Von Willebrand Factor Research Articles

Investigation of von Willebrand factor multimer abnormalities before and after aortic valve replacement using the Hydragel-5 assay

BackgroundSevere aortic stenosis (sAS) is associated with acquired von Willebrand syndrome (AVWS) by loss of high-molecular-weight multimers (HMWM) of von Willebrand factor (VWF), potentially resulting in perioperative bleeding. Analysis of VWF multimers remains challenging. Recently, the new, rapid Hydragel 5 assay has been developed, using electrophoretic protein separation for dividing VWF-multimers into low (LMWM), intermediate (IMWM), and HMWM, the hemostatically active part of VWF. Here, we evaluated its impact on predicting blood loss in presence of AVWS after surgical aortic valve replacement (SAVR). MethodsWe prospectively examined 52 patients (age: 68 ± 7 years; 54 % male) admitted to SAVR. They were divided in two groups (A: normal VWF, n = 28; B: abnormal VWF, n = 24, defined as VWF-activity/antigen (VWF:Ac/Ag)-ratio < 0.7 and/or HMWM loss). Blood samples and echocardiographic data were collected before, seven days and three months after SAVR. Blood loss and transfusions were recorded. ResultsBaseline characteristics and clinical data were similar in both groups. HMWM loss was present in 38.5 % of all patients. HMWM, the VWF:Ac/Ag- and HMWM/(IMWM+LMWM)-ratios were significantly decreased preoperatively in group B but normalized after SAVR. Bleeding, re-thoracotomy and transfusion rates were comparable. HMWM loss was inversely correlated with the peak aortic gradient (Pmax) and positively with the aortic valve area (AVA), while HMWM/(IMWM+LMWM)-ratio negatively correlated with the mean aortic gradient (Pmean). ConclusionHMWM and HMWM/(IMWM+LMWM)-ratio inversely correlate with severity of AS and normalize after SAVR. The Hydragel-5 assay's might be valuable for routine diagnostics to assess bleeding risk and postoperative normalization of AS and VWF abnormalities in SAVR patients.

Innovative approaches to assessing risk factors, diagnostics and treatment of neonatal thrombosis

Compared to children of other ages, neonates especially seriously ill and premature subjects comprise a high thrombotic risk group. A decline in the incidence of neonatal thrombosis may be accounted for by improved treatment of severe conditions in newborns and increased survival of premature infants. Neonatal and adult hemostasis exhibit distinct physiological features: difference in concentration, synthesis rate of blood coagulation factors, metabolic rate, thrombin and plasmin levels. At the same time, neonatal threshold values for natural blood coagulation inhibitors (protein C, protein S, antithrombin, heparin cofactor II) and vitamin K-dependent coagulation factors (FII, FVII, FIX, FX) are quite low, whereas that of FVIII and von Willebrand factor exceeds those found in adults. Thus, newborns have lower plasma fibrinolytic activity. The main risk factors for developing thrombotic complications are as follows: central venous catheters, altered body fluid volume, liver disease, as well as sepsis and inflammatory processes particularly COVID-19. The significance of congenital and acquired maternal and neonatal thrombophilia may pose an additional risk factor for thrombotic complications. Low-molecular weight heparins are the first-choice drugs in treatment and prevention of neonatal thrombosis.

Endothelial biomarkers (Von willebrand factor, BDCA3, urokinase) as predictors of mortality in COVID-19 patients: cohort study

