Karl Egan

Plasma Thrombin Generation and Sensitivity to Activated Protein C Among Patients With Myeloma and Monoclonal Gammopathy of Undetermined Significance

The etiology of the prothrombotic state in myeloma has yet to be definitively characterized. Similarly, while recent evidence suggests that patients with monoclonal gammopathy of undetermined significance (MGUS) may also be at increased risk of thrombosis, the magnitude and the etiology of this risk have also yet to be defined. The present study aims to characterize patterns of plasma thrombin generation and sensitivity to the anticoagulant activity of activated protein C (APC) at the time of initial diagnosis of myeloma and in response to therapy in comparison to that observed among patients with MGUS and matched, healthy volunteers. Patients presenting with newly diagnosed/newly relapsed myeloma (n = 8), MGUS (n = 8), and matched healthy volunteers (n = 8) were recruited. Plasma thrombin generation was determined by calibrated automated thrombography. Peak thrombin generation was significantly higher in patients with myeloma (383.4 ± 33.4 nmol/L) and MGUS (353.4 ± 16.5 nmol/L) compared to healthy volunteers (276.7 ± 20.8 nmol/L; P < .05). In the presence of APC, endogenous thrombin potential was significantly lower in control plasma (228.6 ± 44.5 nmol/L × min) than in either myeloma (866.2 ± 241.3 nmol/L × min, P = .01) or MGUS plasma (627 ± 91.5 nmol/L × min, P = .003). Within the myeloma cohort, peak thrombin generation was significantly higher at diagnosis (353.2 ± 15.9 nmol/L) than following completion of the third cycle of therapy (282.1 ± 15.2 nmol/L; P < .005). Moreover, sensitivity to APC increased progressively with each cycle of chemotherapy. Further study of the etiology and evolving patterns of hypercoagulability among patients with these conditions is warranted and may have future implications for thromboprophylaxis strategies.

Comparative proteomic analysis of trophoblast cell models reveals their differential phenotypes, potential uses and limitations

Trophoblastic cell lines are widely used in in vitro studies of placental function as a surrogate for primary trophoblasts. To date, no reference proteomics dataset exists to directly compare the shared and unique characteristics of these cells. Here, we performed comparative proteomic profiling of the BeWo and HTR8/SVneo cell lines using label‐free quantitative MS. A total of 1557 proteins were identified, which included 338 uniquely attributed to BeWo cells, and a further 304 specifically identified in HTR8/SVneo cells. Raw data are available via ProteomeXchange, identifier PDX005045. Of the 915 proteins expressed by both cell lines, 105 were of higher abundance in BeWo cells, while 199 proteins had a significantly higher expression in HTR8/SVneo cells. Comparative GO of unique and upregulated proteins revealed principal differences in cell junction/adhesion, catenin complex, spindle and microtubule associated complex, as well as cell differentiation. Our data indicate that BeWo cells express an epithelial proteome more characteristic of villous trophoblasts, whereas HTR8/SVneo cells embrace a mesenchymal phenotype, more characteristic of extravillous trophoblasts. This novel comparative proteomic profiling of these trophoblastic cell lines provides a useful platform for future investigations of placental function.

Coagulation indices in very preterm infants from cord blood and postnatal samples

Very premature infants are at high risk of bleeding complications; however, few data exist on ranges for standard coagulation tests.

Elevated venous thromboembolism risk in preeclampsia: molecular mechanisms and clinical impact

Venous thromboembolism (VTE) remains a leading cause of maternal death and morbidity in the developed world. Strategies for prevention of VTE in pregnancy have been the subject of recent guidelines and consensus statements. These guidelines recommend thrombosis prevention in women who have risk factors associated with an elevated VTE risk. Preeclampsia is characterized by maternal hypertension and proteinuria developing after 20 weeks gestation, complicating up to 7% of pregnancies and is associated with a massive annual morbidity and mortality burden. Women with preeclampsia have been shown to be at increased risk of VTE with studies to date suggesting that this risk may be up to 5-fold greater than the risk of pregnancy-associated VTE in the general population. Despite the fact that preeclampsia is so common and potentially devastating, our understanding of its pathogenesis and potential therapeutic strategies remain poor. In addition, the mechanisms underlying the prothrombotic phenotype in preeclampsia are also poorly characterized although a number of potential mechanisms have been postulated. Derangements of platelet and endothelial activation and impairment of endogenous anti-coagulant pathways have been reported and may contribute to the observed VTE risk. Recently, evidence for the role of neutrophil extracellular traps (NETs) and cell-free DNA in the pathogenesis of VTE has emerged and some evidence exists to suggest that this may be of relevance in preeclampsia. Future studies aimed at understanding the diagnostic and potential therapeutic relevance of this procoagulant state are likely to be of enormous clinical benefit for pregnant women affected with this potentially devastating condition.

Effect of platelet-derived β-thromboglobulins on coagulation

BACKGROUND β-thromboglobulins are derived from the cleavage of the CXC chemokine platelet basic protein and are released in high concentrations by activated platelets. Platelet-derived β-thromboglobulins (βTG) share 70% homology with platelet factor 4 (PF4), another CXC chemokine released by activated platelets. PF4 modulates coagulation by inhibiting heparin-antithrombin interactions, promoting protein C activation, and attenuating the activity of activated protein C. In contrast, the effect of βTG on coagulation is unknown. AIM/METHODS Clotting times, thrombin generation, chromogenic clotting factor assays, and surface plasmon resonance (SPR) were used to assess the effect of purified βTG on coagulation. RESULTS In normal pooled plasma, βTG shortened the lagtime and time to peak thrombin generation of tissue factor (TF)-dependent and TF-independent thrombin generation. In factor VIII and factor IX-deficient plasmas, βTG induced thrombin generation in the absence of a TF stimulus and in the presence of anti-TF and factor VIIa inhibitory antibodies. The procoagulant effect was not observed when thrombin generation was independent of factor X activation (supplementation of factor X-deficient plasma with factor Xa). Cleavage of a factor Xa-specific chromogenic substrate was observed when βTG was incubated with factor X, suggesting a direct interaction between βTG and factor X. Using SPR, βTG were found to bind to immobilised factor X in a dose dependent manner. CONCLUSION βTG modulate coagulation in vitro via an interaction with factor X.

