Electron confocal and atomic force microscopic analysis of platelets fibrin and erythrocytes in atopic asthma

Abstract

Background: The underlying risk factor of non-communicable diseases is inflammation. The pathophysiology of asthma and/or allergic asthma is also, no surprisingly, also linked to chronic inflammation. The prevalence and incidence of asthma is on the increase, despite of a variety of treatment modalities. Asthma therefore is a growing burden on healthcare system and on society. Asthma pathophysiology is not only linked to inflammation, but also with activation of coagulation and reduced fibrinolysis due to plasma and locally derived factors in the airways. Alterations to these factors may thus influence the biophysical and mechanical properties of the coagulation, fibrin deposition followed by asthma pathology. Methodology: The objective of this study was to evaluate changes of blood components in asthma patients (n=30) compared to controls (n=30) using routine haematology screening using a haematology analyser; structural changes and axial ratio analysis using light microscopy (erythrocytes), elasticity using atomic force microscopy (erythrocytes); ultrastructural changes using scanning electron microscopy (erythrocytes, platelets and fibrin), transmission electron microscopy (platelets) and confocal microscopy (platelets) as well as the viscoelastic properties of the fibrin clot using thromboelastography; and mRNA levels of FXIII-A in whole blood by quantitative PCR. Results: The haematology findings of controls and asthma patients were within normal clinical ranges. There was, however, a significantly higher level of circulating monocytes (p=0.0066), erythrocytes (p=0.0004), haemoglobin (p=0.0057) and haematocrit (p=0.0049). The analysis of eosin stained erythrocytes by light microscopy showed more echinocytes, acanthocytes and ovalocytes compared to controls and the axial ratio was also significantly higher (controls: 1.2�0.22nm vs. asthma: 1.165�0.16nm, p<0.0001). Atomic force microscopy findings showed significantly reduced erythrocyte membrane elasticity (p=0.001). Morphological changes of erythrocytes were also noted from scanning electron microscopy. Platelet morphology and ultrastructure were qualitatively assessed using scanning electron microscopy, transmission electron microscopy and confocal microscopy and showed morphological changes indicative of platelet activation in asthma samples. In addition, the assessment of clot kinetics by thromboelastography also showed alteration with a tendency to produce stronger fibrin clots in asthma samples. The reaction time was higher (p<0.0001), alpha-angle was lower (p<0.0001), maximum rate of clot formation was higher (p<0.0001) � all indicative of a longer time for clotting to occur but the maximal amplitude which is indicative of clot strength and stability was significantly higher in asthma samples (p=0.0478). As the aggregation and cross-linking of fibrin fibres depends on factor XIII (FXIII), its excessive production may lead to alterations in fibrin polymerisation and crosslinking resulting in stronger fibrin clot formation and resistance to fibrinolysis. Densitometry analysis of scanning electron microscopy images of fibrin fibres showed a significantly lower variance in asthma samples (p<0.0001) indicative of more matt like structures compared to normal fibrin fibres in controls. The analysis of gene expression by qPCR revealed a significant 17.34-fold higher FXIII-A mRNA level in whole blood of asthma patients compared to controls indicating greater potential for de novo production of FXIII-A compared to the control group. These results would facilitate further research possibilities for identifying potential biomarkers in allergic asthma. These findings support the hypothesis of this study: enhanced coagulation may be attributed to the altered morphology and activation of platelets, erythrocyte and fibrin networks in asthma. Conclusion: In conclusion, altered erythrocyte and platelet morphology, excess production of FXIII-A, altered fibrin architecture and clot properties affects the coagulation profile in asthma, systemically. Further research is needed to extrapolate exact mechanism by which increased systemic coagulation contributes to the pathophysiology of the disease locally.