BackgroundSARS-CoV-2 is a systemic disease that affects endothelial function and leads to coagulation disorders, increasing the risk of mortality. Blood levels of endothelial biomarkers such as Von Willebrand Factor (VWF), Thrombomodulin or Blood Dendritic Cell Antigen-3 (BDCA3), and uUokinase (uPA) increase in patients with severe disease and can be prognostic indicators for mortality. Therefore, the aim of this study was to determine the effect of VWF, BDCA3, and uPA levels on mortality.MethodsFrom May 2020 to January 2021, we studied a prospective cohort of hospitalized adult patients with polymerase chain reaction (PCR)-confirmed COVID-19 with a SaO2 ≤ 93% and a PaO2/FiO2 ratio < 300. In-hospital survival was evaluated from admission to death or to a maximum of 60 days of follow-up with Kaplan-Meier survival curves and Cox proportional hazard models as independent predictor measures of endothelial dysfunction.ResultsWe recruited a total of 165 subjects (73% men) with a median age of 57.3 ± 12.9 years. The most common comorbidities were obesity (39.7%), hypertension (35.4%) and diabetes (30.3%). Endothelial biomarkers were increased in non-survivors compared to survivors. According to the multivariate Cox proportional hazard model, those with an elevated VWF concentration ≥ 4870 pg/ml had a hazard ratio (HR) of 4.06 (95% CI: 1.32–12.5) compared to those with a lower VWF concentration adjusted for age, cerebrovascular events, enoxaparin dose, lactate dehydrogenase (LDH) level, and bilirubin level. uPA and BDCA3 also increased mortality in patients with levels ≥ 460 pg/ml and ≥ 3600 pg/ml, respectively.ConclusionThe risk of mortality in those with elevated levels of endothelial biomarkers was observable in this study.

Implications of von Willebrand Factor in Inflammatory Bowel Diseases: Beyond Bleeding and Thrombosis.

Inflammatory bowel disease (IBD) displays an increased venous and arterial thrombotic risk despite the common occurrence of intestinal bleeding. While some of the mechanisms leading to these thrombotic complications have been studied, other specific changes in the hemostasis profile of IBD patients have been less explored. One such example relates to von Willebrand factor (VWF) whose plasma levels have been reported to be modulated in IBD. Von Willebrand factor is a plasma glycoprotein crucial for hemostatic functions via roles both in platelet function and coagulation. High plasma VWF is a known risk factor for venous thromboembolism. In addition to its canonical roles in hemostasis, VWF is known to be directly or indirectly involved in other vascular processes such as maintenance of endothelial barrier integrity or proliferation of vascular smooth muscle cells. The purpose of this review is to recapitulate and update the existing data about VWF biology in IBD and to highlight its role both in the existing procoagulant phenotype and in vascular alterations that may occur in IBD.

Cryptic Extensibility in von Willebrand Factor Revealed by Molecular Nanodissection.

Von Willebrand factor (VWF) is a multimer with a variable number of protomers, each of which is a head-to-head dimer of two multi-domain monomers. VWF responds to shear through the unfolding and extension of distinct domains, thereby mediating platelet adhesion and aggregation to the injured blood vessel wall. VWF's C1-6 segment uncoils and then the A2 domain unfolds and extends in a hierarchical and sequential manner. However, it is unclear whether there is any reservoir of further extensibility. Here, we explored the presence of cryptic extensibility in VWF by nanodissecting individual, pre-stretched multimers with atomic force microscopy (AFM). The AFM cantilever tip was pressed into the surface and moved in a direction perpendicular to the VWF axis. It was possible to pull out protein loops from VWF, which resulted in a mean contour length gain of 217 nm. In some cases, the loop became cleaved, and a gap was present along the contour. Frequently, small nodules appeared in the loops, indicating that parts of the nanodissected VWF segment remained folded. After analyzing the nodal structure, we conclude that the cryptic extensibility lies within the C1-6 and A1-3 regions. Cryptic extensibility may play a role in maintaining VWF's functionality in extreme shear conditions.

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

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

Directed evolution of hyperactive integrases for site specific insertion of transgenes.

The ability to deliver large transgenes to a single genomic sequence with high efficiency would accelerate biomedical interventions. Current methods suffer from low insertion efficiency and most rely on undesired double-strand DNA breaks. Serine integrases catalyze the insertion of large DNA cargos at attachment (att) sites. By targeting att sites to the genome using technologies such as prime editing, integrases can target safe loci while avoiding double-strand breaks. We developed a method of phage-assisted continuous evolution we call IntePACE, that we used to rapidly perform hundreds of rounds of mutagenesis to systematically improve activity of PhiC31 and Bxb1 serine integrases. Novel hyperactive mutants were generated by combining synergistic mutations resulting in integration of a multi-gene cargo at rates as high as 80% of target chromosomes. Hyperactive integrases inserted a 15.7 kb therapeutic DNA cargo containing von Willebrand Factor. This technology could accelerate gene delivery therapeutics and our directed evolution strategy can easily be adapted to improve novel integrases from nature.

