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PubMed1981

Selection of donor platelets for alloimmunized patients using a platelet-associated IgG assay.

A quantitative immunofluorescence platelet-associated immunoglobulins (PA-IgG) assay was used to detect alloimmunity to platelets in 8/12 multitransfused patients and to perform platelet crossmatching in the 8 alloimmunized patients. The correct separation of multitransfused patients into alloimmune and nonalloimmune groups was substantiated with chromium-51-labeled platelet survival studies. For 5 alloimmunized patients, compatible and incompatible donor platelets were demonstrated by PA-IgG crossmatching and were confirmed by platelet survival studies. With the other 3 alloimmunized patients, only studies with 5 of these incompatible donor platelets showed markedly reduced survival times on 4 occasions. PA-Igg compatible donor platelets survived 3.5-8.7 days, while PA-IgG incompatible platelets showed survival times of 0.1-2.4 days. Overall, PA-IgG testing correctly indicated survival results on 15/17 occasions (88%), whereas platelet aggregation, serotonin release, and lymphocytotoxicity testing showed correct predictions for only 41%-59% of the survival studies. PA-IgG testing predicted which times, thus indication patients with platelet-specific alloantibodies. the PA-IgG assay provides a sensitive method to detect platelet alloantibodies and to perform platelet crossmatching, which can complement HLA typing in the selection of donor platelets for alloimmunized patients.

ABO Blood-Group System↗
PubMed1982

Platelet-activating factor stimulates the phosphatidylinositol cycle. Appearance of phosphatidic acid is associated with the release of serotonin in horse platelets.

The addition of platelet-activating factor to horse platelets prelabeled with (32P)orthophosphate induces the rapid formation of [32P]phosphatidic acid. This is subsequently followed by a considerable increase in the labeling of phosphatidylinositol. Activation of the formation of phosphatidic acid by platelet-activating factor is evident at a concentration of 1 nM, is maximal at 0.1 microM, and independent of the presence or absence of plasma in the medium. In horse platelets prelabeled with [14C]arachidonic acid, platelet-activating factor stimulates the rapid formation of [14C]phosphatidic acid, [14C]arachidonic acid, and [14C]arachidonate metabolites. Concomitantly, there is a loss of radioactivity from phosphatidylinostiol., phosphatidylcholine, and phosphatidylethanolamine. Platelet-activating factor at a concentration of 1 nM stimulates formation of phosphatidic acid while the appearance of arachidonate metabolites is seen at a higher concentration (10 nM), without acetate in the 2-position, is unable to induce in platelets the formation of phosphatidic acid, arachidonate metabolites, or the release of [3H]serotonin and, in addition, does not antagonize the action of platelet-activating factor. the release of [3H]serotonin and [14C]arachidonate from platelets stimulated with platelet-activating factor is not affected by indomethacin. Trifluoperazine (50-100 microM) inhibits the platelet-activating factor-stimulated liberation of arachidonic acid from phospholipids, without affecting the formation of phosphatidic acid and the release of serotonin. Prostacyclin, on the other hand, inhibits the platelet-activating factor-induced release of serotonin, arachidonate metabolites, and formation of phosphatidic acid. These data indicate a close relationship between the formation of phosphatidic acid and the release of serotonin in platelets stimulated with the platelet-activating factor.

Animals↗
PubMed1995

Development of a whole platelet ELISA to detect circulating activated platelets.

