Enhanced uptake of blood coagulation factor VIII containing immune complexes by antigen presenting cells.

Essentials Anti-factor (F) VIII antibody formation is a major complication in the treatment of hemophilia A. We investigated uptake of FVIII and FVIII immune complex by bone marrow derived dendritic cells. Immune complex formation increased uptake of FVIII 3-4 fold in a Fcγ receptor dependent manner. FVIII immune complex binding to Fcγ receptors may modulate immune tolerance induction. SUMMARY: Background A major complication in the treatment of hemophilia A is the development of inhibitory antibodies targeting coagulation factor VIII (FVIII). Eradication of these inhibitors can be established by immune tolerance induction (ITI), which consists of daily administration of high dosages of FVIII. FVIII immune complexes (FVIII-IC) could be formed following FVIII infusion in patients with pre-existing anti-FVIII antibodies. Objectives Here we studied endocytosis of FVIII-IC by bone marrow-derived dendritic cells (BMDCs). Methods BMDCs were pulsed with FVIII/FVIII-IC and uptake was assessed by flow cytometry and confocal imaging. Results BMDCs were able to efficiently internalize FVIII-IC in a dose-dependent manner, 3-4-fold more efficiently when compared with equimolar concentrations of non-complexed FVIII. Uptake of FVIII-IC, but not FVIII alone, could be inhibited with anti-Fcγ receptor (FcγR) antibody 2.4G2, indicating functional involvement of FcγR. No internalization of FVIII-IC was observed in BMDCs lacking FcγRI, FcγRIIb, FcγRIII and FcγRIV. Genetic ablation of FcγRIIb, FcγRIII or FcγRIV individually did not affect the ability of anti-FVIII IgG to promote the uptake of FVIII. BMDCs lacking FcγRI showed lower FVIII-IC uptake levels when compared with other single FcγR null BMDCs. Expression of the inhibitory FcγRIIb alone was sufficient to internalize FVIII-IC more efficiently than FVIII. Conclusions FcγR are critical in the internalization of FVIII-IC by BMDCs and multiple FcγR can contribute independently to this process. Our findings provide a basis for future studies to address whether the outcome of ITI is dependent on the interplay between FVIII-IC and inhibitory and activating FcγR.

Tracking down contact activation - from coagulation in vitro to inflammation in vivo.

The contact system is a volatile and versatile enzyme system in blood plasma that responds to the presence of nonphysiological surface materials by spontaneous generation of enzymatic activity. In subsequent steps, it can trigger blood coagulation and is responsible for the generation of the proinflammatory peptide bradykinin. The physiological role of the contact system is presently unknown, but it is commonly used to trigger coagulation in a diagnostic setting. In this three-part review, we will first describe the molecular mechanisms that drive contact activation on nonphysiological materials. Next, we will summarize and compare a number of bioassays, which are commonly used to investigate the contact system in health and disease. Finally, we will discuss recent findings from both fundamental and clinical studies on the contributions of contact system to cardiovascular, infectious, and inflammatory disease.

Shear stress is required for the endocytic uptake of the factor VIII-von Willebrand factor complex by macrophages.

BACKGROUND: Low-density lipoprotein (LDL) receptor family members contribute to the cellular uptake of factor VIII. How von Willebrand factor fits into this endocytic pathway has remained poorly understood. OBJECTIVES: It has been suggested that macrophages contribute to the clearance of the factor VIII (FVIII)-von Willebrand factor (VWF) complex. We now assessed the mechanisms of uptake employing human monocyte-derived macrophages. METHODS: A confocal microscopy study was employed to study the uptake by monocyte-derived macrophages of a functional green fluorescent FVIII-GFP derivative in the presence and absence of VWF. RESULTS: The results revealed that FVIII-GFP is internalized by macrophages. We found that FVIII-GFP co-localizes with LDL receptor-related protein (LRP), and that the LRP antagonist Receptor Associated Protein (RAP) blocks the uptake of FVIII-GFP. However, FVIII-GFP was not detected in the macrophages in the presence of VWF, suggesting that the FVIII-VWF complex is not internalized by these cells at all. Apart from static conditions, we also investigated the effect of shear stress on the uptake of FVIII-GFP in presence of VWF. Immunofluorescence studies demonstrated that VWF does not block endocytosis of FVIII-GFP under flow conditions. Moreover, VWF itself was also internalized by the macrophages. Strikingly, in the presence of RAP, endocytosis of FVIII-GFP and VWF was inhibited. CONCLUSION: The results show that shear stress is required for macrophages to internalize both constituents of the FVIII-VWF complex.