ADAMTS13 Levels Research Articles

The Pro-Adhesive and Reduced Angiogenic Properties of Endothelial Colony Forming Cells (ECFC) from JAK2V617F MPN Patients Are Not Related to the Expression of the JAK2 mutation in These Cells: A Potential Role of ADAM17

INTRODUCTION: JAK2V617F mutation is independently associated with a higher risk of venous thromboembolic events (VTE) in myeloproliferative neoplasms (MPN). We have previously shown that endothelial colony-forming cells (ECFCs) from JAK2V617F MPN patients do not express this mutation but exhibit a pro-adhesive phenotype with a lower in vitro angiogenic potential. Here, we explore the mechanisms through which ECFCs from JAK2V617F MPN patients behave differently from ECFCs from healthy individuals. METHODS: We selected 38 patients with newly or previously diagnosed MPN, regardless of current or prior hydroxyurea therapy, with or without a history of VTE. Plasma levels of ADAMTS13, P-selectin, soluble VCAM, soluble ICAM, PDGF-AA, PDGF-AB/BB, IL-8, IL-1β, and soluble CD40 ligand were measured through the use of a multiplex assay. We then tried to isolate ECFCs from 20 of the JAK2V617F patients as well as 14 healthy individuals. Successfully isolated ECFCs were then submitted to co-culture with red blood cells (RBC), after which RNA was extracted from ECFCs. ADAMTS13, P-selectin, sVCAM, and sICAM were measured in the supernatant from ECFCs and RBC co-culture with multiplex assay. ECFCs' surface expression of P-selectin, VCAM, ICAM, and E-selectin was measured with the use of flow cytometry. Gene expression in RNA from co-cultured ECFCs was assessed with two 84-gene RT-PCR arrays for endothelial cell biology and signal transduction pathways (RT2 Profiler PCR array, Qiagen®). RESULTS: Recruited patients had polycythemia vera (PV) (n=17), primary myelofibrosis (PMF) (n=7) and essential thrombocythemia (ET) (n=14); 34% of them had had a previous VTE (6/17 PV; 3/14 ET and 4/7 PMF). Median age was 59 years (26-88), 23% were male. Compared with JAK2wt, JAK2V617F MPN patients (n=29) showed higher median concentrations of sVCAM (1063 vs. 786.5 ng/ml, p=0.0315), sCD40L (339 vs. 166.4 ng/ml, p=0.0065), PDGF-AA (430.6 vs. 136.5 ng/ml, p=0.03) and PDGF-AB/BB (1068 vs. 365.8 ng/ml, p=0.004). Success in ECFC isolation was 9/20 (45%) in JAK2V617F MPN patients and 8/14 (57%) in healthy individuals. None of the ECFCs from JAK2V617F MPN patients expressed this mutation. In comparison with ECFCs from healthy individuals, ECFCs from JAK2V617F MPN patients did not show a higher expression of either VCAM1, ICAM, P-selectin, and E-selectin, as measured by flow cytometry after co-culture with RBCs. Multiplex assay on the supernatant of this co-culture did not show any difference in the levels of ADAMTS13, sVCAM, sICAM, and sP-selectin. RNA PCR array from the ECFCs after co-culture with RBCs showed no difference in the expression of adhesion molecules when compared with ECFCs from healthy individuals, except for the increased expression of ADAM17 (fold regulation: 5.46, p=0.006), a metalloprotease involved in the shedding of adhesion molecules, among them VCAM1. ECFCs from MPN patients did not show higher activation of the JAK-STAT pathway but had a lower expression of WNT5 (fold regulation: -2.22, p=0.03), indicating a reduced activation of the Hedgehog pathway, suitable with the reduced in vitro angiogenesis capacity of these cells. CONCLUSIONS: JAK2V617F MPNs are associated with an increase in circulating adhesion molecules, such as sVCAM, and platelet activation markers. The pro-adhesive phenotype and lower angiogenic potential of ECFCs JAK2V617F MPN patients are not related to this mutation, which is not expressed in these cells and might be related to an increased expression of ADAM17 and reduced activation of the Hedgehog pathway. Disclosures Ozelo: BioMarin: Research Funding; Pfizer: Research Funding; Takeda: Research Funding; Novo Nordisk: Research Funding; Roche: Research Funding.

Safety of Caplacizumab in Patients Without Documented Severe ADAMTS13 Deficiency During the HERCULES Study

Safety of Caplacizumab in Patients Without Documented Severe ADAMTS13 Deficiency During the HERCULES Study

Plasmic and Plasic Αre Εxcellent Predictors of Severe ADAMTS13 Deficiency in Thrombotic Microangiopathy Patients without Secondary Causes

