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P Lollar

Publications and source records attributed to P Lollar.

At least 19 recordsLinked to original sources
PubMed1997

Loss of tolerance to exogenous and endogenous factor VIII in a mild hemophilia A patient with an Arg593 to Cys mutation.

A 42-year-old patient with mild hemophilia A developed spontaneous muscle hematomas 1 month after intense therapy with factor VIII concentrates. Factor VIII clotting activity was less than 1% and his factor VIII inhibitor was 10 Bethesda units (BU)/mL. The titer peaked at 128 BU despite daily infusions of factor VIII; 1 year later, the titer was 13 BU with no spontaneous bleeding for 4 months. The plasma inhibitor was 95% neutralized by factor VIII A2 domain but less than 15% neutralized by light-chain or C2 domain. His inhibitor did not cross-react with porcine factor VIII and was at least 10-fold less reactive to a series of hybrid factor VIII proteins in which human residues 484-508 are replaced by the homologous porcine sequence (Healey et al, J Biol Chem 270:14505, 1995). The inhibitor patient's DNA encoding his A2 domain and flanking sequences showed a C-T transition predicting Arg593 to Cys. Thirteen patients from 5 unrelated families with Cys593 have not developed inhibitors. Factor VIII clotting activity from one of them was inhibited similarly to diluted normal plasma by inhibitor patient plasma. In an homologous structure, ceruloplasmin (Zaitseva et al, J Biol Inorgan Chem 1:15, 1996), the residue equivalent to Arg593, is in a loop distinct from residues 484-508. On solution phase immunoprecipitation with labeled factor VIII fragments, A2, light chain, and C2 domains bound. In contrast to typical immune responses to factor VIII in patients with severe hemophilia A, this patient's inhibitor was almost entirely reactive with common epitopes within the A2 domain whereas by more sensitive immunoprecipitation testing antibodies to light chain epitopes were also present. Accordingly, immune responsiveness to exogenous factor VIII (antigen burden) appears to be more critical than his endogenous, hemophilic factor VIII to his developing high-titer anti-factor VIII antibodies and loss of tolerance to both native and hemophilic factor VIII proteins.

Adult↗
PubMed1997

Subunit structure and function of porcine factor Xa-activated factor VIII.

Factor Xa and thrombin (factor IIa) activate factor VIII (fVIII) by different proteolytic pathways. Thrombin cleaves fVIII at Arg372 between the A1 and A2 domains, at Arg740 between the A2 and B domains, and at Arg1689 between the B and A3 domains to form an A1/A2/A3-C1-C2 heterotrimer. We now report a stable porcine fVIIIaXa preparation obtained by Mono S HPLC at pH 6. NH2-terminal sequence analysis of purified subunits of fVIIIaXa revealed that factor Xa cleaves fVIII at Arg219 within the A1 domain and at Arg490 within the A2 domain, as well as at Arg372, Arg740, and Arg1689. Analytical ultracentrifugation of the fVIIIaXa preparation yielded results consistent with a single, 148 kDa species, similar to previous results with fVIIIaIIa [Lollar, P., & Parker, C. G. (1989) Biochemistry 28, 666-674]. Thus, the major species in the fVIIIaXa preparation contains five subunits, including fragments of the A1 and A2 domains that remain noncovalently bound. Fluorescence anisotropy measurements indicated there was no difference in the affinity of fVIIIaXa and fVIIIaIIa for a fluorescent dye-labeled, active-site-blocked derivative of porcine factor IXa. Additionally, the fVIIIaXa preparation bound dye-labeled factor IXa with 1:1 stoichiometry, indicating that all fVIIIaXa molecules in the preparation can bind factor IXa. However, fVIIIaXa had 4-fold less procoagulant activity than fVIIIaIIa. Kinetic analysis of fVIIIa cofactor activity using purified factor IXa and factor X suggested this difference is due to greater activity of fVIIIaIIa relative to fVIIIaXa within the intrinsic fXase complex, rather than a difference in their stabilities.

