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PubMed1983

Purification of two heparin-binding proteins from porcine platelets and their homology with human secreted platelet proteins.

Two heparin-neutralizing proteins secreted by thrombin-stimulated platelets were purified to homogeneity by means of heparin-agarose affinity chromatography. These proteins, termed porcine platelet basic protein (PBP) and porcine platelet factor 4 (PF4), were eluted from a heparin-agarose column at 0.6-0.9 M NaCl and at 1-1.4 M NaCl, respectively. The molecular weight of porcine platelet basic protein was 7,000-7,700 daltons, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis. The isoelectric point of this protein was at pH 9.0. The amino acid composition of porcine platelet basic protein resembled that of human low affinity platelet factor 4 (LA-PF4), except that the porcine protein did not contain tyrosine. The molecular weight of porcine platelet factor 4 ranged from 10,000 (estimated from amino acid analysis) to 14,000 (estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis). The amino acid compositions of human platelet factor 4 and of porcine platelet factor 4 were similar. Monospecific antibodies against porcine platelet factor 4 and porcine platelet basic protein were raised in rabbits. Competitive radioimmunoassay demonstrated a low but significant immunologic cross-reactivity between human and porcine platelet factor 4, and between porcine platelet basic protein and a group of human secreted platelet proteins that bind to heparin with low affinity (beta-thromboglobulin [beta TG] and low affinity platelet factor 4). Experiments with direct immuno-precipitation of 125I-labeled antigens suggested that all four proteins investigated (human platelet factor 4, porcine platelet factor 4, human low affinity platelet factor 4 or human beta-thromboglobulin, and porcine platelet basic protein) share common antigenic determinants. However, there was a higher degree of immunologic cross-reactivity between heterologous antigens with similar heparin binding affinity (human platelet factor 4 and porcine platelet factor 4) than between heterologous antigens with different binding affinity (human platelet factor 4 and porcine platelet basic protein). In conclusion, our finding suggests a significant structural homology among the four proteins.

Animals↗
PubMedDate not supplied

Determination of the rate of reduction in platelet counts in recipients of hematopoietic stem and progenitor cell transplant: clinical implications for platelet transfusion therapy.

BACKGROUND: To investigate how a delay between pretransfusion platelet count measurement and actual platelet transfusion affects the assessment of platelet transfusion responses, the rate of reduction in platelet counts was determined in 30 patients with relatively uncomplicated thrombocytopenia. STUDY DESIGN AND METHODS: Fifteen adult and 15 pediatric patients admitted for hematopoietic stem and progenitor cell transplantation were studied. Platelet counts before and after myeloablative conditioning and after prophylactic platelet transfusions were determined and studied as a function of time. The rates of reduction in platelet counts were determined by linear regression analysis. RESULTS: Platelet counts were reduced at linear rates after myeloablative conditioning or prophylactic platelet transfusion in all 30 patients. The average rates of reduction in platelet counts after myeloablation were 1261 +/- 583 and 1070 +/- 492 platelets per microL per hour (mean +/- SD) for adult and pediatric patients, respectively. The average rate of reduction after platelet transfusions during the thrombocytopenic phase was 740 +/- 280 and 820 +/- 288 platelets per microL per hour (mean +/- SD) for adult and pediatric patients, respectively. The rates of reduction in platelet counts between the two phases were significantly different in the two age groups (adult, p < 0.0001; pediatric, p < 0.015) and were proportionally correlated with initial platelet counts immediately before myeloablation and after prophylactic platelet transfusions. CONCLUSION: The rate of reduction in platelet count can have a significant impact on the evaluation of platelet transfusion responses when there is a delay between pretransfusion measurement of platelet count and the initiation of platelet transfusion. In addition, the rate of platelet reduction determined from this study can be used to confirm an accelerated rate of platelet consumption in thrombocytopenic patients.

Adolescent↗
PubMed2004

Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing.

