Biomedical subjects

D Baruch

Publications and source records attributed to D Baruch.

At least 37 records · Page 2Linked to original sources

PubMed1994

Preferential antagonism of the interactions of the integrin alpha IIb beta 3 with immobilized glycoprotein ligands by snake-venom RGD (Arg-Gly-Asp) proteins. Evidence supporting a functional role for the amino acid residues flanking the tripeptide RGD in determining the inhibitory properties of snake-venom RGD proteins.

The inhibitory properties of a panel of snake-venom-derived RGD (Arg-Gly-Asp) proteins, including the disintegrins kistrin, elegantin and albolabrin, and the neurotoxin homologue dendroaspin, were investigated in a platelet-adhesion assay using three immobilized ligands of the glycoprotein IIb-IIIa complex (alpha IIb beta 3), namely fibrinogen, fibronectin and von Willebrand factor (vWF). The snake-venom proteins preferentially inhibited the adhesion of ADP-treated platelets to one or more of the immobilized ligands. Kistrin and dendroaspin exhibited similar inhibitory characteristics, abrogating platelet adhesion to fibrinogen and vWF at nanomolar concentrations, but poorly inhibiting adhesion to fibronectin. Kistrin and dendroaspin share little overall amino-acid-sequence identity, but a considerable level of sequence similarity exists around the RGD tripeptide. Synthetic cyclic peptides corresponding to these regions of kistrin and dendroaspin inhibited platelet adhesion to both fibrinogen and fibronectin with approximately equal potency, but were 100-fold weaker antagonists of the interactions of the alpha IIb beta 3 complex with fibrinogen than their parent proteins. The disintegrins elegantin and albolabrin, which share approx. 60% overall amino-acid-sequence similarity with kistrin but have different residues around the RGD tripeptide, exhibited different antagonistic preferences. Elegantin inhibited platelet adhesion to immobilized vWF and fibronectin, but was significantly less effective at disrupting adhesion to fibrinogen. Albolabrin selectively inhibited platelet adhesion to immobilized vWF and was less effective with fibrinogen and fibronectin as adhesive ligands. In contrast with the behaviour of these venom proteins, the adhesion of ADP-treated platelets to immobilized fibrinogen, fibronectin and vWF was inhibited non-selectively by a range of monoclonal antibodies with specificity for the alpha IIb beta 3 complex. These observations, therefore, define antagonistic preferences in this panel of venom proteins towards the interactions of the alpha IIb beta 3 complex with three immobilized glycoprotein ligands.

Amino Acid Sequence↗

PubMed1994

Influence of mutations and size of multimers in type II von Willebrand disease upon the function of von Willebrand factor.

We compared the properties of plasma von Willebrand factor (vWF) from normal individuals and from two patients with type IIA (Glu875Lys) and type IIB (duplication of Met 540) von Willebrand disease (vWD) with the corresponding fully multimerized recombinant proteins. We included cryosupernatant from normal human plasma and type IIA plasma (Cys509Arg). Functions of vWF were analyzed by binding assays to platelets in the presence of ristocetin or botrocetin. Parameters of binding (number of binding sites per vWF subunit, and dissociation constant Kd) were quantitatively estimated from the binding isotherms of 125I-botrocetin or glycocalicin to vWF, independently of the size of the multimers. We found that ristocetin- or botrocetin-induced binding to platelets was correlated in all cases with the size of vWF multimers. In the absence of inducer, only type IIB rvWF Met-Met540 spontaneously bound to platelets. No significant difference of binding of purified botrocetin to vWF was found between normal and patients' plasma, or between wild-type rvWF (rvWF-WT) and rvWF-Lys875. In contrast, affinity of botrocetin for type IIB rvWF Met-Met540 was decreased. Botrocetin-induced binding of glycocalicin to vWF from all plasma and cryosupernatant was similar. Compared with rvWF-WT, binding of glycocalicin to rvWF-Lys875 was normal. In contrast, the affinity for type IIB rvWF Met-Met540 was 10-fold greater. Thus, our data suggest that, in the patients tested, the abnormal IIA phenotype results from the lack of large-sized multimers and is independent of the point mutations. In contrast, the type IIB mutation is directly involved by providing a conformation to the vWF subunits that allows the high molecular weight multimers to spontaneously interact with platelet glycoprotein Ib.

