Factor XIII levels, clot strength, and impact of fibrinogen concentrate in infants undergoing cardiopulmonary bypass: a mechanistic sub-study of the FIBCON trial.

BACKGROUND: Acquired factor XIII (FXIII) deficiency after major surgery can increase postoperative bleeding. We evaluated FXIII contribution to clot strength and the effect of fibrinogen concentrate administration on FXIII activity in infants undergoing cardiac surgery using cardiopulmonary bypass. METHODS: We conducted a prospectively planned, mechanistic sub-study, nested within the Fibrinogen Concentrate Supplementation in the Management of Bleeding During Paediatric Cardiopulmonary Bypass: A Phase 1B/2A, Open-Label Dose Escalation Study (FIBCON) trial, which investigated fibrinogen concentrate supplementation during cardiopulmonary bypass (ISRCTN: 50553029) in 111 infants (median age 6.4 months). The relationships between platelet number, fibrinogen concentration, and FXIII activity with rotational thromboelastometry clot strength (EXTEM-MCF) in blood taken immediately before cardiopulmonary bypass and after separation from bypass were estimated using multivariable linear regression. Changes in coagulation variables over time were quantified using a generalised linear model comparing three groups: fibrinogen concentrate-supplemented infants, placebo, and a third cohort with lower bleeding risk. RESULTS: Overall, 48% of the variability (multivariable R2) in EXTEM-MCF clot strength was explained by three factors: the largest contribution was from FXIII activity (partial R2=0.21), followed by platelet number (partial R2=0.14), and fibrinogen concentration (partial R2=0.095). During cardiopulmonary bypass, mean platelet count fell by a similar amount in the three groups (-36% to -41%; interaction P=0.98). Conversely, fibrinogen concentration increased in all three groups: 132% in the fibrinogen concentrate-supplemented group, 26% in the placebo group, and 51% in the low-risk group. A similar increase was observed for FXIII activity (61%, 23%, and 25%, respectively; interaction P<0.0001). CONCLUSIONS: FXIII contribution to clot strength is considerable in infants undergoing cardiac surgery. Fibrinogen concentrate supplementation also increased FXIII activity, and hence clot strength. CLINICAL TRIAL REGISTRATION: ISRCTN: 50553029.

Inherited duplications of PPP2R3B predispose to nevi and melanoma via a C21orf91-driven proliferative phenotype.

PURPOSE: Much of the heredity of melanoma remains unexplained. We sought predisposing germline copy-number variants using a rare disease approach. METHODS: Whole-genome copy-number findings in patients with melanoma predisposition syndrome congenital melanocytic nevus were extrapolated to a sporadic melanoma cohort. Functional effects of duplications in PPP2R3B were investigated using immunohistochemistry, transcriptomics, and stable inducible cellular models, themselves characterized using RNAseq, quantitative real-time polymerase chain reaction (qRT-PCR), reverse phase protein arrays, immunoblotting, RNA interference, immunocytochemistry, proliferation, and migration assays. RESULTS: We identify here a previously unreported genetic susceptibility to melanoma and melanocytic nevi, familial duplications of gene PPP2R3B. This encodes PR70, a regulatory unit of critical phosphatase PP2A. Duplications increase expression of PR70 in human nevus, and increased expression in melanoma tissue correlates with survival via a nonimmunological mechanism. PPP2R3B overexpression induces pigment cell switching toward proliferation and away from migration. Importantly, this is independent of the known microphthalmia-associated transcription factor (MITF)-controlled switch, instead driven by C21orf91. Finally, C21orf91 is demonstrated to be downstream of MITF as well as PR70. CONCLUSION: This work confirms the power of a rare disease approach, identifying a previously unreported copy-number change predisposing to melanocytic neoplasia, and discovers C21orf91 as a potentially targetable hub in the control of phenotype switching.

Degradation of the Endothelial Glycocalyx Contributes to Metabolic Acidosis in Children Following Cardiopulmonary Bypass Surgery.

