Polymorphisms of glycoprotein Ib affect the inhibition by aurintricarboxylic acid of the von Willebrand factor dependent platelet aggregation.

Two polymorphisms of platelet glycoprotein Ib, VNTR and Thr/Met(145), regarded as the possible inherited risks factors for thromboembolic complications, have been suggested to underlie platelet response to activating stimuli. This study examined the functional significance of these polymorphisms in platelet reactivity and sensitivity to aurintricarboxylic acid (the antagonist of von Willebrand factor, vWF). To evaluate platelet function at low and high flow conditions we monitored the ristocetin-induced and vWF-mediated aggregation of isolated platelets and the platelet function analyzer collagen/ADP closure time (PFA-100 CT(CADP)), which reflects platelets' ability to adhere and aggregate in whole blood. Aurintricarboxylic acid significantly reduced ristocetin-induced platelet agglutination in a dose-dependent manner (IC(50)=3.5+/-1.9 microM). At the concentration of 100 microM it also markedly prolonged PFA-100 CT(CADP) (up to 147+/-32 s vs. 94+/-17 s in control). The efficacy of this antagonist in the inhibition of vWF-mediated platelet agglutination was approximately 1.5-fold higher in the VNTR B/Met145(+) carriers than in VNTR B/Met145(-) carriers ( P<0.05). Otherwise, no significant differences occurred between VNTR B/Met(145)-positive and B/Met(145)-negative individuals in the prolongation of closure time by ATA. These findings indicate that under certain experimental conditions VNTR-B and Met(145) alleles may contribute to the increased platelet sensitivity to some antagonists of platelet natural ligands.

Effects of fibrinogen receptor antagonist GR144053F and aurintricarboxylic acid on platelet activation and degranulation.

Activated blood platelets play crucial role in restenosis due to their fundamental significance in thrombus formation. Therefore, platelets are attractive targets for the inhibition with a variety of antagonists. In this study, we present direct evidence that GR144053F [non-peptide antagonist of glycoprotein IIb-IIIa complex (GPIIb-IIIa)] inhibits activation and degranulation of human platelets, and opposes the action of aurintricarboxylic acid (ATA), the antagonist of von Willebrand factor, which augments platelet secretion. The effects of both drugs on platelet function were monitored by using various instrumental methods. Platelet-rich plasma and whole-blood aggregation was measured by using ADP and collagen as agonists. Platelet degranulation was assessed based on the expression of surface membrane activation markers: P-selectin, glycoprotein Ib, and activated GPIIb-IIIa complex. Measurements of closure time with platelet function analyzer PFA-100 enabled us to reason on primary hemostatic capacity and reflected both aggregability and adhesiveness. GR144053F markedly reduced primary hemostatic platelet response (IC(50) = 114.0 +/- 9.6 nM) under conditions that closely mimicked natural blood flow in circulation, and inhibited aggregation in platelet-rich plasma (IC(50) = 17.7 +/- 7.0 nM). It was equally potent inhibitor of platelet activation, degranulation, fibrinogen binding, platelet consumption, and aggregate formation. Also, ATA was efficient in inhibition of platelet aggregation and adhesion (by up to 50% at 100 microM), but the combined action of both drugs on primary haemostatic capacity was not additive. GR144053F suppressed the activating effects of ATA on platelet degranulation and secretion. Overall, our data indicate that GR144053F is not only the efficient blocker of fibrinogen binding to GPIIb-IIIa, but also hampers platelet degranulation and may attenuate the activating effects of ATA.

Platelet hyperreactivity after coronary artery bypass grafting: the possible relevance to glycoprotein polymorphisms. A preliminary report.

