DEMONSTRATION OF PLASMA PROTEINASE-INHIBITORS IN BETA-2-MICROGLOBULIN AMYLOID DEPOSITS

Beta-2-microglobulin-related amyloidosis (A-beta-2M) represents a frequent complication in long-term dialysis patients. Although the pathogenetic mechanism has yet to be fully understood, it is known that amyloid fibrils usually consist of intact molecules of beta-2-microglobulin (beta-2m). Plasma proteinase inhibitors (PPI) are a broad family of glycoproteins with the function of eliminating unwanted proteolysis of serine proteases. Their role in amyloidogenesis has become a subject of intense discussion, especially since the recent identification of alpha-1-antichymotrypsin in the beta-protein amyloid deposits of Alzheimer's disease. We evaluated immunohistochemically and biochemically the presence and distribution of several PPIs (alpha-1-proteinase inhibitor, alpha-1-antichymotrypsin, antithrombin III, alpha-2-macroglobulin and tissue inhibitor metalloproteinase) and amyloid P component in A-beta-2M deposits in osteo-articular and visceral tissues from dialysis patients with amyloidosis, as well as two carpal tunnel synovia from non-dialysis patients and one Alzheimer's brain as controls. The immunohistochemical study demonstrated that all but one (anti-alpha-1-antichymotrypsin) of the PPI antibodies tested showed varying degrees of positive reaction against A-beta-2M deposits. All the antibodies (including anti-alpha-1-antichymotrypsin) also reacted to some extent with other non-amyloid visceral and connective tissue elements diffusely and/or selectively. Among them, only the reaction of anti-amyloid P component had significantly distinctive localization to A-beta-2M deposits, which were identified in adjacent serial sections by Congo red staining and immunohistochemical reaction against anti-beta-2m. The biochemical analysis (SDS-PAGE and Western blot analysis) of the solubilized beta-2m-amyloid fibrils confirmed the results of the immunohistochemical study, demonstrating positive reactions with the antibodies directed against all of the tested PPIs. Furthermore, the Western blot analysis of alpha-1-ACT demonstrated the presence of one band of approximate molecular weight of 65 kDa, which comigrated with the band of the alpha-1-ACT standard sample. This discrepancy between the immunohistochemical and biochemical analyses findings on alpha-1-ACT, could be explained by the co-purification of alpha-1-ACT with the amyloid fibrils.