Amino Acid Sequence↗
PubMed1997

Analysis of factor VIII inhibitors using hybrid human/porcine factor VIII.

Recombinant hybrid human/porcine factor VIII molecules have been used to map a major determinant of the epitope recognized by human anti-factor VIII A2 domain inhibitory antibodies to a region bounded by human fVIII residues Arg484-Ile508. This approach is being used to characterize the C2 domain inhibitor epitope. The process of creating hybrid human/porcine factor VIII molecules to map inhibitor epitopes produces procoagulantly active fVIII with reduced reactivity with clinically significant factor VIII inhibitors. This suggests that it may be possible to develop of a hybrid human/porcine factor VIII that is useful in the management of hemophilia A and acquired hemophilia.

Amino Acid Sequence↗
PubMed1996

The cDNA and derived amino acid sequence of porcine factor VIII.

The cDNA corresponding to 137 bp of the 5' untranslated region, the signal peptide, and the A1, A3, C1, and C2 domains of porcine factor VIII (fVIII) have been cloned and sequenced. Along with previously determined sequences of the porcine fVIII B domain and the A2 domain, this completes the sequence determination of the cDNA corresponding to the translated protein. Alignments of the derived amino acid sequence of porcine fVIII with human and murine fVIII indicate that the A1, A2, A3, C1, and C2 domains are more conserved than the B domains or the proteolytic cleavage peptides corresponding to residues 337-372 and 1649-1689. The knowledge of the porcine fVIII cDNA may be useful to understand functional and immunological differences between human and porcine fVIII and may lead to improved fVIII replacement products for hemophilia. A patients through the development of recombinant porcine fVIII or hybrid human/porcine fVIII derivatives.

Amino Acid Sequence↗
PubMed1996

Involvement of thrombin anion-binding exosites 1 and 2 in the activation of factor V and factor VIII.

The role of anion-binding exosites of thrombin in the activation of factor V and factor VIII was studied using thrombin Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala (thrombin RA), a recombinant exosite 2 defective mutant, and a synthetic N-acetylated dodecapeptide, Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-O-SO4Leu (hirugen), which competitively inhibits binding of macromolecules to exosite 1. The catalytic efficiency of the activation of factor VIII or of the first step of factor V activation by thrombin RA was approximately 10% that of wild-type thrombin. The overall rate of conversion to factor Va was not influenced by the mutation. In contrast to factor V, the slow activation of factor VIII by thrombin RA was associated with a decreased rate of cleavage at all three proteolytic sites (Arg372, Arg740, and Arg1689). Hirugen inhibited factor V and factor VIII activation. These results indicate that both anion-binding exosites of thrombin are involved in the recognition of factor V and factor VIII.

Animals↗
PubMed1995

X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B.

Hereditary deficiency of factor IXa (fIXa), a key enzyme in blood coagulation, causes hemophilia B, a severe X chromosome-linked bleeding disorder afflicting 1 in 30,000 males; clinical studies have identified nearly 500 deleterious variants. The x-ray structure of porcine fIXa described here shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for factor X activation by phospholipid-bound fIXa and cofactor VIIIa. The 3.0-A-resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (EGF)-like modules; the N-terminal Gla module is partially disordered. The catalytic module, with covalent inhibitor D-Phe-1I-Pro-2I-Arg-3I chloromethyl ketone, most closely resembles fXa but differs significantly at several positions. Particularly noteworthy is the strained conformation of Glu-388, a residue strictly conserved in known fIXa sequences but conserved as Gly among other trypsin-like serine proteinases. Flexibility apparent in electron density together with modeling studies suggests that this may cause incomplete active site formation, even after zymogen, and hence the low catalytic activity of fIXa. The principal axes of the oblong EGF-like domains define an angle of 110 degrees, stabilized by a strictly conserved and fIX-specific interdomain salt bridge. The disorder of the Gla module, whose hydrophobic helix is apparent in electron density, can be attributed to the absence of calcium in the crystals; we have modeled the Gla module in its calcium form by using prothrombin fragment 1. The arched module arrangement agrees with fluorescence energy transfer experiments. Most hemophilic mutation sites of surface fIX residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary fIXa-FVIIIa-fX complex structure: fIXa and an equivalently arranged fX arch across an underlying fVIIIa subdomain from opposite sides; the stabilizing fVIIIa interactions force the catalytic modules together, completing fIXa active site formation and catalytic enhancement.

