Thrombin-Stimulated Platelets Have Functional Binding Sites For Factor VIIIa That Are Distinct From Phosphatidylserine

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3582-3582
Author(s):  
Jialan Shi ◽  
Valerie A Novakovic ◽  
Steven Pipe ◽  
Shannon Meeks ◽  
John (Pete) S. Lollar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Factor Viii ◽  
Binding Site ◽  
Binding Sites ◽  
Conflicts Of Interest ◽  
Specific Binding ◽  
Phospholipid Vesicles ◽  
C2 Domain ◽  
Phospholipid Binding ◽  
Fviii Activity

Abstract Background Factor VIII (fVIII) functions as a co-factor for factor IXa on the membranes of stimulated platelets. Binding sites for fVIII(a) are expressed at two levels; thrombin induces 3,000 – 20,000 sites/platelet while the combination of collagen and thrombin or A28137 induce >50,000 sites/platelet. Hypothesis We hypothesized that binding sites for fVIII(a) on thrombin-stimulated platelets, are distinct from phosphatidylserine (PS), while those on maximally stimulated platelets are predominantly PS-containing sites. Corollaries were 1) that epitopes on fVIII interact with the non-PS sites and 2) that a macromolecule or a macromolecule complex comprises the binding sites on thrombin-stimulated platelets. Methods Platelets were purified on a density gradient and binding of fluorescein-labeled fVIII (fVIII-fluor) was measured by flow cytometry using a Becton Dickinson LSR-Fortessa flow cytometer. Factor VIII activity was measured in a discontinuous factor Xase assay using extruded phospholipid vesicles of composition PS:PE:PC 4:20:76 or platelets as the membrane source. Oligomeric fibrin was immobilized by incubating thrombin, 1 u/ml, with fibrinogen, 10 µg/ml for 10 min without mixing prior to addition of 59D8-Superose beads. Binding of fVIII-4 Ala to platelets was measured in complex with Alexa-488 labeled mAb GMA-8021, against the A2 domain. Polyphosphate was size-fractionated and recombinant PPX-MBD produced as previously described. Results Lactadherin, a phosphatidyl-L-serine-binding protein, competed for 97% of factor VIII-fluorescein (fVIII-fluor) binding sites on A23187-stimulated platelets but only 30% of binding sites on thrombin-stimulated platelets. Unlabeled fVIII competed with fVIII-fluor for all binding sites. A fVIII C2 domain mutant, with no measurable phospholipid binding - M2199A/F2200A/L2251A/L2252A (fVIII-4Ala) bound to only 3,000 – 5,000 sites on platelets stimulated with A23187 but to a similar number on thrombin-stimulated platelets with a KDof 7 nM. These data indicate that non-PS sites are dominant on thrombin-stimulated platelets but that PS-containing sites comprise at least 95% of sites on A23187-stimulated platelets. We evaluated a panel of mAb’s against the fVIII-C2 domain for platelet-specific inhibition of binding and function. mAb’s ESH4 and I54, with overlapping epitopes, blocked binding of fVIII to thrombin-stimulated platelets but only decreased affinity for PS-containing membranes. In 1-stage and 2-stage commercial aPTT assays ESH4 inhibited 28-33% of fVIII activity. In contrast, ESH4 inhibited 80% of fVIII activity on thrombin-stimulated platelets. mAb’s ESH8 and G99, with partially overlapping epitopes, decreased the affinity of fVIII-fluor for thrombin-stimulated platelets approx. 70% but had no effect on phospholipid binding. ESH8 inhibited 58 ± 8% of fVIII activity on thrombin-stimulated platelets but did not decrease activity supported by phospholipid vesicles. Because oligomeric fibrin is required for expression of most fVIII binding sites on thrombin-stimulated platelets (Phillips et al 2004; JTH 2:1806) we hypothesized that oligomeric, platelet-bound fibrin is a constituent of fVIII binding sites. fVIII-fluor bound to fibrin monomers and oligomers immobilized on mAb 59D8-Superose, detected in solution by flow cytometry. Binding was enhanced by mixing polyphosphate (polyP) with fibrinogen prior to thrombin, with a maximum gain in affinity at 0.1 µM elemental phosphorous. The apparent affinity of fibrin-polyP for fVIII-fluor was 2-12 nM, based on competition studies with unlabeled fVIII. Like binding to platelets, specific binding of fVIII to fibrin-polyP was blocked by mAb’s ESH4, I54 and diminished by ESH8, and G99. Thrombin-stimulated platelets, but not resting platelets, exhibited bound polyP, as detected by PPX-MBP, specific for polyP. Thus, bound polyP is present on thrombin-stimulated platelets under conditions that lead to binding of oligomeric fibrin. Conclusions These data indicate that thrombin-stimulated platelets bind fVIII via a non-PS binding site and that the binding is mediated by epitopes that have greater functional importance on platelets than on phospholipid vesicles. Platelet-bound oligomeric fibrin with polyP is a candidate for the non-PS binding site. These findings have clinical relevance to detection of inhibitory antibodies against fVIII. Disclosures: No relevant conflicts of interest to declare.