P-selectin is a granule membrane protein that is expressed on the surface of activated endothelial cells and platelets. Flow cytometry has been used as a means of detecting activated platelets in the circulation by using antibodies to P-selectin and other surface markers. In the study reported here, we developed a whole platelet ELISA for measuring P-selectin on platelets in platelet-rich plasma. Platelet-rich plasma samples for analysis were isolated from fresh blood by centrifugation, fixed with 1.0% paraformaldehyde, and used within 3 hours or after storage at -70 degrees C for up to 10 months. Paraformaldehyde-fixed, phorbol myristate acetate-activated or thrombin receptor peptide-activated platelets were used to construct a standard calibration curve. These platelets were stable after 10 months of storage at -70 degrees C. Interassay variability showed a high degree of correlation, with r = 0.98 +/- 0.03 (n = 12). The accuracy and specificity of the ELISA was verified by using fluorescence-activated flow cytometric analysis and is as sensitive (< or = 0.5%) as flow cytometry for detecting P-selectin expression on platelets. To assess the ability of the platelet ELISA to detect platelet activation in the systemic circulation, we examined 24 patients with unstable angina and 12 age-matched control subjects. Patients with unstable angina demonstrated significantly higher levels of circulating activated platelets than did age-matched control subjects. Although storage-dependent differences in absolute platelet activation levels were found, platelet ELISA results of samples evaluated within either 3 hours or after 10 months of storage were comparable to results obtained by fluorescence-activated flow cytometric analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Angina, Unstable↗
PubMed1993

Immunolocalization of beta 1 integrins in platelets and platelet-derived microvesicles.

Human platelets contain several adhesion receptors belonging to the integrin superfamily. At least three beta 1 integrins are present on platelets and have been shown to mediate platelet adhesion to collagen, fibronectin, and laminin. To study the cellular localization of the beta 1 integrins in platelets, we produced a polyclonal antibody by immunization of goat 172 with purified beta 1 subunit from HPB-ALL cells. Antibody 172 (Ab172) specifically immunoblotted a 135-Kd protein in a lysate of whole platelets. The reactivity of Ab172 with platelet membrane proteins was further determined by immunoprecipitation of lysates of surface-radioiodinated platelets. Ab172 immunoprecipitates, resolved by nonreducing/reducing two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis consisted of three labeled proteins with migrational properties of platelet glycoprotein (GP)Ia, GPIc and GPIIa. Neither GPIIb/IIIa nor the vitronectin receptor were immunoprecipitated by Ab172, confirming a lack of cross-reactivity with the beta 3 integrins in platelets. Immunofluorescence studies using Ab172 were performed to investigate the cellular distribution of beta 1 integrins in platelets. Fluorescent labeling of intact cells demonstrated the presence of beta 1 antigen on the surface of resting cells. Permeabilization of platelets with Triton X-100 showed the presence of an intracellular pool of beta 1 antigen. Double-label experiments using Ab172 and AP-2 (anti-GPIIb/IIIa) showed identical labeling patterns, suggesting a similar subcellular distribution for these integrins. Following thrombin stimulation, permeabilized cells showed a centralized clearing of both beta 1 antigen and GPIIb/IIIa as well as an intensification of surface labeling for beta 1 antigen. These findings suggest the translocation of intracellular beta 1 antigen to the platelet surface as a result of thrombin stimulation. Because platelet-derived microvesicles have been reported to contain GPIIb/IIIa, we investigated the possible distribution of beta 1 integrins in these structures. Microvesicles, produced as a result of platelet activation, were labeled with Ab172, suggesting the distribution of beta 1 integrins in these structures as well as in intact cells.

Animals↗
PubMed1993

Effects of donor characteristics and platelet in vitro time and temperature on platelet aggregometry.