Amino Acid Chloromethyl Ketones↗
PubMed1995

Immunoblot cross-reactivity of factor VIII inhibitors with porcine factor VIII.

Porcine factor VIII has been used successfully to treat factor VIII inhibitor patients whose plasmas have minimal cross-reactivity to porcine factor VIII. However, some inhibitor plasmas do inhibit porcine factor VIII, and the extent of procoagulant inhibition often increases after treatment with porcine factor VIII. Because there is no information about the porcine factor VIII epitopes with which these antibodies react, we have compared the immunoblot and enzyme-linked immunosorbent assay (ELISA) reactivities with porcine and human factor VIII for 20 inhibitor plasmas (11 from hemophilia A patients and 9 autoantibodies). Immunoblots identified binding to porcine factor VIII for only 2 of the 12 plasmas from patients who had not received porcine factor VIII, but this reactivity could not be predicted from the inhibitor titer to porcine factor VIII. Immunoblot reactivity with porcine factor VIII was detected for 7 of 8 inhibitor plasmas from patients who had been previously treated with porcine factor VIII, and the strength of this reactivity was generally related to the inhibitor titer. Of the 5 plasmas that were immunoblot positive with the porcine factor VIII A2 domain, 4 had inhibitor titers greater than 45 Bethesda units when tested with porcine factor VIII, whereas only 1 of 15 of the other plasmas had this level of inhibitor activity with porcine factor VIII. In contrast, immunoblot reactivity to the porcine factor VIII A1 domain did not correlate with the antiporcine VIII inhibitor titer. We also determined the effect of preincubation with human or porcine factor VIII on immunoblot reactivity. In one case, immunoblot reactivity with porcine factor VIII was absorbed with porcine, but not human, factor VIII, which is consistent with antibody formation after treatment with porcine factor VIII. In no cases did human factor VIII reduce the reactivity of inhibitor plasmas with the porcine A1 domain, suggesting that these antibodies are directed at unique porcine factor VIII determinants. The reactivity to porcine A2 in 2 plasmas probably represented cross-reactivity of similar A2 determinants, because it was absorbed by both human and porcine factor VIII. Although the ELISA assays with porcine factor VIII detected antibodies in some plasmas that could not be identified by inhibitor assay or immunoblot, the level of ELISA reactivity was generally consistent with the titers of the other assays.

Animals↗
PubMed1995

Residues 484-508 contain a major determinant of the inhibitory epitope in the A2 domain of human factor VIII.

The A2 domain (residues 373-740) of human blood coagulation factor VIII (fVIII) contains a major epitope for inhibitory alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine fVIII and mapped a major determinant of the A2 epitope by using a series of active recombinant hybrid human/porcine fVIII molecules. Hybrids containing a substitution of porcine sequence at segment 410-508, 445-508, or 484-508 of the human A2 domain were not inhibited by a murine monoclonal antibody A2 inhibitory, mAb 413, whereas hybrids containing substitutions at 387-403, 387-444, and 387-468 were inhibited by mAb 413. This indicates that the segment bounded by Arg484 and Ile508 contains a major determinant of the A2 epitope. mAb 413 did not inhibit two more hybrids that contained porcine substitutions at residues 484-488 and 489-508, indicating that amino acid side chains on both sides of the Ser488-Arg489 bond within the Arg484-Ile508 segment contribute to the A2 epitope. The 484-508, 484-488, and 489-508 porcine substitution hybrids displayed decreased inhibition by A2 inhibitors from four patient plasmas, suggesting that there is little variation in the structure of the A2 epitope in the inhibitor population.

