The thrombin receptor is a seven transmembrane G-protein-coupled receptor that is activated by thrombin cleavage. In order to characterise the thrombin receptor three mouse monoclonal antibodies were isolated by using a recombinant fusion protein incorporating the extracellular domain of the human thrombin receptor as an antigen.

An affinity matrix prepared from one monoclonal antibody was used in the successful purification of the native receptor from human platelet membranes. Two of the monoclonal antibodies were capable of blocking thrombin-induced platelet aggregation, and this may lead to their use as possible platelet thrombin receptor antagonists. To characterise further the monoclonals, their epitopes were mapped. In addition the cDNA encoding the antibodies variable regions were cloned and sequenced. By using this procedure, the amino acid sequence of the antibody complementarity determining regions (CDRs) involved in binding to the target epitope were determined.

The recently discovered human protease activated-2 receptor (hPAR-2) was studied using rabbit polyclonal antibodies generated with a multiple antigenic peptide comprising a region around the protease cleavage site. Immunocytochemistry and flow cytometry using affinity purified antibodies detected expression of hPAR-2 on human umbilical vein endothelial cells, keratinocytes and granulocytes. The expression of both PAR-2 and thrombin receptor were analysed using Northern analysis in a wide variety of murine haematopoietic cell lines. This analysis revealed the widespread distribution of the thrombin receptor. In contrast expression of PAR-2 was not observed in haematopoietic cells, suggesting that it is a marker of terminal differentiation in granulocytes. To identify potential activators of PAR-2, a variety of serine proteases were tested using a chloromethylketone inhibitor based on the peptide sequence of the cleavage site. These experiments revealed three potential trypsin-like enzymes that may activate PAR-2 in vivo namely, pancreatic trypsin, mast-cell tryptase and acrosin.

Thrombin is a serine protease that plays a central role in blood coagulation. Its specificity is determined by interactions with both the active site and a region distant from the active site called the anion-binding exosite. A surface loop from the thrombin anion-binding exosite, involved in binding to the hirudin-like domain of the thrombin receptor was grafted onto a recombinant single chain antibody using protein engineering. Molecular modelling indicated that incorporation of this loop into a framework region of the well-characterised anti-lysozyme antibody DI.3 would result in a chimeric molecule that would both bind its target antigen hen egg lysozyme, and also bind the platelet thrombin receptor through the engineered loop. The mutant antibody was unfortunately expressed as an incorrectly folded protein.