Is aspirin resistance a real problem in people with type 2 diabetes?
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Aspirin↗
Biomedical subjects
Cezary Watala
Publications and source records attributed to Cezary Watala.
Multivariate relationships between international normalized ratio and vitamin K-dependent coagulation-derived parameters in normal healthy donors and oral anticoagulant therapy patients.
BACKGROUND AND OBJECTIVES: International Normalized Ratio (INR) is a world-wide routinely used factor in the monitoring of oral anticoagulation treatment (OAT). However, it was reported that other factors, e. g. factor II, may even better reflect therapeutic efficacy of OAT and, therefore, may be potentialy useful for OAT monitoring. The primary purpose of this study was to characterize the associations of INR with other vitamin K-dependent plasma proteins in a heterogenous group of individuals, including healthy donors, patients on OAT and patients not receiving OAT. The study aimed also at establishing the influence of co-morbid conditions (incl. accompanying diseases) and co-medications (incl. different intensity of OAT) on INR. DESIGN AND METHODS: Two hundred and three subjects were involved in the study. Of these, 35 were normal healthy donors (group I), 73 were patients on medication different than OAT (group II) and 95 were patients on stable oral anticoagulant (acenocoumarol) therapy lasting for at least half a year prior to the study. The values of INR and activated partial thromboplastin time (APTT) ratio, as well as activities of FII, FVII, FX, protein C, and concentration of prothrombin F1+2 fragments and fibrinogen were obtained for all subjects. In statistical evaluation, the uni- and multivariate analyses were employed and the regression equations describing the obtained associations were estimated. RESULTS: Of the studied parameters, three (factors II, VII and X) appeared as very strong modulators of INR, protein C and prothrombin fragments F1+2 had moderate influence, whereas both APTT ratio and fibrinogen had no significant impact on INR variability. Due to collinearity and low tolerance of independent variables included in the multiple regression models, we routinely employed a ridge multiple regression model which compromises the minimal number of independent variables with the maximal overall determination coefficient. The best-fitted two-component model included FII and FVII activities and explained 90% of INR variability (compared to 93% in the 5-component model including all vitamin K-dependent proteins). Neither the presence of accompanying diseases nor the use of OAT nor any other medication (acetylsalicylic acid, statins, steroids, thyroxin) biased significantly these associations. CONCLUSION: Among various vitamin K-dependent plasma proteins, the coagulation factors II, VII and X showed the most significant associations with INR. Of these variables, the two-component model, including factors II and VII, deserves special attention, as it largely explains the overall variability observed in INR estimates. The statistical power of this model is validated on virtue of the estimation that the revealed associations are rather universal and remain essentially unbiased by other compounding variables, including clinical status and medical treatment. Further, much broader population studies are needed to verify clinical usefulness of methods alternate or compounding to INR monitoring of OAT.
Journal Article↗
Is platelet aggregation a more important contributor than platelet adhesion to the overall platelet-related primary haemostasis measured by PFA-100?
BACKGROUND: Platelet-related primary haemostasis (PRPH), measured in PFA-100 as a closure time (CT), reflects platelets' combined ability to adhere and aggregate under higher shear stress. The inputs of platelet aggregation and platelet adhesion into the real values of CT remain unknown, and this poor discrimination results in the complexity of the PFA-100 measurement. OBJECTIVE: To estimate the particular contributions of two physiological phenomena, platelet aggregation and adhesion, and the importance of various membrane receptors underlying platelets' capability of the plug formation in PFA-100 cartridges. MATERIALS AND METHODS: Effects of various blockers antagonizing ligands binding to platelet surface membrane receptors (antagonists of GPIIb-IIIa complex, collagen receptors and purinoreceptors), and aurintricarboxylic acid (ATA), the antagonist of GPIb-von Willebrand factor (vWF) interaction, were monitored in 47 healthy donors with the use of PFA-100 and whole blood electrical aggregometry (WBEA). RESULTS: PFA-100 collagen/ADP CT was the most sensitive in probing the effect of platelet membrane receptor antagonists acting via the blockade of GPIIb-IIIa complex and those antagonizing GPIb-vWF interaction (GR144053F, Integrilin, ATA), whereas the other blockers, acting on collagen receptors or purinoreceptors, remained much less efficient. For the examined GPIIb-IIIa and GPIb antagonists, the overall variability in WBEA explained a very significant part (30-60%) of the overall variability in PFA-100 CT. CONCLUSIONS: GPIIb-IIIa-mediated platelet aggregation and von Willebrand factor interactions with GPIb and/or GPIIb-IIIa seem to be the major determinants of PFA-100 CT. On the contrary, other platelet receptors participating in platelet aggregation and/or platelet adhesion are of secondary importance and minor significance in blood flow at higher shear stress monitored in PFA-100.
