses. Post-infection blood composition was analyzed by protein spectrometry and ELISA. RESULTS: Compared to the pre-COVID-19 infection period, COVID-19-induced extracorporeal coagulation predominantly occurred in patients with severe/critical symptoms. Further proteomic analysis demonstrated that in patients with severe/critical symptoms, the coagulation cascade reaction, platelet activation, inflammation, and oxidative stress-related pathways were significantly amplified compared to those in patients with no/mild symptoms. Notably, the vWF/FBLN5 pathway, which is associated with inflammation, vascular injury, and coagulation, was significantly upregulated. CONCLUSIONS: Patients with severe/critical COVID-19 symptoms are at a higher risk of extracorporeal coagulation during hemodialysis, which is associated with the upregulation of the vWF/FBLN5 signaling pathway. These findings highlight the importance of early anticoagulant therapy initiation in COVID-19 patients with severe/critical symptoms, particularly those undergoing hemodialysis. Additionally, vWF/FBLN5 upregulation may be a novel mechanism for virus-associated thrombosis/coagulation.

Early-onset indicators of a hypercoagulable state and clinical complications in a cohort of children with sickle cell trait.

Adults with sickle cell trait (SCT) have a procoagulant state with increased risk of thromboembolism, but limited data are available for children. We compared the coagulation profile of children with SCT, different sickle cell disease (SCD) genotypes, and healthy controls. Compared to controls and similarly to HbSC patients, 41 SCT children (mean age 6.85 years; 20 males; 88% Africans) had a characteristic procoagulant profile: higher levels of factor VIII, von Willebrand factor (VWF) Ag and CBA, D-dimer; lower levels of ADAMTS 13 activity, ADAMTS13 activity: VWFAg, plasminogen activator inhibitor, tissue plasminogen activator. Moreover, 13/41 had clinical complications of SCD, five requiring hospitalization.

Vascular study of decellularized porcine long bones: Characterization of a tissue engineering model.

INTRODUCTION: Massive bone allografts enable the reconstruction of critical bone defects in numerous conditions (e.g. tumoral, infection or trauma). Unfortunately, their biological integration remains insufficient and the reconstruction may suffer from several postoperative complications. Perfusion-decellularization emerges as a tissue engineering potential solution to enhance osseointegration. Therefore, an intrinsic vascular study of this novel tissue engineering tool becomes essential to understand its efficacy and applicability. MATERIAL AND METHODS: 32 porcine long bones (humeri and femurs) were used to assess the quality of their vascular network prior and after undergoing a perfusion-decellularization protocol. 12 paired bones were used to assess the vascular matrix prior (N = 6) and after our protocol (N = 6) by immunohistochemistry. Collagen IV, Von Willebrand factor and CD31 were targeted then quantified. The medullary macroscopic vascular network was evaluated with 12 bones: 6 were decellularized and the other 6 were, as control, not treated. All 12 underwent a contrast-agent injection through the nutrient artery prior an angio CT-scan acquisition. The images were processed and the length of medullary vessels filled with contrast agent were measured on angiographic cT images obtained in control and decellularized bones by 4 independent observers to evaluate the vascular network preservation. The microscopic cortical vascular network was evaluated on 8 bones: 4 control and 4 decellularized. After injection of gelatinous fluorochrome mixture (calcein green), non-decalcified fluoroscopic microscopy was performed in order to assess the perfusion quality of cortical vascular lacunae. RESULTS: The continuity of the microscopic vascular network was assessed with Collagen IV immunohistochemistry (p-value = 0.805) while the decellularization quality was observed through CD31 and Von Willebrand factor immunohistochemistry (p-values <0.001). The macroscopic vascular network was severely impaired after perfusion-decellularization; nutrient arteries were still patent but the amount of medullary vascular channels measured was significantly higher in the control group compared to the decellularized group (p-value <0.001). On average, the observers show good agreement on these results, except in the decellularized group where more inter-observer discrepancies were observed. The microscopic vascular network was observed with green fluoroscopic signal in almost every canals and lacunae of the bone cortices, in three different bone locations (proximal metaphysis, diaphysis and distal metaphysis). CONCLUSION: Despite the aggressiveness of the decellularization protocol on medullary vessels, total porcine long bones decellularized by perfusion retain an acellular cortical microvascular network. By injection through the intact nutrient arteries, this latter vascular network can still be used as a total bone infusion access for bone tissue engineering in order to enhance massive bone allografts prior implantation.