Growth factors released from activated platelets initiate and modulate wound healing in both soft and hard tissues. A recent strategy to promote the wound-healing cascade is to prepare an autologous platelet concentrate suspended in plasma, also known as platelet-rich plasma, that contains growth factors and administer it to wound sites. The purpose of this study was to quantitate platelet number and growth factors released from a prepared platelet concentrate. Whole blood was drawn from 10 healthy patients undergoing cosmetic surgery and concentrated into platelet-rich plasma. Platelet counts on whole blood and platelet-rich plasma were determined using a Cell-Dyn 3200. Platelet-derived growth factor-BB, transforming growth factor-beta1, vascular endothelial growth factor, endothelial growth factor, and insulin-like growth factor-1 were measured in the platelet-rich plasma using the enzyme-linked immunosorbent assay method. In addition, platelet activation during the concentration procedure was analyzed by measuring P selectin values in blood serum. An 8-fold increase in platelet concentration was found in the platelet-rich plasma compared with that of whole blood (baseline whole blood, 197 +/- 42 x 10 platelets/microl; platelet concentrate, 1600 +/- 330 x 10 platelets/microl). The concentration of growth factors also increased with increasing platelet number. However, growth factor concentration varied from patient to patient. On average for the whole blood as compared with platelet-rich plasma, the platelet-derived growth factor-BB concentration increased from 3.3 +/- 0.9 ng/ml to 17 +/- 8 ng/ml, transforming growth factor-beta1 concentration increased from 35 +/- 8 ng/ml to 120 +/- 42 ng/ml, vascular endothelial growth factor concentration increased from 155 +/- 110 pg/ml to 955 +/- 1030 pg/ml, and endothelial growth factor concentration increased from 129 +/- 61 pg/ml to 470 +/- 320 pg/ml. No increase was found for insulin-like growth factor-1. In addition, no increase in platelet activation occurred during the concentration procedure as determined by the platelet surface receptor P selectin (45 +/- 16 pg/ml to 52 +/- 11 pg/ml, p = 0.65). In conclusion, a variety of potentially therapeutic growth factors were detected and released from the platelets in significant levels in platelet-rich plasma preparations. Sufficient concentrates and release of these growth factors through autologous platelet gels may be capable of expediting wound healing in a variety of as yet undetermined specific wound applications.

Adult↗
PubMed1998

Neutrophil cathepsin G modulates platelet P-selectin expression and inhibits P-selectin-mediated platelet-neutrophil adhesion.

1. Close contact between platelets and neutrophils modulates their cellular interactions in thrombotic and inflammatory states, with stimulation of P-selectin expression on platelets by agonists such as thrombin and neutrophil-derived cathepsin G being critical in mediating platelet-neutrophil adhesion. This study compared the effects of thrombin and cathepsin G on platelet P-selectin expression and on P-selectin-mediated platelet-neutrophil adhesion. 2. Washed platelets and platelet-neutrophil mixed cell suspensions (platelet/neutrophil ratio, 10:1) were incubated with either the supernatant of activated neutrophils, purified cathepsin G or thrombin. Platelet P-selectin expression and platelet adhesion to neutrophils was quantified by flow fluorocytometric analysis. 3. The supernatant from activated neutrophils stimulated platelet P-selectin expression comparable to that produced by purified cathepsin G or thrombin. P-selectin expression induced by both activated neutrophil supernatant and purified cathepsin G was completely inhibited by alpha 1-antichymotrypsin, a specific inhibitor of cathepsin G. Unlike thrombin, which induced maximum platelet P-selectin expression by 10 min, sustained to 120 min, cathepsin G induced an initial large increase in platelet P-selectin expression, followed by a progressive reduction over 30-60 min to baseline levels. 4. Co-incubation of neutrophils with thrombin-stimulated platelets resulted in a significant increase in P-selectin-mediated platelet-neutrophil adhesion, which was completely inhibited by preincubation of neutrophils with anti-sialyl Lewis(x) monoclonal antibody. Thrombin produced maximum platelet-neutrophil adhesion by 10 min which remained stable over 120 min. In contrast, cathepsin G-stimulated platelets did not adhere to neutrophils over 120 min of co-incubation. Addition of cathepsin G to thrombin-stimulated platelets caused a progressive reduction over 30-60 min to baseline levels of platelet-neutrophil adhesion. 5. Neutrophil-derived cathepsin G is a potent platelet activator, but unlike thrombin it causes a time-dependent loss of platelet P-selectin expression and inhibits P-selectin-mediated platelet-neutrophil adhesion. Therefore, cathepsin G may modulate thrombin-mediated platelet-neutrophil adhesive interactions in inflammation and thrombosis.

Adult↗
PubMed2002

Phosphatidylserine expression on the platelet membrane of patients with myeloproliferative disorders and its effect on platelet-dependent thrombin formation.