OBJECTIVES: Cardiopulmonary bypass surgery is complicated by metabolic acidosis, microvascular dysfunction, and capillary leak. The glycocalyx-a layer of proteins and sugars lining the vascular endothelium-is degraded during cardiopulmonary bypass. We aimed to describe the kinetics of glycocalyx degradation during and following cardiopulmonary bypass. We hypothesized that cleavage of negatively charged fragments of the glycocalyx would directly induce metabolic acidosis through changes in the strong ion gap (defined using Stewart's physicochemical approach to acid-base chemistry). We also investigated whether glycocalyx degradation was associated with failure of endothelial function and cardiovascular dysfunction. DESIGN: Single-center prospective cohort study. SETTING: Twenty-two bed surgical/medical PICU. PATIENTS: Twenty-seven term infants and children requiring cardiopulmonary bypass surgery for the correction/palliation of congenital heart disease. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We recruited 27 patients, 5 days to 57 months old. We prospectively sampled plasma prior to, during, and following cardiopulmonary bypass at predefined time points. We measured plasma concentrations of interleukin-6 (inflammatory marker), heparan sulfate (negatively charged glycocalyx glycosaminoglycan), and syndecan-1 (neutrally charged glycocalyx protein). We defined the following outcome measures: metabolic acidosis (strong ion gap), renal dysfunction (fold change in creatinine), capillary leak (fluid bolus volume), cardiovascular dysfunction (Vasoactive Inotropic Score), and length of ventilation. In linear regression models, maximum measured heparan sulfate concentration (negatively charged) was associated with metabolic acidosis (p = 0.016), renal dysfunction (p = 0.009), and length of ventilation (p = 0.047). In contrast, maximum measured syndecan-1 concentration (neutrally charged) was not associated with these clinical endpoints (p > 0.30 for all). CONCLUSIONS: Our data show that metabolic acidosis (increased strong ion gap) is associated with plasma concentration of heparan sulfate, a negatively charged glycosaminoglycan cleaved from the endothelial glycocalyx during cardiopulmonary bypass. In addition, cleavage of heparan sulfate was associated with renal dysfunction, capillary leak, and global markers of cardiovascular dysfunction. These data highlight the importance of designing translational therapies to protect the glycocalyx in cardiopulmonary bypass.

A prospective observational study of acute traumatic coagulopathy in traumatic bleeding from the battlefield.

BACKGROUND: Acute trauma coagulopathy (ATC) after military trauma has not been comprehensively studied. ATC is defined as a prolonged prothrombin time ratio (PTr) or reduced clot amplitude (A5) in viscoelastic testing. Compared to civilian trauma, military trauma has more injuries from explosions and gunshot wounds (GSWs), potentially leading to a different pathophysiology for traumatic coagulopathy. This study aimed to characterize military ATC on admission to a military hospital in Afghanistan and to explore any differences due to the mechanism of injury. METHODS: Severely injured military casualties were enrolled in the study. Blood samples were taken on admission and after routine testing, waste plasma was prepared, frozen, and transported to the United Kingdom for in-depth hemostatic analysis. RESULTS: Seventy-seven percent of casualties had ATC defined by a PTr greater than 1.2 and 19% when defined by rotational thromboelastometry (ROTEM) A5 less than 36 mm. Coagulation factor depletion correlated with degree of shock, particularly factor V (p < 0.01), factor X (p < 0.01), and fibrinogen levels (p < 0.01). Thrombin generation was well preserved. Fibrinolytic biomarkers were raised correlating with the degree of shock (p < 0.01), and 8% of casualties had hyperfibrinolysis on ROTEM analysis. Plasmin-antiplasmin complexes (p < 0.01) and d-dimer levels (p = 0.01) were higher and clot firmness lower (p = 0.02) in those injured by explosion compared to GSW's. CONCLUSIONS: ATC was present and correlated with shock, similar to civilian trauma. Thrombin generation remained adequate. Fibrinogen and factor V levels were disproportionately low but still sufficient to allow clot formation. Fibrinolysis is a key feature, probably due to a tissue plasminogen activator surge at the time of injury. Blast injuries are associated with a greater activation of fibrinolysis than GSWs.

A comparison of haemostatic biomarkers during low-risk patients undergoing cardiopulmonary bypass using either conventional centrifugal cell salvage or the HemoSep device.

BACKGROUND: Cardiac surgery using cardiopulmonary bypass (CPB) is associated with a coagulopathy due to haemodilution, thrombocytopenia and platelet dysfunction and the activation of coagulation and fibrinolysis, despite the use of large doses of unfractionated heparin. Conventional red cell salvage may exacerbate post-operative bleeding as plasma containing haemostatic factors is discarded. We hypothesized that a novel cell salvage device (HemoSep) may attenuate haemostatic changes associated with red cell salvage. We studied haemostatic markers following autologous transfusion from conventional cell salvage or the HemoSep device. METHODS: This randomised, controlled trial compared haemostatic markers in patients undergoing coronary artery bypass grafting or aortic valve replacement who received autologous blood returned from cell salvage (control) or HemoSep (study). Blood samples were taken pre-operatively, end of CPB, post-transfusion of salvaged blood and 3 hours post-operatively and analysed for full blood count (FBC), prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, D-dimer and endogenous thrombin potential (ETP). RESULTS: Fifty-four patients were recruited (n=28 control, n=26 study). Processed blood volume for transfusion was significantly (p<0.001) higher in the HemoSep group. In the HemoSep group, the PT was shorter (18.7±0.3 vs 19.9±0.3 sec; p<0.05) post-operatively and the aPTT was longer (48.6±3.8 vs 37.3±1.0 sec; p<0.01) following autologous transfusion. In the control group, D-dimer and ETP levels were higher (1903±424 vs.1088±151; p<0.05 and 739±46 vs. 394±60; p<0.001, respectively) following autologous transfusion. CONCLUSIONS: Although centrifuged cell salvage is known to adequately haemoconcentrate and remove unwanted substrates and bacteriological contamination, the process can exacerbate coagulopathy. The HemoSep device demonstrated some increase in haemostatic markers when used in low-risk cardiac surgery patients.

