Distribution and potential biologic function of the thrombin receptor PAR-1 on human keratinocytes

Thrombin has recently been shown not only to exert procoagulant activities, but also to induce mito - genic responses of different cell types involved in wound healing via binding to and cleavage of the thrombin re- ceptor. In order to further explore these aspects of thrombin function, human keratinocytes (HaCaT cell line) were examined for their potential mitogenic re- sponsiveness to thrombin and for the dependency of this process on the expression of the high-affinity thrombin receptor. Quiescent keratinocytes were stim- ulated in the mitogenic assay with α-thrombin and the thrombin receptor activating peptides TRAP 42-55 (SFLLRNPNDKYEPY) and TRAP 42-46 (SFLLR). A strong induction of cell proliferation was noted with α- thrombin, TRAP 42-55 and TRAP 42-46 , but not with the "scrambled" peptide (FSLLR). These findings confirm that keratinocytes express the thrombin receptor and that the sequence of the first two amino acids of the generated neo-N-terminus are important for the ac- tivation of the receptor. Using cDNA fragments of the 5' coding sequence of the receptor, Northern blot analysis confirmed that HaCaT keratinocytes express the thrombin receptor. Expression of the receptor was also detected on normal human keratinocytes by im- munohistochemistry and in situ hybridization. These data demonstrate the expression and biologic function of the human thrombin receptor on human keratino- cytes, suggesting that thrombin, among other media- tors, plays an important part in the orchestration of epidermal growth and repair processes.