Abstract
Abstract 3355
Introduction:
Development of antibodies against factor VIII is a common complication of therapy for hemophilia A and can cause acquired hemophilia A in previously normal subjects. Dominant epitopes for inhibitory antibodies reside on the C2 domain of factor VIII, which has been shown to be important for membrane binding. Although acquired hemophilia A is associated with a prolonged activated partial thromboplastin time (aPTT), the relative bleeding risk does not correlate well with factor VIII activity levels. Thus there is a need for basic insights that explain the discrepancies between factor VIII activity values and bleeding risk in these patients.
mAb ESH4, directed against the factor VIII C2 domain, interferes with membrane binding and is a prototypic factor VIII inhibitor. ESH4 is a type II inhibitor, with residual factor VIII activity in the presence of saturating antibody concentrations. Thus, exploration of the inhibitory mechanism of ESH4 may offer insights into bleeding risk assessment of antibodies that inhibit phospholipid binding.
Methods:
Binding of fluorescein-labeled factor VIII to phospholipid membranes supported on glass microspheres was measured in the presence and absence of ESH4 using flow cytometry. The effect of ESH4 on factor VIII activity was measured using a two-stage amidolytic factor Xase assay and sonicated lipid vesicles with either low (4%) or high (15%) levels of PS. The factor Xase assay was also performed using platelets as the phospholipid source. Factor IXa, with its active site labeled using fluorescein-EGR chloromethyl ketone (Fl-EGRck), was mixed with sonicated phospholipid vesicles, factor VIIIa, and factor × and the anisotropy of the fluorescein molecule was measured to test the effect of ESH4 on the factor IXa active site.
Results:
Saturating concentrations of ESH4 inhibited 40% of factor VIII activity in a commercial aPTT assay. In a defined assay, inhibition of factor VIII activity was directly related to the phospholipid composition and concentration. In the presence of saturating phospholipid and factor X, ESH4 caused over 60% decrease in the Vmax for vesicles with either 4% or 15% PS. To determine the mechanism through which ESH4 inhibits membrane-bound factor VIII activity, we measured the fluorescence anisotropy of factor IXa-Fl-EGRck. ESH4 decreased anisotropy of the factor VIIIa-factor IXa-factor × complex from 0.280 ± 0.002 to 0.272 ± 0.002 on 15% PS vesicles and from 0.275 ± 0.002 to 0.262 ± 0.001 on 4% PS vesicles, indicating that ESH4 alters the Vmax through a change near the factor IXa active site, remote from the C2 domain-membrane interface. ESH4 decreased the apparent affinity 4-fold for membranes of 4% PS (KD = 4.8 ± 0.4 mM without and 21 ± 4 mM with ESH4) but only 2-fold on 15% PS vesicles (KD = 1.3 ± 0.2 mM without and 2.5 ± 0.5 mM with ESH4). Direct membrane binding studies of fluorescein-labeled factor VIII indicated a reduction in affinity and number of binding sites consistent with the results from the factor Xase assay. The apparent affinity for factor × in the presence of saturating phospholipid and ESH4 was higher on 15% PS vesicles (KM = 129 ± 18 nM) than on 4% PS vesicles (KM= 284 ± 30 nM). Together, these results indicate that ESH4 can decrease factor VIII activity through three mechanisms: (1) decreased membrane affinity (2) decreased activity of membrane-bound factor VIII and (3) differential affinity of the factor Xase complex for factor X. Because two of these mechanisms are influenced by membrane composition we asked whether the degree of inhibition by ESH4 might differ on platelets stimulated to different degrees. Platelets stimulated by thrombin express limited PS in a reversible manner while platelets stimulated by > 1 μM A23187 have complete PS exposure. ESH4 showed 80% inhibition of Xase activity on platelets stimulated with thrombin vs. 40% inhibition on platelets stimulated with A23187, similar to the aPTT assay.
Conclusions:
Our results indicate that ESH4 disruption of factor VIII C2 domain engagement with the membrane has a remote effect at the factor IXa active site. Inhibition of factor VIII activity by ESH4 is sensitive to membrane composition and concentration through two mechanisms. These results highlight the need to better understand how membrane binding activates the factor VIIIa-factor IXa complex and to develop clinical assays that measure factor VIII activity on clinically relevant membrane types and concentrations.
Disclosures:
No relevant conflicts of interest to declare.
Factor VIII cross-matches to the human proteome reduce the predicted inhibitor risk in missense mutation hemophilia A