Recently, the asymmetric distribution of phospholipids in eukaryotic cell membranes has been appreciated and been found to be dependent on the activity of a number of enzymes. The expression of phosphatidylserine (PS), a negatively charged phospholipid, on the platelets of patients with polycythemia vera (P vera) and essential thrombocythemia (ET) was compared to that in normal individuals. The effect of platelet aggregation on PS expression was determined. Exposure of PS on platelets obtained from patients with P vera and ET and from age- and sex-matched healthy volunteers was measured by fluorescein-labeled Annexin V binding to platelets and by the platelets' thrombin-generating capacity determined by the prothrombinase assay. PLatelet prothrombinase activity (mean +/- standard deviation [SD]), as measured by thrombin generation, was 2.32+/-2.2 micro/mL in the P vera group and 1.55+/-1.0 micro/mL in the control group (p=0.3). PS expression as measured by Annexin V binding (mean +/- SD) was 2.6+/-2.4 % in the P vera group versus 1.55+/-1.2% among controls (p=0.03). In the ET group, prothrombinase activity (mean +/- SD) was 1.0+/-0.6 micro/mL and 2.1+/-0.9 micro/mL in the control group (p=0.006). Annexin V binding (mean +/- SD) was 4.8+/-4.2% in the ET group and 2.77+/-2.1% among control subjects (p=0.09). When the prothrombinase assay was performed after addition of adenosine diphosphate (ADP) to the platelets, there was a significant increase in thrombin generation in the myeloproliferative disorder (MPD) group (3.1+/-2.0 micro/mL) compared to the thrombin generated by unstimulated myeloproliferative disorder platelets (2.07+/-1.69 micro/mL) (p=0.0006). An increase in thrombin generation was seen in the ADP-stimulated platelet samples in all ten paired samples studied. Likewise, the addition of ADP to control platelets increased thrombin generation from 2.0+/-1.0 micro/mL in unstimulated platelets to 4.3+/-1.6 micro/mL in ADP-treated platelets (p=0.0006). Thrombin generation increased in all of the ADP-stimulated platelet samples compared to the untreated platelets. There was however, no difference in the increased thrombin generation when ADP-stimulated platelets from MPD patient and control subjects were compared (p=0.3). Results indicate that some patients with MPDs may show increased PS expression on platelet surface. When analyzed overall, there was a tendency toward greater PS expression in the P vera and ET patient groups; however, the increase did not reach statistical significance. This increase was noted in both the prothrombinase assay the Annexin V binding assay. We have also shown that stimulation of platelets by addition of the agonist ADP results in enhanced PS expression, which appears increase the thrombogenic potential of the platelets as demonstrated by the enhanced thrombin generation demonstrated by these platelets in the prothrombinase assay. There was no difference in the degree of PS expression in response to ADP stimulation between MPD and control platelets. Results show that PS expression and platelet-dependent thrombin generation is variable in patients with MPDs. This expression is increased after platelet aggregation occurs. The role of PS expression in the thromboembolic complications of MPD patients should be studied further.

Adenosine Diphosphate↗
PubMed1983

Decreased platelet adhesion on vessel segments in von Willebrand's disease: a defect in initial platelet attachment.

The adhesion of platelets to subendothelium exposed to flowing blood involves two distinct morphological stages: (1) platelet contact (C), the initial attachment of unspread, discoid platelets to the subendothelium, and (2) spread platelets (S), the attachment that results as contact platelets spread on the surface and become more firmly bound to it. A defect in either initial platelet attachment or platelet spreading can result in reduced levels of platelet adhesion (C + S). The combined observation of decreased platelet adhesion (C + S) and increased platelet contact (C) has been previously utilized to conclude that a defect exists in the ability of platelets to spread on subendothelium in von Willebrand's disease. In this present investigation, we demonstrate, by modeling the contact and spreading stages of platelet adhesion as a classic set of reactions in series, that the combination of reduced adhesion (C + S) and increased contact (C) is inconclusive with regard to the nature of the adhesion defect in von Willebrand's disease. Decreased adhesion (C + S) coupled with increased platelet contact (C) can result from either decreased rates of initial attachment or decreased rates of spreading. In fact, given the complexity of the temporal behavior of platelet contact (C) and platelet spreading (S), and the relatively small fraction (less than 10%) of the platelets that are in contact (C) at any time, we conclude that a determination of the nature of the adhesion (C + S) defect in von Willebrand's disease is not statistically feasible under conditions in which both contact and spreading occur simultaneously. Experiments were conducted in which blood anticoagulated with EDTA was exposed to subendothelium digested with alpha-chymotrypsin for periods of 10 and 40 min. Under such conditions, platelet spreading (S) was substantially inhibited so that the predominant platelet interaction (greater than 80%) on the subendothelium was platelet contact (C). Values of platelet adhesion (C + S) in von Willebrand's disease were significantly reduced (p less than 0.05) compared with normal values at both exposure times. Thus we conclude that the defect in platelet adhesion (C + S) in von Willebrand's disease appears to be associated with a reduced ability of platelets to attach to the surface rather than their inability to spread on the surface.

