vWF Antigen Levels Research Articles

3233 Von Willebrand Factor is Localized in the Extravascular tissue of Patients with Juvenile Scleroderma

OBJECTIVES/SPECIFIC AIMS: To further explore the role of vWF in the pathogenesis of scleroderma by identifying its location within the tissue of sample biopsies obtained as part of routine diagnosis with the use of immuno-histochemical staining. METHODS/STUDY POPULATION: We examined 8 skin biopsies from 2 patients with systemic sclerosis (SSc), 2 with localized scleroderma (LS) and 4 with JDM. Double immunofluorescence staining was performed in each tissue with antibodies against vWF and collagens type I and III. DAPI (4′, 6-diamidino-2-phenylindole) was also used for counterstaining of inflammatory cells. Tissue staining patterns were compared between groups. RESULTS/ANTICIPATED RESULTS: Biopsies were obtained from the upper extremity of 7 females and the lower extremity of 1 male. Median age, symptom duration, and serum levels of vWF antigen around the time of biopsy was 8 years (IQR 4.5-11), 5.5 months (IQR 2.5-7), and 245% (IQR 203-302 for 7 patients), respectively. All but 1 biopsy was performed prior to initiation of immunosuppressive therapy. Immunofluorescence staining showed a superficial and deep perivascular inflammatory cell infiltrate that co-localized with vWF in all tissues. There was expression of vWF in the extravascular tissue of patients with JScl co-localizing with collagen III in the reticular dermis (Figures 1 and 2). In comparison, vWF expression was restricted to the endothelium and did not co-localize with collagen in the dermis of patients with JDM (Figure 3). Patients with SSc had higher expression of vWF as compared to patients with LS. DISCUSSION/SIGNIFICANCE OF IMPACT: vWF may participate in the pathogenesis of cutaneous inflammatory conditions. We have demonstrated that vWF co-localizes with cellular inflammatory infiltrates in the perivascular areas and in the dermis of patients with JScl and JDM. We additionally speculate that vWF may participate in the pathogenesis of fibrosing skin diseases based on evidence of increased extravascular expression in the tissue of patients with JScl (vs. JDM), and its co-localization with collagen. vWF expression intensity in the dermis of JScl patients may relate to disease extension (SSc vs. LS).

Practice Patterns for Diagnosis of Type 1 Von Willebrand Disease and Effect of Age on Von Willebrand Factor Levels: A Single Institutional Retrospective Study

IntroductionType 1 von Willebrand disease (VWD 1) accounts for ~75% of VWD. The National Heart, Lung, and Blood (NHLBI) VWD guidelines recommend VWF:Ag and VWF:RCo cut-off levels of <30 (IU/dL or %) for definite laboratory diagnosis of VWD 1. However, guidelines indicate that patients with “low VWF” (VWF:Ag and VWF:RCo in the 30-50 IU/dl range) could be candidates for agents to increase VWF levels if they are at risk for bleeding. Here we examine the diagnostic practices for VWD at Mayo Clinic Rochester by determining the proportion of patient currently classified as VWD 1 that meet NHLBI criteria for “low VWF”. In addition, we assess changes in the laboratory phenotype of the aging population of patients with “low VWF” and VWD 1.MethodsPatients with a diagnosis of VWD 1 at our institution were identified through our institutional hemophilia treatment center database (1986 to 2017). All available VWF:Ag and VWF activity (ristocetin cofactor assay [VWF:RCo] and latex immuno-activity assay [VWF:Ab]) levels performed at our institution and available in our electronic medical record (EMR) were recorded. Exclusion criteria included confounding variables known to effect VWF levels (eg,. hormonal therapy, pregnancy, desmopressin, or VWF/VIII concentrate administration) at the time of first laboratory assessment (VWF:Ag and VWF activity (by either VWF:Ab or VWF: RCo) at our institution. Bleeding score, retrospectively assessed using the MCMDM-1 VWD bleeding assessment tool (BAT), at the time of first clinical evaluation was recorded if enough information was available in the EMR. All VWF testing was recorded for comparative analysis between the first and the most recent measurements within the study period. Patients were not included in age-related analysis if the most recent laboratory values were obtained in the presence of the confounding variables noted above. Statistical analysis was performed using JMPfor paired t-tests and STATA for multilevel mixed-effects regression analysis.Results78 patients (50 F) met study criteria. Median age at initial presentation at our institution was 27.5 (0-75) years. Medians (ranges) for VWF:Ag (n=78), VWF:Ab (n=41), and VWF:RCo (n=75) were 39 (5-60)%, 37 (19-53)%, and 36 (11-62)%, respectively (reference ranges 55-200% for VWF and factor VIII values). Coagulation factor VIII activity (FVIII:C) was 63 (10-122)% (n=78). Of 37 patient with available blood type results, 23 (62%) were blood group O. 56% of patients (n=44) met NHLBI criteria for classification as “low VWF” (Figure 1, Panel A). There was no difference in bleeding score between patients with VWD 1 (median bleeding score: 4 (0-14), n=22) compared to those with “low VWF” (median bleeding score: 4 (0-14), n=37).54 patients had repeat laboratory testing at 4.5 (0-29) years after initial testing. VWF antigen and activity levels were compared in those who had repeat testing done >1 year (n=36) after initial lab testing at our institution. Median age of patients at time of most recent measurement was 42.5 (5-87) years compared to 35.5 (0-75) years at first measurement. VWF:Ag was significantly higher in the most recent measurement compared to the first measurement (p< 0.05, Figure 1, Panel B ). There was no statistically significant difference between VWF activity levels in the first versus the most recent measurement.There was a non-significant trend towards increasing VWF:Ag with increasing age; however when stratified into “low VWF” and VWD 1 disease groups, there was a significant relationship between VWF Ag and age in the “low VWF” group with mixed effects regression model (coefficient: 0.207, p:0.043).ConclusionApproximately 55% of patients with a clinical diagnosis for VWD 1 meet criteria for “low VWF.” In our cohort, there was no difference in bleeding (BAT) scores between the two groups; however our analysis is limited by its retrospective nature. We also demonstrate a statistically significant increase in VWF:Ag with aging; however, with regression analysis, this change was only noted in those in the “low VWF” group. Larger prospective studies controlling for confounding factors are needed to further demonstrate the effect of age on VWF antigen and activity levels. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Active and Ultra-Large Von Willebrand Factor Is Significantly Elevated in Patients with Immune Thrombotic Thrombocytopenic Purpura