Animals↗

PubMed1994

Binding of heparin fractions to von Willebrand factor: effect of molecular weight and affinity for antithrombin III.

To investigate the influence of the structure of heparin on its binding to vWF, we compared heparin fractions of different molecular weight (MW) or affinity for antithrombin III (ATIII). We studied the interaction of purified 125I-vWF or plasma vWF, labeled with a pool of 125I-monoclonal antibodies to vWF, with unfractionated heparin immobilized on agarose beads. Fractions were compared as competitors of these interactions and their effect was quantitated by their half-maximal inhibition (IC50). When the MW of the fractions decreased, especially below 7500, their IC50 increased, indicating that the affinity of the fractions for vWF decreased with their MW. Using heparin-derived oligosaccharides, we also demonstrated that a minimal chain length of 18 monosaccharides was required for heparin binding to vWF. In addition, different fractions with low affinity for ATIII were compared as competitors of 125I-vWF binding to heparin-agarose. Despite a very low content of ATIII binding sites, some fractions retained a low IC50. Thus, heparin interaction with vWF is independent of the presence of the ATIII binding site and is mostly dependent on the length of the heparin chain. These data suggest that unfractionated heparin is a more potent inhibitor of vWF-dependent functions than low MW heparin fractions.

Antibodies, Monoclonal↗

PubMed1993

Solid-phase von Willebrand factor contains a conformationally active RGD motif that mediates endothelial cell adhesion through the alpha v beta 3 receptor.

The interaction of von Willebrand factor (vWF) with the alpha v beta 3 integrin of human umbilical vein endothelial cells is dependent on the RGD sequence present at residues 1744-1746 of the mature vWF subunit. We compared vWF and its two dimeric fragments, SpIII (residues 1-1365) and SpII (residues 1366-2050), as adhesion substrates. Solid-phase vWF and SpII supported endothelial cell adhesion, whereas SpIII, which contains the glycoprotein (GP) Ib binding domain, did not. Soluble SpII inhibited adhesion to immobilized ligands, whereas soluble vWF did not, suggesting that exposure of the cell attachment domain involves a conformational modification of vWF. Dendroaspin and albolabrin, two RGD-containing peptides of the disintegrin family, were potent inhibitors of cell adhesion to vWF (IC50 approximately 15 nmol/L). Complete inhibition of endothelial cell adhesion to vWF was obtained in the presence of F(ab')2 of monoclonal antibody 9 to vWF, which blocks vWF binding to platelet GPIIb/IIIa. In contrast, monoclonal antibody 713 to vWF, which blocks its binding to platelet GPIb, did not inhibit cell adhesion to vWF. These results indicate that endothelial cell adhesion to vWF is mediated by an RGD-dependent interaction with alpha v beta 3, but does not seem to involve a GPIb-like receptor, and show the importance of the conformation of the RGD sequence.

Amino Acid Sequence↗

PubMed1993

Localization of von Willebrand factor binding domains to endothelial extracellular matrix and to type VI collagen.