Aptamer Proteomics for Biomarker Discovery in Heart Failure With Preserved Ejection Fraction: The PARAGON-HF Proteomic Substudy.

Prognostic markers and biological pathways linked to detrimental clinical outcomes in heart failure with preserved ejection fraction (HFpEF) remain incompletely defined. We measured serum levels of 4123 unique proteins in 1117 patients with HFpEF enrolled in the PARAGON-HF (Efficacy and Safety of LCZ696 Compared to Valsartan, on Morbidity and Mortality in Heart Failure Patients With Preserved Ejection Fraction) trial using a modified aptamer proteomic assay. Baseline circulating protein concentrations significantly associated with the primary end point and the timing and occurrence of total heart failure hospitalization and cardiovascular death were identified by recurrent events regression, accounting for multiple testing, adjusted for age, sex, treatment, and anticoagulant use, and compared with published analyses in 2515 patients with heart failure with reduced ejection fraction from the PARADIGM-HF (Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure) and ATMOSPHERE (Efficacy and Safety of Aliskiren and Aliskiren/Enalapril Combination on Morbidity-Mortality in Patients With Chronic Heart Failure) clinical trials. We identified 288 proteins that were robustly associated with the risk of heart failure hospitalization and cardiovascular death in patients with HFpEF. The baseline proteins most strongly related to outcomes included B2M (β-2 microglobulin), TIMP1 (tissue inhibitor of matrix metalloproteinase 1), SERPINA4 (serpin family A member 4), and SVEP1 (sushi, von Willebrand factor type A, EGF, and pentraxin domain containing 1). Overall, the protein-outcome associations in patients with HFpEF did not markedly differ as compared with patients with heart failure with reduced ejection fraction. A proteomic risk score derived in patients with HFpEF was not superior to a previous proteomic score derived in heart failure with reduced ejection fraction nor to clinical risk factors, NT-proBNP (N-terminal pro-B-type natriuretic peptide), or high-sensitivity cardiac troponin. Numerous serum proteins linked to metabolic, coagulation, and extracellular matrix regulatory pathways were associated with worse HFpEF prognosis in the PARAGON-HF proteomic substudy. Our results demonstrate substantial similarities among serum proteomic risk markers for heart failure hospitalization and cardiovascular death when comparing clinical trial participants with heart failure across the ejection fraction spectrum. URL: https://www.clinicaltrials.gov; Unique Identifiers: NCT01920711, NCT01035255, NCT00853658.

Excessive cleavage of von Willebrand factor multimers by ADAMTS13 may predict the progression of transplant-associated thrombotic microangiopathy

BackgroundTransplant-associated thrombotic microangiopathy (TA-TMA) is a fatal complication of hematopoietic stem cell transplantation and is characterized by severe thrombocytopenia, hemolytic anemia, and organ dysfunction. In response to several possible triggers, dynamic multimetric change in von Willebrand factor (VWF) may contribute to inducing microthrombi in circulation in TA-TMA. ObjectivesBy performing VWF multimer analysis and measuring VWF-degradation product (DP), we unraveled the relationship between multimeric changes in circulating VWF and the pathogenesis of TA-TMA. MethodsThis study analyzed 135 plasma samples from 14 patients who underwent allogeneic hematopoietic stem cell transplantation at a single institute. VWF-associated markers, namely VWF:antigen (VWF:Ag), VWF-DP/VWF:Ag ratio, VWF:ristocetin cofactor activity, VWF:ristocetin cofactor activity/VWF:Ag ratio, and ADAMTS13 activity, were analyzed in these samples collected every 7 days. ResultsThere were 2 patients with definite thrombotic microangiopathy (TMA) and 6 patients who presented with probable TMA that did not progress to definite TMA. Each plasma sample was classified into 3 groups: definite TMA, probable TMA, and non-TMA. VWF multimer analysis showed the absence of high-molecular-weight VWF multimers in probable TMA, whereas the appearance of unusually large VWF multimers was observed in definite TMA. The median value of the VWF-DP/VWF:Ag ratio in probable TMA was elevated to 4.17, suggesting that excessive cleavage of VWF multimers by VWF cleaving enzyme, ADAMTS13, resulted in the loss of high-molecular-weight VWF multimers. ConclusionDuring the transition from probable to definite TMA, drastic VWF multimer changes imply a switch from bleeding to thrombotic tendencies. Extensive VWF-DP and VWF multimer analyses provided novel insights.

