Mice Lacking PECAM-1 and Ceacam1 Have Enhanced Platelet Secretion and Thrombus Growth: Novel Link with PAR4.

The Ig-ITIM bearing receptors, PECAM-1 and CEACAM1, have been shown net negative regulators of platelet-collagen interactions and hemiITAM signaling pathways. In this study, a double knockout (DKO) mouse was developed with deleted PECAM-1 and CEACAM1 to study their combined contribution in platelet activation by glycoprotein VI, C-type lectin-like receptor 2, protease activated receptor (PAR4), ADP purinergic receptors, and thromboxane receptor (TP) A2 pathways. In addition, their collective contribution was examined in thrombus formation under high shear and microvascular thrombosis using in vivo models. DKO platelets responded normally to ADP purinergic receptors and the TP A2 pathway. However, DKO platelets released significantly higher amounts of P-selectin compared with hyper-responsive Pecam-1-/- or Ceacam1-/- versus wild-type (WT) upon stimulation with collagen-related peptide or rhodocytin. In contrast, DKO platelets showed increased amounts of P-selectin exposure upon stimulation with PAR4 agonist peptide or thrombin but not Pecam-1-/- , Ceacam1-/- , or WT platelets. Blockade of phospholipase C (PLC) or Rho A kinase revealed that DKO platelets enhanced α-granule release via PAR4/Gαq/PLC signaling without crosstalk with Src/Syk or G12/13 signaling pathways. Severely delayed clot retraction in vitro was observed in DKO phenotype. The DKO model revealed a significant increase in thrombus formation compared with the hyper-responsive Ceacam1-/- or Pecam-1-/- versus WT phenotype. DKO platelets have similar glycoprotein surface expression compared with Pecam-1-/- , Ceacam1-/- , and WT platelets. This study demonstrates that PECAM-1 and CEACAM1 work in concert to negatively regulate hemiITAM signaling, platelet-collagen interactions, and PAR4 Gαq protein- coupled signaling pathways. Both PECAM-1 and CEACAM1 are required for negative regulation of platelet activation and microvascular thrombosis in vivo.

A Novel Optical Assay System for Bilirubin Concentration Measurement in Whole Blood.

As a biomarker for liver disease, bilirubin has been utilized in prognostic scoring systems for cirrhosis. While laboratory-based methods are used to determine bilirubin levels in clinical settings, they do not readily lend themselves to applications outside of hospitals. Consequently, bilirubin monitoring for cirrhotic patients is often performed only intermittently; thus, episodes requiring clinical interventions could be missed. This work investigates the feasibility of measuring bilirubin concentration in whole porcine blood samples using dual-wavelength transmission measurement. A compact and low-cost dual-wavelength transmission measurement setup is developed and optimized to measure whole blood bilirubin concentrations. Using small volumes of whole porcine blood (72 µL), we measured the bilirubin concentration within a range corresponding to healthy individuals and cirrhotic patients (1.2-30 mg/dL). We demonstrate that bilirubin levels can be estimated with a positive correlation (R-square > 0.95) and an accuracy of ±1.7 mg/dL, with higher reliability in cirrhotic bilirubin concentrations (> 4 mg/dL) - critical for high-risk patients. The optical and electronic components utilized are economical and can be readily integrated into a miniature, low-cost, and user-friendly system. This could provide a pathway for point-of-care monitoring of blood bilirubin outside of medical facilities (e.g., patient's home).

New generation drugs for treatment of multiple myeloma.

Multiple myeloma (MM), a plasma cell malignancy, is characterised by lesions in multiple bones involving transformed, matured post-follicular B cells. The course of the disease involves an initial development of monoclonal gammopathy of undetermined significance (MGUS), followed by smouldering MM, before the full MM disease emerges. Despite novel therapies, MM remains incurable, managed by combination therapies, including proteasome inhibitors (PIs), immunomodulators (IMiDs) and anti-human CD38 (daratumumab). MM patients have an increased risk of thromboembolic events due to combination treatments with IMiDs, PIs and anti-human CD38 antibody, and steroids. This review will examine the efficacy and pro-thrombotic effects of MM therapies.

Expression of the plasminogen system in the physiological mouse ovary and in the pathological polycystic ovary syndrome (PCOS) state.

BACKGROUND: The fibrinolytic system and its inhibitors play a number of roles, apart from their function in blood haemostasis and thrombosis, namely in ovarian folliculogenesis and in ovulation. Plasminogen is converted to active plasmin at the time of follicular rupture through a decrease in plasminogen activator inhibitor-1 (PAI-1) and an increase in plasminogen activators. Oligo-/anovulation and follicle arrest are key characteristics of PCOS, but studies evaluating fibrinolytic/proteolytic markers within human or animal PCOS ovaries are lacking. We aimed to investigate and compare the expression and distribution of the plasminogen system markers in PCOS and control ovaries. METHODS: A hyperandrogenised PCOS mouse model was used that mimics the ovarian, endocrine and metabolic features of the human condition. Immunohistochemistry and digital image analysis were used to investigate and compare fibrinolytic/proteolytic markers plasminogen, plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator and inhibitor PAI-1 in PCOS and control ovaries. Student's t-test was used to compare data sets for normally distributed data and Wilcoxon-Mann Whitney test for non-normally distributed data. RESULTS: We noted differences in the ovarian distribution of PAI-1 that was expressed throughout the PCOS ovary, unlike the peripheral distribution observed in control ovaries. Plasminogen was present in small follicles only in PCOS ovaries but not in small follicles of control ovaries. When we assessed and compared PAI-1 expression within follicles of different developmental stages we also noted significant differences for both the PCOS and control ovaries. While we noted differences in distribution and expression within specific ovarian structures, no differences were noted in the overall ovarian expression of markers assessed between acyclical PCOS mice and control mice at the diestrus stage of the estrous cycle. CONCLUSIONS: Our novel study, that comprehensively assessed the fibrinolytic/proteolytic system in the mouse ovary, showed the expression, differential localisation and a potential role for the plasminogen system in the physiological mouse ovary and in PCOS. Androgens may be involved in regulating expression of the ovarian plasminogen system. Further studies evaluating these markers at different time-points of ovulation may help to further clarify both physiological and potential pathological actions these markers play in ovulatory processes distorted in PCOS.

