Turbidimetric Platelet Aggregation Research Articles

Allium ursinum L.: Bioassay-Guided Isolation and Identification of a Galactolipid and a Phytosterol Exerting Antiaggregatory Effects

Aims: We wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adenosine diphosphate (ADP)-induced aggregation of human platelets by wild garlic, Allium ursinum L. Methods: For this purpose, a bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. For the bioassay, blood was taken from healthy human volunteers and platelet-rich plasma was prepared for turbidimetric platelet aggregation tests. Platelet-rich plasma, stimulated with 20 µmol/l of ADP, was treated with extracts of different polarities, fractions and isolated single compounds from A. ursinum. The extracts were investigated by thin-layer chromatography (TLC), HPLC, mass spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and 1/2-dimensional ¹H/¹³C-nuclear magnetic resonance (NMR) spectroscopic techniques. Results: Fresh A. ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited ADP-induced aggregation of human platelets. This ethanolic extract was subjected to liquid-liquid partition. Whilst the aqueous phase, containing the moiety of cysteine sulphoxide and thiosulphinate derivatives, showed only weak activity on platelet aggregation, the ethyl-acetate and particularly the chloroform partitions showed the highest aggregation-inhibiting potency. Thus, in our bioassay, the effects of alliins/allicins could be neglected. The chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient-elution open column chromatography on silica gel. The most active fractions 11–17 were separated again, yielding 10 subfractions. This afforded 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol and β-sitosterol-3-O-β-D-glucopyranoside, the structures of which were determined by ESI-MS and 1/2-dimensional ¹H/¹³C NMR spectroscopic techniques. Furthermore, the minute amounts of volatile oil of A. ursinum leaves obtained by steam distillation according to Ph. Eur. could be evaluated as a third aggregation-inhibiting principle. Conclusion: In our study, for the first time, 2 active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on ADP-induced aggregation in human blood platelets. As a major constituent, the galactolipid, 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol, not yet found in Allium sp., appears as a new, highly useful marker substance for A. ursinum drugs, or their pharmaceutical or food preparations, as shown by our orientating TLC analyses.

Functional Evaluation of Platelet Aspirin Resistance after On-Pump Coronary Bypass Grafting Using Multiple Aggregation Tests

The predominant mechanism of early graft failure after coronary artery bypass grafting (CABG) is associated with antiplatelet treatment using drugs such as acetylsalicylic acid (ASA). Impaired hemostasis of multiple etiologies is often present in patients undergoing on-pump cardiac surgery. We investigated the impact of intravenous ASA administration on platelet function in this setting. Forty-two patients were enrolled in the study. Patients received 100 mg oral ASA once daily, beginning in the early postoperative period. Noncompliance was eliminated by the administration of 300 mg ASA intravenously at 6-8 days post-operation. Blood was drawn immediately before, 1 h and 24 h after ASA administration. A platelet function analyzer (PFA-100™) was used to evaluate closure time (CT), turbidimetric platelet aggregation (TPA) and impedance platelet aggregation (IPA) induced by arachidonic acid (AA), collagen and ADP and results were compared with the respective values from 120 healthy individuals. At 1 h and 24 h after administration, we found that intravenous ASA caused CEPI-CT to be significantly prolonged with a reduction of AA and collagen-induced IPA. Despite postoperative oral ASA administration for 6-8 days, PFA-100™ CEPI and CADP-CT were significantly shorter and ADP-TPA and IPA values induced by any agonist were significantly greater in patients than in controls. Intravenous ASA had no significant influence on CADP-CT or ADP-induced IPA (ADP-IPA). Platelet tests for diagnosing patients as aspirin responders (ASA-R) or aspirin non-responders (ASA-NR) were found to be not comparable. Patients after CABG show augmented platelet dysfunction. Intravenous ASA administration may indicate a promising approach to reduce laboratory resistance after CABG procedure. The reason for this is not clear and requires additional clinical studies.

Analysis of a commercial dimethyl‐sulfoxide‐stabilized frozen canine platelet concentrate by turbidimetric aggregometry

To assess platelet function of a commercial dimethyl-sulfoxide (DMSO)-stabilized frozen platelet concentrate (PC) using turbidimetric aggregometry. In vitro analysis. Research laboratory in a school of veterinary medicine. Five units of frozen PC in 6% DMSO were studied. Fresh platelet-rich plasma (PRP), with and without 6% DMSO, from 6 healthy dogs were used as controls. Turbidimetric platelet aggregation was measured after initiation of platelet aggregation by addition of adenosine diphosphate (ADP), collagen, or thrombin at concentrations of 30 μM, 20μg/mL, and 0.5U/mL, respectively. Measures were performed at thaw and repeated 2 hours after thaw for the frozen PC. Compared with PRP, the frozen PC showed decreased aggregation in response to thrombin (amplitude of 84% versus 25%, P=0.01), and collagen (amplitude of 13% versus 3%, P=0.05) but not ADP (6.5% versus 18%, P=0.2). Compared with frozen PC at thaw, the frozen PC at 2 hours after thaw showed decreased aggregation in response to thrombin, collagen, and ADP (P<0.05). There was no difference in aggregation between PRP in 6% DMSO and frozen PC. These in vitro data suggest there is a decrease in platelet response to agonists associated with the freeze-thaw process in the commercially available 6% DMSO canine frozen PC.

