Association of the metabolic syndrome with PAI 1act and clot lysis time over a 10-year follow up in an African population.

BACKGROUND AND AIMS: The association between the metabolic syndrome (MetS) and plasminogen activator inhibitor-1 (PAI-1) has been well established in cross-sectional studies. It is less clear whether this translates into decreased clot lysis rates and very little information is available on non-European populations. Little is known regarding prospective associations and whether clot lysis progressively worsens in MetS individuals over time. We determined the prospective association of MetS with PAI-1 activity (PAI-1act) and clot lysis time (CLT) over a 10-year period. METHODS AND RESULTS: As many as 2010 African men and women aged ≥30 years were stratified according to MetS status and number of MetS criteria (0-5). We also determined the contribution of the PAI-1 4G/5G polymorphism to these associations and identified which MetS criteria had the strongest associations with PAI-1act and CLT. Both PAI-1act and CLT remained consistently elevated in individuals with MetS throughout the 10-year period. PAI-1act and CLT did not increase more over time in MetS individuals than in controls. The 4G/5G genotype did not influence the association of PAI-1act or clot lysis with MetS. Increased waist circumference, increased triglycerides and decreased HDL-C were the main predictors of PAI-1act and CLT. CONCLUSIONS: Black South Africans with MetS had increased PAI-1act and longer CLTs than individuals without MetS. The inhibited clot lysis in MetS did, however, not deteriorate over time compared to controls. Of the MetS criteria, obesity and altered lipids were the main predictors of PAI-1act and CLT and are thus potential targets for prevention strategies to decrease thrombotic risk.

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.

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.

Effect of Progesterone and Synthetic Progestins on Whole Blood Clot Formation and Erythrocyte Structure.

Combined oral contraceptive (COC) use is a risk factor for venous thrombosis (VT) and related to the specific type of progestin used. VT is accompanied by inflammation and pathophysiological clot formation, that includes aberrant erythrocytes and fibrin(ogen) interactions. In this paper, we aim to determine the influence of progesterone and different synthetic progestins found in COCs on the viscoelasticity of whole blood clots, as well as erythrocyte morphology and membrane ultrastructure, in an in vitro laboratory study. Thromboelastography (TEG), light microscopy, and scanning electron microscopy were our chosen methods. Our results point out that progestins influence the rate of whole blood clot formation. Alterations to erythrocyte morphology and membrane ultrastructure suggest the presence of eryptosis. We also note increased rouleaux formation, erythrocyte aggregation, and spontaneous fibrin formation in whole blood which may explain the increased risk of VT associated with COC use. Although not all COC users will experience a thrombotic event, individuals with a thrombotic predisposition, due to inflammatory or hematological illness, should be closely monitored to prevent pathological thrombosis.

The Effect of Endogenous and Synthetic Estrogens on Whole Blood Clot Formation and Erythrocyte Structure.

As erythrocyte and estrogens interact so closely and erythrocytes can indicate the healthiness of an individual, it is essential to investigate the effects of natural estrogens as well as synthetic estrogens on these cells. Whole blood samples were used for thromboelastography (TEG), light microscopy (LM), and scanning electron microscopy (SEM) investigation. Viscoelastic investigation with TEG revealed that estrogens affected the rate of clot formation without any significant effect on the strength or stability of the clot. Axial ratio analysis with LM showed a statistically significant increase in number of erythrocytes with decreased roundness. Morphological analysis with SEM confirmed the change in erythrocyte shape and revealed both ultrastructural membrane changes and erythrocyte interactions. As erythrocyte shape and membrane flexibility correlates to physiological functioning of these cells in circulation, these changes, indicative of possible eryptosis brought on by estrogens, when experienced by individuals with an underlying inflammatory or hematological illness, could impair erythrocyte functioning and even result in obstructions in circulation. In conclusion, we suggest that whole blood analysis with viscoelastic and morphological techniques could be used as assessment of the hematological healthiness of individuals using estrogens.

Synthetic Hormones and Clot Formation.