Animals↗
PubMed2000

P-Selectin expression, platelet aggregates, and platelet-derived microparticle formation are increased in peripheral arterial disease.

Platelet volume has been reported to be increased in vascular disease. Therefore, we studied the relationship of mean platelet volume and platelet count as well as flow cytometrically measured platelet size and platelet function in 50 patients with peripheral arterial disease and 50 healthy volunteers. Platelet activation was measured by P-selectin expression analysis on resting and on stimulated platelets, and the determination of platelet aggregates and platelet-derived microparticles using flow cytometry. P-Selectin expression on platelets was significantly elevated in patients suffering from peripheral arterial disease (all P<0.0001). Platelet aggregates (P<0.0001) and platelet-derived microparticles (P<0.0001) were significantly higher in the patient group compared with controls, whereas mean platelet volume and platelet count showed no significant differences. Platelet count was inversely related to mean platelet volume in patients and controls (r = -0.43, P<0.001). The present study supports the hypothesis of platelet hyperreactivity and circulating activated platelets in peripheral arterial disease. Mean platelet volume, and platelet count cannot be used as predictive markers for platelet activation in peripheral arterial disease patients.

Adult↗
PubMed1985

Platelet size and shape in hereditary giant platelet syndromes on blood smear and in suspension: evidence for two types of abnormalities.

Platelet size on blood smear is compared with platelet size and shape in suspension (i.e., whole blood and citrated platelet-rich plasma [PRP]) for normal donors and 16 patients with hereditary "giant" platelet syndromes (HGPS), including Bernard-Soulier syndrome (BSS) (seven patients), Montreal platelet syndrome (MPS) (three patients), May-Hegglin anomaly (one patient) and Rafael platelet defect (one patient). In whole blood platelet shape is normal for HGPS, but in PRP for 10 of 16 patients with HGPS there is a decrease in the proportion of smooth, discoid-shaped platelets (discocytes [D]). The platelets of all patients with HGPS had abnormally large mean volume (VT) and increased size on peripheral blood smear. Furthermore, 12 of 16 patients with HGPS, including six of seven donors with BSS, had abnormally large discocytes. The measured size of HGPS shape-changed platelets was compared with the size predicted from the size of the D by assuming that the relationship between the size of shape-changed platelets and D was the same as observed for normal donors. In this manner it was shown that for all donors with BSS and MPS, the shape-changed platelets are disproportionately larger than the D. In contrast, in the remaining patients with HGPS the size of the shape-changed platelets was consistent with the size predicted from the D. Examination of VT for MPS as a function of time after addition of 10 mumol/L adenosine diphosphate to PRP revealed an abnormal time course, thereby pointing to an abnormality in the mechanisms that regulate platelet size during shape change. With the lone exceptions of BSS and MPS, the size of platelets on blood smear was well correlated with the total platelet plasma membrane surface area as measured by the osmotic spherocyte method. Our observations point to two distinct abnormalities in platelet size in HGPS: a disproportion between the size of D and "shape-changed" platelets, which may be related to an abnormal shape change and which is observed only for MPS and BSS, and an abnormal increase in platelet size on blood smear, which appears to reflect the increased amount of platelet plasma membrane in other HGPS platelets.

Adenosine Diphosphate↗
PubMed1990

Platelet adhesion to collagen in subtypes of type I von Willebrand's disease is dependent on platelet von Willebrand factor.

Von Willebrand's disease type I, characterized by low levels of factor VIII coagulant activity (VIII: C), von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor activity (RiCof) (1), can be subdivided on the basis of platelet von Willebrand factor into subtype platelet normal, platelet discordant, and platelet low (2). We have investigated the contribution of platelet von Willebrand factor in these various subtypes to platelet adhesion using the rectangular perfusion chamber of Sakariassen et al. (3) with fibrillar collagen or a fibroblast matrix as adhesive surfaces. Platelet adhesion to fibrillar collagen was decreased in all subtypes of von Willebrand's disease, but not as low as in severe von Willebrand's disease. A close correlation was observed between platelet adhesion to collagen and plasma vWF:Ag in severe von Willebrand's disease, subtype platelet low, subtype platelet discordant, and normal controls. The platelet adhesion in subtype platelet normal was higher than expected from the plasma vWF:Ag level. Perfusions in which washed platelets were added to a human albumin solution together with red blood cells gave similar adhesion values in subtype platelet normal and normal controls; adhesion was decreased in subtype platelet discordant, and the lowest values were found in subtype platelet low and in severe von Willebrand's disease. These data indicate that platelet von Willebrand factor may contribute to platelet adhesion, when plasma von Willebrand factor is low. Perfusion studies over a fibroblast matrix gave similar low adhesion values for subtype platelet low and platelet normal, indicating that the contribution of platelet von Willebrand factor can only be observed on a strongly activating surface such as fibrillar collagen.

