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PubMed1989

Synthesis of platelet-activating factor by human blood platelets and leucocytes. Evidence against selective utilization of cellular ether-linked phospholipids.

Synthesis of platelet activating factor (PAF) in blood platelet suspensions may be due to leucocyte contamination. We therefore investigated PAF synthesis in human blood platelet suspensions and granulocyte- (PMN)-enriched leucocyte suspensions upon stimulation by thrombin and Ca2+-ionophore A23187, both in the presence and absence of the presumed PAF catabolism inhibitor phenylmethylsulfonyl fluoride (PMSF). PAF synthesis was measured by aggregation of washed rabbit platelets and by [3H]acetate incorporation. In contrast to A23187, thrombin was unable to stimulate PAF synthesis by leucocytes. As thrombin did induce PAF synthesis by platelet suspensions, this was evidently not due to leucocyte contamination. A23187 also induced PAF synthesis by platelets, but this was dependent upon the platelet isolation method and possibly associated activation. The ratio of [3H]acetate incorporation into 1-alkyl- versus 1-acyl-2-acetylglycerophosphocholine upon stimulation of non-PMSF-treated leucocytes and platelets amounted to 12.8 and 1.2, respectively. These values are at least 10-fold higher than the ratio of 1-alkyl versus 1-acyl species in the cellular phosphatidylcholine precursor for PAF. By PMSF pretreatment, the distribution of incorporated [3H]acetate between 1-ether- and 1-ester-linked species became similar to that in the precursor phosphatidylcholines of the respective cell type, due to increased recovery of [3H]acetate in the acyl compounds. Both leucocyte and platelet homogenates rapidly degraded acylacetylglycerophosphocholine to (acetyl)glycerophosphocholine, and this deacylation was inhibited by PMSF pretreatment of the cells. We conclude that upon cell stimulation a phospholipase A2 converts both alkylacylglycerophosphocholine and diacylglycerophosphocholine to the 2-lysoanalogs in a ratio similar to the occurrence of the parent compounds. The acetyltransferase subsequently acetylates both compounds to acylacetylglycerophosphocholine and alkylacetylglycerophosphocholine (PAF), respectively. Deacylation of the 1-ester-linked species, either before or after acetylation, gives the impression of selective utilization of 1-ether-linked species for PAF production. It is only after inhibition of the deacylation by pretreatment of the cells with PMSF that a mainly nondiscriminative use of 1-ether- and 1-ester-linked species by both phospholipase A2 and acetyltransferase becomes evident.

Acetates↗
PubMed1991

Platelet-mediated cytotoxicity and its enhancement by platelet activating factor.

Platelet cytotoxicity was assessed in 70 cancer patients with various tumor localizations and in 30 normal donors. The data presented reveal that the ACL cell line displays the highest sensitivity to platelet cytotoxicity. Using the ACL cells, we discovered that platelets from oncological patients and normal donors display comparable cytotoxicity. The level of platelet lytic activity is irrelevant to tumor localisation; however, it appears to be dependent on the stage of tumor growth. Incubation of platelets, both from donors and patients, with PAF (concentration range 10 pM to 10 nM) results in a significant rise of the killing activity of platelets. PAF induces greater cytotoxicity enhancement for platelets with lower initial activity, this pattern appearing to be the specific feature of the PAF mediated effect. Hence, platelets can be considered as effector cells relevant to antitumor immunity; PAF-mediated enhancement of platelet cytotoxicity can appear to be useful in the search for new immunotherapeutic drugs.

Adenocarcinoma↗
PubMed2004

Caspase inhibition decreases both platelet phosphatidylserine exposure and aggregation: caspase inhibition of platelets.