Combined oral contraceptives (COCs), colloquially referred to as "the pill," have been regarded as a medical breakthrough, as they have improved the lives of countless women, from simplifying family planning to the treatment of acne, endometriosis, polycystic ovarian syndrome, and dysmenorrhea. Unfortunately, COC usage has been associated with an increased occurrence of venous thrombosis and therefore a systemic hypercoagulable state in susceptible females. Here we discuss the health risks of COC usage and use viscoelastic and morphological techniques to investigate the effect of different COC constituents on clot formation, particularly fibrin network packaging and whole blood viscoelasticity. Viscoelastic properties of whole blood showed gender-specific changes while morphological alterations were person-specific, regardless of gender. Using scanning electron microscopy and thromboelastography provides great insight regarding fibrin packaging and the development of a hypercoagulable state in high-risk individuals. We proposed a three-step approach where (1) an individual's coagulation profile baseline is determined, after which (2) the "ideal" combination of constituents is prescribed, and (3) the coagulation profile of the individual is monitored to assess possible risk of thrombosis. Only in following such an individualized patient-oriented approach will we be able to avoid the many health issues due to COC usage in susceptible females.

Erythrocyte-platelet interaction in uncomplicated pregnancy.

Maternal and fetal requirements during uncomplicated pregnancy are associated with changes in the hematopoietic system. Platelets and erythrocytes [red blood cells (RBCs)], and especially their membranes, are involved in coagulation, and their interactions may provide reasons for the changed hematopoietic system during uncomplicated pregnancy. We review literature regarding RBC and platelet membrane structure and interactions during hypercoagulability and hormonal changes. We then study interactions between RBCs and platelets in uncomplicated pregnancy, as their interactions may be one of the reasons for increased hypercoagulability during uncomplicated pregnancy. Scanning electron microscopy was used to study whole blood smears from 90 pregnant females in different phases of pregnancy. Pregnancy-specific interaction was seen between RBCs and platelets. Typically, one or more platelets interacted through platelet spreading and pseudopodia formation with a single RBC. However, multiple interactions with RBCs were also shown for a single platelet. Specific RBC-platelet interaction seen during uncomplicated pregnancy may be caused by increased estrogen and/or increased fibrinogen concentrations. This interaction may contribute to the hypercoagulable state associated with healthy and uncomplicated pregnancy and may also play a fundamental role in gestational thrombocytopenia.

A descriptive investigation of the ultrastructure of fibrin networks in thrombo-embolic ischemic stroke.

Stroke is one of the leading causes of death worldwide. Formation of a fibrin clot is controlled by a group of tightly regulated plasma proteases and cofactors and a change in the fibrin fiber formation causes an alteration in clot morphology. This plays an important role during thrombotic events. In the current study we investigated the ultrastructure of fibrin networks from fifteen ischemic stroke patients by using scanning electron microscopy. Clot morphology was investigated with and without the addition of human thrombin to the platelet rich plasma. Previously it was shown that, when studying the ultrastructure of fibrin networks, the addition of thrombin is necessary to form an expansive, fully coagulated layer of fibers. Results from the addition of thrombin to the plasma showed thick, matted fibrin fibers and a net covering some of the major fibers in stroke patients. Typical control morphology with major thick fibers and minor thin fibers could be seen in some areas in the stroke patients. In stroke patients, without the addition of thrombin, a matted fibrin network still formed, indicating that the factors responsible for the abnormal fibrin morphology are present in the circulating plasma and is the cause of the observed matted, layered morphology. This is not present in healthy individuals. From the results obtained we suggest that this changed morphology might be useful in a screening regime to identify the possibility of a stroke or even to follow the progress of stroke patients after treatment.

The effect of plant-based diets on thrombotic risk factors.

Plant­based diets are considered to improve cardiometabolic health and to protect against cardiovascular disease. Although they center around plant­based foods, they do not necessarily exclude all animal products and comprise of a range of intakes that vary according to the type and the proportion of animal products included. Numerous metabolic pathways have been identified through which plant­based diets can exert beneficial effects including improved body composition, lipid profile, and glucose metabolism and decreased inflammation and blood pressure. Their effects on thrombosis as a cardiovascular disease pathway are, however, less clear. Ample evidence for the effects of individual dietary components of plant­based diets on thrombotic risk factors exists, but the effect of whole diets and / or dietary patterns remains less­well explored with the existing literature reporting inconsistent and inconclusive findings. Here we aim to review the literature describing the effect of different plant­based diets (vegan, lacto­vegetarian, lacto­ovo­vegetarian, pescatarian, and flexitarian) and dietary patterns (Mediterranean, Nordic, Portfolio, and DASH) on specific thrombotic risk factors (fibrinogen, platelets, factor VII, fibrinolysis) in order to better clarify these relationships and to try to explain the apparent discrepant findings. We demonstrate that a one­size­fits-all conclusion cannot be drawn and that the potential antithrombotic effect of different plant­based diets depends on the nutrient composition, the content of active antithrombotic dietary components, the relative absence of prothrombotic dietary factors as well as the degree of total caloric restriction.