Comparison of thrombomodulin, vWF, and ADAMTS13 levels between preeclampsia and normal pregnancy

Objectives: To determine and compare plasma thrombomodulin, von Willebrand factor and von Willebrand factorcleaving protease levels between pre-eclamptic and healthy pregnant females. METHODS: The cross-sectional, comparative study was conducted at the Department of Haematology, University of Health Sciences, Lahore, Pakistan, from November 2019 to December 2020, and comprised pregnant females who were divided into healthy pregnant group A and pre-eclamptic group B. Plasma thrombomodulin and von Willebrand factor-cleaving protease levels were determined by using commercially available enzyme-linked immunosorbent assay kit, and von Willebrand factor level was determined by using immuno-turbidimetric assay kit. Data was analysed using SPSS 25. RESULTS: Of the 88 participants, there were 44(50%) females with mean age 25.5±6 years in group A and 44(50%) in group B with mean age 26±5 years. Median thrombomodulin level in group B was significantly higher than group A (p=0.003). Median von Willebrand factor-cleaving protease levels were lower in group B compared to group A (p=0.838). A significant difference in von Willebrand factor level was observed between the groups (p=0.038). Conclusion: Females with pre-eclampsia had significantly higher plasma levels of von Willebrand factor and thrombomodulin than healthy pregnant subjects.

Von Willebrand Factor (VWF) multiplex activity assay differentiation of type 1 von Willebrand Disease (VWD) and variant VWD.

INTRODUCTION: VWD diagnosis is challenging requiring multiple VWF activity tests using many individual assays. We have developed an ELISA-based VWF Multiplex Activity Assay (VWF-MAA) to address this concern; however, the ability of the VWF-MAA to discriminate between type 1 VWD, variant VWD, and normal subjects has not been evaluated. AIM: To evaluate the VWF-MAA and its ability to differentiate between type 1 VWD, variant VWD and normal subjects in individuals undergoing an initial laboratory evaluation for bleeding. METHODS: A total of 177 plasma samples from the Zimmerman Program: Comparative Effectiveness in the Diagnosis of VWD were evaluated from 11 centres across the US and Canada. The VWF-MAA was compared to Versiti Blood Research Institute (VBRI) and Local Center (LC) assigned VWD diagnosis. RESULTS: Overall, 129/177 (72.9%) were correctly assigned as normal (non-VWD), type 1, or variant VWD compared to the VBRI assigned diagnosis. VWF-MAA assigned non-VWD accurately in 29/57 (50.9%) samples, and type 1 VWD accurately in 93/110 (84.6%) samples. Considering LC diagnosis where there was agreement with VWF-MAA and not VBRI diagnosis, type 1 VWD was accurate in 105/110 (95.5%) samples. Bland-Altman analysis demonstrated good correlation between laboratory methods. VWD, types 2A, 2B, 1C VWD were also assigned by the VWF-MAA. CONCLUSIONS: We demonstrate that the VWF-MAA has utility in differentiating type 1 VWD, variant VWD and normal subjects in individuals undergoing an initial laboratory evaluation for bleeding.

Prognostic significance of circulating microparticles in IgA nephropathy.

PURPOSE: Endothelial injury, involved in the pathogenesis of renal fibrosis, can generate microparticles (MPs). These are 0.1-1 µm membrane-bound vesicles shed from the damaged or activated cell surfaces. We analyzed the presence of circulating MPs and EnMPs in IgAN and correlated with markers of endothelial injury and disease activity. METHODS: The study included 30 IgAN (mean age 31.5 ± 9 years), 25 healthy controls and Lupus nephritis (n = 10) as disease controls. Circulating MPs were quantitated by Flow cytometry and EnMPs were analyzed using anti-CD31-FITC and anti-CD146-PE antibodies. Their levels were correlated with serum von Willebrand Factor, histological Oxford MEST-C score and renal outcome. A prospective validation group of 20 patients of biopsy-proven IgA nephropathy was also included. RESULTS: IgAN had significantly higher levels of MPs, EnMPs and vWF compared to controls. On multivariate analysis, plasma levels of total MPs, EnMPs and serum vWF correlated significantly with the presence of hypertension and E1 on histology. E1 and high MPs (> 130 counts/µl) were associated with shorter time to doubling of serum creatinine. MPs cutoff level of 130 counts/µl had a sensitivity of 75%, specificity of 93.3% and diagnostic accuracy of 89.5% for E1 in the validation cohort. CONCLUSION: Circulating MPs and EnMPs in IgAN correlate with E1 on histology and have a potential as non-invasive biomarkers to predict disease activity and renal outcome.