Apoptosis of nucleated cells is regulated by caspases, a group of cysteine proteases, and is characterized by phosphatidylserine expression on the outer leaflet of the plasma membrane. Reports indicate that platelets contain caspases. However, the role of caspases in platelet function is not well understood. When platelets become activated, they express phosphatidylserine (PS) on the outer leaflet of the plasma membrane. In addition, platelets aggregate when activated. The aims of this study were to determine if caspase inhibition (using the pan-caspase inhibitor zVAD-fmk): (1) decreased PS expression and (2) decreased platelet aggregation following activation. Flow cytometry was used to determine PS expression and a platelet aggregometer was used to assess aggregation. We found that platelets treated with zVAD-fmk significantly decreased both A23187-induced PS exposure (total fluorescence index, TFI: A23187=791.42+/-174; zVAD+A23187=92.97+/-57, p<==0.05) and ADP-induced PS exposure (TFI: ADP=669.24+/-145, zVAD+ADP=174.6+/-151, p<==0.05). Further, treatment with zVAD-fmk significantly decreased ADP-induced platelet aggregation (%: untreated=80+/-1.5, zVAD treated=69+/-3.0, p<==0.05). These results indicate that caspases play a role in platelet activation, suggesting a unique physiologic role for these proteases.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Effects of a P2Y(12) receptor antagonist on the response of equine platelets to ADP. Comparison with human platelets.

Horses show susceptibility to platelet-related disorders. Equine platelets differ from human platelets in some of their responses, so information available about human platelets must be validated in the horse. Aggregation of platelets by ADP involves both P2Y(1) and P2Y(12) receptors on the platelet surface. We have compared the effect of the P2Y(12) antagonist, AR-C67085, on equine and human platelets in vitro using turbidimetric aggregometry to measure the rate and final extent of aggregation. Aggregation profiles, concentration-response curves and pA(2) values show that the rate of aggregation of equine platelets is much more susceptible to inhibition by AR-C67085 than that of human platelets. This species difference may reflect differences in the relative numbers of P2Y(1) and P2Y(12) receptors, or in intracellular signalling pathways, but will need to be considered by equine clinicians before using P2Y(12) antagonists in the treatment of thrombotic conditions.

Adenosine Diphosphate↗
PubMed1998

Biotinylated platelets have an impaired response to agonists as evidenced by in vitro platelet aggregation tests.

Biotinylation of platelets, using a water soluble biotin analogue which reacts with primary amines, has been proposed to be a reliable technique for study of in vivo survival of platelets and their subpopulations. The information about the influence of this technique on platelet function has been limited. In the present work we studied the effect of in vitro biotinylation on platelet function and activation. Washed human platelets, at a concentration of 1 x 10(9)/L, were biotinylated with five different concentrations of sulfo-NHS-biotin or NHS-LC-biotin, ranging from 0 to 5 mM. The degree of platelet activation during and after biotinylation was monitored by measuring the externalization of P-selectin, and the platelet function was evaluated by aggregometry. It was observed that biotinylated platelets, in a dose dependent manner, displayed an impaired aggregation response. A slight increase in platelet membrane P-selectin occurred during the labelling procedure.

Adenosine Diphosphate↗
PubMedDate not supplied

Characteristics of rat platelets and relative contributions of platelets and blood coagulation to haemostasis.

In order to understand some of the haemostatic mechanisms in rats for the interpretation of toxicological data, basic haemostatic parameters with a special emphasis on platelet functions were first measured in vitro. The results of reactions of rat platelets to many aggregating agents suggest that only ADP may be a consistently significant aggregator. The search for physiologic aggregators revealed ADP to be available from erythrocytes. Adhesion reaction also required ADP. Collagen was not considered to be essential for either reaction. Aggregation and adhesion were probably both reversible in flowing blood, while irreversible thrombi were formed in blood at rest ex vivo. Blood coagulation parameters determined revealed that the intrinsic pathway may be more important than the extrinsic one. The rate of intrinsic coagulation reaction was rapid, and plasma coagulation appeared to be of primary importance while the influence of platelet aggregation was minor. A simple model of rat haemostatic mechanism is proposed based on these results. Additionally, to define the relative contribution of platelets versus other cellular and plasma coagulation in vivo, rats were administered antiplatelet drugs (ticlopidine, suprofen and clopidogrel) and an anticoagulant (warfarin) intraperitoneally. Bleeding times (BTs) were significantly increased in all treated groups. ADP-induced platelet aggregations were significantly depressed by the administration of the three antiplatelet drugs, while kaolon-activated partial thromboplastin time and prothrombin time were greatly increased in the warfarin-treated rats. The increase in BT may be due to the inhibition of platelet activity or blood coagulation defect in rats given antiplatelet drugs or warfarin, respectively. These results suggest that platelets play a key role in haemostasis in the rat. Two possible explanations of the disparity between in vitro and in vivo results may be that functional tests used here are not adequate to cover the properties of rat platelets or that mechanisms leading to the formation of platelet thrombi in rats are ADP-dependent adhesion and ADP-induced aggregation.

