Stabilizing interactions between D666-S1787 and T657-Y1792 at the A2-A3 interface support factor VIIIa stability in the blood clotting pathway.

UNLABELLED: Essentials Factor VIIIa (FVIIIa) is unstable due to loss of A2; D666 and Y1792 contribute to its stability. We conducted a study to identify the interactions made at these residues at the A2-A3 interface. We present evidence for stabilizing interactions between D666-S1787 and T657-Y1792 in FVIIIa. A D666C/S1788C variant with a disulfide A2-A3 linkage has a FVIIIa decay rate that is 1% of wild-type. SUMMARY: Background Factor (F)VIIIa activity and stability depends on the non-covalent association of the A2 subunit with the A1/A3C1C2 dimer, but the interactions that contribute to A2 association are not well defined. Previous work had shown that D666A and Y1792F mutations at the A2-A3 interface resulted in increased FVIIIa decay, suggesting that the residues were involved in bonding interactions important for FVIIIa stability. Objectives Several potential hydrogen bonding partners of D666 and Y1792 across the A2-A3 interface were selected from the low-resolution FVIII crystal structure, and we used mutagenesis and biochemical analysis to examine the bonding interactions occurring at D666 and Y1792. Methods Using a series of stability and functional analyses, we analyzed FVIII variants in which D666 and Y1792 were each swapped with the residues of potential bonding partners. Results and conclusions We present evidence for hydrogen bonds between D666 and S1787 and between Y1792 and T657 that are important for FVIIIa stability. D666S/S1787D and T657Y/Y1792T variants each displayed wild-type (WT)-like FVIIIa stability and performed like WT FVIII in a series of functional analyses, whereas D666S, S1787D, and Y1792T single variants showed increased FVIIIa decay and a T657Y variant had little FVIIIa activity. These results suggest that WT hydrogen bonds are disrupted with the single mutations but maintained in the swap variants. Furthermore, mutation of D666 and S1788 to cysteine resulted in disulfide bond formation across the A2-A3 interface, confirming the close proximity of D666 and S1787, and this covalent attachment of the A2 subunit significantly increased FVIIIa stability.

Enhanced factor VIIIa stability of A2 domain interface variants results from an increased apparent affinity for the A2 subunit. Results from an increased apparent affinity for the A2 subunit.

Factor (F)VIIIa, a heterotrimer comprised of A1, A2, and A3C1C2 subunits, is labile due to the tendency of the A2 subunit to dissociate from the A1/A3C1C2 dimer. As dissociation of the A2 subunit inactivates FVIIIa activity, retention of A2 defines FVIIIa stability and thus, FXase activity. Earlier results showed that replacing residues D519, E665, and E1984 at the A2 domain interface with Ala or Val reduced rates of FVIIIa decay, increasing FXa and thrombin generation. We now show the enhanced FVIIIa stability of these variants results from increases in inter-A2 subunit affinity. Using a FVIIIa reconstitution assay to monitor inter-subunit affinity by activity regeneration, the apparent Kd value for the interaction of wild-type (WT) A2 subunit with WT A1/A3C1C2 dimer (43 ± 2 nM) was significantly higher than values observed for the A2 point mutants D519A/V, E665A/V, and E1984A/V which ranged from ~5 to ~19 nM. Val was determined to be the optimal hydrophobic residue at position 665 (apparent Kd = 5.1 ± 0.7 nM) as substitutions with Ile or Leu at this position increased the apparent Kd value by ~3- and ~7-fold, respectively. Furthermore, the double mutant (D519V/E665V) showed an ~47-fold lower apparent Kd value (0.9 ± 0.6 nM) than WT. Thus these hydrophobic mutations at the A2 subunit interfaces result in high binding affinities for the A2 subunit and correlate well with previously observed reductions in rates in FVIIIa decay.

Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa.