Introduction: ADAMTS13 (A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats) activity remains a key tool in differential diagnosis of thrombotic microangiopathies (TMAs). However, ADAMTS13 testing is not readily available in many hospitals. Recently, PLASMIC and PLASIC scores have been developed to facilitate rapid recognition of TTP. We aimed to evaluate their usefulness compared to ADAMTS13 testing in a real-world cohort of TMA patients. Methods: We enrolled consecutive patients with samples referred to our Center for ADAMTS13 measurement due to TMA over the last 2 years. Samples were collected either at first diagnosis or relapse before initiation of treatment. ADAMTS13 activity was measured with a commercially available and validated ELISA kit (Technozym, Diapharma). Clinical data were retrospectively collected from referring centers. Management was based on treating physicians' decisions. TTP was defined as severe ADAMTS13 deficiency (activity≤10%); while secondary TMAs were diagnosed in patients with cancer, connective tissue disorders or hematopoietic cell transplantation recipients (transplant-associated TMA). Atypical hemolytic uremic syndrome (aHUS) remained a diagnosis of exclusion in patients with ADAMTS13>10%. PLASMIC was calculated based on seven variables: platelets, hemolysis, cancer, transplant, MCV, INR, creatinine; while MCV was not included in PLASIC, as previously described. ROC curve analysis was performed to determine the sensitivity and specificity of scores. Multivariate binary or logistic regression models were performed when appropriate. Results: We studied 50 TMA patients. Combined clinical and laboratory data conferred the following TMA classification: TTP in 36 patients (72%), transplant-associated TMA in 7 (14%), other secondary TMA in 5 and aHUS in 2. PLASMIC score was intermediate in 2 and high in another 4 patients without TTP. The PLASIC score was high in 5 patients without TTP, leading to less false positive results compared to PLASMIC (p<0.001). These patients suffered from secondary TMAs. In the ROC curve analysis, both PLASMIC and PLASIC significantly predicted TTP diagnosis (p<0.001 and area under the curve/AUC 0.891 and 0.892, Figure 1). In patients without secondary TMAs, PLASMIC and PLASIC had an excellent performance (p<0.001 and AUC 1). Plasma exchange was commenced in the majority of patients (42/50, 84%). Among TTP patients, the majority (77%) received rituximab as salvage or prophylactic treatment. Rituximab administration was associated with platelet (p=0.003) and ADAMTS13 (p=0.015) levels at diagnosis. The complement inhibitor eculizumab was administered in 3 patients with TA-TMA, who achieved TMA resolution. With a median follow-up of 2.9 years (range 0.3-26.3), overall survival was significantly lower in patients with secondary TMAs (p<0.001). PLASMIC or PLASIC score were not associated with clinical outcomes in our cohort. Conclusion: PLASMIC and PLASIC scores are excellent tools in TMA patients without secondary causes. While PLASMIC and PLASIC scores conferred similar outcomes, the PLASIC score requires six instead of seven variables, is classified as low/high omitting the intermediate category and leads to less false positive results. Further validation of the PLASIC score might confirm its clinical value. In addition, the role of ADAMTS13 levels in guiding rituximab administration needs to be further investigated. When an underlying etiology is detected, ADAMTS13 testing is necessary to exclude TTP and facilitate further therapeutic decisions. Figure 1 Disclosures Panayiotidis: Bayer: Other: Support of clinical trial.

Outcomes after Treatment with Upfront Cyclophosphamide or Rituximab for Initial Acquired Thrombotic Thrombocytopenic Purpura

Background: Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare, life-threatening emergency caused by auto-antibodies against ADAMTS13. Current standard therapy utilizes plasma exchange (PLEX) and corticosteroids. Although this induces remission, disease relapse remains a common problem. Since more than 90% of aTTP is caused by an antibody to ADAMTS13, additional immunosuppressive therapy with rituximab (RTX) given upfront has been shown to lower relapse rates. An alternative therapy, Cyclophosphamide (CTX) has been used in relapsed disease, but data on its use in upfront management is limited to case reports only. Our study aims to compare outcomes at our institution between upfront CTX, RTX, and standard therapy alone. Our secondary objective is to identify initial presenting features that are predictive of higher relapse risk. Methods: In a retrospective cohort study, we identified all patients at our institution with a diagnosis code of TTP (ICD 9, 10: 446.6, M31.1) between 2009 and 2019. We only included patients with a confirmed diagnosis of aTTP, defined as having an ADAMTS13 level <10%, and either a positive ADAMTS13 inhibitor or antibody. We excluded patients with follow-up of less than 30 days, or thrombotic microangiopathy (TMA) due to other causes. Relapse was defined as recurrence of TMA in addition to an ADAMTS13 level <10%, or "laboratory relapse" evidenced as an acute drop of ADATMS13 level with a positive inhibitor with/without TMA. Time to first relapse was measured from the onset of remission, defined as the time of normalization of both platelets and lactate dehydrogenase (LDH) for at least 48 hours, until the time of relapse. The cumulative incidence of relapse (CIR) was estimated using Kaplan-Meier method, and its difference among treatment groups was examined by log-rank test. The effect of continuous and categorical covariates on relapse rate was estimated by univariate Cox model. The association of categorical variables was examined using chi-square test and the difference of continuous measurements among groups was tested using T-test or ANOVA. Results: A total of 42 patients with a diagnosis of aTTP were identified in our cohort. The average age at diagnosis was 43.7 years. All patients received standard therapy with PLEX and steroids upon initial diagnosis. The prophylaxis group (n=29) received additional upfront therapy with either RTX (n=22) or CTX (n=6), and the control group (n=12) was treated with PLEX and steroids alone. There were no significant differences in the baseline characteristics between both groups. The median time to first relapse was 34.9 months in the control group, and 108.3 months in the prophylaxis group. Treatment with standard therapy alone was associated with a multi-variate hazard risk of 6.38 (95% CI: 1.1-37.7, p<0.05) for time to initial relapse. CIR at 2, 4, and 8 years was higher in the control group (41.7%, 53.3%, and 68.8%) as compared to the prophylaxis group (31.3%, 43.8%, and 43.8%, respectively). No difference was noted in CIR with CTX versus RTX, which was 40% at 4 years with CTX compared to 47% with RTX, p = 0.97 (Figure 1). Similarly, no statistically significant difference was noted in the time to first remission between CTX and RTX treatment (18 days in with CTX versus 14.9 days with RTX, p=0.583). Inhibitor titer at diagnosis correlated inversely with duration of remission (HR: 1.29, 95% CI: 1.03-1.61, p<0.05). Initial presenting platelet count, peak LDH value or co-existing autoimmune disease were not predictive of the duration of remission. Conclusion: Our study is the first to examine outcomes in a subset of patients treated with upfront CTX therapy for initial aTTP, and suggests similar time to first remission and 4-year CIR as compared to RTX. Our institutional experience suggests that CTX may be considered as an alternative therapy in patients intolerant to RTX. However, given the small group of patients treated with CTX, larger, prospective studies are warranted to confirm this observation. Consistent with currently published literature, PLEX and steroid therapy alone was associated with a significantly shorter duration of remission compared to additional immunosuppressive therapy. Furthermore, higher inhibitor titers were predictive of a shorter duration of remission, suggesting a role for aggressive upfront immunosuppressive treatment in these patients. Disclosures No relevant conflicts of interest to declare.