Animals↗
PubMed2002

Comparison of reticulated platelet count and mean platelet volume determination in the evaluation of bone marrow recovery after aplastic chemotherapy.

Reticulated platelet count provides an estimate of thrombopoiesis in the same way as reticulocyte count is a measure of erythropoiesis. We applied thiazole orange (TO) staining, followed by fluorescence-activated flow-cytometric analysis, to platelets in whole-blood samples from normal subjects and 18 aplastic patients after chemotherapy for haematologic malignancies. The percentage of TO-positive platelets in 30 control subjects was 5.7 +/- 2.4% (mean +/- 1 SD), determining the threshold of reticulated platelet positivity as up to 10.5% (mean + 2 SD). In the 18 patients studied, the mean percentage of TO-positive platelets was 4.3 +/- 1.89% during aplasia and 23.3 +/- 9.43% during bone marrow recovery, respectively (P < 0.05). All patients had a percentage of TO-positive platelets of up to 10.5%. In comparison, mean platelet volume during bone marrow recovery increased in 12 cases of the 18 patients studied. We conclude that flow cytometric analysis of reticulated platelets is a sensitive and specific test for evaluating thrombopoiesis recovery during aplastic chemotherapy, and platelet transfusion should be reconsidered in these patients.

Adult↗
PubMed2002

Short-term diets enriched in stearic or palmitic acids do not alter plasma lipids, platelet aggregation or platelet activation status.

OBJECTIVE: To determine whether healthy males who consumed increased amounts of dietary stearic acid compared with increased dietary palmitic acid through the consumption of commercially available foods, exhibited any changes in plasma lipids, platelet aggregation or platelet activation status. DESIGN: A randomised cross-over dietary intervention. SUBJECTS AND INTERVENTIONS: Nine free-living healthy males consumed two experimental diets (stearic acid enriched, diet S, and palmitic acid enriched, diet P) for 3 weeks in a randomised cross-over design separated by a 3 week washout phase. The diets consisted of approximately 30% of energy as fat (30% of which was derived from the treatment diets) providing approximately 13 g/day as stearic acid and 17 g/day as palmitic acid on diet S and approximately 7 g/day as stearic acid and 22 g/day as palmitic acid on diet P. The dietary ratio of stearic to palmitic acids was 0.76 on diet S compared with 0.31 on diet P. Blood samples were collected on days 0 and 21 of each dietary period. RESULTS: LDL cholesterol levels and platelet aggregation response to the agonist ADP were significantly decreased (P<0.025) in subjects on diet S compared with day 0. Apart from that, there were no significant changes in plasma lipids, platelet aggregation, mean platelet volume and platelet reactivity between diets. There were no significant changes in stearic or palmitic acid levels in plasma phospholipid or triacylglycerol. There was a significant difference in palmitic acid levels in platelet phospholipids between the two diets. CONCLUSIONS: Use of commonly available foods led to a 27% increase in stearic acid (diet S) and a 19% increase in palmitic acid (diet P), on diets S and P respectively, and no significant differences between the two diets in plasma lipoprotein concentrations, platelet aggregation or platelet activation status.

Adult↗
PubMed1971

The influence on platelet aggregation of drugs that affect the accumulation of adenosine 3':5'-cyclic monophosphate in platelets.

