Effects of taprostene, a chemically stable prostacyclin analogue, in patients with ischaemic peripheral vascular disease: a placebo controlled double-blind trial

Thirty patients with ischaemic peripheral vascular disease and intermittent claudication were randomly allocated to receive either placebo or taprostene, a chemically stable prostacyclin analogue, intravenously at a rate of 25 ng/kg/min for 6 hours daily on 5 consecutive days. Taprostene produced a significant (p less than 0.05) increase in absolute walking time compared to placebo on one day after infusion and at 1, 4 and 8 weeks (14% vs 2.8%) later. Taprostene also produced a significant (p less than 0.05) increase in the pain-free walking time compared to placebo in the follow-up period (8 weeks after infusion: 23% vs 3.8%). During the infusion period systolic and diastolic blood pressure decreased (p less than 0.05) and heart rate was accelerated (p less than 0.05) in the taprostene treated group whereas no change was monitored in the placebo group. The ankle/brachial Doppler index was unaffected by taprostene. The platelet half-life was significantly (p less than 0.05) prolonged following taprostene-infusion (72.6 +/- 9.35 vs 77.9 +/- 7.44 hours). However, no change on platelet half-life was found in the placebo group (p less than 0.05). Various measures of platelet function parameters followed in vitro (ADP-induced aggregation, platelet sensitivity to PGI2, PGE1, PGD1 and taprostene, concentrations of platelet factor 4 and beta-thromboglobulin) showed no change with taprostene. Measures of circulating platelet aggregates and endothelial cells count showed no changes during the 2 months follow-up period too. It is assumed that taprostene may be of clinical benefit in patients with ischaemic peripheral vascular disease. However, future investigations have to be carried out to assess the optimal dose regime.     

Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E

We studied whether circulating activated platelets and platelet-leukocyte aggregates cause the development of atherosclerotic lesions in apolipoprotein-E-deficient (Apoe(-/-)) mice. Circulating activated platelets bound to leukocytes, preferentially monocytes, to form platelet-monocyte/leukocyte aggregates. Activated platelets and platelet-leukocyte aggregates interacted with atherosclerotic lesions. The interactions of activated platelets with monocytes and atherosclerotic arteries led to delivery of the platelet-derived chemokines CCL5 (regulated on activation, normal T cell expressed and secreted, RANTES) and CXCL4 (platelet factor 4) to the monocyte surface and endothelium of atherosclerotic arteries. The presence of activated platelets promoted leukocyte binding of vascular cell adhesion molecule-1 (VCAM-1) and increased their adhesiveness to inflamed or atherosclerotic endothelium. Injection of activated wild-type, but not P-selectin-deficient, platelets increased monocyte arrest on the surface of atherosclerotic lesions and the size of atherosclerotic lesions in Apoe(-/-) mice. Our results indicate that circulating activated platelets and platelet-leukocyte/monocyte aggregates promote formation of atherosclerotic lesions. This role of activated platelets in atherosclerosis is attributed to platelet P-selectin-mediated delivery of platelet-derived proinflammatory factors to monocytes/leukocytes and the vessel wall.     

A spectrophotometric method for following initial rate kinetics of blood platelet aggregation

A double-beam recording spectrophotometer was used to assay platelet aggregation. Agonist-induced turbidity changes, at 540 nm, in dilute suspensions of platelets (1 ml, 6-8 X 10(7) platelets) were recorded differentially against a reference cuvette, also containing platelets, as a function of time. The curves obtained showed downward pen deflections (decrease of turbidity) abolished by preincubation with the aggregation inhibitor, citrate. The turbidity decrease occurred simultaneously with microscopically determined single platelet recruitment into aggregates and its initial slope (r0) was a linear function of platelet concentration upto approximately 1.5 X 10(8) per ml. At a fixed platelet concentration the r0 values of ADP-induced aggregation of calf platelet-rich plasma varied as a hyperbolic function of ADP concentration at both 32 degrees C and 37 degrees C. The kinetic data at 37 degrees C were comparable to those, in the literature, obtained by following single platelet recruitment into aggregates. The increase in turbidity due to ADP-induced shape-change (6 +/- 1%, mean +/- S.E.M., n = 7) measured in the present study was substantially greater than that (3%) measured in the aggregometer.     

