thromboxane-b2 and Thrombocytopenia

Dose-finding studies and trials of interaction of oral glycoprotein IIb/IIIa antagonists with other antiplatelet agents have been limited. We hypothesized that these detailed assessments could be first performed in patients with stable coronary artery disease (CAD) and then extrapolated to the target population. To this end, we performed 2 sequential studies. The first study examined the dose-related effects on indexes of platelet and vascular function induced by the oral inhibitor RPR 109891, when given alone and in combination with aspirin, in patients (n = 100) with stable CAD. The second study (the Antagonism of the FIbrinogen Receptor after Myocardial Events trial) assessed the pharmacodynamics and safety of derived regimens in patients (n = 320) with unstable coronary syndromes. In patients with stable CAD, platelet aggregation was dose dependently inhibited by RPR 109891, and the dose-response relation was shifted to the right by the concomitant administration of aspirin (p = 0.0001). The degree of platelet inhibition induced by 3 doses of RPR 109891 (plus aspirin) was lower in patients with unstable than stable CAD. No drug-related major bleeding occurred in either study. RPR 109891 treatment was associated with acute and delayed thrombocytopenia. In conclusion, chronic treatment with an oral glycoprotein IIb/IIIa antagonist (1) induces antiplatelet effects that are potentiated by concomitant administration of aspirin, (2) may require dose adjustment in syndromes of platelet activation, (3) is associated with a low rate of clinically significant bleeding when doses inducing incomplete inhibition of platelet aggregation are used, and (4) requires frequent monitoring of platelet count unless reliable predictors of delayed thrombocytopenia become available.

Topics: Administration, Oral; Adult; Aged; Aspirin; Coronary Disease; Dose-Response Relationship, Drug; Double-Blind Method; Drug Therapy, Combination; Humans; Middle Aged; Peptides; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Thrombocytopenia; Thromboxane B2

The discovery of pathogen-recognition receptors such as Toll-like receptors on platelets has led to the emergence of the concept of platelets as important components of the host response to infection. Escherichia coli (E. coli)-mediated sepsis is a serious illness characterized by the occurrence of thrombocytopenia. Whereas there has been a wealth of research on platelet activation by Gram-positive bacteria, little is known about the mechanisms associated with Gram-negative bacteria-induced platelet activation with Gram-negative bacteria.. To determine the mechanisms by which Gram-negative E. coli induces platelet aggregation.. Induction of platelet aggregation with E. coli strain O157:H7 was tested in platelet-rich plasma (PRP), washed platelets, and serum depleted of complement factors. Platelet inhibitors (against αII b β3 , glycoprotein Ibα and FcγRIIa) were used. Platelet thromboxane synthesis was analyzed after E. coli stimulation. Cell binding assays were used to assess the ability of E. coli to support platelet adhesion. Trypsinization was used to determine the role of E. coli surface proteins.. E. coli-induced aggregation in PRP was donor-dependent. E. coli O157:H7 induced aggregation with a lag time of 6.9 ± 1.3 min in an αII b β3 -dependent and FcγRIIa-dependent manner. Furthermore, this interaction was enhanced by the presence of complement, and was dependent on thromboxane synthesis. These results show E. coli to be a potent inducer of platelet aggregation.

Topics: Blood Platelets; Cell Membrane; Escherichia coli Infections; Escherichia coli O157; Humans; Platelet Activation; Platelet Adhesiveness; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Function Tests; Platelet Glycoprotein GPIIb-IIIa Complex; Platelet-Rich Plasma; Receptors, IgG; Sepsis; Thrombocytopenia; Thromboxane B2

Collagen-induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α(2) β(1) . Adhesion and degranulation-promoting adapter protein (ADAP) regulates α(IIb) β(3) in platelets and α(L) β(2) in T cells, and is phosphorylated in GPVI-deficient platelets activated by collagen.. To determine whether ADAP plays a role in collagen-induced platelet activation and in the regulation and function of α(2) β(1).. Using ADAP(-/-) mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation.. Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP(-/-) platelets. However, aggregation and signaling induced by collagen-related peptide (CRP), a GPVI-selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α(2) β(1) -selective ligand GFOGER and to a peptide (III-04), which supports adhesion that is dependent on both GPVI and α(2) β(1), was reduced in ADAP(-/-) platelets. An impedance-based label-free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non-fluorescent differential-interference contrast microscopy, which revealed reduced filpodia formation in ADAP(-/-) platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen-binding integrin α(2) β(1). In addition, we found that ADAP(-/-) mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild-type animals. This may reflect increased removal of platelets from the circulation.