PURPOSE: This study was undertaken to determine whether platelet donor characteristics and the duration and temperature of platelet storage affect platelet aggregation. METHODS: Half of each platelet-rich plasma sample, obtained from 42 healthy volunteers, was maintained at 37 degrees C and the other half at 25 degrees C. Aggregation was stimulated with adenosine diphosphate (ADP), epinephrine, or collagen 1, 2, 4, and 6 hours after platelet donation. The lag time, slope of the aggregation curve, and percent peak aggregation were determined for each aggregation test. RESULTS: Age, sex, time of donation, smoking status, estrogen replacement, and menstruation did not significantly influence platelet aggregation. Storage time and temperature had a major influence on platelet reactivity, with the reactivity decreasing with prolonged storage and higher storage temperatures. Peak ADP-induced aggregation for platelets stored at 37 degrees C was significantly less (p < 0.0002) than the values obtained for ADP aggregation at 25 degrees C. Collagen-stimulated peak aggregation of platelets maintained at 37 degrees C for 4 and 6 hours was also significantly less than the corresponding values at 25 degrees C. Epinephrine-stimulated aggregation produced similar results, with peak platelet aggregation decreasing with time (50.3% +/- 2.9% at 6 hours compared with 38.5% +/- 3.7% at 2 hours at 25 degrees C) and high storage temperatures (there was less aggregation by platelets stored at 37 degrees C at each time period studied). CONCLUSIONS: It is recommended that platelet-rich plasma for platelet aggregation testing be maintained at room temperature and be used between 2 and 4 hours after platelet donation.

Adenosine Diphosphate↗
PubMed1985

Inhibition by CV-3988 of the binding of [3H]-platelet activating factor (PAF) to the platelet.

The inhibitory effects of CV-3988, a specific antagonist of PAF, on the binding of [3H]-PAF to washed platelets of various species including human were examined. The dissociation constant (Kd), binding capacity (Bmax), and the number of receptor/platelet for the specific binding site of rabbit platelets were 2.2 +/- 0.2 nM, 93.7 +/- 8.3 fmoles/10(8) platelets, and 568 +/- 50, respectively. CV-3988 selectively inhibited the specific binding of [3H]-PAF to rabbit platelets with an IC50 of 7.9 X 10(-8) M, and it slightly increased the Kd value (2.5 +/- 0.8 nM) and decreased the binding capacity for PAF (Bmax: 54.3 +/- 16.3 fmoles/10(8) platelets). The Ki value of CV-3988 for the specific binding of [3H]-PAF to rabbit platelets was 1.2 X 10(-7) M. CV-3988 had no effects on the binding of [3H]-5-hydroxytryptamine (5-HT) to rabbit platelets and on the shape change of the platelet induced by 5-HT. CV-3988 also inhibited the specific binding of [3H]-PAF to human and guinea-pig platelets with IC50 values of 1.6 X 10(-7) and 1.8 X 10(-7) M, respectively. CV-3988 inhibited the PAF-induced aggregation in rabbit, guinea-pig, and human platelets. These findings show that CV-3988 is a specific antagonist of PAF at the receptor site(s) of platelets and, in these species, inhibits PAF-induced platelet aggregation by inhibiting the binding of PAF to the "PAF receptor". No specific binding of [3H]-PAF to the platelet of rats and mice was observed, indicating that these species lack a PAF receptor.

Animals↗
PubMed1989

Relative effects of flurbiprofen on platelet 12-hydroxy-eicosatetraenoic acid and thromboxane A2 production: influence on collagen-induced platelet aggregation and adhesion.

Flurbiprofen has been shown to inhibit cyclo-oxygenase metabolism of arachidonic acid to thromboxane A2 (TxA2), resulting in the inhibition of platelet aggregation. Recently, our laboratory reported that the "irreversible" phase of platelet aggregation and adhesion were regulated, in part, by the lipoxygenase metabolism of arachidonic acid to 12-hydroxy-eicosatetraenoic acid (12-HETE) in platelets, and that selective inhibition of one enzyme i.e. either cyclo-oxygenase or lipoxygenase, resulted in paradoxical effects on the metabolism of arachidonic acid and platelet response related to the other pathway. Therefore, we performed experiments to assess the relative effects of flurbiprofen on TxA2 and 12-HETE synthesis, and on collagen-induced platelet aggregation and platelet adhesion to collagen-coated surfaces. "Irreversible" collagen-induced platelet aggregation was only partially inhibited by pre-incubation with 1 x 10(-6) M flurbiprofen, while TxA2 production was elevated and 12-HETE production was maximally inhibited in these platelets. At this concentration of flurbiprofen (1 x 10(-6)M), collagen-induced platelet adhesion was also reduced by 50%. At higher concentrations of flurbiprofen, both platelet aggregation and adhesion were further reduced, with a corresponding inhibition of TxA2 production. Thus it appears that the lipoxygenase pathway of arachidonic acid metabolism in platelets is not only inhibited by flurbiprofen, but is more sensitive to inhibition by flurbiprofen than the cyclo-oxygenase pathway. This differential effect of flurbiprofen on arachidonic acid metabolism in the platelet is related to differential effects on platelet function.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1998