Circulating platelet activation in healthy subjects and in some pathological conditions. Method of study and results

Current clinical methods for evaluating platelet function are artful tests which study the effects of various stimuli on platelets, whereas the clinician is much more interested in methods evaluating the activation of circulating platelets. The hallmark of activation of platelets is their shape change, i.e. the transformation of the platelets from smooth disks into spiny spheres; the aggregation begins when 30% of platelets are activated. In 1138 subjects (384 healthy individuals and 854 patients with various pathological conditions with high thrombotic risk) we have investigated circulating platelet activation and circulating platelet aggregates by fixation of blood cells in a glutaraldehyde mixture and by evaluation of platelet shape change and aggregates on a phase-contrast microscope. The method is precise, accurate and suitable for clinical purposes.     

Flow Cytometric Analysis of Circadian Changes in Platelet Activation Using Anti-Gmp-140 Monoclonal Antibody

The hemostatic activity of blood shows a circadian variation with a higher frequency of acute coronary events in the morning. The thrombotic tendency of blood is influenced by many factors, including platelets. Diurnal changes of in vivo platelet activation were investigated by whole blood flow cytometry in 10 young healthy male volunteers using anti-GMP-140 (anti-α-granule membrane protein 140 kD) monoclonal antibody at 3h intervals from 06:00 to 24:00. We also studied circulating platelet aggregates to investigate whether there exists a similarity between the results of these methods. Results of flow cytometric analysis indicate that there is an increase in platelet activation during the period from 06:00 to 09:00. Platelet activation then decreases gradually during the period from noon to midnight. These changes are accompanied by a similar trend in circulating platelet aggregates. This suggests that GMP-140 expression on platelets is synchronized with or followed by platelet aggregate formation in vivo, and increased platelet activation may predispose individuals to thrombosis at this time.     

Flow cytometric analysis of circadian changes in platelet activation using anti-GMP-140 monoclonal antibody.

The hemostatic activity of blood shows a circadian variation with a higher frequency of acute coronary events in the morning. The thrombotic tendency of blood is influenced by many factors, including platelets. Diurnal changes of in vivo platelet activation were investigated by whole blood flow cytometry in 10 young healthy male volunteers using anti-GMP-140 (anti-alpha-granule membrane protein 140 kD) monoclonal antibody at 3h intervals from 06:00 to 24:00. We also studied circulating platelet aggregates to investigate whether there exists a similarity between the results of these methods. Results of flow cytometric analysis indicate that there is an increase in platelet activation during the period from 06:00 to 09:00. Platelet activation then decreases gradually during the period from noon to midnight. These changes are accompanied by a similar trend in circulating platelet aggregates. This suggests that GMP-140 expression on platelets is synchronized with or followed by platelet aggregate formation in vivo, and increased platelet activation may predispose individuals to thrombosis at this time.     

Influence of a long-term zinc-deficient diet on rat platelet function and fatty acid composition

A reduced zinc intake is associated with numerous abnormalities and, in particular, with hemostasis dysfunction. In this report, we studied the effects of a long-term dietary zinc restriction on platelet function. Three groups of rats were analyzed: a zinc-deficient group (ZD) and two zinc-adequate fed groups, one pair-fed (PF) and one ad libitum fed (AL). We found that ZD diet (0.2 p.p.m.) impaired ADP-induced aggregation of washed platelet after 4 and 8 weeks of diet. Thrombin-induced aggregation was impaired in ZD rats and PF rats after 8 weeks. The thrombin-induced mobilization of radiolabeled arachidonate preincorporated into platelet phospholipids was followed as well as the subsequent formation of labeled cyclooxygenase and lipoxygenase products. Stimulated platelets of ZD rats exhibited a decreased production of cyclooxygenase and lipoxygenase products, particularly after 8 weeks of diet. Moreover, platelet thromboxane generation was decreased in the ZD group as studied using a radioimmunoassay after thrombin stimulation. In addition, we measured the total fatty acid compositions of platelet and plasma. As a whole, 20:5 (n – 3) and 22:5 (n – 3) fatty acids content were significantly increased in platelet lipids after 8 weeks. On the other hand, it is known that enrichment of these fatty acids through dietary studies, both in animal and human as well as in vitro incorporation in platelets, resulted in an inhibition of platelet function. Consequently, these changes in platelet membrane fatty acid composition may contribute to the impaired platelet aggregation observed in ZD rats.     