Differential Effects on Haemostatic Markers by Metformin and the Contraceptive Pill: A Randomized Comparative Trial in PCOS.

Background Polycystic ovarian syndrome (PCOS) affects up to 18% of reproductive-aged women with increased risks of cardiovascular disease and venous thromboembolic disease, related to metabolic and hormonal features, obesity and an apparent hypofibrinolytic state, possibly exacerbated by current PCOS treatments. Objective To investigate and compare haemostatic impacts of common pharmacological treatments and explore relationships with hormonal and metabolic variables in PCOS. Patients/Methods This mechanistic sub-study using biobanked samples from a 6-month randomized comparative trial of pharmacological treatments assessed pro- and anti-thrombotic markers and overall haemostatic activity. Overweight women of mean age 33.9 ± 6.7 years and mean BMI (body mass index) of 36.5 ± 7.0 kg/m2 with PCOS (n = 60) were randomized to either metformin, higher-dose oral contraceptive pill (OCP) or low-dose OCP + spironolactone (OCP + S). Primary outcome measures included changes in plasminogen activator inhibitor 1 (PAI-1), asymmetric dimethylarginine (ADMA), prothrombin fragments 1 and 2 (PF1 and 2), plasminogen, tissue plasminogen activator (tPA), thrombin activatable fibrinolysis inhibitor (TAFI) and thrombin generation (TG). Results PAI-1 activity fell in all groups, ADMA fell in higher-dose OCP, PF1 and 2 increased with metformin and higher-dose OCP, TG rose and tPA fell in both OCP groups, plasminogen increased in all and TAFI increased after higher-dose OCP. Conclusion Endothelial function (primary haemostasis) improved with higher dose with some improvement in low-dose OCP + S and metformin. Aberrant coagulation was noted in both OCP groups, but not with metformin. Fibrinolysis was reduced with higher-dose OCP. Our work suggests an additional dimension of treatment (haemostatic system effects) that favours metformin treatment over the OCP in PCOS.

Comprehensive Assessment of the Hemostatic System in Polycystic Ovarian Syndrome.

Polycystic ovarian syndrome (PCOS) affects 12 to 19% of women and has reproductive and metabolic features (endothelial dysfunction, increased diabetes, and cardiovascular risk factors). It also appears to have altered coagulation and fibrinolysis with a prothrombotic state with epidemiological evidence of increased venous thromboembolism. We aimed to comprehensively assess hemostasis in women with PCOS versus control women. In an established case-control cohort of lean, overweight, and obese women with (n = 107) and without PCOS (n = 67), with existing measures of plasminogen activator inhibitor 1 (PAI-1), asymmetric dimethylarginine (ADMA), hormonal, and metabolic markers, we also assessed prothrombin fragments 1 and 2 (PF1 & 2), plasminogen, tissue plasminogen activator (tPA), and thrombin generation (TG). Higher levels of ADMA (0.70 vs. 0.39 µmol/L, p < 0.01), PAI-1 (4.80 vs. 3.66 U/mL, p < 0.01), and plasminogen (118.39 vs. 108.46%, p < 0.01) were seen in PCOS versus controls, and persisted after adjustment for age and body mass index (BMI). PF1 & 2 was marginally lower (180.0 vs. 236.0 pmol/L, p = 0.05), whereas tPA and TG were not different between groups, after adjustment for age and BMI. Significant relationships were observed between hormonal and metabolic factors with ADMA and PAI-1. We demonstrate impaired fibrinolysis in PCOS. In the context of abnormal endothelial function and known hormonal and metabolic abnormalities, this finding may underpin an increased risk of cardiovascular disease and venous thrombosis in PCOS.

Hemostatic abnormalities and relationships to metabolic and hormonal status in polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS), diagnosed based on hyperandrogenism, ovulatory dysfunction, and polycystic ovaries, is one of the most common disorders of reproductive-aged females. Etiology includes both genetic and environmental/lifestyle factors contributing to both insulin resistance and hyperandrogenism. Clinically, PCOS has reproductive, psychological, and metabolic features, the latter predisposing to cardiovascular disease (CVD). Hemostatic abnormalities have an association with and a demonstrated pathophysiological role in CVD in non-PCOS populations but have not been adequately explored in PCOS. This review focuses on the hemostatic system in PCOS, exploring also relationships to the metabolic and hormonal abnormalities of the syndrome, and aims to identify whether hemostatic abnormalities are present as potential contributors to increased cardiovascular risk. Ultimately, this area may reveal preventative and therapeutic opportunities, which could improve the cardiovascular health of women with PCOS.