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PubMed2001

C4b-binding protein inhibits the factor V-dependent but not the factor V-independent cofactor activity of protein S in the activated protein C-mediated inactivation of factor VIIIa.

Activated protein C (APC) is an important inactivator of coagulation factors Va and VIIIa. In the inactivation of factors Va and VIIIa, protein S serves as a cofactor to APC. Protein S can bind to C4b-binding protein (C4BP), and thereby loses its cofactor activity to APC. By modulating free protein S levels, C4BP is an important regulator of protein S cofactor activity. In the factor VIIIa inactivation, protein S and factor V act as synergistic cofactors to APC. We investigated the effect of C4BP on both the factor V-independent and factor V-dependent cofactor activity of protein S in the factor VIIIa inactivation using a purified system. Protein S increased the APC-mediated inactivation of factor VIIIa to 60% and in synergy with protein S, factor V at equimolar concentrations increased this effect further to 90%. The protein S/factor V synergistic effect was inhibited by preincubation of protein S and factor V with a four-fold molar excess of C4BP. However, C4BP did not inhibit the factor V-independent protein S cofactor activity in the purified system whereas it inhibited the cofactor activity in plasma. We conclude that C4BP-bound protein S retains its cofactor activity to APC in the factor VIIIa inactivation.

Anticoagulants↗
PubMed2000

Role of phosphatidylethanolamine in assembly and function of the factor IXa-factor VIIIa complex on membrane surfaces.

Phospholipid membranes play a significant role during the proteolytic activation of blood coagulation proteins. This investigation identifies a role for phosphatidylethanolamine (PE) during the activation of factor X by the tenase complex, an enzymatic complex composed of the serine protease, factor IXa, a protein cofactor, factor VIIIa, a phospholipid membrane, and Ca(2+). Phospholipid vesicles composed of PE, phosphatidylserine (PS), and phosphatidylcholine support factor Xa generation. The K(m) and k(cat) for factor X activation by the tenase complex are independent of PE in the presence of 20% PS. At lower PS concentrations, the presence of 20 or 35% PE lowers the K(m) and increases the k(cat) as compared to those in vesicles without PE. The effect of PE on the k(cat) of the tenase complex is mediated through factor VIIIa. PE also enhances factor Xa generation by facilitating tenase complex formation; PE lowers the K(d(app)) of factor IXa for both phospholipid/Ca(2+) and phospholipid/Ca(2+)/factor VIIIa complexes in the presence of suboptimal PS. In addition, the K(d)s of factor IXa and factor X were lower for phospholipid vesicles containing PE. N-Methyl-PE increased the k(cat) and decreased the K(d(app)), whereas N,N-dimethyl-PE had no effect on either parameter, indicating the importance of headgroup size. Lyso-PE had no effect on kinetic parameters, indicating the sn-2 acyl chain dependence of the PE effect. Together, these results demonstrate a role for PE in the assembly and activity of the tenase complex and further extend the understanding of the importance of PE-containing membranes in hemostasis.

Binding Sites↗
PubMed2002

Mutations associated with hemophilia A in the 558-565 loop of the factor VIIIa A2 subunit alter the catalytic activity of the factor Xase complex.

The 558-565 loop region in the A2 subunit of factor (F) VIIIa forms a direct interface with FIXa. We have expressed and purified B-domainless FVIII (FVIII(WT)) and B-domainless FVIII containing the hemophilia A-associated mutations Ser558Phe, Val559Ala, Asp560Ala, Gln565Arg, and the activated protein C cleavage site mutant Arg562Ala. Titration of FVIIIa in FXa generation assays showed that the mutant and wild-type proteins had similar functional affinities for FIXa (dissociation constant [K(d)] values approximately 5 nM-20 nM and approximately 100 nM-250 nM in the presence and absence of phospholipid, respectively). The catalytic activities of the factor Xase complex composed of the hemophilia A-associated FVIII species were markedly reduced both in the presence and absence of phospholipid. FVIII(WT) and FVIII(Arg562Ala) showed catalytic rate constant (k(cat)) values of approximately 60 minute(-1) in the presence of phospholipid, whereas the hemophilia A-associated mutants showed k(cat) values ranging from 3.3 minute(-1) to 7.5 minute(-1). In the absence of phospholipid, all k(cat) values were reduced but FVIII(WT) and FVIII(Arg562Ala) retained higher activities as compared with the hemophilic mutant FVIII forms. Fluorescence anisotropy experiments using fluorescein-modified FIXa confirmed that all FVIII forms interacted with FIXa. However, the presence of factor X yielded minimal increases in anisotropy observed with the mutant factor VIII forms, consistent with their reduced activity. These results show that residues within the 558-565 loop are critical in modulating FIXa enzymatic activity but do not contribute significantly to the affinity of FVIIIa for FIXa.