Factor VIII Procoagulant Protein Interacts with Phospholipid Vesicles Via its 80 kDa Light Chain

1988 ◽  
Vol 60 (03) ◽  
pp. 442-446 ◽  
Author(s):  
G Kemball-Cook ◽  
S J Edwards ◽  
K Sewerin ◽  
L O Anderson ◽  
T W Barrowcliffe
Keyword(s):  
Factor Viii ◽  
Binding Site ◽  
Light Chain ◽  
Binding Sites ◽  
Light Chains ◽  
Phospholipid Vesicles ◽  
Polyacrylamide Gels ◽  
Phospholipid Binding

SummaryIn a previous report, we detailed the fractionation of polyclonal human anti-Factor VIII :C into a component directed exclusively against the phospholipid-binding site on Factor VIII (PL-site antibody) and another directed at other sites (non-PL-site antibody). The location on the F.VIII molecule of its PL-binding site has now been studied by two different methods using this fractionated 125I-labelled anti-F.VIII: C Fab’.The first method was modified from that of Weinstein et al. (Proc Natl Acad Sci USA 1981; 78: 5137-41), involving electrophoresis of F.VIII peptide-125I-Fab‘ A/F.VIII immunocomplexes in SDS-polyacrylamide gels. PL-site antibody reacted with F.VIII peptides of apparent Mr approximately 80 kDa and sometimes 160 kDa in plasma and concentrate, but not with larger peptides. Non-PL-site antibody, however, reacted with a range of peptides of apparent Mr 90 kDa to 280 kDa. In addition, when purified F.VIII containing heavy and light chains (HC + LC), and isolated LC peptides were analysed, PL-site antibody bound to LC peptides whereas non-PL-site antibody did not.The second method used the antibody pools in immunoradiometric assays (IRMA’s) of purified F.VIII peptides. Both labels measured similar amounts of F.VIII: Ag in a sample of purified F.VIII containing both HC and LC; on assaying an HC preparation, however, PL-site label measured only 2% of F.VIII: Ag found by non-PL-site label, indicating that PL-binding sites are absent in HC preparations.These results indicate that F.VIII binds to PL via its 80 kDa light chain.

scholarly journals Cryopreserved Platelets Retain Agonist Responsiveness and Support for Factor VIII Function

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-4
Author(s):  
Valerie Anne Novakovic ◽  
Madhumouli Chatterjee ◽  
Gary E Gilbert
Keyword(s):  
Flow Cytometry ◽  
Factor Viii ◽  
Conflicts Of Interest ◽  
Geometric Mean ◽  
Procoagulant Activity ◽  
Macromolecular Complex ◽  
C2 Domain ◽  
Platelet Factor ◽  
Fviii Activity ◽  
Apheresis Platelets