We have recently shown that von Willebrand factor (vWF) binds to endothelial and fibroblastic extracellular matrixes (ECM) in a dose-dependent, specific, and saturable way. To localize the domain on the vWF subunit responsible for this interaction, purified proteolytic fragments of vWF were compared for their ability to inhibit 125I-vWF binding to ECM. A tryptic dimeric fragment of 116 kD (T116), extending from amino acid (aa) residues 449 to 728, produced a significant inhibition of 125I-vWF binding to the ECM. In contrast, P34 (aa 1-272), SpI (aa 911-1,365), and SpII (aa 1,366-2,050) had no significant effect on 125I-vWF binding to the ECM. Using an immunofluorescence technique, we identified type VI collagen and heparan sulfate in the endothelial ECM. 125I-vWF was found to bind specifically to purified type VI collagen. Unlabeled vWF and SpIII were able to completely inhibit 125I-vWF binding to type VI collagen. T116 and SpI appeared as competitors of this interaction, whereas P34 and SpII were not. Our data suggest that vWF binds to the endothelial ECM through the T116 fragment and that T116 and SpI each contain a binding site for type VI collagen. Heparin is known to be a vWF ligand, but did not appear as a competitor of vWF binding to the ECM, nor did heparan sulfate.

Collagen↗

PubMed1991

Role of von Willebrand factor associated to extracellular matrices in platelet adhesion.

The respective role of plasmatic and endothelial extracellular matrix (ECM)-associated von Willebrand factor (vWF) in platelet adhesion was investigated at a high shear rate using a parallel-plate perfusion chamber. Incubation of the endothelial ECM with a monoclonal antibody (MoAb) to vWF, which specifically blocks vWF binding to platelet GP Ib (MoAb 322), inhibited 45% of platelet adhesion. Complete inhibition was achieved by incubating both plasma and endothelial ECM with MoAb 322 at concentrations that blocked only about 50% of adhesion when added separately. The effect of ECM-associated vWF was further demonstrated when a fibroblastic ECM, normally devoid of vWF, was coated with purified plasmatic vWF. Matrix associated-vWF was able to significantly enhance platelet adhesion in both the presence and the absence of plasmatic vWF. In contrast, this effect was not seen on endothelial ECM. Binding of exogenous vWF to the ECM was specific and dose dependent, reached the same value (500 ng/cm2) on both fibroblastic ECM and endothelial ECM, but exhibited a threefold-lower apparent dissociation constant (KD) on fibroblastic than on endothelial ECM. Our studies suggest that vWF deposited by endothelial cells in the ECM may be the most active form in platelet adhesion, whereas plasmatic vWF may only play a secondary role.

Cells, Cultured↗

PubMed1991

Plasmodium falciparum: modulation of surface antigenic expression of infected erythrocytes as revealed by cell fluorescence ELISA.

The surface reactivity of heterologous immune sera with erythrocytes infected with Plasmodium falciparum has been difficult to assess in quantitative terms because of the restricted accessibility of surface epitopes and the lack of sensitive methodologies. In a previous study we showed that cryptic antigens can become exposed on the surface of intact trophozoites if the sterol content of the membranes is increased by means conservative of cell integrity (D. Baruch and Z. I. Cabantchik, Molecular and Biochemical Parasitology 36, 127-138, 1990). In this work we introduce a novel and highly sensitive method of fluorescence cell ELISA for the quantitative estimation of immunoglobulin binding to the surface of P. falciparum-infected erythrocytes. We obtained that elevation of the membrane sterol content markedly increased the (external) surface accessibility of antigenic epitopes of trophozoites as well as rings of various strains of P. falciparum. This treatment induced exposure of similar epitope(s) on the surface of both rings and trophozoites insofar as preadsorption of sera on sterol-treated cells abolished immunoglobulin binding to either stage of infected erythrocytes (treated or not with sterol). These putative epitopes have relatively low but demonstrable accessibility on the surface of untreated rings but become virtually inaccessible at the trophozoite stage. Application of a large variety of sera (98) to sterol-treated infected cells revealed that almost 70% of the tested sera were found to give positive surface reactivity. Relatively higher intensity of binding was obtained with sera originating from clinically immune individuals. Binding of sera to cells infected with five different P. falciparum strains was essentially indistinguishable, strongly suggesting that elevation of membrane visocity induces surface exposure of cryptic epitopes common to different parasite strains.