Functional compensation of the low platelet count by increased individual platelet size in a patient with May-Hegglin anomaly presenting with acute myocardial infarction.

Platelets are known to play a crucial role in normal hemostasis as well as in thrombus formation at sites exposed to blood flow, as in coronary thrombosis. Thus, low platelet count is a strong negative risk factor for the occurrence of arterial thrombosis, such as occurs in acute myocardial infarction. We encountered a patient with May-Hegglin anomaly, presenting with acute myocardial infarction in his sixth decade, even though his platelet counts had always been less than 50 x 10(3)/microl. We investigated the characteristics of his platelets under the effect of shearing and found that shear-induced platelet aggregation and binding of soluble von Willebrand factor (vWF) to platelets could be induced, even when the patient's platelet count was less than 10 x 10(3)/microl, but that virtually no aggregation or vWF binding by normal platelets could be induced by shearing when platelet counts were less than 50 x 10(3)/microl. We conclude that the low platelet counts in a patient with May-Hegglin anomaly can be functionally compensated for by larger individual platelets, in view of the vWF-dependent platelet thrombus formation occurring under the effect of blood flow and that that is why most patients with May-Hegglin anomaly do not have a bleeding tendency, even though their platelet counts are very low.

Blood Platelets↗
PubMed1993

A review of the role of platelet membrane glycoproteins in the platelet-vessel wall interaction.

This review concerns our understanding of the molecular basis of platelet function in haemostasis. In particular, we indicate how research into platelet membrane glycoprotein (GP) receptors is yielding vital information on the mechanisms of platelet adhesion and aggregation. These receptors, nearly always complexes of two or more subunits, are now known to belong to distinct gene families, some of which are unique to platelets while others are widely distributed in mammalian tissues. GP Ib-IX complexes are responsible for the high-shear-rate-dependent adhesion of platelets to von Willebrand factor (vWF) exposed within the subendothelium of damaged vessels. Other adhesion receptors include members of the VLA subclass of the integrin family: VLA-2, VLA-5 and VLA-6, which mediate platelet adhesion to collagen, fibronectin and laminin, respectively. Platelet aggregation is initiated by distinct populations of receptors specific for each physiological agonist. Many of these receptors, including the highly important and recently cloned thrombin receptor, have seven transmembrane domains and possess highly selective agonist-binding determinants. Finally, we highlight platelet aggregation and the role of GP IIb-IIIa complexes which, following platelet activation, bind fibrinogen and other adhesive proteins. The latter, through being polyvalent for GP IIb-IIIa, then form the bridges linking adjoining platelets. The 'ligand-binding pocket' of GP IIb-IIIa contains at least three sequences essential for ligand binding; fibrinogen also binds to the activated complex through identified domains, one of which, the Arg-Gly-Asp (RGD) sequence, is also found in vWF and the other adhesive proteins able to support platelet aggregation. Finally, we further describe how these, and other glycoproteins in both surface and internal membrane systems, constitute a complex receptor network capable of translocation and reorganization after platelet activation. In cardiovascular disease, platelets accumulate within arteries whose luminal surface has been modified through atherosclerosis. Recent molecular advances are yielding exciting opportunities for the development of new, and more powerful, drugs acting as specific inhibitors of thrombotic processes.

Amino Acid Sequence↗
PubMed1997

Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions.