FVIIIa is labile due to the dissociation of A2 subunit. Previously, we introduced hydrophobic mutations at select A1/A2/A3 subunit interfaces yielding more stable FVIII(a) variants. Separately we showed that altering the sequence flanking the primary FXa cleavage site in FVIIIa (Arg336) yielded reduced rates of proteolytic inactivation of FVIIIa. In this study we prepared the FXa-cleavage resistant mutant (336(P4-P3')562) combined with mutations of Ala108Ile, Asp519Val/Glu665Val or Ala108Ile/Asp519Val/Glu665Val and examined the effects of these combinations relative to FVIII thermal stability, rates of FVIIIa decay and proteolytic inactivation of FVIIIa by FXa. Thermal decay rates for 336(P4-P3')562/Ala108Ile, 336(P4-P3')562/Asp519Val/Glu665Val, and 336(P4-P3')562/Ala108Ile/Asp519Val/Glu665Val variants were reduced by ~2- to 5-fold as compared with wild-type (WT) primarily reflecting the effects of the A domain interface mutations. FVIIIa decay rates for 336(P4-P3')562/Asp519Val/Glu665Val and 336(P4-P3')562/Ala108Ile/Asp519Val/Glu665Val variants were reduced by ~25 fold, indicating greater stability than the control Asp519Val/Glu665Val variant (~14-fold). Interestingly, 336(P4-P3')562/Asp519Val/Glu665Val and 336(P4-P3')562/Ala108Ile/Asp519Val/Glu665Val variants showed reduced FXa-inactivation rates compared with the 336(P4-P3')562 control (~4-fold), suggesting A2 subunit destabilisation is a component of proteolytic inactivation. Thrombin generation assays using the combination variants were similar to the Asp519Val/Glu665Val control. These results indicate that combining multiple gain-of-function FVIII mutations yields FVIII variants with increased stability relative to a single type of mutation.

Use of affinity-directed liquid chromatography-mass spectrometry to map the epitopes of a factor VIII inhibitor antibody fraction.

BACKGROUND: Neutralizing factor (F) VIII antibodies develop in approximately 30% of individuals with hemophilia A and show specificity to multiple sites in the FVIII protein. METHODS: Reactive epitopes to an immobilized IgG fraction prepared from a high-titer, FVIII inhibitor plasma were determined after immuno-precipitation (IP) of tryptic and chymotryptic peptides derived from digests of the A1 and A2 subunits of FVIIIa and FVIII light chain. Peptides were detected and identified using highly sensitive liquid chromatography-mass spectrometry (LC-MS). RESULTS: Coverage maps of the A1 subunit, A2 subunit and light chain represented 79%, 69% and 90%, respectively, of the protein sequences. Dot blots indicated that the inhibitor IgG reacted with epitopes contained within each subunit of FVIIIa. IP coupled with LC-MS identified 19 peptides representing epitopes from all FVIII A and C domains. The majority of peptides (10) were derived from the A2 domain. Three peptides mapped to the C2 domain, while two mapped to the A1 and A3 domains, and single peptides mapped to the a1 segment and C1 domain. Epitopes were typically defined by peptide sequences of < 12 residues. CONCLUSIONS: IP coupled with LC-MS identified extensive antibody reactivity at high resolution over the entire functional FVIII molecule and yielded sequence lengths of < 15 residues. A number of the peptides identified mapped to known sequences involved in functionally important protein-protein and protein-membrane interactions.

Combining mutations of charged residues at the A2 domain interface enhances factor VIII stability over single point mutations.

BACKGROUND: Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. Recently we reported that procofactor stability at elevated temperature and cofactor stability over an extended time course were increased following replacement of individual charged residues (Asp(D)519, Glu(E)665 or Glu(E)1984) with either Ala (A) or Val (V) (Wakabayashi et al., Blood, 112, 2761, 2008). OBJECTIVES: In the current study we generated combination mutants at these three sites to examine any additive and/or synergistic effects of these mutations on stability. METHODS: Studies assessing FVIII stability involved monitoring decay rates of FVIII at 55 degrees C or in guanidinium, decay of FVIIIa following A2 subunit dissociation, and thrombin generation at low (0.3 nmol L(-1)) FVIII concentration. RESULTS AND CONCLUSIONS: Similar tendencies were observed within each group of variants. Variants with mutations at D519 and either E665 or E1984 (Group A) generally showed significantly better stability as compared with single mutants. Most variants with mutations at E665 and at E1984 (Group B) did not show significant improvement. Triple mutants with mutations at D519, E665 and E1984 (Group C) showed improvement to a similar degree as the Group A double mutants. Overall, these results indicate that selected combinations of mutations to reduce charge and/or increase hydrophobicity at the A2/A1 and A2/A3 domain interfaces yield FVIII reagents with improved stability parameters.

A3 domain residue Glu1829 contributes to A2 subunit retention in factor VIIIa.

