The effect of combined oral contraceptives containing drospirenone and ethinylestradiol on serum levels of amino acids and acylcarnitines.

INTRODUCTION: Metabolome variations have long been associated with normal hormonal fluctuations, and similar effects, related to the use of early generation synthetic hormones as a means of contraception, have also been identified. OBJECTIVE: We investigated the serum amino acid and acylcarnitine profiles induced by the use of combined oral contraceptives (COCs) consisting of Ethinylestradiol (EE) and a 4th generation progestin, Drospirenone (DRSP). METHOD: Gas chromatography mass spectrometry and liquid chromatography with tandem mass spectrometry was used to identify and quantify the serum amino acids and acyl carnitine levels in 24 controls, 25 DRSP/20EE users and 26 DRSP/30EE users. RESULTS: Of the 26 amino acid compounds measured, 13 showed significant variations in abundance between the control and COC user groups. Although none of the 21 acylcarnitine compounds detected were statistically significant with regards to group variations, a trend, related the EE concentration, was observed. The detected metabolome disparities corresponded to that identified for earlier generation COCs, all pointing toward increased oxidative stress levels in the user groups. CONCLUSION: These findings suggest that the clinical complications associated with these COCs could, to some extent, be alleviated by the simultaneous use of antioxidants. The study also highlights the role that targeted metabolomics could play in the elucidation of the underlying mechanisms of drug-induced severe effects.

The Value of Detecting Pathological Changes During Clot Formation in Early Disease Treatment-Naïve Breast Cancer Patients.

Breast cancer (BC) is one of the most prevalent forms of cancer in women worldwide. Clinical research indicates that BC patients are at an increased risk for thrombotic events, drastically decreasing their quality-of-life and treatment outcomes. There is ample evidence of this in the literature, but it is mainly focused on metastatic BC. Therefore, coagulopathies of nonmetastatic BC are understudied and require in-depth investigation. In this study, clot kinetics and ultrastructure were used to investigate treatment-naïve, nonmetastatic BC patients using scanning electron microscopy, Thromboelastography®, and confocal laser scanning microscopy. It was demonstrated that nonmetastatic BC patients exhibit minimal ultrastructural alterations of the clot components and no changes in the clot kinetics. However, BC patients presented changes to fibrinogen protein structure, compared to matched controls, using an amyloid-selective stain. Together, these findings suggest that coagulation dysfunction(s) in BC patients with early disease manifest at the microlevel, rather than the macrolevel. This study presents novel insights to a method that are more sensitive to coagulation changes in this specific patient group, emphasizing that the coagulation system may react in different forms to the disease, depending on the progression of the disease itself.

Mechanical and Physical Behavior of Fibrin Clot Formation and Lysis in Combined Oral Contraceptive Users.

Combined oral contraceptives (COCs) are commonly prescribed and increase the risk of venous thromboembolism (VTE). We have previously found that two COCs, both containing drospirenone (DRSP) and ethinyl estradiol (EE), cause spontaneous fibrin formation in whole blood. The aim of this study was, therefore, to use platelet-poor plasma (PPP) from the same cohort of DRSP/EE users to determine the impact of these COCs on the fibrin component, specifically the fibrin clot viscoelasticity, turbidimetry, and biophysical traits. PPP from 25 females per test group and a control group (n = 25) were analyzed using thromboelastography (TEG), turbidimetry, and scanning electron microscopy. The results highlight abnormal fibrin clot formation, lysis, and architecture; DRSP/20EE showed the greatest effect. DRSP/EE use increased the fibrin fiber diameter and showed dense matted clots. Only when the influence of COCs on the structural properties and behavior of fibrin fibers during thrombus formation and lysis is better understood are we able to predict and prevent coagulopathies associated with these synthetic hormones. Clinical practitioners should take this into consideration for female patients that either have comorbidities, which could burden the coagulation system, or may be exposed to external factors that could increase their risk for VTE.

Interleukin-12 and its procoagulant effect on erythrocytes, platelets and fibrin(ogen): the lesser known side of inflammation.

