John V. Mitsios

Atorvastatin Does Not Affect the Antiplatelet Potency of Clopidogrel When It Is Administered Concomitantly for 5 Weeks in Patients With Acute Coronary Syndromes

Background—The antiplatelet effect of clopidogrel may be attenuated by short-term coadministration of lipophilic statins. We investigated whether the coadministration of atorvastatin for 5 weeks in patients with acute coronary syndromes (ACS) could affect the antiplatelet potency of clopidogrel. Methods and Results—Forty-five hypercholesterolemic patients with the first episode of an ACS were included in the study. Patients were randomized to receive daily either 10 mg of atorvastatin (n=21) or 40 mg of pravastatin (n=24). Thirty patients who underwent percutaneous coronary intervention (PCI) received a loading dose of 375 mg of clopidogrel, followed by 75 mg/d for at least 3 months. In the remaining 15 patients who refused to undergo PCI, clopidogrel therapy was not administered. Eight normolipidemic patients with the first episode of an ACS were also included and received only clopidogrel. The serum levels of soluble CD40L and the adenosine 5′-diphosphate– or thrombin receptor activating peptide-14–induced platelet aggregation, as well as P-selectin and CD40L surface expression, were studied at baseline (within 30 minutes after admission) and 5 weeks later. Neither atorvastatin nor pravastatin significantly influenced the clopidogrel-induced inhibition of platelet activation, nor did clopidogrel influence the therapeutic efficacy of atorvastatin. Conclusions—Atorvastatin does not affect the antiplatelet potency of clopidogrel when coadministered for 5 weeks in ACS patients.

Human Platelets Secrete the Plasma Type of Platelet-Activating Factor Acetylhydrolase Primarily Associated With Microparticles

Objective—Platelet-activating factor acetylhydrolase (PAF-AH) expresses a Ca2+-independent phospholipase A2 activity and hydrolyzes platelet-activating factor as well as oxidized phospholipids. Two major types of PAF-AH have been described: the plasma type, which is associated with lipoproteins, and the intracellular type II PAF-AH. Methods and Results—We investigated the type(s) of PAF-AH expressed in human platelets as well as the mechanism and the enzyme type secreted from platelets during activation. The majority of the enzyme activity (75.1±14.3% of total) is found in the cytosol, whereas 24.9±7.3% is associated with the membranes. Immunofluorescence microscopy studies and Western blotting analysis showed that platelets contain the plasma type as well as the intracellular type II PAF-AH. Furthermore, platelets contain high levels of the mRNA of plasma PAF-AH, whereas only a small quantity of the type II PAF-AH mRNA was detected. On activation, platelets secrete the plasma type of PAF-AH mainly associated with platelet-derived microparticles (PMPs). The enzyme activity was also detected on circulating PMPs in plasma from normolipidemic healthy subjects. Conclusion—This is the first indication that in addition to lipoproteins, PAF-AH in human plasma is carried by PMPs, suggesting that the PMP-associated PAF-AH may play a role in the dissemination of biological activities mediated by these particles.

The Antiplatelet and Antithrombotic Actions of Statins

Several studies over the last years have demonstrated that statins exhibit actions beyond that of lipid-lowering (pleiotropic effects) ranging from improving endothelial function, modulating the inflammatory response, maintaining plaque stability and preventing thrombus formation. Since the interplay among platelets, cells and other components of atherosclerotic lesions as well as the coagulation system play an important role in the progression of atherosclerosis and in the development of acute coronary syndromes, these non-lipid properties of statins may help to explain the early and significant reduction of cardiovascular events reported in several clinical trials of statin therapy. This review focuses on the experimental and clinical results regarding the antiplatelet/antithrombotic effects of statins.

The inhibitory potency of clopidogrel on ADP-induced platelet activation is not attenuated when it is co-administered with atorvastatin (20 mg/day) for 5 weeks in patients with acute coronary syndromes.