Modulation of cytoplasmic calcium in human platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine

The calcium-sensitive, fluorescent dye Quin 2 was used to quantitate changes in free intracellular calcium [( Ca2+]i) induced in platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine (AGEPC). The Ca2+]i of unstimulated platelets was 91 +/- 18 nM (mean +/- SD, n = 8), and treatment with 1 to 16 nM AGEPC increased [Ca2+]i in a dose-related manner, with 16 nM AGEPC increasing [Ca2+]i by 102 +/- 20 nM. [Ca2+]i was not increased by analogs of AGEPC which do not activate platelets including the lysophospholipid precursor of AGEPC, the optical isomer, and a C-2 benzoyl analog. The capacity of AGEPC to increase [Ca2+]i exceeded that required to induce maximal platelet aggregation. In four experiments, 100% platelet aggregation was induced by 4.5 +/- 2.4 nM AGEPC (mean +/- SD) and was associated with a submaximal increase in [Ca2+]i of 56 +/- 22 nM. Pretreatment of platelets with AGEPC rendered the platelets specifically unresponsive to repeat stimulation with AGEPC in terms of both platelet aggregation and increased [Ca2+]i, whereas the platelet response to thrombin was undiminished by pretreatment with AGEPC. In contrast, the platelet response to 0.5 microM calcium ionophore A23187 was undiminished by pretreatment with the same concentration of ionophore, suggesting that AGEPC does not activate platelets by an ionophore-like mechanism. IgG aggregates and AGEPC in combination activate platelets synergistically, as shown by the observation that a 1-min exposure of platelets to 60 micrograms/ml of IgG aggregates increased the platelet aggregation response to 2 nM AGEPC from 44 to 100%. In contrast, sequential exposure of platelets to IgG aggregates and AGEPC increased [Ca2+]i additively, suggesting that increased [Ca2+]i contributes to but does not fully mediate synergistic platelet activation by IgG aggregates and AGEPC. Quantitation of free intracellular calcium with the fluorescent dye Quin 2 is a highly sensitive technique for delineating the role of calcium in mediating platelet activation.     

Evaluation of platelet function by flow cytometry

Platelet function in whole blood can be comprehensively evaluated by flow cytometry. Flow cytometry can be used to measure platelet reactivity, circulating activated platelets, platelet-platelet aggregates, leukocyte-platelet aggregates, procoagulant platelet-derived microparticles, and calcium flux. Clinical applications of whole blood flow cytometric assays of platelet function in disease states (e.g., acute coronary syndromes, angioplasty, and stroke) may include identification of patients who would benefit from additional antiplatelet therapy and prediction of ischemic events. Circulating monocyte-platelet aggregates appear to be a more sensitive marker of in vivo platelet activation than circulating P-selectin-positive platelets. Flow cytometry can also be used in the following clinical settings: monitoring of GPIIb-IIIa antagonist therapy, diagnosis of inherited deficiencies of platelet surface glycoproteins, diagnosis of storage pool disease, diagnosis of heparin-induced thrombocytopenia, and measurement of the rate of thrombopoiesis.     

Clopidogrel reduces lipopolysaccharide‐induced inflammation and neutrophil‐platelet aggregates in an experimental endotoxemic model

Platelet activation contributes to organs failure in inflammation and plays an important role in endotoxemia. Clopidogrel inhibits platelet aggregation and activation. However, the role of clopidogrel in modulating inflammatory progression of endotoxemia remains largely unexplored. Therefore, we investigated the role of clopidogrel on the activation of platelet and leukocytes in lipopolysaccharide (LPS)‐induced inflammation in mice. Animals were treated with clopidogrel or vehicle before LPS induction. The expression of neutrophil‐platelet aggregates and platelet activation and tissue factor was determined. Immunofluorescence was used to analyze platelet‐leukocyte interactions and tissue factor (TF) expression on leukocytes. Clopidogrel pretreatment markedly decreased lung damage, inhibited platelet‐neutrophil aggregates and TF expression. In addition, clopidogrel reduced thrombocytopenia and affected the number of circulating white blood cell in endotoxemia mice. Moreover, clopidogrel also reduced platelet shedding of CD40L and CD62P in endotoxemic mice. Taken together, clopidogrel played an important role through reducing platelet activation and inflammatory process in endotoxemia.     

Monoclonal antibody against platelet thrombospondin decreases erythrocyte aggregation rate.

The effect of thrombospondin, a major glycoprotein in the platelet alpha-granule, on the erythrocyte aggregation rate was investigated. Venous blood was sampled from 8 healthy male volunteers and anticogulated with 1.1 mg/ml EDTA(K2). The erythrocyte aggregation rate of each blood sample was measured with a whole-blood erythrocyte aggregometer before and after incubation with murine monoclonal antibody against human platelet thrombospondin. After 15 min incubation, the erythrocyte aggregation rate exhibited a significant decrease to 0.055 +/- 0.022/s, representing 71.9 +/- 8.7% of the control value (0.075 +/- 0.028/s) (p less than 0.0005). The results obtained suggest that thrombospondin may participate in the control of erythrocyte aggregability in the circulating blood.     

