Cryopreserved Platelets Retain Agonist Responsiveness and Support for Factor VIII Function

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 CryoPlts were compared to control and outdated apheresis platelets.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 In contrast, outdated apheresis platelets had increased procoagulant activity, minimal agonist response and a shallow curve with varying fVIII concentration.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.

Download Full-text

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

Blood ◽

10.1182/blood.v122.21.3582.3582 ◽

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.

Download Full-text

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

Blood ◽

10.1182/blood.v114.22.3176.3176 ◽

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.

Download Full-text

Lactadherin as a Probe for Phosphatidylserine Exposure and as An Anticoagulant for the Procoagulant Activity in the Study of Stored Platelets.

Blood ◽

10.1182/blood.v114.22.3150.3150 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3150-3150

Author(s):

Jin Zhou ◽

Jinxiao Hou ◽

Wen Li ◽

Xiaoqian Zhang ◽

Yueyue Fu ◽

...

Keyword(s):

Flow Cytometry ◽

Confocal Microscopy ◽

Conflicts Of Interest ◽

Annexin V ◽

Progressive Increase ◽

Procoagulant Activity ◽

Platelet Concentrates ◽

Time Analysis ◽

Coagulation Time ◽

Enzymatic Assays

Abstract Abstract 3150 Poster Board III-87 Background Phosphatidylserine (PS) can support coagulant reactions. However, it is uncertain how the location and extent of PS exposure to the membranes of stored platelets affect such reactions. We compared annexin V with lactadherin as a way of detecting how of PS exposure influences the procoagulant properties of stored platelets in platelet concentrates (PCs). Method PS exposure and the relevant procoagulant activity (PCA) of platelets in 5 different PCs were investigated by flow cytometry, confocal microscopy, coagulation time analysis and enzymatic assays. PS exposure was separately measured using annexin V and lactadherin, respectively. Results Exposure of PS to stored platelets promoted thrombin formation. A progressive increase in PS exposure was detected by flow cytometry. Moreover, using lactadherin, we identifed higher levels of PS exposure on the platelets and platelet-derived microparticles (PMPs) compared to detection using annexin V. The percentage of PS-positive cells was 0.02 % by annexin V versus 0.3 % by lactadherin on day 0, 7.5 % by annexin V versus 12.3 % by lactadherin on day 5, and 29 % by annexin V versus 44.3 % by lactadherin on day 9. Rare microparticles (MPs) were released from fresh platelets, and, the number of PMPs increased approximately 2-fold on day 5 and further progressively increased. Using lactadherin and platelets in the earlier stage of storage, confocal microscopy revealed earlier and localized PS exposure based on plasma membrane staining. For later storage platelets, increased levels of PS-positive platelets and PMPs were clearly detected by both annexin V and lactadherin. Thirty-two nM lactadherin or annexin V prolonged coagulation time 2.4 fold versus 2 fold. The productions of thrombin and intrinsic/extrinsic factor Xase were approximately inhibited 85 % and 60 % by lactadherin and annexin V, respectively. Conclusion PS exposure was localized to the cellular rims, blebbing vesicles and thin elongated filopodia-like areas on banked platelets. Furthermore, lactadherin provides a more accurate measurement of PS exposure and the relevant with PCA, which is an important factor to consider for transfusion medicine. Our findings of elevated PS-positive platelets and PMPs indicate that platelets should not be stored for extended periods of time. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Structural Studies Of The Factor VIII C2 Domain In a Ternary Complex With Two Inhibitory Antibodies Reveals Classical and Non-Classical Epitopes

Blood ◽

10.1182/blood.v122.21.2358.2358 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2358-2358

Author(s):

Justin D Walter ◽

Rachel A Werther ◽

Caileen M Brison ◽

John F. Healey ◽

Shannon L. Meeks ◽

...