BACKGROUND: We conducted a multi-institutional, randomized, blinded trial to determine whether the use of platelets from which leukocytes had been removed by a filter or that had been treated with ultraviolet B irradiation would prevent the formation of antiplatelet alloantibodies and refractoriness to platelet transfusions. METHODS: Patients who were receiving induction chemotherapy for acute myeloid leukemia were randomly assigned to receive one of four types of platelets transfusions: unmodified, pooled platelet concentrates from random donors (control); filtered, pooled platelet concentrates from random donors (F-PC); ultraviolet B-irradiated, pooled platelet concentrates from random donors (UVB-PC); or filtered platelets obtained by apheresis from single random donors (F-AP). All patients received transfusions of filtered, leukocyte-reduced red cells. RESULTS: Of 530 patients with no alloantibodies at base line, 13 percent of those in the control group produced lymphocytotoxic antibodies and their thrombocytopenia became refractory to platelet transfusions, as compared with 3 percent in the F-PC group, 5 percent in the UVB-PC group, and 4 percent in the F-AP group (P< or =0.03 for each treated group as compared with the controls; there were no significant differences among the treated groups). Lymphocytotoxic antibodies were found in 45 percent of the controls, as compared with 17 to 21 percent in the treated groups (P<0.001 for each treated group as compared with the controls; there were no significant differences among the treated groups). Antibodies against platelet glycoproteins developed in 6 to 11 percent of the patients, with no significant differences among the four groups. CONCLUSIONS: Reduction of leukocytes by filtration and ultraviolet B irradiation of platelets are equally effective in preventing alloantibody-mediated refractoriness to platelets during chemotherapy for acute myeloid leukemia. Platelets obtained by apheresis from single random donors provided no additional benefit as compared with pooled platelet concentrates from random donors.

Adolescent↗
PubMed2001

Comparative efficacy of fibrinogen and platelet supplementation on the in vitro reversibility of competitive glycoprotein IIb/IIIa (alphaIIb/beta3) receptor-directed platelet inhibition.

BACKGROUND: Platelet surface glycoprotein (GP) IIb/IIIa (alphaIIb/beta(3)) receptor inhibition, by preventing fibrinogen binding and platelet aggregation, concomitantly attenuates arterial thrombotic capacity and impairs protective hemostasis, 2 divergent platelet-dependent processes. Because the currently available Food and Drug Administration-approved small molecule GP IIb/IIIa receptor antagonists are considered "competitive" inhibitors and because there is limited information on the reversibility of platelet inhibition by fibrinogen or platelet supplementation, the following series of in vitro experiments were performed. METHODS AND RESULTS: Washed platelets from 24 healthy volunteers were suspended in Tyrodes buffer and incubated with achievable (in vivo) steady-state concentrations of either tirofiban or eptifibatide before activation with TRAP (thrombin receptor agonist peptide) (15 micromol/L). Platelet aggregation was inhibited by 40% to 50%, but reversal was achieved by fibrinogen supplementation in a concentration-dependent manner. In a separate series of in vitro experiments, platelet inhibition exceeding 90% was established with tirofiban (average concentration 9.28 microg/L) and eptifibatide (average concentration 95.4 microg/L). Recovery of platelet aggregation to at least 50% was achieved after the addition of fibrinogen (0.76-0.80 g/L), platelets (2.4 x 10(11)/L), or their combination. There was an inverse relationship between plasma baseline fibrinogen and the amount of supplemental fibrinogen required to restore platelet aggregability (r = -0.60, P <.01). CONCLUSIONS: The reversibility of GP IIb/IIIa-directed platelet inhibition is influenced by cell surface receptor availability and the intrinsic pharmacodynamic mechanism of action. Fibrinogen supplementation with fresh frozen plasma or cryoprecipitate either alone or in combination with platelet transfusion, represents an important and readily available treatment consideration for restoring hemostatic potential and managing major hemorrhagic complications associated with the administration of small molecule competitive GP IIb/IIIa receptor antagonists.

