Characterisation of a prothrombinase activator on the hepatoma derived cell-line, PLC/PRF/5.

Abstract

The serine protease thrombin possesses multiple biological activities of which the central role is perceived to be in maintaining haemostasis by promoting blood coagulation and platelet aggregation [ I ] . Thrombin's ability to elicit responses from cells other than the haematopoietic lineage or constituents of vascular walls, e.g. neuroblasts and osteoblasts are thought to be mediated by a novel receptor for thrombin [2,3]. Previous work from our laboratory strongly suggests that several hepatomaderived cell lines express functional copies of this thrombin receptor on their cell surfaces. Thus insights into the way that thrombin activates cells are being unraveled yet the way in which thrombin is delivered to these cells remains obscure. In plasma, the zymogen prothrombin is converted to the twochain active form by the enzyme prothrombinase (factors Xa, Va, anionic phospholipids and Ca ions). Although the main site of prothrombin synthesis is the liver, i t is also expressed in other cells [4]. Therefore, even when a cell is not in contact with blood, i t may have prothrombin in the vicinity, but the prothrombin is unable to be converted to thrombin. Since a number of cell types that possess thrombin receptor are spatially separated from the bloodstream, at least under physiological conditions. How do these cells encounter thrombin? One attractive hypothesis is the existence of an alternative pathway for prothrombin activation that is independent of the coagulation cascade. We herein report that a novel membrane -associated protease capable of activating prothrombin is present on the hepatoma-derived cell line, PLC/PRF/5. This protease can directly convert prothrombin to active thrombin which had fibrinogen converting activity similar to that of authentic a-thrombin. The activation of prothrombin had an absolute requirement for Caions in the physiological range i.e. millimolar and was inhibited b! EDTA (Fig I ) .