Platelet activation supports procoagulant activity through phosphatidylserine exposure, secretion of procoagulant factors, and receptor conformational change. For example, thrombin-stimulated platelets bind factor VIII (fVIII) via a macromolecular complex including oligomeric fibrin and the active αIIbβ3 receptor (Phillips et al, JTH 2004; Gilbert et al, Blood 2015). Thus, coagulation assays in which phospholipid vesicles are substituted for platelets do not fully emulate modulators of fVIII activity. Indeed, inhibition of platelet-supported fVIII activity by a panel of mAbs against the C2 domain was not correlated to inhibition of vesicle-dependent activity (Chatterjee et al, JTH 2020). An obstacle to adoption of a platelet-based assay for fVIII is the need for fresh platelets. Therefore we asked whether cryopreserved platelets might support fVIII activity similarly to fresh platelets. Apheresis platelets were mixed with cryopreservatives with or without calcium chelators, in various aliquot sizes, and frozen on various cooling media. Cryopreserved platelets were compared to non-preserved apheresis platelets with regard to agonist response and support of procoagulant activity. Cryopreservation resulted in an increase in subcellular debris and an unresponsive fraction of platelets with decreased forward scatter judged by flow cytometry. Optimized results were obtained when platelet rich plasma with 5% DMSO, in 1 mL aliquots was frozen on powdered dry ice, and stored at -150C. Purification of thawed platelets using a density gradient removed debris and decreased unresponsive platelets resulting in a forward and side scatter profile comparable to fresh platelets. We refer to these as cryopreserved platelets (CryoPlts). CryoPlts were compared to control and outdated apheresis platelets. As with fresh platelets, procoagulant activity of CryoPlts increased with thrombin receptor agonist peptides (TRAP) 1 & 4 and supported a log-linear relationship between time to initial fibrin strand formation and fVIII activity over a range of 0.0001 - 1 u/mL (Fig 1). Further, the degree of inhibition of fVIII activity by mAbs ESH4 and G99 against the fVIII C2 domain, was the same on control and CryoPlts, but markedly different from inhibition in an aPTT-based inhibitor assay. In contrast, outdated apheresis platelets had increased procoagulant activity, minimal agonist response and a shallow curve with varying fVIII concentration. Flow cytometry studies with lactadherin-FITC indicated that 33 ± 14% of CryoPlts had high PS exposure, and the size of this population was minimally affected by TRAP 1+4. In contrast, the main platelet population had a small, uniform, increment in PS exposure comparable to control platelets. Surprisingly, the PS-rich platelets did not significantly affect the time to fibrin formation, confirming that the viable platelets, with limited PS exposure, provide much of the support for fVIII-related procoagulant activity. Flow cytometry indicated αIIbβ3 activation (PAC1-FITC) and α-granule release (anti-P-selectin-PE) were qualitatively intact on CryoPlts, although staining was decreased 70% for PAC1 and 57% for Psel. We also tested whether CryoPlts may be utilized for evaluating response to anti-PF4-heparin antibodies, relevant to heparin-induced thrombocytopenia (HIT). We evaluated platelet response to platelet factor 4 (PF4) and a platelet-activating anti-PF4 antibody (KKO), a combination that induces activation similar to authentic autoimmune antibodies for HIT. The non-activating PF4 antibody RTO served as a negative control. Geometric mean response, corrected for background, was normalized to response to thrombin activation. Both fresh and CryoPlts responded with increases in PAC1 (Fig 2A) and anti-Psel (Fig 2B) binding in response to KKO/PF4 compared to RTO/PF4 . This data demonstrates that the qualitative αIIbβ3 and P-selectin response to HIT-like antibodies is intact. Our results demonstrate a refined cryopreservation protocol of apheresis platelets. These platelets maintain qualitative agonist responsiveness with near-normal support for factor VIII activity, suggesting that they could be used for other platelet-based laboratory or diagnostic assays. Further, our results suggest that major procoagulant activity is provided by platelets with very limited PS exposure, an area for further investigation. Disclosures No relevant conflicts of interest to declare.

Increasing Hydrophobicity and Disulfide Bridging Between A1 and C2 Subunits Improves Factor VIII Stability.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3176-3176
Author(s):  
Hironao Wakabayashi ◽  
Amy E Griffiths ◽  
Philip Fay
Keyword(s):  
Thermal Stability ◽  
Factor Viii ◽  
Chemical Stability ◽  
Hydrophobic Interaction ◽  
Conflicts Of Interest ◽  
Covalent Modification ◽  
C2 Domain ◽  
Double Mutation ◽  
Fviii Activity ◽  
A1 Domain