Topics: Adaptor Proteins, Signal Transducing; Animals; Blood Platelets; Carrier Proteins; CD36 Antigens; Collagen; Immunoglobulin Variable Region; Integrin alpha2beta1; Mice; Mice, Inbred C57BL; Mice, Knockout; Peptides; Phospholipase C gamma; Phosphorylation; Platelet Activation; Platelet Adhesiveness; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Pseudopodia; Splenomegaly; Thrombocytopenia; Thromboxane A2; Thromboxane B2; Time Factors; Tyrosine

AMG X, a human neutralizing monoclonal antibody (mAb) against a soluble human protein, caused thrombocytopenia, platelet activation, reduced mean arterial pressure, and transient loss of consciousness in cynomolgus monkeys after first intravenous administration. In vitro, AMG X induced activation in platelets from macaque species but not from humans or baboons. Other similar mAbs against the same pharmacological target failed to induce these in vivo and in vitro effects. In addition, the target protein was known to not be expressed on platelets, suggesting that platelet activation occurred through an off-target mechanism. AMG X bound directly to cynomolgus platelets and required both the Fab and Fc portion of the mAb for platelet activation. Binding to platelets was inhibited by preincubation of AMG X with its pharmacological target or with anti-human Fc antibodies or by preincubation of platelets with AMG X F(ab')(2) or human immunoglobulin (IVIG). AMG X F(ab')(2) did not activate platelets. Thus, platelet activation required both recognition/binding of a platelet ligand with the Fab domain and interaction of platelet Fc receptors (i.e., FcγRIIa) with the Fc domain. These findings reflect the complexity of the mechanism of action of mAbs and the increasing awareness of potential for unintended effects in preclinical species.

Topics: Administration, Intravenous; Animals; Antibodies, Monoclonal; Blood Platelets; Humans; Hypotension; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Macaca fascicularis; Male; Papio; Platelet Activation; Platelet Aggregation; Protein Binding; Serotonin; Syncope; Thrombocytopenia; Thromboxane B2

To determine whether platelet-activating factor (PAF)-induced release of cyclooxygenase products might be dependent on G proteins in vivo, we administered pertussis toxin (PTX) (9.7-10.0 micrograms/kg iv) to conscious pigs approximately 48 h before bolus infusions of PAF (10 ng/kg). Autoradiography of ADP-ribosylated lung cell membrane proteins from PTX-treated pigs demonstrated marked reduction in the amount of radiolabel ([32P]NAD) incorporated, indicating that PTX induced ADP-ribosylation of G proteins in vivo. PAF, infused at hourly intervals from 0-4 h, caused increases in plasma concentrations of thromboxane B2 (TxB2) concomitant with pulmonary hypertension and vasoconstriction in anesthetized pigs. These physiological changes were blocked or markedly attenuated by indomethacin, indicating they were dependent on cyclooxygenase products. In PTX-treated pigs, the PAF-induced pulmonary hypertension and vasoconstriction were modestly attenuated, whereas the increases in plasma TxB2 were markedly attenuated. PTX prevented PAF-induced aggregation of platelets in vivo as evidenced by blockade of thrombocytopenia. However, in vitro, PAF-induced aggregation of platelets was independent of PTX. Moreover, incubation of platelet-rich plasma with 50 microM PAF failed to increase TxB2 levels. These findings suggested that a PTX-sensitive cell other than the platelet was responsible for triggering release of TxA2 and thrombocytopenia in vivo. We conclude that PAF-induced release of TxA2, pulmonary vasoconstriction, and thrombocytopenia in anesthetized pigs are dependent on a PTX-sensitive G protein; however, the residual hemodynamic effects indicate involvement of a PTX-insensitive G protein, or alternatively, G protein independent pathways.