Antigens↗
PubMed2005

Do platelet apoptosis, activation, aggregation, lipid peroxidation and platelet-leukocyte aggregate formation occur simultaneously in hyperlipidemia?

OBJECTIVES: The circulating lipoproteins may cause some abnormalities in platelet composition and function in hypercholesterolemia. The aim of this study was to investigate whether platelet apoptosis, platelet activation, platelet aggregation, platelet-leukocyte aggregate (PLA) formation and lipid peroxidation occur simultaneously in hyperlipidemia. DESIGN AND METHODS: Expression of GpIIb/IIIa (CD41a), P-selectin (CD62-P), platelet-bound fibrinogen (antifibrinogen), platelet membrane phosphatidylserine (PS), platelet-monocyte aggregates (mono-PLA) and platelet-neutrophil aggregates (neut-PLA) was measured in eight hyperlipidemic and eight normal subjects using flow cytometry. ADP (10 microM) was used to activate platelets. Furthermore, ADP induced platelet aggregation responses, platelet malondialdehyde (MDA) and glutathione (GSH) levels were determined. RESULTS: Before platelet activation, platelet CD62-P, antifibrinogen, annexin-V, mono-PLA, neut-PLA and platelet MDA levels as well as platelet aggregation responses in the hyperlipidemics were significantly higher than those in the controls (P<0.01, P<0.01, P<0.01, P<0.001, P<0.001, P<0.01, P<0.001, respectively), whereas GpIIb/IIIa expression and GSH levels were not different significantly (P > 0.05). In the control group, CD62-P, antifibrinogen and annexin-V levels increased significantly after ADP activation (P<0.05, P<0.05, P<0.01, respectively). In hyperlipidemic subjects, annexin-V expression increased significantly after activation (P<0.01), whereas expression of GpIIb/IIIa, CD62-P and antifibrinogen remained unchanged (P>0.05). The levels of total cholesterol (T-CHO), low density lipoprotein cholesterol (LDL-C), serum fibrinogen (S-FGN) and high density lipoprotein cholesterol (HDL-C) in patients were found to be correlated with platelet CD62-P, antifibrinogen, annexin-V, mono-PLA and MDA. CONCLUSIONS: In conclusion, it seems that in hyperlipidemia, some platelets are in an activated state in circulation, and that increased lipid peroxidation, early apoptosis, platelet-leukocytes aggregate formation and platelet aggregation altogether accompany this process.

Adult↗
PubMed1991

Dynamic measurements of the platelet membrane glycoprotein IIb-IIIa receptor for fibrinogen by flow cytometry. II. Platelet size-dependent subpopulations.

Platelet aggregation has previously been shown to occur within 1 s of activation with 100 microM adenosine diphosphate (ADP) for both large (L) and small (S) platelet subpopulations, but L platelets were about twofold more sensitive and more rapidly recruited into microaggregates than were S platelets after correcting for differences in platelet surface area. Because platelet aggregation normally requires fibrinogen binding to glycoprotein IIb-IIIa receptors (FbR) expressed on the activated platelet surface, we wished to compare the kinetics and nature of FbR expression induced by ADP for L versus S platelets, and to measure size-dependent differences in FbR expression for platelets maximally activated with phorbol myristate acetate (PMA). We presented the theory and methodology in Part I (Frojmovic, M., T. Wong, and T. van de Ven. 1991. Biophys. J. 59:815-827) for measuring the rate of FbR expression (k1) and both the rate (k2) and efficiency (alpha) of binding of PAC1 to FbR as a function of activation conditions from the initial on-rate of FITC-PAC1 to FbR (V) and the maximal number of FbR expressed: these are measured, respectively, from the initial rate of increase in platelet-bound fluorescence (v) and the maximal increase in mean fluorescence (Flmax). We extended these analyses to L and S platelets, selected by electronic gating of forward scatter profiles (FSC), with corresponding fluorescence (Fl) histograms retrieved analytically. Platelet size (V) and surface area (SA), determined directly for cells separated with a cell sorter, were highly correlated with FSC, allowing v and Flmax values to be expressed per unit area of membrane for L:S comparisons. Surprisingly, ADP activation appeared to express all FbR within 1-3 s of ADP activation for both L and S platelets, whereas k1 was similar for PMA activation. In addition, L platelets maximally expressed two and three times more FbR per unit area than did S platelets when maximally stimulated, respectively, with ADP or PMA. Whereas k2 was independent of platelet size for a given activator, the efficiency of PAC1 binding (alpha), per unit area of membrane, was two times greater for L than for S platelets, for either ADP or PMA activation. Our data suggest that the FbR structure, its microenvironment, or its surface organization may vary with platelet size or activator type. Major reorganization of FbR and/or its environment appears to occur after approximately 5 min of ADP activation equally for both L and S platelets. A model is presented to account for size-dependent differences in FbR expression with implications for regulation of platelet aggregation.