Base Sequence↗
PubMed1992

The active site of factor IXa is located far above the membrane surface and its conformation is altered upon association with factor VIIIa. A fluorescence study.

The topography of membrane-bound blood coagulation factor IXa (fIXa) and the nature of its interaction with its cofactor, factor VIIIa (fVIIIa), were examined using fluorescent derivatives of fIXa. A fluorescein dye was covalently attached to the active-site histidine of fIXa via a D-Phe-Pro-Arg tripeptide tether to form Fl-A-FPR-fIXa; similarly, a 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) dye was covalently attached via Glu-Gly-Arg to form DEGR-fIXa. When either Fl-A-FPR-fIXa or DEGR-fIXa was titrated with phosphatidylcholine-phosphatidylserine vesicles containing octadecylrhodamine in the presence of Ca2+, fluorescence energy transfer was observed. Assuming a random orientation of dyes, the distance of closest approach between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the membrane surface was found to be 89 +/- 3 A for Fl-A-FPR-fIXa and 73 +/- 4 A for DEGR-fIXa. Although the exact distance remains uncertain, it is clear that the active site of fIXa is positioned more than 70 A above the surface, and hence that the elongated fIXa molecule projects approximately perpendicularly from the surface when bound to the membrane. The binding of fVIIIa to membrane-bound Fl-A-FPR-fIXa or DEGR-fIXa did not alter the location of the active site relative to the membrane surface, but did alter both the emission intensity and anisotropy of the fluorescein and dansyl probes and hence their environments. Cofactor stimulation of fIXa activity therefore appears to be mediated, at least in part, by a conformational change in the active site that occurs when fVIIIa binds to the enzyme on the phospholipid surface.

Animals↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed1990

Structural model of porcine factor VIII and factor VIIIa molecules based on scanning transmission electron microscope (STEM) images and STEM mass analysis.

Porcine plasma factor VIII (fVIII) molecules are heterodimers composed of a 76,000-mol wt light chain (-A3-C1-C2) and a heavy chain ranging in molecular weight from 82,000 (A1-A2) to 166,000 (A1-A2-B). Proteolytic activation of fVIII by thrombin results in fVIIIa heterotrimers lacking B domains (A1, A2, A3-C1-C2). In this study, immunoaffinity purified fVIII was further fractionated by mono S or mono Q chromatography to prepare heterodimers containing a light chain and an A1-A2-B heavy chain (fVIII 166/76) or an A1-A2 heavy chain (fVIII 82/76). Mass analysis of scanning transmission electron microscopic (STEM) images of fVIII 166/76 indicated that heterodimers (mass 237 +/- 20 kD) had irregularly globular core structures 10-12 nm across, and frequently displayed a diffuse, occasionally globular to ovoid satellite structure extending 5-14 nm from the core, and attached to it by a thin stalk. Factor VIII 82/76 molecules (mass 176 +/- 20 kD) had the same core structures as fVIII 166/76 molecules, but lacked the satellite structure. These findings indicate that A1-A2 domains of heavy chains and the light chains of the fVIII procofactor molecule are closely associated and constitute the globular core structure, whereas the B domainal portion of heavy chains comprises the peripheral satellite appendage. Factor VIII core structures commonly displayed a finger-like projection near the origin of the B domainal stalk that was also a consistent feature of the free heavy chains (mass 128-162 kD) found in fVIII 166/76 preparations. Factor VIII light chain monomers (mass, 76 +/- 16 kD) were globular to c-shaped particles 6-8 nm across. These chains commonly possessed a v-shaped projection originating from its middle region, that could also be observed at the periphery of fVIII core molecules. Factor VIIIa preparations contained heterotrimers (mass 162 +/- 13 kD) that had the same dimensions as fVIII core structures, lacked the B domainal appendage, and sometimes possessed the same core features as fVIII molecules. Molecular species corresponding to heterodimers (mass, 128 +/- 13 kD) and unassociated subunit chains (40-100 kD) were also observed in fVIIIa preparations, suggesting that heterotrimers have an appreciable tendency to dissociate, a phenomenon that could explain the decay of fVIIIa activity after thrombin activation of fVIII.

