Anti-aggregating Agents Research Articles

The platelet P2Y receptors as targets for new antithrombotic drugs

Drugs that inhibit platelet function are widely used to decrease the risk of occlusive arterial events in patients with atherosclerosis. There are three families of anti-aggregating agents with proven clinical efficacy: (i) cyclooxygenase inhibitors, such as aspirin; (ii) ADP receptor antagonists, such as the thienopyridine compounds ticlopidine and clopidogrel; (iii) glycoprotein IIb/IIIa antagonists. All these drugs are used during coronary interventions and in the medical management of acute coronary syndromes, while only aspirin and the thienopyridine compounds are used in long-term prevention of cardiovascular and cerebrovascular events in patients at risk.

NEW INSIGHTS INTO THE PHARMACOLOGICAL PROPERTIES OF POTENT ANTIPLATELET 2-AMINO-BENZO[d]ISOTHIAZOL-3-ONE DERIVATIVES

A series of 2-amino-benzo[d]isothiazol-3-one derivatives ( 1– 8), previously described as in vitro potent antiaggregatory agents endowed with spasmolytic properties, was evaluated for in vitro antiplatelet activity in guinea-pig platelet rich plasma and for in vivo effects on experimental thrombosis and bleeding time in mice. All the 2-amino-benzo[d]isothiazol-3-one derivatives 1– 8 were more potent antiplatelets against collagen than acetylsalicylic acid and, unlike this drug, strongly inhibited thromboxane agonist U46619-induced aggregation. Subacutely administered (5 mg kg −1 day i.p. for 5 days), compounds 6 and 7 protected mice from collagen/epinephrine induced pulmonary thromboembolism at about 20-fold lower doses than acetylsalicylic acid and they prolonged bleeding time like the most part of the other derivatives. The potent antithrombotic activity was coupled with the absence of any lethal and ulcerogenic effect up to 200 mg kg −1 os.

Evaluation of the effects of anti-thromboxane agents in platelet–vessel wall interaction

We evaluated the capacity of anti-aggregating agents to influence thromboxane A 2 and prostacyclin formation, arachidonic acid-endoperoxide redirection, platelet aggregation and vessel tone, in isolated rabbit aorta incubated with homologous platelets. Picotamide ( N, N′bis(3-pyridinylmethyl)-4-methoxy-isophthalamide), the only dual thromboxane A 2-synthase inhibitor/receptor antagonist in clinical use, inhibited arachidonic acid-induced platelet aggregation with low potency, increased 180-fold by aorta presence. It inhibited thromboxane A 2 formation in platelets and, in aorta presence, increased prostacyclin formation. Ozagrel (OKY-046, ( E)-3-(4-(1-imidazolylmethyl)phenyl)-2-propenoic acid), a pure thromboxane A 2-synthase inhibitor, behaved similarly to picotamide, although the aorta caused a higher (600-fold) shift. The potency of the antagonist SQ 29,548 (1 S-(1α,2β(5 Z),3β,4α))-7-(3((2-((phenylamino)carbonyl)hydrazino)methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid) was unaffected by aorta. In coincubation experiments, arachidonic acid-challenge increased thromboxane A 2-dependent vessel tone; picotamide increased prostacyclin and reduced thromboxane A 2 formation and vasoconstriction. Ozagrel mimicked picotamide; aspirin (acetylsalicylic acid) reduced aorta contractility, thromboxane A 2 and prostacyclin formation. SQ 29,548 reduced vasoconstriction without affecting eicosanoids. We demonstrate the importance of redirection of eicosanoids in the mechanism of action of thromboxane A 2 inhibitors/antagonists within platelet–vascular wall interactions. These findings bear relevance in the development of novel anti-thrombotic drugs.