Topics: Animals; Blood Platelets; Calcimycin; GTP-Binding Proteins; Hemodynamics; Indomethacin; Injections, Intravenous; Lung; Pertussis Toxin; Phosphorylation; Platelet Activating Factor; Prostaglandin-Endoperoxide Synthases; Sodium Chloride; Swine; Thrombocytopenia; Thromboxane B2; Time Factors; Vasoconstriction; Virulence Factors, Bordetella

Previous studies have demonstrated that urinary thromboxane B2 (TXB2) excretion (UTXB2) and glomerular production of TXB2 are enhanced in experimental diabetes and that selective inhibitors of TX synthesis prevent or delay the development of albuminuria. The present study was conducted to examine the contribution of platelet TXB2 production to the enhancement of UTXB2 and glomerular TXB2 production and to the pathogenesis of albuminuria in the partially insulin-treated moderately hyperglycemic (blood glucose, 200 to 400 mg/dL) streptozotocin-diabetic rat (SDR). Treatment of control rats or of SDR with diabetes of 5 months' duration with antiplatelet serum for 4 consecutive days reduced circulating platelet counts and serum TXB2 generation, an index of platelet cyclooxygenase activity, by 80% or greater, but reduced UTXB2 excretion by only 30%. UTXB2 and glomerular production of TXB2 of thrombocytopenic SDR remained markedly elevated compared with corresponding values from age-matched thrombocytopenic or platelet-replete, nondiabetic controls. Similarly, treatment of rats for 180 days with a dose of aspirin (ASA), which selectively inhibited platelet versus renal cyclooxygenase activity, reduced UTXB2 of both SDR and controls by 25% to 35%. The absolute reductions in UTXB2 induced by either ASA or thrombocytopenia in SDR were significantly greater than the absolute decrements in corresponding controls, suggesting that increased platelet TXB2 production in SDR may contribute to the enhanced UTXB2. However, as in the thrombocytopenic SDR, UTXB2 and glomerular production of TXB2 of SDR treated with ASA remained clearly above corresponding control values. Moreover, chronic ASA treatment failed to prevent the development of albuminuria in SDR.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Albuminuria; Animals; Aspirin; Blood Platelets; Diabetes Mellitus, Experimental; Dinoprostone; Female; Glomerular Filtration Rate; Kidney Glomerulus; Rats; Rats, Inbred Strains; Thrombocytopenia; Thromboxane B2

When injected i.v. to guinea pigs, the granulocyte secretagog N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) induces bronchoconstriction (BC), lung platelet sequestration and increased transendothelial albumin exchanges in lungs. We evaluated BC and the variations of the lung contents in radiolabeled platelets, erythrocytes and extravascular albumin, as measurements of platelet lung entrapment, reduction of lung blood volume and increase of transendothelial albumin exchanges, respectively. Trimetoquinol, a thromboxane A2 (TXA2)-endoperoxide receptor antagonist, inhibited BC and platelet entrapment by lungs induced by fMLP, but protection was nonspecific because it also suppressed BC by histamine. The specific TXA2 synthetase inhibitor/endoperoxide receptor antagonist ridogrel suppressed BC and reduced lung platelet entrapment, but failed to prevent the increase of extravascular albumin and the decrease of erythrocyte lung contents due to fMLP. Consequently, the fMLP-induced increase of vascular albumin exchanges and reduction of lung blood volume are TXA2-independent. Aspirin prevented BC, but failed to suppress lung platelet entrapment by fMLP, indicating that in vivo platelet activation is not TXA2-dependent, even though the levels of circulating TXB2, the stable metabolite of TXA2, were increased after fMLP concomitantly with that of 6-keto-prostaglandin (PG)F1 alpha, the stable metabolite of PGI2. The ridogrel-treated animals showed reduced blood level of TXB2 and increased levels of 6-keto-PGF1 alpha after fMLP challenge. Blocking the cyclooxygenase pathway with aspirin prevented ridogrel-induced protection against lung platelet sequestration after fMLP, supporting the concept that rechanneling of arachidonate metabolism toward protective prostaglandins accounts for protection by ridogrel.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acid; Blood Platelets; Bronchoconstriction; Guinea Pigs; Imidazoles; Lung; N-Formylmethionine Leucyl-Phenylalanine; Pentanoic Acids; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Pyridines; Radioimmunoassay; Serum Albumin; Suprofen; Thrombocytopenia; Thromboxane A2; Thromboxane B2; Thromboxane-A Synthase; Tretoquinol