Coronary artery bypass grafting (CABG) surgery impairs platelet function and reactivity to a considerable extent. However, variability in the individual patients' responses makes any generalised statement uncertain. The observed variability is nowadays thought to relate to platelet glycoprotein polymorphisms. Our objective was to investigate the association between platelet reactivity and the restoration of platelet functional response to agonists during the period following cardiosurgical operation and some genetic polymorphisms of selected platelet membrane glycoproteins. Platelet reactivity was monitored in 32 IHD patients (56 +/- 8 years) subjected to CABG surgery by means of whole blood impedance aggregometry and concurrently using the platelet function analyser (PFA-100 at four time intervals: prior to operation (A), 2 h after administration of protamine sulfate (B), 3 days after (C) and 7 days after CABG surgery (D). Three important findings were made. First, in all patients platelet reactivity became decreased 2 h postoperatively (aggregation with 20 microM ADP reduced by up to 49%, P < 0.02) and vastly increased 7 days after CABG surgery (CT(CADP) reduced down to 87% of initial value, P < 0.05, ADP-induced aggregation enhanced up to 167%, P < 0.001, and that with collagen up to 131% of the initial value, P < 0.01). Second, the frequencies of the 'prothrombotic' phenotype variants of platelet membrane glycoproteins were higher in patients referred to as the carriers of more reactive platelets compared to those with less reactive platelets (GPIa (807)T-positive, 50 vs. 28%; GPIIIa Pl(A2)-positive, 27 vs. 21%; GPIb Met(145)-positive and GPIb VNTR B-positive, 13 vs. 0%. Lastly, the restoration in platelet hyperreactivity in CABG surgery patients was recorded more often in patients who underwent postoperative myocardial ischaemic episode(s), and was associated with significantly higher frequency of the 'prothrombotic' allele (807)T of the collagen receptor glycoprotein Ia (GPIa) in these subjects (83 vs. 61%). In conclusion, in patients with ischaemic episodes after CABG, we demonstrated a fast postoperative restoration of haemostatic capacity and evidence of platelet hyperreactivity at 7 days after CABG surgery. The platelet hyperfunction seems to relate to the occurrence of platelet glycoprotein polymorphisms GPIa(807)C/T and GPIIIa PlA(1/A2) and may be important in predicting postoperative vascular complications in CABG patients.

Differentiated reactivity of whole blood neonatal platelets to various agonists.

Neonatal platelets have been occasionally reported to show a reduced response to various agonists. The molecular mechanism(s) of such a depressed reactivity remains unclear. To further address this problem we studied neonatal platelet activation with thrombin, TRAP (thrombin receptor activating peptide, Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe) and ADP in 42 healthy 1-2 day old neonates using a whole peripheral blood flow cytometry. The neonates did not show an increased fraction of P-selectin-positive circulating platelets, whereas the expression of GPIb (glycoprotein Ib) in resting neonatal platelets was significantly lower compared to adults. Neonatal platelets were significantly less reactive than adult platelets to thrombin and TRAP, especially at lower agonist concentrations, but not to ADP or when incubated for 1 h at room temperature. Activation of neonatal platelets with agonists resulted in a marked alterations in the expression of P-selectin, whereas the internalization of GPIb was not affected. The reduced neonatal platelet sensitivity to thrombin and TRAP was accompanied by significantly reduced ATIII (antithrombin III) and increased prothrombin fragment F(1+2) in neonatal plasma. We conclude that various receptor systems potentially able to bind thrombin are relatively insensitive in neonatal platelets. The novelty of our work is that neonatal platelet hyposensitivity is not a generalized phenomenon, but concerns only selected agonists and selected receptor systems.

Low doses of aprotinin in aortocoronary bypass surgery--advantages and disadvantages.