Current Diagnosis of von Willebrand Disease in Italy: 3 Years Following the Release of the International Guidelines.

The American Society of Hematology-International Society on Thrombosis and Haemostasis-National Hemophilia Foundation-World Federation of Hemophilia 2021 International Guidelines (IGL) on von Willebrand disease (VWD) have pointed out many challenges, mainly in the diagnostic approach of VWD patients. To determine the impact of these IGL on the current clinical and laboratory diagnosis of Italian VWD patients, we have recently conducted a survey among 43 centers affiliated with the Italian Association of Hemophilia Centers (AICE). Directors and colleagues responsible for the management of VWD patients were invited to report in a detailed questionnaire how IGL recommendations about the assessment of the specific activities of von Willebrand Factor (VWF) could be applied at their local sites. Results from such a survey showed that bleeding assessment tools, VWF antigen, and factor VIII procoagulant are currently in use in all centers. The automated assays for platelet-dependent VWF activity with or without ristocetin described in IGL have been used since 2021 in 37/43 (86%) centers. Among other laboratory tests, VWF collagen binding, ristocetin-induced platelet agglutination, multimeric analysis, VWF propeptide, VWF:FVIII binding assay were available in 49, 63, 26, 7, and 28% of AICE, respectively. Analyses of VWF gene defects are available only at 3/43 (7%) centers. Desmopressin (DDAVP) infusion trials at diagnosis, with measurements of VWF activities at 1 and 4 hours post-DDAVP, is currently performed at 38/43 (88%) centers. Based on this information, a simplified clinical diagnosis using a few automated tests before and after DDAVP has been proposed. Such a diagnostic approach will be validated prospectively in a large cohort of Italian VWD patients.

Recommendations on Monitoring and Replacement of Antithrombin, Fibrinogen, and Von Willebrand Factor in Pediatric Patients on Extracorporeal Membrane Oxygenation: The Pediatric Extracorporeal Membrane Oxygenation Anticoagulation CollaborativE Consensus Conference.

To derive systematic review informed, modified Delphi consensus regarding monitoring and replacement of specific coagulation factors during pediatric extracorporeal membrane oxygenation (ECMO) support for the Pediatric ECMO Anticoagulation CollaborativE. A structured literature search was performed using PubMed, Embase, and Cochrane Library (CENTRAL) databases from January 1988 to May 2020, with an update in May 2021. Included studies assessed monitoring and replacement of antithrombin, fibrinogen, and von Willebrand factor in pediatric ECMO support. Two authors reviewed all citations independently, with conflicts resolved by a third reviewer if required. Twenty-nine references were used for data extraction and informed recommendations. Evidence tables were constructed using a standardized data extraction form. Risk of bias was assessed using the Quality in Prognosis Studies tool. The evidence was evaluated using the Grading of Recommendations Assessment, Development, and Evaluation system. A panel of 48 experts met over 2 years to develop evidence-based recommendations and, when evidence was lacking, expert-based consensus statements. A web-based modified Delphi process was used to build consensus via the Research And Development/University of California Appropriateness Method. Consensus was defined as greater than 80% agreement. We developed one weak recommendation and four expert consensus statements. There is insufficient evidence to formulate recommendations on monitoring and replacement of antithrombin, fibrinogen, and von Willebrand factor in pediatric patients on ECMO. Optimal monitoring and parameters for replacement of key hemostasis parameters is largely unknown.