Adult↗

PubMed1989

Production in Escherichia coli of a biologically active subfragment of von Willebrand factor corresponding to the platelet glycoprotein Ib, collagen and heparin binding domains.

A full-length cDNA for vWF has been cloned from a human lung cDNA library and a fragment of this cDNA has been modified to allow its expression in E. coli. This fragment, which corresponds to Val 449-Asn 730 of vWF and includes the GPIb-binding domain and binding sites for collagen and heparin, was subcloned into an expression vector containing an inducible lambda PL promoter. On induction, the expressed recombinant vWF subfragment migrated with a mol wt of around 38,000 after SDS-PAGE. It was identified as a vWF fragment by Western blotting using either a polyclonal or a monoclonal antibody which inhibits the binding of vWF to GPIb. Following solubilization in urea, the bacterial extract inhibited ristocetin-induced platelet aggregation and bound to ristocetin-treated platelets, to collagen and to heparin.

Binding Sites↗

PubMed1989

Passive modulation of antigenic expression in the surface of normal and malaria-infected erythrocytes.

The membranes of Plasmodium falciparum-infected human red blood cells contain antigens of demonstrably cryptic character. We show here, by a cell surface radioimmunoassay using anti-human red cell membrane antisera, that raising the membrane microviscosity of intact cells leads to a marked increase in the cell surface antigen reactivity of normal cells, and even more so in cells infected in vitro with two strains of P. falciparum. A variety of sera from adults and children living in endemic areas and from malaria patients, all of which showed no detectable surface reactivity with either normal or infected red cells, were demonstrably surface-reactive to infected cells whose sterol membrane content has been raised by means conservative of cell integrity. New epitopes become exposed on the surface of infected cells after lipid modification. The present studies indicate that the reduced membrane viscosity reported in malaria-infected cells determines to a considerable extent the expression of cell surface antigens of both host and parasite, and could play a significant role in parasite immune evasion.

Adult↗

PubMed1989

von Willebrand factor and platelet function.

vWF is an adhesive protein that binds to two distinct platelet glycoproteins, GP Ib and GP IIb-IIa complex. Its interaction with GP Ib is primarily responsible for platelet adhesion to the subendothelium. The current model is that vWF binds to collagen and/or another component of the subendothelium, after which a conformational change in the vWF molecule exposes the GP Ib binding site. This interaction may not only promote the initial attachment of platelets to the subendothelium but also play a role in thrombus formation through exposure of GP IIb-IIIa to which vWF and fibrinogen can bind. The second important function of vWF is to be a carrier for F. VIII, protecting it from degradation and playing a role in its activation by thrombin. Circulating vWF has a complex multimeric structure that ranges in Mrs from 0.5 to 20 x 10(6) Daltons. The basic subunit has a Mr of 270 kDa. Amino acid sequencing of vWF demonstrated that the basic subunit or mature vWF is made up of 2050 amino acids. Molecular cloning of the vWF cDNA revealed that the primary transcript consists of 8900 base pairs that encode for 2813 amino acids, including a 22 amino acid signal peptide and a propolypeptide of 741 amino acids, called vWF antigen II. Recent studies on the expression of recombinant vWF molecules indicate that the propolypeptide is involved in the multimerization of vWF. The domains on the vWF molecule involved in the interactions of vWF with GP Ib, GP IIb-IIIa, collagen, F. VIII and heparin have been localized to varying extents. It is anticipated that peptide analysis and recombinant DNA techniques, such as in vitro mutagenesis, will further define the structural requirements of these binding domains. vWF is synthesized in a cell-specific manner by endothelial cells and megakaryocytes. It undergoes a complex intracellular biosynthesis involving transcription of a 200 kb gene, splicing out more than 42 introns, translation of a 8900 bp mRNA, glycosylation, disulphide bond formation, sulphatation, multimerization and proteolytic cleavage. The molecule can be secreted in a constitutive or regulated manner upon perturbation of the endothelial cells with physiological and non-physiological secretagogues. The mechanisms that control the synthesis of vWF should be an exciting area of further research. vWD is probably the most common of all congenital disorders of haemostasis. It is an extremely heterogeneous syndrome involving quantitative or qualitative disorders of vWF.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗

PubMed1988

von Willebrand factor-mediated platelet adhesion to collagen involves platelet membrane glycoprotein IIb-IIIa as well as glycoprotein Ib.