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.

ADAMTS13, an Anti-thrombotic Protein: Evidence Outside of Thrombotic Thrombocytopenic Purpura

von Willebrand factor (VWF) is a large multimeric plasma glycoprotein that plays a crucial role in hemostasis and thrombosis. VWF recruits platelets at sites of vascular injury by acting as a molecular bridge between circulating platelets and the site of injured or activated blood vessels. Biosynthesis of VWF is restricted to endothelial cells and megakaryocytes. Endothelial VWF is constitutively secreted into plasma and subendothelium, or is stored as "ultra-large" (UL)-VWF multimers in endothelial Weibel-Palade bodies. VWF produced in megakaryocytes is packaged as UL-VWF in the a-granules of platelets. VWF stored in endothelial and platelet storage organelles is secreted in a regulated process in response to stimulation by secretagogues. Absence or dysfunction of VWF results in bleeding symptoms, as observed in patients with von Willebrand disease. An abnormally high activity of VWF can lead to thrombotic events. Interestingly, the activity of VWF is determined by the size of its multimers and UL-VWF can spontaneously form platelet aggregates. An important regulator of VWF size is the metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which digests large thrombogenic VWF molecules into smaller, less reactive multimers via cleavage of the Y1605-M1606 bond in the VWF A2 domain. ADAMTS13 is mainly synthesized in the liver by hepatic stellate cells, but other sites of synthesis, including renal podocytes, tubular epithelial cell, platelets and endothelial cells, have also been described. ADAMTS13 is released as an active enzyme into the circulation with no physiological inhibitors. Reduced or absent ADAMTS13 activity causes the microangiopathic disorder thrombotic thrombocytopenic purpura (TTP), characterized by VWF and platelet-rich microthrombi that cause multiple organ failure and even death when left untreated. Besides its clear role in the pathophysiology of TTP, the anti-thrombotic and even anti-inflammatory properties of ADAMTS13 have also become apparent in various other thrombotic conditions. High VWF levels and low ADAMTS13 levels are associated with increased risk or even worse outcome of cardiovascular disease, including ischemic stroke and myocardial infarction. Preclinical studies in mouse models showed the beneficial effect of ADAMTS13 in both cerebral and myocardial ischemia/ reperfusion injury by decreasing both thrombosis and inflammation. In addition, ADAMTS13 was shown to also exert a direct thrombolytic effect on VWF-rich thrombi. In a mouse model of ischemic stroke, this thrombolytic activity resulted in efficient lysis of intracranial thrombi that were resistant to standard treatment with tissue plasminogen activator. Hence, ADAMTS13, as a therapeutic agent, could become an interesting avenue, not only to manage TTP, but also to treat other thrombotic complications. Disclosures De Meyer: Fonds voor Wetenschappelijk Onderzoek: Research Funding; KU Leuven: Employment, Research Funding; Ablynx: Consultancy, Research Funding; Cerenovus: Membership on an entity's Board of Directors or advisory committees; WhiteSwell: Consultancy.

Long-lasting prothrombotic state implied by changes of plasma von Willebrand factor parameters after radical prostatectomy for prostate malignancy