The DiPEP (Diagnosis of PE in Pregnancy) biomarker study: An observational cohort study augmented with additional cases to determine the diagnostic utility of biomarkers for suspected venous thromboembolism during pregnancy and puerperium.

This study aimed to estimate the diagnostic utility of biomarkers for suspected venous thromboembolism (VTE) in pregnancy and the puerperium. Research nurses/midwives collected blood samples from 310 pregnant/postpartum women with suspected pulmonary emboli (PE) and 18 with diagnosed deep vein thrombosis (DVT). VTE was diagnosed using imaging, treatment and adverse outcome data. Primary analysis was limited to women with conclusive imaging (36 with VTE, 247 without). The area under the curve (AUC) for each biomarker was: activated partial thromboplastin time 0·669 (95% confidence interval 0·570-0·768), B-type natriuretic peptide 0·549 (0·453-0·645), C-reactive protein 0·542 (0·445-0·639), Clauss fibrinogen 0·589 (0·476-0·701), D-Dimer (by enzyme-linked immunosorbent assay) 0·668 (0·561-0·776), near-patient D-Dimer 0·651 (0·545-0·758), mid-regional pro-atrial natriuretic peptide 0·524 (0·418-0·630), prothrombin fragment 1 + 2 0·562 (0·462-0·661), plasmin-antiplasmin complexes 0·639 (0·536-0·742), prothombin time 0·613 (0·508-0·718), thrombin generation lag time 0·702 (0·598-0·806), thrombin generation endogenous potential 0·559 (0·437-0·681), thrombin generation peak 0·596 (0·478-0·715), thrombin generation time to peak 0·655 (0·541-0·769), soluble tissue factor 0·531 (0·424-0·638) and serum troponin 0·597 (0·499-0·695). No diagnostically useful threshold for diagnosing or ruling out VTE was identified. In pregnancy and the puerperium, conventional and candidate biomarkers have no utility either for their negative or positive predictive value in the diagnosis of VTE.

The effect of hydroxychloroquine on haemostasis, complement, inflammation and angiogenesis in patients with antiphospholipid antibodies.

Objectives: HCQ has been described as having a beneficial effect in patients with APS but its mechanism of action is unclear. We hypothesized that HCQ may have effects on subnormal angiogenesis, inflammation and haemostatic biomarkers seen in APS. The aim of our study was to assess laboratory markers [annexin A5 (AnxA5) anticoagulant activity, tissue factor (TF) levels, thromboelastography (TEG), CRP, Bb, C3a and VEGF] in HCQ-naïve patients with aPL at baseline and after commencing HCQ. Methods: Twenty-two patients with aPL [20 female, 2 male, median age 55 (range 18-70) years] had blood taken pre- and 3 months after starting HCQ 200 mg daily. Results: Soluble TF levels were significantly reduced comparing baseline and 3 months after HCQ commencement [401.8 (152.8) vs 300.9 (108) pg/ml (P = 0.010)]. No significant changes were found in the following [reported as pre- and post-HCQ commencement, mean (s.d.)]: AnxA5 anticoagulant ratio [187.1 (29.5) vs 193 (31) (P = 0.157)], anti-domain1 ß2 glycoprotein1 IgG activity [1.8 (2) vs 1.2 (1.4) µg/ml (P = 0.105)], complement C3a-des-Arg [147.8 (84.5) vs 154.4 (88.1) ng/ml (P = 0.905)], complement Bb [1.3 (0.7) vs 1.1 (0.7) µg/ml (P = 0.422)], VEGF [68.8 (40) vs 59.4 (19.6) pg/ml (P = 0.454)] and CRP [7 (3.5) vs 7 (3.9) µg/ml (P = 0.917)]. TEG results including TEG reaction time, achievement of clot firmness, TEG maximum amplitude and TEG percentage lysis 30 and 60 min after maximum amplitude showed no significant difference. Conclusion: HCQ significantly reduced soluble TF levels in patients with aPL. No significant change was observed in AnxA5 activity, anti-domain 1 IgG activity, TEG, CRP, complement Bb and C3a-des-Arg, and VEGF. Further studies of a larger patient cohort are needed.

Experimentally-induced anti-myeloperoxidase vasculitis does not require properdin, MASP-2 or bone marrow-derived C5.