Abstract Abstract 3176 Poster Board III-100 Factor VIII (FVIII) consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. Previously we have generated FVIII mutants with enhanced stability by mutating residues located at A1-A2 or A2-A3 interfaces (Wakabayashi et al, Blood, 112, 2761-9, 2008, Wakabayashi et al, J. Thromb. Haemost. 7, 438-44, 2009). FVIII X-ray structures show close contacts between the Ca2+ binding site contained within the A1 domain and the C2 domain of LC. In this study we mutated residues located at this interface to examine the effects on FVIII(a) stability. Studies assessing FVIII thermal and chemical stability involved monitoring the rates of loss of FVIII activity by FXa generation assay following incubation of FVIII (4 nM) at 57°C or in various concentrations of guanidinium (0-1.2 M). The rate of decay of FVIIIa was monitored over time at 23°C using FXa generation assays following activation of FVIII (1.5 nM) with thrombin. Data were fitted to single exponential decay equations and rates of decay were compared. In one variant, a disulfide bond was introduced between the two domains by a double mutation at Arg121 in A1 and Leu2302 in the C2 domain to Cys (R121C/L2302C). In addition, based on the finding that there is a gap between the methyl groups of Ala108 (A1 domain) and Ala2328 (C2 domain) we mutated Ala108 to Val, Ile, or Leu to examine whether these mutants increase the stability of FVIII by an improved hydrophobic interaction at this site. Significant increases in FVIII thermal stability, up to 4-fold compared with WT, were observed in R121C/L2302C, Ala108Ile, and Ala108Leu. R121C/L2302C and Ala108Ile retained ∼80% FVIII activity as measured by FXa generation assay compared to WT value, however, that of Ala108Leu was ∼25% the WT value. Only Ala108Ile showed an improvement in chemical stability (10% increase in IC50 value as compared with WT FVIII) and FVIIIa decay due to A2 subunit dissociation was similar to WT FVIII (20-40% reduction in FVIIIa decay rate compared to WT). Ca2+ is necessary for FVIII function and EGTA (2 mM) reduced WT FVIII activity by ∼70%. However, EGTA-treated R121C/L2302C FVIII retained ∼100% activity, suggesting that the Ca2+ requirement for FVIII function may be substituted by covalent bonding between the Ca2+ binding region in A1 and C2 subunit. Furthermore, the Ala108Ile variant showed ∼60% activity remaining after EGTA treatment suggesting partial relief of this Ca2+ dependency for stability of the A1-C2 interaction. Next, we tested whether the mutations at the A1-C2 interface can be combined with mutations at A1-A2 or A2-A3 interfaces to generate a FVIII with further improved stability. Previously characterized FVIII variants, designated A domain mutants, showing up to 2-fold increases in thermal stability compared with WT FVIII included Asp519Ala, Asp519Val, Glu665Ala, Glu665Val, Glu1984Ala, and Glu1984Val. In combining those mutations with either R121C/L2302C or Ala108Ile, we obtained variants with >5-fold increases in thermal stability (9/12 mutants), with the Ala108Ile/Glu665Val variant showing the greatest increase (∼10-fold). Most of the mutants (9/12) showed normal FVIII activity values by FXa generation assay (>60%) and 15-30% increases in IC50 values for chemical stability as compared with WT. In addition, the high FVIIIa stability of the A domain mutants was largely preserved in the combined mutations. Collectively, these results suggest that alterations at this A1-C2 contact region by covalent modification or increasing hydrophobic interaction yields improved FVIII stability that can be combined with other high stability mutations to produce additive effects. Disclosures No relevant conflicts of interest to declare.

Proteolytically Inactivatable Factor VIII is Less Immunogenic than Factor VIII in a Murine Hemophilia A Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 27-27
Author(s):  
Shannon Meeks ◽  
Ernest T Parker ◽  
Amy L. Dunn ◽  
John F Healey ◽  
Pete Lollar
Keyword(s):  
Immune Response ◽  
Immune System ◽  
Factor Viii ◽  
Binding Site ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Foreign Protein ◽  
C2 Domain ◽  
Wild Type ◽  
Antibody Titers