Animals↗
PubMed2004

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.

Binding, Competitive↗
PubMed2006

Intrinsic stability and functional properties of disulfide bond-stabilized coagulation factor VIIIa variants.

BACKGROUND: The utility of purified coagulation factor (F)VIII for treatment of hemophilia A is limited in part by its instability following activation by thrombin, which is caused by spontaneous dissociation of the A2 domain from the activated FVIII (FVIIIa) heterotrimer. To prevent this A2 domain dissociation in FVIIIa, we previously engineered a cysteine pair (C664-C1826) in recombinant FVIII that formed a disulfide bond cross-linking the A2 domain in the heavy chain to the A3 domain in the light chain. This engineered disulfide bond resulted in a more stable FVIIIa. AIMS: Here, we characterize the functional parameters of C664-C1828 FVIII and of a new disulfide bond-stabilized FVIII (C662-C1828 FVIII). METHODS: In order to assess whether these FVIII variants might be good candidates for a new therapeutic agent to treat hemophilia A, we investigated a variety of functional parameters that might affect the in vivo properties of the variants, including half-life of disulfide bond-stabilized FVIII and FVIIIa and the potency of these FVIIIa molecules in the FXase complex. RESULTS: Both disulfide bond-stabilized variants had improved affinity for von Willebrand factor (VWF). In studies of FX activation by purified FIXa and FVIIIa, C662-C1828 FVIIIa had normal activity while C664-C1826 FVIIIa had reduced activity. Both C664-C1826 FVIIIa and C662-C1828 FVIIIa were inactivated by activated protein C (APC) but the rates of inactivation were different. CONCLUSION: Overall, the specific location of the disulfide bridge between the A2 and A3 domains appears to affect functional properties of FVIIIa. In summary, introduction of engineered interdomain disulfides results in FVIIIa variants that resist spontaneous loss of activity while retaining susceptibility to APC proteolytic inactivation and maintaining VWF binding.

Factor IXa↗
PubMed2000

The second laminin G-type domain of protein S is indispensable for expression of full cofactor activity in activated protein C-catalysed inactivation of factor Va and factor VIIIa.

Vitamin K-dependent protein S is a cofactor to the anticoagulant serine protease activated protein C (APC) in the proteolytic inactivation of the procoagulant, activated factor V (FVa) and factor VIII (FVIIIa). In the FVa degradation, protein S selectively accelerates the cleavage at Arg306, having no effect on the Arg506 cleavage. In the FVIIIa inactivation, the APC-cofactor activity of protein S is synergistically potentiated by FV, which thus has the capacity to function both as a pro- and an anticoagulant protein. The SHBG-like region of protein S, containing two laminin G-type domains, is required for the combined action of protein S and FV. To elucidate whether both G domains in protein S are needed for expression of APC-cofactor activities, chimeras of human protein S were created in which the individual G domains were replaced by the corresponding domain of the homologous Gas6, which in itself has no anticoagulant activity. In a plasma-based assay, chimera I (G1 from Gas6) was as efficient as wild-type recombinant protein S, whereas chimera II (G2 from Gas6) was less effective. The synergistic cofactor activity with FV in the inactivation of FVIIIa was lost by the replacement of the G2 domain in protein S (chimera II). However, chimera I did not exert full APC-cofactor activity in the FVIIa degradation, indicating involvement of both G domains or the entire SHBG-like region in this reaction. Chimera I was fully active in the degradation of FVa in contrast to chimera II, which exhibited reduced cofactor activity compared to protein S. In conclusion, by using protein S-Gas6 chimeric proteins, we have identified the G2 domain of protein S to be indispensable for an efficient inactivation of both FVIIa and FVa, whereas the G1 domain was found not to be of direct importance in the FVa-inactivation experiments.

Anticoagulants↗
PubMed1993

Phosphorylation of factor Va and factor VIIIa by activated platelets.