▪Background: Immune thrombotic thrombocytopenic purpura (iTTP), a potentially fatal hematological emergency, is primarily caused by acquired deficiency of ADAMTS13 activity due to autoantibodies. Immunoglobulin G (IgG)-type autoantibodies bind ADAMTS13 and inhibit its ability to cleave endothelium-derived ultra large von Willebrand factor (ULVWF). However, it remains poorly understood whether plasma VWF status can be used as a disease marker for diagnosis and monitoring therapy in patients with acute iTTP.Objective: To address this question, we determined plasma levels of VWF antigen (VWF:Ag), collagen-binding activity (VWF:CB), active forms of VWF (VWF:Ac), and VWF multimers in iTTP patients during acute episode and in early remission.Patients and Methods: From the Alabama registry, we identified 69 unique patients with a confirmed diagnosis of iTTP in whom plasma ADAMTS13 activity was <10 U/dL with positive inhibitors and elevated anti-ADAMTS13 IgGs. Of 69 patients, 21 had longitudinal plasma samples collected. Plasma samples from 56 healthy individuals, who did not have a hematological disease, cancer, and infection, were recruited as controls. Plasma levels of VWF:Ag, VWF:CB, and VWF:Ac were determined by an ELISA-based assay. Plasma VWF multimer distribution was assessed by an in-gel Western blotting assay following electrophoresis on a 1% SDS-agarose gel.Results: The mean age for our cohort iTTP patients was 43.9 ± 13.4 years. Twenty-six patients were male and 43 were female with male to female ratio of 1 to 1.7. Fifty-three patients were African American descents, 14 Caucasians, 1 Hispanic, and 1 unknown race. Plasma levels of VWF:Ag in acute iTTP patients were 289.4 ± 17.7%, significantly increased compared with those in the healthy controls (144.9 ± 7.6%) (p<0.0001); plasma levels of VWF:CB in these patients were 241 ± 17.9%, also significantly elevated compared with those in the healthy controls (149.9 ± 12.01%) (p=0.0001); additionally, plasma levels of VWF:Ac (304.6 ± 23.2%), assessed by its ability to bind anti-VWF-A1 nanobody, were more dramatically elevated compared with those in the controls (101.6 ± 5.9%) (p<0.0001). More interestingly, while the ratios of VWF:CB to VWF:Ag in patients with acute iTTP (0.8 ± 0.04) were lower than those in the healthy controls (1.0 ± 0.05) (p=0.0036), the ratios of VWF:Ac to VWF:Ag were significantly higher in patients with acute episode (1.2 ± 0.1) than those in the controls (0.8 ± 0.05) (p=0.0003). Furthermore, there was no statistically significant difference in the patient plasma levels of VWF:Ag (p=0.69) and VWF:CB (p=0.08) during acute episode and during early remission. However, the plasma levels of VWF:Ac in patients with acute disease were significantly higher than those in the early remission (p=0.002). Surprisingly, 90% (36/40) of out iTTP patients during acute episode showed the presence of ULVWF in their plasma using in-gel Western blotting, which allows the ULVWF to be detected without the transfer step to avoid any potential loss of larger VWF multimers during protein transfer. These ULVWF multimers disappeared in 3/4 iTTP patients in remission when ADAMTS13 activity recovered. In 28 healthy control samples, only one showed ULVWF.Conclusion: Our results demonstrate, for the first time in a large cohort, that active forms of VWF and ultra large VWF multimers are present in iTTP patient's plasma during the acute period, which is reduced or disappears during the early remission. Therefore, measuring active forms of VWF and ultra large VWF multimers may aid in diagnosis of iTTP and help monitoring of disease processes following therapy. Our ongoing study is to determine whether these biomarkers can be used to predict responses to treatment and long-term outcome. DisclosuresZheng:Alexion: Research Funding, Speakers Bureau.

Elevated vWF Antigen Serum Levels Are Associated With Poor Prognosis, and Decreased Circulating ADAMTS-13 Antigen Levels Are Associated With Increased IgM Levels and Features of WM but not Increased vWF Levels in Patients With Symptomatic WM

BackgroundWaldenström's macroglobulinemia (WM) is a rare malignancy characterized by bone marrow infiltration by lymphoplasmacytic cells and the presence of a monoclonal IgM paraprotein. The interactions of lymphoplasmacytic cells with other cells in their microenvironment, including mast cells and endothelial cells, support their survival and proliferation and can induce resistance to therapy. von Willebrand factor (vWF) plays a key role in primary hemostasis but is also a marker of endothelial “stimulation.” High levels of vWF have been associated with an adverse prognosis in patients with symptomatic WM and might reflect the interactions between lymphoplasmacytic cells and other cells of their microenvironment. Materials and MethodsConsidering vWF and ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) as markers of endothelial dysfunction and activation, we evaluated the prognostic importance of vWF and ADAMTS-13 antigen levels in the serum of patients with previously untreated symptomatic WM to validate vWF as a possible prognostic marker for progression-free and overall survival. We also validated the measurement of vWF in the serum instead of citrated plasma and investigated the possible correlations of ADAMTS-13 antigen levels with disease characteristics. The analysis included 42 patients with symptomatic WM and 19 matched healthy controls. ResultsThe serum levels of vWF antigen provided significant prognostic information, and patients with levels ≥ 200 IU/dL had a very poor prognosis compared with patients with lower levels. The ADAMTS-13 antigen levels were decreased in WM patients and correlated with the IgM levels, β2-microglobulin, and extent of bone marrow infiltration. ConclusionvWF levels measured in the serum could become an important prognostic marker in patients with WM and requires further investigation.

