Caroline J. Reddel

Lamin A expression levels are unperturbed at the normal and mutant alleles but display partial splice site selection in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder which is characterised by the very early appearance of accelerated ageing in children. It was first described by Hutchinson1 and Gilford.2 Children with HGPS appear healthy at birth, with distinctive clinical features appearing within the first few years of life, including severe growth retardation, atherosclerosis, incomplete sexual maturation, loss of subcutaneous fat, craniofacial abnormalities, alopecia (hair loss), stiffness of joints, and other skeletal abnormalities including coxa valga (giving a “horse-riding” stance), delayed and abnormal dentition, and scleroderma-like areas of skin, with normal mental and emotional development.3 A specific causal mutation for HGPS in the LMNA gene was identified4,5 and down-regulation of isoforms of the protein arising from the LMNA gene were found in HGPS patients.6 The LMNA gene encodes the A-type lamins, lamins A and C, in which several other laminopathies have their causal mutation.7,8 The heterozygous de novo point mutation is located in exon 11 of the LMNA gene, which is transcribed to lamin A while the shorter lamin C is unaffected by the mutation. The GGC>GGT G608G single-base substitution reveals a cryptic splice site and is proposed to result in a truncated transcript encoding a protein that is 50 amino acid shorter. This mutant protein will be referred to as progerin. A less common mutation in the same codon (GGC>AGC), predicted to have similar effects on lamin A splicing, was also identified.4 Western blots reveal progerin and the normal lamin A protein in HGPS patient cells.5 This hypothesised mechanism raises questions about allelic expression and splicing efficiency. De Sandre-Giovannoli et al 4 postulated that only the mutated allele may be expressed in HGPS cells. We now examine lamin A transcript levels in HGPS cells, identify their allelic sources …

Detection of hypofibrinolysis in stable coronary artery disease using the overall haemostatic potential assay

INTRODUCTION Patients with stable coronary artery disease (CAD) are at risk of arterial thrombosis causing myocardial infarction. Detection of global haemostatic markers of hypercoagulability and hypofibrinolysis may be important for risk stratification and individualised treatment. We examined overall haemostatic potential (OHP) and thrombin generation in a group of stable CAD patients. We also sought to investigate associations between fibrinolytic inhibitors and abnormal global fibrinolysis in these patients. MATERIALS AND METHODS Blood samples were collected from 56 patients defined by coronary anatomy as symptomatically stable CAD. Medications were recorded. Samples were analysed using the global coagulation assays OHP and thrombin generation (calibrated automated thrombogram, CAT), platelet aggregometry measured by Multiplate®, and levels of plasminogen activator inhibitor-1 (PAI-1) antigen measured by ELISA. Results were compared with a reference group of healthy controls. RESULTS Stable CAD patients displayed increased fibrin and thrombin generation and impaired fibrinolysis (decreased overall fibrinolytic potential, OFP, and increased clot lysis time) compared with healthy controls. No effect of antiplatelet agents or other medications on these parameters was observed using platelet-poor plasma. After multivariate adjustment, OFP of healthy individuals was significantly associated with fibrinogen, but in CAD patients PAI-1 became an important determinant. CONCLUSIONS Hypercoagulability of plasma is observed in stable CAD, with both increased thrombin generation and reduced fibrinolytic potential making a significant contribution. The OHP assay may provide a simple method of identifying hypercoagulability in individual patients.

Global hypercoagulability in patients with schizophrenia receiving long-term antipsychotic therapy