INTRODUCTION: Excessive blood loss, as a result of augmented postoperative drainage, is considered one of the most serious cardiosurgical complications. The compounding constitutive anemia seems particularly harmful for patients with coronary artery disease. Aprotinin (Trasylol), a non-specific serine protease inhibitor, is successfully used to reduce excessive postoperative bleeding in such patients. The aim of our study was to verify the hypothesis whether aprotinin used during cardiopulmonary bypass procedure affects hemostatic parameters, which might be crucial for the elevated risk of thromboembolic complications. MATERIAL AND METHODS: The group of 54 patients subjected to coronary artery surgical treatment included 30 patients, who were given intraoperatively 3 million KIU aprotinin each, and 24 subjects non-treated with aprotinin. Aliquots of blood were withdrawn at several time intervals, until the 5th day after the operation. Whole blood platelet activation and reactivity (the expressions of P-selectin and glycoprotein Ib) were monitored by means of flow cytometry. In addition, several plasma parameters, like PAI-1, t-PA, D-dimers, prothrombin fragment F1 + 2, fibrinogen, ATIII activity, troponin I and CK-MB, as well as platelet count were determined at each time point. RESULTS: In this study we confirmed the essential advantage of the use of aprotinin: both the postoperative blood drainage and the blood units to be transfused postoperatively to cardiosurgical patients were vastly reduced in the aprotinin-treated subjects. The enhanced overall frequency of perioperative myocardial infarction events was not attributed to this group of patients, nor the non Q-wave infarctions were observed more often in patients treated with aprotinin. In these patients, fibrinolysis parameters tended to be depressed (with increased PAI-1 dominating over elevated t-PA) on the first day after the operation, and no significant differences with regard to fibrinogen, prothrombin fragment F1 + 2, troponin I and platelet count. There was a continuous rise in D-dimers in all the postoperative patients, which lasted until the third day and tended to reach plateau at the 5th day after the operation. We failed to reveal the preventive effects of aprotinin on platelet function: both platelet activation and reactivity remained apparently unchanged. Overall, our results rather support the reasoning on the advantageous effects of low doses of aprotinin. The use of this inhibitor reduces the risk of postoperative undesirable bleeding and results in a decreased postoperative drainage and reduced transfused blood units. On the other hand, however, a higher incidence of perioperative Q-wave infarction in the aprotinin-treated patients, although purely apparent and not statistically significant, might question the unlimited safety of the use of aprotinin in cardiovascular operations.

[Use of platelet function analyzer PFA-100 and whole blood aggregometry to monitor blood platelet sensitivity to acetylsalicylic acid (aspirin). Is it possible to reliably monitor antiplatelet treatment using routine laboratory diagnostic methods?]. / Zastosowanie analizatora funkcji plytek PFA-100 oraz metody agregacji we krwi pelnej do oceny wrazliwosci plytek krwi na dzialanie kwasu acetylosalicylowego (aspiryny). Czy mozliwa jest wiarygodna laboratoryjna weryfikacja leczenia przeciwplytkowego?

Introduction of the antiplatelet agents of new generations and the occurrence of the phenomenon of "aspirin-resistance" triggered the search for better, simpler and more reliable routine diagnostic methods to monitor platelet reactivity. Our objective was to evaluate the usefulness and reliability of two simple methods: platelet function analyzer (PFA-100) and whole blood platelet aggregometry for monitoring of platelet function in 18 healthy blood donors and 35 patients with ischaemic heart disease (IHD) subjected to small doses/75 mg and 150 mg a day) of acetylsalicylic acid (aspirin). In 50% of healthy blood donors the intake of 75 mg ASA a day resulted in the prolongation of PFA-100 collagen/epinephrine closure time (CEPI = (relevant to reduced platelet reactivity) of over 150 s, whereas 75% donors responded to 150 mg ASA-. Otherwise, the daily dose of 150 mg ASA resulted in a prolonged CEPI merely in 23% of in IHD patients. At both doses ASA completely inhibited the arachidonic acid-induced whole blood platelet aggregation in all healthy donors and in all but 3 IHD patients. Collagen-induced platelet aggregation was only negligibly affected by either dose of ASA. Our results point that the simultaneous monitoring of the PFA-100 collagen/epinephrine closure time and whole blood platelet aggregometry (Chrono-Log) enables to reliably evaluate the inhibition of platelet function by ASA and discriminate the partial or complete platelet insensitivity to aspirin. The phenomenon of more frequent platelet aspirin-resistance in IHD patients requires to be verified in randomised clinical prospective studies.

Release of calcium and P-selectin from intraplatelet granules is hampered by procaine.