Anti-neutrophil cytoplasmic antibody vasculitis is a systemic autoimmune disease with glomerulonephritis and pulmonary haemorrhage as major clinical manifestations. The name reflects the presence of autoantibodies to myeloperoxidase and proteinase-3, which bind to both neutrophils and monocytes. Evidence of the pathogenicity of these autoantibodies is provided by the observation that injection of anti-myeloperoxidase antibodies into mice causes a pauci-immune focal segmental necrotizing glomerulonephritis which is histologically similar to the changes seen on renal biopsy in patients. Previous studies in this model have implicated the alternative pathway of complement activation and the anaphylatoxin C5a. Despite this progress, the factors that initiate complement activation have not been defined. In addition, the relative importance of bone marrow-derived and circulating C5 is not known. This is of interest given the recently identified roles for complement within leukocytes. We induced anti-myeloperoxidase vasculitis in mice and confirmed a role for complement activation by demonstrating protection in C3-deficient mice. We showed that neither MASP-2- nor properdin-deficient mice were protected, suggesting that alternative pathway activation does not require properdin or the lectin pathway. We induced disease in bone marrow chimaeric mice and found that circulating and not bone marrow-derived C5 was required for disease. We have therefore excluded properdin and the lectin pathway as initiators of complement activation and this means that future work should be directed at other potential factors within diseased tissue. In addition, in view of our finding that circulating and not bone marrow-derived C5 mediates disease, therapies that decrease hepatic C5 secretion may be considered as an alternative to those that target C5 and C5a. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Quantification of forces required for stabbing with screwdrivers and other blunter instruments.

In the UK, stabbing is the most common cause of homicide. The weapons used include knives, swords, screwdrivers and glass shards. Quantifying the exact force used in a stabbing incident is considered to be a difficult area due to the large number of variables present, such as sharpness of weapon, angle of attack and relative movements of the people involved. Having quantifiable data would allow a forensic pathologist to make a more informed decision when it comes to answering the commonly posed question in court "what was the degree of force involved in the stabbing incident?" The answer to this question is considered significant in determining an alleged assailant's intent to cause harm. This paper presents results of the first detailed study relating geometry of screwdrivers to the forces required for penetration. Additionally, a range of other blunt weapons such as pens and chisels have also been studied. A silicone rubber-foam analogue has been used as the main skin simulant owing to it having similar mechanical properties to that of human skin and giving highly repeatable results. Different screwdrivers of varying shape and size have been tested (i.e. slotted, Phillips, posidriv and Torx), along with other implements including chisels and pens. The weapon geometry was characterised and related to the peak force required for penetration. Our results show that there is a direct correlation between the cross-sectional area of a screwdriver head and the amount of force required for penetration. Screwdrivers with larger cross-sectional areas require a significantly greater force to penetrate (forces in the region of 100-120 N) but "sharper" slotted screwdrivers penetrate with much lower forces (~30 N). The forces required for penetrating the rubber-foam analogue with screwdrivers are higher than for "sharp" knives, but in some cases similar to the forces required for stabbing with "blunt" knives. For the other weapons such as chisels and biros, the force required for penetration was again high and there was found to be a good relationship between area of the implement making contact and penetration force.

Evaluation of novel coagulation and platelet function assays in patients with chronic kidney disease.

BACKGROUND: Hemostasis evaluation in chronic kidney disease (CKD) is critical for optimal management of thrombotic and bleeding events. Standard coagulation screens are inadequate for predicting coagulopathy in CKD. OBJECTIVE: To evaluate hemostasis parameters in patients with different stages of CKD using novel coagulation assays. PATIENTS/METHODS: Cross-sectional study of 30 healthy controls (HC) and 120 CKD patients (10 Stage 2, 20 Stage 3, 20 Stage 4, 20 Stage 5 not requiring renal replacement therapy, 20 transplant, 10 newly started on hemodialysis [HD], 20 established on HD). Standard laboratory tests were performed in addition to rotational thromboelastometry (ROTEM), multiple electrode aggregometry (MEA), thrombin generation assays, D-dimer, and markers of thrombogenesis (thrombin-antithrombin [TAT]), fibrinolysis, and endothelial activation (intercellular adhesion molecule-1 [ICAM-1]). RESULTS: D-dimer, TAT, and ICAM-1 concentrations were significantly higher in patients with CKD than HC (P < .01). ROTEM maximum clot firmness was significantly higher in patients than in HC (P < .01). In CKD Stage 5 patients (pre-HD and started HD) adenosine diphosphate and thrombin receptor activating peptide MEA tests were significantly lower than HC indicating platelet aggregation defect (P < .05). Multivariate analysis confirmed the direct effect of estimated glomerular filtration rate (eGFR) in the variance of ROTEM and MEA tests. Endogenous thrombin potential and peak thrombin were not statistically different between groups, but Stage 5 CKD patients had prolonged lag time (7.91 vs. 6.33, P < .001) and time to thrombin peak (10.8 vs. 9.5, P < .05) compared to HC. CONCLUSIONS: Patients with CKD exhibit features of concomitant hypercoagulability measured by ROTEM and platelet dysfunction measured with MEA. eGFR was an independent determinant of platelet dysfunction and hypercoagulability.

Modelling acute antibody-mediated rejection of human kidney transplants using ex-vivo warm machine perfusion.

