Characterization of the interaction between the A2 subunit and A1/A3-C1-C2 dimer in human factor VIIIa.

Factor VIIIa is a heterotrimer of the factor VIII heavy chain-derived A1 and A2 subunits plus the factor VIII light chain-derived A3-C1-C2 subunit. While the A1 and A3-C1-C2 subunits can be isolated as a stable dimer, the A2 subunit is weakly associated with the dimer. In the human protein, the association of A2 with dimer is reversible and governed by a pH-dependent dissociation constant. Using the specific activity of factor VIIIa as an indicator of trimer concentration, the Kd (pH 6.0) was determined to be 28 nM whereas at the more physiologic pH (pH 7.4) this value was approximately 260 nM. Results from pH shift experiments confirmed the reversible binding of A2 to dimer as did the capacity for high levels of exogenous A2 subunit to inhibit the spontaneous decay of factor VIIIa activity. A2 subunit associated with the A1 subunit in the A1/A3-C1-C2 dimer based upon the capacity for free A1 subunit to inhibit the reconstitution of factor VIIIa from A2 subunit and dimer. These results indicate that the primary mechanism for the spontaneous decay of human factor VIIIa is the reversible dissociation of A2 subunit from the A1 subunit of the A1/A3-C1-C2 dimer.     

Human factor VIIIa subunit structure. Reconstruction of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit

Heterodimeric human factor VIII was proteolytically activated by catalytic levels of thrombin to yield the (labile) active cofactor factor VIIIa possessing an initial specific activity of approximately 80 units/microgram. Activation paralleled the generation of fragments A1 and A2 derived from the heavy chain and A3-C1-C2 derived from the light chain. Chromatography of factor VIIIa, on Mono-S buffered at pH 6.0 resulted in separation of the bulk of the A2 fragment from a fraction composed predominantly of A1/A3-C1-C2 dimer plus low levels of A2 fragment. Only the latter fraction contained clotting activity (approximately 20 units/microgram) which was stable and represented a less than 10% yield when compared with the peak activity of unfractionated factor VIIIa. Further depletion of A2 fragment from Mono-S-purified factor VIIIA, achieved using an immobilized monoclonal antibody to the A2 domain, yielded a relatively inactive A1/A3-C1-C2 dimer (less than 0.4 unit/microgram). Factor VIIIa (greater than 40 units/microgram) was reconstituted from the A1/A3-C1-C2 dimer plus the A2 fragment in a reaction that was Me(2+)-independent and inhibited by moderate ionic strength. Reassociation of A2 required the A1 subunit in that the A2 subunit associated weakly if at all to A3-C1-C2 in the absence of A1. These results indicated that human factor VIIIa is a trimer represented by the subunits A1/A2/A3-C1-C2 and that the A2 subunit is required for expression of factor VIIIa activity.     

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.     

Factor IXa enhances reconstitution of factor VIIIa from isolated A2 subunit and A1/A3-C1-C2 dimer.

Heterotrimeric factor VIIIa was reconstituted from isolated A2 subunit and A1/A3-C1-C2 dimer of thrombin-activated human factor VIII in a reaction that was sensitive to pH. Maximal levels of reconstituted factor VIIIa at pH 6.0 were as much as 20-fold greater than were values observed at pH 7.5. The presence of factor IXa and phospholipid resulted in a marked increase in factor VIIIa reconstituted at physiologic pH. However, the resultant factor VIIIa was unstable due to slow proteolysis of the A1 subunit. Factor IXa modified by the active site-specific reagent dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone (DEGR-IXa) increased the level of factor VIIIa reconstituted from subunits to a similar extent as was observed for unmodified factor IXa and yielded stable factor VIIIa. This enhancement was saturated above a 1:1 molar ratio of DEGR-IXa to factor VIIIa subunits and could be blocked by an anti-factor IX antibody, suggesting that the DEGR-IXa-dependent increase in factor VIIIa reconstitution correlated with assembly of the factor X-ase complex. At a saturating amount of DEGR-IXa, the level of factor VIIIa reconstitution at pH 7.5 approached values obtained at pH 6.0. Fluorescence polarization measurements indicated that factor VIIIa altered binding of DEGR-IXa to phospholipid. However, neither the A2 subunit nor the A1/A3-C1-C2 dimer alone produced this effect. This result suggested that both A2 and A1/A3-C1-C2 were necessary for association of the cofactor with factor IXa. These results suggest a model in which assembly of the intrinsic factor X-ase complex stabilizes factor VIIIa through inhibition of subunit dissociation.     

