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(Circulation. 1996;94:2728-2734.)
© 1996 American Heart Association, Inc.

Articles

Expression of Kunitz Protease Inhibitor–Containing Forms of Amyloid ß-Protein Precursor Within Vascular Thrombi

Irene M. Lang, MD; Kenneth M. Moser, MD; Raymond R. Schleef, PhD

the Division of Pulmonary and Critical Care Medicine (I.M.L., K.M.M.), University of California at San Diego, and The Department of Vascular Biology (I.M.L., R.R.S.), the Scripps Research Institute, La Jolla, Calif.

Correspondence to Raymond R. Schleef, PhD, Department of Vascular Biology (VB-1), The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037.

Background 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 ß-protein precursor, AßPP) in the organized vascular occlusions harvested from patients with this disease.

Methods and Results Immunohistochemical analysis revealed intense immunoreactivity for AßPP 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 AßPP 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, AßPP₇₅₁ and AßPP₇₇₀). 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.

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

Key Words: thrombosis • amyloid • endothelium • polymerase chain reaction • pulmonary heart disease

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