1. The involvement of intracellular 3':5'-cyclic AMP in the inhibition of platelet aggregation by prostaglandin E(1), isoprenaline and adenosine has been examined by a radiochemical technique. Platelet-rich plasma was incubated with radioactive adenine to incorporate (14)C radioactivity into platelet nucleotides. Pairs of identically treated samples were taken, one for the photometric measurement of platelet aggregation induced by ADP, the other for estimation of the radioactivity of 3':5'-cyclic AMP. 2. Theophylline, papaverine, dipyridamole and 2,6-bis-(diethanolamino)-4-piperidinopyrimido[5,4d]pyrimidine (compound RA233) were found to inhibit 3':5'-cyclic AMP phosphodiesterase from platelets. At concentrations of 3':5'-cyclic AMP greater than 50mum the most active inhibitor was dipyridamole; at 3':5'-cyclic AMP concentrations less than 19mum, papaverine and compound RA233 were more active than dipyridamole. 3. In the presence of compound RA233 (50mum), the effectiveness of prostaglandin E(1) as an inhibitor of platelet aggregation was increased tenfold. Compound RA233 also increased the stimulation by prostaglandin E(1) of the incorporation of radioactivity into 3':5'-cyclic AMP. 4. Compound RA233 (50mum) increased the effectiveness of both adenosine and 2-chloroadenosine as inhibitors of aggregation by 70-100-fold, and in the presence of compound RA233 both adenosine and 2-chloroadenosine stimulated the incorporation of radioactivity into 3':5'-cyclic AMP; the extent of the stimulation was proportional to the logarithm of the nucleoside concentration. 5. Compound RA233 (100-500mum) inhibited platelet aggregation by itself and caused small increases in the radioactivity of 3':5'-cyclic AMP. Partial positive correlations were found between the radioactivity of 3':5'-cyclic AMP in platelets measured at the time of addition of the aggregating agent (ADP) and the extent to which the aggregation was inhibited. 6. The results are interpreted as indicating that adenosine, 2-chloroadenosine, isoprenaline, prostaglandin E(1) and drugs that inhibit platelet 3':5'-cyclic AMP phosphodiesterase all inhibit aggregation by a common mechanism involving intracellular 3':5'-cyclic AMP.

Adenine Nucleotides↗
PubMed1988

Inhibition of agonist-induced platelet aggregation, Ca2+ mobilization and granule secretion by guanosine 5'-[beta-thio]diphosphate and GDP in intact platelets. Evidence for an inhibitory mechanism unrelated to the inhibition of G-protein-GTP interaction.

The effect of guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), reported to be an antagonist of GTP at the G-protein-binding site, on human platelet activation was examined. GDP[beta S] (0.3-3 mM) had significant inhibitory effects on platelet aggregation and 5-hydroxytryptamine (5HT) secretion induced by thrombin, collagen, the thromboxane mimetic U46619 and 1,2-dioctanoylglycerol (diC8) in intact platelets, as well as in saponin-permeabilized platelets. Similar inhibitory effects in intact platelets were also observed with ATP (over similar concentration ranges) and GDP and GTP (at 2- and 10-fold higher concentrations respectively). All four nucleotides also inhibited ADP-induced platelet aggregation in indomethacin-treated platelets under conditions where no 5HT secretion occurred. Inhibition of thrombin-induced aggregation and secretion by GDP[beta S] and ATP in intact platelets was accompanied by a reduction in the thrombin-induced rise in intracellular Ca2+ levels and 45 kDa-protein phosphorylation. The results suggest that at least some of the effects of GDP[beta S] may be unrelated to inhibition of G-protein-GTP interaction, but, instead, may be mediated via an extracellular site, common to all the nucleotides tested and perhaps via inhibition of the effects of endogenous/released ADP. The usefulness of GDP[beta S] as a tool in studying G-protein-GTP interactions in platelets is thus questionable.

Adenosine Triphosphate↗
PubMed1999

Megakaryocyte precursors, megakaryocytes and platelets express the HIV co-receptor CXCR4 on their surface: determination of response to stromal-derived factor-1 by megakaryocytes and platelets.

