vWF Antigen Levels Research Articles

The Use of Thromboelastography in Monitoring Coagulation Status for Patients Treated with Low Molecular Weight Heparin, Warfarin, Apixaban, and Rivaroxaban

Introduction: Thromboelastography (TEG) is used to rapidly assess global coagulation status. A few studies in the literature have explored a possible role for TEG in anticoagulant monitoring, with conflicting observations. We wondered if TEG might be used adjunctively with traditional markers of anticoagulation as a measure of anticoagulant efficacy. We hypothesized that some of these variations might be due to changes in certain coagulation factor levels.Methods: Blood samples were collected from patients on therapeutic doses of warfarin, low molecular weight heparin (LMWH, enoxaparin or dalteparin), or an anti-Xa direct oral anticoagulant (DOAC, apixaban or rivaroxaban) due to a history of thrombosis. TEG was performed on all samples with and without addition of heparinase; R time, K time, alpha-angle, maximum amplitude, and percent of clot lysis after 30 minutes were measured. Standard anticoagulation parameters were obtained at the same time as TEG values [prothrombin time (PT) and activated partial prothrombin time (PTT) for warfarin- and DOAC-treated patients; anti-Xa level for LMWH-treated patients]. For each patient, pertinent medical history was recorded including: age, medical comorbidities, and thrombotic history. Von Willebrand factor (vWF) antigen level, factor VIII (fVIII) activity, anti-Xa, and R time were examined in a follow-up cohort of enoxaparin-treated patients. Correlation between TEG and anticoagulation parameters was analyzed using Spearman correlation coefficient (r).Results: Forty-six patients participated in this study. Median age was 52 years (range, 20-83). The most common indication for anticoagulation was deep venous thrombosis or pulmonary embolism (N = 33); 5 subjects had active cancer at the time of anticoagulation testing (all on LMWH). Among warfarin-treated patients (N = 19), INR was significantly correlated with R time (Figure 1, p=0.005), with 4 patients demonstrating a prolonged R time of over 10 min. Among patients on LMWH (enoxaparin, N = 13; dalteparin, N = 1), 5 had a prolonged R time, but there was no correlation between anti-Xa level and R time, R time ratio or R time difference with or without heparinase, or any other measured TEG parameter when analyzing all LMWH patients or only enoxaparin-treated patients as a function of total daily enoxaparin dose. Among DOAC patients (apixaban, N = 6; rivaroxaban, N = 8), R time was not elevated in any of the samples, but there was a significant correlation between PTT and R time (p=0.006). Four study patients had recurrent thrombotic events (1 with antithrombin deficiency, chronic liver disease, and bowel resection, on warfarin; 1 with May-Thurner syndrome, on LMWH; 2 with metastatic cancer and Trousseau's syndrome, also on LMWH) in spite of therapeutic INR or anti-Xa levels; these patients were all found to have normal R times on TEG. In a follow-up analysis of 8 enoxaparin-treated patients, 6 had normal and 2 had prolonged R times. All 6 with normal R times had elevations in vWF and/or fVIII, while the 2 with prolonged R times had normal levels of vWF and fVIII.Conclusion:TEG R time is significantly correlated with INR in patients on warfarin. TEG R time is also correlated with PTT in patients on DOACs. For both DOACs and warfarin, TEG could be useful in rapidly assessing the coagulation status of patients who present with emergent bleeding and may benefit from reversal agents. In patients on LMWH, TEG R time is affected by circulating levels of vWF and fVIII. For select patients on LMWH, TEG may be helpful as an adjunctive measure of anticoagulant activity in cases where anti-Xa monitoring may not be accurate. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Role of von Willebrand Factor in COVID-19 Associated Coagulopathy.

BackgroundCOVID-19, the disease caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) can present with symptoms ranging from none to severe. Thrombotic events occur in a significant number of patients with COVID-19, especially in critically ill patients. This apparent novel form of coagulopathy is termed COVID-19 associated coagulopathy and endothelial derived von Willebrand factor (vWF) may play an important role in its pathogenesis.ContentvWF is a multimeric glycoprotein molecule that is involved in inflammation, primary and secondary hemostasis. Studies have shown that patients with COVID-19 have significantly elevated levels of vWF antigen and activity, likely contributing to an increased risk of thrombosis seen in CAC. The high levels of both vWF antigen and activity have been clinically correlated with worse outcomes. Furthermore, the severity of a COVID-19 infection appears to reduce molecules that regulate vWF level and activity such as ADAMT-13 and high density lipoproteins (HDL). Finally, studies have suggested that patients with blood group O (a blood group with lower than baseline levels of vWF) have a lower risk of infection and disease severity compared to other blood groups; however, more studies are needed to elucidate the role of vWFSummaryCAC is a significant contributor to morbidity and mortality. Endothelial dysfunction with the release of pro-thrombotic factors, such as vWF, needs further examination as a possible important component in the pathogenesis CAC.

Expression of ADAMTS13 in human endometrium and its potential role in recurrent pregnancy loss

IntroductionThe mechanisms underlying the pathogenesis of recurrent pregnancy loss (RPL) and the effective approaches to treat this disease still remain vague and absent. Proteinases of ADAMTS family play important roles in embryonic growth and development. Our previous study suggest a role of ADAMTS13 during pregnancy. Current Study was to determine the expression of ADAMTS13 in human endometrium and its association with RPL.Material and methodsThe spatiotemporal expression of ADAMTS13 in human endometrium was examined by immunohistochemistry. real-time PCR sand western blot were then employed to determine the mRNA and protein expression levels of ADAMTS13 in human endometrium. Proteolytic cleavage of FRETS-VWF73 were performed to determine the activity of ADAMTS13 in plasma and that secreted by human endometrium. ELISA was carried out to measure plasma VWF antigen.ResultsWe show that proteolytically active ADAMTS13 is expressed in human endometrium throughout the menstrual cycle and pregnancy. The decidual expression levels of mRNA and protein in women with RPL were significantly lower compared with women with uncomplicated pregnancies (P<0.01, P<0.05, respectively). Furthermore, significantly reduced plasma ADAMTS13 activity (median [range] 69.09 [65.2–93.7]% versus 93.62 [88.1–115.6]%, P<0.001) and elevated plasma VWF antigen levels (median [range] of 125.5 [54.2–262.8]% versus 91.9[80.4–138.7]%, P < 0.01) were detected in RPL patients compared with the control group.ConclusionsThese findings suggest that ADAMTS13 may play a role in embryo implantation and the pathogenesis of recurrent pregnancy loss. Further investigation on ADAMTS13 gene knockout animal models is necessary for understanding the molecular mechanisms of the biological roles of ADAMTS13 during gestation.