The common VWF variant p.Y1584C: detailed pathogenic examination of an enigmatic sequence change.

BACKGROUND: As knowledge of the human genome has advanced, so too has the recognition that interpretation of the pathogenic nature of sequence variants can be challenging. The von Willebrand factor (VWF) gene exhibits a significant degree of sequence variability, and the first VWF variant associated with type 1 von Willebrand disease (VWD), c.4751 A>G, p.Y1584C, was described in 2003. However, since that time, the pathogenic nature of this variant has remained unclear, being assigned properties ranging from a risk factor to a pathogenic variant. OBJECTIVES: To provide additional evaluation on the interpretation of pathogenicity for this common VWF variant. METHODS: Fifty-eight subjects with only the p.Y1584C variant were recruited from 2 cohort studies (the Zimmerman Program and the Canadian type 1 VWD study). Clinical and laboratory phenotypes were assessed. RESULTS: The prevalence of the p.Y1584C variant in our cohorts was 23- to 27-fold higher than that in large normal population databases. Significantly more p.Y1584C subjects had an abnormal bleeding score when compared to Y1584 individuals. In comparison with a group of 35 subjects without the p.Y1584C variant, subjects with the variant had lower mean VWF:antigen and VWF:ristocetin cofactor values and significantly higher VWF propeptide/VWF:antigen ratios suggestive of enhanced clearance. CONCLUSION: Collectively, the results of this analysis suggest that p.Y1584C is likely pathogenic, however, due to influences such as incomplete penetrance, variable expressivity, and other genetic modifiers like ABO blood group, the straightforward assignment of pathogenicity to this variant is inevitably challenging.

A multitrait genetic study of hemostatic factors and hemorrhagic transformation after stroke treatment.

BACKGROUND: Thrombolytic recombinant tissue plasminogen activator (r-tPA) treatment is the only pharmacologic intervention available in the ischemic stroke acute phase. This treatment is associated with an increased risk of intracerebral hemorrhages, known as hemorrhagic transformations (HTs), which worsen the patient's prognosis. OBJECTIVES: To investigate the association between genetically determined natural hemostatic factors' levels and increased risk of HT after r-tPA treatment. METHODS: Using data from genome-wide association studies on the risk of HT after r-tPA treatment and data on 7 hemostatic factors (factor [F]VII, FVIII, von Willebrand factor [VWF], FXI, fibrinogen, plasminogen activator inhibitor-1, and tissue plasminogen activator), we performed local and global genetic correlation estimation multitrait analyses and colocalization and 2-sample Mendelian randomization analyses between hemostatic factors and HT. RESULTS: Local correlations identified a genomic region on chromosome 16 with shared covariance: fibrinogen-HT, P = 2.45 × 10-11. Multitrait analysis between fibrinogen-HT revealed 3 loci that simultaneously regulate circulating levels of fibrinogen and risk of HT: rs56026866 (PLXND1), P = 8.80 × 10-10; rs1421067 (CHD9), P = 1.81 × 10-14; and rs34780449, near ROBO1 gene, P = 1.64 × 10-8. Multitrait analysis between VWF-HT showed a novel common association regulating VWF and risk of HT after r-tPA at rs10942300 (ZNF366), P = 1.81 × 10-14. Mendelian randomization analysis did not find significant causal associations, although a nominal association was observed for FXI-HT (inverse-variance weighted estimate [SE], 0.07 [-0.29 to 0.00]; odds ratio, 0.87; 95% CI, 0.75-1.00; raw P = .05). CONCLUSION: We identified 4 shared loci between hemostatic factors and HT after r-tPA treatment, suggesting common regulatory mechanisms between fibrinogen and VWF levels and HT. Further research to determine a possible mediating effect of fibrinogen on HT risk is needed.

