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PubMed1987

Activity of platelet-derived growth factor (PDGF) in platelet concentrates and cryopreserved platelets determined by PDGF bioassay.

The bioassay of platelet-derived growth factor (PDGF) in platelets was established using the rat fibroblast cell line 3Y1. 3Y1 cells (5 X 10(3)/well) were cultured in 24-well microculture plates with RPMI 1640 medium using 1% of PDGF-free serum and 5% of platelet extracts for 4 days. Cell proliferation was measured by the increase of DNA content. This assay made it possible to measure the biological PDGF activity. PDGF activity of platelet concentrates kept the same level as that of fresh platelet samples during preservation for up to 120 h at 22 degrees C; cryopreserved platelets also retained PDGF activity well.

Biological Assay↗
PubMed1990

Functional validation of platelet-activating factor receptor sites characterized biochemically by a specific and reproducible [3H]platelet-activating factor binding in human platelets.

In human platelet membranes, [3H]platelet-activating factor(PAF)-C18 binding sites exhibited high affinity (Kd 0.074 +/- 0.005 nM, n = 28 healthy volunteers), saturability, elevated stereoselectivity, marked pharmacological specificity and small intersubject variability. The maximal binding capacity was 215 +/- 12 fmol/mg protein. Saturation of [3H]PAF binding was obtained with 0.3 nM ligand, and its isotherm was compatible with a single class of binding sites. The stereoselectivity for [3H]PAF was clearly indicated by the low displacing potency of enantio-PAF-C16 (the synthetic enantiomer of PAF) that was 5000-fold less potent than PAF. Specific [3H]PAF binding attained 65% with 0.1 nM ligand and was displaced fully not only by cold PAF but also by RP 59227 (Ki = 6.2 +/- 1.3 nM, n = 7), a novel, potent and specific PAF receptor antagonist in a pure enantiomeric form and several other antagonists such as CV-6209, WEB 2086, L-652,731 and BN 52021. Various classical pharmacological agents did not interfere with the [3H]PAF binding. In intact platelets, [3H]PAF binding shared the same properties as those just described for membrane preparations. A functional role for these binding sites was suggested by the high correlation (r = 0.94, P less than .001) between the Ki values for several known PAF antagonists determined in [3H]PAF binding and the IC50 values obtained against PAF-induced aggregation in whole platelets. Thus, the present [3H]PAF binding in human platelet membranes may be a useful pharmacological tool to study possible changes in [3H]PAF binding parameters induced by pathological states for which PAF may be directly or indirectly responsible.

Adult↗
PubMed1979

Comparison of platelet count and platelet protein methods for determination of platelet MAO activity.

Platelet monoamine oxidase (MAO) activity in 10 normal volunteers was studied as a function of platelet protein or electronically-determined platelet counts. Comparisons of the two methods were made for samples assayed on the same day as well as one week later. The MAO activities resulting from both methods were significantly correlated and reproducible but the results of the platelet count method were, in most instances, slightly but significantly more reliable than the platelet protein method. The relevance of these results to the controversy concerning platelet MAO activity in schizophrenia is discussed.

Blood Cell Count↗
PubMed1979

Thrombocytopenic episodes in patients with well-functioning renal allografts. Inverse relationship between platelet count and platelet size pointing to intermittent platelet destruction.

20 out of 50 patients with well-funcioning renal allograft displayed at least one platelet count below 110 X 10(9)/1 (mean -2 SD of controls). For estimation of platelet production during thrombocytopenic episodes, the percentage of large platelets in the peripheral blood was determined which revealed an inverse relationship (p less than 0.01) to the platelet count, indicating that these thrombocytopenias were due to increased platelet consumption. Immunosuppressive treatment as well as rejection processes could be excluded as major pathogenetic factors whereas anti-platelet autoantibodies may contribute to this phenomenon.

Autoantibodies↗
PubMed1987

ADP-induced platelet shape change and mobilization of cytoplasmic ionized calcium are mediated by distinct binding sites on platelets: 5'-p-fluorosulfonylbenzoyladenosine is a weak platelet agonist.