Blood clot parameters: Thromboelastography and scanning electron microscopy in research and clinical practice.

BACKGROUND: Clotting parameters are informative of overall haematological healthiness of an individual. Particularly, clotting parameters can be used as a measure of the degree of pathology of the coagulation system. Thromboelastography (TEG) is a well-known technique that is an important point-of-care method, as well as research method. Scanning electron microscopy (SEM) is a novel research method, but with possible clinical application. However, there are no clear standardized guidelines for TEG and SEM result interpretation. MATERIALS AND METHODS: We have an extensive database of results from TEG of hypercoagulable, hypocoagulable and healthy whole blood (WB) and platelet poor plasma (PPP). These results were generated using citrated PPP or WB, followed by the addition of CaCl2, to initiate clot formation. We also have an extensive and comprehensive database of thousands of clot micrographs, prepared for SEM. We reanalysed all our data to compile a user-friendly guideline for TEG and SEM. We also discuss the effects of different storage times on both WB and PPP. RESULTS: We provide a quick and informative guide that discusses each TEG parameter, in both WB and PPP. Increases or decreases in the various parameters are indicative of either hyper- or hypocoagulability. We also show how hypo- and hypercoagulable clots look like, compared with healthy clots, using SEM analysis of clots created by adding thrombin to PPP. CONCLUSION: For optimal and speedy interpretation of a patient's coagulation status, it is essential for the clinician to make an informed and precise decision regarding clotting propensity. We believe this guideline will add to the standardization of TEG parameters, and ultimately contribute to the treatment of patients. These guidelines will also allow researchers to standardize their data interpretations and ultimately allow for the use of a global and inclusive database that might be included in precision medicine approaches.

Clinical relevance of hypercoagulability and possible hypofibrinolysis associated with estrone and estriol.

Estrone (E1 ) and Estriol (E3 ) are endogenous female hormones, present in increased concentrations during female specific physiological processes (menopause and pregnancy respectively) that are associated with increased venous thrombotic risk. These hormones are also used as hormone therapies that are also associated with increased thromboembolism risk. Viscoelastic analysis revealed no significant difference to clot formation after hormone addition, however morphological analysis showed that the addition of both E1 and E3 result in fibrin clots composed of thinner fibrin fibers arranged in dense matted networks. These changes to the fibrin network ultrastructure are indicative of a prothrombotic state but may also indicate hypofibrinolysis. We therefore conclude that the increased risk of venous thrombosis during pregnancy and menopause may originate from a combination of hypercoagulation and a possible hypofibrinolytic mechanism of these hormones. Therefore females with a hypercoagulable tendency that fall pregnant or enter menopause need to be monitored to prevent venous thrombotic events. The decision to use hormone therapies during and after menopause should not be taken lightly and the risk-reward scale should be closely examined to ensure it does not tip towards thrombosis and subsequent thrombotic events that ultimately could have been prevented.

Clinical relevance of fibrin fiber diameter during different phases of pregnancy.

INTRODUCTION: Pregnancy-related deep vein thrombosis (DVT) is most common during the late phase of pregnancy and the first 6-weeks postpartum. Pregnancy-related DVT can have long-term complications, specifically post-thrombotic syndrome (PTS). Fibrin network ultrastructure is altered during pregnancy and post-partum. It is therefore essential to evaluate fibrin fiber diameter during and after pregnancy as this may provide insight into pregnancy-related DVT and subsequent PTS. MATERIALS AND METHODS: The fibrin network ultrastructure of females during different phases of pregnancy was compared to that of non-pregnant females to assess possible changes to the fibrin network morphology and fibrin fiber diameter using scanning electron microscopy micrographs. RESULTS: The fibrin network arrangement was more densely packed during different phases of pregnancy, corresponding to earlier findings. Fibrin diameter decreased significantly during pregnancy, with the greatest decrease occurring during the late phase of pregnancy. The fractal dimensions of fibrin micrographs increased significantly during pregnancy compared to nonpregnant females. These changes are indicative of a simultaneous hypercoagulable and hypofibrinolytic state and correspond to the increased risk of DVT and subsequent development of PTS. CONCLUSION: It is critical to identify "vulnerable" females with an inflammatory predisposition to prevent possible DVT and subsequent PTS. Modifiable risk factors like obesity and smoking should be addressed to alleviate the burden on the coagulation system. Morphological and viscoelastic techniques are crucial in assessing the coagulatory health of females during pregnancy.