Adult↗
Inhibition of collagen-induced platelet reactivity by DGEA peptide.
Direct interactions between collagen, the most thrombogenic component of the extracellular matrix, and platelet surface membrane receptors mediate platelet adhesion and induce platelet activation and aggregation. In this process two glycoproteins are crucial: integrin alpha2beta1, an adhesive receptor, and GPVI, which is especially responsible for signal transduction. Specific antagonists of the collagen receptors are useful tools for investigating the complexity of platelet-collagen interactions. In this work we assessed the usefulness of DGEA peptide (Asp-Gly-Glu-Ala), the shortest collagen type I-derived motif recognised by the collagen-binding integrin alpha2beta1, as a potential antagonist of collagen receptors. We examined platelet function using several methods including platelet adhesion under static conditions, platelet function analyser PFA-100TM, whole blood electric impedance aggregometry (WBEA) and flow cytometry. We found that DGEA significantly inhibited adhesion, aggregation and release reaction of collagen activated blood platelets. The inhibitory effect of DGEA on static platelet adhesion reached sub-maximal values at millimolar inhibitor concentrations, whereas the specific blocker of alpha2beta1 - monoclonal antibodies Gi9, when used at saturating concentrations, had only a moderate inhibitory effect on platelet adhesion. Considering that 25-30% of total collagen binding to alpha2beta1 is specific, we conclude that DGEA is a strong antagonist interfering with a variety of collagen-platelet interactions, and it can be recognised not only by the primary platelet adhesion receptor alpha2beta1 but also by other collagen receptors.
Adult↗
Phosphatidylserine content is a more important contributor than transmembrane potential to interactions of merocyanine 540 with lipid bilayers.
Several factors, including the exposure of the negatively charged PL and transmembrane potential (TMP), may affect the binding of merocyanine 540 dye (MC540) to membrane lipids. Our aim was to quantify the significance of each of these two determinants in MC540 interactions with phosphatidylserine:phosphatidylcholine (PS/PC) vesicles. The effects of the altered PS content (PS/PC molar ratio: 5:95, 10:90 and 20:80) and TMP on MC540 binding were monitored using flow cytometry. Rapid [K(+)] flux across the vesicle membrane lipid bilayer was generated using valinomycin. We showed that the increased PS content leads to attenuated MC540 binding, while having no influence on the dynamic parameters of PS/PC vesicle membranes (electron spin resonance (ESR) spectrometry). Higher [K(+)](out) makes PS/PC liposomes bind more MC540, which implies that TMP-which becomes more positive inside the vesicles-favours the interactions of MC540 with the PL bilayer. Overall, the variability attributed to MC540-PL interactions is explained only to a minor extent by the generated TMP (7%) and largely by the variations in PS content (by up to 60%). In conclusion, the content of negatively charged PL is more important than TMP in determining the interactions of MC540 with PS/PC membranes.
Electron Spin Resonance Spectroscopy↗
Merocyanine 540 as a fluorescent probe of altered membrane phospholipid asymmetry in activated whole blood platelets.