Thermoanalytical and Spectroscopic Characterization of Solid State Dipyridamole

A physico-chemical characterization of dipyridamole (C24H40N8O4), a widely used anti-aggregating agent, has been performed by using a combination of thermoanalytical (DSC) and spectroscopic (XRPD and FT-IR/PAS) techniques. A solid state transition, already reported in literature, has been ascribed to the breaking of an intramolecular H-bonds network. The rupture of a network of intermolecular H-bonds is thought to accompany the fusion. The solid state transition has been shown to be reversible provided the sample has not undergone melting.

Development and characterization of cross-linked poly(malate) microspheres with dipyridamole

Biodegradable cross-linked microspheres containing up to 63 wt.% of the active substance were obtained in a polycondensation process between d,l-malic acid and the tetrahydroxy compound dipyridamole. The in vitro release mechanism from biodegradable cross-linked microspheres has been studied. It was found that dipyridamole was released due to two-step hydrolysis of the ester bonds of the network. Initially, the only product of the hydrolytic degradation was found to be an oligomeric ester fraction with M w=1000 Da. The release of the free drug started after 8 days due to a further hydrolysis of the oligomers in solution. It was found that blood plasma enzymes in rats did not affect the hydrolytic processes. Biodegradable poly(malate) microspheres containing an anti-aggregating agent dipyridamole can be considered as a novel drug delivery system for a prolonged period of time implying a future parenteral application.

Medical Management of a Large Aortic Thrombus in a Young Woman With Essential Thrombocythemia

Aortic thrombus formation is rare in the patients with essential thrombocytosis (ET); therefore, no guidelines for its management have been established. Embolism from ET-associated large vessel thrombi is potentially lethal and has been managed surgically in a few reported cases. We describe herein a 45-year-old black woman with ET found to have a 3.5-cm, pedunculated intra-aortic thrombus at the thoracoabdominal junction. How to treat this potentially devastating aortic thrombus was a management dilemma. We believed, based on the patient's diagnosis of ET and the histology of similar thrombi in 1 reported series, that the aortic thrombus was a "white thrombus" consisting primarily of aggregated platelets with a minimal fibrin network and almost no entrapped erythrocytes. The patient was treated with aspirin, 325 mg daily, as a platelet antiaggregating agent and hydroxyurea, 1,500 mg daily, to reduce the platelet count to less than 450 x 10(9)/L. The thrombus resolved without severe thromboembolic events. To our knowledge, this is the first reported case of a large intra-aortic thrombosis associated with ET that has been successfully managed with medical therapy alone.

Inhibition of heat-induced denaturation of albumin by nonsteroidal antiinflammatory drugs (NSAIDs): Pharmacological implications

The activity of nonsteroidal antiinflammatory drugs (NSAIDs) in rheumatoid arthritis is not only due to the inhibition of the production of prostaglandins, which can even have beneficial immunosuppressive effects in chronic inflammatory processes. Since we speculated that these drugs could also act by protecting endogenous proteins against denaturation, we evaluated their effect on heat-induced denaturation human serum albumin (HSA) in comparison with several fatty acids which are known to be potent stabilizers of this protein. By the Mizushimas assay and a recently developed HPLC assay, we observed that NSAIDs were slightly less active [EC50 to approximately 10(-5)-10(-4) M] than FA and that the HPLC method was less sensitive but more selective than the turbidimetric assay, i.e. it was capable of distinguishing true antiaggregant agents like FA and NSAIDs from substances capable of inhibiting the precipitation of denatured protein aggregates. In conclusion, this survey could be useful for the development of more effective agents in protein condensation diseases like rheumatic disorders, cataract and Alzheimers disease.

Medical Management of a Large Aortic Thrombus in a Young Woman With Essential Thrombocythemia

Medical Management of a Large Aortic Thrombus in a Young Woman With Essential Thrombocythemia

Alcohol and atherosclerosis.