Cofactor activities of factor VIIIa and A2 subunit following cleavage of A1 subunit at Arg336.

Factor VIIIa consists of three subunits designated A1, A2, and A3-C1-C2. The isolated A2 subunit possesses limited cofactor activity in stimulating factor IXa-catalyzed activation of factor X. This activity is markedly enhanced by the A1 subunit (inter-subunit K(d) = 1.8 microm). The C-terminal region of A1 subunit (residues 337-372) is thought to represent an A2-interactive site. This region appears critical to factor VIIIa, because proteolysis at Arg(336) by activated protein C or factor IXa is inactivating. A truncated A1 (A1(336)) showed similar affinity for A2 subunit (K(d) = 0.9 microm) and stimulated its cofactor activity to approximately 50% that observed for native A1. However, A1(336) was unable to reconstitute factor VIIIa activity in the presence of A2 and A3-C1-C2 subunits. Fluorescence anisotropy of fluorescein (Fl)-FFR-factor IXa was differentially altered by factor VIIIa trimers containing either A1 or A1(336). Fluorescence energy transfer demonstrated that, although Fl-A1(336)/A3-C1-C2 bound acrylodan-A2 with similar affinity as the native dimer, an increased inter-fluorophore separation was observed. These results indicate that the C-terminal region of A1 appears necessary to properly orient A2 subunit relative to factor IXa in the cofactor rather than directly stimulate A2 and elucidate the mechanism for cofactor inactivation following cleavage at this site.     

Altered interactions between the A1 and A2 subunits of factor VIIIa following cleavage of A1 subunit by factor Xa

Factor VIIIa consists of subunits designated A1, A2, and A3-C1-C2. The limited cofactor activity observed with the isolated A2 subunit is markedly enhanced by the A1 subunit. A truncated A1 (A1(336)) was previously shown to possess similar affinity for A2 and retain approximately 60% of its A2 stimulatory activity. We now identify a second site in A1 at Lys(36) that is cleaved by factor Xa. A1 truncated at both cleavage sites (A1(37-336)) showed little if any affinity for A2 (K(d)>2 microm), whereas factor VIIIa reconstituted with A2 plus A1(37-336)/A3-C1-C2 dimer demonstrated significant cofactor activity ( approximately 30% that of factor VIIIa reconstituted with native A1) in a factor Xa generation assay. These affinity values were consistent with values obtained by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1. In contrast, factor VIIIa reconstituted with A1(37-336) showed little activity in a one-stage clotting assay. This resulted in part from a 5-fold increase in K(m) for factor X when A1 was cleaved at Arg(336). These findings suggest that both A1 termini are necessary for functional interaction of A1 with A2. Furthermore, the C terminus of A1 contributes to the K(m) for factor X binding to factor Xase, and this parameter is critical for activity assessed in plasma-based assays.     

Localization of a pH-dependent, A2 subunit-interactive surface within the factor VIIIa A1 subunit

Factor VIIIa can be reconstituted from A2 subunit and A1/A3-C1-C2 dimer in a reaction that is facilitated by slightly acidic pH. We recently demonstrated that a truncated A1 (A1(37-336)) possessed markedly reduced affinity for A2 compared with intact A1, but retained 30% of native factor VIIIa activity in the presence of A3-C1-C2. We now identify A1-interactive regions for A2 using A1 fragments derived from a limited tryptic digest. Unfractionated trypsin-cleaved A1 inhibited reconstituted factor VIIIa activity. Two fragments, designated A1(37-121) and A1(221-336), markedly inhibited factor VIIIa reconstitution with either native A1 (K(i)=340 and 194 nM, respectively) or with A1(37-336) (K(i)=69 and 116 nM, respectively) at pH 6.0. A third fragment designated A1(122-206) did not possess inhibitory activity. At pH 7.2, the A1(221-336) partially inhibited reconstitution, whereas the A1(37-121) possessed little if any inhibitory activity. Both fragments inhibited factor VIIIa reconstitution as judged by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1 forms at pH 6.0. Furthermore, covalent cross-linking between A2 and A1(37-121) but not A1(221-336) was observed following reaction with a zero-length cross-linker. These findings demonstrate the presence of an extended, pH-dependent A2-interactive surface within regions 37-121 and 221-336 of A1. This interactive surface appears conformationally labile in the truncated A1 as judged by its apparent stabilization following association with A3-C1-C2.     