Inflammation, with its associated inflammatory molecules, is integral to most chronic diseases, including the various cardiovascular diseases. Interleukin 12 (IL12) is one of the inflammatory cytokines that is upregulated during inflammation; however, we know very little about its exact effect on red blood cells (RBCs), platelets and fibrin(ogen). IL12 is an important pleiotropic cytokine in early inflammatory responses and has potent immunomodulatory, antitumour and anti-infection activity. Here we investigate how low levels of circulating IL12, comparable to levels found during chronic inflammation, affect coagulation parameters, platelets and RBCs. We used thromboelastography, scanning electron microscopy, refractometery and wide-field microscopy. Our results show that IL12 caused hypercoagulation, platelet activation and spreading, as well as RBC agglutination. This phenomenon has far-reaching implications for treatment of the plethora of conditions where IL12 is upregulated, since it suggests aberrant haemorheology as agglutination affects blood flow. This information might be used in future to target the lowering of IL12 in inflammatory conditions, as well as address RBC agglutination.

Simultaneous presence of hypercoagulation and increased clot lysis time due to IL-1ß, IL-6 and IL-8.

Circulating cytokines, and particularly the interleukin (IL)-family are known to play an important role in inflammation. These molecules circulate in the blood and therefore have a direct effect on the plasma molecules and the formed elements like the erythrocytes and platelets. Aberrant coagulation (hypercoagulation or blood clots that form too easily) and clot lyses (hypofibrinolysis, where clots do not dissolve properly, with an abnormally low rate of clot lysis time), are usually the hallmarks of many inflammatory conditions. However, the mechanism by which cross-linking augments clot stiffness remains undetermined. IL-1ß; IL-6 and IL-8 has been found to be involved in most chronic and acute inflammatory diseases. In the present study, we investigate clot structure of healthy blood, with the addition of these 3 interleukins, to determine the individual effects at concentrations that mimic low-grade, chronic inflammation. Previous studies showed that clot rheological behavior is regulated by at least the following three factors, fibrinogen concentration, fibrin network architecture and FXIIIa-induced ligation. We investigated clot formation and lysis using thromboelastography (TEG), before and after exposure, and created clots by adding thrombin to whole blood. This allowed us to look at extensive fibrin fiber formation and their interactions with particularly the erythrocytes, using scanning electron microscopy (SEM). Our results showed that IL-1ß; IL-6 and IL-8 causes hypercoagulation and results in a disheveled fibrin clot, with trapped RBCs. IL-8 showed eryptosis (a type of apoptosis in erythrocytes). Our lysis results showed that both clot lysis time and maximum rate of lysis are decreased, with the addition of the interleukins. This is a novel finding and the observations reported in this paper, therefore points to the importance of looking at the effects of individual circulating inflammagens, to better understand the role that each play in the expression of disease. These methods can be used for an individualized patient-orientated approach in healthcare to track blood viscosity in conditions with acute and chronic inflammation.

The Impact of Two Combined Oral Contraceptives Containing Ethinyl Estradiol and Drospirenone on Whole Blood Clot Viscoelasticity and the Biophysical and Biochemical Characteristics of Erythrocytes.

Venous thrombosis is associated with combined oral contraceptive (COC) use. We investigated the impact of two ethinyl estradiol (EE) and drospirenone (DRSP) containing COCs (3 mg DRSP/20 µg EE and 3 µg DRSP/30 µg EE) on the viscoelasticity of whole blood clots along with the biophysical and biochemical characteristics of erythrocytes. Thromboelastography (TEG) analysis showed a tendency toward a hypercoagulable state in the COCs groups that was more pronounced with higher EE concentrations. Light microscopy and scanning electron microscopy (SEM) showed rouleaux formation of erythrocytes and alterations to the erythrocyte shape for both COC groups, which was attributed to membrane damage. SEM analysis showed spontaneous activation of fibrin and platelets in the COC groups, along with interactions between erythrocytes and platelets and/or fibrin. Confocal microscopy confirmed compromised membrane integrity in the COC groups compared to controls. Global thrombosis test analysis showed increased platelet activation and low thrombolysis in both COC groups when compared to controls. In conclusion, DRSP/EE formulations impact erythrocytes' biophysical and biochemical properties to cause a shift in hemostasis to a prothrombotic state. Although these effects are mostly subclinical the long-term effects and risks involved with the use of these hormones should be considered carefully for each individual.