Observational studies have attributed a protective effect to alcohol consumption on the development of atherosclerosis and cardiovascular morbidity and mortality. Alcohol intake in the amount of one to two drinks per day results in an estimated 20-40% reduction in cardiovascular events. An additional protective effect, according to major cohort studies, has been attributed to wine, probably due to antioxidant effects and platelet antiaggregation agents. On the other hand, the influence of different patterns of alcohol consumption and environmental factors may explain a great part of the additional effect of wine. Protection may be mediated by modulation of other risk factors, because alcohol increases HDL-C, produces a biphasic response on blood pressure, and modulates the endothelial function, while it neither increases body weight nor impairs glucose-insulin homeostasis. Alcohol may also have a direct effect on atherogenesis. Despite these favorable effects, the current evidence is not enough to justify prescribing alcohol to prevent cardiovascular disease.

Drug-induced thrombotic microangiopathy: incidence, prevention and management.

The term thrombotic microangiopathy (TMA) describes syndromes characterised by microangiopathic haemolytic anaemia, thrombocytopenia and variable signs of organ damage due to platelet thrombi in the microcirculation. In children, infections with Shigella dysenteriae type 1 or particular strains of Escherichia coli are the most common cause of TMA; in adults, a variety of underlying causes have been identified, such as bacterial and viral infections, bone marrow and organ transplantation, pregnancy, immune disorders and certain drugs. Although drug-induced TMA is a rare condition, it causes significant morbidity and mortality. Antineoplastic therapy may induce TMA. Most of the cases reported are associated with mitomycin. TMA has also been associated with cyclosporin, tacrolimus, muromonab-CD3 (OKT3) and other drugs such as interferon, anti-aggregating agents (ticlopidine, clopidogrel) and quinine. The early diagnosis of drug-induced TMA may be vital. Strict monitoring of renal function, urine and blood abnormalities, and arterial pressure has to be performed in patients undergoing therapy with potentially toxic drugs. The drug must be discontinued immediately in the case of suspected TMA. Treatment modalities sometimes effective in other forms of TMA have been used empirically. Although plasma exchange therapy seems to be of value, the effectiveness of this approach has yet to be proved in multicentre, randomised clinical studies.

Management of patients with essential thrombocythemia: current concepts and perspectives

Essential thrombocythemia must now be regarded as a heterogeneous disease. Recent availability of clonality studies have repeatedly shown that a significant number of female patients diagnosed as E.T. according the most stringent criteria had a definitely polyclonal myelopoiesis. Although the incidence of patients newly diagnosed every year is low, there is in fact a conspicuous population of E.T., followed as outpatients in every department of hematology or internal medicine, including a large number of young females. These eventualities should be integrated in further discussions of the benefit/risk ratio of cytoreduction with the presently available drugs. The combination of several evidence-based data is the basis of a widely accepted stratification of high-risk patients defined by any of the following features. Age >60 to 65. History of thrombosis or embolic or major ischemic events. Platelet counts in excess of 1000 or 1500×10 9/L. In the question of chemotherapy in E.T., growing concern comes from the potential leukemogenic risk associated with the presently available drugs and extends beyond Melphalan, Busulfan and other alkylating agents and includes non-alkylating agents like Hydroxyurea. At the same time, much attention has been paid to the introduction of very precise initial diagnostic criteria directed to elimination of other myeloproliferative or myelodysplastic disorders with an increased risk of transformation. Present treatment of E.T. is a compromise between prevention of E.T. related thrombotic and bleeding complications on one hand and long term side effects and toxicity of the presently available drugs on the other hand. The recent availability of non mutagenic drugs like Interferon and most of all Anagrelid; the recognition of the role of antiaggregating agents in the treatment of platelet related microvascular ischemic events gives the opportunity for further comparative prospective trials. The use of aspirin in the management of pregnant E.T. patients is now widely accepted but there is still controversies concerning the use of Interferon in this situation.

