Loukas D. Tsironis

Lipoprotein (a) Levels and Apolipoprotein (a) Isoform Size in Patients with Subclinical Hypothyroidism: Effect of Treatment with Levothyroxine

The increased risk for ischemic heart disease (IHD) associated with subclinical hypothyroidism (SH) has been partly attributed to dyslipidemia. There is limited information on the effect of SH on lipoprotein (a) [Lp(a)], which is considered a significant predictor of IHD. Serum Lp(a) levels are predominantly regulated by apolipoprotein [apo(a)] gene polymorphisms. The aim of our study was to evaluate the Lp(a) levels and apo(a) phenotypes in patients with SH compared to healthy controls as well as the influence of levothyroxine substitution therapy on Lp(a) values in relation to the apo(a) isoform size. Lp(a) levels were measured in 69 patients with SH before and after restoration of a euthyroid state and in 83 age- and gender-matched healthy controls. Apo(a) isoform size was determined by sodium dodecyl sulfate (SDS) agarose gel electrophoresis followed by immunoblotting and development via chemiluminescence. Patients with SH exhibited increased Lp(a) levels compared to controls (median value 10.6 mg/dL vs. 6.0 mg/dL, p = 0.003]), but this was not because of differences in the frequencies of apo(a) phenotypes. There was no association between thyrotropin (TSH) and Lp(a) levels in patients with SH. In subjects with either low (LMW; 25 patients and 28 controls) or high (HMW; 44 patients and 55 controls) molecular weight apo(a) isoforms, Lp(a) concentrations were higher in patients than in the control group (median values 26.9 mg/dL vs. 21.8 mg/dL, p = 0.02 for LMW, and 6.0 mg/dL versus 3.3 mg/dL, p < 0.001 for HMW). Levothyroxine treatment resulted in an overall reduction of Lp(a) levels (10.6 mg/dL baseline vs. 8.9 mg/dL posttreatment, p = 0.008]). This effect was mainly evident in patients with LMW apo(a) isoforms associated with high baseline Lp(a) concentrations (median values 26.9 mg/dL vs. 23.2 mg/dL pretreatment and posttreatment, respectively; p = 0.03). In conclusion, even though a causal effect of thyroid dysfunction on Lp(a) was not clearly demonstrated in patients with SH, levothyroxine treatment is beneficial, especially in patients with increased baseline Lp(a) levels and LMW apo(a) isoforms.

Efficient enzymatic preparation of hydroxycinnamates in ionic liquids enhances their antioxidant effect on lipoproteins oxidative modification

Biocatalytic lipophilization of hydroxycinnamic acids was performed in several BF(4)(-) and PF(6)(-) imidazolium ionic liquids using immobilized lipases. The influence of various reaction parameters on the performance of the biocatalytic process was pointed out, using as model reaction the esterification of ferulic acid. The biocatalytic lipophilization strongly depended on the ion composition of ionic liquids used. Conversions and initial reaction rates were significantly higher in PF(6)(-) as compared with BF(4)(-) ionic liquids and commonly used organic solvents. The high enzyme stability and the relative solubility of substrate versus product in PF(6)(-) ionic liquids can account for the improved synthesis of lipophilic ferulates. These lipophilic derivatives, when used at a concentration of up to 400-fold lower than the parental compound, efficiently inhibited the oxidation of isolated LDL, HDL and total serum in vitro. Moreover, it has been shown for the first time that the lipophilic ferulates improve the antioxidant efficiency of the HDL(3c) towards LDL in vitro oxidation.

Autoantibody titers against OxLDL are correlated with Achilles tendon thickness in patients with familial hypercholesterolemia

Achilles tendon xanthomas are associated with increased cardiovascular risk in patients with familial hypercholesterolemia (FH). Oxidized low density lipoprotein (OxLDL), the antibodies against OxLDL, and the LDL-associated phospholipase A2 (Lp-PLA2) may play important roles in atherogenesis. We investigated the possible association between plasma levels of OxLDL, Lp-PLA2 activity, and autoantibody titers against various types of mildly OxLDL with Achilles tendon thickness (ATT). ATT was determined by sonography in 80 unrelated heterozygous FH patients. Three different types of mildly OxLDL were prepared: OxLDLL, OxLDLP, and OxLDLD, at the end of the lag, propagation, and decomposition phases of oxidation, respectively. Similar types of OxLDL were also prepared after inactivation of the LDL-associated Lp-PLA2. These types were denoted OxLDL(−)L, OxLDL(−)P, and OxLDL(−)D. FH patients exhibited significantly higher plasma OxLDL levels and serum IgG titers against OxLDLP and OxLDLD compared with 40 normolipidemic apparently healthy controls. ATT values were positively correlated with autoantibody titers against OxLDLP and OxLDLD; however, in multiple regression analysis, ATT was independently associated only with the autoantibody titers against OxLDLD. We conclude that the IgG autoantibody titers against OxLDLD but not OxLDL or Lp-PLA2 may play an important role in the pathogenesis of Achilles tendon xanthomas in FH patients.