ObjectivesThromboembolic complications are present in 0.8%–16.8% of the cases after radical prostatectomy (RP). Association between elevated plasma von Willebrand factor (VWF) levels—as an endothelial activation marker—and increased risk of thrombotic events has been evidenced.We aimed to elicit new data on the VWF after RP in prostate cancer patients and explore the role of it as a thrombotic risk factor. Upon perioperative plasma VWF levels (VWF:Ag) its collagen-binding (CB) activity (VWF:CB), multimerization, and cleaving enzyme (ADAMTS13 [a disintegrin and metalloprotease with thrombospondin type repeats, motif 1, type 13]) of the VWF multimers were quantitated along with Factor VIII and routine laboratory parameters in this observational pilot study. MethodsPlasma samples of 24 prostate cancer patients were collected before (-1 day; D-1) and after RP (1 hour, 6 days, 1 month, and 10 months; H1, D6, M1, and M10). VWF:Ag, VWF:CB, ADAMTS13:Ag were measured by ELISA, and the multimer distribution by electrophoresis and quantitative densitometry. Factor VIII, fibrinogen, D-dimer, and other routine laboratory parameters were determined as well. Preoperative values served as baselines which were compared to controls (24 healthy individuals). ResultsVWF:Ag and CB elevated by 122% and 143% respectively at H1 after RP then plateaued at D6 compared to baseline values. ADAMTS13/VWF:Ag ratio reduced by 41% at H1, and by 46% at D6, meanwhile the ratio of high molecular weight multimers increased as well. Values returned to baseline at M1 and further reduced to the levels of the controls at M10. All of the 24 patients at H1 and D6 and 14 at M10 were in potential prothombotic state according ROC analysis of the VWF parameters as indicators. ConclusionsProstate malignancy and then surgical stress, and inflammatory reactions induced release of VWF from the endothelial cells, along with an increasing amount of large multimers and relative reduction of ADAMTS13 level. Because these changes mark a prothrombotic state even at M1 after RP, more than 1 month follow-up and prophylactic targeting through the thrombotic and inflammatory activity of the VWF is proposed. Evaluation of VWF parameters provides new information about the long-term disturbances of primary hemostasis after radical pelvic oncologic surgery like RP and might improve the understanding the physiological and pathological recovery.

Temporal map of the pig polytrauma plasma proteome with fluid resuscitation and intravenous vitamin C treatment

BackgroundFluid resuscitation plays a prominent role in stabilizing trauma patients with hemorrhagic shock yet there remains uncertainty with regard to optimal administration time, volume, and fluid composition (e.g., whole blood, component, colloids) leading to complications such as trauma‐induced coagulopathies (TIC), acidosis, and poor oxygen transport. Synthetic fluids in combination with antioxidants (e.g., vitamin C) may resolve some of these problems. ObjectivesWe applied quantitative mass spectrometry‐based proteomics [liquid chromatography‐mass spectrometry (LC‐MS/MS)] to map the effects of fluid resuscitation and intravenous vitamin C (VitC) in a pig model of polytrauma (hemorrhagic shock, tissue injury, liver reperfusion, hypothermia, and comminuted bone fracture). The goal was to determine the effects of VitC on plasma protein expression, with respect to changes associated with coagulation and trauma‐induced coagulopathy (TIC). MethodsLongitudinal blood samples were drawn from nine male Sinclair pigs at baseline, 2 h post trauma, and 0.25, 2, and 4 h post fluid resuscitation with 500 mL hydroxyethyl starch. Pigs were treated intravenously (N = 3/treatment group) with saline, 50 mg VitC/kg (Lo‐VitC), or 200 mg VitC/kg (Hi‐VitC) during fluid resuscitation. ResultsA total of 436 plasma proteins were quantified of which 136 changed following trauma and resuscitation; 34 were associated with coagulation, complement cascade, and glycolysis. Unexpectedly, Lo‐VitC and Hi‐VitC treatments stabilized ADAMTS13 levels by ~4‐fold (P = .056) relative to saline and enhanced ADAMTS13/von Willebrand factor (VWF) cleavage efficiency based on LC‐MS/MS evidence for the semitryptic VWF cleavage product (VWF1275‐1286). ConclusionsThis study provides the first comprehensive map of trauma‐induced changes to the plasma proteome, especially with respect to proteins driving the development of TIC.

Biomarkers associating endothelial dysregulation in pediatric-onset systemic lupus erythematous

Background/purposeEndothelium is a key element in the regulation of vascular homeostasis and its alteration can lead to the development of vascular diseases. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with potential extensive vascular lesions, involving skin vessels, renal glomeruli, cardiovascular system, brain, lung alveoli, gastrointestinal tract vessels and more. We aimed to assess endothelial dysregulation related biomarkers in pediatric-onset SLE (pSLE) patient serum and elucidate its correlation with their clinical features, laboratory parameters, and the overall disease activity.MethodsDisease activities were evaluated by SLE disease activity index (SLEDAI). Patient characteristics were obtained by retrospective chart review. Six biomarkers associated with endothelial dysregulation, including Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Tie2, Vascular endothelial growth factor (VEGF), thrombomodulin, and a disintegrin-like and metalloprotease with thrombospondin type 1 motif (ADAMTS13) were tested through enzyme-linked immunosorbent assay (ELISA) measurement.ResultsThis study comprised 118 pSLE patients. Data from 40 age-matched healthy controls were also obtained. The mean diagnostic age was 13 ± 4.12 years-old and 90.7% are females. Serum levels of VEGF, Tie2, thrombomodulin were significantly higher while serum ADAMTS13 was lower in active pSLE patients when compared to those with inactive diseases (all p < 0.05). In organ specific association, serum thrombomodulin level was higher in pSLE patient with renal involvement, and serum ADAMTS13 levels was negatively associated with neurological involvement (p < 0.05). A cutoff of thrombomodulin at 3333.6 pg/ml best correlated renal involvement. (AUC = 0.752, p < 0.01).ConclusionEndothelial dysregulation associating proteins seems to be potent biomarkers for pSLE activity as well as organ involvement in pSLE patients. These biomarkers may be beneficial in understanding of the vascular pathogenesis and disease monitoring.