BACKGROUND: Transplant rejection is a major cause of graft loss and morbidity. Currently, no human models of antibody-mediated rejection (AMR) exist, limiting mechanistic investigation and organ-specific targeted therapy. Here, using 12 human kidneys and ex-vivo normothermic machine perfusion, we demonstrate phenotypes of AMR after addition of antibodies against either human HLA class I or blood group antigens (A, B), thus modelling clinical AMR that can follow HLA incompatible (HLAi) or blood group incompatible (ABOi) transplantation. METHODS: Discarded human kidneys with wide ranging demographics and cold ischaemia times (11-54 h) were perfused with red blood cells and fresh frozen plasma (FFP) as a source of complement/coagulation factors. For the HLAi model, 600 µg of W6/32 anti-class 1 HLA antibody was added to the circuit (time '0'). For the ABOi model, high titre FFP of the relevant blood group antibody was added. Renal blood flow index (RBFi, mL/min/100 g), C3 desArg, prothrombin fragments 1 + 2 and histology were determined. Our endpoints included haemodynamic changes, thrombosis, and biopsy proven complement deposition. FINDINGS: Compared to control kidneys perfused without anti-donor antibodies, both models demonstrated haemodynamic collapse after antibody perfusion with only the HLAi model showing glomerular C4d deposition. INTERPRETATION: We show that a clinically relevant human kidney model of AMR is feasible, and anticipate that these models, with refinements, could provide a basis to test different strategies to prevent AMR. FUNDING: The Rosetrees and Stonygate Trust, The Royal College of Surgeons of England Fellowship Grant, NIHR Biomedical Research Centre/KCL Early Career Grant, Kidney Research U.K.

Coagulation derangement and risk factors for valve thrombosis following transcatheter aortic valve implantation.

AIMS: Durability of transcatheter aortic valve implantation (TAVI) is key to its expansion. We sought to identify incidence of valve thrombosis and predictors of valve thrombosis in our single centre with associated coagulation testing pre-TAVI and post-TAVI. METHODS AND RESULTS: This single-centre observational study comprised patients undergoing transfemoral TAVI discussed in the Heart Team meeting . Patients were followed up with echocardiography at 120 days to identify incidence of elevated transvalvular gradient and multivariable analysis was performed to identify factors associated with an increased odds of developing valve thrombosis. In addition, 11 patients underwent baseline, day 1 and day 120 post-TAVI coagulation testing. Between August 2017 and August 2019, 437 consecutive patients underwent transfemoral TAVI. Of these patients, 207/437 (47.4%) had 3-month follow-up echo data available and were analysed. Of these patients, 26/207 (12.6%) had elevated transvalvular gradients. These patients tended to be younger (80±14 vs 83±6 years; p=0.047) with a lower ejection fraction (49±13 vs 54%±11%; p=0.021), with a greater proportion of the population experiencing atrial fibrillation (14/21, 54% vs 68/181, 38%; p=0.067). Following multivariable analysis, there remained a trend towards higher eccentricity index associated with elevated gradients. Baseline (pre-TAVI) elevation of thrombin antithrombin levels (56±63; reference range 1.0-4.1 ng/L) and PF 1+2 (791±632; reference range 69-229 ng/mL) normalised at 120 days post-TAVI CONCLUSION: This study demonstrated that in the cohort of patients undergoing transfemoral TAVI in our centre: younger age, poor ejection fraction, atrial fibrillation and increased baseline eccentricity of the aortic valve annulus were present to a greater extent in patients exhibiting elevated transvalvular gradients at 3-month follow-up. Further work is required to delineate the extent of coagulation derangement and confirm predictors of thrombosis.

The relationship among thromboelastography, hemostatic variables, and bleeding after cardiopulmonary bypass surgery in children.

BACKGROUND: Mediastinal bleeding is common after pediatric cardiopulmonary bypass (CPB) surgery. Thromboelastography (TEG) may predict bleeding and provide insight into likely mechanisms. We aimed to (a) compare perioperative temporal profiles of TEG and laboratory hemostatic variables between patients with significant hemorrhage (BLEED) and those without (CONTROL), (b) investigate the relationship between TEG variables and routine hemostatic variables, and (c) develop a model for prediction of bleeding. METHODS: TEG and laboratory hemostatic variables were measured prospectively at 8 predefined times for 50 children weighing <20 kg undergoing CPB. RESULTS: Patients who bled demonstrated different TEG profiles than those who did not. This was most apparent after protamine administration and was partly attributable to inadequate heparin reversal, but was also associated with a significantly lower nadir in mean (sd) fibrinogen for the BLEED group compared with CONTROL group: 0.44 (0.18) and 0.71 (0.40) g/L, respectively (P = 0.01). Significant nonlinear relationships were found between the majority of TEG and laboratory hemostatic variables. The strongest relationship was between the maximal amplitude and the platelet-fibrinogen product (logarithmic r(2) = 0.71). Clot strength decreased rapidly when (a) fibrinogen concentration was <1 g/L, (b) platelets were <120 x 10(9)/L, and (c) platelet-fibrinogen product was <100. A 2-variable model including the activated partial thromboplastin time at induction of anesthesia and TEG mean amplitude postprotamine discriminated well for subsequent bleeding (C statistic 0.859). CONCLUSIONS: Hypofibrinogenemia and inadequate heparin reversal are 2 important factors contributing to clot strength and perioperative hemorrhage after pediatric CPB. TEG may be a useful tool for predicting and guiding early treatment of mediastinal bleeding in this group.

