Isolated tumour microparticles induce endothelial microparticle release in vitro Cancer induces a hypercoagulable state, resulting in an increased risk of venous thromboembolism. One of the mechanisms driving this is tissue factor (TF) production by the tumour, released in small lipid bound microparticles. We have previously demonstrated that tumour cell line media-induced procoagulant changes in HUVEC. The aim of this study was to investigate the effect of tumour microparticles and recombinant human TF (rhTF) on the endothelium. Procoagulant microparticles from the PANC-1 cell line were harvested by ultrafiltration. HUVEC were then incubated with these procoagulant microparticles or rhTF. Flow cytometry was used to investigate the effect of endothelial cell surface protein expression and microparticle release. Microparticles but not soluble TF was responsible for the procoagulant activity of cell-free tumour media. We also demonstrated an increase in endothelial microparticle release with exposure to tumour microparticles, with a positive linear relationship observed (R2 = 0.6630 P ≤ 0.0001). rhTF did not induce any of the changes observed with microparticles. Here we demonstrate that procoagulant activity of tumour cell line media is dependent on microparticles, and that exposure of endothelial cells to these microparticles results in an increase in microparticle release from HUVEC. This suggests a mechanism of transfer of procoagulant potential from the cancer to the remote endothelium. Correspondence to Dr Leigh A. Madden, Department of Biomedical Science, The University of Hull, Hardy Building, Cottingham Road, Hull HU6 7RX, UK. Tel: +44 1482466031; e-mail: l.a.madden@hull.ac.uk Received 7 March, 2019 Revised 1 October, 2019 Accepted 5 November, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Long-term outcomes in the treatment of acquired hemophilia A: a 16-year single institution prospective cohort experience Acquired hemophilia A (AHA) is a rare bleeding disorder caused by autoantibodies against coagulation factor VIII. We conducted a single institution prospective cohort study to assess treatment strategies and long-term outcomes in AHA patients and provide further evidence for effective treatment and relapse timing. A total of 25 patients diagnosed with AHA between 2001 and 2017 at Penn State Hershey Medical Center were prospectively followed. Information was collected on factor VIII activity and inhibitor titer at diagnosis, treatment regimen(s), complete remission, and relapse time. For immunosuppressive therapy (IST), 19 patients were treated initially with prednisone and cyclophosphamide, four were treated with prednisone, one with prednisone and rituximab, and one with prednisone and second-line rituximab. 13/17 (76%) evaluable patients treated with prednisone and cyclophosphamide achieved complete remission. Four patients received rituximab as second-line therapy (inhibitor titers 34, 122, 416, and 768 BU); three achieved complete remission and one died from sepsis. Both evaluable patients receiving initial prednisone alone achieved complete remission. Five relapses occurred from 17 days to 7 years; all were treated with prednisone and cyclophosphamide and achieved complete remission. IST with prednisone and cyclophosphamide is highly effective in achieving and maintaining complete remission, even for relapsed patients. Despite dual IST with prednisone and cyclophosphamide, some patients, particularly with extremely high inhibitor titers, required addition of second-line rituximab to achieve complete remission. This supports rituximab as effective salvage treatment, including for patients with inhibitor titers at least 100–200 BU. Those who experienced relapse often did so years after complete remission, signifying importance of continued monitoring and vigilance. Correspondence to Natthapol Songdej, MD, MPH, Department of Medicine, Penn State Health Milton S. Hershey Medical Center, 500 University Drive, C6602, Hershey, PA 17033-0850, USA. Tel: +1 717 531 8399; fax: +1 717 531 0647; e-mail: nsongdej@pennstatehealth.psu.edu Received 17 April, 2019 Revised 8 October, 2019 Accepted 5 November, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Influence of hemolysis, icterus and lipemia on coagulation tests as performed on Cobas t511 new analyzer In the coagulation laboratory, spurious hemolysis, icterus and lipemia (HIL) in test samples represent by far the leading diagnostic prenalytical challenges. The aim of this study was to assess the performance of the preanalytical module on the new hemostasis analyser