Mechanisms of factor Xa-catalyzed cleavage of the factor VIIIa A1 subunit resulting in cofactor inactivation

Activation of factor VIII by factor Xa is followed by proteolytic inactivation resulting from cleavage within the A1 subunit (residues 1-372) of factor VIIIa. Factor Xa attacks two sites in A1, Arg(336), which precedes the highly acidic C-terminal region, and a recently identified site at Lys(36). By using isolated A1 subunit as substrate for proteolysis, production of the terminal fragment, A1(37-336), was shown to proceed via two pathways identified by the intermediates A1(1-336) and A1(37-372) and generated by initial cleavage at Arg(336) and Lys(36), respectively. Appearance of the terminal product by the former pathway was 7-8-fold slower than the product obtained by the latter pathway. The isolated A1 subunit was cleaved slowly, independent of the presence of phospholipid. The A1/A3-C1-C2 dimer demonstrated an approximately 3-fold increased cleavage rate constant, and inclusion of phospholipid further enhanced this value by approximately 2-fold. Although association of A1 or A1(37-372) with A3-C1-C2 enhanced the rate of cleavage at Arg(336), inclusion of A3-C1-C2 did not affect the cleavage at Lys(36) in A1(1-336). A synthetic peptide 337-372 blocked the cleavage at Lys(36) (IC(50) = 230 microm) while showing little if any effect on cleavage at Arg(336). Proteolysis at Lys(36), and to a lesser extent Arg(336), was inhibited in a dose-dependent manner by heparin. These results suggest that inactivating cleavages catalyzed by factor Xa at Lys(36) and Arg(336) are regulated in part by the A3-C1-C2 subunit. Furthermore, cleavage at Lys(36) appears to be selectively modulated by the C-terminal acidic region of A1, a region that may interact with factor Xa via its heparin-binding exosite.     

Activated protein C-catalyzed inactivation of human factor VIII and factor VIIIa. Identification of cleavage sites and correlation of proteolysis with cofactor activity

Human factor VIII and factor VIIIa were proteolytically inactivated by activated protein C. Cleavages occurred within the heavy chain (contiguous A1-A2-B domains) of factor VIII and in the heavy chain-derived A1 and A2 subunits of factor VIIIa, whereas no proteolysis was observed in the light chain or light chain-derived A3-C1-C2 subunit. Reactivity to an anti-A2 domain monoclonal antibody and NH2-terminal sequence analysis of three terminal digest fragments from factor VIII allowed ordering of fragments and identification of cleavage sites. Fragment A1 was derived from the NH2 terminus and resulted from cleavage at Arg336-Met337. The A2 domain was bisected following cleavage at Arg562-Gly563 and yielded fragments designated A2N and A2C. A third cleavage site is proposed at the A2-B junction (Arg740-Ser741) since fragment A2C was of equivalent size when derived either from factor VIII or factor VIIIa. The site at Arg562 was preferentially cleaved first in factor VIII(alpha) compared with the site at Arg336, and it was this initial cleavage that most closely correlated with the loss of cofactor activity. Factor VIIIa was inactivated 5-fold faster than factor VIII, possibly as a result of increased protease utilization of the site at Arg562 when the A2 subunit is not contiguous with the A1 domain. When initial cleavage occurred at Arg336, it appeared to preclude subsequent cleavage at Arg562, possibly by promoting dissociation of the A2 domain (subunit) from the A1/light chain dimer. This conclusion was supported by the failure of protease treated A1/A3-C1-C2 dimer to bind A2 subunit and gel filtration analysis that showed dissociation of the A2 domain-derived fragments, A2N and A2C, from the A1 fragment/light chain dimer. These results suggest a mechanism for activated protein C-catalyzed inactivation of factor VIII(alpha) involving both covalent alteration and fragment dissociation.     

The A1 and A2 subunits of factor VIIIa synergistically stimulate factor IXa catalytic activity.

