THROMBIN STIMULATES FIBROBLAST CHEMOTAXIS AND REPLICATION

The serine protease alpha-thrombin. a product of the circulating zymog en prothrombin, plays multiple roles in homeostasis and coagulation. D uring blood clotting, it is present within fibrin matrices and is like ly to be presented to local cell populations. It is known to be a fibr oblast mitogen, but its effects on fibroblast recruitment have not bee n assessed. Here we compared the effect of human alpha-thrombin on che motaxis and proliferation of human and rat skin fibroblasts and assess ed the mechanism of these actions. Fibroblast chemotaxis was assayed u sing a 48-well Boyden chamber and replication assessed by a spectropho tometric method, based upon the uptake and subsequent elution of methy lene blue by fibroblasts. Two fibroblast cell lines were used; fetal r at skin (FR) and newborn human foreskin (HS68). Human alpha-thrombin s timulated FR fibroblast chemotaxis over a wide range of doses (10(-12) M to 10(-7) M). Maximal migration was seen at 10(-10) M; 39 +/- 2.5 c ells/high power field (h.p.f.) compared with 19 +/- 3 cells/h.p.f. for media control. In the same assay platelet-derived growth factor, a we ll characterized fibroblast chemoattractant, caused a maximal stimulat ion of 44 +/- 5 cells/h.p.f. at a concentration of 3 x 10(-9) M. A sim ilar stimulation was observed with HS68 fibroblasts, although for this cell line maximal chemotaxis (190 +/- 12.5% of control) was seen at 1 0(-8) m thrombin. Fibroblast replication was optimal at 1.25 x 10(-9) m thrombin (134 +/- 4 and 127 +/- 5% of control for FR and HS68, respe ctively). Blocking of the alpha-thrombin proteolytic site with the hig hly specific inhibitor D-Phe-Pro-ArgCh2Cl abolished both the chemotact ic and mitogenic effect of thrombin on fibroblasts. These results show that proteolytically active alpha-thrombin induces both chemotaxis an d replication of fibroblasts in cell culture, suggesting one possible mechanism for fibroblast recruitment and proliferation at the site of tissue in jury.