Grace Gilmore

Background: Thrombotic thrombocytopenia purpura (TTP) is a rare life-threatening disease and timely diagnosis is critical. An ADAMTS13 activity level of < 10 IU/dl is used to differentiate TTP from other thrombotic microangiopathic thrombocytopenia (TMAT) diagnosis. Current test iterations based on FRET, ELISA or chemiluminescence assays are time consuming, have long turnaround times or are not readily available. Aims: To evaluate and compare a rapid flow through screening test (Technoclone, Vienna) against a VWF73 ADAMTS13 activity ELISA (Technoclone, Vienna) and an ADAMTS13 automated chemiluminescence method (Acustar - Werfen). Methods: De- identified suspect and confirmed TTP plasmas (n = 120) from 6 diagnostic laboratories in Australia and New Zealand were collected. Study was approved by MU HREC for assay development and calibration (Permit No. 2013/131). Local and central measurement of ADAMTS13 activity level comparison was made with the flow through test and functional ELISA assay. Additionally, the central study site also included a chemiluminescence method. The semi quantitative screening test assigns ADAMTS13 to one of four level indicator guides: 0, 10, 40 and 80 IU/dl. Results: ADAMTS13 activity levels from local and central study sites were comparable, (r2 = 0.78) as was the correlation with the chemiluminescence and ELISA methods (r2 = 0.86). All but 1 of the rapid flow through results confirmed the quantitative ELISA results of <10 IU/dl, the TTP diagnostic threshold. There were 2 results of 40 IU/dl that were <10 IU/dl by the ELISA assay and no results at 80 IU/dl resulted in activity levels less than <10 IU/dl by the ELISA assay. Conclusion(s): The rapid flow through test provides an initial time critical semi-quantitative ADAMTS13 evaluation. A semi quantitative value of 80 IU/dl does not require an urgent ADAMTS13 assay. The point of care test makes it suitable for all laboratories, especially regional laboratories where quantitative levels are not performed.

Simple Summary A significant number of patients with chronic lymphocytic leukemia (CLL) have an increased risk of bleeding. This risk is further increased when taking ibrutinib, a new effective therapy for CLL. Platelets are the key player in haemostasis and thrombosis. In this study, we first characterized platelet function in untreated stable CLL patients in comparison to age-matched healthy volunteers. Using ex vivo platelets from healthy volunteers, we then investigated a possible mechanism of platelet dysfunction induced by a combination of a CLL-derived factor (adenosine) and ibrutinib. We found that therapeutic concentration of ibrutinib did not affect platelet activation response to collagen. However, the presence of adenosine switched off a central platelet activation pathway leading to increased antiplatelet activity of ibrutinib. Larger studies are needed to draw a correlation between adenosine, platelet function and ibrutinib-associated bleeding in CLL patients. Chronic lymphocytic leukemia patients have an increased bleeding risk with the introduction of Bruton tyrosine kinase (BTK) inhibitors. BTK is a signaling effector downstream of the platelet GPVI receptor. Innate platelet dysfunction in CLL patients and the contribution of the leukemia microenvironment to the anti-platelet effect of BTK inhibitors are still not well defined. Herein, we investigated platelet function in stable, untreated CLL patients in comparison to age-matched healthy subjects as control. Secondly, we proposed a novel mechanism of platelet dysfunction via the adenosinergic pathway during BTK inhibitor therapy. Our data indicate that the nucleotidase that produces adenosine, CD73, was expressed on one-third of B-cells in CLL patients. Inhibition of CD73 improved platelet response to ADP in the blood of CLL patients ex vivo. Using healthy platelets, we show that adenosine 2A (A2A) receptor activation amplifies the anti-platelet effect of ibrutinib (10 nM). Ibrutinib plus an A2A agonist-but not ibrutinib as a single agent-significantly inhibited collagen (10 mu g/mL)-induced platelet aggregation. Mechanistically, A2A activation attenuated collagen-mediated inhibition of p-VASP and synergized with ibrutinib to inhibit the phosphorylation of AKT, ERK and SYK kinases. This manuscript highlights the potential role of adenosine generated by the microenvironment in ibrutinib-associated bleeding in CLL patients.