Flow Cytometry Detection of Platelet Procoagulant Activity and Microparticles in Patients with Unstable Angina Treated by Percutaneous Coronary Angioplasty and Stent Implantation

Platelet activation is known to participate to the pathogenesis of acute coronary syndromes. Aminophospholipid exposure and microparticles shedding are hallmarks of full platelet activation and may account for the dissemination of prothrombotic seats. Using flow cytometry analysis of annexin V binding to externalized aminophospholipids, we followed platelet procoagulant activity (PPA) and platelet microparticles (PMP) shedding in venous and coronary whole blood samples from 30 patients with unstable angina before and after percutaneous coronary angioplasty (PTCA) and stent implantation. Baseline values of PPA and PMP were significantly more elevated in patients than in control subjects (p < 0.005). PMP percentage was significantly higher in coronary than in venous blood, and in coronary blood of patients with proximal instead of mid/distal lesions of coronary arteries. No enhancement of platelet reactivity to TRAP and collagen was induced by procedure. Whereas activated GpIIb-IIIa and P-selectin expression decreased 24 h and 48 h after procedure, PPA and PMP remained as elevated as before. Thus, flow cytometry is a reliable method for detection of fully activated platelets in whole blood samples. Annexin V binding analysis demonstrates the persistance of in vivo platelet activation, despite the use of antiaggregating agents.

A three-residue cyclic scaffold of non-RGD containing peptide analogues as platelet aggregation inhibitors: design, synthesis, and structure--function relationships.

Antagonists of fibrinogen at the GPIIb/IIIa receptor, which is the most abundant membrane protein on the platelet surface, are under active investigation as potential antithrombotics. The critical interaction between GPIIb/IIIa and fibrinogen can be inhibited by either linear or cyclic RGDS-containing peptides, which have been proved as lead compounds in the design of platelet aggregation inhibitors. In this study we present the design and construction of a new class of cyclic (S,S) non-RGD containing peptide sequences, using two Cys as a structural scaffold for the development of antiaggregatory agents. The (S,S)-CDC- sequence was incorporated as a conformational constraint, in molecules bearing at least one positive charge with the general formula (S,S)XCDCZ, where X = Ac-Arg, Pro-Arg, Pro-Ser-Lys, and Pro-Ser-Arg, and Z = -NH(2) and Arg-NH(2). Investigation of the structure-function relationships was performed on the basis of (a) the local conformation induced by the (S,S)-CDC motif, (b) the distance of the positively (R-C(zeta) or K-N(zeta)) and negatively (D-C(gamma)) charged centers, (c) the presence of a second positive or negative charge on the molecule, and (d) the orientation of the basic and acidic side chains defined by the pseudo dihedral angle (Pdo), which is formed by the R-C(zeta), R-C(alpha), D-C(alpha), and D-C(gamma) atoms in the case of (S,S)-RCDC and by the K-N(zeta), K-C(alpha), D-C(alpha), and D-C(gamma) atoms in the case of (S,S)-KCDC.

Aspirin

Acetylsalicylic acid (aspirin) was the first synthetized drug 100 years ago. Whereas other drugs have disappeared a long time ago, aspirin has stood the test of time and is one of the most important drugs at the turn of this century. Initially it was used for its analgesic, antipyretic and antiphlogistic properties. They are due to the acetylation of the cyclooxygenase and hence an inhibition of prostaglandin synthesis. At higher doses aspirin inhibits interleukin expression induced by nuclear factor-kappa B. Aspirin is the prototype of nonsteroidal antiinflammatory drugs, which are currently challenged by the development of specific inhibitors of cyclooxygenase isoform 2 (COX-2), which must, however, first prove their efficacy and safety in clinical trials. The inhibition of platelet aggregation by aspirin has only been recognized in the second half of the century. It is due to an inhibition of thromboxane A2 synthesis because of an irreversible cyclooxygenase blockade in platelets. Aspirin has been found to reduce the incidence and death rate of cardiovascular and cerebrovascular events and is nowadays the cornerstone of any secondary prevention in vascular diseases. Newer antiaggregatory agents such as ticlopidine, clopidogrel or IIb/IIIa-blockers have been developed. Most often they are used in conjunction with aspirin and their place has yet to be defined. New modes of action of aspirin continue to be found. Recent examples are an improvement of endothelial dysfunction or a reduced incidence of colorectal cancers. It is therefore likely that aspirin will continue to be a very useful and cheap drug for a very large population and will meet the interest of researchers for many more decades.