A comparative study of anti–ADAMTS-13 antibody dynamics in immune-mediated thrombotic thrombocytopenic purpura

BackgroundThrombotic thrombocytopenic purpura, particularly its immune-mediated variant (iTTP), necessitates accurate diagnostic approaches for effective management. ObjectivesTo compare a chemiluminescence immunoassay (CLIA) and an enzyme-linked immunosorbent assay (ELISA) for testing ADAMTS-13 activity and detecting anti–ADAMTS-13 autoantibodies (AAbs) in patients with iTTP. MethodsThis study involved 31 paired samples from 12 iTTP patients. ADAMTS-13 activity was measured using the HemosIL AcuStar (Instrumentation Laboratory, CLIA) and Technozym (Technoclone) activity assay (ELISA). The presence of AAbs was assessed using Technozym ADAMTS-13-INH assay (ELISA) and HemosIL AcuStar activity (CLIA) within a Bethesda assay following mixing with normal pool plasma. von Willebrand factor (VWF) multimers were analyzed using the HYDRASYS-2 SCAN system and the HYDRAGEL 5- or 11-VW Multimer kits (Sebia). VWF activity levels were measured with the HemosIL AcuStar VWF:GPIbR on the ACL AcuStar Analyzer (IL). ResultsFor ADAMTS-13 activity, a strong linear relationship and no bias between CLIA and ELISA were confirmed (slope = 1.01 [0.91, 1.11], intercept = 0.00 [−0.47, 0]). However, significant discrepancies were found in AAb detection during remission phases with ADAMTS-13 activity between 10% and 50%, with CLIA and ELISA showing significant divergence (P < .001, Cohen’s g = 0.34). Consistently, VWF multimers and activity levels exhibited significantly different values between remission samples with ADAMTS-13 activity below 50% and above 50%. In longitudinal analysis of patients with multiple iTTP relapses, positivity to CLIA appears to precede ELISA in predicting exacerbations. ConclusionWhile CLIA and ELISA might be interchangeable for assessing ADAMTS-13 activity, they are not equivalent for detecting AAbs, particularly in patients in clinical remission with ADAMTS-13 activity between 10% and 50%.

The effect of ADAMTS13 on graft-versus-host disease.

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) can potentially cure malignant blood disorders and benign conditions such as haemoglobinopathies and immunologic diseases. However, allo-HSCT is associated with significant complications. The most common and debilitating among them is graft-versus-host disease (GVHD). In GVHD, donor-derived T cells mount an alloimmune response against the recipient. The alloimmune response involves several steps, including recognition of recipient antigens, activation and proliferation of T cells in secondary lymphoid organs, and homing into GVHD-targeted organs. Adhesion molecules on T cells and endothelial cells mediate homing of T cells into lymphoid and non-lymphoid tissues. In this study, we showed that Von Willebrand factor (VWF), an adhesion molecule secreted by activated endothelial cells, plays an important role in mouse models of GVHD. We investigated the effect of the VWF-cleaving protease ADAMTS13 on GVHD. We found that ADAMTS13 reduced the severity of GVHD after bone marrow transplantation from C57BL6 donor to BALB/C recipient mice. A recombinant VWF-A2 domain peptide also reduced GVHD in mice. We showed that ADAMTS13 and recombinant VWF-A2 reduced the binding of T cells to endothelial cells and VWF invitro, and reduced the number of T cells in lymph nodes, Peyer's patches and GVHD-targeted organs invivo. We identified LFA-1 (αLβ2) as the binding site of VWF on T cells. Our results showed that blocking T-cell homing by ADAMTS13 or VWF-A2 peptide reduced the severity of the GVHD after allo-HSCT, a potentially novel method for treating and preventing GVHD.