FAKTOR PATOGENESIS DAN DIAGNOSIS PENYAKIT von Willebrand

von Willebrand disease (vWD) is an autosomal inherited bleeding disorder caused by a deficiency or abnormality of von Willebrandfactor (vWF). vWF is a large multimeric glycoprotein that mediates platelet adhesion at the site of vessel injury. It also protects factorVIII from proteolytic degradation in the circulation. vWD has a prevalence of about 1% in the general population but less than 10%have bleeding symptoms. Bleeding symptoms are usually mucocutaneous and post surgical with varying severity. This disorder canresult from either a quantitative (types 1 and 3) or qualitative (type 2) defect in vWF. Type 2 vWD has been further classified into fourdistinct subtypes; 2A, 2B, 2M and 2N. The diagnosis of vWD requires attention to personal and family history of excessive bleeding andconfirmation by laboratory evaluation. A mild chronic thrombocytopenia is often seen in type 2B vWD. Patients with mild vWD oftenhave both a normal bleeding time and normal APTT. Specific tests for vWD diagnosis involve vWF antigen level, vWF activity (ristocetincofactor), and factor VIII activity. Once a diagnosis is established, additional tests that aid in classifying the type of vWD includeristocetin-induced platelet aggregation and vWF multimer analysis.

Colistin dampens fibrinolysis and endothelial activation during endotoxaemia

SummaryColistin electrostatically interacts with lipopolysaccharides (LPS). Preclinical studies demonstrated beneficial effects of colistin on LPS-induced coagulation and fibrinolysis. The objective of this trial was to investigate the effects of colistin during experimental endotoxaemia. In this randomised, double-blind, placebo-controlled, crossover trial 16 healthy volunteers received a 2 ng/kg LPS bolus after infusion of 2.5 million IU colistin or placebo. Plasma levels of F1+2 prothrombin fragments, thrombin-antithrombin complexes (TAT), von Willebrand factor antigen levels (vWF), E-selectin, plasmin-antiplasmin complexes (PAP), tissue-type plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor-1 (PAI-1) were measured. Infusion of colistin significantly reduced peak concentrations of PAP complexes by 70 %, t-PA antigen levels by 63 % and t-PA activity by 48 %, while PAI-1 levels decreased numerically by 63 %. Two hours after the LPS bolus F1+2 levels and TAT complexes were slightly reduced in the colistin period, but peak concentrations were similar in both periods. Colistin blunted the LPS induced four-fold increase in soluble E-Selectin levels by ∼50 % and the two-fold increase in vWF antigen levels by ∼70 %. The LPS-scavenging actions of colistin significantly reduce endothelial activation and fibrinolytic response in the human endotoxaemia model, while the activation of the coagulation system remains largely unaffected.Note: This work was conducted at the Medical University of Vienna.EudraCT-Nr.: 2014–00285720 Supplementary Material to this article is available online at http://www.thrombosis-online.com

Bleeding after tonsillectomy

Oropharyngeal hemorrhage after tonsillectomy is one of the most common postprocedural ENT emergencies that require immediate attention. Management choices depend on the severity of bleeding, underlying platelet or coagulation disorders, and the patient’s maturity and age. Treatment range from simple observation to an emergent need to both secure the airway and stop the hemorrhage. The workup of the hemorrhagic patient will be discussed from the preoperative to postoperative time period. Surgical techniques in response to various acuity will be discussed to fully elucidate options to control the hemorrhage.

VWF correlates with visceral and pericardial adipose tissue in patients with a recent stroke of suspected cardiogenic etiology.

AimsA chronically elevated level of von Willebrand factor (vWF) is a common finding in patients with cardiovascular diseases. Obesity is a well-recognized risk factor for thrombotic cardiovascular complications including ischemic stroke, and it has been linked with increased plasma vWF. We evaluated whether elevated plasma levels of vWF associate with areas of visceral (VAT), pericardial (PAT), and subcutaneous adipose tissue (SAT) compartments in patients with acute/subacute stroke.Methods and resultsA total of 69 patients with stroke of suspected cardiogenic etiology were examined. The plasma level of vWF antigen (vWF-ag) was measured both in the acute phase and in the chronic phase three months after stroke. The areas of VAT and/or PAT were assessed with computed tomography. As expected, in stroke patients, the levels of plasma vWF-ag were significantly higher than in the national reference population both in the acute and in the chronic phase. The level of vWF-ag in the chronic phase correlated with the amounts of VAT and PAT, but not with subcutaneous adipose tissue.ConclusionsThese results agree with previous observations of the chronic inflammation/prothrombotic tendency in patients with cerebrovascular disease. Future studies should seek to clarify the role of visceral type adipose tissue in the pathophysiology of ischemic stroke.

Increased Clot Formation in the Absence of Increased Thrombin Generation in Patients with Peripheral Arterial Disease: A Case-Control Study.