Keyword(s):

Factor Viii ◽

Ternary Complex ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Structural Basis ◽

C2 Domain ◽

Proteolytic Activation ◽

X Ray ◽

Binding Interface ◽

Inhibitory Antibodies

Abstract The factor VIII C2 domain is a highly immunogenic domain, whereby inhibitory antibodies develop following factor VIII replacement therapy for congenital hemophilia A patients. Inhibitory antibodies also arise spontaneously in cases of acquired hemophilia A. The structural basis for molecular recognition by two classes of anti-C2 inhibitory antibodies that bind to factor VIII simultaneously has been investigated by small angle X-ray scattering and X-ray crystallography. The C2 domain/3E6 FAB/G99 FAB stable ternary complex, both in solution and in its crystalline state, illustrates that each antibody epitope resides on opposing faces of the factor VIII C2 domain. The 3E6 epitope is a classical antibody that forms direct contacts to the C2 domain at two loops consisting of Glu2181-Ala2188 and Thr2202-Arg2215, which inhibits the binding of the C2 domain to von Willebrand Factor and phospholipid surfaces. The G99 is a non-classical antibody that prevents proteolytic activation of factor VIII, and its epitope centers on Lys2227 and also makes direct contacts with loops Gln2222-Trp2229, Leu2261-Ser2263, His2269-Val2282 and Arg2307-Gln2311. Each binding interface is highly electrostatic, with positive charges present on both C2 epitopes and complementary negative charges on each antibody. A new model of phospholipid membrane association is also presented, where the 3E6 epitope faces the negatively charged membrane surface and Arg2320 is poised at the center of the binding interface. Furthermore, a 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain has also been determined, which supports the presented model for phospholipid binding. These results illustrate the complex nature of the polyclonal immune response against the factor VIII C2 domain, and further define the epitopes for both classical and non-classical inhibitory antibodies. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Characterization of Interaction of Factor VIII with Engineered Variants of a Single-Chain Variable Antibody Fragment

Blood ◽

10.1182/blood-2018-99-115455 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1170-1170

Author(s):

Svetlana A Shestopal ◽

Leonid A Parunov ◽

Mikhail V Ovanesov ◽

Timothy K Lee ◽

Andrey G Sarafanov

Keyword(s):

Factor Viii ◽

Half Life ◽

Conflicts Of Interest ◽

Antibody Fragment ◽

Density Lipoprotein ◽

Size Exclusion ◽

Single Chain ◽

Study Objective ◽

Thrombin Cleavage ◽

Fviii Activity

Abstract Introduction Replacement therapy for Hemophilia A requires frequent infusions of Factor VIII (FVIII) due to its relatively short half-life of ~12 h in plasma. Previous attempts to extend this half-life by genetic and chemical modification of FVIII met the barrier of ~20 h, which is a half-life of von Willebrand factor (VWF), a carrier of FVIII in plasma. A single-chain variable antibody fragment (scFv) KM33 was shown to inhibit FVIII activity, and interactions with VWF and the low-density lipoprotein receptor-related protein 1 (LRP), the major clearance receptor of FVIII (Bovenschen et al, 2005, Blood, 106:906-12). A study indicated that scFv KM33 may prolong the half-life of FVIII in mice to the level exceeding that of VWF half-life (Mertens et al, US Patent 2008, 20080219983A1). This would make scFv KM33 a promising tool for new designs of the longer-acting FVIII products. Study objective We aimed to generate a scFv KM33 variant that can delay FVIII clearance but can be removed from FVIII during its activation by thrombin. Such antibody fragment may extend the half-life of FVIII above that of VWF. Experimental design We generated three scFv KM33 variants with different linkers connecting the subunits VL and VH of the antibody fragment. The linkers contained variants of thrombin cleavage sites identical to those on FVIII. The proteins were expressed using a baculovirus system, purified by Ni-affinity and size exclusion chromatography (SEC), and tested for their properties. Results The engineered scFv variants, along with the unmodified KM33, were tested for binding to FVIII by surface plasmon resonance (SPR). All scFv versions demonstrated similar affinity for FVIII (~1 nM). In addition, a selected variant of scFv inhibited FVIII binding to LRP. These showed that the modifications of scFv did not affect its binding to FVIII. Thrombin treatment of the engineered scFv variants resulted in dissociation of their VL and VH domains, verified by SEC. However, the respective rates of thrombin cleavage were slower than that of FVIII. The preparation of a thrombin-cleaved scFv still inhibited the interaction of FVIII with LRP by SPR, similarly to that observed for the unmodified KM33. All variants of scFv inhibited FVIII activity in a thrombin generation assay suggesting that their moiety remained in complex with FVIII upon its activation. Conclusions The rate of thrombin cleavage of sites within FVIII is higher than that of the identical sites within the scFv. This suggests that additional determinants of FVIII (e. g. sulfated tyrosines adjacent to the sites) contribute to the higher rate of cleavage. The cleavage of the linker between the VL and VH subunits of scFv KM33 results in dissociation of the subunits and breakdown of the antibody fragment. This mechanism is likely applicable to any scFv, and may be useful in a broad range of applications involving such ligands. Both subunits of thrombin-cleaved scFv KM33, most likely, re-assemble on FVIII and form a tertiary complex FVIII/VL/VH. In turn, thrombin cleavage of the scFv, complexed with FVIII, does not result in its dissociation from FVIII. These indicate that in such design, an scFv should have lower affinity for FVIII to ensure its release from the complex. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Non-Classical Anti-Factor VIII C2 Domain Inhibitors Are Present in the Plasmas of Most Factor VIII Inhibitor Patients.

