EXPRESSION OF KUNITZ PROTEASE INHIBITOR-CONTAINING FORMS OF AMYLOID BETA-PROTEIN PRECURSOR WITHIN VASCULAR THROMBI

Background The presence of patent neovessels within vascular occlusion s in chronic thromboembolic pulmonary hypertension suggests that local mechanisms exist to regulate the coagulation system. This study inves tigated the expression of a potent inhibitor of Factor IXa and Factor XIa (ie, protease nexin2/amyloid beta-protein precursor, A beta PP) in the organized vascular occlusions harvested from patients with this d isease. Methods and Results Immunohistochemical analysis revealed inte nse immunoreactivity for A beta PP in the single layer of cells that U ne the neovessels. A positive signal was also detected by in situ hybr idization analysis with the use of a S-35-UTP-labeled antisense ribopr obe that recognizes the various alternatively spliced mRNA forms of th is molecule. To identify the forms of A beta PP produced within the th rombi, total RNA was extracted from the thrombi, reverse transcribed, and subjected to amplification with the use of the polymerase chain re action (PCR) and primers that Bank the region encoding the alternative ly spliced 56-amino acid Kunitz-type protease inhibitor (KPI) domain. The major PCR products consisted of 255 bp and 312 bp and corresponded to transcripts encoding this domain (ie, A beta PP751 and A beta PP77 0). In situ hybridization analysis with the use of a S-35-UTP-laheled antisense riboprobe complementary to the region encoding the KPI domai n confirmed the presence of these mRNA species in nucleated cells lini ng the neovessels. Conclusions The expression of KPI-containing isofor ms of A beta PP in thrombus endothelial cells: may represent one mecha nism utilized in this disease to shift the local hemostatic balance an d preserve regional vessel patency.