Identification of a plasmin-interactive site within the A2 domain of the factor VIII heavy chain

Factor VIII is activated and inactivated by plasmin by limited proteolysis. In our one-stage clotting assay, these plasmin-catalyzed reactions were inhibited by the addition of isolated factor VIII A2 subunits and by Glu-Gly-Arg-active-site modified factor IXa. SDS-PAGE analysis showed that an anti-A2 monoclonal antibody, recognizing the factor IXa-interactive site (residues 484-509), blocked the plasmin-catalyzed cleavage at Arg(336) and Arg(372) but not at Arg(740). Surface plasmon resonance-based assays and ELISA demonstrated that the A2 subunit bound to active-site modified anhydro-plasmin with high affinity (K(d): 21 nM). Both an anti-A2 monoclonal antibody and a peptide comprising of A2 residues 479-504 blocked A2 binding by approximately 80% and approximately 55%, respectively. Mutant A2 molecules where the basic residues in A2 were converted to alanine were evaluated for binding of anhydro-plasmin. Among the tested mutants, the R484A A2 mutant possessed approximately 250-fold lower affinity than the wild-type A2. The affinities of K377A, K466A, and R471A mutants were decreased by 10-20-fold. The inhibitory effect of R484A mutant on plasmin-catalyzed inactivation of factor VIIIa was approximately 20% of that of wild-type A2. In addition, the inactivation rate by plasmin of factor VIIIa reconstituted with R484A mutant was approximately 3-fold lower than that with wild-type A2. These findings demonstrate that Arg(484) plays a key role within the A2 plasmin-binding site, responsible for plasmin-catalyzed factor VIII(a) inactivation.     

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.     

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.     

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.     

A Glu113Ala mutation within a factor VIII Ca2+-binding site enhances cofactor interactions in factor Xase

We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca(2+), an ion necessary for cofactor activity [Wakabayashi et al. (2004) J. Biol. Chem. 279, 12677-12684]. Mutagenesis studies showed that replacement of residue Glu113 with Ala (E113A) yielded a factor VIII point mutant possessing increased specific activity as determined by a one-stage clotting assay. Mutagenesis at this site suggested that substitution with relatively small, nonpolar residues was well tolerated, whereas replacement with a number of polar or charged residues appeared detrimental to activity. Ala substitution resulted in the greatest enhancement, yielding an approximately 2-fold increased specific activity. Time course experiments following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Results from factor Xa generation assays showed minimal differences in kinetic parameters and factor IXa affinity for E113A and wild-type factor VIIIa when run in the presence of synthetic phospholipid vesicles, whereas factor VIIIa E113A displayed an approximately 4-fold greater affinity for factor IXa compared with factor VIIIa wild type in reactions run on the platelet membrane surface. This latter effect may be attributed, in part, to a 2-fold increased affinity of factor VIIIa E113A for the platelet membrane. Considering that low levels of factors VIIIa and IXa are generated during clotting in plasma, the increased cofactor specific activity observed for E113A factor VIII may result from its enhanced affinity for factor IXa on the physiological membrane.     

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.     

Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule

The primary structure of factor VIII consists of 2332 amino acids that exhibit 3 distinct structural domains, including a triplicated region (A domains), a unique region of 909 amino acids (B domain), and a carboxy-terminal duplicated region (C domains), that are arranged in the order A1-A2-B-A3-C1-C2. The B domain (residues 741-1648) of factor VIII is lost when factor VIII is activated by thrombin, which proteolytically processes factor VIII to active subunits of Mr 50,000 (domain A1), 43,000 (domain A2), and 73,000 (domains A3-C1-C2). To determine if the B domain is required for factor VIII coagulant activity, a variant was constructed by using recombinant DNA techniques in which residues 797-1562 were eliminated. This shortened the B domain from 909 to 142 amino acids. This variant factor VIIIdes-797-1652 was expressed in mammalian cells and was found to be functional. The factor VIIIdes-797-1562 protein was purified and shown to be processed by thrombin in the same manner as full-length factor VIII. The factor VIIIdes-797-1562 variant also bound to von Willebrand factor (vWF) immobilized on Sepharose. These results indicate that most of the highly glycosylated B domain of factor VIII is not required for the expression of factor VIII coagulant activity and its interaction with vWF.     

