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R Sheffer

Publications and source records attributed to R Sheffer.

13 recordsLinked to original sources
PubMed1993

High molecular weight kininogen potentiates the heparin-accelerated inhibition of plasma kallikrein by antithrombin: role for antithrombin in the regulation of kallikrein.

The effects of previously characterized interactions of high molecular weight kininogen (H-kininogen) with plasma kallikrein and with heparin on the regulation of kallikrein by the heparin-activated inhibitor, antithrombin, were investigated. H-kininogen, at levels sufficient to fully complex kallikrein, greatly potentiated the acceleration of antithrombin inhibition of kallikrein produced by heparin with high affinity for antithrombin. At I = 0.15, pH 7.4, 25 degrees C, kininogen thus maximally increased the heparin enhancement of the second-order rate constant for the antithrombin-kallikrein reaction from 13-fold (1.6 x 10(2) M-1 s-1 to 2.1 x 10(3) M-1 s-1) to 1200-fold (1.9 x 10(5) M-1 s-1). In contrast, H-kininogen had no effect on the antithrombin-kallikrein reaction in the absence of heparin, nor did the protein enhance the rate constants of 1.7 x 10(4) and 3.4 x 10(4) M-1 s-1 for kallikrein reactions with its primary plasma inhibitors C1-inhibitor and alpha 2-macroglobulin, respectively, in the absence or presence of heparin. Consistent with these results, SDS gel electrophoresis of the 125I-labeled kallikrein-inhibitor complexes formed in a mixture of these kallikrein inhibitors at their relative plasma concentrations indicated that antithrombin effectively competed with C1-inhibitor and alpha 2-macroglobulin for kallikrein, accounting for 54% of the total kallikrein complexes, only when both heparin and H-kininogen were present. Similarly, the presence of therapeutic levels of heparin (approximately 1 unit/mL) in normal, factor XII-deficient, and prekallikrein-deficient plasmas enhanced the rate of inactivation of added kallikrein by 2.3-fold and significantly altered the partitioning of radiolabeled kallikrein from predominantly C1-inhibitor and alpha 2-macroglobulin complexes (86-92%) to mostly antithrombin complexes (50-53%). Experiments in antithrombin-deficient and H-kininogen-deficient plasmas confirmed that the enhanced kallikrein inactivation rate and predominant formation of antithrombin-kallikrein complexes in heparinized plasma were dependent on antithrombin and H-kininogen. The contribution of antithrombin to kallikrein inhibition in plasma remained significant (approximately 40-70%) at optimal concentrations of unfractionated or size- and antithrombin affinity-fractionated heparin, in the presence of plasma levels of calcium and zinc ions, at 37 degrees C, and with minimal plasma dilution. These results suggest that antithrombin and H-kininogen may play important roles in the regulation of kallikrein activity in the presence of heparin or heparin-like glycosaminoglycans.

Antithrombins↗
PubMed1993

Parallel mechanisms of high molecular weight kininogen action as a cofactor in kallikrein inactivation and prekallikrein activation reactions.

