Biomedical subjects

P Lollar

Publications and source records attributed to P Lollar.

At least 55 records · Page 3Linked to original sources

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↗

PubMed1990

pH-dependent denaturation of thrombin-activated porcine factor VIII.

Thrombin-activated porcine factor VIII (fVIIIaIIa) is a stable, active, 160-kDa heterotrimer at concentrations exceeding 2 x 10(-7) M in 0.7 M NaCl, 0.01 M histidine Cl, 5 mM CaCl2, pH 6.0, at 4 degrees C or 20 degrees C. Two of the subunits, fVIIIA1 and fVIIIA2, are derived from the heavy chain of the plasma-derived, heterodimeric fVIII precursor. The third subunit, fVIIIA3-C1-C2, is derived from the fVIII light chain. We now find that fVIIIaIIa undergoes a sharp decline in coagulant activity between pH 7 and 8. At pH 7.5, the activity of fVIIIaIIa at 3 x 10(-7) M decays within a few hours to a stable level that is approximately 70% of the value at pH 6.0, whereas at pH 8.0, greater than 99% of the activity is lost. The activity cannot be restored by readjusting the pH to 6.0. The loss of activity at pH 8.0 coincides with dissociation of the fVIIIA2 subunit since an inactive fVIIIA1/A3-C1-C2 heterodimer can be isolated by Mono S high performance liquid chromatography. After prolonged incubation at pH 8.0, the fVIIIA1 subunit also dissociates. The free fVIIIA2 fragment appears to be poorly soluble which may explain the irreversible loss of activity. Analytical velocity sedimentation of the pH-inactivated fVIIIaIIa preparation also is consistent with dissociation and precipitation of the fVIIIA2 fragment. We propose that denaturation of fVIIIaIIa by pH-dependent subunit dissociation may provide a major mechanism of inactivation of fVIIIaIIa under physiologic conditions.

Animals↗

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↗

PubMed1989

Subunit structure of thrombin-activated porcine factor VIII.

Factor VIII (fVIII) is synthesized as a single chain having a domainal sequence A1-A2-B-A3-C1-C2. Analysis of the proteolyic cleavage of fVIII by thrombin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) identifies three fragments designated fVIIIA1, fVIIIA2, and fVIIIA3-C1-C2 with fragment(s) derived from the B domain being difficult to visualize. The appearance of these fragments is associated with the development of coagulant activity, but the activity is labile without further apparent proteolysis. In this study, porcine fVIII was reacted with thrombin until peak coagulant activity was obtained and then subjected to cation-exchange (Mono S) high-pressure liquid chromatography. Coagulant activity was recovered in a single peak that contained all three fragments and was stable for weeks at 20 degrees C in 0.65 M NaCl/0.01 M His-HCl/0.005 M CaCl2 at pH 6.0. Analytical ultracentrifugation of activated fVIII was done to test whether all three fragments were associated. The apparent molecular weight of activated fVIII from equilibrium sedimentation increased from 148,000 to 161,000 as the loading concentration was increased from 0.06 to 0.16 mg/mL. This agrees well with the summed apparent molecular weights of fVIIIA1, fVIIIA2, and fVIIIA3-C1-C2 calculated from SDS-PAGE analysis (148,000) or from the amino acid sequence of human fVIII (159,000). This establishes the major species in the preparation as a fVIIIA1/A2/A3-C1-C2 heterotrimer and additionally indicates either weak self-association of the trimer and/or incomplete association of the individual subunits to form the trimer. Velocity sedimentation of activated fViii revealed a single boundry (S020,w = 7.2 S).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin.

Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. We isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO4/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated fVIII to function as a cofactor in the activation of factor X by factor IXa, we found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.

Animals↗

PubMed1988

Association of the factor VIII light chain with von Willebrand factor.

