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At least 217 records · Page 12Linked to original sources
PubMed1987

Platelet-activating factor: mediator of the third pathway of platelet aggregation? A study in three patients with deficient platelet-activating factor synthesis.

Thrombin, collagen, and Ca2+-ionophore A23187 aggregate platelets in the presence of inhibitors of the first (ADP-mediated) and second (cyclooxygenase-dependent) pathway of platelet activation. This aggregation, via a third pathway, was hypothesized to be mediated by the alkoxyether lipid platelet-activating factor (PAF). We recently demonstrated virtual absence of plasmalogen-type alkoxyether lipids and deficiency in key enzymes of their biosynthesis in Zellweger patients. We hypothesized that PAF synthesis might also be impaired. We report two Zellweger patients with an undetectable A23187-induced PAF synthesis of leukocytes (patients, less than 3 pmol PAF/10(8) granulocytes (PMN); four age-matched controls, 249-2,757 pmol PAF/10(8) PMN; five adult controls, 291-5,433 pmol PAF/10(8) PMN). In a third patient, residual PAF synthesis was detected. However in all patients the thrombin-induced third mechanism of platelet aggregation was present. We therefore conclude that PAF may not be the mediator of the third pathway.

Abnormalities, Multiple↗
PubMed1999

Relationship between intracellular calcium-dependent process and protein-tyrosine phosphorylation in human platelets: studies of platelets from a patient with defective A23187-induced platelet aggregation.

We had postulated that in a patient with defective calcium ionophore (A23187)-induced platelet aggregation, whose platelets showed normal intracellular Ca2+ mobilization in either the presence or absence of extracellular Ca2+ in response to A23187. A defect was present in an intracellular calcium-dependent process. We have now investigated whether the agonist-induced protein-tyrosine phosphorylation (PTP) was altered. Protein-tyrosine phosphorylation (PTP)-induced by A23187 in the patient's platelets was greatly diminished but that induced by thrombin was almost normal. These results suggest that an intracellular calcium-dependent process plays a fundamental role in A23187-induced PTP, whereas it does not in thrombin-induced PTP.

Blood Platelet Disorders↗
PubMed1989

Staurosporine potentiates platelet activating factor stimulated phospholipase C activity in rabbit platelets but does not block desensitization by platelet activating factor.

The possible involvement of protein kinase C activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets. PAF (100 nM for 5 seconds) stimulated incorporation of 32P into proteins and caused [3H]InsP3 levels to increase about 260% of control. These responses were compared after platelets were pretreated with either PAF, phorbol 12-myristate 13-acetate (PMA) or staurosporine and also after pretreatments with staurosporine followed by PAF or PMA. Pretreating platelets with staurosporine potentiated PAF-stimulated [3H]InsP3 levels by 54% and blocked protein phosphorylation. Pretreatments with PAF and PMA caused PAF-stimulated [3H]InsP3 levels to decrease to 115 and 136%, respectively. Staurosporine pretreatment blocked the decrease caused by the PMA pretreatment but not that by PAF. This study demonstrates that PAF-stimulated PLC activity is negatively affected by protein kinase C (PKC) activation and that inhibition of PKC activity did not prevent desensitization of PLC by PAF.

Alkaloids↗
PubMed2006

Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part III: leucocyte activation: a new feature for platelet concentrates?

Platelet-rich fibrin (PRF) belongs to a new generation of platelet concentrates, with simplified processing and without biochemical blood handling. In this third article, we investigate the immune features of this biomaterial. During PRF processing, leucocytes could also secrete cytokines in reaction to the hemostatic and inflammatory phenomena artificially induced in the centrifuged tube. We therefore undertook to quantify 5 significant cell mediators within platelet poor plasma supernatant and PRF clot exudate serum: 3 proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha), an antiinflammatory cytokine (IL-4), and a key growth promoter of angiogenesis (VEGF). Our data are correlated with that obtained in plasma (nonactivated blood) and in sera (activated blood). These initial analyses revealed that PRF could be an immune regulation node with inflammation retrocontrol abilities. This concept could explain the reduction of postoperative infections when PRF is used as surgical additive.