Proteomic profiling of serum exosomes reveals acute phase response and promotion of inflammatory and platelet activation pathways in patients with heat stroke.

Background: The pathological mechanism of heat stroke (HS) involves the acute phase response, unbalanced immunological/inflammatory reactions, and coagulation initiation, especially platelet activation. Although exosomes contain proteins involved in these biological processes, their protein cargo levels and potential roles in HS remain unknown. This study explored the serum exosome protein expression patterns after HS and their potential roles in the pathogenesis of HS. Methods: Blood samples were collected from ten patients diagnosed with HS upon admission to the intensive care unit (six with severe HS and four with mild HS). Samples from six healthy volunteers were included as control. Using ultracentrifugation, exosomes were prudently isolated, and their protein contents were profiled using liquid chromatography-tandem mass spectrometry analysis with isobaric tags for relative and absolute quantification-based proteomics. Results: Compared with healthy volunteers, patients with HS showed significant changes in the levels of 33 exosomal proteins (23 upregulated and 10 downregulated). The most upregulated proteins included serum amyloid A-1 (SAA-1), von Willebrand factor (vWF), S100A8, and histone H3. In addition, SAA-1, vWF, platelet membrane glycoprotein, S100A8, and histone H3 were more enriched in the exosomes from patients with severe HS than from those with mild HS. Gene ontology analysis revealed that the HS-modulated exosomal proteins were mostly related to inflammatory response, including the acute-phase response, platelet activation/degranulation, and innate immune response. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed significant enrichment of proteins in the IL-17 signaling pathway, platelet activation, neutrophil extracellular trap formation, Fc epsilon RI signaling pathway, chemokine signaling pathway, and NOD-like receptor signaling pathway, among others. Several serum exosomal proteins, including SAA-1, vWF, and S100A8, which are related to the acute phase, inflammatory response, and platelet activation, were confirmed to be elevated in patients with HS, and were significantly correlated with disease severity, organ dysfunction, and death. Conclusion: Overall, this study explores the potential role of the serum exosomal proteome in the inflammatory response and platelet activation in HS, suggests the pathological mechanisms underlying HS-induced injuries, and recommends reliable exosomal biomarkers for predicting HS prognosis.

[Molecular dynamics simulation of force-regulated interaction between glycoprotein Ib α and filamin].

In resting platelets, the 17 th domain of filamin a (FLNa17) constitutively binds to the platelet membrane glycoprotein Ibα (GPIbα) at its cytoplasmic tail (GPIbα-CT) and inhibits the downstream signal activation, while the binding of ligand and blood shear force can activate platelets. To imitate the pull force transmitted from the extracellular ligand of GPIbα and the lateral tension from platelet cytoskeleton deformation, two pulling modes were applied on the GPIbα-CT/FLNa17 complex, and the molecular dynamics simulation method was used to explore the mechanical regulation on the affinity and mechanical stability of the complex. In this study, at first, nine pairs of key hydrogen bonds on the interface between GPIbα-CT and FLNa17 were identified, which was the basis for maintaining the complex structural stability. Secondly, it was found that these hydrogen bonding networks would be broken down and lead to the dissociation of FLNa17 from GPIbα-CT only under the axial pull force; but, under the lateral tension, the secondary structures at both terminals of FLNa17 would unfold to protect the interface of the GPIbα-CT/FLNa17 complex from mechanical damage. In the range of 0~40 pN, the increase of pull force promoted outward-rotation of the nitrogen atom of the 563 rd phenylalanine (PHE 563-N) at GPIbα-CT and the dissociation of the complex. This study for the first time revealed that the extracellular ligand-transmitted axial force could more effectively relieve the inhibition of FLNa17 on the downstream signal of GPIbα than pure mechanical tension at the atomic level, and would be useful for further understanding the platelet intracellular force-regulated signal pathway.

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

BACKGROUND: Genetic 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. METHOD: The 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. RESULTS: The 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. CONCLUSION: According 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.