Coagulation factor VIII (fVIII) is isolated from porcine blood as a set of three heterodimers because of proteolytic cleavages in the middle, or B region, of the parent single-chain molecule. A single 80-kDa COOH-terminal fragment, the light chain (fVIIILC), is associated with one of three forms of heavy chain (fVIIIHCs) by a calcium-dependent linkage. The purified heterodimers were dissociated using EDTA and fVIIILC, and fVIIIHCs were isolated by high pressure liquid chromatography under nondenaturing conditions. The association of fVIII, fVIIILC, and fVIIIHCs with multimeric human von Willebrand factor (vWF) was studied using analytical velocity sedimentation. A previous study using this method with an intact, single heterodimeric species of fVIII has shown that one molecule of fVIII can bind to each subunit of vWF (Lollar, P., and Parker, C.G. (1987) J. Biol. Chem. 262, 17572-17576). fVIIILC bound vWF as judged by the increase in the plateau height and sedimentation coefficient of the fVIIILC.vWF complex compared to vWF at 42,000 x g and by the decrease in the plateau height of the 4.8 S fVIIILC boundary sedimenting at 240,000 x g. Titration of a fixed concentration of fVIIILC with vWF yielded a stoichiometry of one fVIIILC molecule per subunit of vWF. Proteolytic cleavage by thrombin to remove an acidic 41-residue NH2-terminal peptide from fVIIILC completely abolished its binding to vWF. In contrast, no binding of fVIIIHCs to vWF was observed. Additionally, intact fVIII bound to vWF was completely dissociated after proteolysis by thrombin. These data are consistent with the hypothesis that a critical step in blood coagulation is the release of all regions of fVIII from vWF following a single proteolytic cleavage of fVIIILC.

Edetic Acid↗

PubMed1988

Molecular characterization of commercial porcine factor VIII concentrate.

Commercial porcine factor VIII concentrate (Hyate:C) is effective in treatment of patients with hemophilia A who have circulating antibodies to factor VIII. The molecular forms of factor VIII in the concentrate were identified and evaluated in light of the known properties of porcine and human factor VIII. The factor VIII in the concentrate was isolated by tandem chromatography on gelatin-Sepharose and monoclonal anti-factor VIII-Sepharose. The factor VIII was 1% of the protein mass of the concentrate when calculated by either quantity of protein recovered or by radioimmunoassay. Both functional assay and Western blotting of the crude concentrate indicated that maximum coagulant function was achieved by proteolytic activation of procofactor forms of factor VIII. The factor VIII can be fractionated by cation-exchange high-performance liquid chromatography (HPLC) into two or three species of heterodimers depending on the lot. The specific activity of the purified porcine factor VIII was 550 U/mg using pooled porcine plasma at 1 U/mL as a standard. From this value, a factor VIII concentration in normal pig plasma of 2 micrograms/mL was calculated. This agreed well with a value of 3 micrograms/mL obtained by radioimmunoassay (RIA) of factor VIII in porcine plasma. In contrast, reported values for human factor VIII average 5800 U/mg, resulting in a calculated concentration in plasma of 0.2 microgram/mL. The finding that porcine plasma contains a significantly higher circulating mass of factor VIII than human plasma appears to explain previous difficulties in comparing porcine and human factor VIII in standard assays.

Animals↗

PubMed1987

Stoichiometry of the porcine factor VIII-von Willebrand factor association.

Factor VIII and von Willebrand factor (vWF) are glycoproteins that form a tightly bound complex in plasma. The interaction of porcine factor VIII with porcine vWF was studied by analytical velocity sedimentation. A single approximately 240-kDa species of factor VIII was isolated for use in the analysis. In contrast, when analyzed by agarose/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, vWF consisted of a population of greater than 10 multimers derived from a 270-kDa monomer. A single boundary (So20,w = 7.2 S) was observed during velocity sedimentation of factor VIII at 260,000 x g. A single boundary also was observed for vWF (weight-average So20,w = 21 S) at 42,000 x g. Under condition of excess factor VIII, the weight-average So20,w of the factor VIII-vWF complex was 40 S at 42,000 x g. At 260,000 x g, the factor VIII-vWF complex had sedimented completely, leaving only free factor VIII. The height of the plateau region of the factor VIII sedimentation velocity curve at 260,000 x g was studied as a function of several starting concentrations of vWF. The experiments were done under conditions in which the effect of radial dilution was negligible so that the plateau height was a measure of the concentration of free factor VIII. The plateau height decreased linearly as the concentration of vWF was increased, indicating that the association was essentially irreversible under the conditions used. A stoichiometry of 1.2 vWF monomers/factor VIII molecule was calculated from the slope of the line. Assuming one factor VIII-binding site/vWF monomer, these results indicate that all factor VIII-binding sites are accessible in the vWF multimer.

Animals↗

PubMed1987

Degradation of coagulation proteins by an enzyme from Malayan pit viper (Akistrodon rhodostoma) venom.