P1. Abstract Title: Markers of Coagulation as Potential Indicators for the Diagnosis of Sepsis

<h3>Introduction</h3> The emergency department (ED) is an important venue for early diagnosis of sepsis; however, it remains a clinical challenge due to its heterogenous presentation and lack of a definitive biomarker. Neutrophil extracellular trap formation, with the release of cell free DNA (cfDNA) and activation of the coagulation cascade, and inhibition of the anticoagulant Protein C (PC) through extracellular histone, have been shown to be important in sepsis prognosis. It is unknown whether these or other molecules important in the interface between infection and coagulation, might also be diagnostic. <h3>Objectives</h3> 1) To investigate if coagulation factors known to be involved in sepsis prognosis such as PC, cfDNA can aid in sepsis diagnosis. 2) To explore other potential coagulation markers such as the ADAMTS13. 3) To determine if a panel of biomarkers, in correlation with previously known laboratory tests and clinical scores of abnormal vital signs and organ dysfunction improves sepsis diagnosis in the emergency department. <h3>Materials and Methods</h3> This is a prospective cohort study of patients with suspicion of sepsis presenting to two tertiary hospital EDs in Hamilton, Ontario. Plasma samples were obtained on initial presentation, usually before antibiotic administration, (T1) and 4 hrs later (T2). The demographic and clinical data of the patients admitted to the ED was collected from electronic medical records, including vital signs to calculate an early warning score and evidence of organ dysfunction. Plasma cfDNA levels were determined by silica-membrane-based DNA purification and UV spectrophotometry. PC, PCT and ADAMTS13 antigen were measured by enzyme-linked immunosorbent assay. <h3>Results</h3> We analysed 144 patients included in the study, mean age 68.2 years (SD 17.8), 50% males and a mean highest Hamilton Early Warning Score of 5.05 (SD 3.4). Procalcitonin, a biomarker known to increase in bacterial infections was significantly higher in the cohort at both time points compared to our healthy adult volunteers (p<0.005), while cfDNA levels did not show any change. In contrast, ADAMTS13 levels (T1and T2, p<0.0001) and Protein C levels (T2, p=0.02) were lower among the cohort compared to the healthy controls. Out of the 144 patients, 108 patients with positive body fluid cultures and antibiotic administration, had significantly higher respiratory rate and body temperature compared to culture negative patients. Notably, culture positive patients also showed a notable decline in the levels of ADAMTS13 antigen 4 hours after the initial presentation (p=0.007). <h3>Conclusions</h3> These findings suggest that when ED physicians suspect sepsis there is already evidence of coagulation abnormalities. Evaluating biomarkers associated with increased risk of thrombosis could aid in the diagnosis of sepsis.

Assessment of ADAMTS-13 Level in Hospitalized Children with Serious Bacterial Infections as a Possible Prognostic Marker.

Background and objectives: In children, acute infection is the most common cause of visits in the primary care or emergency department. In 2002, criteria for diagnostics of pediatric sepsis were published, and then revised in 2016 as “life-threatening organ dysfunction due to a dysregulated host response to infection”. In the pathophysiology of sepsis endothelial dysfunction plays a very important role. Deficient proteolysis of von Willebrand factor, due to reduced ADAMTS-13 activity, results in disseminated platelet-rich thrombi in the microcirculation. ADAMTS-13 deficiency has been detected in systemic inflammation. The clinical relevance of ADAMTS-13 during sepsis is still unclear. We aimed to investigate the possible use of ADAMTS-13 as a prognostic marker in children with serious bacterial infection (SBI). Materials and Methods: Inclusion criteria were hospitalized children with SBI, aged from 1 month to 17 years. SBI was defined based on available clinical, imaging, and later also on microbiological data. Sepsis was diagnosed using criteria by The International Consensus Conference. In all the patients, the levels of ADAMTS-13 were measured at the time of inclusion. Results: Data from 71 patients were analyzed. A total of 47.9% (34) had sepsis, 21.1% (15) were admitted to the ICU, 8.5% (6) had mechanical ventilator support, and 4.2% (3) patients had a positive blood culture. The median level of ADAMTS-13 in this study population was 689.43 ng/mL. Patients with sepsis, patients admitted to the Intensive Care Unit, and patients in need of mechanical ventilator support had significantly lower levels of ADAMTS-13. None of the patients had ADAMTS-13 deficiency. In patients with SBI, the area under the curve (AUC) to predict sepsis was 0.67. A cut-off ADAMTS-13 level of ≤730.49 had 82% sensitivity and 60% specificity for sepsis in patients with SBI. Conclusions: ADATMS-13 levels were lower in patients with SBI and sepsis, but AUC and sensitivity were too low to accept it as a prognostic marker.

PF805 INCIDENCE, CLINICAL FEATURES, RISK FACTORS AND OUTCOMES OF REFRACTORY ACQUIRED IMMUNE THROMBOTIC THROMBOCYTOPENIC PURPURA: A CHINESE MULTI‐CENTER EXPERIENCE