A novel von Willebrand factor multimer ratio as marker of disease activity in thrombotic thrombocytopenic purpura.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP), an autoantibody-mediated severe ADAMTS13 deficiency, is caused by insufficient proteolytic processing of von Willebrand factor (VWF) multimers (MMs) and microvascular thrombi. Recurrence of acute iTTP is associated with persistence or reappearance of ADAMTS13 deficiency. Some patients remain in remission despite recurring or persisting severe ADAMTS13 deficiency. In a prospective 2-year observational study, we investigated VWF MM patterns and ADAMTS13 in patients with iTTP in remission and at acute episodes. Of the 83 patients with iTTP, 16 suffered 22 acute episodes whereas 67 remained in clinical remission during follow-up, including 13 with ADAMTS13 <10% and 54 with ADAMTS13 ≥10%. High -molecular weight to low-molecular weight VWF MM ratio based on sodium dodecyl sulfate-agarose gel electrophoresis was compared with ADAMTS13 activity. VWF MM ratio was significantly higher in patients in remission with <10% compared with ≥10% ADAMTS13 activity. Fourteen samples obtained from 13 to 50 days (interquartile range; median, 39) before acute iTTP onset (ADAMTS13 <10% in 9 patients and 10%-26% in 5) showed VWF MM ratios significantly higher than those from 13 patients remaining in remission with ADAMTS13 <10%. At acute iTTP onset, VWF MM ratio decreased significantly and was low in all patients despite <10% ADAMTS13. The VWF MM ratio does not depend exclusively on ADAMTS13 activity. The disappearance of high molecular weight VWF MMs resulting in low VWF MM ratio at iTTP onset may be explained by consumption of larger VWF MMs in the microcirculation. The very high VWF MM ratio preceding acute iTTP recurrence suggests that VWF processing is hampered more than in patients remaining in remission.

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

INTRODUCTION: 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 ß-thalassemia major patients. A possible association between TSP-1 and vWF and ADAMTS-13 was also evaluated. METHODS: The study was conducted on 80 ß-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. RESULTS: 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. CONCLUSION: Our findings highlight the adequacy of patient management protocols for ß-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.

In vitro comparative study of red blood cell and VWF damage on 3D printing biomaterials under different blood-contacting conditions.

Mechanical circulatory support devices (MCSDs) are often associated with hemocompatible complications such as hemolysis and gastrointestinal bleeding when treating patients with end-stage heart failure. Shear stress and exposure time have been identified as the two most important mechanical factors causing blood damage. However, the materials of MCSDs may also induce blood damage when contacting with blood. In this study, the red blood cell and von Willebrand Factor (VWF) damage caused by four 3D printing biomaterials were investigated, including acrylic, PCISO, Somos EvoLVe 128, and stainless steel. A roller pump circulation experimental platform and a rotor blood-shearing experimental platform were constructed to mimic static and dynamic blood-contacting conditions of materials in MCSDs, respectively. Free hemoglobin assay and VWF molecular weight analysis were performed on the experimental blood samples. It indicated that different 3D printing materials and technology could induce different levels of damage to red blood cells and VWF, with acrylic causing the least damage under both static and dynamic conditions. In addition, it was found that blood damage measured for the same material differed on the two platforms. Therefore, a combination of static and dynamic experiments should be used to comprehensively investigate the effects of blood damage caused by the material. It can provide a reference for the design and evaluation of materials in different components of MCSDs.

Construction and Validation of the Nomogram Based on von Willebrand Factor Predicting Mortality in Patients with Heatstroke.

