The Role of Factor VIII C Domains in Sorting to Weibel-Palade Bodies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2241-2241
Author(s):  
Eveline Bouwens ◽  
Maartje van den Biggelaar ◽  
Jan Voorberg ◽  
Koen Mertens
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Conflicts Of Interest ◽  
Factor V ◽  
C2 Domain ◽  
Structural Elements ◽  
Sorting Efficiency ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 2241 Recent studies have shown that factor VIII (FVIII) expressed in endothelial cells sorts with von Willebrand factor (VWF) to secretory Weibel-Palade bodies (WPBs). The sorting mechanism remains controversial although VWF is thought to be essential. However, mutations that lead to impaired FVIII-VWF complex assembly do not reduce the sorting efficiency of FVIII. As factor V (FV) and FVIII are highly homologous in structure, we addressed the possibility that FV sorts to WPBs as well. Our study was designed to identify domains in FVIII that are needed for sorting to WPBs by means of domain deletions and FVIII-FV domain exchange. As the C domains of FVIII contain membrane and VWF binding sites, we particularly focused on comparing the C domains of FVIII and FV. Blood outgrowth endothelial cells (BOECs) were transduced with lentiviral vectors encoding FV, FVIII deletion mutants, or FVIII-FV chimeras. We found by confocal microscopy and subcellular fractionations that FV displays a strong reduction in sorting efficiency (2% sorting efficiency) compared to FVIII (20% sorting efficiency). This indicates that sorting to WPBs is mediated by FVIII-specific structural elements. As the C domains of FVIII are implicated in membrane and VWF binding, these domains could drive sorting to WPBs. Therefore, we constructed FVIII variants lacking C domains to establish their role in WPB sorting. Quantitative determination of the sorting efficiencies demonstrates that the C1 domain is not of major importance for sorting to WPBs (10% sorting efficiency), whereas the C2 domain is (not detectable in WPB fractions). Moreover, exchanging the FVIII C domains for corresponding domains of FV also suggests that the C2 domain drives WPB sorting (3% sorting efficiency). This leads to the conclusion that FVIII sorting to WPBs is driven by FVIII-specific structural elements in both C domains, but in particular the C2 domain. Disclosures: No relevant conflicts of interest to declare.

Factor VIII Inhibitor Antibodies with C2 Domain Specificity Are Less inhibitory to Factor VIII Complexed with von Willebrand Factor

1996 ◽  
Vol 76 (05) ◽  
pp. 749-754 ◽  
Author(s):  
Suzuki Suzuki ◽  
Morio Arai ◽  
Kagehiro Amano ◽  
Kazuhiko Kagawa ◽  
Katsuyuki Fukutake
Keyword(s):  
Complex Formation ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Domain Specificity ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

Identification of Three Non-Overlapping Epitopes in the C2 Domain of Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1007-1007
Author(s):  
Shannon Meeks ◽  
John F. Healey ◽  
John (Pete) S. Lollar
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Structural Complexity ◽  
Basis Set ◽  
Type I ◽  
C2 Domain ◽  
Type Ii ◽  
Secondary Antibodies ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Factor VIII (fVIII) inhibitory antibodies (fVIII inhibitors) are a significant source of morbidity in patients with hemophilia A. Approximately 30% of patients with severe hemophilia A will develop inhibitors. Most inhibitors are directed against either the A2 or C2 domains of fVIII. The repertoire of antibodies to the C2 domain is functionally complex, including antibodies that inhibit fVIII binding to phospholipid and von Willebrand factor and display either type I or type II kinetics. The goal of this study was to investigate the diversity of the immune response to the C2 domain of human fVIII in a murine hemophilia A model and to identify a set of non-overlapping epitopes recognized by these fVIII inhibitors. A panel of 55 murine anti-C2 antibodies was obtained, which included 53 antibodies from anti-fVIII hybridomas produced in our laboratory and previously described antibodies ESH-4 and ESH-8. Nine of the hybridomas were cloned by limiting dilution and the corresponding monoclonal IgG antibodies were purified. IgG from the remaining 44 hybridomas was isolated directly from the hybridoma supernatants and biotinylated. The 9 monoclonal antibodies along with ESH-4 and ESH-8 were used as primary antibodies in a competition ELISA using human fVIII as the antigen. The panel of 55 biotinylated antibodies was used as secondary antibodies. Antibody pairs were classified as having non-overlapping or overlapping epitopes based on whether the binding of the secondary antibody was present or absent, respectively. A basis set of 3 antibodies, 1B5-1B, 3E6-1B, and (2)117-1B, was defined, which consisted of a set of non-overlapping antibodies that as a group competed for binding of human fVIII with all other antibodies. 1B5-1B and 3E6-1B exhibited type I kinetics with Bethesda titers of 950 and 30, respectively. The Bethesda titer of (2)117-1B could not be determined due to its type II behavior. ESH-4 and ESH-8 had the same epitope profiles as 3E6-1B and (2)117-1B, respectively. Of the 52 non-basis set antibodies, 85% overlapped with 1B5-1B, 29% with 3E6-1B, and 56% with (2)117-1B. The majority of the antibodies overlapped with more than one member of the basis set, leading to the identification of five classes of antibodies (see figure). Because of the large number of antibodies characterized, it is unlikely that the frequency of antibodies in any additional classes is high. The elucidation of the structural complexity of the anti-fVIII C2 repertoire should be useful in the characterization of the pathogenicity of C2 inhibitors. Figure Figure