The impact of intravenous aspirin administration on platelet aspirin resistance after on-pump coronary artery bypass surgery

Aspirin resistance continues to be a major challenge in patients after coronary artery bypass grafting (CABG). We investigated the impact of intravenous aspirin on platelet function in this clinical setting. Forty-two patients received 100 mg of oral aspirin once daily, beginning on day 1 after the operation. Between day 6 and 8 post operation one oral dose was replaced by an intravenous dose of 300 mg. Platelet function analyzer (PFA-100™) closure times (CT), turbidimetric platelet aggregation (TPA) and impedance platelet aggregation (IPA) induced by arachidonic acid (AA), collagen and ADP were measured prior to and 1 h and 24 h after intravenous aspirin. Results obtained prior to the intravenous aspirin were compared with respective values from 120 healthy individuals. Despite the postoperative oral aspirin that was given once daily, ADP-induced TPA (ADPTPA) and IPA values induced by AA, ADP or collagen were significantly greater in patients than in controls, while PFA-100™ CT were significantly shorter. Intravenous aspirin induced a significant reduction of platelet aggregability as measured by collagen/epinephrine (CEPI) CT, TPA and IPA induced by AA and collagen 1 h and 24 h after administration. Intravenous aspirin was not found to influence collagen/ADP (CADP) CT and IPA induced by ADP. Concomitantly, the number of patients with laboratory aspirin resistance as measured by CEPI-CT and TPA but not by IPA induced by AA or collagen dropped significantly. Agreement in the detection of aspirin responders and non-responders among platelet function tests was poor. Our findings indicate that the intravenous aspirin appears to be a promising approach for reducing laboratory aspirin resistance during the postoperative phase of CABG.

THIS ARTICLE HAS BEEN RETRACTED The impact of a medium molecular weight, low molar substitution hydroxyethyl starch dissolved in a physiologically balanced electrolyte solution on blood coagulation and platelet function in vitro

Background and Objectives Hydroxyethyl starches (HES) may have the potential to impact negatively on haemostasis. Recent findings suggest that side‐effects on haemostasis stem not only from the physicochemical differences between HES, but also from the composition of the solvent. We compared the effects of a newly developed medium molecular weight (MW) and low molar substitution (MS) HES dissolved in a physiologically balanced electrolyte solution (MW 130, MS 0·42; B‐HES) with a commercially available non‐balanced HES (MW 130, MS 0·4; NB‐HES), and with Ringer's lactate (RL) solution in vitro.Materials and Methods Activated partial thromboplastin time (APTT), factor VIII clotting activity (F VIII:C) and von Willebrand factor (vWF) activity were investigated in 48 healthy individuals. Platelet function as measured by turbidimetric platelet aggregometry and whole blood impedance aggregometry induced by adenosine diphosphate (ADP), collagen and thrombin receptor activating peptide (TRAP), and by ADP and TRAP‐induced expression of activated platelet fibrinogen receptor glycoprotein (GP) IIb/IIIa was determined in 24 participants. Haemodilution (25% and 50%, v/v for blood coagulation analyses and 20% and 40%, v/v for platelet function studies) was performed using the two HES preparations and RL.Results APTT was significantly longer and F VIII and vWF significantly lower at 25% and 50% dilutions with NB‐HES compared to B‐HES and RL. At 20% and 40% dilutions, ADP and TRAP‐induced expression of activated platelet surface GP IIb/IIIa was significantly increased by B‐HES compared to NB‐HES and RL. Percentages of platelet GP IIb/IIIa expression were also significantly greater in samples diluted with B‐HES than in undiluted blood. Neither the diluent (B‐HES, NB‐HES and RL) nor the degree of dilution (undiluted, 20% and 40% dilution) had any significant influence on ADP, collagen or TRAP‐induced turbidimetric platelet aggregation or impedance platelet aggregation.Conclusions In contrast to a non‐balanced 130 kDa, MS 0·4 HES (NB‐HES), a 130 kDa, MS 0·42 HES preparation dissolved in a physiologically balanced electrolyte solution (B‐HES) does not affect APTT, F VIII:C and vWF in vitro. Both types of HES do not affect platelet aggregation induced by ADP, collagen or TRAP. B‐HES but not NB‐HES increases the expression of activated platelet GP IIb/IIIa induced by ADP or TRAP.