Heatstroke (HS), a severe condition, can develop multiple organ dysfunction syndrome and death. However, at present, no early reliable index exists for risk stratification and prognosis. von Willebrand factor (vWF), a marker of vascular endothelial injury, is a key regulatory target of inflammation and coagulation, which is closely associated with the pathogenesis of HS. vWF was reported as a prognostic marker in several infectious and noninfectious severe illness such as COVID-19, sepsis, and trauma. Although early increased level of vWF is seen in HS, the relationship between vWF and mortality is to be elucidated. Clinical data of patients with HS in a tertiary hospital were recorded and analyzed. It was shown that plasma vWF concentrations at admission were significantly increased in the nonsurvivors (351% ± 105%) compared with survivors (278% ± 104%, p = 0.021). After multivariate logistic regression analysis it was shown that vWF (odds ratio [OR] = 1.010; 95% confidence interval [CI], 1.002-1.18; p = 0.017), hemoglobin (Hb) (OR = 0.954; 95% CI, 0.931-0.979; p < 0.001), and hematocrit (HCT) in blood (OR = 0.859; 95% CI, 0.790-0.934; p < 0.001) were independent factors of in-hospital mortality in HS. The nomogram based on vWF and Hb was constructed in patients with HS. The area under curve under the receiver operating characteristic of this prediction model was 0.860 (95% CI, 0.773-0.923) and cutoff was 0.15, with Youden index 0.5840, which were not significantly different to sequential organ failure assessment (p = 0.0644), Acute Physiology and Chronic Health Evaluation II (APACHE II) (p = 0.7976), and systemic inflammatory response syndrome (SIRS) scores (p = 0.3274). The prediction model that integrated vWF and Hb showed a better predicting efficiency than single variable, and a higher specificity (81.48%) than APACHE II (72.84%) and SIRS (72.84%) scores. In summary, vWF, as an independent risk factor for in-hospital mortality, combined with Hb, could effectively prognosis the mortality in HS patients at early stage.

A Fluorescence Cross-Correlation-Spectroscopy-Based Immunoassay for Rapid, Selective, and Accurate Protein Sizing in Human Plasma, Applied to the von Willebrand Factor.

Multimeric abnormalities in plasma von Willebrand factor (VWF) cause bleeding or clotting disorders. Electrophoretic analysis of multimers is used to detect such abnormalities but is qualitative, slow, and difficult to standardize. Fluorescence correlation spectroscopy (FCS) is a good alternative but is affected by low selectivity and concentration bias. Here, we report the development of a homogeneous immunoassay based on dual-color fluorescence cross-correlation spectroscopy (FCCS) that overcomes these challenges. By performing a mild denaturation treatment followed by reacting with polyclonal antibodies, the concentration bias was drastically reduced. The use of a dual antibody assay improved selectivity. Diffusion times of immunolabeled VWF were measured with FCCS and standardized relative to calibrator measurements. The assay measures size changes in VWF using 1 µL of plasma and less than 10 ng of antibody per measurement and was validated over a 16-fold range of VWF antigen concentration (VWF:Ag), with a sensitivity of VWF:Ag 0.8%. Concentration bias and imprecision were less than 10%. Measurements were unaffected by hemolytic, icteric, or lipemic interference. Strong correlations were obtained with reference densitometric readouts (0.97 for calibrators, 0.85 for clinical samples), and significant differences were found between normal (n = 10), type 2A (n = 5), and type 2B (n = 5) von Willebrand's disease and acquired thrombotic thrombocytopenic purpura (n = 10) samples (p < 0.01). This FCCS based immunoassay accurately and selectively determines changes in the multimeric status of plasma VWF and may be used as a simpler, faster, and a standardizable alternative for multimer analysis, following further clinical validation in larger cohorts.

Changes and significance of vascular endothelial injury markers in patients with diabetes mellitus and pulmonary thromboembolism.

BACKGROUND: To investigate the changes and clinical significance of vascular endothelial injury markers in type 2 diabetes mellitus (T2DM) complicated with pulmonary embolism (PE). METHODS: This prospective study enrolled patients with T2DM hospitalized in one hospital from January 2021 to June 2022. Soluble thrombomodulin (sTM) (ELISA), von Willebrand factor (vWF) (ELISA), and circulating endothelial cells (CECs) (flow cytometry) were measured. PE was diagnosed by computed tomography pulmonary angiography (CTPA). RESULTS: Thirty participants were enrolled in each group. The plasma levels of sTM (151.22 ± 120.57 vs. 532.93 ± 243.82 vs. 1016.51 ± 218.00 pg/mL, P < 0.001) and vWF (9.63 ± 2.73 vs. 11.50 ± 2.17 vs. 18.02 ± 3.40 ng/mL, P < 0.001) and the percentage of CECs (0.17 ± 0.46 vs. 0.30 ± 0.08 vs. 0.56 ± 0.18%, P < 0.001) gradually increased from the control group to the T2DM group to the T2DM + PE group. sTM (OR = 1.002, 95%CI: 1.002-1.025, P = 0.022) and vWF (OR = 1.168, 95%CI: 1.168-2.916, P = 0.009) were associated with T2DM + PE. sTM > 676.68 pg/mL for the diagnosis of T2DM + PE achieved an AUC of 0.973, while vWF > 13.75 ng/mL achieved an AUC of 0.954. The combination of sTM and vWF above their cutoff points achieved an AUC of 0.993, with 100% sensitivity and 96.7% specificity. CONCLUSIONS: Patients with T2DM show endothelial injury and dysfunction, which were worse in patients with T2DM and PE. High sTM and vWF levels have certain clinical predictive values for screening T2DM accompanied by PE.