Individualized, Intraoperative Dosing of Fibrinogen Concentrate for the Prevention of Bleeding in Neonatal and Infant Cardiac Surgery Using Cardiopulmonary Bypass (FIBCON): A Phase 1b/2a Randomized Controlled Trial.

BACKGROUND: Mediastinal bleeding is common following pediatric cardiopulmonary bypass surgery for congenital heart disease. Fibrinogen concentrate (FC) represents a potential therapy for preventing bleeding. METHODS: We performed a single-center, phase 1b/2a, randomized controlled trial on infants 2.5 to 12 kg undergoing cardiopulmonary bypass surgery, aimed at (1) demonstrating the feasibility of an intraoperative point-of-care test, rotational thromboelastometry, to screen out patients at low risk of postoperative bleeding and then guide individualized FC dosing in high-risk patients and (2) determining the dose, safety, and efficacy of intraoperative FC supplementation. Screening occurred intraoperatively 1-hour before bypass separation using the rotational thromboelastometry variable fibrinogen thromboelastometry maximum clot firmness (FibTEM-MCF; fibrinogen contribution to clot firmness). If FibTEM-MCF ≥7 mm, patients entered the monitoring cohort. If FibTEM-MCF ≤6 mm, patients were randomized to receive FC/placebo (2:1 ratio). Individualized FC dose calculation included weight, bypass circuit volume, hematocrit, and intraoperative measured and desired FibTEM-MCF. The coprimary outcomes, measured 5 minutes post-FC administration were FibTEM-MCF (desired range, 8-13 mm) and fibrinogen levels (desired range, 1.5-2.5 g/L). Secondary outcomes were thrombosis and thrombosis-related major complications and postoperative 24-hour mediastinal blood loss. RESULTS: We enrolled 111 patients (cohort, n=21; FC, n=60; placebo, n=30); mean (SD) age, 6.4 months (5.8); weight, 5.9 kg (2.0). Intraoperative rotational thromboelastometry screening effectively excluded low-risk patients, in that none in the cohort arm (FibTEM-MCF, ≥7 mm) demonstrated clinically significant early postoperative bleeding (>10 mL/kg per 4 hours). Among randomized patients, the median (range) FC administered dose was 114 mg/kg (51-218). Fibrinogen levels increased from a mean (SD) of 0.91 (0.22) to 1.7 g/L (0.41). The postdose fibrinogen range was 1.2 to 3.3 g/L (72% within the desired range). The corresponding FibTEM-MCF values were as follows: pre-dose, 5.3 mm (1.9); post-dose, 13 mm (3.2). Ten patients (8 FC and 2 placebo) exhibited 12 possible thromboses; none were clearly related to FC. There was an overall difference in mean (SD) 24-hour mediastinal drain loss: cohort, 12.6 mL/kg (6.4); FC, 11.6 mL/kg (5.2); placebo, 17.1 mL/kg (14.3; ANOVA P=0.02). CONCLUSIONS: Intraoperative, individualized dosing of FC appears feasible. The need for individualized dosing is supported by the finding that a 4-fold variation in FC dose is required to achieve therapeutic fibrinogen levels. Registration: URL: https://eudract.ema.europa.eu/; Unique identifier: 2013-003532-68. URL: https://www.isrctn.com/; Unique identifier: 50553029.

Haemostatic Changes in Five Patients Infected with Ebola Virus.

Knowledge on haemostatic changes in humans infected with Ebola virus is limited due to safety concerns and access to patient samples. Ethical approval was obtained to collect plasma samples from patients in Sierra Leone infected with Ebola virus over time and samples were analysed for clotting time, fibrinogen, and D-dimer levels. Plasma from healthy volunteers was also collected by two methods to determine effect of centrifugation on test results as blood collected in Sierra Leone was not centrifuged. Collecting plasma without centrifugation only affected D-dimer values. Patients with Ebola virus disease had higher PT and APTT and D-dimer values than healthy humans with plasma collected in the same manner. Fibrinogen levels in patients with Ebola virus disease were normal or lower than values measured in healthy people. Clotting times and D-dimer levels were elevated during infection with Ebola virus but return to normal over time in patients that survived and therefore could be considered prognostic. Informative data can be obtained from plasma collected without centrifugation which could improve patient monitoring in hazardous environments.

Alterations in fibrin formation and fibrinolysis in early onset-preeclampsia: Association with disease severity.