The antiplatelet potency of clopidogrel may be attenuated by short-term co-administration of lipophilic statins metabolized through the cytochrome P-450, isoform 3A4. We investigated whether the co-administration of atorvastatin (20?mg/day) for 5 weeks, in patients with acute coronary syndromes (ACS) could affect the antiplatelet activity of clopidogrel. Fifty-one patients with the first episode of an ACS were included in the study. All patients underwent percutaneous coronary intervention (PCI) and received a loading dose of 375 mg of clopidogrel, followed by 75 mg/day for at least 3 months. Twenty-six of them presented with low density lipoprotein (LDL) cholesterol levels >100?mg/dl (2.6 mmol/l) (measured within 24 h from the onset of symptoms) and received daily 20 mg/day of atorvastatin. The ADP- or TRAP-induced platelet aggregation, as well as P-selectin and CD40L surface expression, were studied at baseline (within 30 min after admission) and 5 weeks afterwards. Atorvastatin did not influence either the clopidogrel-induced inhibition of platelet aggregation initiated by 5 or 10 microM ADP or the clopidogrel-induced reduction of the membrane expression of P-selectin and CD40L induced by ADP. In conclusion, atorvastatin, even at a dose of 20 mg/day does not affect the antiplatelet efficacy of clopidogrel when co-administered for 5 weeks in ACS patients.

Highly constrained cyclic (S,S) -CXaaC- peptides as inhibitors of fibrinogen binding to platelets

Summary.u2002 The Arg‐Gly‐Asp RGD motif of adhesive proteins is recognized by the activated platelet integrin αIIbβ3. Binding of fibrinogen (Fg) to activated αIIbβ3 causes platelet aggregation and thrombus formation. Highly constraint cyclic (S,S) ‐CXaaC‐ containing peptides incorporating the (S,S) ‐CDC‐ and (S,S) ‐CRC‐ motifs were tested for their ability to inhibit platelet aggregation and Fg binding. Our results suggest that the above cyclic scaffolds stabilize a favorable structure for the antiaggregatory activity (IC50‐values ranged from 1.7 to 570u2003μm). The peptides inhibited Fg binding with IC50‐values up to 30‐fold lower than those determined for the inhibition of the adenosine diphosphate (ADP)‐induced platelet aggregation. Importantly, peptides (S,S) PSRCDCR‐NH2 (peptide 11) and (S,S) PRCDCK‐NH2 (peptide 10) did not inhibit PAC‐1 binding to the activated platelets at a concentration in which they completely inhibited Fg binding. Moreover, (S,S) PSRCDCR‐NH2 (peptide 11), one of the more active peptides, inhibited ADP‐induced P‐selectin exposure. By contrast, peptide (S,S) Ac‐RWDCRC‐NH2, incorporating the inverse (S,S) ‐DCRC‐ sequence (peptide 16), failed to inhibit P‐selectin exposure whereas at the same concentration, it effectively inhibited PAC‐1 and Fg binding. It is concluded that peptides containing the (S,S) ‐CDC‐ as well the (S,S) ‐CRC‐ sequences, exhibit a broad range of activities toward platelets, and could be helpful tools for elucidating the structural interaction of Fg with the integrin receptor αIIbβ3, in its activated form. Furthermore, the (S,S) ‐RCDC‐ sequence can be used as a scaffold for developing potent non‐RGD‐like Fg‐binding inhibitors.

Inhibition of platelet activation by peptide analogs of the beta(3)-intracellular domain of platelet integrin alpha(IIb)beta(3) conjugated to the cell-penetrating peptide Tat(48-60).