Factor VIIIa, the protein cofactor for factor IXa, is comprised of A1, A2, and A3-C1-C2 subunits. Recently, we showed that isolated A2 subunit enhanced the kcat for factor IXa-catalyzed activation of factor X by approximately 100-fold ( approximately 1 min-1), whereas isolated A1 or A3-C1-C2 subunits showed no effect on this rate (Fay, P. J., and Koshibu, K. J. (1998) J. Biol. Chem. 273, 19049-19054). However, A1 subunit increased the A2-dependent stimulation by approximately 10-fold. The Km for factor X in the presence of A2 subunit was unaffected by A1 subunit, whereas the kcat observed in the presence of saturating A1 and A2 subunits ( approximately 15 min-1) represented 5-10% of the value observed for native factor VIIIa (approximately 200 min-1). An anti-A1 subunit antibody that blocks the association of A2 eliminated the A1-dependent contribution to factor IXa activity. Inclusion of both A1 and A2 subunits resulted in greater increases in the fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa than that observed for A2 subunit alone and approached values obtained with factor VIIIa. These results indicate that A1 subunit alters the A2 subunit-dependent modulation of the active site of factor IXa to synergistically increase cofactor activity, yielding an overall increase in kcat of over 1000-fold compared with factor IXa alone.     

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.     

The Gla domain of factor IXa binds to factor VIIIa in the tenase complex

During blood coagulation factor IXa binds to factor VIIIa on phospholipid membranes to form an enzymatic complex, the tenase complex. To test whether there is a protein-protein contact site between the gamma-carboxyglutamic acid (Gla) domain of factor IXa and factor VIIIa, we demonstrated that an antibody to the Gla domain of factor IXa inhibited factor VIIIa-dependent factor IXa activity, suggesting an interaction of the factor IXa Gla domain with factor VIIIa. To study this interaction, we synthesized three analogs of the factor IXa Gla domain (FIX1-47) with Phe-9, Phe-25, or Val-46 replaced, respectively, with benzoylphenylalanine (BPA), a photoactivatable cross-linking reagent. These factor IX Gla domain analogs maintain native tertiary structure, as demonstrated by calcium-induced fluorescence quenching and phospholipid binding studies. In the absence of phospholipid membranes, FIX1-47 was able to inhibit factor IXa activity. This inhibition is dependent on the presence of factor VIIIa, suggesting a contact site between the factor IXa Gla domain and factor VIIIa. To demonstrate a direct interaction we did cross-linking experiments with FIX1-479BPA, FIX1-4725BPA, and FIX1-4746BPA. Covalent cross-linking to factor VIIIa was observed primarily with FIX1-4725BPA and to a much lesser degree with FIX1-4746BPA. Immunoprecipitation experiments with an antibody to the C2 domain of factor VIIIa indicate that the factor IX Gla domain cross-links to the A3-C1-C2 domain of factor VIIIa. These results suggest that the factor IXa Gla domain contacts factor VIIIa in the tenase complex through a contact site that includes phenylalanine 25 and perhaps valine 46.     

Synthetic "interface" peptides alter dimeric assembly of the HIV 1 and 2 proteases.

Retroviral proteases are obligate homodimers and play an essential role in the viral life cycle. Dissociation of dimers or prevention of their assembly may inactivate these enzymes and prevent viral maturation. A salient structural feature of these enzymes is an extended interface composed of interdigitating N- and C-terminal residues of both monomers, which form a four-stranded beta-sheet. Peptides mimicking one beta-strand (residues 95-99), or two beta-strands (residues 1-5 plus 95-99 or 95-99 plus 95-99) from the human immunodeficiency virus 1 (HIV1) interface were shown to inhibit the HIV1 and 2 proteases (PRs) with IC50's in the low micromolar range. These interface peptides show cognate enzyme preference and do not inhibit pepsin, renin, or the Rous sarcoma virus PR, indicating a degree of specificity for the HIV PRs. A tethered HIV1 PR dimer was not inhibited to the same extent as the wild-type enzymes by any of the interface peptides, suggesting that these peptides can only interact effectively with the interface of the two-subunit HIV PR. Measurements of relative dissociation constants by limit dilution of the enzyme show that the one-strand peptide causes a shift in the observed Kd for the HIV1 PR. Both one- and two-strand peptides alter the monomer/dimer equilibrium of both HIV1 and HIV2 PRs. This was shown by the reduced cross-linking of the HIV2 PR by disuccinimidyl suberate in the presence of the interface peptides. Refolding of the HIV1 and HIV2 PRs with the interface peptides shows that only the two-strand peptides prevent the assembly of active PR dimers. Although both one- and two-strand peptides seem to affect dimer dissociation, only the two-strand peptides appear to block assembly. The latter may prove to be more effective backbones for the design of inhibitors directed toward retroviral PR dimerization in vivo.     