Platelet activation leads to the incorporation of 32[PO4(2-)] into bovine coagulation factor Va and recombinant human factor VIII. In the presence of the soluble fraction from thrombin-activated platelets and (gamma-32P) adenosine triphosphate, radioactivity is incorporated exclusively into the M(r) = 94,000 heavy chain (H94) of factor Va and into the M(r) = 210,000 to 90,000 heavy chains as well into the M(r) = 80,000 light chain of factor VIII. Proteolysis of the purified phosphorylated M(r) = 94,000 factor Va heavy chain by activated protein C (APC) gave products of M(r) = 70,000, 24,000, and 20,000. Only the intermediate M(r) = 24,000 fragment contained radioactivity. Because the difference between the M(r) = 24,000 and M(r) = 20,000 fragments is located on the COOH-terminal end of the bovine heavy chain, phosphorylation of H94 must occur within the M(r) = 4,000 peptide derived from the carboxyl-terminal end of H94 (residues 663 through 713). Exposure of the radioactive factor VIII molecule to thrombin ultimately resulted in a nonradioactive light chain and an M(r) = 24,000 radioactive fragment that corresponds to the carboxyl-terminal segment of the A1 domain of factor VIII. Based on the known sequence of human factor VIII, phosphorylation of factor VIII by the platelet kinase probably occurs within the acidic regions 337 through 372 and 1649 through 1689 of the procofactor. These acidic regions are highly homologous to sequences known to be phosphorylated by casein kinase II. Results obtained using purified casein kinase II gave a maximum observed stoichiometry of 0.6 mol of 32[PO4(2-)]/mol of factor Va heavy chain and 0.35 mol of 32[PO4(2-)]/mol of factor VIII. Phosphoamino acid analysis of phosphorylated factor Va by casein kinase II or by the platelet kinase showed only the presence of phosphoserine while phosphoamino acid analysis of phosphorylated factor VIII by casein kinase II showed the presence of phosphothreonine as well as small amounts of phosphoserine. The platelet kinase responsible for the phosphorylation of the two cofactors was found to be inhibited by several synthetic protein kinase inhibitors. Finally, partially phosphorylated factor Va was found to be more sensitive to APC inactivation than its native counterpart. Our findings suggest that phosphorylation of factors Va and VIIIa by a platelet casein kinase II-like kinase may downregulate the activity of the two cofactors.

Amino Acid Sequence↗
PubMed1997

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa.

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690-2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.

Enzyme Activation↗
PubMed2002

Hemophilia A mutations within the factor VIII A2-A3 subunit interface destabilize factor VIIIa and cause one-stage/two-stage activity discrepancy.

Thrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. A homology model (Blood 89:2413, 1997) of the triplicated A domains of factor VIII (FVIII) predicts a pseudo-threefold axis at the tightly packed hydrophobic core with several interdomain interactions. These lie at the interface of A1-A2, A2-A3 and A1-A3. We have previously demonstrated that hemophilia A mutations (R531H, A284E, S289L) within the predicted A1-A2 and A1-A3 interface disrupt potential intersubunit hydrogen bonds and have the molecular phenotype of increased rate of inactivation of FVIIIa due to increased rate of A2 subunit dissociation. Patients with these mutations exhibit a clinical phenotype where the FVIII activity by one-stage(1-st) assay is at least two-fold higher than by two-stage(2-st) assay. We have now also explored mutations within the predicted A2-A3 interface (N694I, R698W and R698L) that also have the phenotype of 1-st/2-st activity discrepancy. These mutations exhibit the same molecular mechanism of increased instability of FVIIIa as those mutations described along the A1-A2 and A1-A3 interfaces. This suggests that the entire tightly packed hydrophobic core within the predicted pseudo-threefold axis contributes to stabilization of FVIIIa.

Factor VIII↗
PubMed2003

An engineered interdomain disulfide bond stabilizes human blood coagulation factor VIIIa.

