HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY In vitro and in vivo evidence for shear-induced activation of latent transforming

Transforming growth factor1 (TGF1) has potent physiologic and pathologic effects on a variety of cell types at subnanomolar concentrations. Platelets contain 40 times as much TGF1 as other cells and secrete it as an inactive (latent) form in complex with latency-associated peptide (LAP), which is disulfide bonded via Cys33 to latent TGFbinding protein 1 (LTBP-1). Little is known about how latent TGF1 becomes activated in vivo. Here we show that TGF1 released from platelets or fibroblasts undergoes dramatic activation when subjected to stirring or shear forces, providing a potential mechanism for physiologic control. Thioldisulfide exchange appears to contribute to the process based on the effects of thiol-reactive reagents and differences in thiol labeling of TGF1 before and after stirring or shear. Activation required the presence of LTBP, as TGF1 contained in complex with only LAP could not be activated by stirring when studied as either a recombinant purified protein complex or in the platelet releasates or sera of mice engineered to contain an LAP C33S mutation. Release and activation of latent TGF1 in vivo was demonstrated in a mouse model 5 minutes after thrombus formation. These data potentially provide a novel mechanism for in vivo activation of TGF1. (Blood. 2008;112:3650-3660)