BACKGROUND: Platelet activation leads to the loss of a natural asymmetry of membrane phospholipids (PL) and the subsequent exposure of negatively charged PL in platelets with procoagulant activity that can be monitored routinely with annexin V (AN-V). METHODS: Flow cytometric analysis of merocyanine 540 (MC540) binding may be the alternate choice for the monitoring of platelet procoagulant activity. Due to the increased partition of negatively charged phosphatidylserine (PS) in the membrane outer leaflet of activated platelets, the interaction with MC540 is reduced. RESULTS: Collagen, which facilitated platelet PL bilayer symmetrization, vastly reduced MC540 fluorescence and augmented AN-V binding to platelets. Such a collagen-induced symmetrization was further augmented in the presence of thrombin receptor-activating peptide (TRAP, SFLLRNPNDKYEPF). In the presence of VO(4) ((-3)) (the inhibitor of aminophospholipid translocase), the rebuilt of membrane asymmetry was attenuated, which resulted in further reduced MC540 fluorescence and enhanced AN-V binding in activated cells. In platelets incubated with thapsigargin, the inhibitor of platelet tubular system Ca(2+) ATP-ase, which elevates intraplatelet Ca(2+) concentration, TRAP increased AN-V and reduced MC540 binding. The chelating of Ca(2+) with EGTA outside of activated platelets reduced AN-V binding, but did not affect MC540-positive platelets. The fluctuations in reduced staining with MC540 paralleled enhanced AN-V binding (r = -0.481, P < 0.01), especially for strong "procoagulant" activating agents. CONCLUSIONS: (1) MC540 may be used in whole blood flow cytometry for the monitoring of platelet membrane symmetrization as an alternate or compounding method to AN-V. (2) Platelet staining with MC540 is sensitive to the fluctuations in the intraplatelet [Ca(2+)] during platelet activation. (3) Use of MC540 is characterized by improved diagnostic precision and reliability compared with AN-V.
Adult↗
Antagonists of platelet fibrinogen receptor are less effective in carriers of Pl(A2) polymorphism of beta(3) integrin.
The polymorphism Leu(33)-->Pro (platelet-specific antigen; Pl(A1/A2)) of platelet GPIIIa is a potential risk factor for arterial thrombosis. However, its influence on platelet function remains controversial and little is known about its impact on platelet sensitivity to GPIIb-IIIa antagonists. Our objective was to compare the effectiveness of various GPIIb-IIIa antagonists in Pl(A2)(+) and Pl(A2)(-) carriers. Platelet aggregation was monitored in healthy donors including Pl(A2)(-) (N=31) and Pl(A2)(+) subjects (N=27; 23 Pl(A1/A2), 4 Pl(A2)/(A2)) using the impedance and turbidimetric aggregation techniques. We evaluated the inhibition of ADP- and collagen-induced aggregation by disintegrins, kistrin and echistatin, and the low-molecular-weight blockers, GR144053F ((4-[4-[4-(aminoiminomethyl]-1-piperazinyl]-1-piperidineacetic acid, hydrochloride trihydrate) and eptifibatide (N(6)-(aminoiminomethyl)-N(2)-(3-mercapto-1-oxopropyl-L-lysylglycyl-L-alpha-aspartyl-L-tryptophyl-L-propyl-L-cysteinamide, cyclic (1-->6)-disulfide). Kistrin (10-30 nmol/l) inhibited ADP- and collagen-induced aggregation stronger in Pl(A2)(-) donors than in Pl(A2)(+) donors; there was a significant difference between 50% inhibitory concentrations (IC(50)). The same tendency occurred with moderate concentrations of eptifibatide (40-100 nmol/l) and also at low concentrations of GR144053F (5-10 nmol/l) and high concentrations of echistatin (80-150 nmol/l), although in the case of the two latter inhibitors, the estimated IC(50) values were not significantly different. In conclusion, GPIIb-IIIa blockers representing various classes are less effective inhibitors of platelet aggregation in Pl(A2)(+) carriers; however, the effect of the genotype is both agonist- and antagonist-dependent.
Adult↗
Cerivastatin, a HMG-CoA reductase inhibitor, reduces plasminogen activator inhibitor-1 (PAI-1) expression in endothelial cells by down-regulation of cellular signaling and the inhibition of PAI-1 promoter activity.
Statins, which competitively inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity and reduce mevalonate synthesis, are believed to exert a plethora of pleiotropic effects. In this report, molecular mechanisms of the inhibitory effect on plasminogen activator inhibitor type 1 (PAI-1) expression produced by cerivastatin (CRV), the most active compound in this class, were studied using monocultures of human endothelial cell line (EA.hy 926). CRV similar to another statin, lovastatin (LOV), significantly inhibited PAI-1 expression and its release from endothelial cells, nonstimulated and stimulated with TNF-alpha. The inhibitory effect of CRV could be detected at the level of PAI-1 promoter in EA.hy 926 cells transfected with plasmid p800 LUC containing PAI-1 promoter fragment (+71 to -800), as well as at the level of PAI-1 mRNA. The PAI-1 promoter activity was markedly suppressed in the nonstimulated cells and almost completely inhibited in TNF-alpha-stimulated cells. In addition, CRV at low doses (IC(50) of 4 - 6 microM) significantly inhibited mitogen-activated protein kinases (MAPKs) phosphorylation. The majority of inhibitory effects occurred at significantly lower concentrations for CRV compared to LOV. The mechanism by which CRV inhibits PAI-1 expression appears to be directly associated with geranylgeranylation of some cell proteins, since the inhibitory effect on PAI-1 expression can be reversed by geranylgeranyl-pyrophosphate but not by farnesyl-pyrophosphate.
