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P Lollar

Publications and source records attributed to P Lollar.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1992

Intrinsic pathway activation of factor X and its activation peptide-deficient derivative, factor Xdes-143-191.

The role of the activation peptide in determining the substrate specificity of intrinsic pathway factor X (fX) activation was studied by using a novel derivative of fX in which 49 residues were removed enzymatically from the NH2 terminus of the 52-residue activation peptide by an enzyme from the venom of the snake Agkistrodon rhodostoma. The modified protein, designated fXdes-143-191, is inactive but is activated to alpha-fXa by either the intrinsic fX activation complex (intrinsic fXase) composed of factor IXa beta, thrombin-activated factor VIII (fVIIIaIIa), and phospholipid vesicles or by the fX coagulant protein from Russell's viper venom (RVV-XCP). Both the Km and kcat for the activation of fX by RVV-XCP were greater than for fXdes-143-191, resulting in less than a 2-fold difference in the catalytic efficiency (kcat/Km) suggestive of nonproductive binding of fXdes-143-191 to RVV-XCP. The activation of each substrate by intrinsic fXase revealed that the kcat was 100-fold greater for fX than fXdes-143-191 (16 and 0.16 s-1, respectively), although there was no detectable difference in Km (60 and 80 nM, respectively). Activations by fIXa beta/phospholipid in the absence of fVIIIaIIa also revealed a difference in kcat but not Km, but the difference in kcat was smaller (kcat of 0.007 and 0.002 s-1 and Km of 220 and 170 nM for fX and fXdes-143-191, respectively). Analysis of product versus time curves demonstrated that fVIIIaIIa promotes formation of the actyl-enzyme intermediate during fX activation. We conclude that the activation peptide plays a critical role during acyl-enzyme formation that is most pronounced in the presence of fVIIIaIIa. The absence of Km differences suggests that residues NH2-terminal to P3 do not contribute to the initial formation of the enzyme-substrate complex.

Amino Acid Sequence↗
PubMed1991

Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog.

The stability of activated human and porcine factor VIII (fVIII) differ, but a direct comparison of their structural and functional properties has not been made. Highly purified, heterodimeric human recombinant and porcine plasma-derived fVIII were exchanged into a common buffer and some minor contaminants were removed by anion-exchange chromatography. The activations of human and porcine fVIII by thrombin were studied by a two-stage coagulation assay using human citrated plasma as the standard. The peak activation of porcine fVIII was 10-fold greater than human fVIII (1.1 x 10(6) unit/mg versus 1.1 x 10(5) unit/mg). The proteolytic fragmentation of fVIII by thrombin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was not different between human and porcine fVIII, yielding previously identified bands corresponding to fragments A1, A2, A3-C1-C2, and the B domain. Following activation by thrombin, human fVIII was subjected to cation-exchange (Mono S) high performance liquid chromatography at pH 6.0 under conditions that yields stable, heterotrimeric (A1/A2/A3-C1-C2) porcine fVIIIaIIa (Lollar, P., and Parker, C.G. (1990) Biochemistry 28, 666-674). Coagulant activity was recovered in a single peak that was less than 0.5% that of porcine fVIIIaIIa (1.2 x 10(4) unit/mg versus 2.6 x 10(6) unit/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the peak fraction revealed bands corresponding to the A3-C1-C2 and A1 fragments but only trace levels of the A2 fragment. In contrast, activation of human fVIII by thrombin followed by Mono S HPLC at pH 5.0 produced a peak with 10-fold greater activity (1.2 x 10(5) unit/mg) than at pH 6.0 and which contained significant amounts of the A2 fragment. We conclude that human fVIIIIIa, like porcine fVIIIIIa, is a heterotrimer and propose that its apparent decreased coagulant activity is due to weaker association of the A2 subunit.

Animals↗
PubMed1991

The association of factor VIII with von Willebrand factor.

Factor VIII (FVIII) and von Willebrand factor (vWF) are plasma glycoproteins that circulate as a tightly associated complex. Because they tend to copurify during procedures designed to isolate the biologic activities associated with them, their identity as distinct entities became unequivocally established only during the past 10 years. Improved procedures for the isolation of FVIII, the deduction of the amino acid sequences of FVIII and vWF by using molecular cloning techniques and by direct sequencing, and the use of a variety of biophysical and immunochemical techniques have enhanced the understanding of the FVIII-vWF association. Each subunit of multimeric vWF potentially can bind a single heterodimeric FVIII molecule, although in vivo most of these binding sites are empty. The binding of FVIII to vWF is primarily, if not exclusively, mediated by the light chain of FVIII to the amino-terminal region of the vWF subunit. Cleavage of a fragment from the amino-terminal region of the FVIII light chain by thrombin results in rapid dissociation of the FVIII-vWF complex, a process that apparently is necessary for development of procoagulant activity. Whether this cleavage is needed for the activation of FVIII in the absence of vWF is controversial. The extracellular association of FVIII with vWF may be necessary for efficient secretion of FVIII from its cell of origin. The thermodynamics, kinetics, and nature of the molecular contacts involved in the interaction have not been studied. The association of FVIII with vWF prolongs the lifetime of FVIII in plasma. Whether the FVIII-vWF interaction has other functional roles, such as restricting the location of procoagulant activity, remains unknown.

Factor VIII↗
PubMed1990

von Willebrand factor is a cofactor for thrombin-catalyzed cleavage of the factor VIII light chain.

The proteolytic activation of highly purified, heterodimeric porcine factor VIII and factor VIII-von Willebrand factor complex by thrombin was compared at I 0.17, pH 7.0, 22 degrees C. During the activation of factor VIII, heavy-chain cleavage is necessary to activate the procoagulant function, whereas light-chain cleavage is required to dissociate factor VIII from von Willebrand factor. The kinetics of activation of free factor VIII and factor VIII-von Willebrand factor complex were identical. The steady-state kinetics of thrombin-catalyzed heavy-chain cleavages and light-chain cleavage of factor VIII either free or in complex with von Willebrand factor were studied using sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis and scanning densitometry of fragments derived from 125I-labeled factor VIII. Association of factor VIII with von Willebrand factor resulted in an 8-fold increase in the catalytic efficiency (kcat/Km) of light-chain cleavage (from 7 x 10(6) to 54 x 10(6) M-1 s-1). The catalytic efficiencies of heavy-chain cleavage at position 372 (approximately 6 x 10(6) M-1 s-1) and position 740 (approximately 100 x 10(6) M-1 s-1) were not affected by von Willebrand factor. We conclude that von Willebrand factor promotes cleavage of the factor VIII light chain by thrombin which is followed by rapid dissociation of the complex, so that the rate-limiting step becomes heavy-chain cleavage at position 372. This accounts for the observation that von Willebrand factor has no effect on the kinetics of activation of factor VIII by thrombin.

Animals↗