Amino Acid Sequence↗
PubMed1994

Inhibition by heparin of the human blood coagulation intrinsic pathway factor X activator.

The effect of heparin and other glycosaminoglycans on the activation of factor X by the phospholipid membrane-bound human factor IXa-factor VIIIa complex (intrinsic fXase) was studied. Standard heparin inhibited purified intrinsic fXase by 50% at approximately 0.08 unit/ml (0.4 microgram/ml), which is below the normal range of heparin concentrations achieved during antithrombotic therapy (0.2-0.7 unit/ml). Kinetic and binding experiments revealed that heparin behaves as a partial noncompetitive inhibitor. The inhibition constant of heparin with low affinity for antithrombin was indistinguishable from heparin with high affinity for antithrombin (Ki = 20 nM). Additionally, "low molecular weight" heparin, which also is used as an antithrombotic drug, was a potent inhibitor of intrinsic fXase (Ki = 60 nM). Dermatan sulfate inhibited intrinsic fXase much more weakly than standard heparin (IC50 = 80 micrograms/ml). The IC50 of the other mammalian glycosaminoglycans, chondroitin sulfate, keratan sulfate, and hyaluronic acid, were greater than 100 micrograms/ml. Purified prothrombinase and extrinsic fXase were not inhibited by heparin. We propose that part of the antithrombotic action of heparin and low molecular weight heparin is due to anti-thrombin-independent inhibition of intrinsic fXase and that heparin with low affinity for antithrombin may be useful as an antithrombotic agent.

Blood Coagulation↗
PubMed1994

Mechanism-based isocoumarin inhibitors for blood coagulation serine proteases. Effect of the 7-substituent in 7-amino-4-chloro-3-(isothioureidoalkoxy)isocoumarins on inhibitory and anticoagulant potency.

A series of 7-amino-4-chloro-3-(3-isothioureidopropoxy)isocoumarin (NH2-CiTPrOIC) derivatives with various substituents at the 7- and 3-positions have been synthesized as inhibitors of several blood coagulation enzymes. Isocoumarins substituted with basic groups such as guanidino or isothioureidoalkoxy groups were previously shown to be potent irreversible inhibitors of blood coagulation enzymes [Kam et al. Biochemistry 1988, 27, 2547-2557]. Substituted isocoumarins with an isothioureidoethoxy group at the 3-position and a large hydrophobic group at the 7-position are better inhibitors for thrombin, factor VIIa, factor Xa, factor XIa, factor IIa, and factor IXa than NH2-CiTPrOIC (4). PhNHCONH-CiTEtOIC (14), (S)-Ph(CH3)CHNHCONH-CiTEtOIC (25), and (R)-Ph(CH3)CHNHCONH-CiTEtOIC (26) inhibit thrombin quite potently and have kobs/[I] values of (1-4) x 10(4) M-1 s-1. Modeled structures of several isocoumarins noncovalently complexed with human alpha-thrombin suggest that H-bonding between the 7-substituent and the Lys-60F NH3+ relates to the inhibitory potency. Thrombin inhibited by 14, 25, or 26 is quite stable, and only 4-16% of enzymatic activity is regained after incubation for 20 days in 0.1 M Hepes, pH 7.5 buffer. However, 100, 67, and 65% of enzyme activity, respectively, is regained with the addition of 0.38 M hydroxylamine. With normal citrated pig or human plasma, these isocoumarin derivatives prolong the prothrombin time ca. 1.3-3.1-fold and also prolong the activated partial thromboplastin time more than 3-7-fold at 32 microM. Thus, these compounds are effective anticoagulants in vitro and may be useful in vivo.

Amino Acid Sequence↗
PubMed1994

Elimination of a major inhibitor epitope in factor VIII.