Adenosine Diphosphate↗
PubMed1999

Effects of nitric oxide on platelet activation during plateletpheresis and in vivo tracking of biotinylated platelets in humans.

BACKGROUND: The use of platelet transfusions has risen considerably over the last few years, which leads to the collection and transfusion of a greater number of donor plateletpheresis units. Plateletpheresis activates platelets in platelet concentrates, which determines the degree of the storage lesion subsequently observed. STUDY DESIGN AND METHODS: As nitric oxide (NO) is a potent inhibitor of platelet aggregation and activation, a placebo-controlled crossover trial was performed in healthy young male volunteers to determine whether the NO-donating compound, sodium nitroprusside (SNP), decreases platelet activation during apheresis and whether activated (p-selectin+) platelets circulate in vivo after transfusion. The study also investigated whether nonradioactive biotin labeling of apheresis platelets is feasible for the study of platelet recovery after transfusion in humans. RESULTS: Platelet activation increased after plateletpheresis in the platelet components, but SNP did not inhibit platelet activation during apheresis, as measured by the percentage of p-selectin expression and the secretion of soluble p-selectin and RANTES. Only a minor increase in p-selectin+ platelets was seen in peripheral blood at 60 minutes after transfusion of the platelets, a rise that was considerably less than that calculated in p-selectin+ platelets if they all were recovered as activated platelets after transfusion. Biotin-labeled platelets averaged 1.5 percent at 10 minutes after transfusion and increased slowly to 2.6 and 3.4 percent after 60 minutes and 24 hours, respectively (p<0.05). CONCLUSION: SNP does not decrease platelet activation during apheresis and subsequent storage, and only a minor proportion of activated (p-selectin+) platelets circulate after transfusion in men. Moreover, biotin labeling of PCs can safely be used in humans for the study of platelet recovery after transfusion, and measuring recovery at 1 hour may lead to an underestimation of the true recovery when activated platelets are transfused.

Biotinylation↗
PubMed1993

Role of platelet-activating factor (PAF) in platelet accumulation in rabbit skin: effect of the novel long-acting PAF antagonist, UK-74,505.