Monoclonal antibodies (MAbs) directed toward distinct functional domains of human von Willebrand factor (vWF) were used to probe the involvement of platelet membrane receptors glycoprotein Ib (GPIb) and glycoprotein IIb-IIIa (GPIIb-IIIa) in vWF-mediated platelet adhesion to collagen in flowing blood. Among nine MAbs to vWF, MAb H9 inhibits binding of vWF to GPIb, MAb 9 blocks binding of vWF to GPIIb-IIIa, and MAbs B200 through B204 inhibit binding of vWF to collagen. Collagen-coated cover slips were exposed to human citrated blood at shear rates varying from 200 to 2600 sec-1 in parallel-plate perfusion chambers. Blood was reconstituted with washed radiolabeled platelets, erythrocytes, and citrated autologous plasma previously incubated with MAb IgG or F(ab')2. Platelet-collagen interactions were estimated by radioactive counting and by quantitative morphometry. Inhibition of 70% of platelet adhesion was observed at a concentration of 20 micrograms/ml MAb 9, similar to that observed with MAbs H9 or B202. The effect of each MAb was dose dependent, and their inhibitory effect on platelet adhesion was also shear rate dependent. Virtually 100% inhibition was observed at 2600 sec-1 shear rate when a mixture of the MAbs (H9 + 9 + B202) was added to reconstituted blood. Thus, blocking three functional domains of vWF virtually abolishes platelet-collagen adhesion at high shear rates, indicating that normal adhesion to collagen is mediated through binding of vWF to collagen and to both platelet membrane GPIb and GPIIb-IIIa.

Adult↗

PubMed1987

Thrombin-induced platelet factor Va formation in patients with a gray platelet syndrome.

The present study was initiated to establish the functional factor V concentration in platelets of patients with a mild bleeding disorder ascribed to a gray platelet syndrome. This inherited platelet disorder has been characterized by a specific deficiency of alpha-granules and subsequent deficiencies in the alpha-granule proteins. We found that the concentration of plasma factor V was slightly decreased (70% of normal values). In contrast, platelet factor Va formation was severely impaired. Besides a much lower factor V content than in control platelets (10-20% of normal), the dependency of platelet factor Va formation on thrombin concentration was altered. Increasing the thrombin concentration 4-fold compared to the concentration that results in maximal factor Va generation from normal platelets did not result in a maximal factor Va formation from gray platelets. When a suspension of washed gray platelets was incubated with a prostacyclin analogue prior to the stimulation with thrombin, a 10-fold lower factor Va activity was measured. Thus, thrombin-induced factor Va formation in a suspension of gray platelets is the result of a release reaction, followed by the thrombin-catalyzed activation of released factor V. Whereas the kinetics of the former reaction are apparently impaired, the kinetics of the latter one were found to be identical to those observed for normal platelet and plasma factor V activation.

Blood Platelet Disorders↗

PubMed1987

Platelet von Willebrand factor: evidence for its involvement in platelet adhesion to collagen.