OBJECTIVE: ; Early-onset preeclampsia is a rare pregnancy-specific disorder associated with significantly increased maternal and fetal morbidity and mortality. Whilst it is known that even normotensive pregnancies are associated with changes in clot formation and dissolution, the nature of how these changes differ in those with early onset preeclampsia has not been well established. We sought to evaluate parameters of fibrin formation and fibrinolysis in individuals with early onset preeclampsia in comparison to both pregnant and non-pregnant controls. Furthermore, such parameters were correlated with markers of disease severity in this patient cohort, including the presence of multiorgan involvement, the rate of disease progression and the extent of the anti-angiogenic state in this condition. STUDY DESIGN: ; Patients with early onset preeclampsia (N = 20) and both pregnant (N = 16) and non -pregnant (N = 16) controls were recruited from the cohort at a large urban maternity hospital which saw over 15,000 deliveries during the study period. Platelet poor plasma was prepared from collected whole blood and analysed for parameters of fibrin formation and fibrinolysis (lagtime to and rate of fibrin formation; PAI-1; PAI-2; D-dimer; plasmin-antiplasmin; tPA) in addition to markers of angiogenesis (sFLT-1; Endoglin) using commercially available specific immunoassays. RESULTS: ; The maximum rate of fibrin formation as well as PAI-1, PAI-2 and D-dimer levels were all significantly increased in those with early onset preeclampsia and pregnant controls when compared to non-pregnant controls without significant differences between the 2 former groups. Plasmin-antiplasmin levels were significantly reduced in a similar manner. tPA levels were significantly elevated in EOP compared to both pregnant and non-pregnant controls. EOP was associated with significantly increased anti-angiogenic factors (sFLT-1; Endoglin) when compared to both pregnant and non-pregnant controls. CONCLUSION: ; Markers of fibrin formation and fibrinolysis are significantly alerted in early onset preeclampsia; furthermore, certain markers correlate with disease severity in this patient cohort.

Rotational thromboelastometry alongside conventional coagulation testing in patients with Crimean-Congo haemorrhagic fever: an observational cohort study.

BACKGROUND: Data describing the coagulopathy of Crimean-Congo haemorrhagic fever are scarce. We did rotational thromboelastometry (ROTEM) and conventional coagulation testing in patients with Crimean-Congo haemorrhagic fever to increase our understanding of the coagulopathy of this infectious disease. METHODS: We did a prospective observational cohort study of adults aged 18 years and older and admitted to hospitals with PCR-confirmed Crimean-Congo haemorrhagic fever in Samsun and Tokat, Turkey. Demographic, clinical, and laboratory data were collected and blood samples for ROTEM analysis and coagulation testing were drawn at admission and during hospital admission and convalescence (up to 30 days after onset of illness). For the ROTEM analysis we recorded the following extrinsically activated ROTEM (EXTEM S) variables, with normal ranges indicated: clotting time (38-79 s), clot formation time (34-159 s), amplitude at 10 min after clotting time (43-65 mm), maximum clot firmness (50-72 mm), and maximum lysis (>15% at 1 h). The following fibrin-specific ROTEM (FIBTEM S) variables were also recorded: amplitude at 10 min after clotting time (normal range 7-23 mm) and maximum clot firmness (9-25 mm). Disease severity was assessed by Swanepoel criteria, severity grading score (SGS), and the severity scoring index (SSI), with mild disease defined as meeting no Swanepoel criteria, graded mild by SSI, and graded low risk by SGS. FINDINGS: Between May 27, 2015, and Aug 2, 2015, 65 patients with confirmed Crimean-Congo haemorrhagic fever were recruited and had blood taken at 110 time points. Most were male (40 [62%] of 65) with mild disease (49 [75%] of 65). Haemorrhage occurred in 13 (20%; 95% CI 11·1-31·8) of 65 patients and 23 (35%) of 65 received blood products (15 received fresh frozen plasma and eight received red blood cell concentrates), and 21 patients received platelet transfusions. At admission, the following EXTEM S variables differed significantly between mild cases and moderate to severe cases: median clotting time 56 s (range 42-81; IQR 48-64) versus 69 s (range 48-164; IQR 54-75; p=0·01); mean amplitude at 10 min after clotting time 45·1 mm (SD 7·0) versus 33·9 mm (SD 8·6; p<0·0001); median clot formation time 147 s (range 72-255; IQR 101-171) versus 197 s (range 98-418; IQR 156-296; p=0·006); and maximum clot firmness 54·4 mm (SD 7·2) versus 45·1 mm (SD 12·5; p=0·003). The EXTEM S variables were compared at different time points; maximum clot firmness (p=0·024) and amplitude at 10 min after clotting time (p=0·090) were lowest on days 4-6 of illness. We found no significant differences in FIBTEM variables between mild and moderate to severe cases (median amplitude at 10 min, 13 mm [range 8-20; IQR 11-15] vs 12 mm [range 6-25; IQR 10-15; p=0·68]; and median maximum clot firmness, 15 mm [range 9-60; IQR 13-21] vs 17 mm [range 7-39; IQR 13-23; p=0·21]); and no hyperfibrinolysis (maximum lysis >15%). INTERPRETATION: Coagulopathy of Crimean-Congo haemorrhagic fever is related to defects in clot development and stabilisation that are more marked in severe disease than in mild disease. The combination of normal and slightly deranged coagulation screens and FIBTEM results with the absence of hyperfibrinolysis suggests that the coagulopathy of Crimean-Congo haemorrhagic fever relates to platelet dysfunction. FUNDING: Wellcome Trust, UK Ministry of Defence, and National Institute for Health Research Health Protection Research Unit.