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.     

A monoclonal antibody to factor IX that inhibits the factor VIII:Ca potentiation of factor X activation

A murine monoclonal antibody (IgG1k, Kd approximately 10(-8) M) specific for an epitope located on the heavy chain of human factor IXa was used to study structure-function relationships of factor IX. The antibody inhibited factor IX clotting activity but did not impair activation of factor IX either by factor XIa/calcium or by factor VIIa/tissue factor/calcium. The antibody also did not impair the binding of factor IXa to antithrombin III. Moreover, the antibody did not prevent calcium and phospholipid (PL) from inhibiting the binding of factor IXa to antithrombin III. The antibody also failed to impair activation of factor VII by factor IXa/calcium/PL. Furthermore, the antibody did not interfere with the very slow activation of factor X by factor IXa/calcium/PL. In contrast, the antibody did interfere with factor X activation when reaction mixtures also contained factor VIII:Ca/von Willebrand factor. The marked acceleration of factor X activation observed in control mixtures was not observed in mixtures containing the antibody. Similar results were obtained in reaction mixtures containing the Fab portion of the antibody and factor VIII:Ca free of von Willebrand factor. In additional experiments, factor VIII:Ca/von Willebrand factor was found to inhibit the binding of the antibody to 125I-factor IXa as determined using an immunosorbent assay. Moreover, the antibody displaced factor VIII:Ca from the factor X activator complex (IXa/calcium/PL/VIII:Ca) as evidenced by an altered elution pattern on gel filtration chromatography. From these observations, we conclude that the antibody impairs the clotting activity of factor IXa through interference with its binding of factor VIII:Ca. This suggests a significant role for the heavy chain (residues of 181-415) of factor IXa in binding factor VIII:Ca.     

Hydrophobic contact between the two epidermal growth factor-like domains of blood coagulation factor IX contributes to enzymatic activity

The three-dimensional structure of activated factor IX comprises multiple contacts between the two epidermal growth factor (EGF)-like domains. One of these is a salt bridge between Glu(78) and Arg(94), which is essential for binding of factor IXa to its cofactor factor VIII and for factor VIII-dependent factor X activation (Christophe, O. D., Lenting, P. J., Kolkman, J. A., Brownlee, G. G., and Mertens, K. (1998) J. Biol. Chem. 273, 222-227). We now addressed the putative hydrophobic contact at the interface between the EGF-like domains. Recombinant factor IX chimeras were constructed in which hydrophobic regions Phe(75)-Phe(77) and Lys(106)-Val(108) were replaced by the corresponding sites of factor X and factor VII. Activated factor IX/factor X chimeras were indistinguishable from normal factor IXa with respect to factor IXa enzymatic activity. In contrast, factor IXa(75-77)/factor VII displayed approximately 2-fold increased factor X activation in the presence of factor VIII, suggesting that residues 75-77 contribute to cofactor-dependent factor X activation. Activation of factor X by factor IX(106-108)/factor VII was strongly decreased, both in the absence and presence of factor VIII. Activity could be restored by simultaneous substitution of the hydrophobic sites in both EGF-like domains for factor VII residues. These data suggest that factor IXa enzymatic activity requires hydrophobic contact between the two EGF-like domains.     

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.     

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.     

Proteolysis at Arg740 facilitates subsequent bond cleavages during thrombin-catalyzed activation of factor VIII