Extending the post-thaw viability of cryoprecipitate.

Cryoprecipitate has a short post-thaw expiry time of 6 h. The aim of this study was to assess the stability and function of cryoprecipitate components (FVIII, fibrinogen, vWF, and FXIII) and cryoprecipitate sterility up to 120 h post-thawing when stored at two temperatures (2-6°C and room temperature [20-24°C]). Twenty batches (110 individual units) of time-expired, thawed cryoprecipitate were collected. Units were sampled at the 6-h expiration mark and then stored at 2-6°C or room temperature (RT). They were resampled every 24 h for 120 h. One unit from each batch was sent for sterility testing at 120 h. Samples had FVIII (one stage and chromogenic), fibrinogen, FXIII, vWFag, and vWF:RCo assays performed in batches. Rotational thromboelastometry (ROTEM) was also performed. FVIII levels declined significantly at 120 h post-thawing at both RT and 2-6°C, but still met international standards for FVIII content. Fibrinogen, vWF antigen, and FXIII levels reduced minimally over 120 h and always met international standard requirements when stored at either temperature. ROTEM analysis demonstrated that fibrinogen function was not compromised at 120 h post-thawing under both storage conditions. vWF:RCo levels declined significantly over 120 h at both storage temperatures. No bacterial contamination was detected in 20 units of cryoprecipitate following storage for 120 h post-thawing. These results demonstrate that extension of the storage time of thawed cryoprecipitate to 120 h, stored at either 2-6°C or RT, is feasible while still maintaining required FVIII, fibrinogen, and vWFag levels. Storage at 2-6°C has the advantage of reduced risk of potential bacterial contamination.

Von Willebrand factor with increased binding capacity is associated with reduced platelet aggregation but enhanced agglutination in COVID-19 patients: another COVID-19 paradox?

Patients with Coronavirus-associated disease-2019 (COVID-19) display alterations of the hemostatic system and the presence of a prothrombotic status frequently leading to vascular complications. However, the impact of COVID-19 on platelet activity, aggregation and agglutination still needs to be clarified. We measured total levels of von Willebrand factor (vWF) and vWF binding to the platelet glycoprotein (Gp) complex (GPIb-IX-V), in a cohort of COVID-19 patients admitted to the intensive care unit of our Institution. Moreover, we evaluated platelet aggregation in response to agonists (ADP, collagen, arachidonic acid) and platelet agglutination in response to ristocetin. We found that levels of vWF antigen and the active form of vWF binding to platelets (vWF:RCo), were markedly increased in these patients. These results were associated with higher agglutination rates induced by ristocetin, thereby indirectly indicating an increased capability of vWF to bind to platelets. Conversely, we found that platelet aggregation in response to both ADP and collagen was lower in COVID-19 patients compared to healthy volunteers. This study shows that COVID-19 is associated with increased vWF-induced platelet agglutination but reduced platelet responsivity to aggregation stimuli. Our findings have translational relevance since platelet adhesion to vWF may represent a marker to predict possible complications and better delineate therapeutic strategies in COVID-19 patients.Electronic Supplementary MaterialThe online version of this article (10.1007/s11239-020-02339-6) contains supplementary material, which is available to authorized users.

Von Willebrand factor:antigen and ADAMTS-13 level, but not soluble P-selectin, are risk factors for the first asymptomatic deep vein thrombosis in cancer patients undergoing chemotherapy

BackgroundThere is a high incidence of deep vein thrombosis (DVT) among cancer patients undergoing chemotherapy. Chemotherapy-induced vascular endothelial cell activation (VECA) is characterized by increased plasma levels of von Willebrand factor (vWF) and soluble P-selectin (sP-selectin), leading to the activation of endothelial cells and signaling cascades. The biological role of a disintegrin-like and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS-13) is to control the activity of vWF and consequently the risk of thrombosis. The objective of this study was to investigate the roles of sP-selectin, vWF, and ADAMTS-13 as risk factors for the first episode of DVT in cancer patients undergoing chemotherapy.MethodsThis prospective cohort study was conducted at Dr. Kariadi Hospital, Indonesia, on 40 cancer patients. Prechemotherapy (baseline) and postchemotherapy sP-selectin, vWF antigen (vWF:Ag), and ADAMTS-13 plasma levels were determined with ELISAs before and 3 months after chemotherapy. The clinical characteristics of the patients, cancer type, cancer stage, chemotherapy regimen, ABO blood type, D-dimer level and Khorana risk score were also analyzed using logistic regression. Patients were observed for the possibility of developing DVT during chemotherapy.ResultsDVT was confirmed in 5 patients (12.5%) after a period of 3 months. In patients with DVT, sP-selectin and vWF were significantly higher while ADAMTS-13 was lower than in their counterparts. The levels of baseline vWF:Ag and ADAMTS-13, with cut-off points ≥ 2.35 IU/mL and ≤ 1.03 IU/mL, respectively, were found to independently predict the incidence of DVT. In the multivariate logistic regression analysis, the relative risk (RR) for DVT in patients with high vWF:Ag was 3.80 (95% CI 1.15–12.48, p = 0.028), and that for patients with low ADAMTS-13 was 2.67 (95% CI 1.22–23.82, p = 0.005). The vWF:Ag/ADAMTS-13 ratio and both vWF:Ag and ADAMTS-13 dynamics during treatment were also able to differentiate those with prospective DVT. However, sP-selectin and other covariates showed no statistical significance.ConclusionWe found that prechemotherapy plasma levels of vWF:Ag ≥ 2.35 IU/mL and ADAMTS-13 ≤ 1.03 IU/mL are independent risk factors for DVT incidence among cancer patients.

