Clinical aspects of control in therapy with platelet inhibitors]

46 patients with ischaemic heart disease were treated with Micristin (20-40 mg/kg) or a combination of Micristin and Propranolol (80-120 mg/die). The values of bleeding time, the platelet factor 4 in lysate of thrombocytes or in plasma of patients as well as the soluble fibrin monomer complexes were investigated. They showed no obvious correlation to the clinical findings.     

Pyrrolo[3,2-c]pyridine derivatives as inhibitors of platelet aggregation

A series of pyrrolo[3,2-c]pyridines, isosteres of the antithrombotic drug ticlopidine, has been synthesized and evaluated in vitro for the ability to inhibit aggregation of human platelet-rich plasma induced by adenosin 5'-diphosphate (ADP). Structure-activity relationships showed their antiplatelet effects to be related to the lipophilicity.     

Interactions between human blood serum inhibitors and native and modified dextran proteinases--terrilytin and trypsin]

Interactions between native terrylytin and trypsin and their derivatives modified by water-soluble dextrans on one hand and human blood serum inhibitors on the other, were studied. It was shown that modification of the enzymes results in changes in the type of their inhibition by blood serum due to a decrease of affinity of polymeric enzyme forms for alpha 2-macroglobulin and alpha 1-antitrypsin. The inhibition constants for native and modified forms of terrylytin and trypsin were calculated. The effects of steric and electrostatic factors on the interaction between inhibitors of blood and polymeric forms of proteinases are discussed.     

Interactions between inhibitors of dihydrofolate reductase.

The binding of substrates and inhibitors to dihydrofolate reductase was studied by steady-state kinetics and high-field 1H-n.m.r. spectroscopy. A series of 5-substituted 2,4-diaminopyrimidines were examined and were found to be 'tightly binding' inhibitors of the enzyme (Ki less than 10(-9) M). Studies on the binding of 4-substituted benzenesulphonamides and benzenesulphonic acids also established the existence of a 'sulphonamide-binding site' on the enzyme. Subsequent n.m.r. experiments showed that there are two binding sites for the sulphonamides on the enzyme, one of which overlaps the coenzyme (NADPH) adenine-ring-binding site. An examination of the pH-dependence of the binding of sulphonamides to the enzyme indicated the influence of an ionizable group on the enzyme that was not directly involved in the sulphonamide binding. The change in pKa value from 6.7 to 7.2 observed on sulphonamide binding suggests the involvement of a histidine residue, which could be histidine-28.     

Interactions of thrombin with benzamidine-based inhibitors.

Trypsin and trypsin-like enzymes cleave C-terminal bonds of the basic amino acids Arg and Lys. Inhibitors of these enzymes have been found not only among Arg and Lys derivatives but also with structurally related benzamidines. Especially cyclic amides of 4-amidinophenylalanine were found to be inhibitors of thrombin. The most potent selective thrombin inhibitor of these type is N alpha-(beta-naphthylsulfonylglycyl)-4-amidinophenylalanine piperidine. From the X-ray crystal structures of thrombin and trypsin-inhibitor complexes the thrombin complexes formed with inhibitors derived from amidinophenylalanine have been modeled. These models allow valuable predictions to design inhibitors of improved selection and binding properties. Most recently, also the X-ray crystal structures of complexes of inhibitors with bovine thrombin have been solved.     

Interactions of prostanoids with the platelet activating factors.

The platelet-activating factor (PAF) induced a marked increase of the thromboxane (TX) B2-formation in the incubation medium of isolated myocardium and tissue from other organs. The content of the 6-oxo-prostaglandin (PG)F1 alpha, the inactive metabolite of PGI2, remained uninfluenced or showed a small decrease. PAF, given in a concentration of 2.10(-9) mol/l or a single dose of 100 ng, significantly reduced the contraction force and the coronary flow of isolated guinea-pig hearts. This effect was connected with a high efflux of TXA2. The PAF-antagonist, WEB 2086, nearly abolished the cardiac effects of PAF, and iloprost or a pretreatment with indomethacin markedly reduced the PAF-influence on the heart. The TXA2-antagonist BM 13177 was ineffective. The results indicate a close interaction between the myocardial PAF-effect and the TXA2-formation of the heart tissue, but gave no suggestion for a mediation of the PAF-effect by TXA2. The PAF-antagonistic action of WEB 2086, iloprost and indomethacin could be of some interest in the therapy of cardiovasculatory diseases.     

New carbamoylpiperidines as human platelet aggregation inhibitors

A series of 3-carbamoylpiperidines (nipecotamides) are designed, synthesized and tested for their inhibitory action against adenosine diphosphate (ADP)-induced aggregation of human platelets. A structure-activity analysis of the bis(nipecotamido)aralkane type showed that a substituent on the piperidine ring should preferably be an amide and that the electronegativity of the carbonyl oxygen and the orientation of the amide group affected activities. Based on the knowledge of factors influencing platelet activation and aggregation, a nitric ester moiety which could release nitric oxide (NO) in situ, is incorporated into the nipecotamide structure. These compounds exhibit increased activity compared to those having no -ONO2 function. They also show stereoselectivity, with the meso isomer being approximately twice as potent as the synthetic diastereomeric mixture. Replacement of the -ONO2 function with hydroxyl, ester or alkyl groups considerably diminishes aggregation-inhibitory potential. Nipecotamides are shown here to inhibit the basal and collagen-induced rise in platelet inositol trisphosphate (IP3) levels, as well as phosphoinositide turnover. A comprehensive mechanism of action is proposed taking earlier results into consideration.     