The blood coagulation disorder, hemophilia A, is caused by deficiency of coagulation factor (F)VIII. Hemophilia A is now treated by infusions of pure FVIII, but the activity of FVIII is limited because it is unstable following activation by thrombin. This instability of activated FVIII is the result of dissociation of the A2 subunit. To obtain increased stability in FVIIIa, a disulfide bond between the A2 domain and the A3 domain, preventing A2 subunit dissociation, has been engineered. Structural analysis of the FVIII A domain homology model allowed us to identify residues 664 and 1826 as a potential disulfide bond pair. A FVIII mutant containing Cys664 and Cys1826 was produced and purified (C664-C1826 FVIII). Immunoblotting showed that a disulfide bond did form to link covalently the A2 and the A3 domains. Following activation of the recombinant C664-C1826 FVIII by thrombin, the mutant FVIIIa had increased stability and retained more than 90% of its clotting activity at a time at which wild-type FVIIIa lost more than 90% of its activity. This remarkably stable C664-C1826 FVIIIa provides a unique approach for studies of the cofactor activity of FVIIIa and also for new, improved therapy for hemophilia A.

Amino Acid Substitution↗
PubMed1999

Mild hemophilia A caused by increased rate of factor VIII A2 subunit dissociation: evidence for nonproteolytic inactivation of factor VIIIa in vivo.

Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional factor VIII (FVIII) protein and are termed cross-reacting material (CRM)-positive. FVIII is a heterodimer (domain structure A1-A2-B/A3-C1-C2) that requires thrombin cleavage to elicit procoagulant activity. Thrombin-activated FVIII is a heterotrimer with the A2 subunit (amino acid residues 373 to 740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIII. Recently, a phenotype of CRM-positive hemophilia A patients has been characterized whose plasma displays a discrepancy between their FVIII activities, where the one-stage clotting assay displays greater activity than the two-stage clotting assay. One example is a missense mutation where ARG531 has been substituted by HIS531. An FVIII cDNA construct was prepared containing the ARG531(HIS) mutation and the protein was expressed in COS-1 monkey cells by transient DNA transfection. Metabolic labeling with [35S]-methionine demonstrated that ARG531(HIS) was synthesized at an equal rate compared with FVIII wild-type (WT) but had slightly reduced antigen in the conditioned medium, suggesting a modest secretion defect. A time course of structural cleavage of ARG531(HIS) demonstrated identical thrombin cleavage sites and rates of proteolysis as FVIII WT. Similar to the patient phenotypes, ARG531(HIS) had discrepant activity as measured by a one-stage activated partial thromboplastin time (aPTT) clotting assay (36% +/- 9.6% of FVIII WT) and a variation of the two-stage assay using a chromogenic substrate (COAMATIC; 19% +/- 6.9% of FVIII WT). Partially purified FVIII WT and ARG531(HIS) proteins were subjected to functional activation by incubation with thrombin. ARG531(HIS) demonstrated significantly reduced peak activity and was completely inactivated after 30 seconds, whereas FVIII WT retained activity until 2.5 minutes after activation. Because the ARG531(HIS) missense mutation predicts a charge change to the A2 subunit, we hypothesized that the ARG531(HIS) A2 subunit could be subject to more rapid dissociation from the heterotrimer. The rate of A2 dissociation, using an optical biosensor, was determined to be fourfold faster for ARG531(HIS) compared with FVIII WT. Because the two-stage assay involves a preincubation phase before assay measurement, an increased rate of A2 dissociation would result in an increased rate of inactivation and reduced specific activity.

Amino Acid Substitution↗
PubMed2006

Disulfide bond-stabilized factor VIII has prolonged factor VIIIa activity and improved potency in whole blood clotting assays.

BACKGROUND: Genetically engineered disulfide bonds in B-domain-deleted factor (F) VIII variants (C662-C1828 FVIII and C664-C1826 FVIII) improve FVIIIa stability by blocking A2 domain dissociation because the new disulfide covalently links the A2 and A3 domains in FVIIIa. AIM: The aim of this study was to assess the hypothesis that these variants have physiologically relevant properties because of prolonged thrombin generation and improved clot formation in whole blood. METHODS: Clot-formation properties in whole blood were measured in thromboelastogram assays. The thrombin generation capabilities of the wild-type (WT) FVIII and FVIII variants were determined, and half-lives of FVIIIa variants were determined in fresh whole blood serum. RESULTS: Thromboelastogram assays were performed with fresh, severe hemophilia whole blood reconstituted with variant and WT FVIII. The two disulfide bond-stabilized FVIII variants and WT FVIII had comparable clotting times at all studied concentrations. However, when compared with WT FVIII at low concentrations, the two FVIII variants required only 10% as much FVIII to achieve comparable clot-formation rates, clot-formation times and clot firmness values. The differences between WT and FVIII variants were quite pronounced at low FVIII concentrations. Measurement of the endogenous thrombin potential in FVIII-deficient plasma supplemented with these FVIII variants confirmed that the disulfide bond-stabilized variants supported high levels of thrombin generation at lower concentrations than did WT FVIII. During the course of clot generation in whole blood, the disulfide bond-stabilized FVIIIa variants had approximately 5-fold increased half-lives relative to WT FVIIIa. CONCLUSION: C662-C1828 FVIII and C664-C1826 FVIII have physiologically relevant superior clot-forming properties in a whole blood environment, most likely due to the increased half-life of these FVIIIa variants in whole blood.

