Study On The Coagulation And Histopathology Induced By Agglutination In Vivo

Objective:Since the 20 th century, the influenza virus caused outbreaks and fatalities in human populations worldwide. There are about three million to five million serious flu cases worldwide each year. Among them the mortality is as high as 60%. The high mortality and the frequent emergence of new human avian influenza virus pose a great threat to human health. And the variation of HA, is a key factor to cause the subtype of the influenza epidemic. Severe infection can cause pathological damage and widespread serious complications, leading to secondary systemic multiple organ dysfunction(MODS), sepsis can appear in critically ill patients, resulting in microcirculation and micro thrombus formation etc. In the past studies, it is believed that the immune pathological damage caused by high viral load and excessive inflammatory reaction is the main pathogenic mechanism of influenza. But so far, there are still many problems such as pathogenesis and host response has not been clarified, so we need to in-depth and comprehensive study. HA localizes to the influenza virus surface and plays a key role in viral infectivity and pathogenesis, and has always been a research hot spot. HA is not only the important components of the influenza virus, but also exists in many bacteria, plants, and other pathogenic microorganism, and has the characteristics of aggregation of red blood cells. As is known to all, HA can cause agglutination of red blood cells in vitro, but the physiological significance of the effect of agglutination in vivo is also unknown. When HA reaches a certain concentration in the body, can cause agglutination, whether in vivo agglutination reaction in blood coagulation disorder in patients with severe secondary pathological injury, microcirculation, and micro thrombosis plays a non negligible role has not been reported yet. For a better understanding of the pathophysiology of flu, identify with the effect of agglutination, we induced agglutination reaction in the body, to explore the effects of coagulation and pathological damage,so as to provide new ideas for the diagnosis and treatment of severe infections.Methods:According to the dosage determination for HA to induce agglutination in vivo, which was determined by haemagglutination assay, the rat blood volume and proportion of red blood cells in the blood, the HA and inactivated HA was diluted to 5 mg/ml, 10 mg/ml and 20 mg/ml and 1.5 ml/100 g of them were injected into the tail vein of the Wistar rats respectively. 0.9% saline as the control group. Then observed agglutination under microscope(10min, 30 min, 2h, 4h, 6h, 12 h, and 24h) and determined hemolysis(30min) by colorimetric method. The 10 mg/ml of HA was used to perform time course experiments(1h, 2h, 4h, 6h, 12 h, 24h) for coagulation evaluation with fibrinogen(FIB),activated partial thromboplastin time(APTT), prothrombin time(PT), thromboplastin time(TT) and International Normalized Ratio(INR), the HA and inactivated HA(5mg/ml, 10 mg/ml, 20 mg/ml) and 0.9% saline groups were used to perform dosage course experiments for coagulation and pathological change by Haematoxylin and eosin(HE).Results:Viewed microscopically, HA obviously induced agglutination and hemolysis, with the increase of HA concentration, the agglutination and hemolysis was enhanced, and the rats showed abnormal behavior including piloerection, arching, curling up, shaking, poor response, and less activity, tachypnea, quadriplegia and even death. In terms of blood coagulation indexes, with HA concentration of 5mg/ml, 10 mg/ml, 20 mg/ml, the serum levels of FIB were(1.25±0.06)g/L,(0.83±0.16)g/L and(0.55±0.23) g/L,which were significantly lower than the control group(1.95 ± 0.29)g/L,(all P<0.05). In the experimental groups with HA 10 mg/ml and 20 mg/ml, the serum levels of PT, TT, APTT and INR were:(11.48 ± 1.43) s,(63.85±8.13)s,(23.62 ±4.06)s,(0.95±0.10)and(14.83±1.00)s,(95.63±2.50)s,(44.50±4.63)s,(1.21±0.11)respecti vely; they were significantly higher than those in control group(9.88 ±0.81)s,(47.97±4.82)s,(17.13±2.14)s and(0.80±0.06)(all P<0.05). In the lungs, there were obvious congestion, edema and inflammatory cell infiltration, the severity were associated with the concentration of HA. The kidney of 20mg/ml HA group were peritubular vessels congestion, hyperemia, the liver of 20mg/ml HA group were hemorrhagic changes, hepatocytes degeneration and liver cells arranged loosely, irregular.No detectable hemolysis and agglutination occurred either in 0.9% saline control group or inactivated HA groups. The inactivated HA groups compared with control, there were no significant differences in the serum levels of coagulation(FIB, PT, TT, APTT, INR) and there was no pathological change in lung, liver, kidney.Conclusions:In the experiment of agglutination in vivo induced by HA, and the responses were dose related, as the concentration of HA increased, agglutination and hemolysis phenomenon gradually enhanced, and the rats had abnormal biological performance and death phenomenon. At the same time, coagulation and fibrinolytic system is activated and accompanied by multiple organ injury and pathological change. Therefore, we inferred that the agglutination in vivo induced by hemagglutinin is significance for the pathogenic mechanism and pathological physiology.