Platelet stimulation with ADP results in several responses, including shape change, increase in cytoplasmic ionized calcium concentration [Ca2+]i, an inhibition of adenylate cyclase. 5'-p-Fluorosulphonyl benzoyladenosine (FSBA), which covalently labels an ADP binding site on platelets, blocks platelet shape change but not the inhibition of cyclic AMP levels by ADP, whereas p-chloromercuribenzenesulfonate (pCMBS), a nonpenetrating thiol reagent, has the opposite effects. We examined the effect of FSBA and pCMBS on ADP-induced increase in [Ca2+]i using platelets loaded with fluorescent Ca2+ indicators quin2 and fura-2. FSBA (50 to 200 mumol/L) induced a dose-dependent rise in [Ca2+]i, indicating that it is a weak platelet agonist. Under conditions of covalent labeling of the ADP binding sites, FSBA (50 to 100 mumol/L) did not inhibit the ADP-induced increase in [Ca2+]i or its inhibition of adenylate cyclase, whereas pCMBS (up to 1 mmol/L) abolished both these responses but not shape change. These findings suggest that ADP-induced Ca2+ mobilization and inhibition of adenylate cyclase are mediated by platelet binding sites distinct from those mediating shape change.

4-Chloromercuribenzenesulfonate↗
PubMed1991

The effect of inhibitors of platelet aggregation on the metabolism of platelet-activating factor (PAF) in washed rabbit platelets.

The rabbit platelet metabolizes platelet-activating factor (PAF) intracellularly. PAF is deacetylated to produce lysoPAF which, in turn, can be acylated to produce 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl GPC). Some PAF receptor antagonists have been shown to inhibit this metabolic conversion. In the present study we examined whether the PAF receptor antagonists SRI 63-441 and WEB 2086 would inhibit the metabolism of PAF by intact rabbit platelets. In addition, we examined whether iloprost, a stable analogue of prostaglandin I2 (PGI2), and a potent inhibitor of platelet activation induced by a range of agonists, would also inhibit PAF metabolism. We found that SRI 63-441 and WEB 2086 caused an almost complete inhibition of the conversion of PAF to alkylacyl GPC. Iloprost caused up to a 50% inhibition of PAF metabolism compared to antagonist-free controls. Iloprost (and PGI2) is thought to inhibit platelet response by elevation of cAMP, while receptor antagonists act by blocking PAF binding to its receptor. Since iloprost caused partial inhibition of PAF metabolism, the results of this study suggest that inhibition of PAF metabolism does not occur solely due to competitive inhibition of PAF binding to its receptor.

Animals↗
PubMed1985

Inhibition of platelet-activating-factor-induced human platelet activation by prostaglandin D2. Differential sensitivity of platelet transduction processes and functional responses to inhibition by cyclic AMP.

It has been proposed that cyclic AMP inhibits platelet reactivity: by preventing agonist-induced phosphoinositide hydrolysis and the resultant formation of 1,2-diacylglycerol and elevation of cytosolic free Ca2+ concentration [( Ca2+]i); by promoting Ca2+ sequestration and/or extrusion; and by suppressing reactions stimulated by (1,2-diacylglycerol-dependent) protein kinase C and/or Ca2+-calmodulin-dependent protein kinase. We used the adenylate cyclase stimulant prostaglandin D2 to compare the sensitivity to cyclic AMP of the transduction processes (phosphoinositide hydrolysis and elevation of [Ca2+]i) and functional responses (shape change, aggregation and ATP secretion) that are initiated after agonist-receptor combination on human platelets. Prostaglandin D2 elicited a concentration-dependent elevation of platelet cyclic AMP content and inhibited platelet-activating-factor(PAF)-induced ATP secretion [I50 (concn. causing 50% inhibition) approximately 2 nM], aggregation (I50 approximately 3 nM), shape change (I50 approximately 30 nM), elevation of [Ca2+]i (I50 approximately 30 nM) and phosphoinositide hydrolysis (I50 approximately 10 nM). A 2-fold increase in cyclic AMP content resulted in abolition of PAF-induced aggregation and ATP secretion, whereas maximal inhibition of shape change, phosphoinositide hydrolysis and elevation of [Ca2+]i required a greater than 10-fold elevation of the cyclic AMP content. This differential sensitivity of the various responses to inhibition by cyclic AMP suggests that the mechanisms underlying PAF-induced aggregation and ATP secretion differ from those underlying shape change. Thus a major component of the cyclic AMP-dependent inhibition of PAF-induced platelet aggregation and ATP secretion is mediated by suppression of certain components of the activation process that occur distal to the formation of DAG or elevation of [Ca2+]i.

Adenosine Triphosphate↗
PubMed1996

Evaluation of platelet function using the in vitro bleeding time and corrected count increment of transfused platelets. Comparison between platelet concentrates derived from pooled buffy coates and apheresis.

