Spatial-temporal reorganization of activated integrins

10.4161/cam.20753Cell Adhesion and Migration63280-28

Investigation of a thromboxane A\u3csub\u3e2\u3c/sub\u3e receptor–based vaccine for managing thrombogenesis

© 2018 The Authors. Background—Despite the well-established role for the thromboxane A2 receptor (TPR) in the development of thrombotic disorders, none of the antagonists developed to date has been approved for clinical use. To this end, we have previously shown that an antibody targeted against TPR’s ligand-binding domain inhibits platelet activation and thrombus formation, without exerting any effects on hemostasis. Thus, the goal of the present studies is to design a novel TPR-based vaccine, demonstrate its ability to trigger an immune response, and characterize its antiplatelet and antithrombotic activity. Methods and Results—We used a mouse keyhole limpet hemocyanin/peptide-based vaccination approach rationalized over the TPR ligand-binding domain (ie, the C-terminus of the second extracellular loop). The biological activity of this vaccine was assessed in the context of platelets and thrombotic diseases, and using a host of in vitro and in vivo platelet function experiments. Our results revealed that the TPR C-terminus of the second extracellular loop vaccine, in mice: (1) triggered an immune response, which resulted in the development of a C-terminus of the second extracellular loop antibody; (2) did not affect expression of major platelet integrins (eg, glycoprotein IIb-IIIa); (3) selectively inhibited TPR-mediated platelet aggregation, platelet-leukocyte aggregation, integrin glycoprotein IIb-IIIa activation, as well as dense and α granule release; (4) significantly prolonged thrombus formation; and (5) did so without impairing physiological hemostasis. Conclusions—Collectively, our findings shed light on TPR’s structural biological features, and demonstrate that the C-terminus of the second extracellular loop domain may define a new therapeutic target and a TPR vaccine-based approach that should have therapeutic applications

Novel α 2

Accumulating experimental evidence indicates that overexpression of α 2 β 1 integrin may correlate with progression in human prostate cancer. The objective of this study was to design a novel imaging probe based on the Asp-Gly-Glu-Ala (DGEA) peptide for near-infrared-fluorescent (NIRF) imaging of α 2 β 1 , integrin expression in prostate cancer. The peptides were conjugated with appropriate fluorescent dyes, and the binding affinity of these probes was evaluated by flow cytometry in three human prostate cell lines (PC-3, CWR-22, and LNCaP). In vivo NIRF imaging of the α 2 β 1 -positive PC-3 xenograft model was performed to evaluate the α 2 β 1 targeted probe. In vitro immunofluorescence staining was carried out to confirm the α 2 β 1 integrin expression level. Flow cytometry analysis showed that PC-3 had the highest probe uptake, followed by CWR-22 and LNCaP tumor cells. In the subcutaneous PC-3 model, the tumor demonstrated prominent uptake with good tumor to background contrast. Immunohistochemistry staining also supported the in vivo optical imaging results. DGEA-based optical agents have been developed for specific imaging of α 2 β 1 , integrin expression. In vitro and in vivo localization demonstrated the potential of this agent to identify tumor subtypes amenable to anti-α 2 β 1 integrin treatment and potentially provide prognostic information regarding tumor progression