Ultrastructural, Confocal and Viscoelastic Characteristics of Whole Blood and Plasma After Exposure to Cadmium and Chromium Alone and in Combination: An Ex Vivo Study.

BACKGROUND/AIMS: Heavy metal pollution is increasing in the environment, contaminating water, food and air supplies. This can be linked to many anthropogenic activities. Heavy metals are absorbed through the skin, inhalation and/or orally. Irrespective of the manner of heavy metal entry in the body, the blood circulatory system is potentially the first to be affected following exposure and adverse effects on blood coagulation can lead to associated thrombotic disease. Although the plasma levels and the effects of cadmium (Cd) and chromium (Cr) on erythrocytes and lymphocytes have been described, the environmental exposure to heavy metals are not limited to a single metal and often involves metal mixtures, with each metal having different rates of absorption, different cellular, tissue, and organ targets. Therefore the aim of this study is to investigate the effects of the heavy metals Cd and Cr alone and whether Cr synergistically increases the effect of Cd on physiological important processes such as blood coagulation. METHODS: Human blood was exposed to the heavy metals ex vivo, and thereafter morphological analysis was performed with scanning electron- and confocal laser scanning microscopy (CLSM) in conjunction with thromboelastography®. RESULTS: The erythrocytes, platelets and fibrin networks presented with ultrastructural changes, including varied erythrocytes morphologies, activated platelets and significantly thicker fibrin fibres in the metal-exposed groups. CLSM analysis revealed the presence of phosphatidylserine on the outer surface of the membranes of the spherocytic erythrocytes exposed to Cd and Cr alone and in combination. The viscoelastic analysis revealed only a trend that indicates that clots that will form after heavy metal exposure, will likely be fragile and unstable especially for Cd and Cr in combination. CONCLUSION: This study identified the blood as an important target system of Cd and Cr toxicity.

Tissue factor levels in type 2 diabetes mellitus.

INTRODUCTION: Type 2 diabetes mellitus is a pandemic associated with disturbance in haemostasis that could contribute to the development of diabetic vascular disease and accelerated atherosclerosis. In this population, hypercoagulation is prevalent, as well as pathological changes to erythrocytes. This is mainly due to upregulated circulating inflammatory markers. MATERIALS AND METHODS: Here we looked at tissue factor (TF) levels using ELISA, in a sample of diabetics, with and without cardiovascular complications. Diabetic subjects were recruited from the diabetic clinic at Steve Biko Academic Hospital, Pretoria, South Africa. 20 diabetics with cardiovascular disease and 22 without were enrolled to participate. RESULTS AND CONCLUSION: TF levels were significantly elevated in both diabetic groups when compared to the controls. We suggest that pathologic plasma TF activity, as marker of increased propensity of clot pathology, should be investigated. Agents that might lower TF levels might also possibly lower thrombotic complications.

A Comprehensive Review on Eryptosis.

Erythrocytes (RBCs) are extremely sensitive cells, and although they do not have nuclei and mitochondria, are important health indicators. This is particularly true because, during inflammation, whether it is systemic or chronic, the haematological system is constantly exposed to circulating inflammatory mediators. RBCs have a highly specialized and organized membrane structure, which interacts and reacts to inflammatory molecule insults, and undergo programmed cell death, similar to apoptosis, known as eryptosis. Over the past years, eryptosis studies have focussed on determining if membrane changes have occurred, particularly whether a phosphatidylserine (PS) flip, Ca2+ leakage into the cell, changes to ceramide and cell shrinkage have occurred. Mostly, flow cytometry is used, but confocal microscopy and ultrastructural studies also confirm eryptosis. Here, we provide a comprehensive overview of eryptosis, where we revisit the biochemical process of the process, review all literature in PUBMED, that is shown under the search word, "eryptosis", and also discuss current methodologies to determine the presence of eryptosis; included in the discussion of the methodologies, we discuss a pitfalls section for each method. This paper is therefore a comprehensive synopsis of current knowledge of eryptosis and discusses how RBCs may provide an essential in vivo cell model system to study not only inflammation in disease, but also track disease progression and treatment regimes.

Transient ischemic attack during smoking: The thrombotic state of erythrocytes and platelets illustrated visually.