Serum Lipoprotein(a) Concentrations and Apolipoprotein(a) Isoforms: Association with the Severity of Clinical Presentation in Patients with Coronary Heart Disease:

Objective The aim of this study was to investigate the possible associations between lipoprotein(a) [Lp(a)] concentrations or apolipoprotein(a) isoforms and the mode of clinical presentation of coronary heart disease (CHD) (acute thrombotic event or not). Methods A total of 131 CHD patients and 71 age- and gender-matched individuals without known CAD (free of symptoms of heart disease) were enrolled in the study. CHD patients were classified into patients with a history of an acute coronary syndrome (ACS, n=94) and patients with stable angina (SA, n=37). Lp(a) levels were measured with an ELISA method, whereas apolipoprotein(a) isoform analysis was performed (in all patients and 33 controls) by electrophoresis in 1.5% SDS-agarose gels followed by immunoblotting. Isoform size was expressed as the number of kringle 4 (K4) repeats. Results ACS patients had higher Lp(a) plasma levels [21.9 (0.8–84.1) mg/dl] and a greater proportion of elevated (≥ 30 mg/dl) Lp(a) concentrations (25.5%) compared with SA patients [9.2 (0.8–50.5) mg/dl, P < 0.01 and 10.8%, P < 0.05] and controls [8.0 (0.8–55.0) mg/dl, P < 0.01 and 11.2%, P < 0.05], while there were no differences between SA patients and controls. The median apolipoprotein(a)-isoform size was 26 K4. In 17 (10%) patients we could not detect any apolipoprotein(a) isoform bands by immunoblotting. ACS patients had a higher proportion of isoforms < 26 K4 (low molecular weight) than SA patients (56/85 vs. 12/33, P < 0.005) and controls (10/29, P < 0.005). Conclusions CAD patients with a history of ACS have higher Lp(a) plasma levels and a significantly higher proportion of low molecular weight apolipoprotein(a) isoforms compared with patients with SA or to controls.

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.

Effect of a Synthetic Peptide Corresponding to Residues 313 to 320 of the αIIb Subunit of the Human Platelet Integrin αIIbβ3 on Carotid Artery Thrombosis in Rabbits

The platelet integrin receptor αIIbβ3 plays a critical role in thrombosis. We have shown previously that the octapeptide YMESRADR, corresponding to sequences 313 to 320 of the human αIIb subunit, inhibits human platelet activation and fibrinogen binding to αIIbβ3, possibly interacting with the ligand. We investigated the effect of YMESRADR on electrically induced carotid artery thrombosis in New Zealand white rabbits. Peptide was administered via the femoral vein, starting 60 min before and continuing for 90 min after the electrical stimulation. Carotid blood flow was monitored for 90 min after the electrical stimulation. The peptide effects on platelet aggregation, in vitro and ex vivo, and on various coagulation, bleeding, and hemostatic parameters were evaluated. YMESRADR significantly inhibited rabbit platelet aggregation in vitro in a dose-dependent manner. It is important that peptide administration in vivo, at doses ranging from 3 to 15 mg/kg, prolonged the duration of the patency of the carotid artery, and no artery occlusion was observed until the end of the study (90 min after electrical stimulation). Furthermore, YMESRADR administration reduced platelet aggregation ex vivo and thrombus weight; however, these reductions reached statistical significance, compared with the control group, at the peptide doses of 12 and 15 mg/kg. YMESRADR did not affect any coagulation parameter studied and the hemostatic response observed in control animals. Thus, YMESRADR represents a novel antiplatelet agent that can inhibit thrombus formation effectively and carotid artery occlusion without causing hemorrhagic complications in a rabbit model of arterial thrombosis.

Lipoprotein(a) as a cardiovascular risk factor.

Evidence for the role of lipoprotein(a) (Lp[a]) in atherosclerosis and thrombosis has considerably increased over the past few years. Therefore, Lp(a) is currently classified as an emerging lipid risk factor for cardiovascular disease. High Lp(a) plasma levels carried in particles with small-sized apolipoprotein(a) isoforms are associated with preclinical vascular changes, cardiovascular disease and the mode of presentation of coronary artery disease (acute coronary syndromes). However, randomized clinical trials with an emphasis on agents that specifically lower plasma Lp(a) do not exist. At present, screening for increases in Lp(a) in the general population is not recommended. The measurement of Lp(a) may be of value in individuals with an increased risk of cardiovascular disease, particularly in patients with high low-density lipoprotein cholesterol plasma levels, since a high Lp(a) concentration in such subjects further increases the risk of coronary heart disease.