Factor XI is a substrate for oxidoreductases: enhanced activation of reduced FXI and its role in antiphospholipid syndrome thrombosis.

Factor XI (FXI), a disulfide-linked covalent homodimer, circulates in plasma, and upon activation initiates the intrinsic/consolidation phase of coagulation. We present evidence that disulfide bonds in FXI are reduced to free thiols by oxidoreductases thioredoxin-1 (TRX-1) and protein disulfide isomerase (PDI). We identified that Cys362-Cys482 and Cys118-Cys147 disulfide bonds are reduced by TRX-1. The activation of TRX-1-treated FXI by thrombin, FXIIa or FXIa was significantly increased compared to non-reduced FXI, indicating that the reduced factor is more efficiently activated than the oxidized protein. Using a novel ELISA system, we compared the amount of reduced FXI in antiphospholipid syndrome (APS) thrombosis patients with levels in healthy controls, and found that APS patients have higher levels of reduced FXI. This may have implication for understanding the contribution of FXI to APS thrombosis, and the predisposition to thrombosis in patients with elevated plasma levels of reduced FXI.

The Fifth Domain of Beta 2 Glycoprotein I Protects from Natural IgM Mediated Cardiac Ischaemia Reperfusion Injury.

Reperfusion after a period of ischemia results in reperfusion injury (IRI) which involves activation of the inflammatory cascade. In cardiac IRI, IgM natural antibodies (NAb) play a prominent role through binding to altered neoepitopes expressed on damaged cells. Beta 2 Glycoprotein I (ß2GPI) is a plasma protein that binds to neoepitopes on damaged cells including anionic phospholipids through its highly conserved Domain V. Domain I of ß2GPI binds circulating IgM NAbs and may provide a link between the innate immune system, IgM NAb binding and cardiac IRI. This study was undertaken to investigate the role of Β2GPI and its Domain V in cardiac IRI using wild-type (WT), Rag-1 -/- and ß2GPI deficient mice. Compared with control, treatment with Domain V prior to cardiac IRI prevented binding of endogenous ß2GPI to post-ischemic myocardium and resulted in smaller myocardial infarction size in both WT and ß2GPI deficient mice. Domain V treatment in WT mice also resulted in less neutrophil infiltration, less apoptosis and improved ejection fraction at 24 h. Rag-1 -/- antibody deficient mice reconstituted with IgM NAbs confirmed that Domain V prevented IgM NAb induced cardiac IRI. Domain V remained equally effective when delivered at the time of reperfusion which has therapeutic clinical relevance.Based upon this study Domain V may function as a universal inhibitor of IgM NAb binding in the setting of cardiac IRI, which offers promise as a new therapeutic strategy in the treatment of cardiac IRI.

Human and mouse mast cells use the tetraspanin CD9 as an alternate interleukin-16 receptor.

Interleukin-16 (IL-16) induces the chemotaxis and activation of mast cells (MCs) and other cell types. While it has been concluded that CD4 is the primary IL-16 receptor on T cells, at least one other IL-16 receptor exists. We now show that the IL-16-responsive human MC line HMC-1 lacks CD4, and that the IL-16-mediated chemotactic and Ca2+ mobilization responses of this cell can be blocked by anti-CD9 monoclonal antibodies (mAbs) but not by mAbs directed against CD4 or other tetraspanins. Anti-CD9 mAbs also inhibited the IL-16-mediated activation of nontransformed human cord blood-derived MCs and mouse bone marrow-derived MCs by 50% to 60%. The chemotactic response of HMC-1 cells to IL-16, as well as the binding of the cytokine to the cell's plasma membrane, was inhibited by CD9-specific antisense oligonucleotides. CD9 is therefore essential for the IL-16-mediated chemotaxis and activation of the HMC-1 cell line. In support of this conclusion, IL-16 bound to CD9-expressing CHO cell transfectants. The ability of wortmannin and xestopongin C to inhibit the IL-16-mediated chemotactic response of these cells suggests that the cytokine activates a phosphatidylinositol 3-kinase (PI3K)/inositol trisphosphate-dependent signaling pathway in MCs. This is the first report of a tetraspanin that plays a prominent role in a cytokine-mediated chemotactic response of human MCs.

Beta 2-Glycoprotein I binds factor XI and inhibits its activation by thrombin and factor XIIa: loss of inhibition by clipped beta 2-glycoprotein I.

Activation of factor XI (FXI) by thrombin in vivo plays a role in coagulation by providing an important positive feedback mechanism for additional thrombin generation. FXI is activated in vitro by thrombin, or FXIIa in the presence of dextran sulfate. In this report, we investigated the effect of beta(2)-glycoprotein I (beta(2)GPI) on the activation of FXI. beta(2)GPI bound FXI in vitro and inhibited its activation to FXIa by thrombin and FXIIa. The affinity of the interaction between beta(2)GPI and FXI was equivalent to the interaction between FXI and high molecular weight kininogen. Inhibition of FXI activation occurred with lower concentrations of beta(2)GPI than found in human plasma. Proteolytic clipping of beta(2)GPI by plasmin abolished its inhibition of FXI activation. The results suggest a mechanism of regulation whereby physiological concentrations of beta(2)GPI may attenuate thrombin generation in vivo by inhibition of FXI activation. Plasmin cleavage of beta(2)GPI provides a negative feedback that counteracts its inhibition of FXI activation.

Biochemical and functional characterization of human transmembrane tryptase (TMT)/tryptase gamma. TMT is an exocytosed mast cell protease that induces airway hyperresponsiveness in vivo via an interleukin-13/interleukin-4 receptor alpha/signal transducer and activator of transcription (STAT) 6-dependent pathway.

Transmembrane tryptase (TMT)/tryptase gamma is a membrane-bound serine protease stored in the secretory granules of human and mouse lung mast cells (MCs). We now show that TMT reaches the external face of the plasma membrane when MCs are induced to degranulate. Analysis of purified recombinant TMT revealed that it is a two-chain neutral protease. Thus, TMT is the only MC protease identified so far which retains its 18-residue propeptide when proteolytically activated. The genes that encode TMT and tryptase betaI reside on human chromosome 16p13.3. However, substrate specificity studies revealed that TMT and tryptase betaI are functionally distinct even though they are approximately 50% identical. Although TMT is rapidly inactivated by the human plasma serpin alpha(1)-antitrypsin in vitro, administration of recombinant TMT (but not recombinant tryptase betaI) into the trachea of mice leads to airway hyperresponsiveness (AHR) and increased expression of interleukin (IL) 13. T cells also increase their expression of IL-13 mRNA when exposed to TMT in vitro. TMT is therefore a novel exocytosed surface mediator that can stimulate those cell types that are in close proximity. TMT induces AHR in normal mice but not in transgenic mice that lack signal transducer and activator of transcription (STAT) 6 or the alpha-chain of the cytokine receptor that recognizes both IL-4 and IL-13. Based on these data, we conclude that TMT is an exocytosed MC neutral protease that induces AHR in lungs primarily by activating an IL-13/IL-4Ralpha/STAT6-dependent pathway.