Expression of Kunitz protease inhibitor--containing forms of amyloid beta-protein precursor within vascular thrombi

The presence of patent neovessels within vascular occlusions in chronic thromboembolic pulmonary hypertension suggests that local mechanisms exist to regulate the coagulation system. This study investigated the expression of a potent inhibitor of Factor IXa and Factor XIa (ie, protease nexin-2/ amyloid beta-protein precursor, A beta PP) in the organized vascular occlusions harvested from patients with this disease.

Immunohistochemical analysis revealed intense immunoreactivity for A beta PP in the single layer of cells that line the neovessels. A positive signal was also detected by in situ hybridization analysis with the use of a 35S-UTP-labeled antisense riboprobe that recognizes the various alternatively spliced mRNA forms of this molecule. To identify the forms of A beta PP produced within the thrombi, total RNA was extracted from the thrombi, reverse transcribed, and subjected to amplification with the use of the polymerase chain reaction (PCR) and primers that flank the region encoding the alternatively 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 PP770). In situ hybridization analysis with the use of a 35S-UTP-labeled antisense riboprobe complementary to the region encoding the KPI domain confirmed the presence of these mRNA species in nucleated cells lining the neovessels.

The expression of KPI-containing isoforms of A beta PP in thrombus endothelial cells may represent one mechanism utilized in this disease to shift the local hemostatic balance and preserve regional vessel patency.