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.

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.

The effect of physiological levels of South African puff adder (Bitis arietans) snake venom on blood cells: an in vitro model.

A significant burden of illness is caused globally by snakebites particularly by the puff adder, Bitis arietans. Presently there is no reliable and rapid method to confirm envenomation on blood chemistry; although coagulation parameters like prothrombin time, partial thromboplastin time, international normalized ratio and also serum electrolytes are tested. Here, we found that direct in vitro exposure of physiological relevant whole venom levels to human healthy blood (N = 32), caused significant physiological changes to platelet activity using a hematology analyzer, and measuring occlusion time, as well as lyses time, with the global thrombosis test (GTT). Disintegrated platelets were confirmed by scanning electron microscopy (SEM). We also confirmed the pathologic effects on erythrocytes (RBCs) (visible as eryptotic RBCs), by looking at both light microscopy and SEM. Thromboelastography showed that no clot formation in whole blood could be induced after addition of whole venom. We propose further clinical studies to investigate the use of light microscopy smears and hematology analyzer results immediately after envenomation, as a possible first-stage of clinical confirmation of envenomation.

Acute induction of anomalous and amyloidogenic blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide.

It is well known that a variety of inflammatory diseases are accompanied by hypercoagulability, and a number of more-or-less longer-term signalling pathways have been shown to be involved. In recent work, we have suggested a direct and primary role for bacterial lipopolysaccharide (LPS) in this hypercoagulability, but it seems never to have been tested directly. Here, we show that the addition of tiny concentrations (0.2 ng l(-1)) of bacterial LPS to both whole blood and platelet-poor plasma of normal, healthy donors leads to marked changes in the nature of the fibrin fibres so formed, as observed by ultrastructural and fluorescence microscopy (the latter implying that the fibrin is actually in an amyloid ß-sheet-rich form that on stoichiometric grounds must occur autocatalytically). They resemble those seen in a number of inflammatory (and also amyloid) diseases, consistent with an involvement of LPS in their aetiology. These changes are mirrored by changes in their viscoelastic properties as measured by thromboelastography. As the terminal stages of coagulation involve the polymerization of fibrinogen into fibrin fibres, we tested whether LPS would bind to fibrinogen directly. We demonstrated this using isothermal calorimetry. Finally, we show that these changes in fibre structure are mirrored when the experiment is done simply with purified fibrinogen and thrombin (±0.2 ng l(-1) LPS). This ratio of concentrations of LPS : fibrinogen in vivo represents a molecular amplification by the LPS of more than 10(8)-fold, a number that is probably unparalleled in biology. The observation of a direct effect of such highly substoichiometric amounts of LPS on both fibrinogen and coagulation can account for the role of very small numbers of dormant bacteria in disease progression in a great many inflammatory conditions, and opens up this process to further mechanistic analysis and possible treatment.

Erythrocytes and their role as health indicator: Using structure in a patient-orientated precision medicine approach.

The relevance of erythrocyte light microscopy analysis (a well-known haematological method) is under the spotlight, however there is a place for innovative electron microscopy, (together with biochemical markers) in a pathology laboratory. Inflammation is a key indicator of the health status and erythrocytes are extremely sensitive to oxidative stress or cytokine upregulation, which typically accompany systemic inflammation in most diseases. They are probably the most adaptable cells, and due to their short lifespan, may form a vital indicator of health, and could play a central part in tracking disease and treatment. As the NIH is proposing a precision medicine approach and because individualised medicine should form an essential part in diagnosis and treatment, biophysical combined with biochemical analysis of erythrocytes may be a novel method to track the inflammatory status before and after treatment. This will allow a fully individualised patient orientated precision medicine approach, where one-medication-regime-fits-all is no longer appropriate.

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.