The synthesis and spectroscopic analysis of the neurotoxic prion peptide 106–126: Comparative use of manual Boc and Fmoc chemistry

A peptide corresponding to residues 106–126 of the human prion protein (PrP) possesses the neurotoxic and amyloidogenic properties of the infectious form of the parental protein. This peptide is now identified as a ‘difficult sequence’ and synthesis using conventional manual Fmoc chemistry was unsuccessful with acylation terminating at a central core of hydrophobic amino acids. The use of tetramethylfluoroformamidinium hexafluorophosphate and 1-methyl-2-pyrrolidone as anti-aggregatory agents in the coupling steps improved the synthesis but still resulted in an incomplete peptide. The incorporation ofN-(2-hydroxy-4-methoxybenzyl)protection at glycine residues 119 and 124 enabled synthesis of the full length peptide in low yield. Synthesis using Boc chemistry within situ neutralisation gave the full length peptide in high yield.

Frozen/Thawed Platelets: Importance of Osmotic Tolerance and Platelet Subpopulations

Me2SO cryopreserved platelets circulatein vivo,reduce bleeding time, and have hemostatic properties but their functional recovery is only half that of the fresh material. Poor osmotic response is often reported as the cause of the freezing injury. Osmotic excursions on 1- and 5-day-old platelets have been studied. Platelets stored for 5 days have a lesser capability to regulate their volume particularly after an initial swelling. This is attributed to the reduction of discoid cell number, 80% vs 62% for 1-day-old and 5-day-old platelets, respectively. After freezing, hypotonic stress response is reduced from 86 to 39% for 1-day-old and 73 to 31% for 5-day-old platelets. This reduction in function is supported by a similar reduction of discoid cells from 80 to 40% for 1-day-old and 62 to 32% for 5-day-old platelets. The integrity of the cytoskeleton is critical for the osmotic response. Freezing recovery is significantly lowered in the presence of propylene glycol, which alters actin. This contrasts with the recovery of platelets treated with anti-aggregating agents. Platelets show a greater viability after freezing and thawing when PGI2is added. It is postulated that freshly collected platelets, which are heterogeneous, contain populations of cells that are more sensitive to freezing than others. More tolerant cells remain discoid after freezing and are also less susceptible to storage lesions. Therefore, the maintenance of the integrity of the membrane and the cytoskeleton should be considered for the development of preservation methodologies.

Tratamiento antiagregante: presente y futuro

To present current information on the use of antiaggregant agents and the possibilities of their further development. Aspirin is an established treatment for the prevention of cerebrovascular accidents (CVA) in patients with transitory ischemic attacks (TIA) or minor CVA. This agent reduces the risk by 20%. Ticlopidine has a slightly greater antiaggregant effect than Aspirin, but has the disadvantage of being more expensive and having serious haematological effects such as thrombotic thrombocytopenic purpura. In combination with Aspirin, ticlopidine is valuable in maintaining coronary stents permeable. Dipyridamole, used in combination with Aspirin reduces the risk of CVA by 37% which is more than either drug used alone. Clopidogrel, chemically related to ticlopidine, has a slightly greater protective effect without the serious haematological side-effects of the latter. Use of Aspirin in CVA, alone or combined with subcutaneous heparin, is effective in the early secondary prevention of CVAs. Future development of antiaggregant treatment includes various aspects, such as the use of Aspirin in primary and secondary prevention of CVA, its value in combination with other antiaggregant, antithrombotic and neuroprotector agents. Antiaggregant agents have meant a great advance in the treatment of CVA. In view of the relatively modest degree of protection given by Aspirin, future strategies will probably include combining it with other antiaggregant agents, antithrombotic drugs and neuroprotectors.