Reduced Cleavage of Von Willebrand Factor By ADAMTS13 Exacerbates Acute Kidney Injury Secondary to Traumatic Injury

Introduction: Increases in plasma von Willebrand Factor (vWF) antigen levels, accompanied by decreases in its respective metalloprotease, ADAMTS13, have been demonstrated following severe injury. We hypothesized that following traumatic injury, endothelial damage and activation would result in the release of high molecular weight multimeric (HMWM) vWF forms into circulation with a concominant decrease in ADAMTS13. We further hypothesized that the alterations in circulating levels and functions of these factors, following traumatic injury, promote an increased thrombogenic and inflammatory response, which exacerbates the risk of developing post traumatic acute kidney injury (AKI). Methods: Plasma samples from a cohort of patients with trauma induced AKI, collected at 0-, 24-, and 72-hours after admission, were evaluated against plasma samples from trauma patients that were matched for injury characteristics, but were without AKI. VWF and ADAMTS13 activity and antigen levels were evaluated. Circulating vWF multimeric composition was determined by vertical agarose gel electrophoresis. We further evaluated the effects of traumatic injury on ADAMTS13 and vWF by means of a pre-clinical model of trauma and hemorrhage, which is comprised of a liver laceration, cardiac puncture, and soft tissue injury. Following this polytrauma model, hepatic ADAMTS13 RNA expression was evaluated by qRT-PCR of total liver RNA; analyzed liver samples were taken immediately before, 24-, and 72-hours after injury. The impact of the polytrauma model on renal function was evaluated via changes in plasma cystatin C, while the effect on the renal microvasculature was evaluated by means of immunofluorescent staining of vWF and fibrinogen in kidney sections. Results: VWF antigen was increased in trauma patients when compared to healthy controls upon admission (11.0 ± 3.72 ug/mL vs. 4.64 ± 0.91 ug/mL), persisting through 72-hours (13.11 ± 3.16 ug/mL). The percentage of HMWM vWF forms in trauma patient samples was elevated at all time points compared to pooled normal plasma (PNP) (27.9 ± 4.5% and 15.9 ± 0.8). Trauma patient ADAMTS13 activity was decreased at all time points (0.48 ± 0.14 IU/mL vs. 1.27 ± 0.26 IU/mL). AKI patients showed a 1.54 ± 0.48-fold increase in vWF antigen levels over the 72-hour period (p=0.016), while there was no change in the control trauma group. Those who developed AKI demonstrated a larger proportion of HMWM vWF at 72-hours than their non-AKI counterparts (32.9 ± 2.4% vs 28.8 ± 3.2%). qRT-PCR of murine total liver RNA, following the polytrauma model, exhibited ADAMTS13 RNA expression levels that were 35% of normal at 24-hours and 60% of normal at 72 hours post-injury. A matched analysis of murine plasma cystatin C levels demonstrated a marked elevation 24-hours post polytrauma model (sham: 510ng/mL (435-561) vs trauma: 550 (479-701)). H&E staining of kidney sections, which were harvested following the polytrauma model, demonstrated generalized renal tubule dilation and denudation, suggestive of post-traumatic acute tubular necrosis. Immunofluorescent studies of kidney sections from the same mice exhibited 8-15µm microvascular plugs that were positive for fibrinogen with a vWF core; renal immunofluorescent studies of control mice demonstrated no microvascular depositions. Conclusions: These data indicate that traumatic injury alters the circulating composition of ADAMTS13 and its target, vWF, shifting their equilibrium to one that promotes inflammation and thrombosis. In the immediate moments following injury, these mechanisms support life-saving hemostasis; however, as these changes extend into the following days, the early hemostatic benefit quickly shifts to a burden that may produce microvascular clot deposition, exacerbating pathologic states such as acute kidney injury. Disclosures No relevant conflicts of interest to declare.

Extensive Characterization of the Hemostatic Derangement Occurring in COVID-19 Patients Admitted to the Bergamo Hospital

