A cell growth factor derived from platelets

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Fibroblasts may be readily cultured in vitro in the presence of foetal calf serum, but this growth is slowed when serum is used which has been prepared from platelet poor plasma. The same is true of smooth muscle cells derived from the arterial wall and in both instances, addition of platelet release material results in rapid recovery of proliferative activity. The role of smooth muscle cell proliferation in the early stages of atheroma has been emphasized by some workers and others have pointed to the importance of platelet subendothelial interaction in the early response to intimal damage. We have studied the properties of platelet release proteins in an endeavour to characterize the active agent. Platelets from cattle, goats, rats and man have all been shown to release a cell proliferation factor following activation by thrombin. Its activity has been demonstrated by stimulation of DNA synthesis in chick embryo fibroblasts arrested in 0.1% foetal calf serum, or by stimulation of contact inhibited cultures of a Balb C 3T3 cell line in serum-free medium. Stimulation ranges in the former to 8 × control values and in the latter, up to 100 × following a latent period of 12–16 hours. Earlier stimulation of 3H uridine incorporation into RNA and of 3H leucine into protein has also been shown following exposure of 3T3 cells to the platelet growth factor. The biological activity is stable to boiling at pH 5.5, and the factor may be frozen and thawed; it is precipitated by 80% acetone. It is non-dialysable and does not pass through an ultrafiltration membrane retaining molecules > 10,000 Daltons. The active principle is absorbed to CM Sephadex at pH7 0.2M and hence is likely to be strongly basic. Gradient elution with NaCl gives a sharp band of activity at 2.5 to 3.5M. SDS electrophoresis of the active fraction coincides with appearance ofa band of M.W. approximately 30,000 Daltons. This was seen with material of both human and bovine origin. The main peak of the CM Sephadex column eluate has the property of increasing capillary permeability to Evans blue in rats, but unabsorbed material from the CM Sephadex column had no such activity. It is likely, therefore, that a second basic protein released from platelets may contribute to the ‘Evans blue’ lesion of endothelial damage. Chemically, the growth factor bears a close resemblance to platelet factor IV. Further purification of this and the permeability factor may provide the basis of a new approach to the study of the role of platelets and platelet activation in the genesis of the early lesion of atheroma.