Enrichment of coenzyme Q10 in plasma and blood cells: defense against oxidative damage

Coenzyme Q10 (CoQ10) concentration in blood cells was analyzed by HPLC and compared to plasma concentration before, during, and after CoQ10 (3 mg/kg/day) supplementation to human probands. Lymphocyte DNA 8-hydroxydeoxy-guanosine (8-OHdG), a marker of oxidative stress, was analyzed by Comet assay. Subjects supplemented with CoQ10 showed a distinct response in plasma concentrations after 14 and 28 days. Plasma levels returned to baseline values 12 weeks after treatment stopped. The plasma concentration increase did not affect erythrocyte levels. However, after CoQ10 supplementation, the platelet level increased; after supplementation stopped, the platelet level showed a delayed decrease. A positive correlation was shown between the plasma CoQ10 level and platelet and white blood cell CoQ10 levels. During CoQ10 supplementation, delayed formation of 8-OHdG in lymphocyte DNA was observed; this effect was long-lasting and could be observed even 12 weeks after supplementation stopped. Intracellular enrichment may support anti-oxidative defense mechanisms.     

Synthesis of CC,CN coupled novel substituted dibutyl benzothiazepinone derivatives and evaluation of their thrombin inhibitory activity

The formation of a thrombus is a key event in thromboembolic disorders, that contribute to high mortality and morbidity in affected patients. In the present study, we synthesized a library of novel substituted 3,3-dibutyl-8-methoxy-2,3-dihydrobenzo [b] [1,4] thiazepin-4(5H)-one derivatives which were tested for their platelet aggregation and thrombin inhibitory activity. Among the tested compounds, 3,3-dibutyl-7-(2-chlorophenyl)-8-methoxy-2,3-dihydrobenzo[b] [1,4]thiazepin-4(5H)-one (DCT) displayed the maximum thrombin inhibition with an IC₅₀ value of 3.85 μM and thus DCT was chosen for further studies. Next, the effect of DCT on primary hemostasis was evaluated using agonist-induced platelet aggregation model. The lead compound inhibited the collagen- or ADP- or thrombin-induced platelet aggregation in a dose-dependent manner. Furthermore, DCT prolonged the process of clot formation when evaluating plasma re-calcification time (320 ± 11 sec at 5 µg DCT), activated partial thromboplastin time (58.0 ± 0.01 sec at 2 µg), and prothrombin time (14.7 ± 0.01 sec at 5 µg). Molecular docking studies suggested that the benzothiazepinones evaluated here consistently display hydrogen bonding with Ser214 of thrombin, which is similar to that of the co-crystallized ligand (1-(2R)-2-amino-3-phenyl-propanoyl-N-(2,5dichlorophenyl)methylpyrrolidine-2-carboxamide). DCT displayed additional hydrogen bonding to Ser195 and π-π interactions between its methoxyphenyl groups and Trp60, thereby providing a structural rationale for the observed biological effect.     

Effect of ASS on platelet function in experimental DIC]

In thrombin-induced DIC, acetylsalicylic acid (ASA) prevents the strong initial fall in platelet count and the obturation of the microvasculature of the lung with platelet aggregates. During the DIC reaction increasing inhibition of aggregability of circulating platelets against collagen and ADP is observed. Furthermore, ASA prevents the increase in the plasma haemoglobin level caused by DIC.     

Pulmonary and anti-platelet effects of intravenous and inhaled prostacyclin in man

Prostacyclin in aerosol was inhaled by three healthy volunteers (0.3 – 30 μg) and by twelve patients with bronchial asthma (200 or 400 μg). Essentially, no changes in pulmonary function were noticed. Intravenous infusion of prostacyclin (2 – 20 ng/kg/min) into six healthy volunteers also remained without effects on respiratory indices studied. Inhaled prostacyclin impaired platelet aggregability to ADP, dispersed circulating platelet aggregates and produced vasodilation comparable to those observed following intravenous administration of the hormone. We suggest that aerosols of prostacyclin might be used in future in treatment of arterial thrombo-embolism.     

Pharmacological actions of Y-24180, a new specific antagonist of platelet activating factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-t hieno [3,2-f][1,2,4]triazolo [4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of 3H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC50 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC50 values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC50 0.84 nM) was more potent than WEB 2086 (IC50 4.21 nM) and etizolam (IC50 998 nM). Y-24180, WEB 2086 and etizolam displaced 3H-PAF binding from the washed-platelets of rabbits with an IC50 value of 3.50, 9.35 and 29.5 nM, respectively. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180 (1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced 3H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 microM. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.     