INTRODUCTION The occurrence of a hypercoagulable state in hospitalized COVID-19 patients is supported by studies conducted with routine coagulation tests, including plasma D-dimer and fibrinogen, and platelet count.AIM In this study we performed an extensive characterization of the hemostatic alterations by both global and specific assays in a cohort of 78 patients hospitalized for COVID-19. The aims were to: 1) clarify mechanisms underlying the coagulopathy, and 2) identify predictive factors of disease severity and thrombotic events (i.e. deep vein thrombosis [DVT], pulmonary embolism [PE] or arterial thromboembolism [ATE]).METHODS COVID-19 patients admitted to the Hospital Papa Giovanni XXIII in Bergamo, Italy, from March 23 to May 30, 2020, were enrolled prospectively, providing informed consent. As a global assay, thromboelastometry (ROTEM) was performed in whole blood by EXTEM, INTEM, and FIBTEM tests. Specific assays included plasma levels of intrinsic and extrinsic pathway coagulation factors, von Willebrand factor (vWF) antigen and activity, anticoagulant proteins (i.e. protein C [PC], free-protein S [PS], and antithrombin [AT]), fibrinolytic proteins (i.e. tissue plasminogen activator [t-PA], and inhibitor [PAI-1]), and hypercoagulation biomarkers (i.e. prothrombin fragment 1+2 [F1+2], and D-dimer). In addition, biomarkers of immunoinflammation (i.e. neutrophil extracellular traps [NETs], CRP and procalcitonin) were measured. Occurrence of thrombotic events and death were monitored during follow up.RESULTS 78 patients (56M/22F), median age 62.7 years (25-87), were analyzed. According to disease severity, 45 were ICU, and 33 non-ICU patients. Sixty-three of them were on thromboprophylaxis. Global hemostasis analysis by ROTEM showed a prothrombotic profile in patients compared to controls, with a significantly shorter clot formation time (CFT), and increased maximum clot firmness (MCF), which were significantly greater in the ICU vs non-ICU patients. The occurrence of an ’in vivo’ hypercoagulable state was confirmed by increased plasma levels of F1+2 and D-dimer, with the highest values of D-dimer in the ICU subjects. Hypercoagulability, rather than factors’ consumption, was also shown by the findings of significantly higher plasma procoagulant factors V, VIII, IX and fibrinogen in ICU compared to non-ICU patients (p<0.001). Endothelium activation was shown by extremely elevated vWF antigen and activity levels in all patients (highest values in ICU subjects). Moreover, the concentrations of fibrinolytic proteins, t-PA, and its inhibitor PAI-1, were elevated (p<0.01) in patients compared to normal controls, without difference between ICU and non-ICU subjects. Finally, the inflammatory parameters’ analysis in the ICU group demonstrated significantly increased plasma levels of NETs, CRP, and procalcitonin, compared to non-ICU patients. Of note, NETs levels significantly (p<0.02) correlated with vWF, D-dimer and t-PA, while CRP and procalcitonin inversely correlated with anticoagulant PC.After a median time of 8.8 days, 19 (24%) patients experienced thrombosis (3 DVT, 8 PE, 8 ATE). Thirteen (17%) patients from total population died after a median time of 33 days of hospitalization. Baseline D-dimer and t-PA levels were significantly higher in patients developing VTE, while baseline FVIII, vWF and D-dimer levels were greater in subjects who died during follow-up. By Cox analysis, high D-dimer and younger age were significantly associated with mortality.CONCLUSIONS Our study provides for the first time an extensive overview of the hypercoagulable state induced by SARSCoV-2 infection, demonstrating alterations in all of the different hemostatic compartments analyzed. The viral infection-induced hemostatic abnormalities are exacerbated by the severity of the disease and strongly correlate with the proinflammatory status, demonstrating the link between coagulation and inflammation. This link is further supported by the clear correlation found between NETosis and markers of endothelial and blood clotting activation. Finally, these data add evidence to the role of D-dimer as a significant predictor of intra-hospital mortality.DisclosuresNo relevant conflicts of interest to declare.

COVID-19 Associated Coagulopathy: A Comprehensive Assessment of the Coagulation Profile in Critically and Non-Critically Ill Patients

INTRODUCTIONCOVID-19 is associated with coagulopathy that correlates with poor prognosis. Although the underlying mechanism of COVID-19 coagulopathy remains unknown, early reports suggested that it may be a form of disseminated intravascular coagulation (DIC). However, recent studies have highlighted the potential role of endothelial cell injury in its pathogenesis.AIMSThe aims of our study were to analyze the coagulation parameters of critically and non-critically ill patients with COVID-19 pneumonia admitted to our hospital, determine if coagulation factors consumption occurs, identify potential prognostic biomarkers of this new disease and explore possible underlying mechanisms of COVID-19 coagulopathy.METHODSWe conducted a retrospective cohort study performed at Gregorio Marañon Hospital in Madrid, Spain. Adult patients with a diagnosis of COVID-19 hospitalized in our center were recruited, including those admitted to the ICU and to general wards. Patients were randomly selected from blood samples that arrived at our Hemostasis laboratory during April 2020. For each patient, we conducted a complete analysis of coagulation parameters, including basic coagulation tests, quantification of coagulation factors and physiological inhibitor proteins, evaluation of the fibrinolytic system and determination of von Willebrand Factor (vWF) and ADAMTS13. Laboratory data were compared with clinical data and outcomes. Data were analyzed using IBM SPSS Statistics for Mac, version 24. This study was approved by our institutional Ethics Committee and it was executed along with the international ethics recommendations for conducting research in humans following the latest revision of Declaration of Helsinki.RESULTSA total of 62 patients (31 ICU, 31 non-ICU) were analyzed. Mean age of the sample was 61.8 (SD 15.2) years and 69.4% of the patients were male. The coagulation parameters assessment demonstrated normal median PT, INR and APTT in our cohort and all coagulation factors were within normal range. Factor VIII showed an increasing trend (194.5±71.9) which could be interpreted as an acute phase reactant, and it was significantly higher in non-survivors (p=0.003). Similarly, we did not observe consumption of physiological inhibitor proteins and platelet counts were also within the normal limits, despite being slightly lower in non-survivor patients (p=0.006) (Table 1).Von Willebrand Factor (vWF) was above the normal range (median 216%, IQR 196-439, normal range 62-175%) in our cohort and higher levels of vWF-antigen (p=0.001) and vWF-activity (p=0.02) were associated with poor prognosis. Likewise, a lower ADAMTS13 activity was observed in non-survivors (p=0.008). Regarding the fibrinolytic pathway, PAI levels were above the normal range (median 52.6ng/ml, IQR 37.2-85.7, normal range 4-40ng/ml), but we found no statistically significant differences based on survival. The remaining parameters of the fibrinolytic pathway (plasminogen and alpha-2 antiplasmin) were within normal range (Table 1).ICU-patients had a poorer prognosis, with a higher rate of mortality (p=0.003). Likewise, they showed more elevated acute phase reactants (p<0.01) and higher levels of D-dimer (p=0.003) than patients hospitalized in general wards. ADAMTS13 activity was also significantly lower in this subgroup (p<0.001), as well as plasminogen levels (p=0.002) and PAI (p=0.003). Criteria of overt disseminated intravascular coagulation (DIC) as defined according to the ISTH DIC score, were met in 14.8% of ICU patients versus 0% of non-ICU patients (p=0.039) (Table 2).CONCLUSIONSCOVID-19 infection is associated with coagulopathy that correlates with poor prognosis. However, coagulation factors, physiological inhibitory proteins and alpha-2-antplasmin levels were preserved in our study. Similarly, we did not observe platelets or fibrinogen consumption, which leads us to assume that COVID-19 coagulopathy is not a form of DIC. Increased vWF and decreased ADAMTS13 activity in our cohort could indicate that the underlying mechanism of this coagulopathy may reside in the endothelial cells and share a similar pathogenesis with thrombotic microangiopathy (TMA), as it has been recently suggested in some scientific reports.DisclosuresNo relevant conflicts of interest to declare.