Cobas Roche t511. We assessed the influence of HIL on prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fib), antithrombin and D-dimer on plasma pools aliquots with different interference degrees. Moreover, we evaluated spontaneous hemolysis by comparing results on 50 paired samples (hemolysed versus nonhemolysed). Spurious hemolysis interference studies highlight the absence of a clinical significant impact on PT, APTT and antithrombin test results at all hemoglobin concentration investigated. For Fib and D-dimer assays a clinically significant difference was observed in the most hemolysed aliquot for Fib and in the two most hemolysed aliquots for D-dimer. Spontaneous hemolysis interference studies showed no clinical significant differences for PT and antithrombin assays, instead for APTT, Fib and D-dimer we found significant statistical and clinical differences between hemolysed and non hemolysed specimens. Bilirubin interference studies and lipemic samples interference studies enable us to confirm that the differences in the results obtained between the different aliquots and reference pool is not clinically significant for all assays. HIL check preanalytical module of Cobas Roche t511 analyzer displaied excellent performance for routine use in clinical laboratories. Regardless of analytical considerations, the type of interference encountered with spurious HIL is substantially different and requires different approaches. Correspondence to Barbara Montaruli, S.C. Laboratorio Analisi, AO Ordine Mauriziano, Largo Turati 62, 10128 Turin, Italy Tel: +39 11 5085; fax: +39 11 5082668; e-mail: bmontaruli@mauriziano.it Received 21 June, 2019 Revised 30 September, 2019 Accepted 5 November, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

A novel mutation in the GP1BA gene in Bernard–Soulier syndrome The Bernard–Soulier syndrome (BSS) is a rare disease with a prevalence of 1/1000 000; it is characterized by macrothrombocytopenia. BSS develops as a result of a defect in the glycoprotein GPIb-IX-V complex on the platelet surface. In this article, we present a pediatric patient with the novel mutation that has been identified for the first time in BSS. A 13-month-old male patient was admitted with severe thrombocytopenia unresponsive to intravenous immunoglobulin in the neonatal period and recurrent mucocutaneous bleeding which initiated at 5 months of age. glycoprotein (GP) IX (CD42a) expression was normal as per flow cytometry results. Genetic analysis revealed a homozygous c.243C>A (p.Cys81*) (p.C81*) mutation. This novel mutation identified by us presents with severe thrombocytopenia and normal GPIX (CD42a) expression and is mistaken for immune thrombocytopenia in the neonatal period. This mutation creates an early stop codon and possibly leads to loss of function of the receptor. Correspondence to Zeynep C. Özdemir, Division of Pediatric Hematology/Oncology, Department of Pediatrics, Eskişehir Osmangazi University Faculty of Medicine, 26480 Eskişehir, Turkey Tel: +90 505 669 11 40; fax: +90 222 239 34 50; e-mail: efecanan@yahoo.com; ORCID: 0000 0002 9172 9627 Received 24 June, 2019 Revised 23 October, 2019 Accepted 25 October, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Neutrophil extracellular traps and NETosis: a report of two autopsies and review of literature Recent studies reveal that neutrophil extracellular traps (NETs) play a significant role in platelet entrapment and consequent activation of the coagulation cascade. Herein we present two autopsy cases of NETosis. The first case is a 76-year-old man, with metastatic squamous cell carcinoma of the lung who expired 5 days post admission. Autopsy revealed extensively necrotic poorly differentiated squamous cell carcinoma of the right lung. A 30-cm cylindrical thrombus was identified, extending from the left ventricle to the thoracic aorta, composed of numerous neutrophils enmeshed in abundant fibrin representing a NET. The second case is a 73-year-old man who suffered a cardiopulmonary arrest of unknown cause and expired 2 days post admission. Autopsy revealed a 5-cm mural thrombus with numerous neutrophils in the descending aorta consistent with NET, bilateral bronchopneumonia and infarcted bowel. These two autopsies highlight the pathogenic role of NET in causing thrombosis. Correspondence to Kritika Krishnamurthy, A.M. Rywlin, MD Department of Pathology, Mount Sinai Medical Center, 4300 Alton Road, Suite 2400, Miami Beach, FL 33140, USA. Tel: +1 305 674 2277; fax: +1 305 674 2999; e-mail: Kritika.Krishnamurthy@msmc.com Received 27 June, 2019 Accepted 5 November, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

