Doctoral Dissertations

Date of Award

12-1992

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Stephen J. Kennel

Committee Members

Eugene Rinchik, Larry Waters, Julian Preston, Steve Kennel

Abstract

Two rat monoclonal antibodies were previously developed that bind separate epitopes on a marine glycoprotein, P112, which is expressed primarily in the lung capillary endothelium. Partial amino acid and cDNA sequence analyses demonstrated a strong similarity between P112 and thrombomodulin (TM), an endothelial cell glycoprotein involved in clot regulation. A subtractive monoclonal antibody assay, followed by Western blot analysis using anti-TM antibodies coupled with a biochemical assay for TM, showed that P112 was indistinguishable from TM. Quantitative analysis of both protein and mRNA levels in different murine tissues showed that the lung contained seven times more TM/cm2 of vessel wall surface than did the kidney, whereas the liver displayed negligible amounts of TM. Analysis of eight different tissues suggests that TM levels are highest on continuous type endothelia, and much less prevalent on fenestrated and sinusoidal endothelia. Sequence analysis of the promoter region of the TM gene demonstrated a significant difference in two strains of inbred mice. One strain, BALB/cBD, contains an intracisternal-A particle (lAP) proviral DNA sequence upstream of the initiation codon while another strain (that used for the MUSTHROMBO sequence listed in Genbank) lacked this insertion. Examination of ten different strains of laboratory mice showed that five contained the insert while the rest did not. Northern blot analysis for TM mRNA levels in these strains, showed that there were different levels of TM expression in six-week-old mice, but these differences were not correlated with the presence or absence of the lAP proviral DNA. In adult mice, 70% of the total body levels of TM are found in the lung microvasculature. The other 30% is distributed in the vessels of other organs, except the brain and liver, which have no detectable TM. Immunohistochemical studies of developing BALB/cBD (containing the TAP proviral insertion) and A/J (lacking the provirus) embryos exhibited an indistinguishable pattern of TM expression. As early as Day 6.5 post coitum to mid-gestation, TM is located in high amounts in the parietal endoderm of the placenta. High levels of TM are also found in the lung bud epithelium, and the meninges of the brain and spinal cord, suggesting that TM may play a role in development that is different from the role of clot regulation in the adult vasculature.

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