The current status of remote diagnosis of von Willebrand disease in children in Russia

The diagnosis of von Willebrand disease (vWD) in children remains a challenge in many regions of our country. This encouraged the Russian Hemophilia Society to create, in 2019, a special diagnostic programme offering remote diagnosis of vWD to patients in regions. Objectives: An interim evaluation of the effectiveness of the programme for remote diagnosis of vWD in children. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of Ministry of Healthcare of the Russian Federation. Under this programme, if advised by a hematologist at the place of residence, a patient's peripheral blood was collected, frozen and sent to the Clinical Hemostasis Laboratory at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. Over the period from 11.04.2019 to 07.08.2020, we received 72 frozen peripheral blood samples from children under the age of 18 sent from 21 regions of the Russian Federation. Von Willebrand factor (vWF) ristocetin cofactor activity, vWF antigen levels as well as coagulation factor VIII activity were measured. Out of the 72 samples, only one (1.4%) was inadequate. Reduced vWF activity (30–50%) was observed in 16.7% of cases, and another 16.7% of patients had vWF activity &lt; 30%. VWF antigen levels were 30–50 % in 13.9 % of patients and &lt; 30% in 9.7% of cases. Among the patients with vWF activity &lt; 30%, 1 patient (8.3%) had type 1 vWD, 10 patients (83.3%) – type 2 vWD, 1 child (8.3%) – type 3 vWD. Six children (8.3%) demonstrated reduced factor VIII activity while maintaining normal vWF activity and antigen levels. The coagulation factor VIII activity/vWF antigen ratio was decreased in 8 children (11.1%). The vWD remote diagnosis programme has allowed us to detect abnormalities in 30 patients (41.7%), 16.7% of whom demonstrated laboratory features characteristic of vWD. Eighteen children (25%) require further, more comprehensive laboratory testing. This programme has great potential, especially in underpopulated regions where the development of local diagnostic capabilities may not be economically viable.

Von Willebrand Factor and ADAMTS13 and long-term outcomes in patients undergoing percutaneous coronary intervention

BackgroundVon Willebrand factor (VWF) and its cleaving protease a disintegrin-like and metalloprotease with thrombospondin type I repeats 13 (ADAMTS13) are pivotal mediators of thrombosis and are associated with the progression of atherosclerosis. We investigated the impact of VWF, ADAMTS13 and VWF/ADAMTS13 on long-term major adverse cardiovascular outcomes (MACE) in patients undergoing percutaneous coronary intervention (PCI). MethodsWe analysed 701 patients undergoing PCI between 2003 and 2006. VWF and ADAMTS13 antigen levels were measured before PCI. As primary endpoint, we investigated MACE, a composite of all-cause mortality, myocardial infarction or ischemic stroke during 8 years of follow-up. As secondary endpoint, we investigated all-cause mortality. ResultsMean age was 63.8 years, 496 (70.8%) were male. Acute coronary syndrome (ACS) was diagnosed in 347 (49.5%) patients, stable coronary artery disease (SCAD) in 354 (50.5%). During follow-up 228 (32.5%) patients experienced MACE, and 161 (23.0%) died. In ACS patients, VWF was significantly associated with MACE (HR 1.402 (95%CI 1.003–1.959), p = 0.048), whereas ADAMTS13 and VWF/ADAMTS13 had no predictive value. In SCAD, neither VWF, ADAMTS13, nor VWF/ADAMTS13 correlated with MACE. VWF was significantly associated with all-cause death in ACS patients (HR 1.841 (95%CI 1.187–2.856), p = 0.006), but not in SCAD (1.394 (95%CI 0.856–2.269), p = 0.181). ADAMTS13 and VWF/ADAMTS13 were not correlated with ACS and SCAD, respectively. ConclusionVWF but not ADAMTS13 and VWF/ADAMTS13 was associated with MACE and mortality in patients with ACS but not SCAD. This finding highlights the importance of VWF as an essential marker of risk in patients with ACS.

Von Willebrand factor (vWF): marker of endothelial damage and thrombotic risk in COVID-19?

COVID-19 infection is characterised, among other features, by a prothrombotic state with high rate of venous thromboembolism (VTE), D-dimer, and fibrinogen levels. Clinical observations have also highlighted that these patients have elevated von Willebrand factor (vWF) and factor VIIIc. 24 consecutive COVID-19 positive patients were selected from the intensive care unit (ICU) or the high acuity ward of Brighton and Sussex University Hospitals NHS Trust. The rate of VTE was 25% and mortality rate was 16.7%. Fibrinogen and D-Dimers were elevated, 7.9 (1.6) g/L and 2.4 (2.02) ug/ml respectively. Factor VIIIc and von vWF antigen levels were both extremely elevated at 279 (148) u/dL and 350 (131) % respectively, which are comparable to levels seen in ICU patients with severe sepsis. vWF levels were significantly higher in patients that died (p=0.017) and showed a positive correlation with age. There was a statistically significant association between COVID-19 disease and non-O blood group (p=0.02); 80% (4/5) of COVID-19 patients with VTE were blood group A. Very high levels of vWF and factor VIIIc are common in COVID-19 patients, comparable to levels in severely septic non-COVID ICU patients. This could contribute to the hypercoagulable state and increased VTE rate in COVID-19. Further studies are needed to evaluate the use of vWF for stratifying thrombotic risk in COVID-19 and to determine if elevated vWF is contributing to disease pathogenesis.