Thrombocytopenia is a late complication of human immunodeficiency virus (HIV) infection. The chemokine receptor CXCR4 has been shown to be a co-receptor for lymphocyte-tropic HIV-1 strains. CXCR4 is also a natural receptor for the chemokine SDF-1. We have previously shown that CXCR1 and CXCR2 are present on megakaryocytes and platelets. Although interleukin-8 (IL-8) and other chemokines that bind to these two receptors do not activate platelets, they are able to inhibit megakaryocytopoiesis, presumably through these receptors. We therefore examined whether CXCR4 is present on developing and mature megakaryocytes and on platelets. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated the presence of CXCR4 message. Immature and mature alphaIIbbeta3+ megakaryocytes, and platelets were also positive for CXCR4 by flow cytometric studies using a CXCR4-specific antibody. We then tested whether SDF-1 can affect the biology of these cells. CD34+ cells and immature alphaIIbbeta3+ cells responded to SDF-1 as indicated by Ca2+ mobilization and chemotaxis. However, mature megakaryocytes failed to demonstrate either of these responses, in spite of their continued ability to bind 125I-SDF-1. Further, SDF-1 failed to inhibit megakaryocyte colony growth. Platelets bound 125I-SDF-1 with a K(D) similar to the affinity seen for CXCR4 on other cells, yet SDF-1 did not aggregate washed platelets nor augment aggregation by low-dose ADP or thrombin. SDF-1 also failed to stimulate Ca2+ mobilization, granular release or expression of P-selectin in platelets. Accordingly, although our studies demonstrate that CD34+ precursors, megakaryocytes and platelets all express CXCR4 and bind SDF-1, biological effects were only demonstrable of SDF-1 on CD34+ precursors. The potential biological implications of CXCR4 expression on maturing megakaryocytes and platelets in normal individuals and following HIV infection are discussed.

Acquired Immunodeficiency Syndrome↗
PubMed1999

The cytoskeletal linker protein moesin: decreased levels in Wiskott-Aldrich syndrome platelets and identification of a cleavage pathway in normal platelets.

The Wiskott-Aldrich syndrome (WAS) is a severe disease of platelets (small size, thrombocytopenia) and lymphocytes (immunodeficiency) arising from mutations of the X-chromosome gene WASP. Because of the prominent role of cytoskeletal abnormalities, particularly the paucity of surface microvilli, in the cellular pathology of this disease, blood cells from WAS patients were examined for moesin, a cytoskeletal linker protein that stabilizes cell surface microvilli, filopodia and lamellipodia. Comparison of patient and normal lymphocytes by immunofluorescence microscopy and immunoblotting showed normal levels and distribution of moesin in lymphocytes of WAS patients. In contrast, platelets from WAS patients stained only dimly for moesin relative to normal platelets. Quantitation by immunoblot revealed significantly decreased moesin levels in WAS patient platelets relative to normal platelets (63.5 +/- 4.9% of normal levels, n = 8, P < 0.0001). A novel reaction of normal platelets was discovered that may play a role in the depletion of moesin in patient platelets, namely the cleavage of moesin as a late event in platelet activation in response to certain platelet agonists.

Blood Platelets↗
PubMed2003

Role of Galphaq and phospholipase C-beta2 in human platelets activation by thrombin receptors PAR1 and PAR4: studies in human platelets deficient in Galphaq and phospholipase C-beta2.