Although it is well established that plasma von Willebrand Factor (vWF) is essential to platelet adhesion to subendothelium at high shear rates, the role of platelet vWF is less clear. We studied the respective role of both plasma and platelet vWF in mediating platelet adhesion to fibrillar collagen in a parallel-plate perfusion chamber. Reconstituted blood containing RBCs, various mixtures of labeled washed platelets and plasma from controls or five patients with severe von Willebrand disease (vWD), was perfused through the chamber for five minutes at a shear rate of 1,600 s-1. Platelet-collagen interactions were estimated by counting the radioactivity in deposited platelets and by quantitative morphometry. When the perfusate consisted of normal platelets suspended in normal plasma, platelet deposition on the collagen was 24.7 +/- 3.6 X 10(6)/cm2 (mean +/- SEM, n = 6). Significantly less deposition (16 +/- 2.3) was observed when vWD platelets were substituted for normal platelets. In mixtures containing vWD plasma, significantly greater deposition (9 +/- 2.2) was obtained with normal than with vWD platelets (1 +/- 0.4) demonstrating a role for platelet vWF in mediating the deposition of platelets on collagen. Morphometric analysis confirmed these data. Our findings indicate that platelet, as well as plasma, vWF mediates platelet-collagen interactions at a high shear rate.

Antibodies, Monoclonal↗

PubMed1986

Thrombin generation and inactivation in the presence of antithrombin III and heparin.

We have determined the rate constants of inactivation of factor Xa and thrombin by antithrombin III/heparin during the process of prothrombin activation. The second-order rate constant of inhibition of factor Xa alone by antithrombin III as determined by using the synthetic peptide substrate S-2337 was found to be 1.1 X 10(6) M-1 min-1. Factor Xa in prothrombin activation mixtures that contained prothrombin, and either saturating amounts of factor Va or phospholipid (20 mol % dioleoylphosphatidylserine/80 mol % dioleoylphosphatidylcholine, 10 microM), was inhibited by antithrombin III with a second-order rate constant that was essentially the same: 1.2 X 10(6) M-1 min-1. When both factor Va and phospholipid were present during prothrombin activation, factor Xa inhibition by antithrombin III was reduced about 10-fold, with a second-order rate constant of 1.3 X 10(5) M-1 min-1. Factor Xa in the prothrombin activation mixture that contained both factor Va and phospholipid was even more protected from inhibition by the antithrombin III-heparin complex. The first-order rate constants of these reactions at 200 nM antithrombin III and normalized to heparin at 1 microgram/mL were 0.33 and 9.5 min-1 in the presence and absence of factor Va and phospholipid, respectively. When the prothrombin concentration was varied widely around the Km for prothrombin, this had no effect on the first-order rate constants of inhibition. It is our conclusion that factor Xa when acting in prothrombinase on prothrombin is profoundly protected from inhibition by antithrombin III in the absence as well as in the presence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Kinetics of thrombin-induced release and activation of platelet factor V.

The kinetics of thrombin-induced platelet factor V activation were studied in suspension of washed human platelets. The effect of thrombin in stimulating the release reaction could be separated from its effect on factor V activation by use of a potent inhibitor of the release reaction, the prostacyclin analogue ZK 36374. When platelets were incubated with ZK 36374 prior to stimulation with thrombin, the amount of ZK 36374 required to inhibit 50% of factor Va formation was 15 pM. ZK 36374 at a final concentration of 1 nM was found to block instantaneously and completely the release of factor Va, whereas it has no effect neither on platelet factor V activation nor on the factor Va assay. By varying the time interval between the addition of thrombin (0.5 nM) and ZK 36374 to suspensions of 4.6 X 10(6) platelets/ml the rate of factor V release was found to be 12 pM factor V/min. In the absence of ZK 36374 the total amount of factor V released was 8 pM, whereas Triton X-100-treated platelets gave 13 pM factor V. It appeared that the amount of factor V that could be released was dependent on the thrombin concentration. Maximum release was obtained at 1 nM thrombin. The rate of factor V release increased in proportion to the thrombin concentration. The rate of factor V activation was found to be proportional to the thrombin concentration as well as to the amount of released factor V. When 4.6 X 10(6) platelets/ml were activated by 0.5 nM thrombin, the rates of factor V activation were found to be 0.3 pM and 1.2 pM factor Va/min at 20% and 90% completion of the release reaction. Therefore, the rate of factor V release was at least one order of magnitude faster than the rate of factor V activation. The kinetics of thrombin-induced platelet factor V activation were compared to those of plasma factor V activation in platelet-rich and platelet-free plasma. The results clearly demonstrate that platelets have no effect on the rate of factor V activation and that the kinetics of plasma factor V activation are identical to those of platelet factor V activation.