Abstract Abstract 27 Patients with hemophilia A have a congenital deficiency of the factor VIII (fVIII) protein due to a mutation in the fVIII gene that frequently leads to absence of detectable expression of fVIII. Accordingly, the therapeutic replacement fVIII protein potentially is recognized as non-self by the immune system. Thirty percent of patients with severe hemophilia A develop detectable inhibitory anti-fVIII antibodies (inhibitors). Additionally, greater than 90 percent of hemophilia A mice treated with human fVIII develop inhibitors using dosing schedule that mimics use in humans. Because fVIII is an immunologically foreign protein, it might be expected that a hemophilia A patient would make a fVIII inhibitor. However, intravenous injection of soluble proteins in either humans or rodents usually results in tolerance rather than a humoral immune response. One major difference between fVIII and other proteins is that it is released from its large carrier protein von Willebrand factor (VWF) and is potentially exposed to the immune system at sites of active hemostasis and inflammation. Heat-inactivated, denatured fVIII, which maintains all T-cell epitopes but lacks several B-cell epitopes, is less immunogenic than native fVIII, suggesting that fVIII-dependent thrombin generation along the intrinsic pathway of blood coagulation may provide co-stimulatory signals necessary for the immune response (Skupsky BS, Zhang A, Scott DW Blood 2008; 112:1220a). We constructed a B domain-deleted human fVIII mutant, designated fVIIIi, which contains alanine substitutions at two critical thrombin cleavage sites, Arg372 and Arg1689, and purified it to homogeneity. FVIIIi does not develop procoagulant activity and is not released from VWF in response to thrombin. Therefore fVIIIi is less likely than wild-type fVIII to be exposed to the immune system at sites of active hemostasis and inflammation. Additionally, VWF binds to the immunodominant fVIII C2 domain and potentially hides part of fVIII from the immune system. FVIIIi was antigenically intact judging from intact binding to a panel of11 mouse anti-fVIII monoclonal antibodies whose epitope specificity was represented by all five domains of BDD fVIII. The immunogenicity of wild-type fVIII and fVIIIi was compared in a murine hemophilia A model in which groups of 25 mice received 8 weekly injections of physiologic doses of fVIII. Plasma was collected weekly for total anti-fVIII antibody titers by ELISA and one week following the last injection for total anti-fVIII antibody titers, inhibitor titers by Bethesda assay and for epitope mapping. Mice treated with fVIIIi had significantly lower levels of inhibitory as well as total anti-fVIII antibodies than mice treated with wild-type fVIII. Domain mapping using single human domain hybrid human/porcine molecules as ELISA antigens revealed that hemophilia A mice broadly recognized all fVIII domains in response to either wild-type or fVIIIi, although fVIIIi produced less anti-light chain antibodies. Mice in both the wild-type fVIII and fVIIIi groups produced antibodies that recognized the phospholipid-binding site of the C2 domain, even though this site overlaps the VWF binding site on fVIII. There was no difference in the isotype spectrum of the antibodies made to fVIII or fVIIIi. This study indicates that inactivatable fVIII is less immunogenic than native fVIII and suggests that the immunogenicity of fVIII is related either to its interaction with VWF or to events triggered by activation of the coagulation mechanism. Disclosures: No relevant conflicts of interest to declare.

Crystal Structure of the C2 Domain of Lactadherin and Computational Docking of Phosphatidyl-L-Serine.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 195-195
Author(s):  
Chenghua Shao ◽  
James F. Head ◽  
Barbara A. Seaton ◽  
Gary E. Gilbert
Keyword(s):  
Factor Viii ◽  
Binding Sites ◽  
Specific Binding ◽  
Lower Surface ◽  
Head Group ◽  
Factor V ◽  
C2 Domain ◽  
Computational Docking ◽  
Specific Binding Sites ◽  
Acyl Chains