Background:Acquired immune thrombotic thrombocytopenic purpura(TTP) is an autoimmune disorder that is characterized by microangiopathic hemolytic anemia and thrombocytopenia with associated organ dysfunction. Refractory TTP that fails to respond to standard therapeutic plasma exchange(TPE) and prednisone continues to be associated with high mortality. Although recent evidence had identified age, renal involvement, and cardiac involvement as predictors of high mortality, little information is available on clinical factors capable of predicting the treatment response that may be able to guide the initiation of early and targeted aggressive therapy.Aims:To identify the incidence and early indicators of refractory TTP and risk factors of exacerbation, relapse and mortality.Methods:The study population consisted of all patients with clinically suspected TTP from 14 large academic medical centers in China from January 2005 to January 2018. We excluded patients who were diagnosed congenital TTP or other thrombotic microangiopathies. Final diagnosis of acquired immune TTP was confirmed by at least one senior hematologist. Data(demographic, clinical, laboratory and treatment information) obtained at diagnosis and prior to treatment were collected from electronic medical records. Stepwise regression was used to identify independent risk factors.Results:Among 261 patients referred to the 14 medical centers, 126 patients satisfied the inclusion criteria and were included in the analysis. Overall, 41/126 patients were regarded as having refractory TTP. Compared with those of the responsive group, the patients in the refractory group were older and had higher d‐dimer, C‐reactive protein(CRP) and serum creatinine levels and more acute kidney insufficiency. The ADAMTS13 levels were comparable between the two groups. In multivariate logistic analysis, we identified albumin&lt;35 g/L(OR 3.939, 95% CI 1.050–14.782; p = 0.042) and CRP &lt; 15 mg/L(OR 0.280, 95% CI 0.085–0.928, p = 0.037) as independent predictors of refractory TTP. All patients underwent a median of 5 TPE sessions, and corticosteroids were given to most of the patients. When combined with first‐line treatments, alternative treatments, such as intravenous immunoglobulin, rituximab, cyclosporine A and vincristine, each showed better outcomes, although the differences were not statistically significant. With a median follow‐up time of 1131 days, the 1‐year overall survival(OS) and progression‐free survival of all TTP patients were 74.1% and 60.3%, respectively. Refractory and responsive TTP had significantly different outcomes(p &lt; 0.001). In the multivariate analysis, an older age(OR 1.082, 95% CI 1.017–1.151, p = 0.013), proteinuria(OR 13.628, 95% CI 1.406–132.072, p = 0.024) and increased indirect bilirubin(OR 1.067, 95% CI 1.022–1.114, p = 0.03) were independent risk factors of exacerbation/relapse, but exacerbation/relapse had no influence on OS(p = 0.566). Cox regression indicated that two factors were significantly associated with OS: age&gt;45 years(HR 14.127, 95% CI 1.404–142.136, p = 0.025) and acute kidney insufficiency(HR 9.100, 95% CI 1.838–45.052, p = 0.007).Summary/Conclusion:This novel observation showed that 32.5% of TTP cases were refractory and that serum albumin and elevated CRP might be early predictors of these cases. These factors may have the potential to guide intensification of therapy and improve survival outcomes, which should be reevaluated in larger prospective studies.

Risk stratification and prognostic evaluation of endothelial cell-specific molecule1, von Willebrand factor, and a disintegrin-like and metalloprotease with thrombospondin type 1 motif for sepsis in the emergency department: An observational study.

This study evaluated endothelial cell-specific molecule1 (endocan), von Willebrand factor (vWF), and A disintegrin-like and metalloprotease with thrombospondin type 1 motif (ADAMTS-13), alone or in combination, in the risk stratification and prognosis prediction of patients with sepsis. Clinical data of 301 patients were prospectively analyzed, and divided into systemic inflammatory reaction syndrome, sepsis, severe sepsis, and septic shock groups. A total of 40 healthy individuals were studied as the control group. Endocan, vWF, ADAMTS-13, vWF/ADAMTS-13, and procalcitonin levels were measured, and Acute Physiology and Chronic Health Evaluation (APACHE II) score, Mortality in Emergency Department Sepsis (MEDS) score as well as Sequential Organ Failure Assessment (SOFA) score were calculated. The all-cause death or survival of each patient was recorded during the 28-day follow-up. The endocan, vWF, and vWF/ADAMTS-13 levels significantly increased in patients and were positively correlated with disease severity. On the first day of admission, MEDS score, ADAMTS-13, and vWF/AMAMTS-13 ratio were independent predictors for 28-day mortality from sepsis. Moreover, the combination of vWF/ADAMTS-13 ratio with MEDS score improved the accuracy in predicting the 28-day mortality from sepsis. On day 5, endocan, vWF, ADAMTS-13, and vWF/ADAMTS-13 ratio were independent predictors for the 28-day mortality from sepsis, while the combined use of endocan and vWF/ADAMTS-13 ratio improved the prognostic value of individual indicators. Endocan, vWF, ADAMTS-13, and vWF/ADAMTS-13 ratio are valuable in the risk stratification and prognostic evaluation of sepsis as novel biomarkers.

A Multimodality Approach to Assessing Factor I Genetic Variants in Atypical Hemolytic Uremic Syndrome

A Multimodality Approach to Assessing Factor I Genetic Variants in Atypical Hemolytic Uremic Syndrome

Prevention of relapse in patients with acquired thrombotic thrombocytopenic purpura undergoing elective surgery: a case series