Blood Coagulation↗
PubMed1996

Ca2+ binding to the first epidermal growth factor-like domain of human blood coagulation factor IX promotes enzyme activity and factor VIII light chain binding.

Ca2+ binding to the first epidermal growth factor (EGF)-like domain of factor IX is known to be required for biological activity, but the mechanism by which Ca2+ contributes to factor IX function has remained unclear. We have studied recombinant factor IX mutants which lack Ca2+ binding to the first EGF-like domain, due to a replacement of Asp64 by Glu, Lys, or Val. The purified mutants (factors IX D64E, D64K, and D64V), were compared to plasma-derived and recombinant wild-type factor IX with regard to a number of metal-ion dependent functional parameters. In the presence of Mg2+, the activated mutants were indistinguishable from normal factor IXa in hydrolyzing the synthetic substrate CH3-SO2-Leu-Gly-Arg-p-nitroanilide. Replacing Mg2+ by Ca2+ further stimulated the activity of normal factor IXa but not of mutant factor IXa. In factor VIII-independent factor X activation, factor IXa D64K and D64E displayed reduced catalytic activity compared to normal factor IXa (apparent kcat/Km approximately 1, 2, and 4 x 10(3) M-1 s-1, respectively). In the presence of factor VIIIa, factor X activation rates by normal and mutant factor IXa were stimulated by factor VIIIa to a different extent ( approximately700- and 200-fold, respectively), indicating that Asp64 replacements affect the interaction with factor VIIIa. This possibility was addressed in inhibition studies employing synthetic peptides comprising the factor IXa-binding motifs of factor VIII heavy or light chains. Whereas the heavy chain peptide (Ser558-Gln565) inhibited factor VIII-dependent factor X activation by normal and mutant factor IXa with similar efficiency, the light chain peptide (Lys1804-Lys1818) inhibited normal factor IXa 2-3-fold more efficiently than did mutant factor IXa. This indicates that the reduced response to factor VIIIa may be due to impaired binding of mutant factor IXa to the factor VIII light chain. This was further explored in direct binding studies. In the presence of Mg2+, normal and mutant factor IXa were similar in binding to the factor VIII light chain. However, in the presence of Ca2+, factor IXa mutants were less efficient than normal factor IXa, which was illustrated by a 4-5-fold lower affinity than normal factor IXa for factor VIII light chain. Collectively, our data demonstrate that a number of factor IXa functions, including enzymatic activity and assembly into the factor IXa-factor VIIIa complex, are dependent on Ca2+ binding to the first EGF-like domain of factor IX.

Amino Acid Sequence↗
PubMed2006

The influence of sodium ion binding on factor IXa activity.

Sodium plays an important role in modulating both the amidolytic and proteolytic activities of thrombin. By contrast, while the optimal amidolytic activity of factor Xa requires Na(+), the proteolytic activity of factor Xa in the prothrombinase complex is minimally affected by the monovalent cation. In this study, we analyzed the effect of Na(+) on the amidolytic and proteolytic activity of factor IXa in the absence and presence of factor VIIIa. Factor IXa exhibited normal activity towards a fIXa-specific chromogenic substrate and antithrombin in the presence of physiological concentrations of Ca(2+) with no obvious requirement for Na(+) in either reaction. Further studies revealed that factor IXa binds to its cofactor factor VIIIa with a normal affinity in the absence of Na(+) and that the catalytic function in the intrinsic Xase complex is also independent of Na(+) in the presence of physiological concentrations of Ca(2+). These results suggest that unlike the important role that Na(+) plays in modulating the macromolecular substrate specificity of thrombin, the monovalent cation is not required for the physiological function of factor Ixa in the intrinsic Xase complex.

Allosteric Regulation↗