Abstract Lactadherin is a phosphatidyl-L-serine (Ptd-L-Ser)-binding protein that decorates the membranes of milk fat globules. Lactadherin is also secreted by macrophages, engaging Ptd-L-Ser on the membranes of apoptotic cells and hastening clearance by the macrophages. Sequence homology between the lectin-like C1 and C2 domains of lactadherin and those of factor VIII and factor V correlates with the capacity of lactadherin to compete for sites on Ptd-L-Ser-containing membranes and function as a potent anticoagulant. The C2 domain of lactadherin contains a major Ptd-L-Ser binding motif. Solution studies have shown that binding of the phospho-L-serine head group exhibits stereospecificity, consistent with the existence of one or more specific binding sites on the protein. Neither the three-dimensional structure of the lactadherin C2 domain nor the identity of specific residues that interact with Ptd-L-Ser have been determined. We have solved the crystal structure of the bovine lactadherin C2 domain (Lact-C2) at 1.67 Å resolution. The β-barrel protein core, composed of eight anti-parallel strands, is homologous with the previously published structures of the factor VIII C2 domain (fVIII-C2) and factor V C2 domain (fV-C2). The loops on the lower surface of Lact-C2 display four water-exposed hydrophobic amino acids, reminiscent of the membrane-interacting residues of fVIII-C2 and fV-C2. These hydrophobic amino acids, Trp26, Leu28, Phe31, and Phe81, form a diamond-shaped hydrophobic patch on the lower surface of Lact-C2. This hydrophobic patch is well positioned to interact extensively with the phospholipid membrane. The longest loop, predicted to participate in membrane binding, has a W-like shape that contrasts with the simpler shapes adopted by the corresponding loops of fV-C2 and fVIII-C2. Furthermore, in Lact-C2 glycine residues within putative membrane-interacting loops appear likely to provide conformational flexibility that may facilitate Lact-C2 binding to the membrane. To investigate potential Ptd-L-Ser binding sites on the protein, computational docking studies were performed, utilizing the program AutoDock. A Ptd-L-Ser analog with acetyl moieties replacing both acyl chains was utilized as the docking ligand. This approach avoided spurious identification of non-specific binding sites for acyl chains, which would naturally be immersed in a membrane bilayer. We identified two candidate Ptd-L-Ser binding sites that could engage the hydrophilic head group of Ptd-L-Ser in a stereoselective manner. The binding pocket for the better of the two solutions is not homologous with the Ptd-L-Ser binding sites previously proposed for fVIII-C2 or fV-C2. These results provide the basis for a testable hypothesis rationalizing the efficient phospholipid binding of lactadherin vs. factor VIII and factor V and the mechanism for Ptd-L-Ser stereoselectivity.

Reduced Affinity To VWF Induced By a Novel Mutation At Pro1809Leu (factor VIII-Tenri) Is The Cause Of Mild Hemophilia A Developing Anti-Factor VIII Inhibitor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2343-2343
Author(s):  
Koji Yada ◽  
Keiji Nogami ◽  
Masahiro Takeyama ◽  
Hiroaki Minami ◽  
Philip J. Fay ◽  
...  
Keyword(s):  
Factor Viii ◽  
Binding Site ◽  
Conformational Change ◽  
Binding Affinity ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Novel Mutation ◽  
Fold Increase ◽  
Factor Viii Inhibitor ◽  
C2 Domain

Abstract The development of inhibitory antibodies to factor (F)VIII in patients with mild hemophilia A is a rare, but significant event. We reported the mild hemophilia A associated with Pro(P)1809Leu(L) mutation in FVIII gene (FVIII-Tenri), developing the type II inhibitor (peak 5.6BU/ml), which inhibited allogeneic but not autologous FVIII recognizing the epitope(s) overlapping with that of anti-FVIII monoclonal antibody (mAb) ESH8 in the C2 domain remote from the mutated site in the A3 domain, at the 54th ASH meeting (#52283). However, the haemostatic characteristics of FVIII-Tenri remain unclear. In this study, to elucidate the characteristics of FVIII-Tenri, we prepared and stably expressed a recombinant, B-domainless FVIII mutant, P1809L. FVIII:C of the mutant reconstituted with FVIII-deficient plasma (0.3 nM) was 31.5 IU/dl, whilst that of the wild-type (WT) (0.3 nM) was 88.6 IU/dl, similar to the level of patient’s plasma. The polyclonal IgG immune-purified from this patient’s plasma decreased FVIII:C of WT by ∼60% at the maximum concentration of IgG, whilst any little affected in the mutant. The mutant was activated by thrombin and FXa, showing ∼16- and 7-fold increase in FVIII:C compared to baseline control, respectively, similar to WT. Furthermore, purified FXa generation assays showed that the mutant was any little different in Km for FIXa compared to WT (0.74 ± 0.07 and 0.72 ± 0.13 nM, respectively), supporting that this mutation little affected the association with FIXa. Interestingly, the mutant showed an ∼3-fold weak binding affinity (Kdapp 0.92 ± 0.23nM) to VWF compared to WT (Kdapp 0.33 ± 0.01nM), whilst any little difference was observed in binding affinity to phospholipid between the mutant and WT, in an ELISA. To further investigate the haemostatic mechanism(s) associated with less binding affinity to VWF, the residual FVIII:C of the mutant or WT (0.05-0.25nM) was measured in a one-stage clotting assay, after incubation with several anti-FVIII mAbs including anti-C2 mAbs ESH8 (residues 2248-2285) and ESH4 (2303-2332) with individual epitope overlapping the VWF-binding site(s) in the C2 domain. ESH8 conferred the ∼60% lower inhibition of FVIII:C in the mutant compared to that in WT, whilst ESH4 showed the similar inhibition of FVIII:C between in the mutant and WT. Anti-A2 mAb JR8 showed any little difference in the inhibition between in the mutant and WT. These results demonstrated that the mutation P1809L in the A3 domain did little affect upon activation by thrombin and FXa as well as association with FIXa and phospholipid, but attenuated the binding to VWF through the conformational change in the C2 domain. Taken together with the development of the allogeneic inhibitor associated with this mutation, these results strongly suggested that the conformational change occurred in the C2 domain remote from the mutated site in the A3 domain hampered the binding to VWF, resulting in the haemostatic impairment due to fragility of the mutant FVIII molecule as well as the change of the immunogenicity in the epitopes overlapping with the FVIII-VWF binding site (residue 2248-2285) presented on allogeneic FVIII. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1811-1819 ◽  
Author(s):  
D Scandella ◽  
GE Gilbert ◽  
M Shima ◽  
H Nakai ◽  
C Eagleson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Binding Site ◽  
Light Chain ◽  
Intrinsic Factor ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Amino Acid Residues ◽  
Fviii Inhibitor ◽  
Human Factor Viii