Prognostic value of von Willebrand factor in adult patients with congenital heart disease

Objectivesvon Willebrand factor (vWF) has prognostic value in patients with heart failure (HF) and in those with liver disease. Liver congestion, due to right-sided HF (RHF), is one of the...

Intracardiac Fibrinolysis and Endothelium Activation Related to Atrial Fibrillation Ablation with Different Techniques.

Objective The effect of pulmonary vein isolation (PVI) on fibrinolytic and endothelial activation with currently applied periprocedural anticoagulation has not been explored. We measured markers of fibrinolysis and endothelium activation before and after PVI with the second-generation cryoballoon (Cryo), pulmonary vein ablation catheter (PVAC-Gold), and irrigated radiofrequency (IRF). Methods Markers of fibrinolysis and endothelium activation in left atrial (LA) blood samples were measured in 31 patients before and after PVI (Cryo:10, PVAC-Gold: 7, IRF: 14). Periprocedural anticoagulation included uninterrupted vitamin K antagonist and iv heparin (ACT≥300 sec) during LA dwelling. Results Levels of D-dimer (median; interquartile range, mgFEU/L) increased with all techniques (PVAC: 0.34; 0.24–0.50 versus 0.70; 0.61–1.31; p=0.0313, Cryo: 0.33; 0.28–0.49 versus 0.79; 0.65–0.93; p=0.0313, Cryo: 0.33; 0.28–0.49 versus 0.79; 0.65–0.93; p=0.0313, Cryo: 0.33; 0.28–0.49 versus 0.79; 0.65–0.93; PAP complex level (ng/ml) increased after Cryo (247.3, 199.9–331.6 versus 270.9, 227.9–346.7; p=0.0313, Cryo: 0.33; 0.28–0.49 versus 0.79; 0.65–0.93; p=0.0313, Cryo: 0.33; 0.28–0.49 versus 0.79; 0.65–0.93; p=0.0313, Cryo: 0.33; 0.28–0.49 versus 0.79; 0.65–0.93; PAI-1 activity (%) decreased with the PVAC (1.931; 0.508–3.859 versus 0.735, 0.240–2.707; p=0.0313, Cryo: 0.33; 0.28–0.49 versus 0.79; 0.65–0.93; p=0.0313, Cryo: 0.33; 0.28–0.49 versus 0.79; 0.65–0.93; p=0.0313, Cryo: 0.33; 0.28–0.49 versus 0.79; 0.65–0.93; VWF antigen levels and FVIII activity increased after PVI with all the 3 techniques. The levels of soluble VCAM-1 (ng/ml) did not change after PVAC procedures, but increased after Cryo (542, 6; 428.5–753.1 versus 619.2; 499.8–799.0; p=0.0313, Cryo: 0.33; 0.28–0.49 versus 0.79; 0.65–0.93; p=0.0313, Cryo: 0.33; 0.28–0.49 versus 0.79; 0.65–0.93; Conclusion PVI with contemporary ablation techniques and periprocedural antithrombotic treatment induces coagulation and endothelium activation of similar magnitude with different ablation methods.

Prolonged Circulation of Ultra-Large Von Willebrand Factor and a Reduction in ADAMTS13 Activity Promotes Microvascular Disease Following Traumatic Injury