The imbalance between von Willebrand factor and ADAMTS13 and the risk of venous thromboembolism

The imbalance between von Willebrand factor and ADAMTS13 and the risk of venous thromboembolism

Combined Intracoronary Prourokinase Thrombolysis on Myocardial Perfusion and Vascular Endothelial Function in STEMI.

This study aimed to investigate the effects of intracoronary prourokinase thrombolysis combined with emergency percutaneous coronary intervention (PCI) on myocardial perfusion and vascular endothelial function in patients with acute ST-segment elevation myocardial infarction (STEMI). A total of 104 patients with STEMI were collected from August 2020 to August 2022, and were divided into control group and observation group in a random manner. The control group received PCI directly, and the observation group received intracoronary prourokinase thrombolytic therapy before PCI. The treatment effects were evaluated by measuring the cardiac function indexes, including left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), and left ventricular ejection fraction (LVEF), the TIMI myocardial perfusion grade, the vascular endothelial indexes, including soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1), the von Willebrand factor (vWF), the myocardial injury indexes, including cardiac troponin I (cTnI), creatine kinase isoenzyme MB (CK-MB), and lactate dehydrogenase (LDH), and the inflammatory factors, including myeloperoxidase (MPO), C-reactive protein (CRP), and interleukin-6 (IL-6). Furthermore, the treatment safety was assessed by recording the incidence of major MACE events, 6 months after the operation. After treatment, LVEDD and LVESD were lower in the observation group than in the control group, and LVEF was higher (p < 0.05). The TIMI myocardial perfusion grade in the observation group was higher than in the control group, after treatment (p < 0.05). The levels of sICAM-1, sVCAM-1, and vWF were higher in the observation group than in the control group (p < 0.05). The levels of cTnI, CK-MB, and LDH in the observation group were lower than those in the control group, 24 hours after surgery. At 3 days after surgery, MPO was lower in the observation group than in the control group, and CRP and IL-6 were higher (p < 0.05). The incidence of major MACE events in the observation group was lower than that in the control group, 6 months after surgery (p < 0.05). There was 1 case of puncture site bleeding in the observation group, 1 case of puncture site bleeding and 1 case of subcutaneous ecchymosis in the control group, but no serious bleeding events, such as internal bleeding or cerebral hemorrhage, in the two groups. Intracoronary prourokinase thrombolytic therapy combined with emergency PCI can promote the recovery of cardiac function, improve myocardial perfusion and vascular endothelial function, and reduce inflammation and the incidence of major postoperative MACE events in acute STEMI patients.

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®.

Spontaneous bleeding in chronic kidney disease: global coagulation assays may predict bleeding risk

BackgroundChronic kidney disease (CKD) is associated with increased bleeding and thrombotic risks. Standard blood tests do not sufficiently quantify these risks. Global coagulation assays (GCAs) provide a more comprehensive assessment of coagulation. ObjectivesWe aimed to evaluate if GCAs are predictive of spontaneous major bleeding (sMB) in CKD. MethodsAdult patients with CKD (estimated glomerular filtration rate, <30 mL/min/1.73m2) were recruited to this pilot prospective observational study. Testing with GCAs (thromboelastography, overall hemostatic potential, calibrated automated thrombogram, and plasminogen activator inhibitor-1) was performed, and the results were correlated to sMB events. ResultsEighty-seven CKD patients (median age, 67 years; 67.8% male) were included, with median follow-up of 3.1 years. CKD patients demonstrated elevated fibrinogen, factor VIII, and von Willebrand factor antigen levels, while other conventional coagulation test results were within reference intervals. Ten episodes of sMB (11.5%) were captured (3.0/100 person-years), with no significant association demonstrated between sMB and antiplatelet use (P = .36), platelet count (P = .14), or renal function (urea, P = .27; estimated glomerular filtration rate, P = .09). CKD patients with sMB had more hypocoagulable GCA parameters compared with those without sMB. The lowest quartiles of endogenous thrombin potential (subhazard ratio [sHR], 7.11; 95% CI, 1.84-27.45), overall hemostatic potential (sHR, 6.81; 95% CI, 1.77-26.16), and plasminogen activator inhibitor-1 (sHR, 5.26; 95% CI, 1.55-17.91) were associated with sMB. ConclusionThis pilot study demonstrates that GCAs such as thrombin and fibrin generation may predict sMB risk in patients with CKD, which has potential to be practice-changing. Larger studies are required to validate these findings.