BACKGROUND: Factor VIII (FVIII) is activated by thrombin to the labile FVIIIa, a heterotrimer of A1, A2 and A3C1C2 subunits, which serves as a cofactor for FIXa. A primary reason for the instability of FVIIIa is the tendency for the A2 subunit to dissociate from FVIIIa leading to an inactive cofactor and consequent loss of FXase activity. OBJECTIVE: Based on our finding of low-specific activity and a fast decay rate for a FVIII point mutation of Glu1829 to Ala (E1829A), we examined whether residue Glu1829 in the A3 subunit is important for A2 subunit retention. RESULTS: The rate of activity decay of E1829A was approximately fourteen fold faster than wild-type (wt) FVIIIa and this rate was reduced in the presence of added A2 subunit. Specific activity values for E1829A measured by one-stage and two-stage assays were approximately 14% and approximately 11%, respectively, compared with wt FVIII. Binding affinity for the A1 subunit to E1829A-A3C1C2 was comparable to wt A3C1C2 (K(d) = 20.1 +/- 3.4 nM for E1829A, 15.3 +/- 3.7 nM for wt), whereas A2 subunit affinity for the A1/A3C1C2 dimer forms was reduced by approximately 3.6-fold as a result of the mutation (K(d) = 526 +/- 107 nM for E1829A, 144 +/- 21 nM for wt). CONCLUSION: As modeling data suggest that Glu1829 is located at the A2-A3 domain interface these results are consistent with Glu1829 contributing to the interactions involved with A2 subunit retention in FVIIIa.

Epitope mapping factor VIII A2 domain by affinity-directed mass spectrometry: residues 497-510 and 584-593 comprise a discontinuous epitope for the monoclonal antibody R8B12.

The murine monoclonal antibody R8B12 recognizes the C-terminal region (residues 563-740) of the A2 subunit of factor VIIIa [J Biol Chem 266: 1991; p. 20139], as judged by Western blotting. However, the location of the epitope within this region is not known. In the present study, we used affinity-directed mass spectrometry to map the epitope. A2 subunit was digested with trypsin or chymotrypsin and then subjected to immunoprecipitation (IP) using R8B12 IgG. Masses of the affinity-selected peptides were determined directly from the immune complexes by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Proteolysis of A2 with the two proteases generated a pre-IP peptide fingerprint that covered approximately 70% of the A2 domain sequence. Analysis of the post-IP tryptic peptide fingerprint showed two masses, 1309 and 1653 Da representing residues 584-593 and 497-510, respectively, determined from a theoretical database search and confirmed by direct sequencing. Results using a chymotryptic digest yielded a single, weakly reactive fragment consistent with residues 577-586, suggesting the importance of residues Ser584-Tyr586 in forming the epitope. A synthetic peptide to residues 584-593 was immunoprecipitated by the IgG and blocked R8B12-directed blotting to A2 subunit. The 497-510 and 584-593 segments were observed to be adjacent and surface exposed in the A2 domain model, and together with the above results suggest that A2 domain residues 497-510 and 584-593 represent a discontinuous epitope for R8B12. Furthermore, based upon blotting specificity, we speculate that residues 584-593 make a substantially greater contribution to the binding energy for this interaction.

Clustered basic residues within segment 484-510 of the factor VIIIa A2 subunit contribute to the catalytic efficiency for factor Xa generation.

Residues 484-510 of factor (F)VIIIa A2 subunit comprise a prominent epitope for inhibitor antibodies, suggesting that this region is critical for cofactor function. To address the role of this region in catalysis, FVIIIa forms were evaluated following conversion of conserved charged residues to Ala, either in clusters or individually. The two cluster mutants, Lys496Ala/Lys499Ala/Asp500Ala and Glu507Ala/Lys510Ala, were indistinguishable from wild type. The mutation Arg489Ala/Arg490Ala/Lys493Ala (489-3A) possessed near-normal affinity for FIXa and showed no effect on the Km for FX, but exhibited approximately 3-fold and approximately 30-fold reduced kcat values for FXase in the presence and absence of surface, respectively. However, the single-site mutants Arg489Ala, Arg490Ala and Lys493Ala exhibited affinity and kcat values similar to wild type. Furthermore, the 489-3A mutant showed a marked reduction in the positive electrostatic potential within this region of A2, consistent with the hypothesis that the cumulative basic charge in this region of A2 subunit modulates cofactor function.

Metal ion-independent association of factor VIII subunits and the roles of calcium and copper ions for cofactor activity and inter-subunit affinity.