Thrombin responses in human platelets are mediated by the protease-activated receptors (PAR), PAR1 and PAR4. The signalling pathways mediating PAR activation have not been fully delineated for human platelets. We assessed cytoplasmic Ca2+ mobilization in response to activation with thrombin and PAR1 (SFLLRN) and PAR4 (GYPGKF) peptides in two patients whose platelets were deficient in two major signalling proteins, Galphaq or phospholipase (PLC)-beta2. In normal platelets, thrombin induced a biphasic Ca2+ response with a rapid rise to a peak followed by a sustained elevation in Ca2+. The peak Ca2+ rise was impaired in both patients at lower thrombin concentrations. At higher concentrations, it was decreased in PLC-beta2-deficient platelets; the sustained Ca2+ elevation observed in normal and Galphaq-deficient platelets was reduced in PLC-beta2-deficient platelets. The response to SFLLRN was decreased in both patients at lower concentrations. The peak Ca2+ in response to GYPGKF was reduced in both patients; the sustained Ca2+ increase was markedly decreased in PLC-beta2-deficient platelets. These studies provide evidence that, in human platelets, both Galphaq and PLC-beta2 play a major role in responses to PAR1 and PAR4 activation, and that PLC-beta2 is required for the sustained Ca2+ rise upon thrombin activation.

Blood Platelets↗
PubMed2002

Evaluation of platelet parameters on the ADVIA 120 as the quality indicator for stored platelets.

In order to investigate the possible use of platelet parameters on the ADVIA 120 hematologic analyzer as the routine quality control indicator for preparation and storage of platelets, platelet parameters, pH and CD62P expression were determined in stored platelet concentrates. Platelet component distribution width (PCDW) was decreased progressively on days 1 and 3 of storage for 5 days when compared with 0 day. PCDW correlated with CD62P expression on unstimulated platelets and the difference in CD62P expression following agonistic stimulation (a measure of functional reserve). Mean platelet component (MPC) was decreased on day 1 of storage. It did not however, show a progressive decrease over storage time and did not correlate with CD62P, although MPC has been known to be a useful screening test for platelet activation. Therefore, PCDW is considered to be a simple, convenient and cost-effective quality indicator for determining the viability and storage lesion of platelets for transfusion.

Blood Preservation↗
PubMedDate not supplied

Extended storage of platelets in a new plastic container. II. In vivo response to infusion of platelets stored for 5 days.

A new blood container material (PL 1240 plastic) made of polyvinyl chloride containing a tri(2-ethylhexyl) trimellitate plasticizer was evaluated in three laboratories. When platelet concentrates (50-60 ml) were stored on a variety of agitators for 7 days at 22 +/- 2 degrees C, poststorage pH (mean +/- SD) ranged from 7.29 +/- 0.05 (6 rpm elliptical rotator) to 6.87 +/- 0.8 (70 cycles per minute flatbed agitator). The platelet counts ranged from 1.51 +/- 0.12 to 0.95 +/- 0.36 X 10(6) per microliter. Morphology scores and hypotonic shock response values of platelets stored 7 days in PL 1240 plastic containers were better than those noted following 3-day storage of control platelets in PL 146 plastic containers. The percent discharge of lactic dehydrogenase from platelets stored 7 days in PL 1240 plastic containers for 3 days (p less than 0.05). Mean platelet recoveries of 44 +/- 15 percent (n = 11; 111Indium) and 39 +/- 8 percent (n = 29; 51Chromium) were seen when autologous platelets were infused following 5-day storage in PL 1240 plastic bags. Platelet half-lives of 3.6 +/- 0.4 (n = 9) 4.1 +/- 0.4 (n = 20) days were reported in the two laboratories which used 51Cr labeling, while survival values of 7.0 +/- 1.0, 2.8 +/- 0.8, and 5.4 +/- 1.9 days were seen when data from the 111Indium studies (n = 11) were analyzed using linear, exponential, and multiple hit programs, respectively. Platelets stored for 5 days also were administered to 13 thrombocytopenic oncology patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗
PubMed1989

Monitoring of platelet morphology during storage of platelet concentrates.