The A2 and C2 domains of human blood coagulation factor VIII (fVIII) contain the epitopes targeted by most inhibitory allo- and autoantibodies. Human inhibitors usually display limited or no reaction with porcine fVIII. We constructed an active, recombinant hybrid human/porcine fVIII molecule by replacing the putative human fVIII A2 domain epitope with the homologous porcine sequence. The hybrid retained full activity in the presence of antibodies with specificity restricted to the human A2 epitope. In contrast, the hybrid was neutralized by an anti-C2 antibody. These findings provide a basis for fine epitope mapping and for therapy of the inhibitor patient.

Amino Acid Sequence↗
PubMed1994

Isolation and characterization of thrombin-activated human factor VIII.

Recombinant human factor VIII (fVIII) was activated by thrombin at pH 7.4, followed by CM-Sepharose chromatography at pH values ranging from 3.5 to 7.4. Optimal coagulant activity was recovered at pH 5.5 and was associated with the isolation of an A1/A2/A3-C1-C2 heterotrimer. The activity was stable at -80 degrees C, but decayed slowly (t1/2 approximately 1 week) and nonproteolytically at room temperature or 4 degrees C. The coagulant activity of the pH 5.5 fVIIIa preparation assayed in human hemophilia A plasma was only 20% that of porcine factor VIIIa. However, its activity was approximately 75% that of porcine fVIIIa in a plasma-free assay, indicating that human fVIIIa is unstable relative to porcine fVIIIa during the coagulation assay. The first-order rate constant for spontaneous, nonproteolytic loss of activity of human fVIIIa at pH 7.4 was decreased 8-fold by fIXa and phospholipid, indicating that human fVIIIa is stabilized when incorporated into the intrinsic pathway factor X activation complex.

Animals↗
PubMed1994

Inhibition by heparin of thrombin-catalyzed activation of the factor VIII-von Willebrand factor complex.

The activation of factor VIII (fVIII) by thrombin is associated with heavy chain cleavages at Arg372 and Arg740 and light chain cleavage at Arg1689. In a defined, plasma-free assay of fVIII activation and at physiological ionic strength and pH, heparin inhibited the rate of activation of either human or porcine fVIII by thrombin in either the presence or absence of von Willebrand factor (vWf). The inhibitory effect of heparin was associated with inhibition of all three thrombin-catalyzed bond cleavages. At plasma concentrations of fVIII (approximately 1 nM) and vWf (approximately 35 nM), the rate of fVIII activation was inhibited by 50% at approximately 0.1 unit/ml heparin, which is below the normal range of heparin concentrations in plasma during therapeutic anticoagulation (0.2-0.7 unit/ml). We propose that, in addition to catalyzing the inhibition of thrombin and other intrinsic pathway coagulation proteases by antithrombin, heparin functions as an anticoagulant by direct inhibition of the activation of the fVIII-vWf complex by thrombin.

Animals↗
PubMed1994

Inhibition of human factor VIIIa by anti-A2 subunit antibodies.

Human inhibitory alloantibodies and autoantibodies to Factor VIII (FVIII) are usually directed toward the A2 and/or C2 domains of the FVIII molecule. Anti-C2 antibodies block the binding of FVIII to phospholipid, but the mechanism of action of anti-A2 antibodies is not known. We investigated the properties of a patient autoantibody, RC, and a monoclonal antibody, 413, that bind to the region which contains the epitopes of all anti-A2 alloantibodies or autoantibodies studied to date. mAb 413 and RC were noncompetitive inhibitors of a model intrinsic Factor X activation complex (intrinsic FXase) consisting of Factor IXa, activated FVIII (FVIIIa), and synthetic phospholipid vesicles, since they decreased the Vmax of intrinsic FXase by > 95% at saturating concentrations without altering the Km. This indicates that RC and mAb 413 either block the binding of FVIIIa to FIXa or phospholipid or interfere with the catalytic function of fully assembled intrinsic FXase, but they do not inhibit the binding of the substrate Factor X. mAb 413 did not inhibit the increase in fluorescence anisotropy that results from the binding of Factor VIIIa to fluorescein-5-maleimidyl-D-phenylalanyl-prolyl-arginyl-FIXa (Fl-M-FPR-FIXa) on phospholipid vesicles in the absence of Factor X, indicating it does not inhibit assembly of intrinsic FXase. Addition of Factor X to Fl-M-FPR-FIXa, FVIIIa, and phospholipid vesicles produced a further increase in fluorescence anisotropy and a decrease in fluorescence intensity. This effect was blocked completely by mAb 413. We conclude that anti-A2 antibodies inhibit FVIIIa function by blocking the conversion of intrinsic FXase/FX complex to the transition state, rather than by interfering with formation of the ground state Michaelis complex.