Blood ◽

10.1182/blood.v110.11.786.786 ◽

2007 ◽

Vol 110 (11) ◽

pp. 786-786

Author(s):

Shannon L. Meeks ◽

John F. Healey ◽

Rachel T. Barrow ◽

Ernest T. Parker ◽

Pete Lollar

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Factor Viii Inhibitor ◽

C2 Domain ◽

Proteolytic Activation ◽

Autoimmune Antibodies ◽

Fviii Inhibitor ◽

Fviii Activity ◽

Viii Inhibitor ◽

Murine Mabs

Abstract Approximately 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). The immune response to fVIII currently is the most significant complication in the management of patients with hemophilia A. In addition, autoimmune antibodies to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. These inhibitors primarily are directed against the A2 or C2 domains of fVIII. The human response to the C2 domain of fVIII classically has been thought to inhibit fVIII activity by blocking its binding to phospholipid. We recently characterized the antibody response to the C2 domain of human fVIII in a murine hemophilia model and described 5 structural groups of antibodies. Groups A, AB, and B are classical anti-C2 antibodies. Groups BC and C consist of non-classical anti-C2 antibodies that inhibit the proteolytic activation of fVIII but do not block the binding of fVIII to phospholipid. Most non-classical antibodies have inhibitor titers greater than 10,000 Bethesda units/mg IgG. To determine if non-classical antibodies are present in fVIII inhibitor patients, patient plasmas were tested in an ELISA for their ability to block the binding of representative antibodies from the different anti-human fVIII C2 antibody groups. Classical and non-classical monoclonal antibodies (MAbs) were biotinylated and serially diluted into either fVIII deficient plasma or patient inhibitor plasma and then added to microtiter wells coated with fVIII. The ability of patient plasma to block the binding of the murine MAbs to fVIII was determined. A total of 16 patient plasmas were assessed: 4 from patients with a C2 predominant response, 2 with a non-C2 predominant response, and 10 with unknown specificities. Three of the 4 patients with C2 predominant responses had non-classical anti-C2 antibodies, while the 2 with non-C2 predominant responses did not. In the unknown plasmas, 6 of 10 had evidence of non-classical antibodies. Figure 1 shows representative results of the effect of 3 patient plasmas on the binding of a biotinylated non-classical MAb to fVIII. Patient plasmas 1 and 2 blocked MAb binding while patient plasma 3 did not. This study indicates that the majority of patients with fVIII inhibitors have non-classical anti-C2 antibodies in their response to fVIII. Figure Figure

Download Full-text

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

Blood ◽

10.1182/blood.v114.22.27.27 ◽

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.

