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Studies on the Mechanism of Ristocetin-Induced Platelet Agglutination: EFFECTS of STRUCTURAL MODIFICATION of RISTOCETIN and VANCOMYCIN

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Studies on the Mechanism of Ristocetin-Induced Platelet Agglutination: EFFECTS of STRUCTURAL MODIFICATION of RISTOCETIN and VANCOMYCIN Studies on the Mechanism of Ristocetin-Induced Platelet Agglutination: EFFECTS OF STRUCTURAL MODIFICATION OF RISTOCETIN AND VANCOMYCIN Barry S. Coller, Harvey R. Gralnick J Clin Invest. 1977;60(2):302-312. https://doi.org/10.1172/JCI108778. The mechanism by which ristocetin induces platelet agglutination in the presence of the von Willebrand factor was studied by chemically altering ristocetin and a similar antibiotic, vancomycin, by reaction with a water-soluble carbodiimide in the presence of glycine methyl ester at pH 4.75. Altering ristocetin's phenolic groups (which are thought to be important in its peptide-binding properties) resulted in a loss of both platelet-agglutinating and antibiotic activities. Restoring the phenolic groups with hydroxylamine restored both activities. Vancomycin has antibiotic and peptide-binding properties similar to ristocetin's, but differs structurally in having a free carboxyl group and thus a less positive charge at neutral pH. It does not induce platelet agglutination and actually inhibits ristocetin-induced agglutination. Reacting vancomycin with the water-soluble carbodiimide resulted in alteration of phenolic groups and permanent conversion of the carboxyl to a neutral derivative. Restoring the phenolic groups with hydroxylamine (but leaving the carboxyl neutralized) produced a compound with charge properties similar to ristocetin's which induced platelet agglutination as ristocetin does. These data suggest both a binding requirement (mediated through phenolic groups) and a strong positive charge requirement for ristocetin-induced agglutination. The data are consistent with a model wherein positively charged ristocetin binds, via its phenolic groups, to sites on the platelet surface and reduces the platelet's negative charge. This could reduce the electrostatic repulsion between platelets and/or […] Find the latest version: https://jci.me/108778/pdf Studies on the Mechanism of Ristocetin-Induced Platelet Agglutination EFFECTS OF STRUCTURAL MODIFICATION OF RISTOCETIN AND VANCOMYCIN BARRY S. COLLER and HARVEY R. GRALNICK From the Hematology Service, Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda, Maryland 20014 A B S T R A C T The mechanism by which ristocetin in- wherein positively charged ristocetin binds, via its duces platelet agglutination in the presence of the phenolic groups, to sites on the platelet surface von Willebrand factor was studied by chemically and reduces the platelet's negative charge. This could altering ristocetin and a similar antibiotic, vancomy- reduce the electrostatic repulsion between platelets cin, by reaction with a water-soluble carbodiimide and/or between platelets and the negatively charged in the presence of glycine methyl ester at pH 4.75. von Willebrand factor, and permit the macromolecular Altering ristocetin's phenolic groups (which are von Willebrand factor to cause agglutination by bridg- thought to be important in its peptide-binding ing between platelets. properties) resulted in a loss of both platelet- agglutinating and antibiotic activities. Restoring the INTRODUCTION phenolic groups with hydroxylamine restored both activities. In 1971, Howard and Firkin observed that the anti- Vancomycin has antibiotic and peptide-binding biotic ristocetin induced platelet aggregation in normal properties similar to ristocetin's, but differs struc- platelet-rich plasma (1). It is now known that this turally in having a free carboxyl group and thus a phenomenon requires: (a) a plasma factor activity ab- less positive charge at neutral pH. It does not induce sent or decreased in von Willebrand's disease (1-3), platelet agglutination and actually inhibits ristocetin- and (b) a platelet factor missing or decreased in the induced agglutination. Reacting vancomycin with the Bernard-Soulier syndrome (4). This platelet factor is water-soluble carbodiimide resulted in alteration of not affected by formalinization (5) but is decreased by phenolic groups and permanent conversion of the proteolytic enzyme treatment (6). Whereas previous carboxyl to a neutral derivative. Restoring the phenolic studies (7-9), including our own (10, 11), support an groups with hydroxylamine (but leaving the carboxyl electrostatic mechanism of aggregation, the precise neutralized) produced a compound with charge proper- manner in which ristocetin interacts with these two ties similar to ristocetin's which induced platelet factors remains unknown. agglutination as ristocetin does. The molecular structure of ristocetin (which actually These data suggest both a binding requirement is composed of two