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.

Purification of mast cell proteases from murine skin.

Different subpopulations of mast cells are characterized by their abundant contents of either tryptase or in addition chymase. These two neutral proteases are found in mast cells and may thus hold a key to the understanding of mast cell dependent reactions. Such studies are however hampered by the lack of readily available supplies of chymase. We have therefore studied the simultaneous purification of both proteases from hairless moro hr/hr mouse skin, using a sequence of salt extractions and chromatographic separations. After three steps of extraction, a 13-fold purification with an 82% yield was obtained for tryptase and a 15-fold purification with a 90% yield for chymase. Further one step purification on conventional sephadex, sephacryl and octyl sepharose columns was unsatisfactory because of further protein contamination of the fractions. Heparin affinity chromatography caused a high loss of tryptase and residual protein contamination. Gradient elution on a benzamidine sepharose 6B column resulted however in a single, low yield (17.9%) tryptase peak and a broader, high yield (>90%) chymase peak, and a 34% yield high purity fraction. The proteases thus purified exhibited their typical inhibitor profile. On Western blot analysis and on autoradiography in the presence of the serine protease inhibitor diisopropylfluorophosphate (DFP), only one 28 kD molecule with chymase activity was identified, whereas a broad 32-38 kD band of tryptase monomers was noted. Taken together, these data show that, after salt extraction and a single benzamidine affinity chromatography step, both mast cell chymase and tryptase can be separated and in case of chymase also highly purified, allowing thus for the study of biological activities of this molecule.