Adult↗
PubMedDate not supplied

Platelet antibody determination by platelet factor 3 assay (comparison with radiolabelled serotonin release and platelet aggregometry).

Platelet antibody determination by the PF3 test was carried out in 96 thrombocytopenic patients with various disorders, 31 repeatedly transfused patients with or without thrombocytopenia and 24 patients with autoimmune disease (SLE and myasthenia gravis) without thrombocytopenia. The frequency of a positive test was greatest in the patients with ITP (61%), SLE (50%) or a history of numerous blood transfusions (60%). The patients with myasthenia gravis also showed a considerable frequency (20%) of platelet antibodies detectable by the PF3 test. The PF3 test is less sensitive than the serotonin release test in detecting autoantibodies, but it is more sensitive than aggregometry in detecting isoantibodies and drug-related antibodies.

Autoantibodies↗
PubMed1997

A system for the supply of platelets suspended in a storage medium including buffy-coat-derived platelet concentrates in combination with "split" apheresis platelets.

Our objective was to create a system for the supply of platelet concentrates (PCs) based on leukocyte-filtered PCs suspended in a storage medium (PAS-II) including: (1) pooled buffy-coat-derived (BC) PCs and (2) "split" apheresis PCs. The same standards were intended for the two preparations with regard to composition and blood cell counts. In preliminary studies, similar in vitro data were found for leukocyte-filtered and non-filtered reference BC PCs. Slightly inferior in vitro results than for BC PCs restricted the shelf-life of apheresis PCs to 5 days compared to 7 days for BC PCs. With the present platelet supply system in use as a routine service for one year, the experience was very satisfactory, meeting the demands for PCs very efficiently and resulting in a low out-dating rate (5%).

Blood Platelets↗
PubMed1986

Ionic and GTP regulation of binding of platelet-activating factor to receptors and platelet-activating factor-induced activation of GTPase in rabbit platelet membranes.

Specific binding of 3H-labeled platelet-activating factor (PAF) to rabbit platelet membranes was found to be regulated by monovalent and divalent cations and GTP. At 0 degrees C, inhibition of [3H]PAF binding by sodium is specific, with an ED50 of 6 mM, while Li+ is 25-fold less effective. On the contrary, K+, Cs+, and Rb+ enhance the binding. The divalent cations, Mg2+, Ca2+, and Mn2+ enhance the specific binding 8-10-fold. From both Scatchard and Klotz analyses, the inhibitory effect of Na+ is apparently due to an increase in the equilibrium dissociation constant (KD) of PAF binding to its receptors. However, the Mg2+-induced enhancement of the PAF specific binding may be attributed to an increased affinity of the receptor and an increased availability of the receptor sites. In the presence of Na+, PAF receptor affinity decreased with increasing temperature with a 100-fold sharp discontinuous decrease in receptor affinity at 24 degrees C. In contrast, the Mg2+-induced increase is independent of temperature suggesting that the Mg2+ regulatory site is different from Na+ regulatory site. [3H]PAF binding is also specifically inhibited by GTP; other nucleotides have little effect. PAF also stimulates hydrolysis of [gamma-32P]GTP with an ED50 of 0.7 nM, whereas 3-O-hexadecyl-2-O-acetyl-sn-glyceryl-1-phosphorylcholine showed no activity even at 10 microM. Moreover, such stimulatory effect of PAF is dependent on Na+ and can be abolished by the PAF-specific receptor antagonist, kadsurenone, but not by an inactive analog, kadsurin B. These results suggest that the PAF receptor may be coupled with the adenylate cyclase system via an inhibitory guanine nucleotide regulatory protein.

Animals↗
PubMed1985

Inactivation of platelet activating factor by rabbit platelets. Lyso-platelet activating factor as a key intermediate with phosphatidylcholine as the source of arachidonic acid in its conversion to a tetraenoic acylated product.

