Cyclic nucleotides and platelet aggregation. Effect of aggregating agents on the activity of cyclic nucleotide-metabolizing enzymes.

The activities of adenylate and guanylate cyclase and cyclic nucleotide 3':5'-phosphodiesterase were determined during the aggregation of human blood platelets with thrombin, ADP, arachidonic acid and epinephrine. The activity of guanylate cyclase is altered to a much larger degree than adenylate cyclase, while cyclic nucleotide phosphodiesterease activity remains unchanged. During the early phases of thrombin-and ADP-induced platelet aggregation a marked activation of the guanylate cyclase occurs whereas aggregation induced by arachidonic acid or epinephrine results in a rapid diminution of this activity. In all four cases, the adenylate cyclase activity is only slightly decreased when examined under identical conditions. Platelet aggregation induced by a wide variety of aggregating agents including collagen and platelet isoantibodies results in the "release" of only small amounts (1-3%) of guanylate cyclase and cyclic nucleotide phosphodiesterase and no adenylate cyclase. The guanylate cyclase and cyclic nucleotide phosphodiesterase activities are associated almost entirely with the soluble cytoplasmic fraction of the platelet, while the adenylate cyclase if found exclusively in a membrane bound form. ADP and epinephrine moderately inhibit guanylate and adenylate cyclase in subcellular preparations, while arachidonic and other unsaturated fatty acids moderately stimulate (2-4-fold) the former. It is concluded that (1) the activity of platelet guanylate cyclase during aggregation depends on the nature and mode of action of the inducing agent, (2) the activity of the membrnae adenylate cyclase during aggregation is independent of the aggregating agent and is associated with a reduction of activity and (3) cyclic nucleotide phosphodiesterase remains unchanged during the process of platelet aggregation and release. Furthermore, these observations suggest a role for unsaturated fatty acids in the control of intracellular cyclic GMP levels.     

Impaired ristocetin aggregation in myeloma due to binding of the aggregating agent by monoclonal protein--presentation of a case

In a case of severe IgG kappa myeloma with cryoglobulinaemia usual concentrations of epinephrine, collagen, ADP, arachidonic acid, thrombin and ristocetin caused no aggregation of platelets in platelet rich plasma. However, in contrast to other agents ristocetin induced platelet aggregation in higher concentrations. The investigations showed, that the aggregating activity was inhibited by binding of ristocetin to the abnormal protein. Following saturation of the monoclonal protein, the surplus ristocetin caused normal aggregation. This indicates that platelets actually preserved their responsiveness to ristocetin. Possible causes of the phenomenon are discussed.     

Preparation of 6-125I-labeled amiloride derivatives

Amiloride and certain of its derivatives are effective inhibitors of Na/H antiporters and of epithelial Na channels. We describe a simple method for the preparation of a variety of pharmacologically active 6-iodoamiloride derivatives that are labeled with 125I at high specific radioactivity. 6-Dechloroamiloride derivatives (bearing a hydrogen atom instead of the chlorine at the 6 position of the amiloride molecule) are reacted with 125ICl, prepared by the oxidation of the iodide in Na125I preparations. The 125I-labeled derivatives are separated from free 125I by anion exchange chromatography, or purified by thin layer chromatography. Both 6-dechloroamiloride and 5-(N-alkyl)-6-dechloroamiloride derivatives can be labeled by this method, with yields varying between 10 and 70%, depending on the ICl concentration and the structure of the 5-N-alkyl group. Efficient radiolabeling at high specific radioactivity also depends on the use of freshly prepared batches of 125I. Using carrier-free 125I, [125I]6-iodoamiloride and [125I]6-iodo-5-(N-tert-butyl)amiloride were prepared with yields of 27 and 22%, respectively. Potential applications of the 125I-labeled amiloride derivatives include ligand binding and affinity labeling experiments.     

Preparation and characterization of an 125I-labeled Sendai virus ligand

In an attempt to prepare a radioviral ligand that was effective both as an antigen in binding to antibody and as a ligand that effectively binds to receptor-bearing cells, inactivated Sendai virus was adsorbed to immobilized fetuin at 4°C and recovered by temperature elevation at 37°C. The eluted virus was iodinated using the chloramine-T procedure and free iodine and labeled virus were separated on a Sepharose 4B column. Radiolabeted virus contained 1000 to 5000 cpm per hemagglutination unit and over 90% of the counts were sedimented following high-speed centrifugation. Radioviral ligand was 80% reactive with immobilized Sendai virus antibody, 70% reactive with sheep red blood cells,and 1% reactive with receptor-negative horse red blood cells. Pretreatment of the cells with neuraminidase completely inhibited the virus binding reaction. The reaction with receptor-bearing cells was competitively inhibited by unlabeled Sendai virus, but not by murine leukemia virus or T-2 coliphage. Radioviral ligand binding to human lymphoblastic cell line CCL-119 was a saturable reaction, a result that demonstrates the absence of virus-virus aggregates. The preparation of an effective cell-reactive radioviral ligand was dependent on the initial purification from immobilized receptor-containing proteins.     