Background: High estradiol (E2) levels are linked to an increased risk of venous thromboembolism; however, the underlying molecular mechanism(s) remain poorly understood. We previously identified an E2-responsive microRNA (miR), miR-494–3p, that downregulates protein S expression, and posited additional coagulation factors, such as tissue factor, may be regulated in a similar manner via miRs. Objectives: To evaluate the coagulation capacity of cohorts with high physiological E2, and to further characterize novel E2-responsive miR and miR regulation on tissue factor in E2-related hypercoagulability. Methods: Ceveron Alpha thrombin generation assay (TGA) was used to assess plasma coagulation profile of three cohorts. The effect of physiological levels of E2, 10 nM, on miR expression in HuH-7 cells was compared using NanoString nCounter and validated with independent assays. The effect of tissue factor-interacting miR was confirmed by dual-luciferase reporter assays, immunoblotting, flow cytometry, biochemistry assays, and TGA. Results: Plasma samples from pregnant women and women on the contraceptive pill were confirmed to be hypercoagulable (compared with sex-matched controls). At equivalent and high physiological levels of E2, miR-365a-3p displayed concordant E2 downregulation in two independent miR quantification platforms, and tissue factor protein was upregulated by E2 treatment. Direct interaction between miR-365a-3p and F3-3′UTR was confirmed and overexpression of miR-365a-3p led to a decrease of (1) tissue factor mRNA transcripts, (2) protein levels, (3) activity, and (4) tissue factor-initiated thrombin generation. Conclusion: miR-365a-3p is a novel tissue factor regulator. High E2 concentrations induce a hypercoagulable state via a miR network specific for coagulation factors.

Introduction: Haemophilia A(HA) is a complex bleeding disorder resultant from mutations in the Factor VIII protein. Factor VIII is encoded by the F8 gene, and mutations within exon14 contribute to the aetiology of HA in a subset of patients (˜14%), resulting in an incorrectly spliced mRNA transcript and subsequent mutant protein. The Factor VIII B‐domain is mostly encoded by exon14, however, there exists a small degree of functional redundancy in the B‐domain which may be potential targets for nucleic acid‐based therapies. This study aims to target non‐canonical splicing sequences in the F8 exon14 sequences using antisense‐oligonucleotides (AON) incorporating mixer chemistry to induce a truncated but functional Factor VIII transcript.

Materials and Method: Splice inducing sequences can be difficult to predict, we targeted predicted spliceosome elements in the F8 exon14 in HuH7 cells using mixmer chemistries. Following 24 h transfection, total RNA was extracted, converted into cDNA and PCR‐assessed using primers spanning exon13&14 and exon 14&15 boundaries. Positive PCR products were sequenced for identity. Factor VIII protein was analysed by immunoblotting with anti‐factor VIII antibodies.

Results: The entire F8 Exon14 can be deleted by targeting the canonical donor and acceptor splice sites. Human splicing finder 3.1 predicted several sequences for mixmer AON targeting the 3‐prime end of the F8 Exon14 sequence. Different mixmer AON could induce novel F8 pre‐mRNA splicing in HuH7 hepatocarcinoma cells. A wild‐type product (3.5Kb) was present in all treatments, and excision events were observed in mixmer AON‐ #7‐17. Three main F8 products were confirmed by Sanger sequencing. All three contained novel splice inducing sites (U1‐ and U12‐like sequences) and the truncated F8 exon14 transcript is in‐frame with F8‐exon15. All novel transcripts retained the furin‐cleavage site.

Conclusion: Gene skipping of mutated regions of the Factor VIII B‐domain at the pre‐mRNA stage is a potential novel treatment strategy for Haemophilia A. This study successfully excised exon14 of the F8 gene to generate a complete B domain deletion, as well as several partially deleted B‐domain Factor VIII transcripts by inducing spliceosome binding in exonic sequences with mixmer antisense‐oligonucleotides.

