Etheresia Pretorius

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 patients 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.

Substantial fibrin amyloidogenesis in type 2 diabetes assessed using amyloid-selective fluorescent stains

BackgroundWe have previously shown that many chronic, inflammatory diseases are accompanied, and possibly partly caused or exacerbated, by various coagulopathies, manifested as anomalous clots in the form of ‘dense matted deposits’. More recently, we have shown that these clots can be amyloid in nature, and that the plasma of healthy controls can be induced to form such clots by the addition of tiny amounts of bacterial lipopolysaccharide or lipoteichoic acid. Type 2 diabetes (T2D) is also accompanied by raised levels of LPS.MethodsWe use superresolution and confocal microscopies to investigate the amyloid nature of clots from healthy and T2D individuals.ResultsWe show here, with the established stain thioflavin T and the novel stains Amytracker™ 480 and 680, that the clotting of plasma from type 2 diabetics is also amyloid in nature, and that this may be prevented by the addition of suitable concentrations of LPS-binding protein.ConclusionThis implies strongly that there is indeed a microbial component to the development of type 2 diabetes, and suggests that LBP might be used as treatment for it and its sequelae.

To What Extent Are the Terminal Stages of Sepsis, Septic Shock, Systemic Inflammatory Response Syndrome, and Multiple Organ Dysfunction Syndrome Actually Driven by a Prion/Amyloid Form of Fibrin?

A well-established development of increasing disease severity leads from sepsis through systemic inflammatory response syndrome, septic shock, multiple organ dysfunction syndrome, and cellular and organismal death. Less commonly discussed are the equally well-established coagulopathies that accompany this. We argue that a lipopolysaccharide-initiated (often disseminated intravascular) coagulation is accompanied by a proteolysis of fibrinogen such that formed fibrin is both inflammatory and resistant to fibrinolysis. In particular, we argue that the form of fibrin generated is amyloid in nature because much of its normal α-helical content is transformed to β-sheets, as occurs with other proteins in established amyloidogenic and prion diseases. We hypothesize that these processes of amyloidogenic clotting and the attendant coagulopathies play a role in the passage along the aforementioned pathways to organismal death, and that their inhibition would be of significant therapeutic value, a claim for which there is considerable emerging evidence.

Both lipopolysaccharide and lipoteichoic acids potently induce anomalous fibrin amyloid formation: assessment with novel Amytracker™ stains

In recent work, we discovered that the presence of highly substoichiometric amounts (10−8 molar ratio) of lipopolysaccharide (LPS) from Gram-negative bacteria caused fibrinogen clotting to lead to the formation of an amyloid form of fibrin. We here show that the broadly equivalent lipoteichoic acids (LTAs) from two species of Gram-positive bacteria have similarly (if not more) potent effects. Using thioflavin T fluorescence to detect amyloid as before, the addition of low concentrations of free ferric ion is found to have similar effects. Luminescent conjugated oligothiophene dyes (LCOs), marketed under the trade name Amytracker™, also stain classical amyloid structures. We here show that they too give very large fluorescence enhancements when clotting is initiated in the presence of the four amyloidogens (LPS, ferric ions and two LTA types). The staining patterns differ significantly as a function of both the amyloidogens and the dyes used to assess them, indicating clearly that the nature of the clots formed is different. This is also the case when clotting is measured viscometrically using thromboelastography. Overall, the data provide further evidence for an important role of bacterial cell wall products in the various coagulopathies that are observable in chronic, inflammatory diseases. The assays may have potential in both diagnostics and therapeutics.

Lipopolysaccharide-binding protein (LBP) reverses the amyloid state of fibrin seen in plasma of type 2 diabetics with cardiovascular co-morbidities

Type 2 diabetes (T2D) has many cardiovascular complications, including a thrombotic propensity. Many such chronic, inflammatory diseases are accompanied (and may be exacerbated, and possibly even largely caused) by amyloid fibril formation. Recognising that there are few strong genetic associations underpinning T2D, but that amyloidogenesis of amylin is closely involved, we have been seeking to understand what might trigger the disease. Serum levels of bacterial lipopolysaccharide are raised in T2D, and we recently showed that fibrin(ogen) polymerisation during blood clotting can be affected strongly by LPS. The selectivity was indicated by the regularisation of clotting by lipopolysaccharide-binding protein (LBP). Since coagulopathies are a hallmark of T2D, we wondered whether they might too be caused by LPS (and reversed by LBP). We show here, using SEM and confocal microscopy, that platelet-poor-plasma from subjects with T2D had a much greater propensity for hypercoagulability and for amyloidogenesis, and that these could both be reversed by LBP. These data imply that coagulopathies are an important feature of T2D, and may be driven by ‘hidden’ LPS. Given the prevalence of amyloid formation in the sequelae of diabetes, this opens up novel strategies for both the prevention and treatment of T2D.

Revisiting the safety of aspartame

Aspartame is a synthetic dipeptide artificial sweetener, frequently used in foods, medications, and beverages, notably carbonated and powdered soft drinks. Since 1981, when aspartame was first approved by the US Food and Drug Administration, researchers have debated both its recommended safe dosage (40 mg/kg/d) and its general safety to organ systems. This review examines papers published between 2000 and 2016 on both the safe dosage and higher-than-recommended dosages and presents a concise synthesis of current trends. Data on the safe aspartame dosage are controversial, and the literature suggests there are potential side effects associated with aspartame consumption. Since aspartame consumption is on the rise, the safety of this sweetener should be revisited. Most of the literature available on the safety of aspartame is included in this review. Safety studies are based primarily on animal models, as data from human studies are limited. The existing animal studies and the limited human studies suggest that aspartame and its metabolites, whether consumed in quantities significantly higher than the recommended safe dosage or within recommended safe levels, may disrupt the oxidant/antioxidant balance, induce oxidative stress, and damage cell membrane integrity, potentially affecting a variety of cells and tissues and causing a deregulation of cellular function, ultimately leading to systemic inflammation.