Essentials Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease. Surgery is a possible trigger of acute TTP episodes and no guidelines are available. Six patients with severe ADAMTS-13 deficiency during remission underwent elective surgery. Patients were prophylactically treated to restore ADAMTS-13 activity and no relapses occurred. SUMMARY: Background Severe ADAMTS-13 deficiency has been recognized as the main risk factor for recurrence of thrombotic thrombocytopenic purpura (TTP). Several conditions, including surgery, may influence the levels of ultra-large von Willebrand factor and ADAMTS-13, acting as a trigger for an acute TTP event. Objectives To report our experience of management of six patients with acquired TTP who underwent elective surgery after prophylactic treatment to restore ADAMTS-13 activity levels. Patients Six patients followed for acquired TTP with severe ADAMTS-13 deficiency during remission were candidates for seven elective surgeries (inguinal hernioplasty, cholecystectomy, laparoscopic hysterectomy, oophorectomy, parotidectomy and two total hip arthroplasties). Results Four patients were treated with prophylactic plasma exchange (PEX) therapy immediately before surgery. One patient was treated with PEX therapy before her first surgery and with preemptive rituximab once her second surgery was scheduled. Because rituximab increased ADAMTS-13 levels only partially, she required one PEX procedure the day before her second surgery. One patient was treated with azathioprine after rituximab failure, obtaining a progressive increase of ADAMTS-13 activity to more than 40%. This level allowed her to undergo total hip arthroplasty without additional treatment. All surgeries were successful and no complications or relapses occurred. Conclusions Six patients with acquired TTP underwent seven successful surgical procedures using prophylaxis to restore ADAMTS-13 activity. Further observational studies or randomized clinical trials are needed to confirm whether prophylactic PEX could be the key factor in preventing relapse.

Association between fibrinogen and fibrinogen γ’ and atherosclerotic plaque morphology and composition in symptomatic carotid artery stenosis: Plaque-At-RISK study

IntroductionVon Willebrand Factor (VWF), ADAMTS13, fibrinogen and fibrinogen γ’ are associated with an increased risk of ischemic stroke. Carotid atherosclerosis is an important risk factor for ischemic stroke. Characteristics of the vulnerable plaque; intraplaque hemorrhage (IPH), plaque ulceration and lipid-rich necrotic core (LRNC) can be visualized with imaging techniques. Since atherosclerosis might attribute to the association between coagulation factors and ischemic stroke risk, the aim of this study is to investigate the association between coagulation factors and atherosclerotic plaque characteristics in more detail. Materials and methodsIn 182 patients of the Plaque-At-RISK study (prospective multicenter cohort study) with a recent transient ischemic attack (TIA) or ischemic stroke and a symptomatic mild-to-moderate carotid artery stenosis, we measured VWF antigen (VWF:Ag), ADAMTS13 activity, fibrinogen (Clauss), and fibrinogen γ’. Presence of plaque ulceration, IPH volume and LRNC volume were determined by Multidetector-Row Computed Tomography (MDCTA, n = 160) and Magnetic Resonance Imaging (MRI, n = 172). Linear regression analysis was used to assess the association between imaging biomarkers and coagulation factors. ResultsVWF:Ag or ADAMTS13 levels were not significantly associated with plaque ulceration, IPH and LRNC. We found an inverse association between fibrinogen and fibrinogen γ’ and IPH volume (B = −23.40 mm3/g/L, p = 0.01 and B = −161.73 mm3/g/L, p = 0.01) and between fibrinogen and fibrinogen γ’ and LRNC volume (B = −38.89 mm3 g/L, p < 0.01 and B = −227.06 mm3 g/L, p = 0.01). Additional adjustments for C-reactive protein (CRP) did not change the results. ConclusionsFibrinogen and fibrinogen γ’ are inversely associated with IPH volume and LRNC volume, independent of inflammation.Clinical Trial Registration: clinicaltrials.govNCT01208025

Comparison of sepsis severity markers in emergency department

Objective This study was to evaluate the values of endothelial cell-specific molecule1 (endocan),von Willebrand factor (vWF),andA disintegrin-like and metalloprotease with thrombospondin type 1 motif(ADAMTS-13), alone or in combination, in the risk stratification and disease severity assessment of patients with sepsis via comparing the differences of these markers in patients with systemic inflammatory reaction syndrome (SIRS), sepsis, severe sepsis, or septic shock, and healthy volunteers. Methods Clinical data of 301 patients with SIRS or sepsis treated in our Emergency Department from October 2014 to October 2015, were prospectively analyzed. These patients were divided into SIRS, sepsis, severe sepsis, and septic shock groups. 40 healthy individuals were selected as control. Endocan, vWF, ADAMTS-13, vWF/ADAMTS-13, and Procalcitonin (PCT) levels were measured, and APACHEⅡ score, MEDS score as well as SOFA score were calculated. The all-cause death or survival of each patient was recorded during the 28-day follow-up. Results The endocan, vWF, and vWF/ADAMTS-13 levels significantly increased in patients with SIRS, sepsis, severe sepsis, or septic shock and were positively correlated with disease severity, and were also positively correlated with MEDS score, APACHEⅡ score, SOFA score. On the other way, the ADAMTS-13 level gradually declined during disease progression and was negatively correlated with the disease severity, it was also negatively correlated with MEDS score, APACHEⅡ score, SOFA score. Conclusions Endocan, vWF, ADAMTS-13, and vWF/ADAMTS-13 ratio are valuable in the risk stratification and disease severity evaluation of sepsis, providing novel sepsis biomarkers in clinic. Key words: Endothelial cell specific molecule-1; von Willebrand factor; A disintegrin-like and metalloprotease with thrombospondin type 1 motif; Sepsis; Risk stratification