Primary hemostasis dysfunctions and bleeding risk in newly diagnosed acute myeloid leukemia.

BACKGROUND: Acute myeloid leukaemia carries the risk of complications associated with dysfunctions in haemostasis system. The purpose of this study was to investigate the factors associated with the risk of bleeding in patients with newly diagnosed acute myeloid leukaemia (AML). METHODS: This study involved the methods of immunoenzymatic analysis and classical coagulation studies. The number of biochemical parameters important for establishing coagulative dysfunction in acute myeloid leukaemia was determined, the main ones being the level of von Willebrand factor, the Ristocetin-cofactor activity of von Willebrand factor and factor VIII activity, prothrombin time, platelet count, and fibrinogen concentration. RESULTS: According to the results of the present study, the reduced activity of von Willebrand factor in patients with AML was associated with severe bleeding. The authors observed an increase in the number of platelets count in patients with AML who experienced haemorrhages compared to patients with no bleeding signs. The study also established an increase in the concentration of fibrinogen in cancer patients, compared to the control sample. Symptoms and quantitative indicators for diagnosing the severity of haemorrhagic syndrome were grouped. The authors considered the advantages and disadvantages of many therapeutic preparations and focussed on specific markers of activated haemorrhage-predicting platelets. CONCLUSION: Further studies concern the search for effective markers and therapeutic approaches to minimize haemorrhagic syndrome. The results were statistically processed using the functions ANOVA, t test, CORREL, determination of the value of reliability, and mean square deviation.

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

BACKGROUND: Hereditary 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. OBJECTIVES: To 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. METHODS: This 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. RESULTS: Fourteen 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. CONCLUSION: Elevated 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.

Identification of thrombotic biomarkers in orthopedic surgery patients by plasma proteomics.

BACKGROUND: Due to the poor specificity of D-dimer, more accurate thrombus biomarkers are clinically needed to improve the diagnostic power of VTE. METHODS: The plasma samples were classified into low-risk group (n = 6) and high-risk group (n = 6) according to the Caprini Thrombosis Risk Assessment Scale score. Data-independent acquisition mass spectrometry (DIA-MS) was performed to identify the proteins in the 12 plasma samples. Bioinformatics analysis including volcano plot, heatmap, KEGG pathways and chord diagram analysis were drawn to analyze the significantly differentially expressed proteins (DEPs) between the two groups. Then, another 26 plasma samples were collected to verify the key proteins as potential biomarkers of VTE in orthopedic surgery patients. RESULTS: A total of 371 proteins were identified by DIA-MS in 12 plasma samples. Volcano plotting showed that there were 30 DEPs. KEGG pathway enrichment analysis revealed that the DEPs were majorly involved in the blood coagulation pathway. The chord diagram analysis demonstrated that proteins SAA1, VWF, FLNA, ACTB, VINC, F13B, F13A and IPSP in the DEPs were significantly related to blood coagulation. VWF and F13B were selected for validation experiments. ELISA test showed that, as compared with those in the low-risk group, the level of VWF in the high-risk sera was significantly increased. CONCLUSIONS: The level of VWF in the high-risk group of thrombosis after orthopedic surgery was significantly higher than that in the low-risk group of preoperative thrombosis, suggesting that VWF may be used as a potential thrombus biomarker in orthopedic surgery patients.