Introduction: Increases in plasma von Willebrand Factor (vWF) levels, accompanied by decreases in its respective metalloprotease, ADAMTS13, have been demonstrated in diseases of microvascular injury. We hypothesized that following severe trauma, a burst of ultra-large vWF (UL-vWF) is released into the bloodstream by damaged endothelium, resulting in increased thrombogenicity due to circulating vWF multimers. We further hypothesized that traumatic injury would lead to a deficit of ADAMTS13, promoting the accumulation of UL-vWF forms and, ultimately, the increased risk of microvascular disease, such as acute kidney injury (AKI). Methods: A cohort of 37 severely injured trauma patients was analyzed for antigen levels of plasma vWF and ADAMTS13 at 0- and 24-hours after admission. Circulating vWF multimeric composition from both time points was determined by vertical agarose gel electrophoresis. Multivariate analyses were performed with data abstracted from the electronic medical records to identify further dependences. A similar analysis was also performed on plasma from a cohort of 8 patients with trauma induced AKI at 0-, 24-, and 72-hours after admission; these patients were well matched against trauma patients without AKI. Finally, we utilized a murine model of polytrauma and hemorrhage, in conjunction with qRT-PCR of ADAMTS13 in total liver RNA, to specifically address how the expression of ADAMTS13 is altered by the systemic effects of traumatic injury. Results: Circulating vWF levels were increased in severe trauma patients when compared to healthy controls at presentation (189% (110-263) vs. 95% (74-120)) and persisted through 24-hours (213% (146-257) vs. 132% (57-160)). Ultra-large vWF forms were elevated at both 0- and 24-hours when compared to pooled normal plasma ((10.0% (8.9-14.3) and 11.3% (9.1-21.2), respectively, vs 0.6%). The largest vWF forms within trauma patient plasma circulated at 33±4 dimers vs 18±1 dimer in length within pooled normal plasma. Severe trauma patient ADAMTS13 activity was decreased at 0-hours (66% (47-86) vs. 100% (98-100)) and at 24-hours (72.5% (56-87.3) vs 103% (103-103)) when compared to healthy patients. Furthermore, the proportion of circulating low molecular weight multimeric (LMWM) vWF to total circulating vWF forms was directly dependent upon ADAMTS13 activity at 24-hours (Decreased ADAMTS13 Activity: 20.4% (15.0-22.7) LMWM vWF vs Normal Activity: 25.8% (22.7-35.2) LMWM vWF). Strikingly, ADAMTS13 activity independently predicted the development of coagulopathy, correlating with presentation INR (ρ =-0.63), activated clotting time of thromboelastography (TEG) (ρ=-0.36), and TEG maximum amplitude (ρ=0.36). ADAMTS13 activity also closely correlated with injury severity (ISS) (ρ=-0.34) and blood product transfusion (ρ =-.45). The cohort of 8 trauma patients who went on to develop AKI showed a 1.54-fold (1.02-2.05) increase in plasma vWF antigen levels between 0 and 72 hours, while those who did not develop AKI showed no change in vWF levels over this time period. Furthermore, those who developed AKI demonstrated a smaller proportion of LMWM vWF in plasma than those who did not (25.4% (23.4-28.0) vs 31.2% (27.2-35.6)), suggesting the increased thrombogenicity of their circulating vWF forms. Finally, qRT-PCR of total liver RNA in 6 mice demonstrated a 2-fold decrease in ADAMTS13 RNA expression levels between the times immediately before and 24-hours after trauma. Altogether, these data indicate that both circulating ADAMTS13 and its production are deficient in the days following severe injury. Conclusions: Severe traumatic injury alters the circulating composition of ADAMTS13 and its target, vWF, shifting their equilibrium to one that promotes thrombosis. Not only is the concentration of circulating ADAMTS13 decreased following traumatic injury, but hepatic expression of the enzyme is lacking as well. In the immediate moments following injury, these mechanisms contribute to life-saving hemostasis; however, as these changes extend into the following days, the early hemostatic benefit quickly shifts to burden that may exacerbate microvascular disease. Disclosures Ragni: Bioverativ/Sanofi: Consultancy, Research Funding; Sangamo: Research Funding; Shire/Takeda: Consultancy, Other: Study drug; Alnylam/Sanofi: Consultancy, Research Funding; Bayer: Consultancy; Spark Therapeutics: Consultancy, Research Funding; ICER: Consultancy; OPKO: Research Funding; Biomarin: Consultancy, Research Funding. Rollins-Raval:Bayer, Inc: Membership on an entity's Board of Directors or advisory committees. Raval:Sanofi: Membership on an entity's Board of Directors or advisory committees; Bayer, Inc: Research Funding. Neal:Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

Von Willebrand factor antigen levels predict major adverse cardiovascular events in patients with carotid stenosis of the ICARAS study

Background and aimsVon Willebrand factor (VWF) plays an important role in thrombogenesis and mediates platelet adhesion particularly under high shear stress. Such conditions are generally found in stenotic arteries and can eventually cause myocardial infarction or stroke. We aimed to study whether levels of VWF antigen (VWF:Ag) predict future major adverse cardiovascular events (MACE) in patients suffering from carotid artery stenosis. MethodsPatients with atherosclerotic carotid artery disease defined by the presence of nonstenotic plaques or any degree of carotid stenosis were prospectively enrolled. Concentrations of VWF were measured by enzyme immunoassay. ResultsVWF:Ag levels were more stable after 4 freeze-thaw cycles, when compared to VWF activity, and we showed similar concentrations of VWF in citrated plasma and serum (±4%). Levels of VWF:Ag predicted future cardiovascular events in 811 patients with carotid stenosis independent of known cardiovascular risk factors. Patients with VWF:Ag concentrations in the 4th quartile had a 44% event rate after an average 3-year follow up and a hazard ratio of 2.15 (95% confidence interval 1.46–3.16; p < 0.001). ConclusionsHigh concentrations of VWF:Ag predict major cardiovascular events in patients with carotid stenosis, and given their high event rate may be useful for risk stratification of such patients.

Ischaemia–reperfusion injury with Pringle's maneuver induces unusually large von Willebrand factor multimers after hepatectomy

Introductionvon Willebrand factor (VWF) is synthesised in vascular endothelial cells and released into the plasma as unusually large VWF multimers (UL-VWFMs). Sinusoidal endothelial cells are a major target of ischaemia–reperfusion injury due to liver surgery. This study aimed to clarify the effect of hepatectomy on UL-VWFMs. Materials and methodsThirty-five patients who underwent hepatectomy were eligible for the study. Plasma ADAMTS13 activity and VWF antigen levels were measured by enzyme-linked immunosorbent assay and multimer analysis of plasma VWF was performed according to Ruggeri and Zimmerman's method. For analyses, patients were categorised according to UL-VWFM positivity after hepatectomy. ResultsPlasma ADAMTS13 activity significantly decreased from 61.0% (27.7%–126.2%) before operation to 37.4% (20.2%–71.4%) on postoperative day 7 (p < 0.001). Plasma VWF antigen levels significantly increased from 172.1% (80.5%–412.8%) before operation to 361.0% (154.7%–745.8%) on postoperative day 2, which remained high until postoperative day 7 (p < 0.001). Seven patients remained UL-VWFMs-negative and 22 patients became UL-VWFMs-positive after operation. Pringle's maneuver duration was significantly longer and blood loss volume was significantly higher in the UL-VWFMs-positive group (p = 0.001 and p = 0.003, respectively). By multivariable analysis, Pringle's maneuver duration [odds ratio 1.049, 95% confidence interval (CI) 1.001–1.098; p = 0.043] was significantly associated with increased UL-VWFMs level after hepatectomy. UL-VWFMs index was significantly correlated with Pringle's maneuver duration (r = 0.444, p = 0.017). ConclusionsPlasma UL-VWFMs levels increased after hepatectomy due to ischaemia–reperfusion injury with Pringle's maneuver.