Thrombin generation correlates with disease duration in multiple sclerosis (MS): Novel insights into the MS-associated prothrombotic state:

Background Thrombin is well recognised for its role in the coagulation cascade but it also plays a role in inflammation, with enhanced thrombin generation observed in several inflammatory disorders. Although patients with multiple sclerosis (MS) have a higher incidence of thrombotic disease, thrombin generation has not been studied to date. Objectives The aim of this study was to characterise calibrated automated thrombography parameters in patients with relapsing–remitting MS (RRMS) and primary progressive MS (PPMS) in comparison to healthy controls (HCs). Methods Calibrated automated thrombography was performed on platelet poor plasma from 15 patients with RRMS, 15 with PPMS and 19 HCs. Results We found that patients with RRMS generate thrombin at a significantly faster rate than the less inflammatory subtype, PPMS or HCs. In addition, the speed of thrombin generation was significantly correlated with time from clinical diagnosis in both subtypes. However, in RRMS the rate of thrombin generation was increased with increased time from clinical diagnosis, while in PPMS the rate of thrombin generation decreased with increased time from clinical diagnosis. Conclusions These data likely reflect the differential active proinflammatory states in each MS subtype and provide novel mechanistic insights into the clinically relevant prothrombotic state observed in these patients.

Early onset preeclampsia is associated with an elevated mean platelet volume (MPV) and a greater rise in MPV from time of booking compared with pregnant controls: results of the CAPE study

Abstract Objective: To characterise Mean platelet volume (MPV) in patients with early onset preeclampsia (EOPE) and unaffected controls from time of first antenatal visit until the postpartum. Materials and methods: Retrospective secondary analysis of an observational study in an Irish tertiary referral centre with 9000 deliveries annually. The MPV of 27 women with EOPE was compared to 19 unaffected controls. The inclusion criteria for the disease state was the development of EOPE defined by the National Institute for Health and Care Excellence (NICE) guideline, as new onset hypertension presenting after 20 weeks and prior to 34 weeks with significant proteinuria. Between October 2013 and July 2015 we recruited 27 women with EOPE and 19 pregnant controls. Statistical analysis was performed using paired T-test of Mann-Whitney test where appropriate and a P-value <0.05 was deemed significant. Results: At time of diagnosis and late in the third trimester MPV was significantly increased to 9.0 (±0.3) fL in cases of EOPE in comparison to 8.5 (±0.6) fL in normotensive controls (P<0.05). There was no significant difference during the first trimester or postpartum when comparing the MPV in EOPE to controls. Conclusion: Despite an increased MPV at time of diagnosis of EOPE this study did not demonstrate a potential use for increased MPV as a first trimester screening tool.

The platelet releasate is altered in human pregnancy

Healthy pregnancy is characterized by an increase in platelet activation and a decrease in the number of circulating platelets with gestation. Despite this recognized importance, proteomic studies investigating platelets in healthy pregnancy have not been performed. As platelet cargo can be altered in different conditions, it is hypothesized that platelets may store a relevant and bespoke collection of molecules during pregnancy.

Dual endothelin-1 receptor antagonism attenuates platelet-mediated derangements of blood coagulation in Eisenmenger syndrome

Essentials Eisenmenger syndrome is characterised by thrombotic and hemorrhagic risks of unclear aetiology. Calibrated automated thrombography was used to assess these coagulation derangements. Platelet activity supported abnormalities in procoagulant and anticoagulant pathway function. Endothelin‐1 antagonism appeared to ameliorate these derangements.

Elevated plasma TFPI activity causes attenuated TF-dependent thrombin generation in early onset preeclampsia

Early onset preeclampsia (EOP) is a pregnancy-specific proinflammatory disorder that is characterised by competing thrombotic and bleeding risks. It was the aim of this study to characterise thrombin generation, a major determinant of thrombotic and bleeding risk, in order to better understand the haemostatic balance in patients with EOP. Patients with EOP were recruited at the Rotunda Hospital, Dublin. Twenty-six cases of EOP were recruited over a 21-month period, out of 15,299 deliveries at the Rotunda. Blood samples were collected into sodium citrate plus corn trypsin inhibitor anticoagulated vacutainers, platelet-poor plasma was prepared, and calibrated automated thrombography was used to assess thrombin generation. Results were compared to age and sex-matched non-pregnant controls (n=13) and age- and gestation-matched pregnant controls (n=20). The rate and extent of thrombin generation triggered by low-dose tissue factor (TF) was significantly reduced in patients with EOP compared to pregnant controls, most significantly in cases of severe EOP. EOP patients displayed a trend towards an increased response to endogenous activated protein C and thrombomodulin relative to pregnant controls. Plasma tissue factor pathway inhibitor (TFPI) activity was increased in EOP patients. Inhibition of TFPI abolished the attenuation of thrombin generation stimulated by low-dose TF. In conclusion, patients with EOP are characterised by an attenuated coagulation response characterised by reduced thrombin generation stimulated by low-dose TF and elevated plasma TFPI activity. These changes in coagulation may modulate thrombotic risk and bleeding risk in patients with EOP.