Lipopolysaccharide-binding protein (LBP) can reverse the amyloid state of fibrin seen or induced in Parkinson's disease

The thrombin-induced polymerisation of fibrinogen to form fibrin is well established as a late stage of blood clotting. It is known that Parkinson’s Disease (PD) is accompanied by dysregulation in blood clotting, but it is less widely known as a coagulopathy. In recent work, we showed that the presence of tiny amounts of bacterial lipopolysaccharide (LPS) in healthy individuals could cause clots to adopt an amyloid form, and this could be observed via scanning electron microscopy (SEM) or via the fluorescence of thioflavin-T. This could be prevented by the prior addition of lipopolysaccharide-binding protein (LBP). We had also observed by SEM this unusual clotting in the blood of patients with Parkinson’s Disease. We hypothesised, and here show, that this too can be prevented by LBP in the context of PD. This adds further evidence implicating inflammatory microbial cell wall products as an accompaniment to the disease, and may be part of its aetiology. This may lead to novel treatment strategies in PD designed to target microbes and their products.

No effects without causes: the Iron Dysregulation and Dormant Microbes hypothesis for chronic, inflammatory diseases: Iron Dysregulation and Dormant Microbes

Since the successful conquest of many acute, communicable (infectious) diseases through the use of vaccines and antibiotics, the currently most prevalent diseases are chronic and progressive in nature, and are all accompanied by inflammation. These diseases include neurodegenerative (e.g. Alzheimers, Parkinsons), vascular (e.g. atherosclerosis, pre‐eclampsia, type 2 diabetes) and autoimmune (e.g. rheumatoid arthritis and multiple sclerosis) diseases that may appear to have little in common. In fact they all share significant features, in particular chronic inflammation and its attendant inflammatory cytokines. Such effects do not happen without underlying and initially ‘external’ causes, and it is of interest to seek these causes. Taking a systems approach, we argue that these causes include (i) stress‐induced iron dysregulation, and (ii) its ability to awaken dormant, non‐replicating microbes with which the host has become infected. Other external causes may be dietary. Such microbes are capable of shedding small, but functionally significant amounts of highly inflammagenic molecules such as lipopolysaccharide and lipoteichoic acid. Sequelae include significant coagulopathies, not least the recently discovered amyloidogenic clotting of blood, leading to cell death and the release of further inflammagens. The extensive evidence discussed here implies, as was found with ulcers, that almost all chronic, infectious diseases do in fact harbour a microbial component. What differs is simply the microbes and the anatomical location from and at which they exert damage. This analysis offers novel avenues for diagnosis and treatment.

Lipopolysaccharide-Binding Protein (LBP) Can Reverse The Amyloid State Of Fibrin Seen Or Induced In Parkinson's Disease: Implications For Its Aetiology

The thrombin-induced polymerisation of fibrinogen to form fibrin is well established as a late stage of blood clotting. In recent work, we showed that the presence of tiny amounts of bacterial lipopolysaccharide (LPS) could cause these clots to adopt an amyloid form, that could be observed via scanning electron microscopy (SEM) or via the fluorescence of thioflavin-T. This could be prevented by the prior addition of lipopolysaccharide-binding protein (LBP). We had also observed by SEM this unusual clotting in the blood of patients with Parkinson’s disease (PD). We here show that this too can be prevented by LBP, thereby implicating such inflammatory microbial cell wall products in the aetiology of the disease. This may lead to novel treatment strategies in PD designed to target microbes and their products.

Characterizing pathology in erythrocytes using morphological and biophysical membrane properties: Relation to impaired hemorheology and cardiovascular function in rheumatoid arthritis

The inflammatory burden of the complex rheumatoid arthritis (RA) disease affects several organ-systems, including rheological properties of blood and its formed elements. Red blood cells (RBCs) are constantly exposed to circulating dysregulated inflammatory molecules that are co-transported within the vasculature; and their membranes may be particularly vulnerable to the accompanying oxidative stress. In the current study, we investigate biophysical and ultrastructural characteristics of RBCs obtained from a cohort of patients using atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal microscopy (CM). Statistical analyses of AFM data showed that RA RBCs possessed significantly reduced membrane elasticity relative to that of RBCs from healthy individuals (P-value <0.0001). SEM imaging of RA RBCs revealed increased anisocytes and poikilocytes. Poikilocytes included knizocytes, stomatocytes, dacryocytes, irregularly contracted cells, and knot cells. CM imaging of several RA RBCs, spectrin, and band 3 protein networks portrayed the similar morphological profiles. Analyses of CM images confirmed changes to distribution of band-3 skeletal protein, a protein critical for gaseous exchange functions of the RBC and preventing membrane surface loss. Decreased membrane deformability impairs the RBCs capacity to adequately adapt its shape to navigate blood vessels, especially microvasculature, and this decrease is also reflected in the cells morphology. Changes to morphology and deformability may also indicate loss of functional domains and/or pathological protein and lipid associations. These findings suggest that RA disease and/or its concomitant factors impact on the RBC and its membrane integrity with potential for exacerbating pathological cellular function, hemorheology, and cardiovascular function.