Cells, Cultured↗
The contribution of phosphatidylserine partition and transmembrane potential to the interaction of merocyanine 540 with lipid bilayers.
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Fluorescent Dyes↗
Blood platelet membrane fluidity and the exposition of membrane protein receptors in Alzheimer disease (AD) patients--preliminary Study.
We estimated membrane fluidity by the (ESR) spectroscopy and the expression of membrane P-selectin and glycoproteins GP Ib alpha and GP IIb/IIIa by flow cytometry in platelets (plts) from 12 AD sufferers. In AD patients membrane fluidity was significantly increased at two different depths (p < 0.05 or less). Platelet reactivity was significantly decreased, as reflected by reduced expression of GP Ib alpha in the resting (p < 0.0001) and activated (p < 0.005) platelets, as well as the expression of P-selectin and beta 3 subunit of GP IIb/IIIa in activated platelets (p < 0.0001; p < 0.04).
Aged↗
Effect of the 807 c/t polymorphism in glycoprotein ia on blood platelet reactivity.
The 807 C/T dimorphism in the GPIa gene has been shown to affect GPIaIIa receptor density, which can affect platelet adhesiveness to collagens. In this work, we studied platelet function mediated by GPIaIIa. The 807 T/T genotype was associated with increased platelet adhesion to monomeric collagen after activation with ADP, but not following activation with thrombin and U46619. Adhesion to fibrillar collagen and PFA-100 closure time were not different between carriers of the C/C and T/T genotypes. Also, to monitor the role of the 807 C/T polymorphism in the sensitivity to platelet antagonists, anti-GPIaIIa monoclonal antibodies (Gi9) were used. Irrespective of the 807 C/T genotype, Gi9 inhibited the ADP-induced platelet adhesion to the monomeric collagen-coated surface stronger than adhesion evoked by thrombin. Moreover, Gi9 significantly inhibited platelet adhesion to both monomeric and fibrillar collagen in 807 T/T carriers, whereas in 807 C/C subjects, Gi9 blocked only adhesion to monomeric collagen. Our results indicate that the 807 T/T genotype is related to increased platelet activation induced by ADP and higher platelet sensitivity to GPIaIIa antagonists.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Genetic factors underlying differential blood platelet sensitivity to inhibitors.
Blood platelets are not only the primary defence mechanism involved in physiological hemostasis, but also their disorders constitute a crucial risk factor in arterial thrombosis. As arterial thrombi are composed of predominantly platelets formed under conditions of elevated shear stress at sites of atherosclerotic vascular injury and disturbed blood flow, the prevention of arterial thrombosis has been for years the main target for antiplatelet therapy. Individual differences in the rate of platelet activation and reactivity markedly influence normal hemostasis and the pathological outcome of thrombosis. Such an individual variability is largely determined by environmental and genetic factors. These are known to either hamper platelets' response to agonists, and thereby mimic the pharmacological modulation of platelet function or mask therapy effect and sensitize platelets. Some clinical studies have indicated that platelet glycoprotein polymorphisms are genetic factors contributing to arterial thrombosis. In spite of some discrepancies between different studies, there is substantial evidence that the integrin beta3 P1(A2) allele, the variants GPIbalpha Met145 and GPIbalpha (-5C) haplotype or the integrin alpha2 haplotype 1 (807T) each contribute to the risk for and morbidity of thrombotic disease. In this article, we reviewed a role of the aforementioned polymorphisms in modulating platelet function and platelet response to inhibitors. The paper focuses on the association between Pl(A1/A2) polymorphism and sensitivity (or resistance) to aspirin and the inhibitory efficacy of GPIIb-IIIa antagonists. Additionally, a potential role of 807C/T polymorphism (GPIa), polymorphisms of GPIb and platelet purinoreceptor P2Y12 in affecting platelet sensitivity to blocking agents is discussed.
Blood Platelets↗