The mechanism by which high molecular weight kininogen (H-kininogen) potentiates the heparin-accelerated inhibition of plasma kallikrein by antithrombin [Olson, S. T., Sheffer, R., & Francis, A. M. (1993) Biochemistry (preceding paper in this issue)] was investigated at I = 0.15, pH 7.4, 25 degrees C. Single-chain, two-chain, and light-chain, but not heavy-chain, forms of H-kininogen were similarly effective in potentiating the heparin-accelerated antithrombin-kallikrein reaction, indicating that the light-chain region of the protein was responsible for promoting kallikrein inactivation and that cleavage of H-kininogen did not significantly affect this promoting activity. H-kininogen potentiation increased in a saturable manner with increasing kininogen concentration, reflecting a KD (23 +/- 8 nM) similar to that previously measured for H-kininogen binding to kallikrein by equilibrium methods. Both H-kininogen-stimulated and unstimulated heparin rate enhancements initially increased with increasing heparin concentration in a manner corresponding to the saturation of antithrombin with heparin (KD = 10-30 nM). However, at higher heparin concentrations, the stimulated but not the unstimulated heparin rate enhancement decreased in parallel with the saturation of a protein-heparin interaction with a KD (0.4 +/- 0.2 microM) comparable to that directly measured for the H-kininogen-heparin interaction (2.0 +/- 0.2 microM). These results implied that H-kininogen stimulation required the formation of a quaternary complex in which antithrombin and H-kininogen-kallikrein complex were bound to the same heparin chain. In keeping with this interpretation, a synthetic heparin pentasaccharide representing the antithrombin binding sequence accelerated the antithrombin-kallikrein reaction to an extent similar to that of full-length heparin chains containing this sequence, but the pentasaccharide acceleration was not stimulated by H-kininogen. The importance of H-kininogen-kallikrein complex binding to heparin for kininogen stimulation was further indicated from the marked salt dependence of the second-order rate constant for the association of H-kininogen-kallikrein complex but not free kallikrein with antithrombin-heparin complex, under conditions where saturation of the two binary complexes was maintained. Kinetic analyses of antithrombin-kallikrein reactions as a function of the inhibitor concentration indicated that the KD for an initial antithrombin-kallikrein encounter complex was decreased 20-fold by heparin binding to antithrombin and an additional 200-fold by H-kininogen also binding to kallikrein. By contrast, rate constants for the conversion of the encounter complex to a stable complex were comparable for all reactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Heparin↗
PubMed1993

Mutations in the paired domain of the human PAX3 gene cause Klein-Waardenburg syndrome (WS-III) as well as Waardenburg syndrome type I (WS-I).

Waardenburg syndrome type I (WS-I) is an autosomal dominant disorder characterized by sensorineural hearing loss, dystopia canthorum, pigmentary disturbances, and other developmental defects. Klein-Waardenburg syndrome (WS-III) is a disorder with many of the same characteristics as WS-I and includes musculoskeletal abnormalities. We have recently reported the identification and characterization of one of the first gene defects, in the human PAX3 gene, which causes WS-I. PAX3 is a DNA-binding protein that contains a structural motif known as the paired domain and is believed to regulate the expression of other genes. In this report we describe two new mutations, in the human PAX3 gene, that are associated with WS. One mutation was found in a family with WS-I, while the other mutation was found in a family with WS-III. Both mutations were in the highly conserved paired domain of the human PAX3 gene and are similar to other mutations that cause WS. The results indicate that mutations in the PAX3 gene can cause both WS-I and WS-III.

Amino Acid Sequence↗
PubMed1992

Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement.

The synthetic antithrombin-binding heparin pentasaccharide and a full-length heparin of approximately 26 saccharides containing this specific sequence have been compared with respect to their interactions with antithrombin and their ability to promote inhibition and substrate reactions of antithrombin with thrombin and factor Xa. The aim of these studies was to elucidate the pentasaccharide contribution to heparin's accelerating effect on antithrombin-proteinase reactions. Pentasaccharide and full-length heparins bound antithrombin with comparable high affinities (KD values of 36 +/- 11 and 10 +/- 3 nM, respectively, at I 0.15) and induced highly similar protein fluorescence, ultraviolet and circular dichroism changes in the inhibitor. Stopped-flow fluorescence kinetic studies of the heparin binding interactions at I 0.15 were consistent with a two-step binding process for both heparins, involving an initial weak encounter complex interaction formed with similar affinities (KD 20-30 microM), followed by an inhibitor conformational change with indistinguishable forward rate constants of 520-700 s-1 but dissimilar reverse rate constants of approximately 1 s-1 for the pentasaccharide and approximately 0.2 s-1 for the full-length heparin. Second order rate constants for antithrombin reactions with thrombin and factor Xa were maximally enhanced by the pentasaccharide only 1.7-fold for thrombin, but a substantial 270-fold for factor Xa, in an ionic strength-independent manner at saturating oligosaccharide. In contrast, the full-length heparin produced large ionic strength-dependent enhancements in second order rate constants for both antithrombin reactions of 4,300-fold for thrombin and 580-fold for factor Xa at I 0.15. These enhancements were resolvable into a nonionic component ascribable to the pentasaccharide and an ionic component responsible for the additional rate increase of the larger heparin. Stoichiometric titrations of thrombin and factor Xa inactivation by antithrombin, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of these reactions, indicated that pentasaccharide and full-length heparins similarly promoted the formation of proteolytically modified inhibitor during the inactivation of factor Xa by antithrombin, whereas only the full-length heparin was effective in promoting this substrate reaction of antithrombin during the reaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)

Antithrombin III↗
PubMed1992

Autosomal dominant inheritance of Klein-Waardenburg syndrome.