Transient ischemic attack (TIA) is an important predictor of future ischemic events, including stroke. Due to the typically brief period of neurologic dysfunction, patients often overlook the importance of reporting a TIA. We have recently shown that platelet activation plays an important role in TIA pathology. In a similar vein, smoking is associated with a hypercoagulable state and is also one of the important risk factors for stroke. Here we present an interesting case where a 61-year-old male, with hypercholesterolemia, and a previous heart valve replacement, developed a TIA 5 months after he started smoking. Subsequent to the event, Warfarin dosage was monitored monthly using the international normalized ratio (INR). We compared erythrocyte and platelet morphology of healthy individuals, that of smokers, individuals who had a diagnosed TIA (without smoking), and the patient. The erythrocytes from the case study are ultrastructurally similar to that of a smoker, while his platelets are similar to that of smokers and TIA patients who do not smoke. We conclude that smoking exacerbated the chronic inflammation induced by hypercholesterolemia, causing changes in his erythrocyte morphology and platelet activation, and suggest that ultrastructure here explains the clinical manifestations of the thrombotic state of this patient.

Poorly controlled type 2 diabetes is accompanied by significant morphological and ultrastructural changes in both erythrocytes and in thrombin-generated fibrin: implications for diagnostics.

We have noted in previous work, in a variety of inflammatory diseases, where iron dysregulation occurs, a strong tendency for erythrocytes to lose their normal discoid shape and to adopt a skewed morphology (as judged by their axial ratios in the light microscope and by their ultrastructure in the SEM). Similarly, the polymerization of fibrinogen, as induced in vitro by added thrombin, leads not to the common 'spaghetti-like' structures but to dense matted deposits. Type 2 diabetes is a known inflammatory disease. In the present work, we found that the axial ratio of the erythrocytes of poorly controlled (as suggested by increased HbA1c levels) type 2 diabetics was significantly increased, and that their fibrin morphologies were again highly aberrant. As judged by scanning electron microscopy and in the atomic force microscope, these could be reversed, to some degree, by the addition of the iron chelators deferoxamine (DFO) or deferasirox (DFX). As well as their demonstrated diagnostic significance, these morphological indicators may have prognostic value.

Atypical erythrocytes and platelets in a patient with a pro-thrombin mutation.

Prothrombin mutation G20210A, anti-phospholipid syndrome as well as iron overload has previously been shown to cause thrombotic events. The main reason for this is the involvement of these anomalies in causing hypercoagulability of the coagulation system, which frequently leads to venous and arterial thrombotic events. We report the case of a 37-year-old white female with prothrombin mutation G20210A, anti-phospholipid syndrome, as well as an increased serum ferritin level, who experienced two transient ischemic attacks and suffers from regular amaurosis fugax. We present an ultrastructural depiction of erythrocytes, platelets, and the fibrin network, to explain the clinical manifestations of the thrombotic state seen in this patient.

Exploring the ex vivo effects of Naja mossambica venom on the ultrastructure and viscoelastic properties of human blood.

Background: Venomous snake bites have been listed as a neglected tropical disease by the World Health Organization. The Mozambique spitting cobra (Naja mossambica) is found in Sub-Saharan African countries, and its venom has been identified to predominantly result in cytotoxic effects. However, there is limited evidence on the possible hemotoxic effects of this venom on human blood. Objectives: In this cross-sectional study, we investigated how Mozambique spitting cobra venom affects blood clot formation. Methods: Cell morphology and clot architecture were studied by using microscopy techniques. We also studied the effects of the venom on platelets by measuring platelet activity with the global thrombosis test, followed by analyzing the viscoelasticity with thromboelastography using a 0.025 ng/µL venom concentration. Results: The most prominent findings indicated that the viscoelastic profile in the venom-treated blood samples formed an unstable and elastic clot. The clot architecture seen with the scanning electron microscopy analysis showed an altered fibrin network and red blood cells, confirmed by the increased axial ratios, and aggregated platelets with spreading. Conclusion: These findings may offer insights into the species-specific effects of snake venom on human blood and add value to the clinical workup in confirming envenomation. Further research is needed to correlate the 20 minute whole blood clotting test with measurable values from the thromboelastography within the context of snake envenomation. This may offer a bridge between cost, early diagnosis, and treatment of snake envenomation in resource-constrained countries.

