Transforming growth factor-␤1 (TGF-␤1) has potent physiologic and pathologic effects on a variety of cell types at subnanomolar concentrations. Platelets contain 40 times as much TGF-␤1 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 TGF-␤ binding protein 1 (LTBP-1). Little is known about how latent TGF-␤1 becomes activated in vivo.Here we show that TGF-␤1 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 TGF-␤1 before and after stirring or shear. Activation required the presence of LTBP, as TGF-␤1 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 IntroductionTransforming growth factor-␤1 (TGF-␤1) has potent physiologic and pathologic effects on a variety of cell types at subnanomolar concentrations, including cells of the immune and hematopoietic systems, as well as malignant cells and fibroblasts, 1 with the latter responding with increased collagen production leading to tissue fibrosis. 2 Nearly all cellular TGF-␤1 exists, however, in a biologically inactive (latent) form in a noncovalent complex with the remaining portion of its precursor molecule, latency-associated peptide (LAP), which is disulfide bonded to a latent TGF-␤ binding protein 3,or 4), forming the large latent complex (LLC). 3 Although much is known about the signaling mechanisms and downstream effects of TGF-␤1, 4 and several latent TGF-␤1 activating mechanisms have been identified in vitro (reviewed in Annes et al 3 ), little is known about the physiologic mechanisms that control latent TGF-␤1 activation in vivo. Recent data support activation of LLC TGF-␤1 via a traction mechanism, 3,[5][6][7][8][9][10][11] with LAP binding to integrins ␣V␤5, ␣V␤6, and ␣V␤8, and LTBP-1 binding to extracellular matrix.Blood platelets are a rich source of TGF-␤1, containing 40 to 100 times as much as other cells, 12 and releasing it when activated by a variety of agents, including thrombin, which is produced during blood clotting. [13][14][15][16][17][18][19] Virtually all of the TGF-␤1 released from platelets is in the LLC. 3,16 Because platelet latent TGF-␤1 is released into the circulation, and because traction on the molecule has been proposed as a mechanism of latent TGF-␤1 activation, 5,6 we hypothesized that intravascular shear force may serve as an analog of traction mediated by cellular contraction and contribute to the activation of latent TGF-␤1 released from platelets. We therefore tested this hypothesis by both in vitro and in vivo experiments. Methods Antibodies and reagentsAnti-TGF-␤1 (polyclonal chi...

Thrombospondin 1 (TSP-1), which is contained in platelet α-granules and released with activation, has been shown to activate latent TGF-β1 in vitro, but its in vivo role is unclear as TSP-1-null (Thbs1−/−) mice have a much less severe phenotype than TGF-β1-null (Tgfb1−/−) mice. We recently demonstrated that stirring and/or shear could activate latent TGF-β1 released from platelets and have now studied these methods of TGF-β1 activation in samples from Thbs1−/− mice, which have higher platelet counts and higher levels of total TGF-β1 in their serum than wild type mice. After either two hours of stirring or shear, Thbs1−/− samples demonstrated less TGF-β1 activation (31% and 54% lower levels of active TGF-β1 in serum and platelet releasates, respectively). TGF-β1 activation in Thbs1−/− mice samples was normalized by adding recombinant human TSP-1 (rhTSP-1). Exposure of platelet releasates to shear for one hour led to near depletion of TSP-1, but this could be prevented by preincubating samples with thiol-reactive agents. Moreover, replenishing rhTSP-1 to human platelet releasates after one hour of stirring enhanced TGF-β1 activation. In vivo TGF-β1 activation in carotid artery thrombi was also partially impaired in Thbs1−/− mice. These data indicate that TSP-1 contributes to shear-dependent TGF-β1 activation, thus providing a potential explanation for the inconsistent in vitro data previously reported as well as for the differences in phenotypes of Thbs1−/− and Tgfb1−/− mice.