Thrombin activates factor VIII by proteolysis at three P1 residues: Arg372, Arg740, and Arg1689. Cleavage at Arg372 and Arg1689 are essential for procofactor activation; however cleavage at Arg740 has not been rigorously studied. To evaluate the role for cleavage at Arg740, we prepared and stably expressed two recombinant B-domainless factor VIII mutants, R740H and R740Q to slow and eliminate, respectively, cleavage at this site. Specific activity values for the variants were approximately 50 and 20%, respectively, that of wild-type factor VIII. Activation of factor VIII R740H by thrombin showed an approximately 40-fold reduction in the rate of A2 subunit generation, which reflected an approximately 20-fold reduction in cleavage rate at Arg372. Similarly, a approximately 40-fold rate reduction in cleavage at Arg1689 and consequent generation of the A3-C1-C2 subunit were observed. Rate values for A2 and A3-C1-C2 subunit generation were reduced by >700-fold and approximately 140-fold, respectively, in the R740Q variant. These results suggest that initial cleavage at Arg740 affects cleavage at both Arg372 and Arg1689 sites. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed more modest rate reductions (<10-fold) in generating A2 and A3-C1-C2 subunits from either variant, suggesting that factor Xa-catalyzed activation of factor VIII was significantly less dependent upon prior cleavage at residue 740 than thrombin. Overall, these results support a model whereby cleavage of factor VIII by thrombin is an ordered pathway with cleavage at Arg740 facilitating cleavages at Arg372 and Arg1689, which result in procofactor activation.     

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.     

Amphipathic helices support function of blood coagulation factor IXa

Blood coagulation factor IXa gains proteolytic efficiency upon binding to a phospholipid membrane. We have found that an amphipathic, membrane-binding peptide from the C2 domain of factor VIII, fVIII(2303)(-23), enhances proteolytic efficiency of factor IXa in the absence of phospholipid membranes. This enhancement is the result of a reduction in the K(M) for the substrate, factor X, with little effect on the k(cat). Enhanced function requires interaction of the gamma-carboxyglutamic acid (Gla) domains of factor IXa and factor X since (i) a synthetic peptide comprising the Gla domain of factor IXa and antibodies directed to the Gla domain of factor IXa inhibit this acceleration, (ii) the acceleration is Ca(II) dependent, and (iii) conversion of Gla-domainless factor X is not affected by the presence of fVIII(2303)(-23). The effect of fVIII(2303)(-23) on factor IXa parallels the enhanced function produced by phosphatidylserine-containing bilayers, and fVIII(2303)(-23) does not further enhance function of factor IXa when phospholipid vesicles are present. The critical feature of fVIII(2303)(-23) is apparently its amphipathic helix-forming structure [Gilbert, G. E., and Baleja, J. D. (1995) Biochemistry 34, 3022-3031] because other alpha-helical peptides such as a homologous peptide from the C2 domain of factor V and melittin have similar effects. Diastereomeric analogues of fVIII(2303)(-23) and melittin, which have reduced helical content, do not support factor IXa activity. A truncated peptide of fVIII(2303)(-23) with three C-terminal residues deleted retains alpha-helical content but loses capacity to enhance factor X cleavage, suggesting that a minimum length of alpha-helix is required. Although these results probably do not illuminate the physiologic function of the factor VIII peptide corresponding to fVIII(2303)(-23), they demonstrate a novel, membrane-mimetic role of amphipathic helical peptides in supporting function of factor IXa.     

Identification of residues contributing to A2 domain-dependent structural stability in factor VIII and factor VIIIa

Factor VIII circulates as a heterodimer composed of heavy (A1A2B domains) and light (A3C1C2 domains) chains, whereas the contiguous A1A2 domains are separate subunits in the active cofactor, factor VIIIa. Whereas the A1 subunit maintains a stable interaction with the A3C1C2 subunit, the A2 subunit is weakly associated in factor VIIIa and its dissociation accounts for the labile activity of the cofactor. In examining the ceruloplasmin-based factor VIII A domain model, potential hydrogen bonding based upon spatial separations of <2.8A were found between side chains of 14 A2 domain residues and 7 and 9 residues in the A1 and A3 domains, respectively. These residues were individually replaced with Ala, except Tyr residues were replaced with Phe, and proteins stably expressed to examine the contribution of each residue to protein stability. Factor VIII stability at 55 degrees C and factor VIIIa activity were monitored using factor Xa generation assays. Fourteen of 30 factor VIII mutants showed >2-fold increases in either or both decay rates compared with wild type; whereas, 7 mutants showed >2-fold increased rates in factor VIIIa decay compared with factor VIII decay. These results suggested that multiple residues at the A1-A2 and A2-A3 domain interfaces contribute to stabilizing the protein. Furthermore, these data discriminate residues that stabilize interactions in the procofactor from those in the cofactor, where hydrogen bonding in the latter appears to contribute more significantly to stability. This observation is consistent with an altered conformation involving new inter-subunit interactions involving A2 domain following procofactor activation.     