An in vitro study of the effects of t-PA and tranexamic acid on whole blood coagulation and fibrinolysis.

AIMS: Acute traumatic coagulopathy is characterised by fibrinolysis and low fibrinogen. It is unclear how much fibrinogenolysis contributes to reduce fibrinogen levels. The study aim was to: investigate in vitro the effects of tissue-plasminogen activator (t-PA) and tranexamic acid (TXA) on coagulation and fibrinolysis. METHODS: Whole blood was spiked with varying t-PA concentrations. Clauss fibrinogen levels and thrombelastography (TEG, Haemonetics) were performed, including functional fibrinogen level (FLEV). TXA effects were assessed using four TXA concentrations. Recorded parameters from kaolin activated TEG included maximal amplitude (MA), clot strength (G), percentage lysis (LY). Plasmin-antiplasmin complex (PAP), endogenous thrombin potential (ETP), prothrombin fragment 1+2 (PF1+2), factor V and factor VIII levels were all measured. RESULTS: t-PA induced fibrinolysis: it increased PAP and LY, but decreased MA and G. t-PA induced fibrinogenolysis, with a concentration-dependant decrease in fibrinogen from 2.7 (2.6-3.1) to 0.8 (0.8-0.9) g/L with 60 nM t-PA. FLEV and fibrinogen levels were well correlated. High t-PA doses increased PF1+2, decreased ETP of 19% and FVIII of 63% but not FV. TXA had no effect on plasmin generation as evidenced by no change in PAP. It corrected LY, MA and G and partly protected fibrinogen against fibrinogenolysis: 0.03 mg/mL TXA reduced the fibrinogen fall induced by t-PA 20 nM from 43% to 14%. TXA halved the FVIII fall and increased ETP. CONCLUSIONS: t-PA induced plasminogen activation and fibrinogenolysis in a concentration-dependant manner. TXA did not affect plasmin activation but reduced fibrinogenolysis. These results suggest that TXA given early in bleeding patients may prevent fibrinogenolysis.

The utility of thromboelastography and thrombin generation in assessing the prothrombotic state of adults with sickle cell disease.

INTRODUCTION: Previous studies have suggested a chronic hypercoagulable state in SCD, and that thrombosis also plays a role in the pathophysiology of sickle cell vaso-occlusive pain crises (VOC). Studies looking at thrombin generation have produced conflicting results. In this study we aimed to assess and compare whole blood thromboelastography (TEG) and plasma Calibrated Automated Thrombogram (CAT) in SCD versus healthy controls and in four different SCD subgroups. MATERIALS AND METHODS: In this prospective observational study, TEG and 1pM TF activated CAT assays were performed in citrated blood samples from 77 adult (18-66years old) SCD patients (HbSS and HbSB) and 22 healthy (HbAA) ethnically-matched controls. RESULTS AND CONCLUSIONS: SCD was associated with a prothrombotic state in all TEG parameters. CAT results showed that the upslope of the CAT in SCD displayed a hypercoagulable state with shorter time to peak and higher velocity index, but the downslope was also faster leading to an overall lower endogenous thrombin potential (ETP) compared to healthy controls. TEG subgroup analyses showed that during VOC the prothrombotic state is greater compared to patients on disease ameliorating therapy. CAT did not display statistically significant differences between the SCD subgroups. This study shows that the prothrombotic state in SCD is best displayed with TEG, and suggests the hypercoagulable changes of SCD rely at least in part in the cellular components of blood, which can only be detected in whole blood assays.

An observational study of haemostatic changes, leptin and soluble endoglin during pregnancy in women with different BMIs.

Obesity increases the risk of venous thromboembolism (VTE) in pregnancy. The pathogenesis is hypothesized to be because of multiple factors including prothrombotic changes, but there has been minimal haemostatic research looking at the combined state of obesity and pregnancy. We aimed to determine whether variation in BMI in the third trimester of pregnancy was associated with prothrombotic changes. We recruited 110 women into four groups depending on their BMI at first antenatal appointment: normal, overweight, obese and morbidly obese. Women with increased risk of VTE, and/or receiving thromboprophylaxis, and/or more than 35 years and those in labour were excluded. Thromboelastography, platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen, prothrombin fragment 1 + 2, free and total protein S, plasminogen activator inhibitor type 1, tissue plasminogen activator antigen, D-dimers, soluble endoglin and leptin levels were measured. There were no significant differences in haemostatic measures with changing BMI. There was a positive correlation between BMI and both platelet count (correlation coefficient r = 0.214, P = 0.036) and leptin (r = 0.435, P < 0.001), but only leptin had a significant association with BMI once adjusted for age, gestation and parity. Despite recruitment into the morbidly obese group being suboptimal, these findings suggest that in pregnancy, the increased risk of VTE seen in obese mothers is not mediated through increased prothrombotic changes, and thus the increased risk of VTE in obese pregnant women may be because of other mechanisms, for example endothelial dysfunction, inflammation and venous stasis.