In peripheral arterial disease (PAD), activation of the hemostatic system may contribute to atherosclerosis progression and atherothrombotic events. This case-control study assesses the overall coagulation status in PAD patients by evaluating coagulation markers in combination with thrombin generation potential, whole blood (WB) clot formation, and fibrinolysis. In blood from 40 PAD patients (n = 20 with cardiovascular event within 1 year after initial diagnosis, n = 20 without) and 40 apparently healthy controls, thrombin generation was determined in WB and platelet-poor plasma. Whole blood rotational thromboelastometry (ROTEM) measurements were triggered with tissue factor with/without tissue plasminogen activator. We observed increased levels of erythrocyte sedimentation rate, leukocytes, eosinophil granulocytes, vWF antigen, fibrinogen, and D-dimer in PAD patients (p < 0.05). Markers of thrombin generation potential showed no difference between patients and healthy controls. In PAD patients with event compared to patients without, WB-thrombin generation showed a lower thrombin potential when triggered with 0 and 2.5 pM tissue factor. The ROTEM clotting assay showed significantly faster clot formation and increased clot firmness in PAD patients compared to controls. No significant differences were found for parameters of clot degradation. There are no significant differences between the thrombin generation profiles of PAD patients and healthy controls. Between PAD patients with and without cardiovascular event, the WB thrombin generation appears to differ. Mechanistically, PAD patients show an increased ability to form a stable clot in WB in comparison to healthy controls. This is most likely due to the increased fibrinogen levels related to the inflammation in atherosclerosis, confirming the importance of the inflammation-coagulation axis.

THU-449 - Preoperative von willebrand factor – antigen predicts clinical outcome after liver resection: a prospective, international, multicenter trial

Backgroundand Aims:vWF-antigenhasbeenshowntobeincreased in patients with portal hypertension and to predict mortality in patients with chronic liver disease. This study aimed to assess the clinical utility of preoperative vWF-antigen levels to predict poor postoperative outcome in patients undergoing liver resection in a routine clinical setting. Methods: 95patientsundergoingliverresectionservedasprospective exploration cohort and results were validated in an independent cohort of 133 patients for 4 different institutions. VWF-Ag was evaluated perioperativelyand postoperative outcomewas recorded. Results: Preoperative vWF-antigen levels significantly predicted postoperative liver dysfunction (LD, area under the curve [AUC]: 0.725, P=0.009). Furthermore, a cut-off of vWF-antigen ≥182% was defined to identify patients with a higher incidence of postoperative LDormorbidity(LD:≥182%:33.3%,<182%:5.9%,P<0.001;morbidity: ≥182%: 74.1%, <182%: 44.1%, P=0.008). We confirmed our results within a prospective validation cohort (LD: ≥182%: 20.0%, <182%: 5.2%, P=0.008; morbidity: ≥182%: 56.4%, <182%: 35.1%, P=0.015). Analyzing the entire cohort, we found that patients exceeding the cut-off suffered from a significantly increased incidence of postoperative LD, morbidity, prolonged hospitalization, intensive care unit stayand mortality. Conclusions: Within this study we were able to reveal and subsequently validate the potential of preoperative vWF-antigen levels to predict poor postoperative outcome in patients undergoing liver resection. As vWF-antigen is easily determined and available in most standard laboratories, it seems to be avaluable clinical marker to allow for preoperative risk-stratification of patients undergoing liver resection.

Superimposed Hypercoagulable and Hypocoagulable States: Elevated Von Willebrand Factor (vWF) Levels Secondary to Polycystic Ovary Syndrome Masking Von Willebrand Disease

Von Willebrand Disease (vWD) is the most common inherited bleeding diathesis and can present with easy bruising, cutaneous bleeding, prolonged bleeding from mucosal surfaces after surgical procedures, and menorrhagia. However, laboratory confirmation of vWD can be challenging, as vWF and Factor VIII are acute-phase reactants, and levels will be elevated in inflammation, stress, physical activity, estrogen therapy, and hyperthyroidism.Polycystic Ovary Syndrome (PCOS) induces a state of both chronic inflammation and hyperestrogenism. PCOS is further characterized by a prothrombotic state, with elevated circulating levels of vWF, fibrinogen, and PAI-1. The precise origin of PCOS-induced hypercoagulability is yet to be elucidated but is most likely multifactorial, with insulin resistance, hyperestrogenism, hyperandrogenism, LH excess, age, and BMI imparting varying degrees of influence and synergism.We present a case of a morbidly obese 23 year-old female with PCOS presenting with menorrhagia. She has a history of prolonged bleeding after surgical procedures and was self-referred due to concern for bleeding risk during an upcoming dental extraction. Laboratory evaluation revealed she was blood group A positive with a normal PT and aPTT. Evaluation for vWD was within normal limits, with vWF antigen of 84% (55-200%), vWF activity of 68% (55-200%), Factor VIII activity assay of 75% (55-200%), and normal vWF multimers. Factor IX assay was significantly elevated at 218% (60-150%). Thromboelastography (TEG) showed elevated MA and G, consistent with platelet hypercoagulability and increased clot strength.Subsequent review of remote external records determined that the patient had a previous evaluation for a bleeding disorder at age three following prolonged bleeding after tonsillectomy. At that time, she had a prolonged aPTT of 36.1 sec (21.0-31.0 sec) that corrected after mixing study to 29.9 sec. vWF antigen and ristocetin cofactor levels were both low. Factors VIII, IX, XI, and XII were within normal limits. The patient was diagnosed with type I von Willebrand disease at that time.PCOS is a hypercoagulable state associated with hyperestrogenism and elevated vWF levels, which may lead to a false negative workup for vWD. In this case, a patient with PCOS and a remote diagnosis of type I vWD had elevation of vWF levels into the normal range on current laboratory studies. The patient's current laboratory studies are consistent with a hypercoagulable state with an elevated Factor IX level and do not reflect the underlying bleeding diathesis. Management of such patients with coexisting hypercoagulable and hypocoagulable states, particularly perioperatively, may be complex. DisclosuresNo relevant conflicts of interest to declare.