Factor VIII circulates as a divalent metal ion-dependent heterodimer comprised of a light chain (LC) and a heavy chain (HC). Reassociation of factor VIII subunits was assessed using fluorescence energy transfer where LC and HC were labeled with acrylodan (Ac; fluorescence donor) and fluorescein-5-maleimide (Fl; fluorescence acceptor), respectively. The reduction of donor fluorescence due to the acceptor was used as an indicator of binding. Subunits associated with high affinity (K(d) = 53.8 nM) in the absence of metal ion and presence of EDTA. However, this product showed no cofactor activity, as measured by a factor Xa generation assay. In the presence of 25 mM Ca(2+), no increase in the intersubunit affinity was observed (K(d) = 48.7 nM) but specific activity of the cofactor was approximately 30% that of native factor VIII. At saturating levels of Fl-HC relative to Ac-LC, donor fluorescence decreased to 79.3 and 73.5% of its original value in the absence and presence of Ca(2+), respectively. Thrombin cleaved the heterodimers that were associated in the absence or presence of Ca(2+) with similar efficiency, indicating that the lack of activity was not the result of a defect in activation. Cu(2+) (0.5 microM) increased the intersubunit affinity by approximately 100 fold (K(d) = 0.52 nM) and the specific activity to approximately 60% of native factor VIII. The former effect was independent of Ca(2+), whereas the latter effect required Ca(2+). These results indicate that the intersubunit association in factor VIII is primarily metal-ion independent while divalent metal ions serve specific roles. Ca(2+) appears essential to promote the active conformation of factor VIII while Cu(2+) primarily enhances the intersubunit affinity.

The kissing hairpin sequence promotes recombination within the HIV-I 5' leader region.

The role of RNA-RNA template interactions in facilitating recombination during reverse transcription of minus strand DNA has been examined. The tested hypothesis is that template switching by reverse transcriptase is promoted at sites where homologous regions of two RNAs are brought in close proximity via stable intertemplate interactions. Frequency and distribution of template switching between homologous donor and acceptor RNAs were examined within the human immunodeficiency virus type I (HIV-I) 5'-untranslated region (UTR) containing the dimer initiation sequence (DIS). Results were compared with control nondimerizing templates from the pol region. The dimerizing UTR templates displayed a 4-fold higher transfer efficiency than the control. A striking 53% of transfers in the UTR mapped near the DIS, of which two-thirds occurred immediately 5' to this sequence. In the UTR template, deletion of the DIS hairpin disrupted template dimerization and caused a 4-fold drop in transfer efficiency. Insertion of the DIS within the pol template increased both dimerization and transfer efficiency. Transfer distributions revealed that in both sets of templates, DIS-induced dimerization increased the efficiency of transfers across the whole template, with the transfers peaking around the dimerization site. Overall, these results suggest that template dimerization facilitated by the unique geometry of the DIS-promoted kissing interactions effectively promotes recombination within the HIV-I 5'-UTR.

Factor VIIIa cofactor activity shows enhanced ionic strength sensitivity in the absence of phospholipid.

Factor VIIIa, a cofactor for the protease factor IXa, is a trimer of A1, A2 and A3-C1-C2 subunits. In the absence of phospholipid (PL), the k(cat) for factor VIIIa-dependent, factor IXa-catalyzed conversion of factor X was markedly less than that observed in the presence of PL (approx. 150 min(-1)) and decreased as the ionic strength of the reaction increased. At low salt concentration, the k(cat) (5.5 min(-1)) was approx. 8-fold greater than observed at near physiologic ionic strength (0.7 min(-1)). However, this level of salt showed minimal effects on the intermolecular affinities of factor VIIIa (or isolated A2 subunit) for factor IXa or on the K(m) for factor X. Alternatively, the association of A2 subunit with A1 subunit was sensitive to increases in salt and paralleled the reduction in k(cat) observed with factor VIIIa. This instability was not observed in PL-containing reactions. Fluorescence energy transfer between acrylodan-A2 and fluorescein-A1/A3-C1-C2 dimer showed a requirement for both PL and factor IXa for maximal association of A2 with dimer. These results indicate that in the presence of factor IXa, the salt-dependent dissociation of factor VIIIa subunits is significantly enhanced in the absence of PL, promoting a reduced k(cat) for the cofactor-dependent generation of factor Xa.

Cleavage of factor VIII heavy chain is required for the functional interaction of a2 subunit with factor IXA.

