Evaluation of plasma alpha-2-macroglobulin and interactions with tumour necrosis factor-alpha in horses with endotoxemic signs.

The electrophoretic position and behavior of the native and activated forms of equine plasma alpha-2-macroglobulin (alpha 2M) were characterized and compared to human alpha 2M by nondenaturing polyacrylamide-gel electrophoresis (PAGE). Plasma alpha 2M was also compared between 6 normal horses and 6 horses with clinical signs of colic and endotoxemia due to volvulus or enteritis. Native and activated forms of alpha 2M were quantified by PAGE and densitometry. Binding of radio-labeled recombinant human tumour necrosis factor-alpha (125I-rhTNF-alpha) to native and activated forms of equine alpha 2M was also evaluated by autoradiography and densitometry of PAGE. Equine plasma alpha 2M migrated as a single band at a position equivalent to native human alpha 2M. Methylamine-reacted equine plasma samples resulted in faster migration of alpha 2M in a similar position to activated human alpha 2M. However, in methylamine-reacted equine plasma, an intermediate alpha 2M band was consistently present between the bands corresponding to native and activated alpha 2M. Amounts of plasma alpha 2M were similar in normal and endotoxemic horses, and remained in the electrophoretically slow or unreacted native form. The vast majority of 125I-rHuTNF-alpha did not bind to alpha 2M or other equine plasma proteins. 125I-rHuTNF-alpha bound weakly to both native and fast methylamine-reacted equine forms of alpha 2M, although binding was better to the activated form. This study indicates that: (1) equine plasma alpha 2M behaves similarly to human alpha 2M on PAGE, (2) plasma alpha 2M of horses can be activated to electrophoretically fast forms, but it is neither activated nor depleted during endotoxemia, and (3) the binding interactions between equine alpha 2M and TNF-alpha are too low to implicate equine alpha 2M as a regulator of TNF-alpha during endotoxemia in horses.