Paulina B. Szklanna

Analysis of the proteins associated with platelet detergent resistant membranes.

Proteomic studies have facilitated the identification of proteins associated with the detergent‐resistant membrane (DRM) fraction in a variety of cell types. Here, we have undertaken label‐free quantitative (LFQ) proteomic profiling of the proteins associated with detergent‐resistant plasma and internal membranes from resting and activated platelets. One hundred forty‐one proteins were identified and raw data is available via ProteomeXchange with identifier PXD002554. The proteins identified include a myriad of important platelet signaling and trafficking proteins including Rap1b, Src, SNAP‐23, syntaxin‐11, and members of the previously unattributed Ragulator complex. Mean LFQ intensities calculated across three technical replicates for the three biological donors revealed that several important platelet signaling proteins altered their detergent solubility upon activation, including GPIbα, GPIbβ, Src, and 14‐3‐3ζ. Altered detergent solubility for GPIbα, following activation using a variety of platelet agonists, was confirmed by immunoblotting and further coimmunoprecipitation experiments revealed that GPIbα forms a complex with 14‐3‐3ζ that shifts into DRMs following activation. Taken together, proteomic profiling of platelet DRMs allowed greater insight in the complex biology of both DRMs and platelets and will be a useful subproteome to study platelet‐related disease. All MS data have been deposited in the ProteomeXchange with identifier PXD002554 (http://proteomecentral.proteomexchange.org/dataset/PXD002554).

Proteomic Analysis Reveals a Strong Association of β‐Catenin with Cadherin Adherens Junctions in Resting Human Platelets

It was previously demonstrated that the WNT/β‐catenin pathway is present and active in platelets and established that the canonical WNT ligand, WNT‐3a, suppresses platelet adhesion and activation. In nucleated cells, β‐catenin, the key downstream effector of this pathway, is a dual function protein, regulating the coordination of gene transcription and cell–cell adhesion. The specific role of β‐catenin in the anucleate platelet however remains elusive. Here, a label‐free quantitative proteomic analysis of β‐catenin immunoprecipitates from human platelets is performed and nine co‐immunoprecipitating proteins are identified. Three of the co‐immunoprecipitating proteins (α‐catenin‐1, cadherin‐6, and β‐catenin‐interacting protein 1) are common to both resting and activated conditions. Bioinformatics analysis of proteomics data reveal a strong association of the dataset with both cadherin adherens junctions and regulators of WNT signaling. It is then verified that platelet β‐catenin and cadherin‐6 interact and that this interaction is regulated by the activation state of the platelet. Taken together, this proteomics study suggests a novel role for β‐catenin in human platelets where it interacts with platelet cadherins and associated junctional proteins.

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.

A Protocol for Improved Precision and Increased Confidence in Nanoparticle Tracking Analysis Concentration Measurements between 50 and 120 nm in Biological Fluids

Nanoparticle tracking analysis (NTA) can be used to quantitate extracellular vesicles (EVs) in biological samples and is widely considered a useful diagnostic tool to detect disease. However, accurately profiling EVs can be challenging due to their small size and heterogeneity. Here, we aimed to provide a protocol to facilitate high-precision particle quantitation by NTA in plasma, the supernatant of activated purified platelets [the platelet releasate (PR)] and in serum, to increase confidence in NTA particle enumeration. The overall variance and the precision of NTA measurements were quantified by root mean square error and relative standard error. Using a bootstrapping approach, we found that increasing video replicates from 5 s × 60 s to 25 s × 60 s captures led to a reduction in overall variance and a reproducible increase in the precision of NTA particle-concentration quantitation for all three biofluids. We then validated our approach in an extended cohort of 32 healthy donors. Our results indicate that for vesicles sized between 50 and 120 nm, the precision of routine NTA measurements in serum, plasma, and PR can be significantly improved by increasing the number of video replicates captured. Our protocol provides a common platform to statistical compare particle size distribution profiles in the exosomal-vesicle size range across a variety of biofluids and in both healthy donor and patient groups.

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.

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.