Type B Transforming Growth Factor in Human Platelets : Release during Platelet Degranulation and Action on Vascular Smooth Muscle Cells

A specific radioimmunoassay for type beta transforming growth factor (TGF-/3) was developed and used to show that human platelets treated with thrombin release TGF-~ as a consequence of degranulation. The thrombin concentrations required to induce release of TGF-t3 parallel those concentrations that release the alpha-granule marker, beta-thromboglobulin. Related studies showed that TGF-/3 acts on early passage, explant cultures of bovine aortic smooth muscle cells by inhibiting the effect of mitogens on proliferation of subconfluent cell monolayers yet synergizing with mitogens to stimulate growth of the same cells when cultured in soft agar. The results show that primary cultures of bovine aortic smooth muscle cells and established normal rat kidney cells behave similarly with regard to TGF-~3 action. Moreover, the data suggest that platelet-mediated proliferation of aortic smooth muscle cells in vivo may not result solely from the stimulatory effect of plateletderived growth factor (PDGF), but rather from an interaction of platelet factors which has the intrinsic ability to limit as well as stimulate mitosis. T HE rate of fibroblast and smooth muscle cell division is controlled, in large part, by the action of peptide growth factors present in platelets. Physiologically, these peptides are released as a consequence of platelet degranulation at sites of injury; together with growth factors in plasma, they stimulate mitosis of connective tissue cells (36, 37, 47). The best studied of these platelet peptides is plateletderived growth factor (PDGF), ~ a component of alpha-granules ( 16, 17) that is a chemoattractant (13, 4 l, 42) and mitogen (36, 37, 47) for fibroblasts and vascular smooth muscle cells. It is important that PDGF has also been implicated in the etiology of proliferative diseases. Expression of PDGF-like proteins is a common feature of many transformed cells (7) and the hallmark of transformation by simian sarcoma virus (l I, 52). Similarly, exposure of vascular smooth muscle ceils to PDGF (35) or PDGF-like proteins (10, 40, 43) may be an important component in the proliferation of these cells during atherogenesis. We and others have shown that PDGF is only one of several growth factors in human platelets (4-6, 9, 14, 26); type beta transforming growth factor (TGF-/~) can be purified (5) from platelets with yields similar to that of PDGF. TGF-/~ is a widely distributed growth factor in animal tissues (27), but platelets contain ~100-fold more TGF-fl than do the other cells or tissues examined to date (5). Consistent with the role of platelet factors as participants in wound repair, TGF-~ can Abbreviations used in this paper. " DME, Dulbeceo's modified Eagle's medium; EGF. epidermal growth factor; NRK, normal rat kidney; PDGF, plateletderived growth factor; ~-TGF, type 3 transforming growth factor. stimulate cell proliferation, protein synthesis, and collagen production in an in vivo wound healing system (46). A functional, epidermal growth factor (EGF)-like peptide is also present in human platelets (4, 6, 26), but lack of the purifed protein has precluded detailed studies of its structure and action. The initial in vitro studies with TGF-~ focused on its role in cellular transformation (28, 30); this peptide stimulates growth of non-neoplastic fibroblastic cells suspended in medium with 10% serum and agar (conditions of anchorageindependent growth). In particular, NRK fibroblasts grow efficiently in soft agar when both TGF-/3 and EGF (or TGFalpha, an EGF-like peptide) are added to the culture medium (1, 28, 30). AKR-2B fibroblasts require only the addition of TGF-/3 (50). Recent studies with TGF-/3 have demonstrated that this growth factor is actually a bifunctional regulator of cell proliferation; in addition to its stimulatory effects on anchorage-independent growth of mesenchymal cells, TGF-¢~ can inhibit the soft agar growth of many epithelial tumor cells and several fibroblastic cell lines (29, 49). In NRK cells, TGFinhibits monolayer growth of subconfluent cultures yet stimulates the growth of confluent monolayers and cells suspended in agar (29). The inhibitory effects of TGF-~ on monolayer cultures of NRK cells have not been observed in all studies (21), suggesting that culture conditions can alter the cellular response to TGF-#. The widespread use of subcloned cell lines has further complicated the interpretation of TGF-~ action. Moses et ai. (24) have recently studied the effects of TGF-~ on early The Journal of Cell Biology, Volume 102, April 1986 1217-1223 1217 on M ay 9, 2017 D ow nladed fom Published April 1, 1986