Identification of Platelet Releasate Proteins that Bind to Mastoparan, a Peptide that Inhibits Shear-Induced TGF-β1 Activation,

Abstract 3271

Transforming growth factor β1 (TGF-β1) is a disulfide-bonded, 25 kD homodimeric protein produced by most cell types, including platelets, that functions as a cytokine in many physiologic and pathologic processes. Platelets contain 40–100 times more TGF-β1 than other cells and release it as an inactive large latent complex (LLC) comprised of TGF-β1 non-covalently associated with its latency-associated peptide (LAP), which is, in turn, disulfide-bonded to latent TGF-β binding protein 1 (LTBP-1). Thrombospondin-1 (TSP1), proteases, and reactive oxygen species have all been shown to activate TGF-β1 in vitro and a role for integrins in vivo has been inferred from studies of transgenic mice. Recently, we discovered that shear force can activate latent TGF-β1 released from platelets in vitro and that thiol-disulfide exchange contributes to shear-dependent TGF-β1 activation. A number of thiol isomerase enzymes that can catalyze thiol-disulfide exchange have been identified in platelets, including protein disulfide isomerase (PDI), ERp5, ERp57, ERp72, ERp44, ERp29, and TMX3. As shear-induced activation of TGF-β1 is partially thiol-dependent, we investigated if thiol isomerases can affect this process. Mastoparan is a non-thiol-containing wasp venom peptide known to inhibit the chaperone activity of PDI, ERp5, and perhaps other thiol isomerases. We recently showed that mastoparan, (INLKALAALAKKIL), inhibits stirring-induced TGF-β1 activation by more than 90% (100 μM; n=3, p=0.03), whereas no inhibition was observed with an inactive mastoparan-like control peptide (INLKAKAALAKKLL) at 100 μM (n=3, p=0.66). To identify the proteins that bind to mastoparan, either directly or indirectly, platelet releasates were chromatographed on a mastoparan affinity column prepared from N-hydroxysuccinimide Sepharose. Two control columns were employed: 1. unconjugated Sepharose, and 2. Sepharose conjugated with the mastoparan-like control peptide. Elution of bound proteins was achieved by increasing the NaCl concentration. Proteins identified by mass spectrometry as specifically binding to the mastoparan peptide column included LTBP-1, TGF-β1 precursor, clusterin, coagulation Factor V, multimerin-1, 14-3-3 protein zeta/delta, and α-actinin 4. These results were confirmed by immunoblotting. Furthermore, the thiol isomerases PDI, ERp5, ERp57, and ERp72 were all found to bind specifically to mastoparan as confirmed by immunoblotting. We conclude that mastoparan affinity chromatography identified a number of proteins in platelet releasates that may contribute to shear-induced TGF-β1 activation.