A novel homozygous mutation (Gly1715Ser) causing hereditary factor V deficiency in a Chinese patient To explore the phenotype and genotype of a Chinese family with hereditary factor V deficiency. Routine blood coagulation indexes were detected by one-stage clotting method, whereas factor V antigen was detected by ELISA. All exons and intron–exon boundaries of F5 gene were amplified by PCR and sequenced directly. The suspected mutation was confirmed by reverse sequencing. Bioinformatics softwares were used to analyze the possible impact of this mutation. Phenotypic analysis showed that the proband had significantly prolonged prothrombin time and activated partial thromboplastin time, and his factor V clotting activity was decreased to 3%. Genetic analysis revealed a homozygous missense mutation c.5227G>A (p.Gly1715Ser) in exon 16 of F5 gene. Bioinformatics and structural analysis demonstrated this mutation was deleterious and could affect the integrity of local intermolecular structures. The missense mutation (Gly1715Ser) was responsible for the decrease of factor V clotting activity and factor V antigen in this family, and caused type I hereditary factor V deficiency. Correspondence to Mingshan Wang, Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Shangcai Village, Ouhai District, Wenzhou 325000, China. Tel: +86 57788069594; fax: +86 57788069596; e-mail: wywms@126.com Received 30 July, 2019 Revised 21 October, 2019 Accepted 5 November, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Clinico-hematological and thromboelastographic profiles in glanzmann's thrombasthenia Glanzmann's thrombasthenia is a rare inherited bleeding disorder characterized by the quantitative or qualitative defect of glycoprotein IIb/IIIa receptor on platelets which leads to ineffective aggregation. Light transmittance aggregometry is considered as the gold standard for diagnosis of Glanzmann's thrombasthenia. Thromboelastography (TEG) is a global hemostatic assay which measures clot formation, clot strengthening and fibrinolysis. This study evaluates the clinical, laboratory and TEG profiles in patients with Glanzmann's thrombasthenia. Bleeding score by (International Society on Thrombosis and Haemostasis) ISTH-bleeding assessment tool (bleeding score), laboratory tests to diagnose Glanzmann's thrombasthenia, and TEG parameters were correlated in 11 Glanzmann's thrombasthenia patients. Seventeen participants with normal bleeding score were included as controls. Bleeding score was increased in all patients. The highest bleeding score was in an adult female (26), whereas the lowest score (4) was in two children of less than 1 year. Majority of TEG parameters (except R-time) showed a statistically significant difference between Glanzmann's thrombasthenia patients and controls (K-time: P < 0.001, angle: P < 0.001, maximum amplitude: P < 0.001). The average time required to record the maximum amplitude was 23 min. Maximum amplitude was markedly reduced in all Glanzmann's thrombasthenia patients with an average of 20.9 mm (reference range 44–68 mm) having 100% sensitivity. The thromboelastographic profile of Glanzmann's thrombasthenia showed a consistently reduced maximum amplitude. Hence reduced maximum amplitude with a normal platelet count, significant bleeding score and prolonged bleeding time could potentially be used as a preliminary algorithm for the diagnosis of Glanzmann's thrombasthenia. Correspondence to Dr Annamma Kurien, Professor and Head, Department of Pathology, Melaka Manipal Medical College, Manipal Academy of Higher Education, Manipal 576104, Karnataka, India Tel: +91 8202933015/+91 9845821201; e-mail: annamma.kurien@manipal.edu Received 15 August, 2019 Revised 18 October, 2019 Accepted 5 November, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

Purification and characterization of a thrombin-like enzyme isolated from Vipera lebetina venom: its interaction with platelet receptor Snake venoms contain various molecules that can be used as tools in the diagnosis and in the treatment of hemostatic disorders. This study reports the isolation and functional characterization of a new thrombin-like enzyme and its role in the modulation of platelet aggregation and coagulation. The molecule was purified by gel filtration, anion exchange chromatography and reverse-phase-HPLC on C8 column; its molecular weight was determined. Natural and synthetic substrates were used to evaluate its enzymatic activities. The fibrinogenolytic activity was tested electrophoretically and by reverse-phase-HPLC