The Growth Differentiation Factor-15 Levels Are Increased in Patients with Compound Heterozygous Sickle Cell and Beta-Thalassemia, Correlate with Hepcidin-25/Ferritin Molar Ratio and with Markers of Hemolysis, Endothelial Dysfunction and Angiogenesis

Background: The clinical manifestations of Sickle Cell Disease (SCD) include episodes of vascular occlusion, chronic hemolytic anemia and frequent infections. SCD is also characterized by the presence of chronic inflammation manifested by leukocytosis and monocytosis and increased circulating levels of pro-inflammatory cytokines and chemokines. Growth Differentiation Factor-15 (GDF-15), also known as macrophage inhibitory cytokine 1 (MIC-1) or non-steroidal anti-inflammatory drug-activated gene (NAG-1) is a member of the transforming growth factor- superfamily. Expression of the GDF-15 gene in cardiomyocytes, vascular smooth muscle cells, and endothelial cells is strongly upregulated in response to oxidative stress, inflammation and tissue injury. Recently, GDF-15 and Twisted Gastrulation Protein (TWSG1) have been proposed to be erythroblast-derived factors, although not erythroblast specific, that mediate Hepcidin-25 suppression under conditions of increased erythropoietic activity. High levels of GDF15 associate with ineffective erythropoiesis and may reflect a certain type of bone marrow stress or erythroblast apoptosis. However, the role of GDF15 in Hepcidin-25 regulation under physiologic conditions and in other disorders is unclear and has been debated.Aims: The aim of this study was to evaluate the GDF-15 levels in patients with compound heterozygous HbS and beta-thalassemia (HbS/βthal) and to explore possible associations with disease features, such as Hepcidin-25 production, hemolysis, inflammation, endothelial dysfunction and angiogenesis.Methods: Seventy-five adult Caucasian patients with HbS/βthal were included in the study, while 20 healthy individuals served as controls. Patients with HbS/βthal divided in two groups: group A included 36 patients under hydroxycarbamide (HC+) treatment and group B included 39 patients without hydroxycarbamide (HC-) treatment. Along with hematology and blood chemistry parameters determination, measurements of circulating levels of GDF-15, hepcidin-25, hs-CRP, vWF-antigen, hs-TnT and Placental Growth Factor (PlGF) were performed in both patients with HbS/βthaland controls using immunoenzymatic techniques.Results: The main results of the study included: GDF-15 levels were elevated in patients with HbS/βthal compared to controls ((1,980.7±159.8 vs 665.4±50.9 pg/mL, p<0.001). Regarding hydroxycarbamide treatment, GDF-15 levels were elevated in (HC+) patients compared to (HC-) patients (p=0.002), or 30/36 vs 21/39 patients had elevated GDF-15 levels (p=0.002). In contrast, Hepcidin-25 levels were significantly lower in patients with HbS/βthal compared to controls (57.2±12.3 vs 67.3±9.1 ng/mL, p<0.01). The reduction of Hepcidin-25 levels was more pronounced in (HC-) patients compared to (HC+) patients (p=0.005). In addition, a markedly low Hepcidin-25/Ferritin molar ratio was obsereved in patients with HbS/βthal compared to controls (p<0.001). Whilst, no direct correlation was found between GDF-15 and hepcidin-25 levels, a significant negative correlation between GDF-15 levels and Hepcidin-25/Ferritin molar ratio was detected in patients with HbS/βthal(p=0.002). GDF-15 levels also correlated significantly with markers of erythropoiesis, such as Hb, HbF, ferritin and reticulocytes (p<0.05), with markers of hemolysis, such as LDH and uric acid (p<0.01), and with markers of endothelial dysfunction and angiogenesis such as vWF-antigen and PlGF (p<0.01). Surprisingly, no correlation was found between GDF-15 and hs-CRP levels.Conclusions: These findings demonstrate a multifactorial role of GDF-15 in patients with HbS/βthal as it correlates with erythropoiesis, hemolysis, endothelial dysfunction and angiogenesis. However, we found no direct specific function, despite the negative correlation with Hepcidin-25/Ferritin molar ratio. Interestingly, the higher GDF-15 levels measured in patients treated with hydroxycarbamide may reflect possible drug induced sub-clinical cardiotoxicity, although this has not been described to-date. To this end, our knowledge is restricted only to doxorubicin-induced cardiotoxicity, where GDF-15 up-regulation seems to be more sensitive than that of hs-TnT, LDH and NT-proBNP. Further studies will reveal the role of GDF-15 in the biology of HbS/βthal. DisclosuresOstland:Intrinsic LifeScienc s: Employment, Equity Ownership. Westerman:Intrinsic LifeSciences: Employment, Equity Ownership. Terpos:Genesis: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Honoraria.

