Thrombin peptide, TP508, induces differential gene expression in fibroblasts through a nonproteolytic activation pathway

Prior studies have shown that synthetic peptides representing the domain of thrombin responsible for high-affinity binding to fibroblasts stimulate ch emo tactic and cell proliferative signals through a nonproteolytic mechanis m. One of these peptides, TP508, has recently been shown to be chemotactic for neutrophils, to enhance collagen accumulation in wounds, to enhance rev ascularization of wounds, and to accelerate the healing of incisional and o pen wounds in normal animals and in animals with impaired healing To determ ine whether TP508 activates the proteolytically activated receptor for thro mbin (PARI), or the signals that are activated by PAR1, we treated human fi broblasts with TP508 and the PAR1-activating peptide, SFLLRNP, and analyzed the effects of these peptides on gene expression using differential displa y reverse transcriptase polymerase chain reaction. TP508 induces expression of a number of specific message fragments with short tyrosine kinase-like domains that are not induced by SFLLRNP. Sequencing full-length clones prep ared by Marathon extension of TP508-induced fragments revealed that among t he induced transcripts, there was a sequence with 88% homology to human ann exin V. Northern analysis with authentic annexin V cDNA confirms that TP508 , but not SFLLRNP, induces expression of annexin V in human fibroblasts. Th ese results demonstrate that TP508 activates a cellular response separate f rom that activated through PAR1 and supports the hypothesis that TP508 acts through a separate nonproteolytically activated thrombin receptor that may be responsible for high-affinity thrombin binding and for nonproteolytic s ignals that are required for thrombin stimulation of cell proliferation. (C ) 1999 Academic Press.