Albumin stabilizes fibrin fiber ultrastructure in low serum albumin type 2 diabetes.

Serum albumin is an essential plasma protein that serves an important function in maintaining osmotic pressure. Low levels of this protein are associated with the kidney failure and hemodialysis that are often seen in diabetic patients who are at high risk of thrombotic events. In diabetes, fibrin fiber nets are changed to form dense matted deposits (DMDs, or parafibrin). Here the authors investigate whether parafibrin is also present in diagnosed low-albumin diabetes patients and whether the addition of human albumin to plasma from low-albumin diabetes type 2 individuals may change the architecture of the fibrin nets. The authors show that the addition of albumin to plasma of low-albumin diabetes patients progressively caused the DMDs typically found in these patients to revert back to ultrastructure typically seen in healthy individuals. This disease has an extremely complicated pathophysiology and thus cannot be considered as a simple condition. This study shows that serum albumin levels may play an important role in the structure of fibrin fibrils, making them more susceptible to the fibrinolytic degradation and elimination from the circulation.

A novel method for assessing the role of iron and its functional chelation in fibrin fibril formation: the use of scanning electron microscopy.

AIMS: Inflammatory diseases associated with iron overload are characterized by a changed coagulation profile, where there is a persistent presence of fibrin-like material of dense-matted deposits (DMDs). It is believed that one source of such material is a result of the activation of blood coagulation without the generation of thrombin, causing clots to become resistant to fibrinolytic dissolution. The aim of the current manuscript therefore is to apply a novel scanning electron microscopy method for assessing the role of functional chelation in the prevention or reversal of iron-induced fibrin formation. METHODS AND RESULTS: Purified fibrinogen and platelet-rich plasma were exposed to chelating agents followed by iron, to determine the chelating effects. We show that there is another, pathological pathway of fibrin formation initiated by free iron (initially as Fe (III)), leading to the formation of highly reactive oxygen species such as the hydroxyl radical that can oxidize and insolubilize proteins, a process that might be inhibited by iron-chelating compounds. The final product of such a pathway is a fibrin-like material, termed DMDs that are remarkably resistant to proteolytic degradation. CONCLUSIONS: Scanning electron microscopy shows that iron-chelating agents are effective inhibitors of DMD formation. The most active inhibitors of DMD formation proved to be Desferal, Clioquinol and Curcumin, whereas Epigallocatechin gallate and Deferiprone were less effective. The functional model we describe may point the clinical utility of various substances in iron-mediated degenerative diseases.

Metabolomic Profiling of Hormonal Contraceptive Use in Young Females Using a Commercially Available LC-MS/MS Kit.

Oral hormonal contraceptive users carry the risk of venous thrombosis and increased mortality. This study aimed to comprehensively profile the serum metabolome of participants using a combination of drospirenone (DRSP) and ethinyl estradiol (EE) containing oral contraceptives (COCs). The MxP Quant 500 kit for liquid chromatography mass tandem spectrometry (LC-MS/MS) was used to analyse the 22 controls and 44 COC users (22 on a low EE dose (DRSP/20EE) and 22 on a higher EE dose (DRSP/30EE)). The kit's results were compared to our internally developed untargeted and targeted metabolomics methods previously applied to this cohort. Of the 630 metabolites included in the method, 277 provided desirable results (consistently detected above their detection limits), and of these, 5 had p-values < 0.05, including betaine, glutamine, cortisol, glycine, and choline. Notably, these variations were observed between the control and COC groups, rather than among the two COC groups. Partial least squares-discriminant analysis revealed 49 compounds with VIP values ≥ 1, including amino acids and their derivatives, ceramides, phosphatidylcholines, and triglycerides, among others. Ten differential compounds were consistent with our previous studies, reinforcing the notion of COCs inducing a prothrombotic state and increased oxidative stress. Although only a limited number of compounds were deemed usable, these were quantified with high reliability and facilitated the identification of meaningful biological differences among the sample groups. In addition to substantiating known drug-induced variations, new hypotheses were also generated.