closely related compounds, (mediated through phenolic groups) and a strong ristocetins A and B) is not completely known, but positive charge requirement for ristocetin-induced current data suggest that it consists of a cyclic dipep- agglutination. The data are consistent with a model tide aglycone to which various sugars (including a unique aminosugar) are attached (12). Titration anal- ysis revealed phenolic and amino groups, with an Dr. Coller's current address is the Division of Hematology, isoionic point of 8.2-8.3 (13). Department of Medicine, State University of New York at Studies of ristocetin's antimicrobial activity showed Stony Brook, Health Sciences Center, Stony Brook, N.Y. 11794. that it can bind to specific peptides with configura- Received for publication 16 December 1976 and in revised tions similar or identical to intermediates in bac- form 24 March 1977. terial cell wall synthesis (14). This binding had an 302 The Journal of Clinical Investigation Volume 60 August 1977 -302-312 absolute requirement for a terminal, ionized carboxyl phile glycine methyl ester. Under these conditions, group on the peptide, and titration analysis indi- both phenolic and carboxyl groups will react (21, 22), cated that ristocetin's phenolic groups take part in the yielding an 0-aryl isourea and the neutral glycine binding reaction. McFarlane recently showed that methyl ester derivative of the carboxylic acid, re- these ristocetin-binding peptides can inhibit risto- spectively (Fig. 1). Both reactions consume hydrogen cetin-induced platelet agglutination (15), suggesting ions so that the amount of acid required to main- that the peptides can compete for the available tain a constant pH can be used as a rough measure of ristocetin. the extent of the reaction. The phenolic group can be Vancomycin is an antibiotic similar to ristocetin in regenerated from the 0-aryl isourea by reaction with structure (16), antimicrobial spectrum (17), mechanism hydroxylamine (22), but the carboxylic acid derivative of action, and peptide-binding properties (18). We have is not reversible with hydroxylamine. Thus, with previously shown that it does not aggregate plate- ristocetin, which contains phenolic but not carboxylic lets as ristocetin does (19), and a preliminary report acid groups, the reaction will alter the phenolic indicated that it actually inhibits ristocetin-induced groups which can then be regenerated with hydroxyl- platelet aggregation (20). Vancomycin, unlike risto- amine. Vancomycin, on the other hand, will undergo cetin, contains a single ionizable carboxyl group, in modification of both its phenolic and carboxylic addition to its amino and phenolic groups (13). This acid groups. Subsequent reaction with hydroxylamine results in vancomycin having a less positive net will regenerate the phenolic groups but not the car- charge than ristocetin at neutral pH. Since we previ- boxylic acid group. Since the carboxyl derivative is ously postulated that ristocetin's ability to induce uncharged at neutral pH, the net charge of the vanco- aggregation depended upon it having a net positive mycin will be made more positive, that is, more like charge (based on the coincidence of the upper pH limit ristocetin's. Thus, these reactions permitted us to in- of ristocetin-induced platelet aggregation [8.3-8.5] vestigate the importance of the phenolic groups (pre- with its isoionic point [8.2-8.3]), this charge differ- sumed to be important in the binding reactions) and ence may account for vancomycin's inability to induce the net molecular charge in ristocetin-induced ag- agglutination. glutination. To investigate the mechanism of ristocetin-induced agglutination, we modified both ristocetin and vanco- METHODS mycin by reaction with the water-soluble carbodi- Ristocetin (lot 54-182-BW) was obtained from Abbott Labor- imide 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide atories (North Chicago, Ill.) and vancomycin hydrochloride (EDC),1 at pH 4.75, in the presence of the nucleo- (Vancocin HCI) was obtained from Eli Lilly and Company (Indianapolis, Ind.). The manufacturer states that this risto- cetin preparation contains more than 90%o ristocetin A and IAbbreviations used in this paper: EDC, 1-(3-dimethyl- the remainder is ristocetin B as determined by paper aminopropyl)-3-ethyl carbodiimide; OD, optical density; chromatography. Before reaction with the carbodiimide, PRP, platelet-rich plasma. vancomycin hydrochloride was purified by ion-exchange R'HN - C=NHR" OH R' 0 OH N 11 Hydroxylamine + IIC 11 N R" R' HX 0 N 0 11 11 H+ II Hydroxylamine RC-OH + C \t, RC-X -~ NRNo Reaction 11 N R" U R'NH - C -NHR" FIGURE 1 Reactions occurring between water-soluble carbodiimide and phenolic and carboxyl groups. HX represents any nucleophile. Ristocetin-Induced Platelet Agglutination 303 chromatography on CM-Sephadex C-50 (Pharmacia, Fine were reduced by a factor of 0.38 to make the total micro-
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