Adult↗
PubMed2002

Studies of fibrinogen binding to porcine platelets by flow cytometry: a method for studies of porcine platelet activation.

Platelets have a central function in haemostasis. They also participate in arterial thrombus formation in vascular disorders. Platelets have an important role in initiating and mediating ischaemia and related complications of ischemic heart disease. Several research groups are thus studying platelet activation and developing new platelet inhibitors. Platelet function is dependent upon membrane receptors and their interaction with other proteins. Binding of fibrinogen to the platelet glycoprotein (GP) IIb/IIIa receptor is a prerequisite for platelet aggregation and thrombus formation. Thus, several GPIIb/IIIa inhibitors have been developed of which abciximab is the clinically most widely used. Pigs are often used for experimental studies. We have developed a flow cytometry assay for measuring porcine platelet activation utilising an FITC-labelled chicken anti-fibrinogen antibody. ADP, ristocetin and thrombin induce fibrinogen binding to porcine platelets similarly to human platelets. Ristocetin induces platelet aggregation and microvesicle formation from porcine platelets as well as from human platelets.

Adenosine Diphosphate↗
PubMed1990

Influence of plasma protein content and platelet number on the potency of PAF and its antagonist RP 59227 in rabbit platelet preparations.

1. The potency of platelet activating factor (PAF) as a pro-aggregatory agent and of RP 59227, a selective antagonist of PAF-induced platelet aggregation, was determined in several types of rabbit platelet preparations. 2. PAF produced concentration-dependent responses irrespective of whether the suspension medium for the platelets (200,000 per microliter) was undiluted plasma (PRP), saline-diluted plasma (dil. PRP) or a salt solution (WP: washed platelets). The potency of PAF, expressed as pD2, was 3 fold higher in WP than PRP or diluted PRP (dil. PRP) for which the ratio (v/v) of total plasma to saline was 1:1.5. In PRP and WP preparations, an increase in the number of platelets in the reaction medium from 200,000 to 600,000 enhanced the potency of PAF slightly (2.3 fold). Furthermore, PAF was 3 times more potent in WP than PRP when studied in preparations containing either 200,000, 400,000 or 600,000 platelets per microliter. 3. RP 59227, like the reference compounds WEB 2086 and CV-6209, behaved as a competitive antagonist against PAF-evoked platelet aggregation in PRP, WP and dil. PRP (200,000 platelets per microliter). Their potency was slightly greater (1.6 to 2.6 fold more) in dil. PRP than in WP, but in PRP it was 3.5 to 4.3 times lower than in WP. RP 59227 was 2.3 and 5.0 times less potent when the platelet number of the PRP suspension was increased from 200,000 to 400,000 and 600,000 per microliter, respectively, whereas the potency of RP 59227 in WP was not modified by these changes in platelet number. Furthermore, CV-6209, RP 59227 and WEB 2086 were found to be 2.8 to 3.3 times more potent when studied after 10 rather than 1 min incubation time. 4. In conclusion, methodological variables such as platelet number, plasma protein content and incubation time can modify the potency not only of PAF, as an aggregatory agent, but also of its antagonists. Thus, only platelet aggregation studies carried out by using well-defined experimental conditions afford an appropriate investigational approach to establish a potency rank order for PAF-receptor antagonists. Furthermore, dil. PRP which can be easily and rapidly prepared appears to be as appropriate as WP to determine pA2 values for PAF antagonists.

Animals↗
PubMed2003

Platelets of female mice are intrinsically more sensitive to agonists than are platelets of males.