1. The contribution of platelet-activating factor (PAF) to platelet deposition and oedema formation induced by exogenous soluble mediators, zymosan particles and associated with a reversed passive Arthus (RPA) reaction in rabbit skin was investigated by use of a novel long-acting PAF receptor antagonist, UK-74,505. 2. Oedema formation and platelet accumulation were simultaneously measured by i.v. injection of [125I]-albumin and 111In-labelled rabbit platelets. UK-74,505 was either administered i.v. or used to pretreat radiolabelled platelets in vitro before their injection into recipient animals. Platelets pretreated with UK-74,505 were also labelled with the fluorescent calcium indicator, Fura-2, to assess their ex vivo reactivity to PAF at the end of the in vivo experiment. 3. UK-74,505 (0.5 mg kg-1), administered i.v., inhibited PAF-induced oedema formation, but did not affect oedema induced by zymosan particles, bradykinin (BK), histamine, formyl-methionyl-leucylphenylalanine (FMLP), zymosan-activated plasma (ZAP, as a source of C5a des Arg), leukotriene B4 (LTB4) or interleukin-8 (IL-8). 4. UK-74,505, administered i.v. also suppressed the small platelet accumulation induced by exogenous PAF, but had no effect on accumulation induced by IL-8 or ZAP. Although oedema induced by zymosan was not affected by i.v. UK-74,505, zymosan-induced platelet accumulation was significantly attenuated by the antagonist. 5. The RPA reaction in rabbit skin was associated with marked oedema formation and platelet accumulation which were both inhibited by i.v. UK-74,505. 6. In vitro, UK-74,505 inhibited aggregation and the increase in intracellular calcium concentration induced by PAF in rabbit washed platelets in a concentration-dependent manner (IC50 = 1.6 x 10-8 M and 1.1 x 10-8 M, respectively). Platelets pretreated with 10-6 M UK-74,505, and maintained at 37 degrees C,were unresponsive to PAF, whilst responding normally to thrombin, for up to 4 h.7. In a second series of in vivo experiments, platelets were labelled with 111In and loaded with Fura-2.The platelets were then pretreated with 10-6 M UK-74,505, washed, and injected into recipient rabbits.These platelets, prepared from blood samples taken at the end of the in vivo experiments, exhibited an 80% reduction in their response to PAF as measured ex vivo with Fura-2. However, in contrast to the effects of i.v. UK-74,505, platelets pretreated with the antagonist did accumulate effectively in the RPA reaction, a significant reduction only being observed in responses at the lowest antibody dose. In addition, pretreatment of platelets had no effect on the small platelet accumulation induced by PAF.8. These results suggest that PAF is an important mediator of oedema formation and platelet accumulation in the RPA reaction in rabbit skin. However, they question the role of PAF receptors on platelets in this model. The results also indicate that PAF may be involved in platelet accumulation induced by zymosan in rabbit skin.

Animals↗
PubMed1993

Roles of prostacyclin, EDRF and active oxygens in leukocyte-dependent platelet adhesion to endothelial cells induced by platelet-activating factor in vitro.

1. The mechanism of polymorphonuclear leukocyte (PMN)-dependent platelet adhesion to cultured endothelial cells induced by platelet-activating factor (PAF) was investigated to determine whether PMNs release or generate any factor(s) capable of inducing platelet adhesion, and the roles of prostacyclin and endothelium-derived relaxing factor (EDRF). 2. Cell-free supernatants, sonicates or rapid filtrates of PAF-stimulated PMN suspensions did not induce platelet adhesion to endothelial cells, but the PMN sonicates induced platelet adhesion when endothelial cells were pretreated with both aspirin and NG-nitro-L-arginine (L-NOARG). Its microphotograph showed that mainly platelet aggregates adhered to the endothelial cell surface. 3. Platelet adhesion induced by the PMN sonicates to aspirin- and L-NOARG-pretreated endothelial cells was dose-dependently prevented by OP-41483 (1-100 nM), and slightly by L-arginine (1 mM). The inhibition of platelet adhesion by OP-41483 and L-arginine was potentiated by their combination. 4. WEB 2170 (3 microM), a PAF antagonist, inhibited platelet adhesion induced by the PMN sonicates. However, PAF alone did not induce significant platelet adhesion to aspirin- and L-NOARG-treated endothelial cells. 5. Platelet adhesion induced by the PMN sonicates was not suppressed by AA-861 and indomethacin. However, both superoxide dismutase and catalase significantly inhibited platelet adhesion, and, in combination, their inhibitory effect was synergistically potentiated. Mannitol had no effect. It was also significantly inhibited by alpha 1-antitrypsin, whereas chymostatin and elastatinal had no effect. 6. PAF-induced platelet adhesion to endothelial cells in the presence of intact PMNs was not suppressed by indomethacin and AA-861, or by protease inhibitors. SOD alone, and in combination with catalase, caused a slight but significant inhibition, while catalase and mannitol by themselves had no effect.7. PMN-induced platelet adhesion was slightly inhibited by OP-41483 (100 nM). L-Arginine (1 mM)alone had no effect, but slightly potentiated the effect of OP-41483. This platelet adhesion was not accompanied by suppression of prostacyclin synthesis.8. The results with the PMN sonicates show that prostacyclin, EDRF and active oxygens are important modulators of intercellular interactions between platelets and endothelial cells. These results further suggest that the mechanism of intact PMN-dependent platelet adhesion is primarily through platelet endothelial cell interactions in which leukocyte-derived active oxygens play a role, but does not involve platelet-platelet interactions inhibitable by prostacyclin.

Animals↗
PubMed1985

Inhibition and subsequent enhancement of platelet responsiveness by prostacyclin in the rabbit. Relationship to platelet adenosine 3',5'-cyclic monophosphate.