The inhibition of platelets by some local anaesthetics has been related to the modulation of platelet membrane lipid fluidity, and one of these compounds, procaine, has been proven to be particularly effective inhibitor. In the present study, we examined the effect of procaine on the mobilization of intracellular granule contents in isolated washed platelets. We revealed that the presence of 10 mg/ml procaine significantly hampered platelet release reaction, as demonstrated by the significant reduction in the expression of platelet P-selectin (CD62) on one hand, and significantly enhanced expression of GPIb alpha (CD42b) antigen on the other, following either 1 hour incubation of washed platelets at room temperature (%CD62: 37.1+/-6.8% of control incubated without procaine, p<<0.0001; %CD42b: 116.2+/-6.3% of control, p<0.0001) or activation of whole blood platelets with ADP, TRAP, or thrombin. Procaine, which acted as a rigidizer, significantly decreased platelet membrane fluidity (ESR h(+1)/h0 ratio of 5-DOXYL-Ste reduced down to 93.1+/-3.7% of control, p<0.001). In washed Fura-2-loaded platelets procaine not only brought about the significantly reduced Ca2+ release from intraplatelet storage pools after platelet stimulation with 15 micromol/l ADP (25.3+/-12.5% of control, p<0.001), but also it significantly increased the reduction in Ca2+ concentration upon the addition of Ca2+ chelator, EDTAK2 (48.9+/-13.5% vs. 40.9+/-12.1% of initial [Ca2+]i concentration, p(1,alpha)<0.025). Overall, procaine considerably reduced calcium mobilization from intraplatelet storage pools and Ca2+ efflux across platelet membrane. Based on these data, we suggest that the preventive effects of procaine on platelet release reaction and calcium mobilization might relate to the changes in the organization of membrane components embedded into a lipid bilayer, which are crucial in triggering of platelet release reaction. Procaine-mediated dislocations of some membrane components and/or distortion of lipid-protein interactions could generate a steric hindrance, which might interfere with platelet signal transduction, thus leading to impaired mobilization of Ca2+ and other components from intraplatelet storage pools.

Platelet membrane lipid fluidity and intraplatelet calcium mobilization in type 2 diabetes mellitus.

The aim of the present study was to relate the impairments in calcium mobilization and/or release to the altered membrane dynamics in platelets from patients with type 2 diabetes mellitus. Higher expression of P-selectin (1.4-fold, NS) and the reduction in GPIb alpha expression (by 27.8+/-16.7%, p < 0.0002), as well as the increased fractions of platelet microparticles (p < 0.03), reflected more intensified platelet release reaction in diabetic platelets. Overall, diabetic platelets appeared more vulnerable to stimuli facilitating calcium mobilization (by 41%, p < 0.01) and less susceptible to preventive effects of the agents hampering calcium release from intraplatelet storage pools (by 38%, p < 0.01). Both the increased calcium mobilization from intraplatelet storage pools and higher levels of intracellular free calcium in the presence of procaine in diabetic platelets correlated with the reduced platelet membrane lipid fluidity (resp. pR < 0.03 and pR < 0.015). We conclude that the biophysical state of platelet membrane components in diabetes mellitus is the crucial determinant of platelet hyperfunction and probably contributes to the intensified calcium mobilization in diabetic platelets. The depressed preventive effects of procaine on platelet release reaction and calcium mobilization in diabetic platelets may result from the primary dislocations and/or distortions of membrane components caused by the diabetic state.

Membrane lipid fluidity of blood platelets: a common denominator that underlies the opposing actions of various agents that affect platelet activation in whole blood.