Background Investigation of hemostasis is problematic when patients are on anticoagulant therapy. Rivaroxaban especially causes substantial interference, extending many clot-based tests, thereby leading to false positive or negative events. In particular, rivaroxaban affects some assays for activated protein C resistance (APCR). Methods We assessed, in an international setting, cross laboratory (n = 31) testing using four samples to evaluate rivaroxaban induced interference in APCR testing, and whether this interference could be neutralised. The samples comprised: (A) pool of normal plasma (APCR-negative control); (B) this normal pool spiked with rivaroxaban (200 ng/mL) to create rivaroxaban-induced interference (potential ‘false’ positive APCR event sample); (C) the rivaroxaban sample subsequently treated with a commercial direct oral anticoagulant ‘DOAC-neutraliser’ (DOAC Stop), or (D) treated with andexanet alfa (200 μg/mL). Testing was performed blind to sample type. Results The rivaroxaban-spiked sample generated false positive APCR results for some, but unexpectedly not most APCR-tests. The sample treated with DOAC Stop evidenced a correction in the rivaroxaban-affected APCR assays, and did not otherwise adversely affect the rivaroxaban ‘unaffected’ APCR assays. The andexanet alfa-treated sample did not evidence correction of the false positive APCR, and instead unexpectedly exacerbated false positive APCR status with many tests. Conclusions DOAC Stop was able to neutralise any APCR interference induced by rivaroxaban. In contrast, andexanet alfa did not negate such interference, and instead unexpectedly created more false-positive APCR events.

Introduction
Investigation of factors (F) VIII and IX is common, with testing important for diagnosis or exclusion of haemophilia A or B, associated acquired conditions and factor inhibitors. Rivaroxaban, a common direct anti‐Xa agent, causes significant interference in clotting assays, including substantial false reduction of factor levels.

Aim
To assess whether rivaroxaban‐induced interference of FVIII and FIX testing could be neutralized.

Materials and methods
An international, cross‐laboratory exercise for FVIII (n = 84) and FIX (n = 74), using four samples: (A) pool of normal plasma; (B) pool spiked with rivaroxaban (200 ng/mL); (C) rivaroxaban sample subsequently treated with ‘DOAC Stop’ and; (D) rivaroxaban sample treated with andexanet alfa (200 μg/mL). Testing performed blind to sample type.

Results
All laboratories reported normal FIX and 94% reported normal FVIII in the pool sample. Instead, 55% and 95%, respectively, reported abnormal FIX and FVIII levels for the rivaroxaban sample. DOAC Stop treatment evidenced a correction in most laboratories (100% reported normal FIX and 86% normal FVIII). Andexanet alfa provided intermediate results, with many laboratories still reporting abnormal results (59% for FVIII, 18% for FIX). We also identified reagent‐specific issues.

Conclusions
As expected, rivaroxaban caused false low values of FVIII and FIX. This might lead to increased testing to identify the cause of low factor levels and potentially lead to false identification of (mild) haemophilia A or B if unrecognized by clinicians/laboratories. DOAC Stop effectively neutralized the rivaroxaban effect, but andexanet alfa less so, with reagent‐related effects evident, and thus, false low values sometimes persisted.

Introduction Lupus anticoagulant (LA) investigation in patients on anticoagulant therapy is problematic. Rivaroxaban in particular causes significant interference, prolonging both LA screening and confirmation tests, and falsely raising LA screen/confirm ratios, leading to potential false identification of LA. The Russell Viper Venom Time (RVVT) assay, key to the investigation of LA, is especially sensitive to rivaroxaban. Materials and methods We assessed cross laboratory (n = 82) testing of four samples to investigate whether rivaroxaban induced interference in LA testing could be neutralised. Testing was performed blind to sample type. The samples comprised: (A) A pool of normal plasma (LA-negative control); (B) sample A spiked with rivaroxaban (200 ng/mL) to create rivaroxaban-induced interference (LA ‘false’ positive sample); (C) sample B subsequently treated with a commercial ‘DOAC-neutraliser’ (DOAC Stop); (D) sample B treated with andexanet alfa (200 μg/mL). Results As expected, the rivaroxaban-spiked sample (B) caused prolongation of most LA-tests, and also generated a falsely prolonged RVVT screen/confirm ratio (median 1.37, compared to 0.97 for sample A). The sample (C) treated with DOAC Stop evidenced a correction in LA-test clotting times, as well as neutralising the false positive LA (median RVVT screen/confirm ratio of 0.99). Although the andexanet alfa treated sample (D) also yielded a low median RVVT screen/confirm ratio of 0.88, it did not fully correct LA-test clotting times. Consistent with test findings, all laboratories interpreted samples A and C as being LA-negative. For sample B (rivaroxaban), 45.3% identified this as LA positive, and 38.7% identified LA interference. Most (61.3%) also identified sample D as LA negative, with the remainder (38.7%) identifying LA interference. Conclusions DOAC Stop was able to neutralise the false LA activity induced by rivaroxaban, both in terms of clot-times and LA ratios. In contrast, whilst andexanet alfa negated the rivaroxaban-prolonged LA-ratio, it did not fully correct clot-times, leaving some residual LA interference, and requiring additional testing to investigate prolonged clotting times.

