Anti-thrombin activity in cultured aortic and capillary endothelial cells: binding, internalization and degradation of thrombin

Abstract

Thrombin (Th) binds specifically to confluent cultures of adult bovine aortic (ABAE) and bovine brain capillary (BBC) endothelial cells. Saturation of 125I-Th binding is observed after 1 h exposure to the ligand and at an extracellular concentration of 0.5 and 1.0 microgram/ml for ABAE and BBC cells, respectively. Under optimal conditions both ABAE and BBC cultures bind about 2 to 5 ng/10(6) cells, which represents about 20% of Th binding to bovine corneal endothelial (BCE) cells. Under optimal conditions less than 30% of the total cell associated 125I-Th is internalized in ABAE and BBC cells, while in BCE cells the extent of internalization is more than 50%. The internalized 125I-Th is degraded both in ABAE and BBC cells as previously demonstrated in BCE cells. As analyzed by SDS-PAGE, 17%, 22% and 77% of the bound 125I-Th is in complex with anti-thrombins (anti-Ths) in BBC, ABAE and BCE cultures, respectively. ABAE cells possess 3 types of complexes, one which appears only on the cell surface with a molecular weight of 78 kDa, and two others which appear only in the conditioned medium (CM) with molecular weights of 84 and 85 kDa. BBC and BCE cells demonstrate only one type of complex with a molecular weight of 77 kDa which appears both on the cell surface and in the CM. The 125I-Th 77 kDa complex formed in the CM of BCE cells is recognized and bound by BBC cells and ABAE cells.(ABSTRACT TRUNCATED AT 250 WORDS)