BACKGROUND Patients with schizophrenia are at increased risk of venous thromboembolism. The mechanisms underlying this association are poorly understood. AIMS We investigated whether there is a global hypercoagulable state in patients with schizophrenia utilising the overall haemostatic potential (OHP) assay which assesses overall coagulation potential (OCP), haemostatic potential (OHP) and fibrinolytic potential (OFP). METHOD Citrated plasma was collected for OHP assays from patients with schizophrenia on long-term antipsychotic treatment and compared with healthy age- and sex-matched controls. Time courses of fibrin formation and degradation were measured by spectrophotometry (absorption of 405nm) after the addition of tissue factor and tissue plasminogen activator to plasma. RESULTS Ninety patients with schizophrenia (antipsychotic treatment-15.9±9.7years) and 30 controls were recruited. Patients with schizophrenia had higher rates of smoking and levels of inflammatory markers (high-sensitivity C-reactive protein and neutrophil-to-lymphocyte ratio) than controls. Whilst D-dimer, fibrinogen and platelet count did not differ between patients with schizophrenia and controls, the OCP (54.0±12.6 vs 45.9±9.1, p=0.002) and OHP (12.6±5.8 vs 7.2±3.7, p<0.001) were higher, and OFP was lower (76.6±9.8% vs 84.9±6.4%, p<0.001) in patients with schizophrenia, implying both a hypercoagulable and hypofibrinolytic state in these patients. Importantly, abnormalities in overall coagulation were independently predicted by levels of plasminogen-activator-inhibitor-1, fibrinogen, platelet count, inflammatory markers and plasma triglycerides, suggesting a multifactorial aetiology. CONCLUSION Patients with schizophrenia have evidence of a global hypercoagulable and hypofibrinolytic state which may contribute to their increased risk of venous thromboembolism.

Persistent global hypercoagulability in long-term survivors of acute pulmonary embolism.

Hypercoagulable and/or hypofibrinolytic states are risk factors for venous thromboembolism (VTE) including acute pulmonary embolism. Current screening for thrombophilia is targeted towards identifying a specific defect and guidelines recommend a population-based rather than individualized strategy for anticoagulation treatment. We investigated whether there is a global hypercoagulable state in long-term survivors of pulmonary embolism no longer receiving therapeutic anticoagulation utilizing the overall haemostatic potential (OHP) assay, which assesses overall coagulation potential (OCP), OHP and overall fibrinolytic potential (OFP). Long-term survivors of acute pulmonary embolism were identified from a local registry and OHP assays were performed and compared with age and sex-matched controls without pulmonary embolism. Time courses of fibrin formation and degradation were measured by spectrophotometry (absorption 405 nm) after addition of tissue factor and tissue plasminogen activator to plasma. OHP assays were performed in 67 long-term survivors of single pulmonary embolism (7.9 ± 1.4 years after pulmonary embolism) and 20 age (61.7 ± 11.2 vs 56.6 ± 6.4 years, P = 0.06) and sex (P = 0.45)-matched controls. Survivors of pulmonary embolism were more hypercoagulable as reflected by significantly higher OCP (56.4 ± 13.0 vs 49.9 ± 6.9, P = 0.03) and had impaired fibrinolysis with higher OHP (12.6 ± 7.0 vs 5.9 ± 2.0, P < 0.001) and lower OFP (78.1 ± 9.4 vs 88.2 ± 2.9, P < 0.001) compared with controls. Importantly, these abnormalities in overall coagulation were independently predicted by levels of fibrinogen, platelet count, shortened activated partial thromboplastin time and inflammatory markers suggesting a multifactorial cause. Long-term survivors of pulmonary embolism demonstrate enhanced global coagulation and reduced fibrinolytic potential. Assessment of global coagulation may provide new insights into the aggregate effects of multiple prothombotic factors and long-term risk of VTE recurrence.

The rebound phenomenon after aspirin cessation: The biochemical evidence

Aspirin is the most commonly used anti-platelet agent in the primary and secondary prevention of cardiovascular events, with approximately 5% of middle-aged adults on long-term therapy [1]. Despite the clinical observations of a clustering of events following cessation of aspirin treatment, there are few prospective studies investigating the potential mechanisms. There are two possible explanations for these events: the first is that the withdrawal of aspirin allows the prothrombotic manifestations of the underlying disease process to re-emerge, with clinical consequences. The second is that aspirinwithdrawal is associatedwith a “rebound” phenomenon that is prothrombotic and/or proinflammatory, and plays a causative role in adverse events. This rebound hypothesis, as a scientific entity, can be defined as an increase in platelet reactivity following aspirin withdrawal, to a level exceeding that at baseline prior to initiation of aspirin therapy [2]. The primary aim of this study was to investigate the effect of aspirin withdrawal on platelet function. Specifically, we assessed whether aspirin cessation resulted in a “rebound” phenomenon of platelet hyperaggregation, or merely a recovery of normal platelet function. We also sought to describe the haemostatic balance after aspirin withdrawal using global haemostatic assays. Eleven healthy volunteers were enrolled in this prospective study of platelet function. Participants were given aspirin for one week (300 mg loading dose on day 1, 150 mg on days 2–7). Blood sampling was performed in all subjects at baseline (before the first dose of aspirin therapy), and at days 7 (the day of the last dose of aspirin therapy), 14 and 21. The institutional Human Research and Ethics Committee approved the study protocol and written informed consent was obtained from all participants. Peripheral venous blood samples were drawn through a short venous catheter inserted into a forearm vein. Platelet-rich plasma (PRP) was prepared by centrifugation of citrated blood and the resulting plasmawas used to dilute the PRP 2:3 (a platelet count of approximately 250 × 10 International Journal of Cardiology 174 (2014) 376–463