ADAMTS13 Content of Plasma-Derived Factor VIII-Von Willebrand Factor Concentrates

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3383-3383
Author(s):  
Flora Peyvandi ◽  
Pier Mannuccio Mannucci ◽  
Carla Valsecchi ◽  
Silvia Pontiggia ◽  
Jonathan Bernstein ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Limit Of Detection ◽  
Analysis Data ◽  
Current Treatment ◽  
Adamts13 Activity ◽  
Pilot Studies ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 3383 Introduction: Anecdotal and published reports on the use of specific plasma-derived Factor VIII (FVIII)-von Willebrand Factor (vWF) concentrates in the treatment of congenital thrombotic thrombocytopenic purpura (TTP) led to the determination of the functional and antigenic content of ADAMTS13 in a number such concentrates commercially available in the US and/or the EU. Deficiency or inhibition of ADAMTS13 is the putative cause of TTP. TTP is a rare disorder causing microvascular thrombosis resulting in low platelet counts. It affects 1 –5 patients per 1,000,000 population. The current treatment of this disorder consists of regular infusions of fresh frozen plasma (FFP) for inherited TTP and exchange plasmapheresis for the acquired version of the disorder. The possibility of utilizing FVIII-vWF concentrates that incorporate robust virucidal treatments in their manufacturing and the possible utilization of lower infusion volumes prompted the evaluation of these concentrates for ADAMTS13 content. Methods: For this analysis, we obtained 5 lots of Koate®-DVI, 2 lots of Human Bioplasma, and 1 lot of each of the following concentrates: Humate® P, Wilate®, Alphanate®, Emoclot® and Fractogel.® Following reconstitution of these concentrates in 10 mL of water for injection, we determined the concentration of ADAMTS13 antigen by an ELISA method previously described by Feys et al. (J Thromb Haemost. 2006; 4: 955–62) with minor modifications (Peyvandi et al. Haematologica 2008; 93: 232–239). The lower limit of detection was 1% while the lower value of the normal range was 45%. The ADAMTS13 activity was measured using collagen binding (CBA) and FRET assays with minor modifications (Peyvandi et al. Haematologica 2008; 93: 232–239); in both assays, serially diluted normal human plasma (NHP) and plasma samples were diluted 1:10 in assay buffer and incubated 1:1 with vWF substrate in a final volume of 100 μl. In addition to the determination of ADAMTS13, we evaluated the amount of vWF antigen (vWF:Ag) using a commercial kit (Instrumentation Laboratory, IL US, Bedford, Massachusetts, USA) in these concentrates and the multimeric composition of this protein. Utilizing densitometry, we determined the percentage of ultra-large vWF multimers in each of these concentrate. Results: The analysis showed that among the tested concentrates, Koate®-DVI had the highest ratio of ADAMTS13/FVIII. This concentrated presented ratios of 9.1 ± 0.7% (Mean ± SD) and 8.4 ± 1.1% (Mean ± SD) per IU FVIII for ADAMTS13 activity and antigen respectively. The remainder of the concentrates contained only nominal amounts of ADAMTS13; only Alphanate® reached a ratio of 0.23% ADAMTS13 activity/IU FVIII. These data are in agreement with previous reports (Qorraj et al. Blood 2001, 116: 3677). A summary analysis data are shown in Table 1. Koate®-DVI displayed a ratio of vWF:Ag/FVIII of approximately 2.81, very similar to the ratio of 2.77 included in the single lot of Humate®-P. The ratio of ultra-large multimers to total multimers in Humate® P was higher, at 0.66 vs. 0.39 for Koate-DVI. However, the lower content of ultra-large multimers in Koate®-DVI may be advantageous in its possible utilization in the treatment of TTP. Conclusion: On the basis of these results, Koate-DVI may have a role to play in the management of congenital and, possibly, idiopathic TTP, especially on the basis of observations that FVIII accelerates the breakdown of vWF (Cao et al. PNAS 2008, 105: 7416–7421). Additional characterization, testing in pertinent animal models and pilot studies in humans would be required to determine the extent to which this concentrate can assist in the treatment of TTP. Disclosures: Guazzini: Kedrion S.p.A: Consultancy. Retzios:Kedrion Biopharma, Inc.: Consultancy.