We report on 2 sibs with the Klein-Waardenburg syndrome; they had dystopia canthorum, blepharophimosis, and bilateral flexion contractures of the fingers. The children's father and paternal aunt are also affected. This report confirms that the Klein-Waardenburg syndrome is an autosomal dominant syndrome.

Adult↗
PubMed1992

Parallel procoagulant and anticoagulant pathways for high molecular weight kininogen coagulant function.

High molecular weight kininogen (HK) or its procoagulant light-chain but not the heavy chain potentiated the heparin enhancement of antithrombin III inactivation of plasma kallikrein and factor XIa from 10-50-fold to approximately 1000-fold at I 0.15, pH 7.4, 25 degrees C. This potentiation resulted in antithrombin becoming a predominant inhibitor of kallikrein and factor XIa in heparinized normal but not HK-deficient plasmas. The heparin chain-length and salt dependence of this potentiation suggested an anticoagulant action of HK analogous to its procoagulant action.

Antithrombin III↗
PubMed1992

[Two-phase effect of endothelin-1 on resistance of coronary vessels in anesthesized rats with intact chest].

Effects of intracoronary infused (2 pM/kg/min for 5 min) endothelin-I on coronary blood flow was studied using modification of the method of Vetterlein and Schmidt. Blood flow in extracorporeal circuit was measured by 20 MHz pulsed Doppler flowmeter. One end of the circuit was connected to the left common carotid artery and the other was connected to the especially curved glass cannule which was placed to the origin of the coronary artery and through the right common carotid artery. Five-minute infusion of endothelin was followed by transitory dilatation and then by constriction of coronary vessels. Blockade of dihydropyridine-sensitive Ca-channels potentiated endothelin-induced vasodilation and decreased the constrictor response three-fold.

Animals↗
PubMed1991

Quantitative evaluation of solution equilibrium binding interactions by affinity partitioning: application to specific and nonspecific protein-heparin interactions.

A variation of the quantitative affinity chromatography (QAC) method of Winzor, Chaiken, and co-workers for the analysis of protein-ligand interactions has been developed and used to characterize sequence-specific and nonspecific protein-heparin interactions relevant to blood coagulation. The method allows quantitation of the binding of two components, A and B, from the competitive effect of one component, B, on the partitioning of the other component, A, between an immobilized acceptor phase and solution phase at equilibrium. Under the conditions employed, the differences in total A concentrations yielding an equivalent degree of saturation of the immobilized acceptor in the absence and presence of B defines the concentration of A bound to B in solution, thereby enabling conventional Scatchard or nonlinear least-squares analysis of the A-B equilibrium interaction. Like the QAC method, quantitation of the competitor interaction does not depend on the nature of the affinity matrix interaction, which need only be described empirically. The additional advantage of the difference method is that only the total rather than the free competitor ligand concentration need be known. The method requires that the partitioning component A be univalent, but allows for multivalency in the competitor, B, and can in principle be used to study binding interactions involving nonidentical, interacting, or nonspecific overlapping sites. Both the binding constant and the stoichiometry for the specific antithrombin-heparin interaction as well as the apparent binding constant for the nonspecific thrombin-heparin interaction at low thrombin binding densities obtained using this technique were in excellent agreement with values determined using spectroscopic probes.

Antithrombin III↗
PubMed1988

Disturbed patterns of globin chain synthesis in childhood monosomy 7 myeloproliferative syndrome.

Two children with typical clinical and haematological features of monosomy 7 myeloproliferative syndrome are presented. Both children displayed decreased production of beta-globin chains and unbalanced high alpha/non-alpha synthetic ratios similar to those characteristic of homozygous beta-thalassaemia. These provide further evidence for the involvement of the erythroid line as part of the malignant clone, indicating neoplastic transformation of a pluripotential stem cell in this disease.

Bone Marrow↗