Factor VIII circulates as a noncovalent heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B domains) and light chain (LC). Cleavage of HC at the A1-A2 and A2-B junctions generates the A1 and A2 subunits of factor VIIIa. Although the isolated A2 subunit stimulates factor IXa-catalyzed generation of factor Xa by approximately 100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa and X resulted in an increase in factor IXa activity because of conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of approximately 1% observed for thrombin. HC showed little inhibition of the A2 subunit-dependent stimulation of factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa in the presence of factor X, whereas thrombin-cleaved HC yielded a marked increase in this parameter. These results indicate that HC cleavage by either thrombin or factor Xa is essential to expose the factor IXa-interactive site(s) in the A2 subunit required to modulate protease activity.

Factor IXa:factor VIIIa interaction. helix 330-338 of factor ixa interacts with residues 558-565 and spatially adjacent regions of the a2 subunit of factor VIIIa.

The physiologic activator of factor X consists of a complex of factor IXa, factor VIIIa, Ca(2+) and a suitable phospholipid surface. In one study, helix 330 (162 in chymotrypsin) of the protease domain of factor IXa was implicated in binding to factor VIIIa. In another study, residues 558-565 of the A2 subunit of factor VIIIa were implicated in binding to factor IXa. We now provide data, which indicate that the helix 330 of factor IXa interacts with the 558-565 region of the A2 subunit. Thus, the ability of the isolated A2 subunit was severely impaired in potentiating factor X activation by IXa(R333Q) and by a helix replacement mutant (IXa(helixVII) in which helix 330-338 is replaced by that of factor VII) but it was normal for an epidermal growth factor 1 replacement mutant (IXa(PCEGF1) in which epidermal growth factor 1 domain is replaced by that of protein C). Further, affinity of each 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Glu-Gly-Arg-IXa (dEGR-IXa) with the A2 subunit was determined from its ability to inhibit wild-type IXa in the tenase assay and from the changes in dansyl fluorescence emission signal upon its binding to the A2 subunit. Apparent K(d(A2)) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) approximately 100 nm, dEGR-IXa(R333Q) approximately 1.8 micrometer, and dEGR-IXa(helixVII) >10 micrometer. In additional experiments, we measured the affinities of these factor IXa molecules for a peptide comprising residues 558-565 of the A2 subunit. Apparent K(d(peptide)) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) approximately 4 micrometer, and dEGR-IXa(R333Q) approximately 62 micrometer. Thus as compared with the wild-type or PCEGF1 mutant, the affinity of the R333Q mutant for the A2 subunit or the A2 558-565 peptide is similarly reduced. These data support a conclusion that the helix 330 of factor IXa interacts with the A2 558-565 sequence. This information was used to model the interface between the IXa protease domain and the A2 subunit, which is also provided herein.

Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H.

HIV-1 reverse transcriptase (RT) degrades the plus strand viral RNA genome while synthesizing the minus strand of DNA. Many RNA fragments, including the polypurine tracts, remain annealed to the new DNA. Several RTs are believed to bind after synthesis to degrade all RNA fragments except the polypurine tracts by a polymerization-independent mode of RNase H activity. For this latter process, we found that RT positions the RNase H active site approximately 18 nt from the 5' end of the RNA, making the primary cut. The enzyme rebinds or slides toward the 5' end of the RNA to make a secondary cut creating two products 8-9 nt long. RT then binds the new 5' end of the RNA created by the first primary or the secondary cuts to make the next primary cut. In addition, we observed another type of RNase H cleavage specificity. RT aligns the RNase H active site to the 3' end of the RNA, cutting 5 residues in. We determined the relative rates of these cuts, defining their temporal order. Results show that the first primary cut is fastest, and the secondary and 5-nt cuts occur next at similar rates. The second primary cuts appear last. Based on these results, we present a model by which RT progressively cleaves RNA fragments.

The sequential mechanism of HIV reverse transcriptase RNase H.

Synthesis of the minus strand of viral DNA by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase is accompanied by RNase H degradation of the viral RNA genome. RNA fragments remain after synthesis and are degraded by the polymerase-independent mode of RNase H cleavage. Recently, we showed that this mode of cleavage occurs by a specific ordered mechanism in which primary cuts are first, secondary and 5-nucleotide cuts are next, and second primary cuts occur last (Wisniewski, M., Balakrishnan, M., Palaniappan, C., Fay, P., J., and Bambara, R., A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 11978-11983). Ultimately the RNAs are cleaved into small fragments that can dissociate from the DNA template. Because the cleavage mechanism is an ordered series of events, we determined in this study whether any earlier cut is required for a later cut. By precisely inhibiting cleavage at each site, we examined the dependence of later cuts on cleavage at that site. We found that each cut is independent of the other cuts, demonstrating that the order of this stepwise mechanism is based on the rates of each cut. A mechanism for unlinked ordered cleavage consistent with these results is presented.