The clinical relevance of altered fibrinogen packaging in the presence of 17ß-estradiol and progesterone.

BACKGROUND: The effect of endogenous hormone concentrations, specifically 17ß-estradiol and progesterone, on fibrin network formation has not been established. OBJECTIVES: It is essential to understand natural hormone mechanisms since these hormones are still present in circulation while hormonal contraceptives, which are associated with increased risk of venous thromboembolism, are used. METHODS: Due to the fact that these hormones are known to increase hypercoagulability and the prothrombotic state scanning electron microscopy (SEM), atomic force microscopy (AFM), thromboelastography (TEG) and turbidimetry were employed to investigate the morphology, surface roughness, viscoelastic properties and formation and lysis of fibrin. RESULTS: 17ß-estradiol and progesterone showed hypercoagulable viscoelastic properties and decreased the diameter and surface roughness of fibrin while increasing dense matted deposit occurrence. Our results suggest that the additional burden of hormonal load, together with the presence of endogenous estrogen and progesterone, may result in a prothrombotic and hypercoagulable state in females with an inflammatory predisposition. CONCLUSION: Our results are of clinical importance when considering hormones as either pathological agent or therapeutic intervention as will be assessed in future investigation.

Ultrastructural analysis of platelets during three phases of pregnancy: a qualitative and quantitative investigation.

OBJECTIVES: In the past, platelet morphology during normal pregnancy has not been given much attention. METHODS: Electron microscopy analysis of platelets from 60 pregnant individuals (30 early pregnancy (weeks 8-14) participants and 30 late pregnancy (weeks 36-40) participants which were followed up 6-8 weeks post-partum) were compared to platelets from 30 non-pregnant individuals as well as each other to establish whether differences in platelet morphology exist during pregnancy. RESULTS: Ultrastructural changes pertaining to the external and internal arrangements of platelets were visible. Fixated platelets showed pseudopodia formation and membrane blebbing. Increased and enlarged open canalicular system pores, pseudopodia formation, platelet spreading, and membrane blebbing were visible in vital platelets. Platelets from pregnancy were tightly packed and internal structures were different from the non-pregnant group. The internal granules showed modification in their occurrence within the cell. The α- and lysosomal granule counts were significantly increased during pregnancy while dense granule and mitochondrial numbers were significantly decreased. DISCUSSION: This may point to a pregnancy-specific modification. The changes in platelet ultrastructure discerned in this study attribute to the hypercoagulable state associated with pregnancy. All ultrastructural alterations associated with pregnancy persist for at least 2 months after birth.

Prevention and follow-up in thromboembolic ischemic stroke: Do we need to think out of the box?

Stroke is one of the most debilitating thrombotic diseases, and world-wide it is estimated that by, 2030, 23 million people will be affected. Except for the impact on the individual families, the world economy is also affected adversely. Although the medical treatment and knowledge of stroke are both increasing and well-researched, we still do not see a decrease in stroke prevalence. Currently various diagnostic tests are employed to determine the specific type of ischemic stroke as classified by the TOAST criteria. However, these tests are done after the stroke has occurred and therefore only contribute to the unquestionably crucial aspect of treating that particular stroke patient, but it does not improve prevention of future events. Prevention strategies regarding first-time stroke need urgent attention given the alarming present and future incidence of stroke. Therefore, here we discuss the importance of stroke prevention and suggest a more inclusive, perhaps "new" comprehensive approach for pre-stroke screening. Ultrastructural tests, particularly scanning electron microscopy, provide an innovative and novel advance in preventative and individualized patient-centered precision medicine. This precise technique when used in combination with well-established methods, as well as viscoelastic methods like thromboelastography (TEG), as a screening tool to prevent stroke can ultimately alleviate the financial and economical burden of stroke and also improve quality of life. Although we appreciate the fact that this suggestion might be difficult to accept by clinicians, a bold new approach is needed to address this pandemic we call stroke.

Viscoelasticity and Ultrastructure in Coagulation and Inflammation: Two Diverse Techniques, One Conclusion.