A Shear Micro-Gradient Microfluidic to Monitor Platelet Aggregation Dynamics in the Context of Von Willebrand Disease

Background:Virchow's triad identifies the three principle parameters driving haemostasis and thrombosis as: i. Changes in vessel wall properties and exposure of subendothelial matrix proteins; ii. Presentation of blood borne chemical activators (hypercoaguability); and iii.Blood-flow dependent mechanical factors (haemodynamics). Studies identifying a key role for micro-scale shear gradients in driving the earliest stages of platelet thrombus formation have informed the development of a novel set of microfluidic devices that have potential utility as rapid and efficient screening tools of shear dependent platelet function.Aim:The aim of this project was to undertake a small scale clinical and laboratory based characterisation study of a microfluidic platform designed by us and to assess its ability to identify differences in platelet aggregation dynamics in citrated whole blood taken from control subjects and subjects with clinically diagnosed or undiagnosed von Willebrand disease (Types 1, 2 and 3).Method:Patients with VWD were recruited from the haemophilia outpatient clinic, Alfred hospital. Whole blood samples (250mL) or samples treated ex vivo to block the canonical platelet amplification loop pathways were perfused at a defined flow rate (45ml/mL) through a set of well defined micro-shear gradient geometries pre-coated with purified VWF to initiate platelet capture. Microfluidic geometries were characterised by an entry shear rate of 1,800.s-1, that was accelerated to a peak shear rate of either 45,000.s-1 or 150,000.s-1, returning to an exit shear rate of 1,800.s-1. The rate of initial shear acceleration was varied using a series of geometries with contraction entry angles varying from 15 - 85o.Results:The microfluidic platform was able to identify patients with Types 1 (VWF antigen < 30%), 2A and 3 VWD. ROC analysis of control versus VWD samples determined that the device sensitivity approached 94.4%, with a specificity of 100% for VWD. A statistically significant difference (p< 0.05) was observed when comparing control blood samples to type 1VWD (p< 0.001) and type 2A VWD samples (p=0.004), with both subtypes showing minimal to no platelet aggregation in the device. Patients presenting with bleeding symptoms but found not to have VWD (normal VWF:Ag levels) showed no significant difference (p=0.907) to controls. Furthermore, exogenous titration of Type 3 (n=2) VWD blood samples with purified VWF (10 - 100mg/mL) recapitulated platelet aggregation in a concentration dependent manner. Head to head comparison with standard laboratory based tests including, VWF:Ag, VWF:CB and VWF:RCo demonstrated a strong linear correlation with device output. In addition, time-course (0, 2 and 4 Hrs) trials demonstrate that the device is a sensitive measure of DDAVP treatment of VWD.Conclusion:The Studies presented demonstrate that haemodynamically sensitive platelet aggregation within our prototype platform is critically dependent on blood VWF antigen levels and demonstrates good proof-of-concept that the microfludic can selectively identify VWD dependent defects in whole blood platelet aggregation. Taken together these data suggest that a microfluidic platform with discrete haemodynamic control can operate as a sensitive screen for VWD. Future studies will focus on defining the particle and key haemodynamic parameters that shift both device selectivity and sensitivity. DisclosuresNo relevant conflicts of interest to declare.

Changes of ADAMTS13 Activity and TSP1 Level in Patients with Hematologic Malignancies

To investigate the changes of thrombospondin 1(TSP1) level and von Willebrand factor cleaving protease(ADAMTS13) activity in the patients with hematologic malignancies before and after treatment and to evaluate their clinical significance. Eighty-two patients with hematologic malignancies were enrolled in this study, among them 20 patients were with acute leukemia, 48 patients were with lymphoma and 14 patients were with multiple myeloma. The plasma samples of 82 patients with hematologic malignancies and 45 healthy controls were collected. The activities of ADAMTS13 were evaluated by residue collagen binding assay(R-CBA), the levels of TSP1 and vWF antigen were measured by enzyme-linked immunosorbent assay(ELISA). The activity of plasma ADAMTS13 in patients with hematologic malignancies was lower than that of normal controls(P<0.05). The levels of vWF antigen and TSP1 in the patients with hematologic malignancies were higher than those in normal controls(P<0.05). After standard induction chemotherapy, the ADAMTS13 activity of the patients with hematologic malignancies at the complete remission was higher than that before therapy(P<0.05); the vWF antigen level was significantly lower than that in the patients with hematologic malignancies before therapy(P<0.05), but still higher than that in controls(P<0.05). There were 25 infection patients in 82 cases of hematologic malignancies, and the ADAMTS13 activity in the patients with newly diagnosed hematologic malignancies complicated with infection before therapy was obviously lower than that in the patients with hematologic malignancies without infection(P<0.05), the levels of vWF antigen and TSP1 were significantly lower than that in patients without infection (P<0.05). In the process of treatment, 8 patients have been speculated to suffer from thrombus, and the ADAMTS13 activity in the patients with thrombus was obviously lower than that in the patients without thrombus(P<0.05). Low ADAMTS13 activity and high TSP1 level may participate in the progress of hematologic malignancies, the infection and thrombotic events may lead to further reduction of the ADAMTS13 activity. Assaying the level of ADAMTS13 activity in the patients with malignant tumor may be helpful to prevent the infection and thrombosis in the patients with hematologic malignancies.

Evaluation of von Willebrand factor and ADAMTS-13 antigen and activity levels in sickle cell disease patients in Kuwait.

Sickle cell disease (SCD) is a severe form of hemolytic anemia characterized by chronic hemolysis and is associated with increased thrombotic risk. Elevated von Willebrand factor (vWF) levels in SCD have been attributed to increased secretion and impaired processing by its cleaving protease ADAMTS-13. In this study we measured vWF and ADAMTS-13 antigen and activity levels in our SCD patients. Hematological and biochemical parameters for 59 SCD patients (20 children and 39 adults) were analyzed and compared to 59 age- and sex-matched controls. Commercially available ELISA kits were used to measure vWF and ADAMTS-13 antigen and activity levels in patients and controls. Patients had significantly higher levels of vWF (p < 0.006) and ADAMTS-13 activity (p < 0.006) compared to controls. When patients were analyzed according to age and genotype, adult patients (23SS and 16Sβ0thal) maintained higher vWF antigen levels (p < 0.001), but with reduced ADAMTS-13 activity to vWF:Ag ratio (p < 0.003) compared to controls. Pediatric patients (8SS and 12Sβ0thal) had comparable vWF antigen levels to controls (p > 0.05), but had higher levels of ADAMTS-13 activity (p < 0.011) and ADAMTS-13 activity to vWF:Ag ratio (p < 0.038). Age is an important factor to consider when vWF and ADAMTS-13 proteins are analyzed among our patients. Increased vWF in adult patients may be attributed to increased production and resistance of vWF to proteolysis rather than ADAMTS-13 deficiency. This outcome was not seen in pediatric patients as higher ADAMTS-13 activity maintained vWF antigen at comparable levels to normal controls.