scholarly journals Inflammation Drives Coagulopathies in Sars-Cov-2 Patients

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-35
Author(s):  
Martha MS Sim ◽  
Meenakshi Banerjee ◽  
Melissa Hollifield ◽  
Hammodah Alfar ◽  
Xian Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Similar Process ◽  
Aids Patients ◽  
Thrombotic Risk ◽  
S Protein ◽  
Von Willebrand ◽  
Willebrand Factor

Background:A hypercoagulable state has been consistently reported in patients with severe Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), characterized by elevated D-dimer, prolonged PT, and mild thrombocytopenia, though the mechanism is unclear. We have previously shown that human immunodeficiency virus (HIV) infection causes depletion of the anticoagulant protein S and virus-mediated platelet activation. Based on early reports, we hypothesized that a similar process contributed to COVID-19-associated thrombosis. Aim:To probe platelet activation and coagulation factor activity in SARS-CoV-2-infected patients. Methods:Blood was collected from consenting patients with differing COVID-19 severity: outpatients (15), hospitalized inpatients (15), and healthy controls (8). Platelet-leukocyte aggregate (PLA) formation and monocyte profiling were measured by flow cytometry. Coagulation factors were assessed by enzymatic assays. PS, von Willebrand Factor (vWF), PC, cytokines, and anti-S-Protein (viral spike protein) IgG were measured by ELISAs. Results:Ninety percent of SARS-CoV-2+ out-patients and in-patients had circulating anti-S-Protein IgG, but plasma IL-6 and TNFα were only elevated in three in-patients, consistent with reports that systemic inflammation is relatively rare in this population. Immune response did not correlate with disease severity. Unlike in HIV1+/AIDS patients, total PS was not reduced in SARS-CoV-2+ patients. However, the anticoagulant pool of PS ("free PS") was reduced in plasma samples from in-patients compared to controls (47.2%±23.3% vs. 100.8±42.6%, p=0057), while out-patients had an intermediate concentration (73.1%±28.9%). Specific loss of free PS is likely mediated by an increase in C4-binding protein (C4bp), which binds PS. In-patients also had a trend toward elevated plasma tissue factor (TF) compared to controls (79.5±121.4 fM vs. 37.8±39.7 fM, p = 0.32). Endothelial cells and monocytes can express TF under inflammatory conditions. We evaluated endothelial damage and dysfunction by measuring E-Selectin, which was unchanged in either in-patients or out-patients, and von Willebrand Factor (vWF), which was elevated in in-patients compared to controls (143±29.8 ng/mL vs. 56.2±41.9 ng/mL, p=0.0023). Plasma from in-patients also had elevated myeloperoxidase (524±187 ng/mL vs. 127±35 ng/mL, p=0.0026) and had a trend toward increased platelet-leukocyte aggregates (14.6±11.7% vs. 5.2±3.7%, p=0.24), indicating platelet and leukocyte stimulation. Unlike in the HIV1+/AIDS patients, no virus was detectable in any of the SARS-CoV-2+ patient plasmas. Consistent with a lack of direct platelet-virus interaction, plasma PF4 and platelet Akt phosphorylation were unchanged in the patient samples. We also observed a trend toward increased TF on TF+/CD64+/CD11b+ monocytes from in-patients compared to controls (MFI = 3244±2340 vs. 1741±382, p=0.18). Two inpatients were followed until they were SARS-CoV-2-negative. In both, PLAs, IL-6, vWF, and plasma TF remained elevated out to 28 days and PS remained reduced, suggesting that hemostatic dysregulation persists after SARS-CoV-2 is undetectable. Conclusions:We propose that localized inflammation in SARS-CoV-2+ patients results in a decrease in anticoagulant PS, through a shift of the free and C4bp-bound forms. At the same time, this inflammation causes stimulation of endothelial cells, which secrete procoagulant vWF, monocytes, which express TF and release it into plasma on microvesicles, and platelets, which form platelet-leukocyte aggregates. These changes may not return to baseline post-infection, suggesting that long-term monitoring of thrombotic risk may be necessary for SARS-CoV-2+ patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Platelets, Endothelial Cells and von Willebrand Factor

1977 ◽  
Author(s):  
Z.M. Ruggeri
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Marked Reduction ◽  
Normal Values ◽  
Crossed Immunoelectrophoresis ◽  
Cellular Compartments ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor ◽  
Normal Controls

