Splenic Synthesis of Factor VIII: Basis for Transplant Therapy of Hemophilia A.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1615-1615
Author(s):  
Matthew Y. Suh ◽  
Shirine Dada ◽  
Humberto Azpurua ◽  
Evelyn Flynn ◽  
David M. Briscoe ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Liver Transplantation ◽  
Endothelial Cells ◽  
Factor Viii ◽  
Mononuclear Cells ◽  
Hemophilia A ◽  
Sinusoidal Endothelial Cells ◽  
Murine Spleen ◽  
Splenic Cells ◽  
Red Pulp

Abstract Synthesis of factor VIII by liver sinusoidal endothelial cells has been documented, and liver transplantation for end stage liver disease has been shown to cure concomitant hemophilia A. Given the irreversible nature of liver transplantation, the spleen is an alternate organ of interest in transplant therapy to cure hemophilia A. Factor VIII gene expression in the spleen has been documented by RT-PCR (Hollestelle, M. J. et al, 2001, Thromb Haemost86:855–61), and a recent report of living related splenic transplantation for hemophilia A has shown symptom-free survival up to 4 years (Jiang, H. C. et al, 2006, Transplant Proc38:1483). However, it is unclear which cell populations in the spleen synthesize factor VIII. Immunohistochemistry of murine spleen showed factor VIII staining of the sinusoidal endothelial cells as expected. Surprisingly, clusters of large mononuclear cells in the red pulp also intensely stained for factor VIII. CD31 staining revealed same pattern of sinusoidal endothelial cells and large mononuclear cells in the red pulp. Single cell suspensions of murine spleen analyzed by flow cytometry corroborated significant factor VIII expression (47–70% of all splenic cells). At least 60% of splenic cells were CD31+ CD45+, and these CD31+ CD45+ cells showed significant factor VIII expression (38–48%). When CD31+ cells were depleted by magnetic cell sorting, there was still a subgroup of cells expressing factor VIII (approximately 50%). In summary, we have shown that a significant proportion of splenic cells synthesize factor VIII by flow cytometry and immunohistochemistry. In addition to the sinusoidal endothelial cells, CD31+ CD45+ cells (possibly the large mononuclear cells seen on immunohistochemistry) contribute to splenic synthesis of factor VIII. Based on CD31 depletion study, there is still an unidentified group of cells (CD31-) that contributes to factor VIII synthesis. Further characterization of these cells and an in vivo study in a murine hemophilia A model have been initiated. We conclude that the spleen is a viable source of factor VIII and may be useful in cell-based and/or organ transplant therapy of hemophilia A.

scholarly journals Localization of factor VIII-procoagulant antigen: an immunohistological survey of the human body using monoclonal antibodies

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 222-227 ◽  
Author(s):  
TH van der Kwast ◽  
HV Stel ◽  
E Cristen ◽  
RM Bertina ◽  
EC Veerman
Keyword(s):  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Lymph Nodes ◽  
Factor Viii ◽  
Mononuclear Cells ◽  
Immunoperoxidase Staining ◽  
Sinusoidal Endothelial Cells ◽  
Morphological Criteria ◽  
Hepatic Sinusoidal Endothelial Cells ◽  
Red Pulp

Abstract Various organs, including liver, spleen, heart, lung, kidney, intestines, lymph nodes, pancreas, bone marrow, and thymus, were investigated for the presence of factor VIII-procoagulant antigen (VIIICAg) and factor VIII-related antigen (VIIIRAg), using a panel of monoclonal antibodies directed to factor VIII-von Willebrand factor in combination with a sensitive immunoperoxidase staining technique. In addition to hepatic sinusoidal endothelial cells, the presence of VIIICAg was demonstrated in mononuclear cells sporadically present in lymph nodes, in the alveolar septa of lung, and in the red pulp of spleen. The identity of these mononuclear cells could not be unequivocally determined. Based on morphological criteria, however, it is tentatively concluded that these cells are nonlymphoid and belong to the mononuclear phagocyte system. The presence of VIII-RAg was confined to vascular endothelial cells, hepatic sinusoidal endothelial cells, cells lining the venous sinuses of the red pulp of the spleen, cells lining renal glomeruli and lung capillaries, platelets, and megakaryocytes.