Sites in the A2 subunit involved in the interfactor VIIIa interaction.

Factor VIIIa is a trimer of the A1, A2, and A3-C1-C2 subunits. Regions in the A2 subunit that interact with the A1/A3-C1-C2 dimer were localized using synthetic peptides derived from A2 sequences showing high probability of being surface exposed. Peptides were restricted to residues 373-562 of A2 based on the earlier observation that this region of A2 reacts with A1 using a zero length cross-linker. Peptides were assessed for their capacity to inhibit the reconstitution of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit. Reconstitution was monitored using both regeneration of factor VIIIa activity and fluorescence quenching of an acrylodan-labeled A2 (Ac-A2) by fluorescein-labeled A1/A3-C1-C2. The activity assay identified four peptides as inhibitors, residues 373-395 (IC(50) = 65 micrometer), 418-428 (IC(50) = 25 micrometer), 482-493 (IC(50) = 325 micrometer), and 518-533 (IC(50) = 585 micrometer). The 373-395 and 518-533 peptides eliminated the fluorescence quenching of Ac-A2, whereas the 418-428 peptide reduced but did not eliminate Ac-A2 quenching. Peptide 482-493 had no effect on the fluorescence quenching of Ac-A2 suggesting that the peptide did not directly affect reassociation of the factor VIIIa subunits. These results identify three regions in the A2 subunit (373-395, 418-428, and 518-533) that interact with the A1/A3-C1-C2 dimer. Furthermore, comparison of results obtained using the two assays distinguish inhibition of the intersubunit interactions from intermolecular interactions.

Regulation of factor VIIIa by human activated protein C and protein S: inactivation of cofactor in the intrinsic factor Xase.

Factor VIIIa is a trimer of A1, A2, and A3-C1-C2 subunits. Inactivation of the cofactor by human activated protein C (APC) results from preferential cleavage at Arg336 within the A1 subunit, followed by cleavage at Arg562 bisecting the A2 subunit. In the presence of human protein S, the rate of APC-dependent factor VIIIa inactivation increased several-fold and correlated with an increased rate of cleavage at Arg562. (Active site-modified) factor IXa, blocked cleavage at the A2 site. However, APC-catalyzed inactivation of factor VIIIa proceeded at a similar rate independent of factor IXa, consistent with the location of the preferential cleavage site within the A1 subunit. Addition of protein S failed to increase the rate of cleavage at the A2 site when factor IXa was present. In the presence of factor X, cofactor inactivation was inhibited, due to a reduced rate of cleavage at Arg336. However, inclusion of protein S restored near original rates of factor VIIIa inactivation and cleavage at the A1 site, thus overcoming the factor X-dependent protective effect. These results suggest that in the human system, protein S stimulates APC-catalyzed factor VIIIa inactivation by facilitating cleavage of A2 subunit (an effect retarded in the presence of factor IXa), as well as abrogating protective interactions of the cofactor with factor X. (Blood. 2000;95:1714-1720)

Human inhibitor antibodies specific for the factor VIII A2 domain disrupt the interaction between the subunit and factor IXa.

Factor VIIIa, a heterotrimer of the A1, A2, and A3-C1-C2 subunits, increases the catalytic efficiency for factor IXa-catalyzed activation of factor X. A significant fraction of naturally occurring, anti-factor VIII inhibitor antibodies reacts with the A2 domain. Utilizing the capacity for isolated A2 subunit to stimulate factor IXa activity, we show that a panel of these inhibitors block this activity. Inhibition of activity parallels the antibody potency as measured in the Bethesda assay. These antibodies also block the A2-dependent increases in fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa. Similar to the IgG fractions, a peptide representing the sequence of the inhibitor epitope (A2 residues 484-509) blocked the A2-dependent stimulation of factor IXa. These results indicate that antibodies possessing this specificity directly inhibit the interaction of A2 subunit with factor IXa, thus abrogating the contribution of this subunit to cofactor activity. Furthermore, these results also suggest that factor VIII residues 484-509 contribute to a factor IXa-interactive site.