How to test the effect of aspirin and clopidogrel in patients on dual antiplatelet therapy?

Dual antiplatelet therapy with clopidogrel and aspirin is frequently used for the prevention of recurrent ischemic events. Various laboratory methods are used to detect the effect of these drugs administered in monotherapy, however their value in dual therapy has not been explored. Here, we determined which methods used for testing the effect of clopidogrel or aspirin are influenced by the other antiplatelet agent. One arm of the study included 53 ischemic stroke patients being on clopidogrel monotherapy showing effective inhibition of the P2Y12 ADP receptor. Laboratory tests routinely used for the detection of aspirin resistance (arachidonic acid (AA)-induced platelet aggregation/secretion, AA-induced thromboxane B2 (TXB2) production in platelet-rich plasma and VerifyNow Aspirin assay) were carried out on samples obtained from these patients. The other arm of the study involved 52 patients with coronary artery disease being on aspirin monotherapy. Methods used for testing the effect of clopidogrel (ADP-induced platelet aggregation and secretion, flow cytometric analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation and a newly developed P2Y12-specific platelet aggregation (ADP[PGE1] test)) were performed on samples obtained from these patients. Clopidogrel monotherapy significantly inhibited AA-induced platelet aggregation and secretion, moreover, AA-induced TXB2 production was also significantly decreased. VASP phosphorylation and AA-induced platelet aggregation showed fair correlation in patients taking clopidogrel only. Clopidogrel did not inhibit the VerifyNow Aspirin test significantly. Aspirin monotherapy influenced ADP-induced platelet aggregation and secretion, but did not have an effect on VASP phosphorylation and on the ADP[PGE1] platelet aggregation test.

Potential links between platelets and amyloid-ß in the pathogenesis of Alzheimer's disease: Evidence from in vitro, in vivo, and clinical studies.

Cerebral amyloid angiopathy (CAA) is a prevalent comorbidity among patients with Alzheimer's disease (AD), present in up to 80% of cases with varying levels of severity. There is evidence to suggest that CAA might intensify cognitive deterioration in AD patients, thereby accelerating the development of AD pathology. As a source of amyloids, it has been postulated that platelets play a significant role in the pathogenesis of both AD and CAA. Although several studies have demonstrated that platelet activation plays an important role in the pathogenesis of AD and CAA, a clear understanding of the mechanisms involved in the three steps: platelet activation, platelet adhesion, and platelet aggregation in AD pathogenesis still remains elusive. Moreover, potential therapeutic targets in platelet-mediated AD pathogenesis have not been explicitly addressed. Therefore, the aim of this review is to collate and discuss the in vitro, in vivo, and clinical evidence related to platelet dysfunction, including associated activation, adhesion, and aggregation, with specific reference to amyloid-related AD pathogenesis. Potential therapeutic targets of platelet-mediated AD pathogenesis are also discussed. By enriching the understanding of the intricate relationship between platelet dysfunction and onset of AD, researchers may unveil new therapeutic targets or strategies to tackle this devastating neurodegeneration.

From Snake Venom's Disintegrins and C-Type Lectins to Anti-Platelet Drugs.

Snake venoms are attractive natural sources for drug discovery and development, with a number of substances either in clinical use or in research and development. These drugs were developed based on RGD-containing snake venom disintegrins, which efficiently antagonize fibrinogen activation of αIIbß3 integrin (glycoprotein GP IIb/IIIa). Typical examples of anti-platelet drugs found in clinics are Integrilin (Eptifibatide), a heptapeptide derived from Barbourin, a protein found in the venom of the American Southeastern pygmy rattlesnake and Aggrastat (Tirofiban), a small molecule based on the structure of Echistatin, and a protein found in the venom of the saw-scaled viper. Using a similar drug discovery approach, linear and cyclic peptides containing the sequence K(R)TS derived from VP12, a C-type lectin protein found in the venom of Israeli viper venom, were used as a template to synthesize Vipegitide, a novel peptidomimetic antagonist of α2ß1 integrin, with anti-platelet activity. This review focus on drug discovery of these anti-platelet agents, their indications for clinical use in acute coronary syndromes and percutaneous coronary intervention based on several clinical trials, as well as their adverse effects.

Molecular spectroscopic and thermodynamic studies on the interaction of anti-platelet drug ticlopidine with calf thymus DNA.

Ticlopidine is an anti-platelet drug which belongs to the thienopyridine structural family and exerts its effect by functioning as an ADP receptor inhibitor. Ticlopidine inhibits the expression of TarO gene in S. aureus and may provide protection against MRSA. Groove binding agents are known to disrupt the transcription factor DNA complex and consequently inhibit gene expression. Understanding the mechanism of interaction of ticlopidine with DNA can prove useful in the development of a rational drug designing system. At present, there is no such study on the interaction of anti-platelet drugs with nucleic acids. A series of biophysical experiments were performed to ascertain the binding mode between ticlopidine and calf thymus DNA. UV-visible and fluorescence spectroscopic experiments confirmed the formation of a complex between ticlopidine and calf thymus DNA. Moreover, the values of binding constant were found to be in the range of 103M-1, which is indicative of groove binding between ticlopidine and calf thymus DNA. These results were further confirmed by studying the effect of denaturation on double stranded DNA, iodide quenching, viscometric studies, thermal melting profile as well as CD spectral analysis. The thermodynamic profile of the interaction was also determined using isothermal titration calorimetric studies. The reaction was found to be endothermic and the parameters obtained were found to be consistent with those of known groove binders. In silico molecular docking studies further corroborated well with the experimental results.

Comparative effects of the anti-platelet drugs, clopidogrel, ticlopidine, and cilostazol on aspirin-induced gastric bleeding and damage in rats.

AIMS: The present study compared the effects of frequently used anti-platelet drugs, such as clopidogrel, ticlopidine, and cilostazol, on the gastric bleeding and ulcerogenic responses induced by intraluminal perfusion with 25 mM aspirin acidified with 25 mM HCl (acidified ASA) in rats. MAIN METHODS: The stomach was perfused with acidified ASA at a rate of 0.4 ml/min for 60 min under urethane anesthesia, and gastric bleeding was measured as the concentration of hemoglobin in the luminal perfusate, which was collected every 15 min. Clopidogrel (10-100mg/kg), ticlopidine (10-300 mg/kg), or cilostazol (3-30 mg/kg) was given p.o. 24h or 90 min before the perfusion of acidified ASA, respectively. KEY FINDINGS: Perfusion of the stomach with acidified ASA alone led to slight bleeding and lesions in the stomach. The pretreatment with clopidogrel, even though it did not cause bleeding or damage by itself, dose-dependently increased the gastric bleeding and ulcerogenic responses induced by acidified ASA. Ticlopidine also aggravated the severity of damage by increasing gastric bleeding, and the effects of ticlopidine at 300 mg/kg were equivalent to those of clopidogrel at 100mg/kg. In contrast, cilostazol dose-dependently decreased gastric bleeding and damage in response to acidified ASA. SIGNIFICANCE: These results demonstrated that clopidogrel and ticlopidine, P2Y12 receptor inhibitors, increased gastric bleeding and ulcerogenic responses to acidified ASA, to the same extent, while cilostazol, a phosphodiesterase III inhibitor, suppressed these responses. Therefore, cilostazol may be safely used in dual anti-platelet therapy combined with ASA, without increasing the risk of gastric bleeding.