scholarly journals Localization of factor VIII-procoagulant antigen: an immunohistological survey of the human body using monoclonal antibodies

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 222-227 ◽  
Author(s):  
TH van der Kwast ◽  
HV Stel ◽  
E Cristen ◽  
RM Bertina ◽  
EC Veerman
Keyword(s):  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Lymph Nodes ◽  
Factor Viii ◽  
Mononuclear Cells ◽  
Immunoperoxidase Staining ◽  
Sinusoidal Endothelial Cells ◽  
Morphological Criteria ◽  
Hepatic Sinusoidal Endothelial Cells ◽  
Red Pulp

Various organs, including liver, spleen, heart, lung, kidney, intestines, lymph nodes, pancreas, bone marrow, and thymus, were investigated for the presence of factor VIII-procoagulant antigen (VIIICAg) and factor VIII-related antigen (VIIIRAg), using a panel of monoclonal antibodies directed to factor VIII-von Willebrand factor in combination with a sensitive immunoperoxidase staining technique. In addition to hepatic sinusoidal endothelial cells, the presence of VIIICAg was demonstrated in mononuclear cells sporadically present in lymph nodes, in the alveolar septa of lung, and in the red pulp of spleen. The identity of these mononuclear cells could not be unequivocally determined. Based on morphological criteria, however, it is tentatively concluded that these cells are nonlymphoid and belong to the mononuclear phagocyte system. The presence of VIII-RAg was confined to vascular endothelial cells, hepatic sinusoidal endothelial cells, cells lining the venous sinuses of the red pulp of the spleen, cells lining renal glomeruli and lung capillaries, platelets, and megakaryocytes.

Cellular Uptake of rFVIIIFc in Liver Is Primarily Contributed by Sinusoidal Endothelial Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2219-2219
Author(s):  
Siyuan Tan ◽  
Kai Chen ◽  
Arjan van der Flier ◽  
Zhan Liu ◽  
David R. Light ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Cellular Uptake ◽  
Kupffer Cells ◽  
Half Life ◽  
Hemophilia A ◽  
Liver Cells ◽  
Whole Body ◽  
Sinusoidal Endothelial Cells ◽  
The Impact