[3H]PAF (platelet activating factor or 1-alkyl-2-acetyl-GPC) is converted to 1-alkyl-2-lyso-GPC and 1-alkyl-2-acyl-GPC by rabbit platelets (GPC is sn-glycero-3-phosphocholine). The deacetylation reaction does not involve the transfer of the acetate of PAF to any other lipid class and added exogenous lyso-PAF readily mixes with the cellular pool of the [3H]lyso-PAF intermediate formed from [3H]PAF. [3H]1-Alkyl-2-acyl-GPC produced during the inactivation of [3H]PAF contained primarily the tetraenoic acyl species (approximately 80% of the 3H in this fraction). The source of the arachidonic acid used for the reacylation of the lyso-PAF intermediate is the diacyl species, phosphatidylcholine.

Acetylation↗
PubMed1987

Platelet-binding immunoglobulins in pregnancy-induced hypertension. I. Platelet-associated IgM on fetal platelets: evidence of a fetal autoimmune reaction?

Pregnancy-induced hypertension (PIH) can be complicated by maternal or fetal thrombocytopenia, or both. In order to investigate possible immunologic causes of these thrombocytopenias, platelet-associated IgG (PAIgG) and IgM (PAIgM) were measured in mothers with PIH and in their infants and compared with those from patients with autoimmune thrombocytopenic purpura (ATP), a known immunodestructive platelet disorder. Many PIH patients (33.3%) and most ATP patients (68.1%) had elevated levels of maternal PAIgG. In both diseases, the amount of PAIgG was directly proportional with the degree of thrombocytopenia (r = 0.446 in PIH and r = 0.668 for ATP). But in neither disease did the degree of maternal thrombocytopenia correlate with the degree of neonatal thrombocytopenia (r = 0.153 for PIH and r = 0.175 for ATP). Umbilical cord samples from PIH patients contained PAIgG (53.3%) and PAIgM (53.8%), whereas the umbilical cord samples from ATP patients had elevated amounts of PAIgG but not PAIgM. PAIgM in the umbilical cord blood could not be accounted for by IgM rheumatoid factors, IgM-containing immune complexes, or non-specific adsorption because of elevated total IgM levels. The umbilical cord blood PAIgM was probably not of maternal origin because it was observed even when the maternal blood contained no PAIgM and maternal IgM is not normally transported transplacentally. Therefore, the PAIgM appears to be of fetal origin. These results suggest that both maternal and fetal immunologic mechanisms may be involved in PIH-induced thrombocytopenia; if so, this is one of the first reported examples of a possible fetal autoimmune response.

Autoantibodies↗
PubMed1994

Sensitivity of the platelet immunofluorescence test (PIFT) and the MAIPA assay for the detection of platelet-reactive alloantibodies: a report on two U.K. National Platelet Workshop exercises.

This report presents the results of two National Workshop exercises which were designed to evaluate interlaboratory and interassay variation in sensitivity of techniques used for the detection of platelet-reactive alloantibodies. Most workshop participants used the platelet immunofluorescence test. Sensitivity of this assay was improved when fluorescence was measured by flow cytometer rather than by microscope. The MAIPA assay was found to be highly sensitive but required considerable technical expertise, and the choice of antiglycoprotein IIb/IIIa (CD61/41) monoclonal antibody was found to have a significant effect on its ability to detect anti-HPA-1a.

Antibodies, Monoclonal↗
PubMed2005

Characterization of the alloreactive helper T-cell response to the platelet membrane glycoprotein IIIa (integrin-beta3) in human platelet antigen-1a alloimmunized human platelet antigen-1b1b women.