Interactions of papaya proteinase IV with inhibitors.

Papaya proteinase IV (PPIV) is not inhibited by chicken cystatin, or human cystatins A or C, unlike most other proteinases of the papain superfamily. The enzyme inactivates chicken cystatin and human cystatin C by limited proteolysis of the glycyl bond previously shown to be involved in the inhibitory inactivity of the cystatins, but has no action on cystatin A. Contamination of commercial crystalline papain with PPIV accounts for the limited proteolysis of cystatins by 'papain' reported previously. PPIV is slowly bound by human alpha 2-macroglobulin. The enzyme is irreversibly inactivated by E-64, and by peptidyl diazomethanes containing glycine in P1 and a hydrophobic side-chain in P2. The reaction of PPIV with iodoacetate is extremely slow. PPIV is inhibited by peptide aldehydes despite the presence of bulky sidechains in P1, suggesting that these reversible inhibitors do not bind as substrate analogues.     

Interactions among substrates and inhibitors of nitrogenase.

Examination of interactions among various substrates and inhibitors reacting with a partially purified nitrogenase from Azotobacter vinelandii has shown that: nitrous oxide is competitive with N2; carbon monixide and acetylene are noncompetitive with N2; carbon monoxide, cyanide, and nitrous oxide are noncompetitive with acetylene, whereas N2 is competitive with acetylene; carbon monoxide is noncompetitive with cyanide, whereas azide is competitive with cyanide; acetylene and nitrous oxide increase the rate of reduction of cyanide. The results are understandable if nitrogenase serves as an electron sink and substrates and inhibitors bind at multiple modified sites on reduced nitrogenase. It is suggested that substrates such as acetylene may be reduced by a less completely reduced electron sink than is required for the six-electron transfer necessary to reduce N2.     

Elastase inhibitors]

The serine proteinase elastase is located in the azurophil granules of mature circulating polymorphonuclear neutrophils. This neutrophil elastase or NE is a potent non specific serine protease which plays a role as bactericidal agent and in the degradation of immune complexes by intraphagosomal processes. It promotes inflammation when the granule contents are secreted in the extracellular environment. In certain pathological circumstances, an imbalance between NE and its major plasmatic inhibitor alpha 1-PI (formerly, alpha 1-antitrypsin) leads to abnormal tissue destruction and disease development. Genetic or acquired alpha 1-PI deficiency is thought to be involved in the pathogenesis of pulmonary emphysema. A variety of degenerative and degradative disorders are also associated to uncontrolled proteolysis by NE (rheumatoid arthritis, glomerulonephritis, adult respiratory distress symptom, psoriasis, cancer). Numerous inhibitors of NE have been reported. Various molecules are currently undergoing clinical trials for emphysema and other pulmonary diseases.     

Thienopyrimidine-based P2Y12 platelet aggregation inhibitors

Herein we describe the design and synthesis of a novel series of potent thienopyrimidine P2Y₁₂ inhibitors and the negative impact protein binding has on the inhibition of platelet aggregation.     

Anticoagulants and inhibitors of platelet aggregation derived from leeches

Increased life expectancy is associated with aging populations in the developed countries, and we can expect an increased incidence of cardiovascular and inflammatory diseases and cancers. A priority for medical research is to reduce such morbidity. Leeches have been demonstrated to be a useful source of drugs to treat cardiovascular diseases, as they have evolved highly specific mechanisms to feed on their hosts by blocking blood coagulation. Powerful molecules acting at different points in the coagulation cascade or in the inhibition of platelet aggregation have been purified from these animals. Moreover, clinical trials confirm their potential to treat cardiovascular diseases.     

The effects of platelet secretion inhibitors on protein phosphorylation

Protein phosphorylation was investigated in human platelets after stimulation to secretion by thrombin. After stimulation by thrombin at 4 degrees C (in which secretion is inhibited), phosphorylations of the 80, 56, and 38 kDa polypeptides and dephosphorylation of the 67 kDa phosphopeptide eventually occurred. The phosphorylations of the 27 and 20 kDa polypeptides remained inhibited until the temperature was increased to 37 degree C, which also resulted in secretion. Various stimulants and inhibitors of platelet function were used to characterize individual protein phosphorylations. The divalent-cation ionophore, A23187, induced the phosphorylations (or dephosphorylation) of the same proteins as thrombin with the exception of the 80 kDa protein, which remained incompletely phosphorylated. The intracellular calcium antagonist, TMB-8, inhibited thrombin-stimulated secretion and phosphorylation of all the polypeptides except the 80 kDa protein. The dephosphorylation of the 67 kDa phosphoprotein was not affected by TMB-8. Incubation of platelets with prostaglandin E1 and isobutylmethylxanthine inhibited thrombin-stimulated secretion and the phosphorylation of the 38 and 20 kDa protein and increased the phosphorylation of the 67 and 27 kDa phosphoproteins. These observations may be used to correlate protein phosphorylation with secretion, suggesting a possible sequence of intracellular events that mediate thrombin-stimulated secretion.