PB2206 HEMOSTATIC MARKERS OF HYPERCOAGULABILITY IN PRIMARY MYELOFIBROSIS PATIENTS, RELATION WITH JAK2V617F ALLELE BURDEN

Background:Thrombotic complications contribute significantly to morbidity and mortality in Ph‐negative myeloproliferative neoplasms, including primary myelofibrosis (PMF). The majority of PMF patients have JAK2V617F mutation that is associated with increased thrombotic risk, CALR gene mutations are also common in PMF. Clonal myeloproliferation in PMF followed by secondary inflammation with pathologic cytokine production induce endothelium, leukocytes and platelets activation resulting in coagulation activation. Natural anticoagulants protein C (PC) and protein S (PS) deficiencies with activated protein C resistance development are supposed among the crucial procoagulant features in Polycythemia Vera and Essential Thrombocythemia, while the data on hemostatic abnormalities in PMF and their relationship with JAK2V617F burden is scarce.Aims:To evaluate the hemostatic parameters in PMF patients, predisposing high thrombosis risk, and their relationship with JAK2V617F presence and allele burden.Methods:The study included 36 PMF patients (age 30‐86 years, median 60 years). Somatic mutations (JAK2V617F and CALR) presence and JAK2V617F allele burden were assessed using allele specific polymerase‐chain reaction. The control group consisted of 68 healthy persons (30‐73 years, median 46 years). Coagulation assays (factors VIII, V, von Willebrand (VWF), antithrombin and PC activities, fibrinogen concentration, VWF antigen and free PS level) were performed by standard clotting, chromogenic and immunoturbidimetric techniques. STATISTICA 6.0 was used for data analysis. The results were presented as median with 95% confidence interval (CI). Mann‐Whitney and Spearman rank tests were applied, the differences considered statistically significant with p &lt; 0.05(∗).Results:23 (63.9%) PMF patients in the study were JAK2V617F‐positive, 8 (22.2%) had mutation in CALR gene. JAK2V617F allele burden data was available in 13 PMF patients with median 8.1% (95%CI 3.4‐43.8%). The results of hemostatic parameters in PMF patients and controls are shown in Table 1.Unexpectedly there were no significant differences in studied parameters between PMF patients with JAK2V617F and CALR mutations. However, a statistically significant inverse correlation was revealed between JAK2V617F allele burden and PC activity (r = ‐0.68301, p = 0.01).imageSummary/Conclusion:PC and, especially, PS activities were decreased in PMF, predisposing hypercoagulation. The increased FVIII activity and fibrinogen in PMF patients confirm the hypercoagulability and also reflect high inflammation level, while VWF:Ag increase suggests the endothelium damage and dysfunction, enhancing prothrombotic state in PMF. An inverse relationship between JAK2V617F allele burden and PC activity can serve as one of the mechanisms contributing to the higher thrombosis incidence depending on JAK2V617F burden.

Blood group antigen A on von Willebrand factor is more protective against ADAMTS13 cleavage than antigens B and H

BackgroundADAMTS13 specifically cleaves the peptide bond between Y1605 and M1606 within the VWF‐A2 domain. ObjectiveThe VWF contains ABO(H) blood group antigens, which may influence the susceptibility of VWF to ADAMTS13. MethodsUsing a unique monoclonal antibody recognizing the Y1605 residue, we have developed a sandwich ELISA to analyze the generation of a VWF‐DP by ADAMTS13 quantitatively. ResultsProduction of VWF‐DP after exposure to four different degrees of high shear stress was validated in comparison to the reduction in high‐molecular‐weight multimers using VWF multimer analysis. In analysis of plasma from 259 healthy individuals, plasma levels of VWF antigen (VWF:Ag) were significantly lower in blood group O than in the other groups and were significantly correlated with plasma VWF‐DP levels. The ratio between VWF‐DP and VWF:Ag was significantly higher in blood group O than in blood groups A and AB. The ratio in blood group B was also significantly higher than those in A and AB, but did not differ from blood group O. Finally, to examine whether ABO(H) blood group antigens contributed to VWF cleavage, 82 plasma samples were exposed to high shear stress using a cone‐plate shear stress inducer. The difference in the VWF‐DP/VWF:Ag ratio before and after high shear stress in blood group O was significantly greater than those in groups A and AB. ConclusionThese results indicate that blood group antigen A on VWF was more protective against ADAMTS13 cleavage than antigens B and H.

Blood platelet function abnormalities in cirrhotic patients with esophageal varices in relation to the variceal bleeding history*

Objective: The study aimed at assessing the effect of thrombocytopenia and platelet function abnormalities on the occurrence of variceal bleeding in patients with cirrhosis.Methods: The results of impedance aggregometry, von Willebrand factor antigen level and thromboelastometry (TEM) with and without the addition of a platelet inhibitor (FIBTEM®, EXTEM® test, respectively) were compared in two patient groups: Group 1 (n = 32) – patients with moderate or large esophageal or gastric varices, who had never had symptoms of acute gastrointestinal bleeding and Group 2 (n = 26) – patients with history of variceal bleeding.Results: Standard clotting test indicated more hypocoagulable profile in Group 2 compared to Group 1. However, no differences in any TEM component were observed between groups in EXTEM® test. The contribution of platelets to clot strength was significantly higher in Group 2 than in Group 1 [PLT% = 74.2 (67.5–80.4) versus 68.8 (63.7–76.5) %; p = .039]. The aggregation index was also higher in Group 2 compared to Group 1, although not statistically significant [% of healthy = 96.9 (73.2–140.1) versus 67.6 (52.5–118.8) %, p = .195]. No differences in vWF antigen levels were observed between groups.Conclusions: The results of thromboelastometry and aggregometry indicate increased contribution of platelets in clot formation in patients with a history of variceal bleeding compared to cirrhotic patients who never bled. Comparable effectiveness of hemostasis in both groups is most likely associated with the compensatory role of platelets. Increased platelet activity in this group of patients is probably due to a mechanism independent of the von Willebrand factor antigen level.