The functional capacity of transfused platelets was evaluated with in vitro bleeding time (IVBT) and corrected count increment (CCI) in order to compare platelet concentrates (PCs) derived from pooled buffy coats (BC-PCs) with PCs collected by apheresis (A-PCs). The suspension medium in the BC-PCs was 30% CPD plasma and 70% of an additive solution (containing sodium and potassium chloride, sodium citrate and phosphate, mannitol), and in the A-PCs the medium was 100% CPD plasma. IVBT was evaluated using a Thrombostat 4000/2. BC-PC and A-PC were transfused 57 and 41 times, respectively to 36 patients with chemotherapy-induced thrombocytopenia. PCs transfused within 2 days of donation were considered fresh, and those transfused within 3-5 days were considered stored. IVBT was determined before, as well as 10-30 min and 24 h after transfusion; CCI was determined 10-30 min and 24 h after transfusion. The median pretransfusion IVBT value was 486 s. It was measurable in 21 of 98 (21%) of the transfusions, i.e. below the cutoff limit of 486 s. Ten to 30 min after transfusion, the IVBT showed a measurable reduction in 90% of the transfusions with fresh BC-PCs, 92% of those with fresh a-PCs, 63% of those with stored BC-PCs and 79% of those with stored A-PCs. After 24 h, the corresponding values were 63% for fresh BC-PCs, 50% for fresh A-PCs, 26% for stored BC-PCs and 38% for stored A-PCs. The median value of CCI 10-30 min after transfusion was 20 for fresh BC-PCs, 17 for fresh A-PCs, 16 for stored BC-PCs and 14 for stored A-PCs. The difference in IVBT between fresh and stored BC-PCs was significant (p = 0.032), unlike that between fresh and stored A-PC. After 24 h the corresponding values were 7 for fresh BC-PCs, 4 for fresh A-PCs, 4 for stored BC-PCs and 3 for stored A-PCs. When all transfusions with fresh PCs (BC-PCs + A-PCs) were compared with all transfusions with stored PCs, a statistical difference was demonstrated in both CCI (p = 0.027) and IVBT (p = 0.043). Spearman's rank correlation coefficient (rs) was -0.41 between CCI and IVBT < 486 s 10-30 min after transfusion, and -0.55 between the posttransfusion platelet count and IVBT, indicating a relatively poor correlation between CCI and IVBT, and a slightly better correlation between platelet count and IVBT. In conclusion, BC-PCs showed a slightly higher CCI and a better response in IVBT than A-PCs. No statistical difference was demonstrated between BC-PCs and A-PCs transfused within 2 days after donation, with respect to function and recovery in vivo. BC-PCs stored for 3 days or more showed about the same CCI and IVBT as stored A-PC but significantly lower CCI and higher IVBT than fresh BC-PCs. This may indicate that the preparation and/or storage conditions were not optimal. IVBT seems to be a useful possibility to test the in vivo behavior of transfused platelets.

Adolescent↗
PubMed1989

Abolition of in vivo platelet thrombus formation in primates with monoclonal antibodies to the platelet GPIIb/IIIa receptor. Correlation with bleeding time, platelet aggregation, and blockade of GPIIb/IIIa receptors.

We studied the dose-response effects of the F(ab')2 fragments of murine monoclonal antibodies to the platelet GPIIb/IIIa receptor (7E3 and 10E5) on in vivo platelet thrombus formation in a well-characterized monkey model in which the carotid artery is stenosed and thrombus formation is provoked and augmented by intimal damage and the infusion of subaggregating doses of epinephrine. Both antibodies abolished thrombus formation with a mean dose of -0.2 mg/kg. Ex vivo platelet aggregation was not always abolished at doses that abolished thrombus formation; similarly, bleeding times were only moderately prolonged (9.1 +/- 1.4 minutes) at these doses. Increasing the dose above that required to abolish thrombus formation consistently produced abolition of ex vivo platelet aggregation, marked prolongation of the bleeding time (14.2 +/- 1.5 minutes), and nearly quantitative blockade of GPIIb/IIIa receptors. We conclude that in a significant percentage of animals, the extent of GPIIb/IIIa blockade required to prevent vasoocclusive thrombus formation in this model is less than that required for abolition of platelet aggregation, and that the preservation of only a minority of functional GPIIb/IIIa receptors might be adequate to maintain a nearly normal bleeding time.

Animals↗
PubMed2000

Platelets expressing P-selectin and platelet-derived microparticles in stored platelet concentrates bind to PSGL-1 on filtrated leukocytes.