Variables influencing Multiplate™ whole blood impedance platelet aggregometry and turbidimetric platelet aggregation in healthy individuals

We investigated the relationship between impedance platelet aggregometry (IPA) as measured by the Multiplate™ system and turbidimetric platelet aggregation (TPA) induced by ADP, arachidonic acid (AA), and collagen; blood cell counts; platelet function analyzer (PFA-100™) closure times (CT), and von Willebrand factor (VWF) in 120 well-characterized healthy individuals. Pre-analytical and analytical conditions were standardized comprehensively. Analytical reliability of IPA and TPA and the influence of pre-analytical variables on assay results were also examined. IPA and TPA did not change significantly between 0.5 and 5 hours after blood collection when samples were stored at room temperature. TPA and IPA showed significantly greater intra-assay imprecision than respective TPA induced by the same agonists. Intra-individual variation did not differ significantly between IPA and TPA. The lower limits of reference range (2.5th percentiles) of AAIPA, ADPIPA and collagen IPA determined AM were 37, 20 and 40 AU, respectively. ADPIPA showed significantly lower maximum aggregation values than AAIPA and collagen IPA (P < 0.0001). There were no significant differences in any parameter between males and females. No significant differences between blood group 0 and non-0 individuals were noted with respect to IPA and TPA. IPA did not change significantly during the day. In contrast, TPA measured PM was significantly lower than corresponding values determined a.m. (p < 0.0001). CEPI-CT, CADP-CT and leukocyte counts increased significantly from a.m. to p.m. (P = 0.008 and P > 0.0001, respectively). Donors had significantly greater IPA induced by any agonist than non-donors (P-values < 0.0001, 0.0001 and 0.001, respectively), whereas TPA was not significantly different between donors and non-donors. IPA did not correlate significantly with TPA nor with PFA-100™ CT. ADPIPA and collagen IPA correlated significantly with platelet count. TPA was not associated with platelet count. An inverse significant correlation was observed between TPA induced by any agonist and leukocyte count, whereas leukocyte count did not influence IPA. CEPI-CT and CADP-CT correlated significantly with VWF:CBA and with each other but not with TPA. We concluded that IPA and TPA measure different aspects of platelet function. IPA results reflect interactions between platelets, red and white cells, while TPA does not. This explains discrepancies in associations of IPA and TPA with cell counts, time of day and blood donation. The clinical significance of IPA determined using the Multiplate™ device remains to be determined in studies on patients with platelet dysfunction and under treatment with antiplatelet agents.

Pharmacodynamic profile of the direct thrombin antagonist bivalirudin given in combination with the glycoprotein IIb/IIIa antagonist eptifibatide

Background Because of the adverse characteristics associated with heparin, direct antagonists of thrombin have been investigated as anticoagulants during percutaneous coronary interventions. However, the hematologic and clinical interactions between direct thrombin antagonists and inhibitors of platelet glycoprotein IIb-IIIa are incompletely explored. Methods Forty-two patients who underwent elective percutaneous coronary intervention were randomized to receive a bivalirudin 1.0 mg/kg bolus followed by a 4-hour infusion at 2.5 mg/kg/h; a bivalirudin 0.75 mg/kg bolus followed by a 4-hour infusion at 1.75 mg/kg; or a heparin 60 U/kg bolus. All the patients also received eptifibatide, given as 2 sequential boluses of 180 μg/kg followed by a 2 μg/kg/min infusion for 18 to 24 hours, and aspirin. Results After the bolus dose of the study drug, turbidimetric platelet aggregation in response to 5 μmol/L adenosine diphosphate increased in patients assigned to heparin but not those assigned to bivalirudin. After eptifibatide, platelet aggregation was eliminated in all 3 treatment groups. The effect of heparin and the effects of both bivalirudin regimens on the formation of thrombin antithrombin complexes and prothrombin fragment 1.2 were comparable. Neither agent affected the formation of platelet-monocyte complexes or expression of CD 63 lysosomal antigen. There were no major bleeding events, and a single non–Q-wave myocardial infarction (MI) occurred in a patient treated with bivalirudin. Conclusion These findings show the feasibility of combining the direct thrombin antagonist bivalirudin with a potent antagonist of platelet glycoprotein IIb-IIIa. Clinical trials are needed to assess the safety and efficacy of this combination. (Am Heart J 2002;143:585-93.)