OBJECTIVE: It has been reported that women fare worse after ischemic coronary events, but the mechanisms remain unclear. Because platelets play a central role in the formation of occlusive thrombi at sites of ruptured atherosclerotic plaques, we studied male/female paired mouse littermates for sex differences in platelet function. METHODS AND RESULTS: We compared platelet reactivity in male/female mouse littermates by monitoring agonist-induced fibrinogen (FGN) binding and platelet aggregation. Compared with the platelets from males, platelets from females bound more FGN in response to low concentrations of thrombin and collagen-related peptide. Female platelets also demonstrated greater aggregation in response to adenosine diphosphate and collagen-related peptide. Platelet protein tyrosine phosphorylation on activation also showed small differences between sexes. These differences are independent of platelet size and surface expression of alphaIIbbeta3 and GPIb-IX-V, and they were not blocked by apyrase or aspirin. The sex differences we observed were intrinsic to platelets, because they were observed in washed platelets, but not when platelets were in plasma. CONCLUSIONS: The platelets of female mice were more reactive than those of males in a manner independent of COX-1 and secreted ADP.

Animals↗
PubMed1997

Platelet activation by superoxide anion and hydroxyl radicals intrinsically generated by platelets that had undergone anoxia and then reoxygenated.

BACKGROUND: Platelet activation has been demonstrated in experimental and clinical models of ischemia-reperfusion, but the underlying mechanism is still unclear. We mimicked the ischemia-reperfusion model in vitro by exposing platelets to anoxia-reoxygenation (A-R) and evaluated the role of oxygen free radicals (OFRs), which are usually produced during the reperfusion phase, in inducing platelet activation. METHODS AND RESULTS: Human platelets were exposed to 15 and 30 minutes of anoxia and then reoxygenated. Compared with control platelets kept in atmospheric conditions, platelets exposed to A-R showed spontaneous platelet aggregation (SPA), which was maximal after 30 minutes of anoxia. Superoxide dismutase (SOD) (-74%, P < .005), catalase (-67%. P < .005). SOD plus catalase (-82%, P < .005), and the hydroxyl radical (OH0) scavengers mannitol (-66%, P < .005) and deoxyribose (-55%, P < .005) inhibited SPA. Platelets that had undergone A-R released superoxide anion (0-2), as detected by lucigenin chemiluminescence. Also, platelets exposed to A-R and incubated with salicylic acid generated 2.3- and 2,5-dihydroxybenzoates, which derive from salicylic acid reaction with OH0. SPA was significantly inhibited by the cyclooxygenase enzyme inhibitors aspirin and indomethacin: by SQ29548, a thromboxane (Tx) A2 receptor antagonist; by diphenyliodonium an inhibitor of flavoprotein-dependent enzymes: and by arachidonyl trifluoromethyl ketone, a selective inhibitor of cytosolic phospholipase A2. Platelets exposed to A-R markedly generated inositol 1,3,4-trisphosphate and TxA2, which were inhibited by incubation of platelets with SOD plus catalase. CONCLUSIONS: This study shows that platelets exposed to A-R intrinsically generated 0-2 and OH0, which in turn activate arachidonic acid metabolism via phospholipases A2 and C, and provides further support for the use of antioxidant agents as inhibitors of platelet function in ischemia-reperfusion models.

Acridines↗
PubMed1993

Role of platelet-activating factor in hypotension and platelet activation induced by infusion of thrombolytic agents in rabbits.