BACKGROUND: The aims were to characterize the helper T-cell response to platelet (PLT) glycoprotein (GP) IIIa, which stimulates the alloimmune antibody response to human PLT antigen (HPA)-1a, to identify immunodominant epitopes and to examine the HLA Class II associations. STUDY DESIGN AND METHODS: Peripheral blood mononuclear cells (PBMNCs) were obtained from 21 HPA-1b1b women who had an HPA-1a-mismatched pregnancy, 14 of whom developed anti-HPA-1a, and 11 control donors. PBMNCs were stimulated with two panels of 15-mer peptides corresponding to the HPA-1a/1b polymorphic region, with either Leu33 (-1a) or Pro33 (-1b) at each possible position, and the proliferative responses were measured. HLA Class II and HPA genotyping was by conventional polymerase chain reaction-sequence-specific priming. RESULTS: Peptides with Leu33 at, or near, the C-terminus contained an immunodominant epitope, stimulating proliferation by helper T cells from all nine women who had anti-HPA-1a at the time of testing; peptide L1 (Val19-Leu33) stimulated a response in 50 percent of these women. Their T cells did not respond to the corresponding HPA-1b Pro33 peptides, and responses to either peptide panel were rare in unimmunized women and controls. HLA-DRB3*01+ was significantly overrepresented (p = 0.014) in alloimmunized women whose T cells responded to the major HPA-1a Leu33-containing epitope. Conversely, HLA-DRB1*15 was negatively associated (p = 0.014) with this response. CONCLUSIONS: The HPA-1a polymorphic region of GPIIIa contains both the linear T-cell and the conformational B-cell epitopes. The immunodominant T-cell epitope is constrained by HLA-DRB3*01+, and if presented by a tolerogenic route, a peptide containing this epitope may form the basis for the prevention or reversal of the alloimmune response to HPA-1a.

Antigens, Human Platelet↗
PubMed2005

Platelet glycolytic flux increases stimulated by ultraviolet-induced stress is not the direct cause of platelet morphology and activation changes: possible implications for the role of glucose in platelet storage.

BACKGROUND: Stress-enhanced platelet (PLT) storage lesions include increased glycolysis, discoid-to-sphere morphology change, and spontaneous PLT activation. It is not clear if reduction in glycolysis can alleviate storage lesion development. STUDY DESIGN AND METHODS: Apheresis PLT concentrates were exposed to 17.2 J/mL UV light and 50 microM riboflavin, followed by storage with various concentrations of 2-deoxyglucose (2-DOG) for 5 days. The control had no UV or 2-DOG exposure. RESULTS: Lactate production and glucose consumption were increased significantly to 0.1371 +/- 0.0281 and 0.0724 +/- 0.0151 mmol per 10(12) cells per hour for UV-treated PLTs, respectively, when compared to control samples. UV treatment induced a decline in pH to 6.55 +/- 0.26 for treated PLTs on Day 5, hypotonic shock response (HSR) 33 +/- 25 percent, extent of shape change (ESC) to 3.8 +/- 3.6 percent, swirl 1.0 +/- 1.0 and increased P-selectin expression 85.2 +/- 9.4 percent. Addition of 2-DOG up to 20 mmol per L significantly reduced lactate production to 0.0515 +/- 0.0045 mmol per 10(12) cells per hour (p < 0.05) and glucose consumption to 0.0293 +/- 0.0060 mmol per 10(12) cells per hour and increased pH to 7.35 +/- 0.09 in a dose-dependent manner. 2-DOG, however, had no effects on HSR, ESC, swirl, and P-selectin expression. Furthermore, an exaggeration of UV-stressed PLT aggregation by addition of 2-DOG was also observed. CONCLUSIONS: Increased glycolytic flux is not a direct cause for PLT morphology change and spontaneous activation during storage lesion development. Reduction of glucose utilization may increase PLT loss during storage.

Blood Glucose↗
PubMed1989

Effect of aspirin on platelet-von Willebrand factor surface expression on thrombin and ADP-stimulated platelets.