Methods were developed for measuring changes in platelet sensitivity to a release-inducing stimulus and in platelet cyclic AMP in fresh whole blood samples from rabbits. These techniques permitted detection of the effects of exogenous and endogenous prostacyclin on circulating platelets. In these methods, rabbit platelets were labeled in vitro by incubation with [14C]serotonin and [3H]adenine and then transfused into other rabbits. Release of platelet [14C]serotonin by a standard dose of synthetic platelet-activating factor (40 pmol/ml) and the platelet cyclic [3H]AMP levels were then measured in citrated blood from the conscious animals within 2 min of arterial puncture. Bolus intravenous injections of prostacyclin (1-10 nmol/kg) caused concentration-dependent increases in platelet cyclic AMP after 2 min, which decreased approximately 75% by 5 min, and disappeared after 30 min. Significant inhibition of the platelet release reaction was detected 2 min but not 5 min after injection of 10 nmol of prostacyclin per kilogram. With lower doses, significant enhancement of the release of [14C]serotonin was observed after 5 min. Similar changes in platelet responsiveness and cyclic [3H]AMP were observed after release of endogenous prostacyclin by intravenous injection of angiotensin II (5 nmol/kg); inhibition of the release of [14C]serotonin after 2 min was followed by potentiation after 5 min, though platelet cyclic [3H]AMP remained above control values. In these experiments, the time course of the changes in platelet cyclic [3H]AMP correlated closely with values for blood prostacyclin obtained previously (Haslam, R.J., and M.D. McClenaghan, 1981, Nature [Lond.]., 292:364-366). Prostacyclin also had a biphasic effect on the release of [14C]serotonin when added to citrated blood in vitro, though both the increase in sensitivity to platelet-activating factor and the return of platelet cyclic [3H]AMP towards control values took place more slowly. At all times, addition of platelet-activating factor decreased platelet cyclic [3H]AMP towards but not below the control level observed in the absence of prostacyclin. Our results indicate that although transient increases in platelet cyclic AMP cause an immediate decrease in platelet responsiveness in vivo or in vitro, a period of enhanced platelet sensitivity follows as platelet cyclic AMP falls.

Adenosine Triphosphate↗
PubMed1991

Endogenous platelet fibrinogen surface expression on activated platelets.

Intracellular platelet fibrinogen surface expression was studied in arabinogalactan-purified, resting, and thrombin-stimulated platelets. Platelet fibrinogen is derived from endocytosis of plasma fibrinogen by megakaryocytes. Like a variety of other adhesive proteins, it is stored in the platelet alpha-granule. Platelet fibrinogen surface expression was studied by using the antigen-binding fragments of a murine monoclonal antibody to platelet fibrinogen, F26, and an immunopurified polyclonal antifibrinogen antibody. Studies correlating platelet fibrinogen surface expression with the presence of the glycoprotein IIb-IIIa (GPIIb-IIIa) complex showed that in the presence of ethylene glycol tetraacetic acid (EGTA) at 37 degrees C, neither the GPIIb-IIIa complex nor platelet fibrinogen was expressed on the surface of thrombin-activated platelets. Similar experiments performed in the presence of EGTA and calcium showed proportional expression of the GPIIb-IIIa complex and platelet fibrinogen. The addition of Arg-Gly-Asp-Ser-containing peptides, the pentadecapeptide of the fibrinogen gamma-chain carboxy terminus, or the monoclonal antibody 10E5, when directed against the GPIIb-IIIa complex before thrombin activation, inhibited 65% to 94% of the platelet fibrinogen expression, as determined with the polyclonal and monoclonal antigen-binding fragments. When these same inhibitory agents were added immediately after or 5 minutes after thrombin, the amount of inhibition decreased significantly. Similar studies with a washed platelet system revealed that when the inhibitors of platelet fibrinogen expression were added before thrombin stimulation, the degree of inhibition observed was only 24% to 38%. This suggests that the major portion of platelet fibrinogen expression involves the release of platelet fibrinogen and its subsequent binding to GPIIb-IIIa. This binding may occur within the open canalicular system or on the platelet surface; in either case, wherever the site of released platelet fibrinogen binding occurs, it can be markedly inhibited by the RGD-containing peptides and the gamma-chain fibrinogen peptides. Approximately 10% to 30% of platelet fibrinogen may be expressed prebound to a platelet receptor, or else it is released and binds to a platelet receptor other than the GPIIb-IIIa complex.

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