During storage, human platelet concentrates progressively lose the capacity to survive and function in vivo after transfusion. A shape transformation from disc to sphere is the most reliable in vitro determinant for the loss of the in vivo survival of platelets. To find an objective measurement for platelet morphology, we studied the effect of anticoagulant, temperature, and storage on the apparent median platelet volume (MPV) as determined by a particle counter and on changes in platelet shape as measured and by light microscopy. Changes in MPV, light transmission, and morphology score by light microscopy were observed within 1 minute after collection of blood in CPDA. As compared to blood immediately fixed on withdrawal, in CPDA blood, the MPV increased from 4.1 to 5.7 fl, and light transmission difference decreased from 22 to 7 percent. A partial restoration of these determinants was found when the whole blood was incubated for 30 minutes at 37 degrees C, before preparation of platelet-rich plasma. In the first 5 days of platelet storage, the MPV increased from 4.6 to 5.0 fl; thereafter, it started to decrease. An increase in fragmented platelets after 5 days was observed on light microscopy. The light transmission difference showed a slow disc-to-sphere transformation during storage. This transformation accelerated from Day 5 to Day 7; after 11 days, only spheres were detected. After 7 days the swirling pattern scores were still in accordance with the presence of discs, whereas the other structure-associated determinants showed already spheric and even fragmented platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1989

Stability of platelet and plasma HLA concentrations in healthy adults or random-donor platelet concentrates.

The quantitative differences between the platelet HLA antigens of different individuals are potentially significant in determining the survival of transfused platelets in allosensitized patients. To learn whether platelet HLA concentration is sufficiently stable to be documented for each donor and used for donor selection, platelet and plasma HLA concentrations in five healthy adults were measured monthly for 5 consecutive months. HLA concentrations were measured by using a competitive enzyme-linked immunoassay. The results showed that both platelet and plasma HLA concentrations varied within +/- 20 percent of the mean values in each individual. Changes in plasma and platelet HLA concentrations were also studied in 5 units of random-donor platelets during in vitro storage. HLA concentrations were determined every other day over a 5-day period. There was no significant quantitative change in the platelet and plasma HLA concentrations during the 5 days of in vitro storage. Thus, platelet HLA concentrations in healthy individuals are stable and may be quantitatively documented for donor selection.

Adult↗
PubMed1998

Molecular basis for ADP-induced platelet activation. II. The P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets.

ADP is an important platelet agonist causing shape change from smooth discoid shape to spiculated spheres and platelet aggregation. However, the molecular mechanisms involved in ADP-induced platelet activation have not been elucidated. We demonstrated earlier the existence of two distinct ADP receptors on platelets, one coupled to phospholipase C, P2TPLC, and the other to inhibition of adenylyl cyclase, P2TAC (Daniel, J. L., Dangelmaier, C., Jin, J., Ashby, B., Smith, J. B., and Kunapuli, S. P. (1998) J. Biol. Chem. 273, 2024-2029), in addition to the previously described P2X1 receptor. Here we report the cloning of a cDNA clone encoding the P2Y1 receptor from a human platelet cDNA library by homology screening with radiolabeled P2Y1-P2Y6 receptor cDNAs. ADP or 2-methyl(thio)-ADP-induced intracellular calcium increases were inhibited by the P2Y1 receptor-specific antagonists, adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS), adenosine 3'-phosphate 5'-phosphate (A3P5P), and adenosine 2'-phosphate 5'-phosphate (A2P5P), in a concentration-dependent manner, but not by ARL 66096 or alpha, beta-MeATP. A3P5PS, A3P5P, and A2P5P also inhibited the shape change of aspirinated platelets induced by 10 microM ADP or 3 microM 2-methyl-(thio)-ADP in a concentration-dependent manner, with complete inhibition occurring at 300 microM. On the other hand ARL 66096 (100 nM), a potent P2TAC antagonist and alpha, beta-methylene-ATP (40 microM), a P2X1 receptor agonist, had no effect on ADP-induced platelet shape change. On the contrary, ADP-induced inhibition of adenylyl cyclase was blocked by ARL 66096, but not by alpha, beta-MeATP or the P2Y1 receptor-specific antagonists, A3P5PS, A3P5P, or A2P5P. These results demonstrate the role of the P2Y1 receptor in ADP-induced platelet shape change and calcium mobilization and support the idea that several P2 receptors are involved in the regulation of different aspects of platelet stimulus-response coupling.

Adenosine Diphosphate↗