Chemokine receptors are rapidly desensitized and internalized following ligand binding, a process that attenuates receptor-mediated responses. However, the physiological settings in which this process occurs are not clear. Therefore, we examined the fate of CXCR3, a chemokine receptor preferentially expressed on activated T cells following contact with endothelial cells. By immunofluorescence microscopy and flow cytometry, we found that CXCR3 was rapidly internalized when T cells were incubated with IFN-γ-activated human saphenous vein endothelial cells (HSVEC), but not with resting HSVEC. Similar results were obtained using human CXCR3-transfected murine 300-19 B cells. CXCR3 down-regulation was significantly more pronounced when T cells were in contact with HSVEC than with their supernatants, suggesting that CXCR3 ligands were efficiently displayed on the surface of HSVEC. Using neutralizing mAbs to IFN-induced protein-10 (CXCL10), monokine induced by IFN-γ (CXCL9), and IFN-inducible T cell α chemoattractant (I-TAC; CXCL11), we found that even though I-TAC was secreted from IFN-γ-activated HSVEC to lower levels than IFN-induced protein-10 or the monokine induced by IFN-γ, it was the principal chemokine responsible for CXCR3 internalization. This correlated with studies using recombinant chemokines, which revealed that I-TAC was the most potent inducer of CXCR3 down-regulation and of transendothelial migration. Known inhibitors of chemokine-induced chemotaxis, such as pertussis toxin or wortmannin, did not reduce ligand-induced internalization, suggesting that a distinct signal transduction pathway mediates internalization. Our data demonstrate that I-TAC is the physiological inducer of CXCR3 internalization and suggest that chemokine receptor internalization occurs in physiological settings, such as leukocyte contact with an activated endothelium.

Key Points• DDAVP is the drug of choice for mild hemophilia A and von Willebrand disease and (by unclear mechanisms) for platelet function disorders.• In vivo DDAVP selectively and markedly enhances the ability to form procoagulant platelets by enhancing intracellular Na 1 and Ca 21 fluxes.is clinically efficacious in patients with mild platelet function disorders but it is not known which mechanisms mediate this effect. Our aim was to evaluate the impact of in vivo DDAVP administration in these patients. We assessed von Willebrand factor (VWF), factor VIII, platelet activation and aggregation, platelet-dependent thrombin generation, and platelet intracellular Na 1 /Ca 21 fluxes, before and 2 and 4 hours after DDAVP (0.3 mg/kg). We found (1) no significant changes for P-selectin expression, PAC-1 binding, d-granule content and secretion, and platelet-aggregation; (2) significant decreases of secretion of a-granules and GPIIb-IIIa activation induced by adenosine 59-diphosphate, convulxin, and thrombin; (3) significant increases of procoagulant platelets induced by convulxin/thrombin and platelet-dependent thrombin generation; and (4) significant increases of intracellular Na 1 /Ca 21 concentrations. We show that in vivo DDAVP selectively and markedly enhances the ability to form procoagulant platelets and increases platelet-dependent thrombin generation by enhancing Na

Background: A clinically relevant bleeding diathesis is a frequent diagnostic