Effect of Ca2+ Chelation on the Platelet Inhibitory Ability of the GPIIb/IIIa Antagonists Abciximab, Eptifibatide and Tirofiban

Enhanced GPIIb/IIIa binding and inhibition of platelet aggregation of eptifibatide by the reduction of ionized plasma calcium concentrations have been reported. The present study compared the importance of Ca2+ chelation on the in vitro platelet inhibitory profiles of the GPIIb/IIIa antagonists abciximab, eptifibatide and tirofiban. Turbidimetric platelet aggregation dose response curves of the various GPIIb/IIIa antagonists were performed using platelet rich plasma (PRP) anticoagulated with either trisodium citrate, or the non-chelating anticoagulant, PPACK. The concentrations of antagonist that resulted in 50% inhibition of TRAP-induced (10 microM) platelet aggregation (IC50) were measured in the presence of either citrate or PPACK. In addition, the influence of Ca2+ chelation on the binding properties (relative affinity, on- and off-rates) of abciximab for the GPIIb/IIIa receptor on platelets was measured. For all three agonists, the IC50 concentrations were lower for platelets treated with citrate than PPACK, but the degree of difference varied among the agents. The mean TRAP IC50 values for citrate and PPACK were 88.2 +/- 12.2 nM and 126.1 +/- 28.4 nM for abciximab (1.4 fold enhancement; p = 0.0007), 75.9 +/- 13.3 nM and 142.6 +/- 32.6 nM for tirofiban (1.9-fold enhancement; p = 0.001), and 260.2 +/- 62.5 nM and 810.3 +/- 182.5 nM for eptifibatide (3.1-fold enhancement; p = 0.001). A similar shift in effective inhibitor concentrations for abciximab was observed with ADP (10 microM). The relative affinities (EC50), on- and off-rates of abciximab for the platelet GPIIb/IIIa receptor in the presence of trisodium citrate and PPACK were equivalent. These data confirm previous observations that Ca2+ chelation afforded by citrate decreases the effective inhibitor concentrations of GPIIb/IIIa antagonists, as assessed by turbidimetric platelet aggregation. However, the extent of decrease was less for abciximab and tirofiban, compared to eptifibatide.

Rapid platelet-function assay: an automated and quantitative cartridge-based method.

The platelet glycoprotein (GP) IIb/IIIa receptor is important in mediating platelet thrombus formation, and the GP IIb/IIIa antagonist abciximab (c7E3 Fab; ReoPro) is effective in preventing thrombotic ischemic cardiovascular complications of unstable angina and percutaneous coronary interventions. Small-molecule antagonists of GP IIb/IIIa based on the Arg-Gly-Asp (RGD) sequence show similar benefit, and some of these agents are orally active. However, there may be significant interindividual variation in response to such antagonists, especially with chronic oral therapy. It will be essential to balance the beneficial antithrombotic effect of these drugs with their potential for causing bleeding. In response to this need, we have developed a rapid platelet-function assay (RPFA), a point-of-care system that provides a quantitative measure of the competence of the GP IIb/IIIa receptor as reflected in the ability of platelets to agglutinate fibrinogen-coated beads. Polystyrene beads were coated with fibrinogen and placed in a cartridge along with a lyophilized peptide that activates the thrombin receptor. Anticoagulated whole blood was added to the cartridge, and then a microprocessor-controlled operation mixed the reagents and detected agglutination between platelets and coated beads. Quantitative digital results were displayed within 3 minutes. Because there is no dilution of the blood, the assay can be used to measure platelet activity in samples that have been treated with GP IIb/IIIa antagonists with high dissociation rates. RPFA results of whole-blood samples treated with different GP IIb/IIIa antagonists correlated well with both conventional turbidimetric platelet aggregation (r2=0.95) and the percentage of free GP IIb/IIIa molecules in the sample (r2=0.96). The mean difference in measurements between RPFA and aggregometry was -4% (+/-4% SD), and the mean difference in measurements between RPFA and free GP IIb/IIIa receptors was -2% (+/-6% SD). The RPFA provides rapid information on platelet function that mirrors turbidimetric platelet aggregation and reflects GP IIb/IIIa receptor blockade.

Acute and chronic regulation of alpha 2-adrenoceptor number and function in man.

Agonist regulation of platelet alpha 2-adrenoceptors was examined in human volunteers after acute elevations of adrenoceptor agonist and during chronic elevation of plasma catecholamines in two patients with phaeochromocytoma. Platelet alpha 2-adrenoceptor number was measured by radioligand binding [( 3H]yohimbine) and alpha 2-adrenoceptor function measured by turbidimetric platelet aggregation. Short term infusion of adrenoceptor agonists with alpha 2 activity caused reductions in the platelet response to adrenaline in vitro; conversely an increase in activity was observed postoperatively in two patients after removal of phaeochromocytoma. The changes in platelet response were not accompanied by changes in alpha 2-adrenoceptor number. It is proposed that a process of receptor inactivation occurs during desensitization and this is responsible for the dynamic regulation of platelet responses.