Platelets contain a pool of endogenous platelet-von Willebrand factor (vWF) that becomes expressed on the platelet surface when platelets are stimulated by a variety of agonists. Maximal platelet-vWF expression occurs in concert with platelet alpha-granule secretion. Aspirin (ASA) is known to impair platelet activation and alpha-granule secretion by irreversible inhibition of platelet cyclo-oxygenase. We studied native and ASA-treated platelets for their ability to mobilize and to express platelet-vWF in response to adenosine diphosphate (ADP) or thrombin. We found that each agonist was effective in promoting increased platelet-vWF surface expression on native and ASA-treated platelets. ASA-treated platelets responded identically to native platelets to low (0.01 U/mL) and high (1.0 U/mL) concentrations of thrombin, while the ADP-induced increase in ASA-treated platelets was only 50% to 60% of that for control platelets. Measurement of secreted platelet-vWF and beta-thromboglobulin indicated that the increase seen with ADP was largely independent of alpha-granule secretion. Using monoclonal antibodies (MoAbs) against the platelet glycoproteins (GP) IIb/IIIa and Ib (MoAbs 10E5 and 6D1, respectively), we demonstrated that the ADP-induced increase in platelet-vWF expression on control platelets primarily involved the binding of secreted platelet-vWF to the platelet GPIIb/IIIa. In contrast, the increase in platelet-vWF that occurred following ADP stimulation of ASA-treated platelets was largely insensitive to GPIIb/IIIa blockade. No effect of GPIb blockade in platelet-vWf expression was noted for either control or ASA-treated platelets. When platelet shape change was prevented by the addition of cytochalasin D, ADP-induced platelet-vWf surface expression on ASA-treated platelets was reduced by more than 80%. Our data indicate that platelets in which the cyclooxygenase pathway is blocked by the action of aspirin can increase surface expression of platelet-vWf as a consequence of platelet shape change. We speculate that this process exposes platelet-vWf bound to GPIIb/IIIa, or possibly GPIb, within the surface connected canalicular system.

Adenosine Diphosphate↗
PubMed2001

Standardisation of platelet counting accuracy in blood banks by reference to an automated immunoplatelet procedure: comparative evaluation of Cell-Dyn CD4000 impedance and optical platelet counts.

Prophylactic and therapeutic platelet transfusions are increasingly used for patients with conditions associated with thrombocytopenia in order to prevent the development of potentially life threatening bleeding. These clinical strategies have led to a significant expansion in platelet unit manufacture, and this now represents a major resource and cost commitment for blood banks. As part of the manufacturing process, blood banks are required to implement control procedures, and the determination of platelet counts in particular is necessary to confirm that the quality of platelet unit production meets the standards defined by national or international guidelines. Apart from linearity analysis and comparisons of platelet counts given by different instruments, there has been no systematic standardisation of platelet counting methods in blood bank practice because to date there has been no suitable reference method for counting platelets in citrate anticoagulants. The recent introduction of an automated immunoplatelet procedure on the Cell-Dyn CD4000 provides a means of determining a true platelet count that is unaffected by changes induced either by storage or anticoagulant. The CD4000 in its routine configuration also provides simultaneous impedance and optical platelet counts and this study was therefore undertaken in order to compare all three different platelet counting methods in parallel with a representative series of platelet units. Platelet counts determined after sub-sampling of platelet units into EDTA vs plain non-anticoagulated tubes revealed no differences in impedance or immunoplatelet counts but generally lower optical counts when aliquoted into tubes that did not contain EDTA. This study therefore routinely used EDTA for platelet unit sub-samples. Comparative results of platelet counts for buffy coat platelet units (n = 36) aliquoted into EDTA indicated that the impedance count was higher than the reference immunoplatelet count by a mean factor of 1.25 while the optical count was lower by a mean factor of 0.87. The degree of impedance count overestimation was particularly consistent while the optical count underestimation was more variable. Linearity studies of 10 fresh platelet units showed no deviation in the range 0-2305 x 10(9) l(-1) for impedance and 0 to 1420 x 10(9) l(-1) for the optical counts, and the relative numerical relationships between impedance and optical counts were conserved throughout the range of dilutions tested. In the CD4000 optical analysis, blood samples anticoagulated with EDTA showed a distinctive elliptical population distribution that fell within the system thresholds. In contrast, the optical pattern observed for platelet units (in CPD) and ACD-anticoagulated venous blood showed a wider 90 degrees scatter with a population of platelet events above the upper parallel discriminator. As these were excluded from the optical count (but were still identified as platelets by the immunoplatelet method) it meant that the optical counts of samples in citrate-based anticoagulants were systematically lower than immunoplatelet counts. Platelet units (n = 15) analysed daily over a seven day period of storage revealed that the greatest decline in platelet counts was with the optical measurement while the most stable value was obtained by impedance analysis. The results of the immunoplatelet analysis further suggested a progressive increase in small platelets with increasing storage time. The use in this study of a standardised immunoplatelet reference method to examine the question of analyser suitability for determining platelet counts/yields of platelet units thus provided a number of important findings. An impedance platelet counting method is utilised by the great majority of haematology instruments in current use, and in common with the CD4000 analyser, a correction factor is employed to take account of RBC/platelet coincidence. This study found that when analysed samples such as platelet units were RBC-free, that an inappropriate correction factor was applied. Consequently, the CD4000 impedance platelet count will provide reliable platelet counts, irrespective of the day of platelet unit storage, when a factor of 1.25 is applied to the system-reported result. By comparison, optical methods are more likely to be affected by subtle morphological changes that may result from anticoagulants or progressive storage time. The method limitations documented by this study may well affect many other analysers and mean that the implementation of process control statistics related to platelet counts may be less reliable than previously assumed. It is suggested that standardisation could be much better achieved if there was some form of system cross-calibration that was referenced to an independent method such as an immunoplatelet assay. It is proposed that studies of this type should be extended to a wide assessment of platelet count accuracy of blood bank instruments in order to standardise data within national organisations. If consistent inter-instrument correction factors such as those documented here can be identified, it would considerably increase the relevance of determining platelet counts in production control processes.