The finding that human factor VIII (fVIII) inhibitor antibodies with C2 domain epitopes interfere with the binding of fVIII to phosphatidylserine (PS) suggested that this is the mechanism by which they inactivate fVIII. We constructed a recombinant C2 domain polypeptide and demonstrated that it bound to all six human inhibitors with fVIII light chain specificity. Thus, some antibodies within the polyclonal anti-light chain population require only amino acids within C2 for binding. Recombinant C2 also partially or completely neutralized the inhibitor titer of these plasmas, demonstrating that anti-C2 antibodies inhibit fVIII activity. Immunoblotting of a series of C2 deletion polypeptides, expressed in Escherichia coli, with inhibitor plasmas showed that the epitopes for human inhibitors consist of a common core of amino acid residues 2248 through 2312 with differing extensions for individual inhibitors. The epitope of inhibitory monoclonal antibody (MoAb) ESH8 was localized to residues 2248 through 2285. Three human antibodies and anti-C2 MoAb NMC-VIII/5 bound to a synthetic peptide consisting of amino acids 2303 through 2332, a PS- binding site, but MoAb ESH8 did not. These antibodies also inhibited the binding of fVIII to synthetic phospholipid membranes of PS and phosphatidylcholine, confirming that the blocked epitopes contribute to membrane binding as well as binding to PS. In contrast, MoAb ESH8 did not inhibit binding. As the maximal function of activated fVIII in the intrinsic factor Xase complex requires its binding to a phospholipid membrane, we propose that fVIII inhibition by anti-C2 antibodies is related to the overlap of their epitopes with the PS-binding site. MoAb ESH8 did not inhibit fVIII binding to PS-containing membranes, suggesting the existence of a second mechanism of fVIII inhibition by anti-C2 antibodies.

Specific binding of endothelin-1 to canine tracheal epithelial cells in culture

1995 ◽  
Vol 268 (3) ◽  
pp. L424-L431 ◽  
Author(s):  
H. Ninomiya ◽  
X. Y. Yu ◽  
Y. Uchida ◽  
S. Hasegawa ◽  
E. W. Spannhake
Keyword(s):  
Epithelial Cells ◽  
Binding Site ◽  
Binding Sites ◽  
Specific Binding ◽  
Receptor Subtype ◽  
Control Mechanisms ◽  
Endothelin 1 ◽  
Tracheal Epithelial Cells ◽  
Eta Receptor ◽  
Tracheal Epithelial