Three hydrolases from the crude venom of the Malayan pit viper (Akistrodon rhodostoma) can be differentiated. The first, which we designate ARH alpha, is the well-known fibrinogenolytic enzyme ancrod. The second, ARH beta, which has not been described previously, is identified by its electrophoretic mobility after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by its ability to hydrolyze H-D-phenylalanyl-L-piperyl-L-arginyl-rho-nitroanilide, and by inhibition of its activity by diisopropyl phosphorofluoridate. The third, ARH gamma, also previously not described, has been purified by using gel permeation and ion-exchange chromatography and preparative PAGE. Chemical, electrophoretic, and hydrodynamic data indicate that it is a single-chain, nonglobular glycoprotein with a molecular weight of 25,600. ARH gamma catalyzes the degradation of several plasma vitamin K dependent coagulation factors, including factor IX, factor X, prothrombin, and protein C. The products are electrophoretically similar to factor IXa beta, factor Xa, thrombin, and activated protein C, respectively. However, these products contain little or no enzymatic activity. ARH gamma-degraded factor IX, factor X, prothrombin, and protein C can be subsequently activated by factor XIa, Russell's viper venom X coagulant protein, crude taipan snake venom, and thrombin, respectively. The N-terminal sequence of the peptides resulting from the ARH gamma digest of porcine factor IX shows that at least three bonds are hydrolyzed: (1) at position 152, seven residues from the Arg145-Ala146 factor XIa cleavage site; (2) at position 167 within the factor IX activation peptide; and (3) at position 177, three residues from the Arg180-Val181 factor XIa cleavage site. The degradation of factor IX by ARH gamma is not affected by several serine protease inhibitors. ARH gamma catalyzes the degradation of both the heavy and light chains of porcine factor VIII which results in the inability of thrombin to activate factor VIII. ARH gamma also catalyzes the degradation of porcine antithrombin III which abolishes its ability to inhibit thrombin. These findings may have relevance to studies of hemostatic derangements following envenomation by this snake. Additionally, several novel coagulation factor derivatives have been generated for structure-function studies.

Amino Acids↗

PubMed1987

Heterogeneous, ideal associations at sedimentation equilibrium.

A general approach to analyze associations of the type mA + nB = AmBn at sedimentation equilibrium under ideal conditions was derived. A simple transform to generate a function Q was defined in which the optical measurement (absorbance or refractive index increment) at a given radial distance is divided by the value that would result if no association occurred. Explicit calculations for the cases of m = 1, n = 1 and m = 2, n = 1 were used to simulate centrifuge experiments. Nonlinear least-squares regression of Q vs. the optical measurement showed that accurate estimation of parameters could be obtained in selected instances for both types of association only if the appropriate model was used. Additionally, the effect of random experimental and systematic error (e.g., the presence of contaminants) was evaluated.

Biopolymers↗

PubMed1985

Activation of porcine factor VIII:C by thrombin and factor Xa.

The activation of porcine factor VIII:C by thrombin and by factor Xa was studied by a chromogenic substrate assay and by sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis of 125I-labeled factor VIII:C activation products. In the chromogenic assay, the kinetics of factor VIII:C dependent activation of factor X by factor IXa in the presence of calcium and phosphatidylserine/phosphatidylcholine vesicles were measured with N-benzoyl-L-isoleucyl-L-glutamylglycyl-L-arginine p-nitroanilide (S2222) as substrate. Substrate dependence of initial rates of the reaction at fixed factor IXa, factor VIII:C, lipid, and calcium obeyed Michaelis-Menten kinetics. At fixed factor IXa, factor X, lipid, and calcium the initial rates of the reaction varied linearly with lower factor VIII:C concentrations and plateaued at higher concentrations. The linear initial rate dependence formed the basis of a rapid, plasma-free assay of activated factor VIII:C. The activation of factor VIII:C by thrombin or factor Xa and the enzyme-independent rate of spontaneous inactivation were studied under conditions of excess enzyme. A model of the activation kinetics was developed and fit to the data by a nonlinear least-squares technique. From the model, the catalytic efficiencies (kcat/Km) of factor VIII:C activation by thrombin and factor Xa were 5.0 X 10(6) M-1 s-1 and 1.1 X 10(6) M-1 s-1, respectively. By comparison with published values of the catalytic efficiencies of several other coagulation enzymes for various substrates, both thrombin and factor Xa are efficient enzymes toward factor VIII:C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Isolation and characterization of native adult osteonectin.