The levels of interleukin-6 and platelet-derived microparticles (PMPs) were measured in the blood of 137 patients with side effects from platelet concentrate (PC) transfusion with leukocyte removal filtration, P-selectin-expressing platelet and PMPs in stored PC before and after the filtration, and filtered leukocytes positive for P-selectin glycoprotein ligand-1. The side effects, which were observed in 203 transfusions for 84 patients with hematologic disease and 53 patients with nonhematologic disease with no significant difference between the two groups, included urticaria (75.9%), erythema (18.7%), and fever (17.2%), but no anaphylactic reactions. The levels of interleukin-6 and PMP correlated in both groups, and were significantly higher in the hematologic disease group than in the nonhematologic disease group. The level of PMP, but not interleukin-6, was significantly higher for patients testing positive for allergic reaction than for those testing negative. In the stored PC prior to filtration, the level of interleukin-6 was normal. The level of P-selectin-expressing platelets and PMPs was elevated before filtration, but was significantly lower after filtration. Taken together, the results suggest that PMP is involved in the generation of transfusion reactions, and indicate that both platelets and PMP displaying P-selectin bind to P-selectin glycoprotein ligand-1 of leukocytes retained by the leukocyte filter.

Blood Platelets↗
PubMed1987

Platelet deposition at angioplasty sites and platelet survival time after PTA in iliac and femoral arteries: investigations with indium-111-oxine labelled platelets in patients with ASA (1.0 g/day)-therapy.

In this study we have assessed the deposition of 111-In-oxine-labelled platelets--using a dual radiotracer method--at angioplasty sites of the lower extremities in 20 patients (14 male, 6 female; median age: 60 years) with ASA (1.0 g/day)-therapy. The platelet survival time (PST)--using the multiple hit model--was evaluated before and after percutaneous transluminal angioplasty, and we also measured the plasma levels of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF 4) before and after PTA. Before PTA, scintigraphy was positive in only one patient, while 24 hours after PTA a positive scintigraphic result was observed in 16/20 patients. The median target/non target-ratio was 1.0 (0.66-1.3) before PTA, and this ratio increased significantly (p less than 0.0005) to 1.53 (1.0-3.3) after PTA. The median PST decreased significantly (185.0 hours before PTA----145.2 hours after PTA; p less than 0.001), while the median platelet turnover increased from 34,000/microliter/day to 47,900/microliter/day (p less than 0.01). The median plasma levels of the platelet specific proteins increased significantly immediately after PTA (p less than 0.001), but one day later they were not significantly different from the pretreatment values. The quantitative methods used in this study seem a valuable tool to evaluate the effects of different therapeutical--especially antiplatelet--interventions after PTA in humans, thus helping to find the best antithrombotic regimen for this widely used therapeutical procedure.

Adult↗
PubMedDate not supplied

Combined platelet-leukapheresis: a technique for preparing separate platelet and granulocyte-platelet units from single donors.

One donor can provide separate platelet and granulocyte-platelet units that have the potential to fulfill the needs of two recipients. Combined platelet and leukapheresis were performed in the presence of hyddroxyethyl starch and citrate employing the Haemonetics Model 30 Blood Separator. Platelets and granulocytes were separated subsequently by centrifugation and two individual units were prepared for transfusion. The platelet units contained a mean of 4.4 x 10(11) platelets/unit and were nearly devoid of leukocytes. The combined granulocyte-platelet units contained a mean of 7.38 x 10(9) neutrophils/unit and 3.06 x 10(11) platelets/unit. Thus two separate units containing sufficient numbers of cells for transfusion can be obtained from pheresis of one donor.

Blood Donors↗
PubMed1998

Automated analysis of feline platelets in whole blood, including platelet count, mean platelet volume, and activation state.

A new whole-blood flow cytometric method has been developed for counting and sizing platelets in samples from cats, a species in which platelet and red blood cell sizes overlap significantly. The method is a modified version of the two-angle laser light scattering technology used by Bayer H*System hematology analyzers. The new method provided accurate platelet counts and mean platelet volumes (MPV, fl) for cats. The method also measured mean platelet component concentration (MPC, g/dl), a parameter which was shown to be sensitive to platelet activation state, and which decreased in value as activation progressed.

Journal Article↗
PubMed1987

Platelet storage lesion: formation of platelet fragments with platelet factor 3 activity.

