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Protein disulfide isomerase (PDI) derived from intravascular cells is required for thrombus formation. However, it remains unclear whether platelet PDI contributes to the process. Using platelet-specific PDI-deficient mice, we demonstrate that PDI-null platelets have defects in aggregation and adenosine triphosphate secretion induced by thrombin, collagen, and adenosine diphosphate. Such defects were rescued by wild-type but not mutant PDI, indicating that the isomerase activity of platelet surface PDI is critical for the regulatory effect. PDI-deficient platelets expressed increased levels of intracellular ER protein 57 (ERp57) and ERp72. Platelet PDI regulated αIIbβ3 integrin activation but not P-selectin exposure, Ca(2+) mobilization, β3-talin1 interaction, or platelet spreading on immobilized fibrinogen. Inhibition of ERp57 further diminished αIIbβ3 integrin activation and aggregation of activated PDI-deficient platelets, suggesting distinct roles of PDI and ERp57 in platelet functions. We found that platelet PDI is important for thrombus formation on collagen-coated surfaces under shear. Intravital microscopy demonstrates that platelet PDI is important for platelet accumulation but not initial adhesion and fibrin generation following laser-induced arteriolar injury. Tail bleeding time in platelet-specific PDI-deficient mice were not significantly increased. Our results provide important evidence that platelet PDI is essential for thrombus formation but not for hemostasis in mice.

β2 integrins play a crucial role during neutrophil recruitment into the site of vascular inflammation. However, it remains unknown how ligand-binding activity of the integrin is regulated. Using fluorescence intravital microscopy in mice generated by crossing protein disulfide isomerase (PDI) floxed mice with lysozyme-Cre transgenic mice, we demonstrate that neutrophil PDI is required for neutrophil adhesion and crawling during tumor necrosis factor-α–induced vascular inflammation in vivo. Rescue experiments show that the isomerase activity of extracellular PDI is critical for its regulatory effect on neutrophil recruitment. Studies with blocking anti-PDI antibodies and αLβ2 or αMβ2 null mice suggest that extracellular PDI regulates αMβ2 integrin-mediated adhesive function of neutrophils during vascular inflammation. Consistently, we show that neutrophil surface PDI is important for αMβ2 integrin-mediated adhesion of human neutrophils under shear and static conditions and for binding of soluble fibrinogen to activated αMβ2 integrin. Confocal microscopy and biochemical studies reveal that neutrophil surface PDI interacts with αMβ2 integrin in lipid rafts of stimulated neutrophils and regulates αMβ2 integrin clustering, presumably by changing the redox state of the integrin. Thus, our results provide the first evidence that extracellular PDI could be a novel therapeutic target for preventing and treating inappropriate neutrophil sequestration.

Platelet-leukocyte interactions on activated endothelial cells play important roles in mediating pathological thrombosis and inflammation. Heterotypic platelet-leukocyte aggregation is mediated by the interaction of two crucial receptors and counter receptors; P-selectin-P-selectin glycoprotein ligand-1 and glycoprotein Ibalpha-alphaMbeta2 integrin. In spite of extensive understanding of receptor-counter receptor interactions, it remains unclear how heterotypic cell-cell interactions are regulated under thrombo-inflammatory conditions. Using real-time fluorescence intravital microscopic analysis of Akt isoform-specific knockout (KO) mice, we have demonstrated that Akt2, but not Akt1 or Akt3, plays an important role in neutrophil adhesion to the site of TNF-alpha-induced vascular inflammation. Further, heterotypic platelet-neutrophil interactions on the activated endothelium were markedly reduced in Akt2 but not Akt1 or Akt3 KO mice. Studies with chimeric mice generated from bone marrow transplants on wild-type and Akt2 KO mice revealed that hematopoietic but not endothelial cell Akt2 regulates neutrophil recruitment and platelet-neutrophil interactions during vascular inflammation. Using in vitro reconstituted systems in which platelets and neutrophils were treated with an Akt2 specific inhibitor or cells were isolated from WT and Akt KO mice, we observed that both platelet and neutrophil Akt2 play an important role in platelet-neutrophil aggregation under shear conditions. In particular, neutrophil Akt2 was critical for membrane translocation, activation, and adhesive function of alphaMbeta2 integrin. We found that the basal phosphorylation levels of Akt isoforms are significantly increased in neutrophils and platelets of patients with sickle cell disease (SCD), which is an inherited hematological disorder with vascular inflammation and occlusion. Also, SCD patients’ neutrophils show increased alphaMbeta2 integrin activation in the absence of an agonist, in comparison with healthy donors’ cells. Inhibition of Akt2 dose-dependently reduced heterotypic aggregation of patients’ neutrophils and platelets in vitro and inhibited neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow. Our results provide important genetic and pharmacologic evidence that neutrophil Akt2 regulates alphaMbeta2 integrin function and thus plays a critical role during neutrophil recruitment and neutrophil-platelet interactions under thrombo-inflammatory conditions such as SCD. Disclosures: No relevant conflicts of interest to declare.