Identification of a brain acetylcholine receptor alpha subunit able to bind alpha-bungarotoxin

Peptides corresponding to sequence segments homologous to an alpha-bungarotoxin (alpha-BGT) binding region on the alpha subunit of the Torpedo nicotinic cholinergic receptor (nAChR) were synthesized for each identified nAChR alpha subunit of the rat nervous system (alpha 1, which is expressed in muscle, and alpha 2, alpha 3, alpha 4, and alpha 5, which are expressed by neurons). The peptides were tested for their ability to directly bind 125I-alpha-BGT and to compete for 125I-alpha-BGT with Torpedo nAChR and with the alpha-BGT-binding component expressed by PC12, a sympathetic neuronal cell line. In addition to peptides of the muscle alpha 1 subunit, peptides corresponding to the sequence of a neuronal subunit, alpha 5, were able to bind 125I-alpha-BGT. Peptides containing the sequence segments 182-201 of the alpha 1 subunit and 180-199 of the alpha 5 subunit competed with Torpedo nAChR for 125I-alpha-BGT binding with IC50 values of 0.5 and 3.5 microM, respectively. Both of these peptides were also able to compete for 125I-alpha-BGT binding with native Torpedo nAChR and with the alpha-BGT-binding protein(s) expressed on PC12 cells. To determine if other sequence segments contribute to form the neuronal alpha-BGT-binding site, overlapping peptides corresponding to the putative extracellular domain of the alpha 5 subunit were synthesized and used both in direct binding assays and in competition experiments. Peptides corresponding to amino acids 16-35 and 180-199 of the alpha 5 subunit directly bound 125I-alpha-BGT and inhibited the binding of toxin to both Torpedo nAChR and PC12 cells. The results of these studies strongly support identification of the alpha 5 subunit as a component of a neuronal alpha-BGT-binding nAChR.     

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.     

Contribution of factor VIIIa A2 and A3-C1-C2 subunits to the affinity for factor IXa in factor Xase

Contributions of factor (F) VIIIa subunits to cofactor association with FIXa were evaluated. Steady-state fluorescence resonance energy transfer using an acrylodan-labeled A3-C1-C2 subunit and fluorescein-Phe-Phe-Arg-FIXa yielded K(d) values of 52 +/- 10 and 197 +/- 55 nM in the presence and absence of phospholipid vesicles, respectively. A3-C1-C2 was an effective competitor of FVIIIa binding to FIXa as judged by inhibition of FXa generation performed in the absence of vesicles (K(i) approximately 1.6K(d) for FVIIIa-FIXa). However, the capacity for A3-C1-C2 to inhibit FVIIIa-dependent FXa generation in the presence of phospholipid was poor with a K(i) values (approximately 400 nM) that were approximately 100-fold greater than the K(d) for FVIIIa-FIXa interaction (4.2 +/- 0.6 nM). These results indicated that a significant component of the interprotein affinity is contributed by FVIIIa subunits other than A3-C1-C2 in the membrane-dependent complex. The isolated A2 subunit of FVIIIa interacts weakly with FIXa, and recent modeling studies have implicated a number of residues that potentially contact the FIXa protease domain (Bajaj et al. (2001) J. Biol. Chem. 276, 16302-16309). Site-directed mutagenesis of candidate residues in the A2 domain was performed, and recombinant proteins were stably expressed and purified. Functional affinity determinations demonstrated that one mutant, FVIII/Asp712Ala exhibited an 8-fold increased K(d) (35 +/- 1.5 nM) relative to wild-type suggesting a contribution by this residue of approximately 10% of the FVIIIa-FIXa binding energy. Thus both A2 and A3-C1-C2 subunits contribute to the affinity of FVIIIa for FIXa in the membrane-dependent FXase.     