Abstract Abstract 2219 rFVIIIFc is a recombinant fusion protein consisting of human B-domain deleted factor VIII covalently linked to the Fc domain of IgG1. In hemophilia A patients, rFVIIIFc has been shown to display a ∼1.6-fold longer half-life than recombinant full length FVIII (Advate®) (Powell et al., 2012. Blood). This half-life extension can be attributed to a natural pathway mediated by the neonatal Fc receptor (FcRn) that re-circulates IgG molecules into the vascular system, as the long-lasting activity of rFVIIIFc is not observed in FcRn knockout mice. To identify the cell type that takes up and subsequently protects and recycles rFVIIIFc, we have recombinantly replaced the missing B-domain with a Halo tag in rFVIIIFc (rFVIIIFc-Halo) to allow visualization of the protein in the presence of fluorescently labeled Halo-ligand using confocal microscopy. Purified rFVIIIFc-Halo protein displayed similar specific activity and pharmacokinetic properties as rFVIIIFc in hemophilia A (HemA) mice, indicating that the addition of the Halo tag does not alter the functionality and the clearance mechanisms of rFVIIIFc. In quantitative whole body autoradiography studies (QWBA) in HemA mice with radiolabeled rFVIIIFc, we observed that 125I-rFVIIIFc is predominately distributed to the liver. Therefore, we selected primary liver cells isolated from HemA mice to study cellular uptake of rFVIIIFc. A co-culture of hepatocytes and non-parenchymal cells was isolated from HemA mice and prepared at a 1:1 ratio. Liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) in this culture were identified by fluorescently labeled antibodies to CD31 and F4/80 respectively. Both cell types effectively took up the fluorescently labeled AcLDL, confirming that the isolated LSECs and KCs retained the capacity for functional endocytosis in vitro. It was found that LSECs, as opposed to Kupffer cells or hepatocytes, are predominantly responsible for the cellular uptake of rFVIIIFc, as the localization of rFVIIIFc-Halo is apparent only in LSECs within 5 minutes after exposing 10 nM of rFVIIIFc-Halo to primary co-culture freshly isolated from HemA mice. In contrast, even with longer exposure time (up to 1 hour) and higher protein concentration (up to 40 nM), the localization of rFVIIIFc-Halo in Kupffer cells and hepatocytes still remains undetectable. Analysis of recombinant Halo-tagged factor VIII (rFVIII-Halo) yielded similar results, suggesting that the Fc-fusion does not alter the cellular uptake pathway of FVIII, which is consistent with the notion that the interaction of Fc with FcRn occurs at the intracellular level. Therefore, interestingly, both rFVIII-Halo and rFVIIIFc-Halo are internalized by LSEC that are the same cells reported to express FVIII by in situ hybridization studies (Hollestelle et al. 2001 Thromb Haemost). This study, together with recent findings that somatic cells in the liver are primarily responsible for rFVIIIFc recycling (Abstract by van der Flier et al), highlights the critical role of LSECs in the clearance of rFVIIIFc and suggests that rFVIIIFc is primarily recycled by FcRn in LSECs. The impact of VWF on the cellular uptake and recycling of the rFVIIIFc-VWF complex in liver cells may also be assessed utilizing this system. Disclosures: Tan: BiogenIdec: Employment. Chen:BiogenIdec: Employment. van der Flier:BiogenIdec: Employment. Liu:BiogenIdec: Employment. Light:biogenidec: Employment. Jiang:biogenidec: Employment.

T Lymphocyte Proliferative Responses Induced by Recombinant Factor VIII in Hemophilia A Patients with Inhibitors

1996 ◽  
Vol 76 (01) ◽  
pp. 017-022 ◽  
Author(s):  
Sylvia T Singer ◽  
Joseph E Addiego ◽  
Donald C Reason ◽  
Alexander H Lucas
Keyword(s):  
T Lymphocytes ◽  
Factor Viii ◽  
Cellular Proliferation ◽  
Mononuclear Cells ◽  
Hemophilia A ◽  
Normal Subjects ◽  
Normal Male ◽  
Cell Fractionation ◽  
Severe Hemophilia ◽  
Human Factor Viii

SummaryIn this study we sought to determine whether factor VUI-reactive T lymphocytes were present in hemophilia A patients with inhibitor antibodies. Peripheral blood mononuclear cells (MNC) were obtained from 12 severe hemophilia A patients having high titer inhibitors, 4 severe hemophilia A patients without inhibitors and 5 normal male subjects. B cell-depleted MNC were cultured in serum-free medium in the absence or presence of 2 µg of recombinant human factor VIII (rFVIII) per ml, and cellular proliferation was assessed after 5 days of culture by measuring 3H-thymidine incorporation. rFVIII induced marked cellular proliferation in cultures of 4 of 12 inhibitor-positive hemophilia patients: fold increase over background (stimulation index, SI) of 7.8 to 23.3. The remaining 8 inhibitor-positive patients, the 4 hemophilia patients without inhibitors and the 5 normal subjects, all had lower proliferative responses to rFVIII, SI range = 1.6 to 6.0. As a group, the inhibitor-positive subjects had significantly higher proliferative responses to rFVIII than did the inhibitor-negative and normal subjects (p < 0.05 by t-test). Cell fractionation experiments showed that T lymphocytes were the rFVIII-responsive cell type, and that monocytes were required for T cell proliferation. Thus, rFVIII-reactive T lymphocytes are present in the peripheral circulation of some inhibitor-positive hemophilia A patients. These T cells may recognize FVIII in an antigen-specific manner and play a central role in the regulation of inhibitor antibody production

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