Identification of a thrombin sequence with growth factor activity on macrophages

In contrast to fibroblasts, the exposure of G0/G1-arrested J774 cells, a murine macrophage-like tumor cell line, with either active or esterolytically inactive diisopropyl phosphorofluoridate-conjugated alpha-thrombin (the enzymatically active form of thrombin, EC 3.4.21.5) results in a mitogenic response as measured by increased [3H]thymidine incorporation. This response to thrombin is optimal at 10 nM and is specifically blocked by hirudin, a high-affinity thrombin inhibitor. When prethrombin 1 [a single-chain prothrombin derivative lacking fragment 1, resulting from the action of thrombin on prothrombin] is cleaved with cyanogen bromide, a fragment (peptide CB67-129) is produced that, like the parent thrombin molecule, is mitogenic for J774 cells but not for fibroblasts. Limited tryptic digests of this fragment retain the ability to stimulate macrophages--a function that can be mimicked by a synthetic tetradecapeptide homologue of CB67-129 (representing residues 367-380 of the human thrombin B chain sequence) but not by any of a series of well-known growth promoters, including platelet-derived growth factor, epidermal growth factor, nerve growth factor, and fibroblast epidermal growth factor, nerve growth factor, and fibroblast growth factor. The mitogenic effects of this peptide are not limited to J774 cells but can be expressed in other macrophage-like tumor cell lines, including P388D1, RAW, and PU5. In addition to increased [3H]thymidine incorporation, the synthetic B chain peptide stimulates cell proliferation as evidenced by a dose-dependent increase in total protein per culture well and cell number. We conclude that the thrombin molecule contains a macrophage growth factor domain that is separate and distinct from its active center. Thus, thrombin, in addition to its major role in hemostasis and thrombosis, may also have important functions in such basic processes as the inflammatory response and monocytopoiesis.