ABO blood group status and Von Willebrand Factor antigen levels in a cohort of 100 blood donors in an African population

Von Willebrand Factor (vWF) antigen levels are known to vary widely in healthy individuals. Approximately 30% of the variation in plasma vWF is due to the effect of ABO blood group. The aim of this study was to determine the plasma vWF levels in different ABO blood groups in an African population. Using blood samples of 100 blood donors at the Blood Bank Unit, University of Uyo Teaching Hospital, Uyo, Nigeria, the ABO blood group phenotype was determined by standard tube method while von Willebrand factor antigen (vWF: Ag) was determined by ELISA Method. There were 63 male subjects and 37 female subjects with a mean age of 31.7 ± 6.39 years. The frequency of O, A, B and AB blood groups were 50%, 31%, 16% and 3%, respectively. The mean plasma vWF: Ag concentration of the subjects was 1.38 ± 1.02IU/ml with blood group A having the lowest mean vWF: Ag level (1.087 ± 0.402) followed by group O (1.2896 ± 1.005), followed by group AB (1.687 ± 1.061), followed by group B (2.176 ± 1487) having the highest level. This study reaffirms the well-known notion that ABO blood group influences the plasma level of vWF: Ag and also demonstrates that vWF levels in group O individuals are lower than non-group O individuals in Nigeria, Africa.

Influence of HSCT Preconditioning on ADAMTS-13 Activity and vWF level and Its Clinical Significance

To explore the influence of precondintioning on ADAMTS-13 activity and vWF level, and its clinical significance by measuring the alterations of ADAMTS-13 activity and vWF antigen levels before and after preconditioning of hematopoietic stem cell transplantation (HSCT) in the patients. A total of 113 patients received HSCT in the First Hospital affiliated to Soochow University were investigated, 20 healthy volunteers were used as the control. The ADAMTS-13 activity and vWF antigen level were measured by FRETS-vWF73 and ELISA respectively. Modified BU/CY, TBI/CY or BEAM were used as preconditioning regimen. (1) out of all the patients enrolled in this study, 8 patients were diagnosed as thrombotic disorders, and 49 patients were with occurence of acute graft-versus-host disease (aGVHD); (2) comparing with the control group, the ADAMTS-13 activity were lower and vWF antigen was higher in the patients after preconditioning (P < 0.01). Among all the patients, 69 (59.3%) cases showed the decrease of ADAMTS-13 activity after preconditioning, including 9 patients with more than 60% (9/113, 8.0%) decrease, in the meantime, the average plasma vWF antigen level of these 69 patients was significantly increased after preconditioning (P < 0.05); (3) the plasma ADAMTS-13 activity in 8 patients with thrombotic complications decreased after preconditioning, and there was significant difference in comparision with that of patients without thrombotic complication (P < 0.01). The patients with decrease of ADAMTS-13 activity more than 60% accounted for 37.5% (3/8), at the same time, the the level of vWF antigen increased (P < 0.01); (4) The medium level of ADAMTS-13 activity was dropped in 49 patients with aGVHD after preconditoning, but there was no abvious difference in comparision with that of patients without aGVHD. Among them 25 patients were with significant drop actvity of ADAMTS-13 at that time when aGVHD occurred (P < 0.01). Out of them, the patients with drop more than 60% before preconditioning accounted for 60% (2/35). The logistic regression analyse showed that the drop of ADAMTS-13 activity more than 60% before preconditioning was the risk factor for occurence of thrombosis at the later stage (P < 0.01), but the drop of ADAMTS-13 activity after preconditioning did not was the risk factor for occurence of aGVHD. The decrease of ADAMTS-13 activity after preconditioning of HSCT confirmed to be lower than that befor preconditioning of HSCT, and the level of vWF antigen after preconditioning of HSCT could be higher than that before precondifioning, especially in patients with thrombosis. The decrease of ADAMTS-13 activity after preconditioning has been found to be mone than 60%, then this decrease can be considered as an independent risk factor for later thrombogenesis, but the decrease of ADAMTS-13 after precoditioning activity did not associate with occurence of aGVHD, so the decrease of ADAMTS-13 activity can be used as an important predictor of thrombosis after HSCT.

Thrombotic microangiopathy without renal involvement: two novel mutations in complement‐regulator genes