Molecular cloning and expression of a functional snake venom serine proteinase,with platelet aggregating activity,from the Cerastes cerastes viper

The venoms of Viperidae snakes contain numerous serine proteinases that have been recognized to possess one or more of the essential activities of thrombin on fibrinogen and platelets. Among them, a platelet proaggregant protein, cerastocytin, has been isolated from the venom of the Tunisian viper Cerastes cerastes. Using the RACE-PCR technique, we isolated and identified the complete nucleotide sequence of a cDNA serine proteinase precursor. The recombinant protein was designated rCC-PPP (for C. cerastes platelet proaggregant protein), since its deduced amino acid sequence is more than 96% identical to the partial polypeptide sequences that have been determined for natural cerastocytin. The structure of the rCC-PPP cDNA is similar to that of snake venom serine proteinases. The expression of rCC-PPP in Escherichia coli system allowed, for the first time, the preparation and purification of an active protein from snake venom with platelet proaggregant and fibrinogenolytic activities. Purified rCC-PPP efficiently activates blood platelets at nanomolar (8 nM) concentrations, as do natural cerastocytin (5 nM) and thrombin (1 nM). It is able to clot purified fibrinogen and to hydrolyze alpha-chains. Thus, rCC-PPP could be therefore considered a cerastocytin isoform. By comparison with other snake venom serine proteinases, a Gly replaces the conserved Cys(42). This implies that rCC-PPP lacks the conserved Cys(42)-Cys(58) disulfide bridge. A structural analysis performed by molecular modeling indicated that the segment of residues Tyr(67)-Arg(80) of rCC-PPP corresponds to anion-binding exosite 1 of thrombin that is involved in its capacity to induce platelet aggregation. Furthermore, the surface of the rCC-PPP molecule is characterized by a hydrophobic pocket, comprising the 90 loop (Phe(90)-Val(99)), Tyr(172), and Trp(215) residues, which might be involved in the fibrinogen clotting activity of rCC-PPP.     

Preparation of 125I-labeled secretin of high specific radioactivity.

A simple and rapid method of preparing ¹²⁵I-labeled secretin of high specific radioactivity has been developed. Synthetic porcine secretin was iodinated with Na[¹²⁵I] by a modification of the chloramine T method. Purification and separation of labeled from unlabeled secretin was achieved by chromatography on SP-Sephadex C-25 column. The labeled secretin possessed specific radioactivity as high as 500–550 μCi/μg.     

Interaction of lectins with human platelets. Effects on platelet stimulation by thrombin and ristocetin.

Wheat germ agglutinin induced aggregation and secretion of fresh platelets. Aggregation, but not secretion of serotonin by platelets in plasma, by the lectin was inhibited by 5 mM EDTA. Further, the lectin-induced stimulation of fresh platelets was blocked by prostaglandin E1. Thus, this lectin stimulates platelets by a mechanism which closely mimics thrombin activation and is independent of intercellular crosslinking. Lentil lectin did not stimulate platelets. Each platelet contained about 6 . 10(-5) binding sites for the lectins with an apparent dissociation constant of 3.0 . 10(-7) M. Wheat germ agglutinin, which binds mainly to glycoprotein I (Mr 150 000), increased the subsequent binding of thrombin to fixed platelets while lentil lectin was without effect. It appears that thrombin and wheat germ agglutinin bind to independent but interacting sites. Wheat germ agglutinin, but neither thrombin nor lentil lectin, inhibited the agglutination of platelets by ristocetin. Further, rat platelets were not aggregated by either ristocetin or wheat germ agglutinin. It appears that the interaction sites of ristocetin and wheat germ agglutinin on platelets are overlapping.     

A high molecular weight platelet aggregating factor in Crocus sativus

Bulbs of Crocus sativus, variety Cartwrightianus contain a protein factor with aggregating properties on human platelets. This factor was purified by different chromatographic techniques and shows a molecular weight of 42 000, as it was estimated by Sephadex G-75 column chromatography and sodium dodecyl sulfate (SDS) polyacrylamide slab gel electrophoresis.     

Preparation of an 125I-labeled red pigment-concentrating hormone analog.

The red pigment-concentrating hormone (RPCH) from crustacea is an octapeptide with the following structure: pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH₂, [4-Tyr]RPCH has been synthesized and was 4 times as active as RPCH in the Leander assay. Iodination of the hormone analog using chloramin-T, lactoperoxidase, solid-state lactoperoxidase, and thallium chloride was carried out and the results were compared. ¹²⁵I-labeled [4-Tyr]RPCH was prepared using a modificated chloramin-T method and was purified from unlabeled and diiodinated peptide by chromatography on Sephadex LH-20.     

Amino group modification inhibits ristocetin cofactor activity of human von Willebrand factor

Purified von Willebrand factor rapidly loses activity when treated under mild conditions with the highly specific amino group reagent trinitrobenzenesulfonic acid. Greater than 90 percent inhibition of activity is achieved by modification of only 7 percent of the amino groups. Other modifications such as acetylation and succinylation also abolish activity. It is unlikely that the essential rapidly reacting amino groups function simply in an electrostatic manner since modifications such as amidination and methylation which produce derivatives which retain positive charge are also inactive or nearly so.     

Non enzymatic glycosylation increases platelet aggregating potency of collagen from placenta of diabetic human beings

Pepsin extracted collagen and an acid soluble glycoprotein were purified from placentas of normal and diabetic human beings. Diabetic samples exhibit a significant increase in ketoamine-linked glucose whereas both amino acid and carbohydrate composition were unaffected. This excess non enzymatic condensation of glucose on free amino-groups was found to increase platelet aggregating potency of these proteins independently of any modification in fiber morphology.     

Quantitation of platelet adhesion to Ti and DLC-coated Ti in vitro using (125)I-labeled platelets

Measurement of platelet adhesion in vitro is a good indicator of its reactivity to implant devices in vivo. Platelets were labeled with I-125 without affecting its normal morphology and function and the labeled platelets were suspended in platelet poor plasma and exposed to Ti and diamond like carbon-coated (DLC) Ti discs, under dynamic conditions, using a parallel plate flow chamber. The test materials were washed, dried, exposed to a phosphor screen and scanned to get the images. The number of platelets that adhered to Ti was found to be higher than those that adhered to DLC coated Ti sample, irrespective of the shear stress which was varied between 2 and 16 dynes/cm(2).