Membrane lipid fluidity (MLF) is thought to play a crucial role in signal transduction and is believed to affect the responsiveness of blood platelets. In a recent study it was demonstrated that EDTA, used as the blood anticoagulant, brought about a significant increase in expression of GMP-140 antigen, and this effect was accompanied by a significant increase in platelet MLF. Moreover, this spontaneous EDTA-driven platelet activation was vastly attenuated in the presence of tissue-type plasminogen activator, which is also known to affect platelet MLF. The hypothesis was raised that the modulation of platelet membrane fluidity by EDTA might underlie platelet spontaneous activation in the presence of EDTA. To further explore the possible molecular mechanism(s) of the EDTA-dependent triggering of signal transduction pathway(s) in human blood platelets, we monitored the extent of spontaneous platelet activation in the presence of EDTA and selected platelet membrane 'fluidizers' and 'rigidizers'. A reduction in the EDTA-dependent platelet release and activation was noted, not only in the presence of rt-PA (by over 50%, P < 0.001), which acted as a rigidizer of platelet membrane fluidity (ESR h+1/h0 ratios of 5-DOXYL-Ste and 12-DOXYL-Ste decreased by 6.2%, P<< 0.0001, and 3.8%, P < 0.02, respectively), but also in the presence of other modulators of MLF, regardless of their fluidizing or rigidizing effects. Both rigidizers (procaine and lidocaine, 5-DOXYL-Ste h+1/h0 reduced by up to 6.5%, 12-DOXYL-Ste h+1/h0- by up to 4.5%, P < 0.02 or less) and fluidizers (benzyl alcohol, ethanol, 12-DOXYL-Ste h+1/h0 increased by 17.8% and 6.1%, respectively, P <<0.0001) of platelet membranes significantly depressed platelet activation (respectively, down to 1.1%, 7.7%, 6.7% and 8.5% vs control EDTA 22.9% of CD62-positive platelets). We suggest that EDTA induces alterations in membrane glycoprotein structure and affect MLF by altering lipid-protein interactions, and thus triggers signal transduction in the course of platelet activation. The resulting displacements in platelet membrane proteins, dislocation of membrane components and/or distortion of lipid-protein interactions could generate an 'outside-in' signalling that is mediated by the altered platelet MLF. Overall, it is likely that interference with the structure and conformation of selected domains of platelet membrane proteins might be the crucial mechanism by which EDTA leads to exaggerated activation of platelets in whole blood.

The effects of in vivo and in vitro non-enzymatic glycosylation and glycoxidation on physico-chemical properties of haemoglobin in control and diabetic patients.

The erythrocyte deformability, which is related to erythrocyte internal viscosity, was suggested to depend upon the physico-chemical properties of haemoglobin. In the present study we employed ESR spectroscopy on order to explore further the extent to which the in vivo or in vitro glycation and/or glycoxidation might affect haemoglobin structure on conformation. We revealed that under both in vivo and in vitro conditions the attachment of glucose induced a mobilization of thiol groups in the selected domains of haemoglobin molecules ( the increased h+1/h0 parameter of maleimide spin label, MSL; 0.277 +/- 0.021 in diabetics vs 0.338 +/- 0.017 in controls, n = 12, P < 0.0001). The relative rotational correlation time (tau c) of two spin labels, TEMPONE and TEMPAMINE, respectively, in erythrocyte insides (5.22 +/- 0.42 in diabetics, n = 21 vs 4.79 +/- 0.38, n = 16 in controls, P < 0.005) and in the solutions of in vitro glycated haemoglobin, were increased. Neither oxidation nor crosslinking of thiol groups was evidenced in glycated and/or oxidized haemoglobin. In addition, erythrocyte deformability was found to be reduced in type 2 diabetic patients (6.71 +/- 1.08, n = 28 vs 7.31 +/- 0.96, n = 21, P < 0.015). In conclusion, these observations suggest that: the attachment of glucose to haemoglobin might have decreased the mobility of the Lys-adjacent Cys residues, thus leading to the increased h+1/h0 parameter of MSL. Such structural changes in haemoglobin owing to non-enzymatic glycosylation may contribute to the increased viscosity of haemoglobin solutions (r = 0.497, P < 0.0035) and the enhanced internal viscosity of diabetic erythrocytes (r = 0.503, P < 0.003). We argue that such changes in haemoglobin, and consequently in red blood cells, might contribute to the handicapped oxygen release under tissue hypoxia in the diabetic state.