Off-Pump Coronary Artery Bypass Surgery Induces Prolonged Alterations to Host Neutrophil Physiology

ABSTRACT Persistent alteration to host polymorphonuclear cell (PMN) physiology has been demonstrated after cardiac surgery performed with cardiopulmonary bypass (CPB). However, to date, PMN physiology and function beyond the first 24 h have not been investigated after cardiac surgery performed without CPB (off-pump coronary artery bypass grafting [OPCAB]). Blood samples of 15 patients were collected preoperatively and on days 1, 3, and 5 after OPCAB. Expression of CD11b, CD18, CBRM1/5, and CD62L were assessed by flow cytometry under resting conditions and after stimulation with formyl methionyl-leucyl-phenylalanine (fMLF), and respiratory burst activity was also measured. Under resting conditions, PMN CD11b, CBRM1/5, and CD62L expressions were minimally altered by surgery. Compared with the response of preoperative PMNs, PMNs assayed on days 3 and 5 after OPCAB demonstrated a significantly blunted increase in the expression of CD11b and CBRM1/5 after fMLF, significantly diminished shedding of CD62L in response to platelet-activating factor and fMLF, and diminished superoxide production after stimulation on day 3. The alteration of PMN function after OPCAB implies that cardiac surgical trauma without CPB directly modulates host PMN physiology.

Increased thrombin generation in a mouse model of cancer cachexia is partially interleukin‐6 dependent

Essentials Cancer cachexia and cancer‐associated thrombosis have not previously been mechanistically linked. We assessed thrombin generation and coagulation parameters in cachectic C26 tumor‐bearing mice. C26 mice are hypercoagulable, partially corrected by blocking tumor derived interleukin‐6. Coagulability and anti‐inflammatory interventions may be clinically important in cancer cachexia.

Procoagulant Effects of Low-Level Platelet Activation and Its Inhibition by Colchicine

Platelets play an important role in diseases such as cardiovascular disease and cancer, especially through their release of extracellular vesicles (EVs) and role in thrombosis. The effects of the anti-inflammatory drug colchicine on platelets are not well understood. We investigated the effect of colchicine on the release of pro-coagulant EVs from platelets under low-level activation. Citrated platelet-rich plasma (PRP) from healthy donors was incubated with 2 mM colchicine or vehicle at 37°C for 30 minutes with gentle rotation. The incubation conditions caused mild platelet activation (expression of CD62P and increased surface lactadherin binding) and release of EVs expressing phosphatidylserine (PS, measured by binding of lactadherin), CD61 and CD62P, both of which were attenuated by colchicine. The incubation conditions shortened the delay to fibrin generation and this correlated with elevated levels of PS+/CD61+ EVs. Removal of EVs from plasma abrogated clot formation in the overall haemostatic potential (OHP) assay. Colchicine decreased levels of both PS+/CD61+ and CD62P+ EVs and abrogated the shortened delay to fibrin generation achieved by platelet activation. Similar results were observed after incubation of PRP with 200 µM vinblastine, suggesting a microtubular effect. An alternative method of platelet activation using platelet agonists 20 µM ADP or 10 µM epinephrine also increased CD62P+ EV levels, and this too was attenuated by prior incubation with colchicine. Our novel findings demonstrate procoagulant effects of low-level platelet activation and EV formation which are inhibited by colchicine.