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 mitogenic responses of different cell types involved in wound healing via binding to and cleavage of the thrombin receptor. In order to further explore these aspects of thrombin function, human keratinocytes (HaCaT cell line) were examined for their potential mitogenic responsiveness to thrombin and for the dependency of this process on the expression of the high-affinity thrombin receptor. Quiescent keratinocytes were stimulated in the mitogenic assay with alpha-thrombin and the thrombin receptor activating peptides TRAP42-55 (SFLLRNPNDKYEPY) and TRAP42-46 (SFLLR). A strong induction of cell proliferation was noted with alpha-thrombin, TRAP42-55 and TRAP42-46, 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 activation 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 immunohistochemistry and in situ hybridization. These data demonstrate the expression and biologic function of the human thrombin receptor on human keratinocytes, suggesting that thrombin, among other mediators, plays an important part in the orchestration of epidermal growth and repair processes.