Molecular insights into the anticoagulant-induced spontaneous activation of platelets in whole blood-various anticoagulants are not equal.

The spontaneous anticoagulant-dependent platelet activation in vitro may potentially interfere with the determination of haemostatic parameters. The effects of various blood anticoagulants on platelet activation were monitored using flow cytometry. Regardless of a blood anticoagulant used (EDTAK2, heparin, citrate or PPACK), platelet activation began immediately after blood withdrawal and was most pronounced in the EDTAK2-anticoagulated blood samples. The progressing expression of GMP-140 antigen was accompanied by the enhanced abundance of the subunit beta 3 of the platelet membrane integrin alpha IIb beta 3 without parallel changes in the fluorescence attributed to the complex form of the integrin alpha IIb beta 3. The increased expression of GMP-140 was paralleled by the enhanced platelet clumping in the samples anticoagulated with either EDTAK2 or heparin, and the raised platelet microparticles in blood withdrawn into citrate. The EDTAK2-induced platelet activation was markedly reduced by methyl 2,5-dihydroxycinnamate, tyrosine kinase inhibitor. The influence of disodium EDTA on platelet membrane dynamics closely mimicked the alterations induced upon the interaction of fibrinogen with platelet GPIIb-IIIa. Thus, the EDTAK2-induced platelet activation might result from an interference with platelet membrane protein structure and conformation and possibly related to an "unspecific" trigerring of a signal transduction pathway. Overall, EDTAK2 and heparin appeared the least suitable anticoagulants, particularly with the regard to the expression of GMP-140 antigen. The failure to recognize the importance of a spontaneous anticoagulant-induced platelet activation may result in misdiagnoses during the monitoring of coagulation parameters.

Does reduced membrane lipid fluidity underlie the altered thrombin-induced expression of integrin α(IIb)ß(3) and PADGEM-140 in membranes of platelets from diabetic juveniles?

In diabetic patients, where the membrane lipid microviscosity of blood platelets is altered, the availability of platelet membrane receptors may change concomitantly. Platelet hypersensitivity in diabetic subjects was previously hypothesized to result from the nonenzymatic glycosylation-induced loss in platelet membrane fluidity. In our present study juvenile type 1 diabetic subjects were compared with their relevant controls with respect to thrombin-stimulated platelet activation in relation to glycation-induced impairments of platelet membrane dynamics. Our results indicate that: (a) the mean steady-state fluorescence polarization (p) of both 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-anilino-8-naphthalenesulphonate (ANS) in membranes from diabetic subjects were significantly greater than for control subjects, thus indicating reduced membrane lipid fluidity in diabetic platelets in various membrane regions; (b) the significantly higher [(3)H]NaBH(4) reduction, indicating the increased attachment of glucose to protein amino groups, was attributed to the proteins extracted from diabetic platelet membranes; (c) CD62-positive resting platelets were not significantly more abundant in diabetic patients; (d) basically, unaltered amounts of PADGEM-140 membrane antigen (CD62) copies were detected in resting diabetic platelets; (e) significantly higher numbers of membrane glycoprotein ß(3) were found in diabetic platelets; (f) thrombin-induced elevations in the expression of CD61 (ß(3)) and CD62 (PADGEM-140) occurred to much higher extent in platelets of diabetic patients, thus pointing to more profound activation of diabetic platelets by thrombin; (g) the total amounts of platelet membrane glycoprotein ß(3) was significantly reduced in platelet lysates from diabetic subjects. We conclude that glycation-induced rigidization of platelet membranes might hypersensitize diabetic platelets to aggregating agents by rendering platelet membrane receptors more exposed to the external environment. Thus, thrombin may bind more efficiently to the exposed glycoprotein receptors (due to glycation) in diabetic platelets. Such excessive exposure and displacements toward the external environment might favour the accelerated shedding of some membrane proteins in diabetic platelets. We further suggest that their subsequent replacements would render platelet intrinsic storage pools exhausted and thus, might explain the diminished total amount of ß(3) found in platelets of diabetic patients.