Noncollagenous bovine bone proteins were obtained from EDTA-solubilized extracts of adult bovine bone in the absence of denaturants. Native osteonectin was isolated from the noncollagenous bone proteins by ion-exchange chromatography using DEAE-Sephadex A-50 and DEAE-Sephadex A-25, followed by gel filtration on Sephadex G-100. Comparison of the physical and chemical properties (i.e. electrophoric mobility, amino acid composition and pI) of this protein with those reported by Termine et al. (Termine, J.D., Belcourt, A.B., Conn, K.M., and Kleinman, H.K. (1981) J. Biol. Chem. 256, 10403-10408) indicate that this protein is osteonectin. Sedimentation equilibrium analyses in the presence of 6 M guandinium chloride, 10 mM Ca2+, 10 mM EDTA, or 0.15 M NaCl all yielded a molecular weight of 29,100 +/- 900. 125I-Osteonectin underwent saturable and exchangeable binding to hydroxyapatite and calf skin collagen. Eleven mg of 125I-osteonectin bound to 1.0 g of hydroxyapatite with a Kd of 8 X 10(-8) M. The intrinsic fluorescence of bovine osteonectin was partially quenched when micromolar Ca2+ was added, indicating a high affinity Ca2+ interaction. Native osteonectin was found to reduce the rate of hydroxyapatite crystal seeded growth by 50% (1 IU) at a concentration of 1.6 X 10(-7) M at pH 7.4, 37 degrees C in 0.15 M NaCl. This makes osteonectin one of the most potent inhibitors of hydroxyapatite formation presently known and more than 5 times as effective as bone Gla protein (1 IU = 8 X 10(-7) M).

Amino Acids↗

PubMed1984

Reaction of antithrombin III with thrombin bound to the vascular endothelium. Analysis in a recirculating perfused rabbit heart preparation.

A recirculating perfused rabbit heart preparation is used to study the reaction of antithrombin III (ATIII) with thrombin bound to the surface of the microvascular endothelium. Addition of ATIII to the system after thrombin is equilibrated with its binding sites results in inhibition of the enzyme as measured by disappearance of thrombin enzymatic activity from the circulation or by appearance of 125I-thrombin-ATIII complexes. The rate of inhibition of thrombin as reflected by either method is independent of the bound state of thrombin. Comparable results are obtained with ATIII modified at a single tryptophan residue. This modification does not alter the reaction rate of ATIII with thrombin but abolishes the capacity of heparin or heparan sulfate to enhance the reaction rate. From the kinetics and structural studies and the fit of the kinetics to a theoretical model relating binding equilibrium to thrombin inhibition, it is concluded that glycosaminoglycans are not involved in the reversible, high affinity, high capacity binding of thrombin to the vascular endothelium.

Animals↗

PubMed1984

Inhibition of activated porcine factor IX by dansyl-glutamyl-glycyl-arginyl-chloromethylketone.

Activated porcine Factor IX is irreversibly inhibited by an active site histidine-directed serine protease inhibitor, dansyl-glutamyl-glycyl-arginyl-chloromethylketone (DEGR-CK). The kinetics of inhibition are second order up to inhibitor concentrations of 10(-5) M. The apparent second-order rate constant (in 0.20 M NaCl, pH 8.0) is 1.7 X 10(4) M-1 min-1, which is considerably lower than values reported for Factor Xa, thrombin, plasmin, and kallikrein. Reaction of increasing concentrations of DEGR-CK with Factor IXa, followed by analysis of residual enzymatic activity, yields 1.2 mol DEGR-CK/mol protein, indicating 1:1 stoichiometry for the DEGR-CK/Factor IXa interaction. DEGR-Factor IXa is a potent anticoagulant in vitro. A concentration of 1 nM causes 50% inhibition of the ability of normal porcine-citrated plasma to correct either Factor VIII- or Factor IX-deficient plasmas (intrinsic pathway factors). In contrast, more than 100 nM DEGR-Factor IXa is required to cause 50% inhibition of Factor VII (extrinsic pathway) or Factor X (common pathway) assays. Activation of porcine Factor VIII:C by thrombin in the presence of DEGR-Factor IXa and phosphatidylcholine-phosphatidylserine vesicles reveals that DEGR-Factor IXa markedly stabilizes the spontaneous loss of Factor VIII:Ca activity as does unmodified Factor IXa [P. Lollar, G.J. Knutson, and D. N. Fass (1984) Blood 63, 1303-1308]. These results suggest that DEGR-Factor IXa incorporates into the intrinsic pathway Factor X-activator enzymatic complex, and also that stabilization of Factor VIII:Ca by this complex is independent of the active site of Factor IXa. Inhibition of Factor IXa by DEGR-CK results in the first reported irreversible active-site-modified derivative of this enzyme. DEGR-CK promises to be a useful reagent in the study of the Factor X activator complex. Conceivably, its specific anticoagulant properties could have future clinical benefit.

Amino Acid Chloromethyl Ketones↗