Blood Platelets↗

PubMed1986

Inhibition of thrombin-catalyzed reactions in blood coagulation and platelet activation by heparin fractions in the absence of antithrombin III.

The antithrombin-III-independent effect of heparin was studied in the following thrombin-catalyzed reactions: activation of purified plasma factor V and partially purified plasma factor VIII:C, generation of factor Va from the platelets and, in the presence of collagen, of the platelet procoagulant activity. Five heparin fractions and a heparinoid were compared to crude heparin. Crude heparin was a more potent inhibitor of these reactions than the fractions or the heparinoid. The inhibitory action of heparin (fractions) appeared to be the result of the formation of a complex between heparin and thrombin that alters the specificity of thrombin towards high molecular weight substrates. The inhibition of these thrombin-dependent feedback reactions in blood coagulation might be of importance in the mechanisms for the dissociation between the antithrombotic and hemorrhagic properties of low molecular weight heparins.

Antithrombin III↗

PubMed1986

The modulatory effect of membrane viscosity on structural and functional properties of the anion exchange protein of human erythrocytes.

The sterol content of human erythrocyte membranes was modified by polyvinylpyrrolidone (PVP)-mediated enrichment or depletion of cholesterol (CHL) or incorporation of cholesteryl hemisuccinate (CHS). The effects of these modifications on osmotic fragility and anion exchange protein (AEP) disposition and function were evaluated. CHS enrichment was fast (1 hr, 37 degrees C) and led to a concentration-dependent crenation as well as a decrease in osmotic cell fragility, in parallel with increased membrane microviscosity. CHL caused similar but considerably less marked effects due to slower incorporation rates into membranes. CHS enrichment of cells induced susceptibility of AEP to trypsin, a protease which otherwise does not affect AEP in intact cells. Although transport rates of monosaccharides, nucleosides, and anions were markedly slowed down by CHS enrichment of cells in parallel with increased membrane viscosity, anion transport was the most affected. The temperature profile of anion transport in CHS-enriched cells revealed a 10-kcal/mol increase in the enthalpy of activation relative to normal cells. Anion transport measured in heteroexchange conditions (Cl in--pyruvate out) and (Cl in-sulfate out) was relatively more susceptible to CHS modification than when it was measured in homoexchange conditions (Cl in-Cl out). The results of these measurements indicate that CHS-mediated increase in membrane viscosity affects AEP translocation capacity and transmembrane disposition via changes in lipid compressibility. Specific effects of CHS on AEP function, however, could not be ruled out.

Anion Transport Proteins↗

PubMed1985

Effect of heparin and low molecular weight heparins on thrombin-induced blood platelet activation in the absence of antithrombin III.

We have investigated the antithrombin III independent effect of crude heparin, two heparin fractions and a heparinoid on in vitro thrombin-induced platelet activation. Thrombin-induced platelet factor Va generation and thrombin plus collagen-induced platelet prothrombin converting activity were tested. Crude heparin was a more potent inhibitor of these reactions than the fractions or the heparinoid. The inhibitory action of the heparins was found to be the result of a direct effect on thrombin and not of an effect either on platelet activation functions or on the assembly or functioning of the prothrombinase complex. Probably this heparin inhibition is due to the masking of secondary macromolecular substrate binding sites on the thrombin molecule. We found no correlation between IC50 values and the antithrombin III-dependent antithrombin specific activities of the heparins. This supports the notion that heparin properties other than their affinity for antithrombin III may contribute to the action of this drug in blood coagulation.

Antithrombin III↗