Infusion of the thrombolytic agents streptokinase (SK, 666 units/kg per minute for 60 minutes) and tissue-type plasminogen activator (t-PA, 10 micrograms/kg per minute for 15 minutes) in rabbits induced a significant hypotension and decrease in platelet count that were completely prevented by treatment with platelet-activating factor (PAF) receptor antagonists SDZ 63-675 and WEB 2170. PAF synthesis by vascular tissue was suggested by its extraction from blood-free heart and aorta of rabbits treated in vivo with SK or t-PA but not of control rabbits. In contrast, PAF was not detected in peripheral blood. Ex vivo studies on platelet aggregation response to ADP and PAF performed on platelet-rich plasma obtained before and after SK and t-PA infusion demonstrated an early hyperaggregable phase, abrogated by PAF receptor antagonists and followed by reduced sensitivity of platelets to PAF. The ED50 values for the aggregation of washed rabbit platelets induced by PAF but not thrombin were significantly increased at 60 and 120 minutes after SK and t-PA infusion, suggesting a specific desensitization of platelets to PAF. In contrast to PAF receptor antagonists, aspirin did not significantly modify the hypotension and the platelet hyperaggregability induced by SK or t-PA or the platelet hypoaggregability induced by t-PA. Thrombocytopenia induced by t-PA, but not by SK, was partially prevented by aspirin. The effect of SK, t-PA, and plasmin on the aggregation of washed platelets from untreated rabbits and from humans was also studied. Whereas SK and t-PA were inactive, plasmin induced dose-dependent platelet aggregation that was inhibited by platelet pretreatment with PAF receptor antagonists. In conclusion, the effect of PAF receptor antagonists observed in the present experimental model suggests that the hypotension and activation of platelets induced by SK and t-PA infusion are mediated by PAF.

Animals↗
PubMed1997

Studies on platelet membrane glycoproteins and platelet function during hemodialysis.

Hemodialysis only partially corrects the defects in platelet function associated with uremia. Platelet contact with the artificial surfaces of the dialysis filter during hemodialysis can itself cause platelet activation, degranulation, and loss of platelet membrane glycoproteins. Although the transient platelet dysfunction that occurs after platelet contact with foreign surfaces during cardiopulmonary bypass has been well characterized, there has been no such investigation of hemodialysis. In this study of hemodialysis patients, bleeding times (BT) and the response of their platelets to thrombin, ristocetin, and collagen were measured before, immediately after, and in some patients, the day after hemodialysis. In addition, membrane glycoproteins in platelets obtained at these time intervals were studied using fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies (mAb) CD42b (anti-GPIb), CD41a (anti-GPIIb/IIIa), and CD62 (anti-P-selectin), with flow cytometry. BT was significantly prolonged, and response to thrombin and ristocetin was significantly decreased immediately after hemodialysis (P < 0.01). Binding of CD42b mAb to the platelet membrane was decreased in platelets obtained immediately after hemodialysis. Most patients had shortened BT and demonstrated increased response of their platelets to thrombin and increased CD42b binding to their platelets the day after hemodialysis. These findings suggest that in uremic patients, hemodialysis is associated with transient platelet dysfunction and decreased membrane expression of GPIb.

Antibodies, Monoclonal↗
PubMed2005

Clopidogrel inhibits platelet-leukocyte adhesion and platelet-dependent leukocyte activation.

Clopidogrel is considered to be an important therapeutic advance in anti-platelet therapy. We investigated whether inhibition by clopidogrel results in a reduced capacity of platelets to adhere and stimulate pro-atherothrombotic and inflammatory functions in polymorphonuclear leukocytes (PMN) and in monocytes (MN). An eventual effect on these processes could further substantiate anti-atherothrombotic properties of this drug. The effects of clopidogrel or of its active metabolite were investigated on ADP or thrombin receptor-induced platelet activation and on platelet-leukocyte interactions ex vivo in the mouse or in vitro in isolated human cells or whole blood, respectively. Clopidogrel inhibited platelet aggregation, expression of P-selectin, platelet-PMN adhesion and platelet-dependent ROS production in mouse PMN. Similarly pretreatment of human platelets with the active metabolite of clopidogrel in vitro resulted in a profound inhibition of platelet P-selectin expression, platelet-PMN adhesion and production of ROS by PMN. Pretreatment with the active metabolite of clopidogrel significantly impaired the ability of platelets to up-regulate the expression of TF procoagulant activity in MN, in a washed cell system. Moreover, the active metabolite of clopidogrel inhibited rapidTF exposure on platelet as well as on leukocyte surfaces in whole blood. By reducing platelet-dependent up-regulation of inflammatory and pro-atherothrombotic functions in leukocytes, clopidogrel may reduce inflammation that underlies the chronic process of atherosclerosis and its acute complications.

Animals↗