Pathophysiological Changes in Erythrocytes Contributing to Complications of Inflammation and Coagulation in COVID-19.

Higher thrombotic burden in the acute phase of COVID-19 relies on a complex interplay between pro-inflammatory cytokine/chemokine release, increased endothelial dysfunction/damage, and potential sepsis-induced coagulopathy development in severe cases, all promoting coagulation activation. Plasma levels of cytokines and chemokines are known to be increased in COVID-19 however, are much higher in severe infections. Increased levels of IL-1ß, IL-6, and IL-8 are known to play an important role in both acute and chronic inflammation, resulting in pathological clotting. However, little has been published on the effects of these interleukins on red blood cells (RBCs). Evidence shows that cytokines have a negative effect on the RBCs ultrastructure and introduce signs of eryptosis. Eryptosis can be described as a form of suicidal death of RBCs characterized by distinct findings of cell shrinkage, membrane blebbing, activation of proteases, and phosphatidylserine exposure at the outer membrane leaflet. Red blood cells from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, in particular, short- and medium-chain saturated fatty acids, acyl-carnitines, and sphingolipids. Emerging research suggests that RBCs may contribute to a precision medicine approach to sepsis and have diagnostic value in monitoring complement dysregulation in COVID-19-sepsis and non-COVID sepsis as research indicates that complement activation products and viral antigens are present on RBCs in patients with COVID-19.

Ex vivo Vitamin D supplementation improves viscoelastic profiles in prostate cancer patients.

BACKGROUND: Increased risk of thromboembolic events is associated with prostate cancer, specifically linked to activation of tissue factor. Vitamin D has potential anticoagulant effects by the downregulation of tissue factor expression. OBJECTIVES: To evaluate the effects on clot formation, the morphological and viscoelastic profiles of prostate cancer patients, before and after ex vivo supplementation of Vitamin D was studied. METHODS: Participants were recruited into a metastatic, non-metastatic and reference group. Whole blood samples were treated ex vivo with a dose of 0.5µg/kg Calcitriol. Clot kinetics were assessed using Thromboelastography®. Morphology of the blood components were studied using scanning electron microscopy (SEM). RESULTS: Results from the Thromboelastography® and SEM indicated no major differences between the non-metastatic group before and after treatment compared to the reference group. The Thromboelastography® showed that the metastatic group had an increased viscoelastic profile relating to a hypercoagulable state. Visible changes with regards to platelet activation and fibrin morphology were demonstrated with SEM analysis of the metastatic group. The viscoelastic and morphological properties for the non-metastatic group after treatment improved to be comparable to the reference group. CONCLUSION: Vitamin D supplementation may lead to a more favorable viscoelastic profile, with less dangerous clots forming.

Serum Metabolome Changes in Relation to Prothrombotic State Induced by Combined Oral Contraceptives with Drospirenone and Ethinylestradiol.

The association between hypercoagulability and use of drospirenone (DRSP) and ethinylestradiol (EE) containing combined oral contraceptives (COCs) is an important clinical concern. We have previously reported that the two formulations of DRSP combined with EE (namely, DRSP/20EE and DRSP/30EE) bring about a prothrombotic state in hemostatic traits of female users. We report here the serum metabolomic changes in the same study cohort in relation to the attendant prothrombotic state induced by COC use, thus offering new insights on the underlying biochemical mechanisms contributing to the altered coagulatory profile with COC use. A total of 78 healthy women participated in this study and were grouped as follows: control group not using oral contraceptives (n = 25), DRSP/20EE group (n = 27), and DRSP/30EE group (n = 26). Untargeted metabolomics revealed changes in amino acid concentrations, particularly a decrease in glycine and an increase in both cysteine and lanthionine in the serum, accompanied by variations in oxidative stress markers in the COC users compared with the controls. Of importance, this study is the first to link specific amino acid variations, serum metabolites, and the oxidative metabolic profile with DRSP/EE use. These molecular changes could be linked to specific biophysical coagulatory alterations observed in the same individuals. These new findings lend evidence on the metabolomic substrates of the prothrombotic state associated with COC use in women and informs future personalized/precision medicine research. Moreover, we underscore the importance of an interdisciplinary approach to evaluate venous thrombotic risk associated with COC use.