The process of blood clotting has been studied for centuries. A synopsis of current knowledge pertaining to haemostasis and the blood components, including platelets and fibrin networks which are closely involved in coagulation, are discussed. Special emphasis is placed on tissue factor (TF), calcium and thrombin since these components have been implicated in both the coagulation process and inflammation. Analysis of platelets and fibrin morphology indicate that calcium, tissue factor and thrombin at concentrations used during viscoelastic analysis (with thromboelastography or TEG) bring about alterations in platelet and fibrin network ultrastructure, which is similar to that seen in inflammation. Scanning electron microscopy indicated that, when investigating platelet structure in disease, addition of TF, calcium or thrombin will mask disease-induced alterations associated with platelet activation. Therefore, washed platelets without any additives is preferred for morphological analysis. Furthermore, morphological and viscoelastic analysis confirmed that thrombin activation is the preferred method of fibrin activation when investigating fibrin network ultrastructure.

Eryptosis as a marker of Parkinson's disease.

A major trend in recent Parkinson's disease (PD) research is the investigation of biological markers that could help in identifying at-risk individuals or to track disease progression and response to therapies. Central to this is the knowledge that inflammation is a known hallmark of PD and of many other degenerative diseases. In the current work, we focus on inflammatory signalling in PD, using a systems approach that allows us to look at the disease in a more holistic way. We discuss cyclooxygenases, prostaglandins, thromboxanes and also iron in PD. These particular signalling molecules are involved in PD pathophysiology, but are also very important in an aberrant coagulation/hematology system. We present and discuss a hypothesis regarding the possible interaction of these aberrant signalling molecules implicated in PD, and suggest that these molecules may affect the erythrocytes of PD patients. This would be observable as changes in the morphology of the RBCs and of PD patients relative to healthy controls. We then show that the RBCs of PD patients are indeed rather dramatically deranged in their morphology, exhibiting eryptosis (a kind of programmed cell death). This morphological indicator may have useful diagnostic and prognostic significance.

Part 2: ultrastructural changes of fibrin networks during three phases of pregnancy: a qualitative investigation.

INTRODUCTION: Normal pregnancy is characterized by significant alterations in the haemostatic system accompanied by an augmented risk of thrombosis. MATERIALS AND METHODS: The fibrin network ultrastructure of different phases of pregnancy, namely early pregnancy (week 8-14), late pregnancy (week 36-40) as well as post-partum (week 6-8 after birth) were compared with nonpregnant fibrin networks as well as each other to establish whether differences in fibrin network morphology exist during pregnancy. Scanning electron microscopy was employed to analyse fibrin network morphology. RESULTS: The fibrin networks from all phases of pregnancy appeared similar to each other, exhibiting prominent coagulant formation, an increase in the formation of minor, thin fibers, and the presence of granular globules. All three phases, however, differ from the typical fibrin network ultrastructure exhibited by the fibrin networks from nonpregnant individuals. The increase in estrogen associated with pregnancy may cause the increase in coagulation factors and ultimately the prothrombotic state characteristic of pregnancy. CONCLUSIONS: Since no differences were apparent between the different phases of pregnancy it suggests that activation of the coagulation system commences with pregnancy and this pro-thrombotic state continues till at least 8 weeks after birth. These results may shed light on possible pathological mechanisms employed in the development of abnormal or ailing pregnancy.

Estrogen causes ultrastructural changes of fibrin networks during the menstrual cycle: a qualitative investigation.

INTRODUCTION: Hormonal fluctuations may influence fibrin structure. During the menstrual cycle, plasma fibrinogen levels change, mainly due to the variations of estrogen. Throughout the menstrual cycle estrogen levels peak twice, first during the mid-follicular phase and then a lower second peak during the luteal phase. MATERIALS AND METHODS: In order to investigate the possible changes in the fibrin network throughout the menstrual cycle, the fibrin network ultrastructure of six healthy female participants were studied at different intervals in the menstrual cycle where differences in estrogen levels are prevalent. Blood plasma smears were prepared for scanning and transmission electron microscopy analysis. RESULTS: The external and internal structure of the fibrin fibers showed different morphologies throughout the menstrual cycle. The fibrin fibers were smooth during days 1-5. However, during days 12-14 of the menstrual cycle the fibrin fiber morphology started to change, becoming less smooth. During the luteal phase of the cycle (days 20-25), the network appears sticky, where the minor, thin fibers are more prominent between the thick fibers when compared to the menstrual phase. CONCLUSION: The two estrogen peaks of the menstrual cycle coincide with the changes seen in the current qualitative research, where the fibrin morphology changes during the same time as the estrogen peaks occur. Purified fibrinogen confirmed that it is indeed estrogen that causes the altered fibrin network morphology. This research is the first to show ultrastructural changes in fibrin fiber morphology resulting from estrogen changes during the menstrual cycle.