Point-of-Care Testing in Patients with Hereditary Disorders of Primary Hemostasis: A Narrative Review.

Inherited disorders of primary hemostasis, such as von Willebrand disease and congenital platelet disorders, can cause extensive, typically mucocutaneous bleeding. Assays to diagnose and monitor these disorders, such as von Willebrand factor activity assays and light transmission aggregometry, are performed in specialized hemostasis laboratories but are commonly not available in local hospitals. Due to the complexity and relative scarcity of these conventional assays, point-of-care tests (POCT) might be an attractive alternative in patients with hereditary bleeding disorders. POCTs, such as thromboelastography, are increasingly used to assess hemostasis in patients with acquired hemostatic defects, aiding clinical decision-making in critical situations, such as during surgery or childbirth. In comparison, the use of these assays in patients with hereditary hemostasis defects remains relatively unexplored. This review aims to give an overview of point-of-care hemostasis tests in patients with hereditary disorders of primary hemostasis. A summary of the literature reporting on the performance of currently available and experimental POCTs in these disorders is given, and the potential utility of the assays in various use scenarios is discussed. Altogether, the studies included in this review reveal that several POCTs are capable of identifying and monitoring severe defects in the primary hemostasis, while a POCT that can reliably detect milder defects of primary hemostasis is currently lacking. A better understanding of the strengths and limitations of POCTs in assessing hereditary defects of primary hemostasis is needed, after which these tests may become available for clinical practice, potentially targeting a large group of patients with milder defects of primary hemostasis.

On-slide clearing and imaging of 100-µm-thick histological sections using ethyl cinnamate and epifluorescence.

Thick histological samples are difficult to image without proper tissue clearing methods. Among these methods ethyl cinnamate (ECi)-based clearing preserves antigenicity and is compatible with immunofluorescent labeling. In contrast to many other clearing protocols, ECi-based clearing is fast and is done as a final step after standard immunofluorescent labeling protocols.

Hemostatic management of von Willebrand disease during childbirth with a plasma-derived von Willebrand factor/factor VIII concentrate

BackgroundFemales with von Willebrand disease (VWD) do not show the same increases in von Willebrand factor and factor (F)VIII levels during pregnancy as females without VWD and are at higher risk of excessive bleeding associated with childbirth. Data on hemostatic management for childbirth in VWD patients are limited. ObjectivesTo evaluate the dosing, efficacy, and safety of plasma-derived von Willebrand factor/FVIII (wilate) for prevention of excessive bleeding associated with childbirth in females with any type of VWD. MethodsData for females with VWD who received wilate for hemostatic coverage for childbirth during 2 prospective clinical studies were analyzed. ResultsTen females with VWD and a mean age at enrolment of 29.6 years were treated with wilate to prevent excessive bleeding associated with childbirth. Two patients had type 1, 4 had type 2 (2 2A, 1 2B, and 1 2M), and 4 had type 3 VWD. Of the 10 deliveries, 5 were by cesarean section. Patients received a mean of 9.5 infusions of wilate over 6.8 exposure days, with a mean total dose of 234 IU/kg per delivery and 25 IU/kg per infusion. Hemostatic management for all deliveries was rated excellent or good, with no excessive bleeding during delivery and no postpartum bleeding during the period of wilate treatment in any patient. Two patients experienced 8 possible or probable treatment-related adverse events; all were mild or moderate and resolved. No thromboembolic events were observed. ConclusionThe results of this case series indicate that wilate provided effective hemostatic cover for childbirth in females with VWD during delivery and postpartum.