Animals↗
PubMed1992

Coagulant properties of hybrid human/porcine factor VIII molecules.

Human and porcine factor VIII (fVIII) are activated by thrombin to form a heterotrimer composed of subunits designated A1 and A2 derived from the fVIII heavy chain (HC) and a subunit designated A3-C1-C2 derived from the fVIII light chain (LC). Human and porcine fVIII were activated at the same rate to the same peak levels but dissociation of the A2 subunit and concomitant loss of fVIIIa activity at pH 7.4 and 22 degrees C was 3-fold faster with human fVIIIa compared to porcine fVIIIa (0.35 min-1 versus 0.12 min-1, respectively). To determine structural requirements for the increased activity of porcine fVIII, plasma-derived hybrid human/porcine fVIII molecules were isolated. Porcine HC/human LC (pHC/hLC) fVIII had 44-fold higher coagulant activity than reconstituted human fVIII (hHC/hLC), 40-fold higher activity than hHC/pLC, and slightly (1.4-fold) higher activity than reconstituted porcine fVIII (pHC/pLC). Additionally, human and porcine A2 subunits and inactive A1/A3-C1-C2 human and porcine dimers were isolated and reconstitution experiments were done. Addition of the porcine A2 subunit to the human A1/A3-C1-C2 dimer produced coagulant activity similar to that found with porcine fVIIIa and superior to human fVIIIa. These results suggest that human fVIII has weaker coagulant activity than porcine fVIII due to faster dissociation of the A2 subunit and that the A2 subunit itself is responsible for the difference.

Animals↗
PubMed1992

Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles.

The activation of factor X by factor IXa (fIXa) in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles is markedly accelerated by thrombin-activated factor VIII (fVIIIa). The interaction between highly purified fVIIIa and fIXa in this complex was studied fluorometrically at 25 degrees C by using a derivative of D-phenylalanyl-prolyl-arginyl-fIXa which was modified at the active site with fluorescein-5-maleimide (Fl-M-FPR-fIXa). Titration of Fl-M-FPR-fIXa with fVIIIa at fixed PCPS resulted in a large, saturable increase in anisotropy (delta r = 0.09). The titration data were fit to a model assuming a reversible equilibrium between fVIIIa and fIXa, resulting in an apparent dissociation constant of 2 nM and a stoichiometry of 1 mol of fVIIIa/mol of Fl-M-FPR-fIXa. The initial velocity of factor X activation was measured under identical conditions except that active fIXa and factor X were included, which yielded binding parameters similar to those determined fluorometrically. Thus, the fluorescence method accurately reflects complex formation between fVIIIa and fIXa on the phospholipid surface, and the fVIIIa-fIXa interaction is not influenced by the presence of the substrate, factor X. Addition of fVIII to Fl-M-FPR-fIXa and PCPS produced a small, saturable increase in anisotropy (delta r = 0.03), followed by a larger increase (delta r = 0.07) upon addition of thrombin to activate fVIII. Thus, fVIII binds fIXa, but proteolytic modification of fVIII must occur before the complete fVIIIa-dependent structural change in the active site of fIXa, as reflected in the anisotropy change, occurs

Animals↗