We previously reported on a novel compound (Compound 1; RUC-1) identified by high-throughput screening that inhibits human ␣IIb␤3. RUC-1 did not inhibit ␣V␤3, suggesting that it interacts with ␣IIb, and flexible ligand/rigid protein molecular docking studies supported this speculation. We have now studied RUC-1's effects on murine and rat platelets, which are less sensitive than human to inhibition by Arg-Gly-Asp (RGD) peptides due to differences in the ␣IIb sequences contributing to the binding pocket. We found that RUC-1 was much less potent in inhibiting aggregation of murine and rat platelets. Moreover, RUC-1 potently inhibited fibrinogen binding to murine platelets expressing a hybrid ␣IIb␤3 receptor composed of human ␣IIb and murine ␤3, but not a hybrid receptor composed of murine ␣IIb and human ␤3. Molecular docking studies of RUC-1 were consistent with the functional data. In vivo studies of RUC-1 administered intraperitoneally at a dose of 26.5 mg/kg demonstrated antithrombotic effects in both ferric chloride carotid artery and laser-induced microvascular injury models in mice with hybrid h␣IIb/m␤3 receptors. Collectively, these data support RUC-1's specificity for ␣IIb, provide new insights into the ␣IIb binding pocket, and establish RUC-1's antithrombotic effects in vivo. (Blood. 2009;114:195-201) IntroductionWe previously published data on the identification of a novel inhibitor of ␣IIb␤3 (Compound 1; now referred to as RUC-1). 1 We speculated that it interacted exclusively with the ␣IIb portion of the Arg-Gly-Asp (RGD) binding site based on its specificity for ␣IIb␤3 compared with ␣V␤3 and molecular docking studies into the human ␣IIb␤3 headpiece suggesting that the positively charged piperazinyl nitrogen of RUC-1 interacts with the carboxyl group of D224 in ␣IIb and that the heterocyclic fused ring of RUC-1 interacts with one or more of the 3 aromatic residues that line the ␣IIb pocket. RUC-1 also is too short to span between D224 of ␣IIb and the ␤3 metal ion-dependent adhesion site (MIDAS) and lacks a carboxyl group to coordinate the MIDAS metal ion, which is an invariant feature of all other small molecule ␣IIb␤3 antagonists. [2][3][4] In the present study, we further tested whether RUC-1 demonstrates specificity for ␣IIb by taking advantage of known differences in the abilities of ␣IIb␤3 antagonists to inhibit ␣IIb␤3-mediated platelet aggregation in different species. Consistent with these data, we also found that RUC-1 could inhibit thrombus formation in vivo in transgenic mice expressing human (h) ␣IIb in complex with murine (m) ␤3, but not wild-type (WT) mice. Estimates of electrostatic and van der Waals interaction energies of RUC-1 docked into the crystal structure of human ␣IIb␤3 or molecular models of rat ␣IIb␤3, mouse ␣IIb␤3, or hybrid human ␣IIb/mouse␤3 were consistent with the functional data. In aggregate, these data have important implications for understanding the structure of the ␣IIb binding pocket and the potential antiplatelet effects of ␣IIb-specific ␣IIb␤3 antagonists. Me...

CIITA is the primary factor activating the expression of the class II MHC genes necessary for the exogenous pathway of Ag processing and presentation. Strict control of CIITA is necessary to regulate MHC class II gene expression and induction of an immune response. We show in this study that the nuclear localized form of CIITA is a predominantly phosphorylated form of the protein, whereas cytoplasmic CIITA is predominantly unphosphorylated. Novel phosphorylation sites were determined to be located within a region that contains serine residues 286, 288, and 293. Double mutations of these residues increased nuclear CIITA, indicating that these sites are not required for nuclear import. CIITA-bearing mutations of these serine residues significantly increased endogenous MHC class II expression, but did not significantly enhance trans-activation from a MHC class II promoter, indicating that these phosphorylation sites may be important for gene activation from intact chromatin rather than artificial plasmid-based promoters. These data suggest a model for CIITA function in which phosphorylation of these specific sites in CIITA in the nucleus serves to down-regulate CIITA activity.

Berkeley sickle cell mice are used as an animal model of human sickle cell disease but there are no reports of platelet studies in this model. Since humans with sickle cell disease have platelet abnormalities, we studied platelet morphology and function in Berkeley mice (SS). We observed elevated mean platelet forward angle light scatter (FSC) values (an indirect measure of platelet volume) in SS compared to wild type (WT) (37 ± 3.2 vs. 27 ± 1.4, mean ± SD; p <0.001), in association with moderate thrombocytopenia (505 ± 49 × 10 3 /μl vs. 1151 ± 162 × 10 3 /μl; p <0.001). Despite having marked splenomegaly, SS mice had elevated levels of Howell-Jolly bodies and "pocked" erythrocytes (p <0.001 for both) suggesting splenic dysfunction. SS mice also had elevated numbers of thiazole orange positive platelets (5 ± 1 % vs. 1 ± 1%; p <0.001), normal to low plasma thrombopoietin levels, normal plasma glycocalicin levels, normal levels of platelet recovery, and near normal platelet life spans. Platelets from SS mice bound more fibrinogen and antibody to Pselectin following activation with a threshold concentration of a protease activated receptor (PAR)-4 peptide compared to WT mice. Enlarged platelets are associated with a predisposition to arterial thrombosis in humans and some humans with SCD have been reported to have large platelets. Thus, additional studies are needed to assess whether large platelets contribute either to pulmonary hypertension or the large vessel arterial occlusion that produces stroke in some children with sickle cell disease.