Certain samples of stored platelet concentrates exhibited marked platelet factor 3 activity. This activity was associated almost completely with platelet fragments formed during storage. Platelet concentrates containing high levels of these fragments and hence high platelet factor 3 activity were characterized after storage by alkaline plasma pH values, high levels of extracellular lactate dehydrogenase activity and relatively low cell counts.

Blood Platelets↗
PubMed1984

[Anti-leuko-platelet allo-immunization in blood replacements. Do transfusions of platelets from single donors present an advantage over platelets from multiple donors?].

42 patients with acute leukaemia, treated with cytotoxic drugs, have been evaluated retrospectively: --group I: 11 patients received packed red blood cells and platelets from single donors; --group II: 6 patients received packed red blood cells and platelets from multiple donors; --group III: 25 patients received packed red blood cells and platelets from single or multiple donors and granulocytes transfusions. There was no difference in age, sex, time of follow up, number of transfusions, in the three groups. The rate of alloimmunization defined as lymphocytotoxicity against more than 20% of a panel of 24 lymphocytes, was 33% (36% group I--33% group II--32% group III). This study shows that platelets from single donors are of no use in preventing or delaying alloimmunization. On the other hand, their major interest is to provide alloimmunized patients with compatible platelets.

Adolescent↗
PubMed1976

Activation of platelets by platelet activating factor (PAF) derived from IgE-sensitized basophils. IV. PAF does not activate platelet factor 3 (PF3).

Platelet activating factor (PAF) derived from antigen-stimulated, IgE-sensitized rabbit basophils acts on platelets to induce aggregation and secretion of their content of granule-bound vasoactive amines. Despite this, PAF did not activate platelet factor 3. In contrast, collagen induced aggregation, secretion and PF3 activation in the washed platelets. Other stimuli (ADP, C3b, thrombin) also initiated both secretion and PF3 activation. A wide dose range of PAC, including those giving maximal secretion and aggregation, were ineffictive in making PF3 available and the possibility that PAF inhibited PF3, or its generation, was also excluded. It is concluded that PAF is a unique stimulus for platelets and that secretion and aggregation are not necessarily accompanied by PF3 generation.

Animals↗
PubMed2000

Smooth muscle cell migration induced by shear-loaded platelets and endothelial cells. Enhanced platelet-derived growth factor production by shear-loaded platelets.

BACKGROUND: Uncontrolled migration and proliferation of smooth muscle cells (SMC) are the main mechanisms of development of atherosclerotic neointima. However, it has not yet been elucidated how mechanical stress is involved in this process. We investigated smooth muscle cell mitogenic activity induced by shear-loaded platelets and endothelial cells. METHODS EXPERIMENTAL DESIGN: in vitro experimental study. We devised four types of conditioned media; supernatant of mixed culture of platelets and endothelial cell (ST), supernatant of shear-loaded mixed culture of platelets and endothelial cell (SH), ST medium neutralised with anti-PDGF antibody (ST+), and SH medium neutralised with anti-PDGF antibody (SH+). Smooth muscle cells were cultured in each conditioned medium, and their spreading activity was determined under a microscope. RESULTS: Smooth muscle cells spreading activity in the SH group was significantly greater than that in the ST group. Their spreading activity was suppressed by anti-PDGF antibody under shear conditions (SH+), but it was not by anti-PDGF antibody under static conditions (ST+). CONCLUSIONS: Our results demonstrate that platelet-derived growth factor is produced by shear-loaded platelets and endothelial cells, and local mechanical forces may play an important role in cardiovascular disease.

Blood Platelets↗
PubMedDate not supplied

[Effect of dietary linoleic acid on the platelet aggregation, fatty acid composition of platelet phospholipids and synthesis of platelet prostaglandins in rats].

Three groups of Wistar-Kyoto rats were kept on diets containing different quantity of linoleic acid (LA) in prenatal and postnatal periods up to 18 weeks age: I. less than 0.1 cal.% of LA, 2. 9.0 cal.% of LA and 3. 16 cal.% of LA. Aggregation of washed platelets with ADP and A23187, biosynthesis of prostaglandins from 14C-arachidonic acid in platelets and fatty acid composition of platelet phospholipids were studied using gas liquid chromatography. Modification of fatty acid composition of platelet phospholipids and changes in biosynthesis of platelet prostaglandins are the base for proaggregation effect of LA deficient and for antiaggregation effect of LA enriched diets. The effects of dietary LA were mediated not only via altered metabolism of fatty acids of linoleic family but also via metabolism of fatty acids of oleic and linolenic series.

Animals↗