Download Full-text

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

Blood ◽

10.1182/blood.v118.21.2241.2241 ◽

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.

Download Full-text

Novel Techniques for Measurement of Variable Sized PF4/H Complexes.

Blood ◽

10.1182/blood.v120.21.2204.2204 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2204-2204

Author(s):

Benjamin Espinasse ◽

Manali Joglekar ◽

Giancarlo Valiente ◽

Gowthami M. Arepally

Keyword(s):

Flow Cytometry ◽

Electrostatic Interactions ◽

Conflicts Of Interest ◽

Correlation Spectroscopy ◽

Size Exclusion ◽

Size Determination ◽

Becton Dickinson ◽

Cationic Protein ◽

Platelet Factor ◽

Laser Illumination

Abstract Abstract 2204 Electrostatic interactions between Platelet factor 4 (PF4), a cationic protein, and heparin, an anionic carbohydrate result in the formation of ultra-large complexes (ULCs) that are immunogenic in mice (Suvarna, Blood 2007) and contribute to the immune pathogenesis of Heparin-induced thrombocytopenia (HIT). Previous studies (Rauova, Blood 2005; Greinacher, Arterioscler Thromb Vasc Biol, 2006) have shown that the size of ULCs is determined by the concentration and the molar ratios of PF4:H (PHRs) of each compound. Size determination of PF4/H complexes has been problematic due to technical limitations of two commonly employed methods for sizing complexes, photon correlation spectroscopy (PCS) and size exclusion chromatography (SEC). PCS is a technique for measuring particles in solution using laser illumination is based on principles of Brownian motion. PCS performs optimally with monodisperse populations and is biased by the presence of large aggregates. SEC, a liquid chromatography method, is technically cumbersome, requires sample labeling and not feasible for measuring large numbers of samples. To address these limitations, we examined two novel approaches for measuring a broad range of PF4/H complex size (100–3000 nm) in vitro: Nanosight and flow cytometry (FC). Nanosight (Nanosight Ltd, Wiltshire, United Kingdom),was employed for measuring small-sized complexes using physiologic concentrations of hPF4 (10 ug/mL). Nanosight uses proprietary software to track nanoparticles (range 10–1000nm) in solution by laser illumination with real-time tracking of the motion of individual particles by a camera. Analysis parameters provided by the software include: 1) Particle size distributions displayed as histograms 2) direct visualization of particles 3) particle counting and sizing and 4) particle scatter intensity vs. count and size. For measuring intermediate to large sized particles, formed at high hPF4 concentrations (95 ug/mL), we used flow cytometry calibrated with sizing beads on side scatter channel (SSC). FC was performed using a BD LSRII cell analyzer (Becton Dickinson, Franklin Lakes, NJ), a high throughput flow analyzer with the threshold channel for SSC set to 200 and a flow rate of 1 ul per second. The instrument was calibrated using sizing beads ranging from 0.3–6 μm in size (Figure A). For both techniques, PF4/H ULCs were formed by adding hPF4 (10 or 95 ug/mL)and various UFH concentrations in HBSS to yield the indicated PHRs. Complexes were incubated for 60 minutes and measured by NanoSight or FC. Results of experiments using Nanosight are shown in Table 1 with results showing size and particle counts for each PHR. Results of FC are shown in Figure B and Table 2 (median, 5% and 95% size in nm). Both studies showed reproducibility for measurements for a given concentration and showed changes in complex size as a function of PHR (Figure B). Both methodologies are technically simple and provide complementary approaches to PCS for PF4/H complex size determination. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Coagulation Factor VIII Is Synthesized In Endothelial Cells

Blood ◽

10.1182/blood.v122.21.26.26 ◽

2013 ◽

Vol 122 (21) ◽

pp. 26-26

Author(s):