on C18 column. Otherwise, the effect on blood coagulation and deficient plasma factors were also evaluated. The mechanism by which a thrombin-like enzyme VLCV (thrombin-like enzyme)-induced platelet aggregation was explored in presence of ticlopidin, clopidogrel and aspirin. VLCV (45 kDa) isolated from Vipera lebetina as a thrombin-like enzyme seems to be able to modulate platelet function. This enzyme showed an amidolytic activity by hydrolyzing the chromogenic-specific substrate of thrombin and the α-chain of fibrinogen. It is also able to clot human plasma and the deficient human plasma in factor X, suggesting that it is involved in the intrinsic and common pathways. The aggregating effect of VLCV is more sensitive to ticlopidine than to the clopidogrel suggesting the involvement of ADP/P2Y12/PI3K pathway. VLCV seems to be able to promote human platelet aggregation suggesting an interaction between P2Y12 and PAR1. Due to its ability to replace the missing factor X and its proaggregating activity, VLCV could be used as molecular tool to better understand the hemostasis mechanism. Correspondence to Fatima Laraba-Djebari, USTHB, Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, BP32 El-Alia, Bab Ezzouar, Algiers, Algeria. Tel: +21321336076; fax: +21321336077; email address: flaraba@hotmail.com/flaraba@usthb.dz Received 15 February, 2019 Revised 5 September, 2019 Accepted 5 September, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

The utility of thromboelastography and tranexamic acid in plasminogen activator inhibitor deficiency during pregnancy: a rare case report Complete plasminogen activator inhibitor-1 (PAI-1) deficiency is a very rare genetic disorder that is associated with an increased risk of bleeding diathesis. Patients with PAI-1 deficiency are also known to be at increased risk for massive postpartum hemorrhage. We discuss one such rare case of PAI-1 deficiency in a young pregnant patient at 22 weeks of gestation with history of prolonged bleeding. Tranexamic acid was administered for menorrhagia and resumed later for labor and continued into the postpartum period since antifibrinolytics have been the mainstay in the management of PAI-1 deficiency. The patient delivered a healthy infant at 39 weeks. As PAI-1 deficiency causes increased fibrinolysis, the patient's coagulation panel was monitored by performing serial thromboelastograms to monitor for any increase in fibrinolysis. We believe that thromboelastograms might be a useful tool in the monitoring and management of fibrinolytic conditions such as PAI-1 deficiency. Correspondence to Nitya Prabhakaran, MD, Resident, Post Graduate Year 4, Pathology and Anatomic Sciences, Department of Pathology and Anatomical Sciences, University of Missouri School of Medicine, M263 Medical School Building, One Hospital Drive, Columbia, MO 65212, USA Tel: +1 573 882 1201; e-mail: prabhakarann@health.missouri.edu Received 29 May, 2019 Revised 21 October, 2019 Accepted 25 October, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.

von Willebrand factor alloantibodies in type 3 von Willebrand disease The development of neutralizing antibodies is a rare complication of von Willebrand disease treatment. In major surgical procedures for severe forms of the disease, the recognition of ineffective therapy and alternative treatment protocols are lifesaving. We report the case of a 6-year-old girl with type 3 von Willebrand disease in whom inhibitors were sought due to ineffective haemostasis together with lower than expected von Willebrand factor (VWF) recoveries after a surgical procedure. Replacement therapy first with recombinant factor VIIa and then with high doses of recombinant factor VIII in continuous infusion successfully stopped the bleeding. A high level of anti-VWF antibodies was determined by the immunological method. A frameshift mutation associated with premature termination codon (c.2435delC, p.Pro812ArgfsTer31) was determined in our patient. Although the reports on association of this mutation with inhibitor risk are inconsistent, it represents an evidence-based diagnostic and management practice in recognition of high-risk VWF genotype. Correspondence to Barbara Faganel Kotnik, MD, PhD, Department of Haematology and Oncology, University Medical Centre Ljubljana, University Children's Hospital, Bohoričeva 20, 1000 Ljubljana, Slovenia. Tel: +386 1 522 9215; fax: +386 1 522 4038; e-mail: barbara.faganel@kclj.si Received 16 May, 2019 Revised 22 August, 2019 Accepted 26 September, 2019 Copyright © 2019 YEAR Wolters Kluwer Health, Inc. All rights reserved.