ESSENTIALS: The differential diagnosis among thrombotic microangiopathies (TMAs) is challenging. We studied a case of TMA with neurologic symptoms, no renal impairment and normal ADAMTS-13 levels. Two novel mutations in complement factor I and thrombomodulin genes were identified. Complement-regulator genes can be involved in TMAs with normal ADAMTS-13 regardless of renal damage. Thrombotic microangiopathies (TMAs) often represent a challenge for clinicians, because clinical, laboratory, and even genetic features are not always sufficient to distinguish among different TMAs. The aim of this study was to investigate the pathogenetic mechanisms underlying an acute case of TMA with features of both thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS). We report the case of a 49-year-old woman who developed an acute TMA with neurologic involvement and no renal impairment. ADAMTS-13, von Willebrand factor, and complement-system biochemical characterization was performed on acute phase samples. Exome sequencing and direct Sanger sequencing of previously aHUS-associated genes were performed. The functional consequences of the thrombomodulin (THBD) mutation were investigated by in vitro expression studies. Despite a clinical diagnosis of TTP, the patient had normal ADAMTS-13 levels and increased VWF antigen levels with ultra-large von Willebrand factor multimers. C3, C4, and complement factors H and I (CFI) were normal. Molecular analysis confirmed two novel heterozygous mutations in CFI (c.805G>A, p.G269S) and THBD (c.1103C>T, p.P368L), and in vitro expression studies showed a reduction in the generation of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) caused by mutated THBD. This proinflammatory condition, associated with the p.G269S mutation in CFI, probably leads to a complement-mediated endothelial activation, with a relevant prothrombotic potential in case of transient environmental triggers. This study identified the first case of acute TMA without renal involvement but with neurological damage carrying two novel mutations in complement-regulator genes, highlighting the possible role of the complement system as a common pathogenetic mechanism in TMAs.

Quality of Clotting Factor Activity in Fresh Frozen Plasma at Thaw with a Microwave System and after Storage at 4 degrees C for 48 Hours.

Uncontrolled hemorrhage in polytrauma patients usually results in rapid need of blood products. Despite the shorter thawing times of microwave devices for heating fresh frozen plasma (FFP), their use has remained controversial, and just a few laboratory analyses have been published on this topic. The aim of this study was to analyse the quality of clotting factors immediately after thawing FFP with a microwave device and after 48-hour post thaw storage at 4 degrees C. 24 FFP units of all four ABO blood groups (six of each blood group) were thawed with a Transfusio-therm 2000 and later stored at 4 degrees C for 48 hours. Samples were drawn aseptically and investigated on various clotting factors and protein proteases (fibrinogen, antithrombin, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, vWF antigen and activity, protein S, and protein C) using standard coagulation and chromogenic assays immediately after thawing and again after a 48-hour storage period at 4 degrees C. All units were tested for both anaerobic and aerobic microbial contamination using standard operating procedures immediately after thawing. After thawing, all coagulation factors and protein protease activities were within normal ranges. Blood group O individuals had approximately 25% lower plasma levels of vWF antigen and activity. After a 48-hour storage period at 4 degrees C, FVIII and FIX activities declined significantly in all blood groups, whereas the remaining clotting factors remained comparably stable. Immediately after rapid thawing using a microwave system, all FFP units contained adequate coagulation factor activities to maintain hemostatic activity at the time of product thaw. The post thaw refrigerated storage caused an anticipated decrease in factor VIII and IX activities, but retained normal coagulation factor levels of many plasma proteins. Therefore we conclude that the Transfusio-therm 2000 has no clinically significant influence on the activity of clotting factors and plasma proteases in FFP units.

Assessing Bleeding Risk in Pediatric Patients Undergoing Major Spinal Surgery

Preoperative screening for bleeding disorders in pediatric patients is problematic due to children's limited exposures to significant hemostatic challenges and the inherent difficulty in obtaining blood samples from young patients. Overcoming these challenges is of particular importance for surgical procedures that carry a significant bleeding risk, such as spinal surgeries. Many pediatric surgeons, including the Pediatric Orthopedic team at our Institution, rely on an unfocused history and measurement of general markers of hemostasis for preoperative screening. In order to improve preoperative screening of pediatric patients undergoing spinal procedures, we instituted the use of a detailed semi-quantitative questionnaire based on the ISTH Bleeding Assessment Tool (BAT), in combination with evaluation of PT, aPTT, platelet count, and PFA. The BAT gives positive points for a personal or family history of bleeding, and negative points for significant hemostatic challenges that did not result in bleeding complications. It was decided a priori that a BAT score of ≥3 would result in referral to Pediatric Hematology. A total of 212 patients presenting for major spinal surgeries (e.g., spinal fusion, growth rod placement) ranging in age from 3 to 25 years were prospectively evaluated in this fashion. A total of 41 patients (19.3%) had a prolongation of the PT and/or aPTT, none of which had a high BAT score. The majority of the abnormal PT/aPTT values were minimal prolongations that were not reproducible on repeat testing. Prolongation of the PT and/or aPTT revealed 3 patients with mild deficiencies of either factors VII, X, or XI, none of which were felt to be clinically significant. Prolonged PFAs were observed in 32 patients (16%), 1 of which was diagnosed with type I VWD (BAT score = 1), and the other with "possible VWD" based on a borderline VWF antigen level (BAT score = 0). Both were treated with Humate P. The remainder of the patients with a prolonged PFA were determined not to have a significant bleeding disorder after further testing. A total of 15 patients were referred to Hematology based on a high BAT score. Of these, 2 had a history of thrombocytopenia (1 with known DiGeorge syndrome and 1 with Depakote-related thrombocytopenia). Neither required platelet transfusion. One patient with a high BAT score was known to have type I VWD and was treated with Humate P, another was diagnosed with low expression of glycoprotein GP1b and was treated with Humate P and platelet transfusion. The remainder of the patients with high BAT scores were not felt to have a clinically significant bleeding disorder based on a Hematologist's assessment. None of the 212 patients evaluated were felt to have excessive intraoperative bleeding by the surgical team, suggesting that none of the patients had a significant undiagnosed hemostatic defect. Together, these results suggest that reliance on history or screening labs alone may not be sufficient for many pediatric surgery patients. While the PFA identified 2 patients with mild/possible VWD that would have been missed by the BAT, the PFA also had a significant number of apparent false positives. The combination of a BAT and a platelet count, as well as assessment of VWF activity for patients without previous hemostatic system challenges, may provide a more effective screening methodology for institutions with ready access to VWF activity measurement. DisclosuresNo relevant conflicts of interest to declare.