Synthesis of angelicin heteroanalogues: preliminary photobiological and pharmacological studies

A series of angelicin heteroanalogues, in which the furan was replaced by thiophene or a 1-substituted pyrazole moiety, was synthesised in order to obtain potential therapeutic agents with antiproliferative and/or other biological activities. In general, the antiproliferative activity of the new thioangelicin, tested in different biological substrates, appeared to be higher than that of the angelicin, the natural parent compound, but lower than that of 8-MOP, the furocoumarin ordinarily used in PUVA therapy and photopheresis. Thioangelicin 6 induced strong inhibition of T2 bacteriophage infectivity and was able to significantly repress the DNA synthesis in Ehrlich ascites cells and the clonal growth in HeLa cells. The pyrazolocoumarins did not show any noticeable effect upon UVA irradiation in all the biological systems considered. All the new angelicin heteroanalogues appeared to be free of the known phototoxicity of furocoumarins on the skin. The pyrazolocoumarins have also been tested as anti-inflammatory, analgesic, antipyretic, local anaesthetic, anti-arrhythmic and platelet anti-aggregating agents by standard procedures. In this class of derivatives, 10a showed good anti-inflammatory and antipyretic properties, while 9a and 11a showed significant local anaesthetic activity.

Estrogen and postmenopausal estrogen/progestin therapy: effect on endothelium-dependent prostacyclin, nitric oxide and endothelin-1 production

It is well documented that postmenopausal estrogen/progestin therapy (HRT) protects women against cardiovascular disorders. However, the mechanism(s) by which this protection is mediated remains largely unresolved, because beneficial effects of estrogen on the blood lipid profile account for only 20–30% of the overall protection. Growing evidence suggests that estrogen has direct effects on the blood vessel wall indicating that vascular endothelium may play a key role in mediating these effects by producing vasoactive factors, such as prostacyclin (PGI 2), nitric oxide (NO) and endothelin-1 (ET-1). In vitro estrogen stimulates endothelial PGI 2 and NO production, whereas ET-1 production is not affected. Moreover, in vivo studies indicate that estrogen and HRT increase PGI 2 and NO production, whereas ET-1 production decreases. These effects are evidently mediated through estrogen receptors in endothelial cells. Thus, estrogen and HRT lead to the dominance of vasodilatory and antiaggregatory agents released by the endothelial cells. This may be an important new mechanism in the cardiovascular protection mediated by estrogen and HRT.

Inhibition of Platelet-Mediated Clot Retraction by Integrin Antagonists

The effects of the platelet αIIbβ3 integrin (GPIIb/IIIa) antagonists XV459 (non-peptide), c7E3 (Fab monoclonal antibody) and DMP728 (cyclic peptide) as well as the αvβ3 integrin antagonists, LM609 (monoclonal antibody) and XT199 (non-peptide) on clotting and platelet-mediated clot retraction were examined. While 30 nM of XV459 had no significant effect on the kinetics of coagulation, platelet-mediated clot retraction was nearly fully inhibited at this concentration (Relative Retraction Rate = 0.09). XV459 resulted in a concentration related-response curve. Other experiments demonstrated that platelet aggregation was maximally inhibited at XV459 concentrations ranging from 30–50 nM. Similarly, c7E3 demonstrated comparable inhibitory efficacy in inhibiting either clot retraction or platelet aggregation. In contrast, DMP728, an equally potent anti-aggregatory agent with an IC 50 of 20–50 nM in inhibiting platelet aggregation induced by various agonists, was found to be a less potent inhibitor of platelet-mediated clot retraction with a half-maximal inhibition of clot retraction at ∼0.7 μM, and maximum effects at concentrations of 10 μM. The αvβ3 integrin antagonists, LM609 or XT199 were without any significant effects on either platelet-mediated clot retraction or platelet aggregation. In conclusion, these data suggest a differential efficacy among different GPIIb/IIIa antagonists in inhibiting platelet-mediated clot retraction in spite of the equivalent anti-aggregatory potency. Additionally, the αvβ3 integrin antagonists do not affect platelet-mediated clot retraction or aggregation. Further studies with the previously described αIIbβ3 integrin antagonists as well as others revealed a distinct correlation between the Kd to resting and activated platelets and the efficacy in inhibiting platelet-mediated clot retraction.