Mode of inhibition of smooth muscle myosin light chain kinase by synthetic peptide analogs of the regulatory site

The functions associated with the inhibitory region and calmodulin binding region of smooth muscle myosin light chain kinase (MLCK) were studied using various synthetic peptide analogs. Peptides 480-501 and 483-498 strongly inhibited 61 kDa Ca2+/calmodulin-independent MLCK activity with Ki of 25 nM. Peptides 493-512 and 493-504 were considerably less effective as inhibitor of the Ca2+/calmodulin-independent MLCK and Kiapp. were 2 and 3 microM, respectively. Inhibition of Ca2+/calmodulin-independent MLCK by the peptides 480-501 and 483-498 were competitive with ATP and 20,000 dalton smooth muscle myosin light chain. The inhibition of native MLCK by peptide 493-512 was explained by the calmodulin depletion model in which the peptide binds to free calmodulin and prevents it from activating MLCK. On the other hand, the inhibition of native MLCK by the peptides 480-501 and 483-498 was explained by the binding of these peptides to the MLCK-calmodulin complex. The present study suggests that the inhibitory region of MLCK directly binds to MLCK active site and competes with both ATP and 20,000 dalton light chain so as to inhibit the enzyme.     

Synthesis and antibody recognition of synthetic antigens from MUC1.

In the altered form of MUC1 mucin associated with breast cancer, the highly immunogenic sequence PDTRPAP is exposed, and may be an immunologically relevant target for the development of diagnostics or cancer immunotherapy. In this study, we report the preparation and antibody binding properties of monomeric and dimeric MUC1 peptides containing the epitope region recognized by monoclonal antibody (mAb) C595. Peptides contained a single or two copies of the whole 20-mer repeat unit (VTSAPDTRPAPGSTAPPAHG) of MUC1 protein. MUC1 40-mer peptides were prepared by the condensation of semi-protected fragments of the repeat unit, in solution or by chemical ligation. In the first case, cyclohexyl-type protecting groups were used for the synthesis of semi-protected fragments by the Boc/Bzl strategy. Unprotected fragments were used in the chemical ligation to produce thioether linkages. In one of the fragments, a Gly residue was replaced by Cys at the C-terminus and the other fragment was chloroacetylated at the N-terminus. In addition, the short peptide APDTRPAPG, and its disulfide dimer, (APDTRPAPGC)(2) were produced. The antibody binding properties of these MUC1 peptide constructs were tested by competition enzyme-linked immunosorbent assay (ELISA). The short epitope region peptide, APDTRPAPG and its dimer (APDTRPAPGC)(2) showed higher IC(50) values (IC(50) = 56.3 and 53.2 micromol/l, respectively). While the 20-mer peptide (IC(50) = 25.9 micromol/l) and more markedly its 40-mer dimers (IC(50) = 0.62 and 0.78 micromol/l) were recognized better. CD data obtained in water or in TFE indicated no significant conformational differences between the 20-mer and 40-mer peptides. We found a high level of similarity between the binding properties of the 40-mer peptides with amide or thioether links, providing a new possibility to build up oligomeric MUC1 peptides by thioether bond formation.     

Procyanidin structure defines the extent and specificity of angiotensin I converting enzyme inhibition

Recent reports have shown a decrease in blood pressure associated with the consumption of flavanol-containing foods. However, the mechanism behind this effect is not yet known. Previously we demonstrated that the flavanol epicatechin and its related oligomers, the procyanidins, inhibit angiotensin I converting enzyme (ACE) activity in vitro. In this study, we further characterized epicatechin monomer, dimer, tetramer and hexamer ACE inhibitory effect, by performing fluorescence quenching and kinetic assays, using angiotensin I as substrate. Assessment of ACE activity in cultured human umbilical vein endothelial cells (HUVEC) indicated that the tetramer was the most active inhibitor decreasing the formation of angiotensin II by 52% (P<0.001). When ACE activity was measured using isolated rabbit lung ACE, dimer, tetramer and hexamer inhibited angiotensin II production at IC(50) values of 97.0, 4.4, and 8.2 microM, respectively. The quenching of ACE tryptophan fluorescence was assayed to evaluate the molecular interaction between ACE and procyanidins. The hexamer was the most active quencher decreasing ACE fluorescence by 56%, followed by the tetramer and the dimer, decreasing ACE fluorescence by 37% and 36%, respectively. ACE activity was evaluated in the presence of different concentrations of the ACE activator chloride ion (Cl(-)). Increased Cl(-) concentrations reduced IC(50) values for the dimer and tetramer. Finally, ACE inhibition was determined in the presence of different albumin concentrations. The presence of albumin did not reverse the ACE inhibition by dimer and tetramer, but decreased hexamer inhibition by 65%. In summary, the inhibitory effect of procyanidins on ACE and the extent of this inhibition were largely dependent on procyanidin structure. ACE inhibition by procyanidins in vivo might provide a mechanism to explain the benefits of flavonoid consumption on cardiovascular diseases.