In plasma and platelets (PI) from 10 patients (pts) with “severe” recessive von Willebrand’s disease (vWD) factor VIII-related antigen (VIIIR:AG) was unmeasurable in 7 and extremely low in 3 using a very sensitive immunoradiometric assay (IRMA); ristocetin cofactor (VIIIR:RCo) was always unmeasurable and VIM:AG was not detectable on immunofluorescence on endothelial cells (EC) of the vessel wall. In 12 pts with “classical”, dominant vWD characterized by reduced plasma level of VIIIR:AG and VI I IR:RCo, normal values were found in PI and the mobility of PI VIIIR:AG on crossed Immunoelectrophoresis was not different from that of normal controls; VIIIRr AG was normal on EC. In 7 pts showing a faster electrophoretic mobility of plasma VIIIR:AG (variant: vWD), the same abnormality was found in PI. VIIIR:AG concentration was normal both in plasma and in PI when measured by electroimmunodiffusion, whereas IRMA gave lower values and VI I IR:RCo was decreased; VIIIR:AG was normal on EC. These findings show that “severe” vWD is the expression of a marked reduction of factor VIII synthesis fully expressed in EC, PI and plasma. In “classical” vWD the plasma defects are not reflected in the cellular compartments, suggesting a defective transfer from EC and PI to plasma. Patients with “variant” vWD are the expression of a qualitative alteration of the factor VIII molecule, functionally defective both in PI and in plasma.

Contributions of Asn2198, Met2199, and Phe2200 in the factor VIII C2 domain to cofactor activity, phospholipid-binding, and von Willebrand factor-binding

2003 ◽  
Vol 89 (05) ◽  
pp. 795-802 ◽  
Author(s):  
Deborah Lewis ◽  
Mary Pound ◽  
Thomas Ortel
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Hydrophobic Surface ◽  
C2 Domain ◽  
Mutant Proteins ◽  
Phospholipid Binding ◽  
Cofactor Activity ◽  
The Individual ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe crystal structure of the factor VIII C2 domain consists of a β-sandwich core from which β-hairpins and loops extend to form a hydrophobic surface. The hydrophobic surface includes M2199 and F2200 at the tip of the 1st β-hairpin. To determine the individual contributions of residues N2198, M2199, and F2200 to phospholipid and von Willebrand factor (vWF) binding properties of factor VIII, we prepared mutant proteins with single alanine substitutions. We found that single mutations at N2198 and M2199 had relatively little impact on cofactor activity, or phospholipid and vWF binding. However the F2200A mutant had slightly lower cofactor activity at subsaturating phospholipid concentrations. Competitive ELISAs suggested that F2200 plays a more important role in both phospholipid-binding and vWF-binding than N2198 and M2199. All mutant proteins were still recognized by a monoclonal antibody and two factor VIII inhibitors that neutralized cofactor activity and blocked factor VIII binding to phospholipids.Presented in part at the XVIII Congress of the International Society on Thrombosis and Haemostasis, Paris, France, 6-12 July 2001, and the 43rd Annual Meeting of the American Society of Hematology, Orlando, Florida, 7-11 December 2001

Functional mapping of factor VIII C2 domain

2011 ◽  
Vol 106 (07) ◽  
pp. 121-131 ◽  
Author(s):  
Jean-Luc Pellequer ◽  
Shu-wen W. Chen ◽  
Didier Saboulard ◽  
Marc Delcourt ◽  
Claude Négrier ◽  
...  
Keyword(s):  
Factor Viii ◽  
Functional Relation ◽  
Functional Mapping ◽  
Coagulation Cascade ◽  
Structure Stability ◽  
C2 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Β Sheet ◽  
In Silico Analyses

SummaryThe factor VIII (FVIII) is a cofactor of the coagulation cascade. The FVIII C2 domain is a critical domain that participates in the interactions with the von Willebrand factor and the phospholipidic surfaces. To assess the importance of each residue of this domain in the maintenance of the structure and the function of FVIII, a number (n=139) of mutants were generated by substituting the original residues, from Ser2173 to Gly2325, by an alanine. Mutants were built within a complete B domain- deleted FVIII and expressed in COS-1 cells. Mutant antigen levels and procoagulant activities were measured. Two in silico analyses, a sliding average procedure and an analysis of the mutation energy cost were conducted in parallel on the FVIII structure. Both results were in agreement with the functional data, and illustrated the benefit of using such strategies prior to targeting specific residues in the aim of generating active recombinant molecules. The functional assays identify the residues that are important to maintaining the structure of the C2 domain, mainly those forming β-sheet, and those that can afford substitution, establishing a detailed functional relation with the available crystallographic data. This study provided a comprehensive functional mapping of the FVIII C2 domain and discussed the implication of specific residues in respect to the maintenance in the activity and structure stability, the efficiency in secretion, the binding to phospholipids and the formation of epitope.

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