Aptamers are short synthetic DNA or RNA oligonucleotides that adopt secondary and tertiary conformations based on Watson–Crick base-pairing interactions and can be used to target a range of different molecules. Two aptamers, HD1 and HD22, that bind to exosites I and II of the human thrombin molecule, respectively, have been extensively studied due to their anticoagulant potentials. However, a fundamental issue preventing the clinical translation of many aptamers is degradation by nucleases and reduced pharmacokinetic properties requiring higher dosing regimens more often. In this study, we have chemically modified the design of previously described thrombin binding aptamers targeting exosites I, HD1, and exosite II, HD22. The individual aptamers were first modified with an inverted deoxythymidine nucleotide, and then constructed bivalent aptamers by connecting the HD1 and HD22 aptamers either through a triethylene glycol (TEG) linkage or four consecutive deoxythymidines together with an inverted deoxythymidine nucleotide at the 3′-end. The anticoagulation potential, the reversal of coagulation with different antidote sequences, and the nuclease stability of the aptamers were then investigated. The results showed that a bivalent aptamer RNV220 containing an inverted deoxythymidine and a TEG linkage chemistry significantly enhanced the anticoagulation properties in blood plasma and nuclease stability compared to the existing aptamer designs. Furthermore, a bivalent antidote sequence RNV220AD efficiently reversed the anticoagulation effect of RNV220 in blood plasma. Based on our results, we believe that RNV220 could be developed as a potential anticoagulant therapeutic molecule.

The small noncoding RNAs, microRNAs (or miRNAs), have been implicated in a myriad of diseases and accumulating evidence indicate their potential high value as diagnostic biomarkers. Although their roles in hemostasis and coagulation pathways are less defined, many studies have demonstrated their participation in regulating key factors of hemostasis. However, the mounting challenges associated with the accurate measurement of circulating miRNAs and the involvement of platelet activation in contributing to the circulating miRNA expression profile introduce further complexity to the study of thrombosis-associated miRNAs. This review outlines the current knowledge of miRNAs that have been postulated to regulate key hemostatic factors, and miRNA diagnostic panels in thrombotic disease, with a focus on experimental fundamentals, such as selecting condition-specific reference controls, considerations that are crucial for accurate evaluation of miRNAs in the context of disease biomarkers.

Functional nucleic acids, such as microRNAs (miRNAs), have been implicated in the pathophysiology of many diseases. The miRNA expression profiles of various cancers including haematological malignancies are well defined, but the role of miRNAs in haemostasis and the regulation of coagulation is poorly understood. We identified that miR-494 is oestrogen responsive and directly targets the anticoagulant protein, Protein S, as a mechanism for acquiring Protein S deficiency under high oestrogenic conditions such as during pregnancy and oral contraceptive use. Furthermore, previous studies have also characterised miR-494 to be involved in many biological processes. This paper reviews the current knowledge in the role of miRNAs in regulating haemostatic proteins and the known biological functions of miR-494, highlighting miR-494 as an emerging therapeutic target, with an overview of the strategy we have employed in identifying functional nucleic acids such as miRNAs that target haemostatic factors and the therapeutic potential of miR-494-directed therapy for the treatment of thrombotic disorders.