Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate.

Abstract

Abstract. Studies with various thrombin derivativeshave shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selected antibodies that blocked the binding of t25I-thrombin to high affinity receptors on hamster fibroblasts. One of these antibodies, TR-9, inhibits from 80 to 100% of t25I-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubilized thrombin receptors. By itself, TR-9 did not initiate DNA synthesis nor did it block thrombin initiation, but TR-9 addition to cells in the presence of alpha-thrombin, gammathrombin (0.5 gg/ml), or PMA stimulated thymidine incorporation up to threefold over controls. In all cases, maximal stimulation was observed at concentrations of TR-9, ranging from 1 to 4 nM corresponding to concentrations required to inhibit from 30 to 100% of ~25I-thrombin binding. These results demonstrate that the binding of the monoclonal antibody to the alpha-thrombin receptor can mimic the effects of thrombin's high affinity interaction with this receptor in stimulating cell proliferation.

Mouse fibroblasts defective in thrombin mitogenesis possess functional proteolytically activated receptor for thrombin: Requirement for a second signaling pathway

“…Interestingly, much of this binding appears as a punctate pattern suggesting the aggregation of PART or association of PART with multicomponent receptor clusters. This pattern is similar to that identified by direct thrombin binding to fibroblasts (Carney, 1983;Carney and Bergmann, 1982), or the binding of thrombin receptor monoclonal antibody TR-9 (Frost et al, 1987). In parallel experiments, preincubation of this antibody with TRP-14 diminishes the apparent intensity of this stain-…”

“…4). It should be noted that the size of this component corresponds to the predicted molecular mass of the receptor with approximately 10% of its mass accounted for by glycosylation (Vu et al, 1991), t o the size of the original thrombin receptor component identified by photoaffinity crosslinking with active a-thrombin (Carney et al, 19791, and to the size of the major component of the thrombin receptor complex identified in fibroblastic cells by immunoaffinity chromatography (Frost et al, 1987). Thus, it is clear that both B11-A and B11-B cells synthesize and express the thrombin receptor component protein.…”

“…It remains to be determined how high-affinity interaction of thrombin initiates a second set of signals or if this second set of signals involves a second thrombin-activated receptor. Initial identification of thrombin receptors using monoclonal antibodies and various photoaffinity labeling studies showed that thrombin may interact with at least two different sized molecules at the cell surface, and that these molecules may exist as a complex (Frost et al, 1987). Thus, it is possible that thrombin-mediated mitogenic signal production involves interaction with two receptor components.…”

“…These cell surface interactions appear to activate distinct sets of intracellular signals. For example, occupancy of high-affinity receptors on fibroblasts by either diisopropyl fluorophosphate (DIP)-inactivated a-thrombin Glenn et al, 1980;Gordon et al, 19861, wound-healing accelerating peptides derived from thrombin (Glenn et al, 1988;Carney et al, 19921, or receptor monoclonal antibodies (Frost et al, 1987) initiates a subset of signals, but proteolytic receptor cleavage or direct activation of protein kmase C is required to complete the proliferative response .…”

Enhancement of incisional wound healing and neovascularization in normal rats by thrombin and synthetic thrombin receptor-activating peptides.

“…Many of these cellular postclotting effects of thrombin appear to be mediated through interaction with high affinity receptors which have been identified on fibroblasts (37)(38)(39)(40), capillary endothelial cells (34), keratinocytes (McCaully, R. L., M. C. Robson, and D. H. Carney, unpublished observations), monocytes (24, 41), and neutrophils (42). A unique functional receptor for thrombin has now been cloned confirming the specificity and potential physiological significance of these interactions (43).…”