Lesley Everett ◽

Audrey C.A. Cleuren ◽

Rami Khoriaty ◽

David Ginsburg

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Factor Viii ◽

Knockout Mice ◽

Conflicts Of Interest ◽

Coagulation Factor ◽

Fold Enrichment ◽

Fviii Activity ◽

Cargo Receptor ◽

Cellular Source

Abstract Combined deficiency of coagulation factors V and VIII (F5F8D) is an autosomal recessive bleeding disorder resulting from mutations in Lman1. This gene encodes a cargo receptor in the early secretory pathway that is responsible for the efficient secretion of factor V (FV) and factor VIII (FVIII) to the plasma. F5F8D is characterized by levels of both FV and FVIII reduced to ∼5-30% of normal. In contrast, Lman1 knockout mouse models of F5F8D exhibit FV and FVIII activities that are ∼50% of normal, relative to wildtype mice. Though FV and FVIII are synthesized at markedly different levels and potentially in different tissues, loss of the LMAN1 cargo receptor leads to parallel reductions in both FV and FVIII activity. FV is synthesized in hepatocytes (as well as megakaryocytes in the mouse). However, the primary cellular source of FVIII biosynthesis is controversial, with contradictory evidence supporting an endothelial or hepatocyte origin. We took advantage of the dependence of efficient FV and FVIII secretion on LMAN1 to examine the cellular source of each protein. FV and FVIII secretion profiles of conditional Lman1 knockout mice were characterized, relative to that of wildtype mice and ubiquitous Lman1 null mice (Lman1-/-). In order to generate mice with Lman1 expression specifically deleted in the endothelium or the hepatocytes, either a Tie2-Cre or Albumin-Cre transgene was crossed into Lman1 conditional mice (Lman1fl). FV and FVIII activity levels were measured by functional coagulation activity assays. Though Lman1fl/fl/Tie2-Cre+ mice (endothelial-specific knockout) exhibit normal plasma FV activity (99.9%) relative to wildtype mice (set to 100%), FVIII activity is reduced to 53.5% (p < 2.5 x 10-6). In contrast, Lman1fl/fl/Alb-Cre+ (hepatocyte-specific knockout) mice demonstrate normal FVIII activity (89.0%) and reduced FV activity (37.0%) (p < 1.4 x 10-10). To confirm endothelial cells as the biosynthetic source of FVIII, we took advantage of the previously reported RiboTag mouse (Sanz et al., 2009. PNAS 106(33):13939-44) to isolate endothelial cell RNA for qPCR analysis from various murine tissues. RiboTag mice carry a hemaglutinin-tagged ribosomal protein that can be used for cell-type specific immunoprecipitation of polyribosomes and subsequent RNA analysis when crossed with a Cre-recombinase expressing animal. qPCR analyses of endothelial cell RNA isolated from total liver lysates of five RiboTag/Tie2-Cre+ mice demonstrated 10-20 fold enrichment for gene transcripts that are known to be endothelial-specific, such as Cdhs (12.1 fold enrichment, p < 8.0 x 10-3), Vcam1 (13.4 fold enrichment, p <1.1 x 10-5), and Vwf (15.3 fold enrichment, p < 7.0 x 10-4), as well as for FVIII transcripts (11.4 fold enrichment, p < 4.0 x 10-5). In contrast, this analysis demonstrated a statistically significant depletion (5-10 fold) of transcripts from many known hepatocyte-specific genes, including multiple coagulation factor genes. Similar examination of kidney endothelial cell RNA also demonstrated enrichment for FVIII transcripts, thereby demonstrating that endothelial cells from multiple tissues and vascular beds contribute to the plasma FVIII pool in the mouse. These results explain the successful reversal of hemophilia A by both liver and kidney transplants. Taken together, these results definitively demonstrate that endothelial cells are the primary source of FVIII biosynthesis in the mouse, and that hepatocytes make no significant contribution to the plasma FVIII pool. Disclosures: No relevant conflicts of interest to declare.

Download Full-text