Adult↗
PubMed1978

Heterogeneity of rabbit platelets. VI. Further resolution of changes in platelet density, volume, and radioactivity following cohort labelling with 75Se-selenomethione.

Rabbits were injected intravenously with a cohort platelet label, 75Se-selenomethionine Platelet-rich plasma was separated into five different platelet density fractions on each of seven days by repetitively centrifuging the same sample of platelet-rich plasma at increasing gravitational force. The heaviest platelet sediment fraction was enriched with smaller platelets. Incorporation of isotope into the heaviest platelet fraction was considerably greater than incorporation into the highest platelet fraction, i.e. heavy--light specific activity ratio was 9.63, on day 1. The ratio of the heaviest platelet fraction specific activity to the specific activities of the platelets sedimented at higher g forces (lighter-smaller platelets), generally decreased on each day for 4--6 d by a factor of 2.8--7.0-fold. Whereas incorporation of isotope into the heaviest platelet fraction declined on days 3--4, incorporation into the lightest three fractions remained the same. Cohort platelet survival curves revealed a significant lag period of 1--2 d for the incorporation of isotope into the lightest three platelet fractions compared to the heaviest two platelet fractions. The mean platelet survival of the lightest two fractions was significantly shorter than the mean platelet survival of the heaviest three fractions. SDS-polyacrylamide gel electrophoresis of the platelet cell sap of the five platelet fractions generally revealed 10 prominent protein bands for the heaviest platelet fractions. The lightest platelet fraction, which comprised 16% of the harvested platelet population, had six absent to markedly diminished platelet proteins of modecular weights between 142 000 and 46 000 daltons. The data are compatible with two models: (1) Heavy-large platelets are, on average, young platelets which become lighter-smaller platelets while losing platelet membrane and cell sap components with time. (2) Heavy-large platelets and light-small platelets are produced independently by specific megakarocytes. The heavy-large platelets incorporate more isotope than lighter-smaller platelets (possibly because of their megakarocyte precursor). However, they are released earlier into the circulation than lighter-smaller platelets and are therefore younger platelets.

Animals↗