Platelet size distribution in mice following immunothrombopenia and vincristine administration

In the present study platelet size distribution was investigated after induction of immunothrombocytopenia by rabbit-anti-mouse-platelet-serum (RAMPS) and after vincristine-induced thrombocytopenia. The platelet size distribution after a single dose of RAMPS showed a shift to larger volumes at day 1 and 2, and a decrease to slightly smaller volumes than normal at day 8. These differences, however, were not statistically significant. After vincristine administration, a dose-dependent increase of the platelet size distribution was demonstrated, which lasted from day 1-7. It is suggested that in immune-induced thrombocytopenia the young platelets released from bone marrow megakaryocytes are not exclusively large platelets. On the other hand the early appearance of large platelets after vincristine administration points to a toxic or segregating effect of vincristine on circulating platelets. Therefore, in our experiments, the platelet size is not suitable for the differentiation of young and old platelets.     

Effect of vitamin E on the function of blood platelets in patients with diabetes mellitus

An effect of vitamin E on blood platelets functioning was studied in 39 patients with diabetes mellitus type 1. Control group included 20 healthy blood donors. Vitamin E in a daily dose of 1000 mg produced statistically significant decrease in platelets aggregation, number of circulating platelet aggregates and release of the platelet factory 4 in diabetics after 7 days of treatment. No adverse reactions were seen in any patient treated with vitamin E. The obtained results indicate that vitamin E inhibits increased platelets activity in the patients with diabetes mellitus type 1 and does not exert toxic reactions during the treatment.     

Efficient flow cytometric assay for platelet-leukocyte aggregates in whole blood using fluorescence signal triggering.

BACKGROUND: Platelet-leukocyte aggregates (PLAs) may be important in thrombotic and inflammatory disease states, but accurate assessment of PLA formation in vivo is hampered by the propensity for in vitro artefacts caused by sample manipulation. A whole blood flow cytometric assay for circulating PLAs, based on minimal sample manipulation, was thus developed. METHODS: Citrated whole blood was labeled with a RPE-CD45 MAb (leukocyte marker) and an FITC-CD42a (GPIX) MAb (platelet marker). The latter was used to avoid possible influences of platelet glycoprotein proteolysis by neutrophil-derived proteases. The samples were mildly fixed with 0.5% formaldehyde saline. The cytometer was triggered by RPE-CD45 fluorescence. Leukocyte subpopulations were separated according to their typical light scattering and CD45 expression. RESULTS: Minimal sample manipulation and mild sample fixation resulted in minor in vitro artefacts and good sample stability. Fluorescence triggering increased the efficiency of the flow cytometric analysis approximately 5-fold compared with triggering with light scatter, and allowed discrimination of leukocyte subpopulations. The majority of PLAs involved monocytes and neutrophils, rather than lymphocytes, both without and with in vitro stimulation by ADP or thrombin. A cocktail of blocking MAbs to CD62P, CD15, GPIIb/IIIa and the CD11b/CD18 complex had no effect on unstimulated samples, whilst totally inhibiting aggregation induced by 10(-5) M ADP, suggesting that the PLAs in unstimulated blood were preformed in vivo. CONCLUSIONS: This whole blood flow cytometric assay for PLAs is simple and efficient, and appears to reflect closely platelet-leukocyte aggregates in circulating blood in vivo.     

Morphologic alterations of blood cells in the impedance aggregometer

The scanning (SEM) and transmission (TEM) electron microscopic appearance of blood cells was studied in correlation with the aggregometer tracing recorded after activation of whole blood samples by collagen or ADP. Early morphologic alterations of platelets characterized by the formation of marginal pseudopods and bulbous protrusions were not indicated by the aggregometer. The initial increase in impedance was caused by the attachment of platelets displaying typical shape change morphology at the surface of the electrode wires joint with collagenous fibrils in collagen activated specimens. During further increase in impedance, aggregates were detectable in the blood suspension and at the electrode, the number and size of which increased up to the maximal extension of the aggregometer tracing. Using low doses of ADP (2-3 microM), dissociation of aggregates in the blood suspension was detectable by SEM, which was not recorded by the aggregometer tracing indicating further limitation of the impedance aggregometer. In collagen activated samples, platelet aggregates were covered by PMN and monocytes that in TEM displayed distinct phagocytosis of platelet fragments and fibrin masses. In ADP specimens, activation of leukocytes was only rarely detectable. The detection of mixed aggregates may be important for further employment of the impedance aggregometer in the diagnosis of hematologic diseases.