Activation of the platelet integrin-receptor αIIbβ3 is the final pathway of platelet aggregation, regardless of the initiating stimulus. Many studies suggest that there are several cytoplasmic proteins such as talin and β3-endonexin that bind to N744PLY747 and N756ITY759 motif of the β3 cytoplasmic tail and play the major role in the receptor activation. In this study, we investigated the role of the membrane distal region of human β3 cytoplasmic tail and specifically the N743NPLYKEA750 and T755NITYRGT762 sequence that contains an NXXY motif, in platelet aggregation, secretion, αIIbβ3 activation (PAC-1 binding) and fibrinogen binding. We synthesized two peptides corresponding to the above sequences as well as their conjugates with the Tat(48–60) cell-penetrating peptide. The capability of conjugates to penetrate the platelet membrane was investigated with confocal laser scanning microscopy using carboxyfluorescein (CF)-labeled peptides. Our results showed that the conjugated with the Tat(48–60) sequence peptides penetrate the platelet membrane and inhibit platelet aggregation in both PRP and washed platelets in a dose-dependent manner. The Tat-β3743–750 conjugate exhibited similar inhibitory activity in PRP and in washed platelets whereas the Tat-β3755–762 conjugate was more potent inhibitor of aggregation in washed platelets than in PRP. Both conjugated peptides were also able to inhibit P-selectin membrane expression as well as PAC-1 and fibrinogen binding to the platelets, the Tat-β3755–762 conjugate being more potent than Tat-β3743–750. The Tat(48–60) peptide and the peptides β3743–750 and β3755–762, which were not conjugated to the Tat(48–60) sequence, did not exhibit any inhibitory effect on the above parameters. In conclusion, the present study shows for the first time that the peptide analogs of the intracellular domain of the β3 subunit β3743–750 and β3755–762 conjugated to the cell-penetrating peptide Tat(48–60) are capable of penetrating the platelet membrane and expressing biological activity by inhibiting the activation of αIIbβ3, the fibrinogen binding to the activated receptor as well as platelet aggregation. Further studies are necessary to support whether such conjugated peptides may be useful tools for the development of potent antiplatelet agents acting intracellularly through the platelet integrin αIIbβ3.

Investigation of the role of adjacent amino acids to the 313–320 sequence of the αIIb subunit on platelet activation and fibrinogen binding to αIIbβ3

The platelet integrin receptor αIIbβ3 plays a critical role in thrombosis and haemostasis by mediating interactions between platelets and several ligands, primarily fibrinogen. We have previously shown that the synthetic peptide YMESRADRKLAEVGRVYLFL corresponding to residues 313–332 of αIIb, is a potent inhibitor of platelet aggregation and fibrinogen binding to αIIbβ3, interacting with fibrinogen rather than the receptor. Furthermore, we have demonstrated that the biological activities of the above peptide are due to the sequence YMESRADR, which corresponds to residues 313–320. By using new synthetic peptide analogues we investigated the structural characteristics responsible for the biological activity of YMESRADR as well the possible influence of the adjacent amino acids on the peptides biological potency. According to our results, the synthetic octapeptide YMESRADR, is a potent inhibitor of platelet aggregation and P-selectin expression. Furthermore, YMESRADR inhibits fibrinogen binding but it does not significantly influence the binding of PAC-1 to ADP-activated platelets. The inhibitory potency of YMESRADR was gradually diminished by deleting the YMES sequence from the amino terminus and prolonging the carboxyl terminus of this peptide with the KLAE sequence. Extension of YMESRADR towards the amino terminus with the GAPL sequence (GAPLYMESRADR) does not modify the biological activity of YMESRADR. Furthermore, extension of GAPLYMESRADR at its carboxy terminus with the KLAE sequence (GAPLYMESRADRKLAE) significantly diminished its biological potency. Substitution of E315 with D significantly enhances antiaggregatory potency and completely abolishes the inhibitory effect on P-selectin expression. Importantly, the D315-containing peptides inhibit to a similar extent both fibrinogen and PAC-1 binding to activated αIIbβ3 in contrast to the E315-containing peptide which only inhibits fibrinogen binding. In conclusion, the present study suggests that the YMESRADR sequence 313–320 of αIIb, is an important functional region of the insert connecting the β2 and β3 antiparallel β-strands of the W5 blade of the αIIb subunit. Structural changes significantly modify the biological properties of this region.