We have studied the binding of endothelin-1 (ET-1) to cultured canine tracheal epithelial cells. A single specific binding site for 125I-labeled ET-1 was identified with an apparent dissociation constant (Kd) of 0.2 nM, maximal binding sites (Bmax) of 6.7 x 10(3) sites/cell, and half-maximal inhibition (IC50) of 0.3 nM during a 2-h incubation period. The binding of 125I-ET-1 to these cells was inhibited by the presence of unlabeled ET-1, ET-2, or BQ-123, whereas ET-3 and sarafotoxin S6c did not compete for this binding site. These binding characteristics are consistent with those of the ETA receptor. At 37 degrees C, specific binding continuously increased over 18 h, while at 4 degrees C, it reached a plateau by 2 h. The increase in binding at 37 degrees C was not associated with DNA synthesis but was dependent upon protein synthesis, suggesting that epithelial binding sites were produced continuously under these incubation conditions. Our results indicate that canine tracheal epithelial cells possess specific binding sites for ET-1 with characteristics similar to those of the ETA receptor subtype. Because these cells are demonstrated to both release and bind ET-1, the results further suggest that ET-1 is involved in paracrine and/or autocrine control mechanisms in the airway epithelium.

Factor V Binding to Multimerin 1: Modulation by Factor V Activation and Binding Sites in the Factor V C1 and C2 Domains.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 193-193
Author(s):  
Samira B. Jeimy ◽  
Mary Ann Quinn-Allen ◽  
Nola Fuller ◽  
Kenneth Segers ◽  
Alan R. Stafford ◽  
...  
Keyword(s):  
Binding Sites ◽  
Thrombin Generation ◽  
Factor V ◽  
C2 Domain ◽  
Binding Studies ◽  
C2 Domains ◽  
Phospholipid Binding ◽  
Factor Va ◽  
Plasma Factor ◽  
C1 Domain

Abstract Platelets and endothelial cells store the polymeric factor V(a) binding protein, multimerin 1 (MMRN1), for release upon agonist stimulation. In human megakaryocytes, factor V binding to MMRN1 follows plasma factor V endocytosis, resulting in stored complexes of MMRN1 and factor V in platelet α-granules. The C2 domain of the factor V light chain contains a MMRN1 binding site; however, the affinity and stoichiometry of factor V-MMRN1 binding have not been determined, direct comparisons of factor V and Va binding to MMRN1 have not been done, and potential homologous roles of C1 and C2 domain structures in MMRN1 binding have not been studied. To further explore the mechanism of factor V and Va binding to MMRN1, and the roles of B domain release and C1 domain residues in MMRN1 binding, we used surface plasmon resonance and solid-phase binding studies. Functional consequences of factor V-MMRN1 binding were tested in competitive binding assays with the soluble phospholipid 1,2-Dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), and calibrated automated thrombinography (CAT). Factor V bound to MMRN1 with a higher affinity than factor Va (approximately 2 nM versus 12 nM), and a stoichiometry consistent with binding to MMRN1 trimers. The higher affinity of factor V for MMRN1 was mainly due to differences in rates of formation of a more stable, secondary complex with MMRN1. Factor V activation by thrombin dissociated bound factor V from MMRN1, consistent with the reduced affinity of factor Va for MMRN1. A panel of point mutated, B domain deleted factor V constructs were used to identify MMRN1 binding residues in the C1 domain of factor V and Va. On a three dimensional model of factor Va, these residues mapped to a large, predominantly contiguous region between the C1 and C2 domains, that overlapped residues critical for factor Va phospholipid binding and procoagulant function. Consistent with the lowered affinity of factor Va for MMRN1, C6PS significantly inhibited factor Va-MMRN1, but not factor V-MMRN1 binding (p<0.05). Overlap between the MMRN1 and phospholipid binding sites was verified by CAT assays, as MMRN1 caused dose-dependent, significant reductions in plasma thrombin generation in these assays, by increasing lag time (p<0.01), and reducing peak (p<0.01) and total thrombin generation (p<0.01). Taken together, these data indicate that the functional homologies between the C domains of factor V extend to their MMRN1 binding sites. Moreover, thrombin has modulating effects on factor V-MMRN1 binding that mimic its effects on factor VIII-von Willebrand factor binding. The affinity of factor V-MMRN1 binding could be important to promote the association of MMRN1 with factor V in platelets, until factor V release and activation for prothrombinase assembly.

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