Thrombotic thrombocytopenic purpura in a 13 year boy – a case report

Thrombotic thrombocytopaenic purpura (TTP) is a very rare presentation in the paediatric population.1 We report a previously healthy 13-year-old boy of Korean heritage, who presented to the Children’s Hospital Westmead with prolonged epistaxis, thrombocytopenia (platelet count of 5×109/L) and haemoglobinuria. There were no neurological or renal manifestations present. There was no significant haematological family history. On examination he had widespread petechiae and ecchymosis, dark coloured urine and skin pallor. He was anaemic and the blood film demonstrated red cell fragmentation consistent with microangiopathic haemolytic anaemia (MAHA). The coagulation studies were normal. He was treated with plasmapheresis, and subsequently commenced on rituximab and prednisolone therapy. He suffered recurrent allergic reactions to blood products requiring a tailored premedication regime. Overall he tolerated the treatment well. The prednisolone has been weaned over six months, on the basis of clinical and haematological improvement. On investigation, he had a deficiency of the vWF cleaving protease ADAMTS13 (2%) and high levels of vWF Ag (333%). Autoantibodies inhibiting the protease were detected suggesting an autoimmune cause of his TTP.1 Interestingly, 6 months later he was found to have a large goitre, with associated biochemical hypothyroidism and autoantibodies. This is under ongoing investigation.

The ADAMTS13-VWF axis is dysregulated in chronic thromboembolic pulmonary hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is an important consequence of pulmonary embolism that is associated with abnormalities in haemostasis. We investigated the ADAMTS13–von Willebrand factor (VWF) axis in CTEPH, including its relationship with disease severity, inflammation, ABO groups and ADAMTS13 genetic variants.ADAMTS13 and VWF plasma antigen levels were measured in patients with CTEPH (n=208), chronic thromboembolic disease without pulmonary hypertension (CTED) (n=35), resolved pulmonary embolism (n=28), idiopathic pulmonary arterial hypertension (n=30) and healthy controls (n=68). CTEPH genetic ABO associations and protein quantitative trait loci were investigated. ADAMTS13–VWF axis abnormalities were assessed in CTEPH and healthy control subsets by measuring ADAMTS13 activity, D-dimers and VWF multimeric size.Patients with CTEPH had decreased ADAMTS13 (adjusted β −23.4%, 95% CI −30.9– −15.1%, p<0.001) and increased VWF levels (β +75.5%, 95% CI 44.8–113%, p<0.001) compared to healthy controls. ADAMTS13 levels remained low after reversal of pulmonary hypertension by pulmonary endarterectomy surgery and were equally reduced in CTED. We identified a genetic variant near the ADAMTS13 gene associated with ADAMTS13 protein that accounted for ∼8% of the variation in levels.The ADAMTS13–VWF axis is dysregulated in CTEPH. This is unrelated to pulmonary hypertension, disease severity or markers of systemic inflammation and implicates the ADAMTS13–VWF axis in CTEPH pathobiology.

Analysis of survival and risk factors of DVT in patients with cervical cancer

Objective To investigate the relationship between vWF levels and ADAMTS13 levels of DVT (venous thrombosis) in patients with cervical cancer, and then to evaluate the predictive value of vWF levels and ADAMTS13 levels of cervical cancer patients with DVT. Methods A total of sixty-six patients diagnosed as cervical cancer and treated from June 2015 to June 2016 in Weinan Maternal and Child Health Hospital were enrolled, and their baseline data, pathological type, tumor classification, TNM staging, vWF, ADAMTS13 levels and Autar score were recorded.All patients were followed up for 1 year and the patients with adverse outcomes were defined as poor prognosis group during the follow-up period while the patients whose disease progression free were defined as good prognosis group.Univariate analysis showed that the incidence of venous thrombosis in patients with cervical cancer was affected; Cox proportional hazard model further assessed the impact of all statistically significant factors on the occurrence of venous thrombosis in patients; t test showed the distribution of influential factors between the two groups; ROC curve was used to analyze the sensitivity and specificity of vWF and ADAMTS13 levels in predicting the occurrence of venous thrombosis in cervical cancer patients. Results At the end of the follow-up, among the 66 patients, Six were not followed and 40 had good prognosis without venous thrombosis, which was a good prognosis group; Twenty had poor prognosis, i.e.venous thrombosis, which was a poor prognosis group, including 5 deaths.The poor prognosis rate was 30.30%.Univariate Cox proportional hazard model results showed that vWF (P=0.023, (95%CI: 1.743-1.215)) and ADAMTS13 (P=0.037, (95%CI: 1.158-1.566)) had a significant effect on the patients with venous thrombosis.The levels of vWF ((0.535±0.075) μg/L) and ADAMTS13 ((69.453±10.284) %) in good prognosis group were significantly lower than those in poor prognosis group((0.592±0.082)μg/L, (79.245±11.477) %), and the differences between the two groups were statistically significant (t=2.690, 3.345, P<0.05). Under ROC curve, the AUC of vWF level was 0.841, the sensitivity and specificity were 55% and 97.5% respectively, and that of ADAMTS13 level was 0.906, the sensitivity and specificity were 85% and 80%, respectively. Conclusion The levels of vWF and ADAMTS13 have a good predictive effect on the occurrence of venous thrombosis in patients with cervical cancer.It is expected to be used as a routine evaluation index to predict the occurrence of venous thrombosis in patients with cervical cancer after treatment. Key words: Cervical cancer; Venous thrombosis; Von Willebrand factor vWF; A distintegrin-like and metalloproteinase with thrombospond in type-1 domain 13