Comparative interactions of factor IX and factor IXa with human platelets

Both factor IX and factor IXa were bound to gel filtered platelets in the presence of CaCl2 (2-20 mM) and human alpha-thrombin (0.06-0.2 units/ml) with maximal binding occurring in 10-20 min at 37 degrees C, and rapid reversibility was observed when unlabeled ligands were added in 100-fold molar excess. Competition studies with various coagulation proteins revealed that neither factor XI nor high molecular weight kininogen, at 300-fold molar excess, could compete with 125I-labeled factor IXa for binding sites on thrombin-activated platelets, whereas prothrombin and factor X, in 450-fold molar excess, could displace approximately 15 and 35%, respectively, of bound factor IXa in the absence of added factor VIII. Analysis of saturation binding data in the presence of CaCl2 and thrombin without factors VIII and X indicated the presence of 306 (+/- 57) binding sites per platelet for factor IX (Kd(app) = 2.68 +/- 0.25 nM) and 515 (+/- 39) sites per platelet for factor IXa (Kd = 2.57 +/- 0.14 nM). In the presence of thrombin-activated factor VIII (1-5 units/ml) and factor X (0.15-1.5 microM), the number of sites for factor IX was 316 (+/- 50) with Kd = 2.44 (+/- 0.30) nM and for factor IXa 551 (+/- 48) sites per platelet (Kd = 0.56 +/- 0.05 nM). Studies of competition for bound factor IXa by excess unlabeled factor IX or factor IXa, and direct 125I-labeled factor IXa binding studies in the presence of large molar excesses of factor IX, confirmed the conclusion from these studies that factor IX and factor IXa share approximately 300 low-affinity binding sites per thrombin-activated platelet in the presence of Ca2+ and in the absence of factor VIII and factor X, with an additional 200-250 sites for factor IXa with Kd(app) similar to that for factor IX. The presence of factor VIII and factor X increases by 5-fold the affinity of receptors on thrombin-activated platelets for factor IXa that participate in factor X activation.     

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.     

Mechanisms of plasmin-catalyzed inactivation of factor VIII: a crucial role for proteolytic cleavage at Arg336 responsible for plasmin-catalyzed factor VIII inactivation

Plasmin not only functions as a key enzyme in the fibrinolytic system but also directly inactivates factor VIII and other clotting factors such as factor V. However, the mechanisms of plasmin-catalyzed factor VIII inactivation are poorly understood. In this study, levels of factor VIII activity increased approximately 2-fold within 3 min in the presence of plasmin, and subsequently decreased to undetectable levels within 45 min. This time-dependent reaction was not affected by von Willebrand factor and phospholipid. The rate constant of plasmin-catalyzed factor VIIIa inactivation was approximately 12- and approximately 3.7-fold greater than those mediated by factor Xa and activated protein C, respectively. SDS-PAGE analysis showed that plasmin cleaved the heavy chain of factor VIII into two terminal products, A1(37-336) and A2 subunits, by limited proteolysis at Lys(36), Arg(336), Arg(372), and Arg(740). The 80-kDa light chain was converted into a 67-kDa subunit by cleavage at Arg(1689) and Arg(1721), identical to the pattern induced by factor Xa. Plasmin-catalyzed cleavage at Arg(336) proceeded faster than that at Arg(372), in contrast to proteolysis by factor Xa. Furthermore, breakdown was faster than that in the presence of activated protein C, consistent with rapid inactivation of factor VIII. The cleavages at Arg(336) and Lys(36) occurred rapidly in the presence of A2 and A3-C1-C2 subunits, respectively. These results strongly indicated that cleavage at Arg(336) was a central mechanism of plasmin-catalyzed factor VIII inactivation. Furthermore, the cleavages at Arg(336) and Lys(36) appeared to be selectively regulated by the A2 and A3-C1-C2 domains, respectively, interacting with plasmin.