1. The effects of alkaline phosphatase on platelet aggregation, secretion and thromboxane B2 (TxB2) generation induced by the full dose-range of common platelet agonists were studied in human platelet-rich plasma and washed platelets. 2. Platelet aggregation and adenosine 5'-triphosphate (ATP) secretion induced by threshold and supramaximal concentrations of arachidonate and stable TxA2 and prostaglandin endoperoxide-mimetics (compounds U46619 and EP171) were abolished in the presence of alkaline phosphatase (0.5-1 u ml-1), even though the synthesis of TxB2 persisted. In contrast, platelet aggregation by PAF-acether and by supramaximal concentrations of thrombin as well as the primary wave of aggregation by adenosine diphosphate (ADP) and adrenaline were unaffected by alkaline phosphatase under conditions where the secondary wave of aggregation by ADP was blocked. 3. Alkaline phosphatase, unlike prostacyclin, failed to raise the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content of the platelets. Also, the pretreatment of platelets by inorganic phosphate or by ATP plus creatine phosphate/creatine phosphokinase reversed the inhibitory effect of alkaline phosphatase. 4. Experiments performed in the guinea-pig in vivo showed that alkaline phosphatase was effective on thrombocytopenia induced by arachidonate. 5. Our results provide the first direct evidence for a specific inhibitory effect of alkaline phosphatase at a site sensitive to TxA2 and prostaglandin endoperoxides and suggest that its phosphorylation/dephosphorylation state may play an important role in modulating platelet activation. These results also suggest the presence of ecto-protein kinases on membrane platelets.

Topics: Alkaline Phosphatase; Blood Platelets; Bronchoconstriction; Cyclic AMP; Humans; Phosphorylation; Platelet Aggregation Inhibitors; Radioimmunoassay; Thrombocytopenia; Thromboxane B2; Thromboxanes

Arthritis induced in hyperimmune rabbits by the intra-articular injection of the specific antigen was associated with a fall in circulating platelet number that lasted up to 60 days. Pretreatment of the animals with indomethacin and econazol at doses that significantly decreased thromboxane levels in the synovial fluids reduced the arthritis-related thrombocytopenia in the acute phase of arthritis. A similar inhibition was seen when L-655,240, a specific Thromboxane A2 antagonist, and BN 52021, a Platelet Activating Factor antagonist were used. The results suggest that both thromboxane and PAF are involved in the mechanisms leading to thrombocytopenia in this experimental model of arthritis.

Topics: Animals; Arthritis, Experimental; Diterpenes; Econazole; Female; Ginkgolides; Indoles; Indomethacin; Lactones; Male; Platelet Activating Factor; Rabbits; Synovial Fluid; Thrombocytopenia; Thromboxane A2; Thromboxane B2

Thrombin has been shown to increase pulmonary transvascular permeability in vivo. This permeability change appears to be dependent on polymorphonuclear leukocytes (PMNs). In vitro, thrombin has been demonstrated to increase PMN adherence to endothelial cells coincident with generation of platelet activating factor (PAF) by endothelial cells. These observations have led to the suggestion that PAF mediates, in part, the attachment of PMNs to endothelial cells. We examined this hypothesis in vivo and in vitro with a specific PAF receptor antagonist, WEB 2086. Prior infusion of WEB 2086 into conscious sheep significantly attenuated the drop in peripheral blood PMN counts observed during and after infusion of alpha-thrombin (30 NIH U/kg). These data suggest that WEB 2086 prevented PMN margination on endothelial cells. WEB 2086 also attenuated the thrombocytopenia seen after thrombin infusion and ameliorated the thrombin-induced hypoxemia and hemoconcentration. WEB 2086 did not affect the thrombin-induced hemodynamic response, the degree of intravascular coagulation as assessed by fibrin degradation product generation, or thromboxane B2 generation. In vitro, WEB 2086 prevented the augmented adherence of sheep PMNs to sheep endothelial cell monolayers after thrombin stimulation. The results of the present study are consistent with the hypothesis that PAF mediates, at least in part, thrombin-induced leukopenia and thrombocytopenia in vivo.

Topics: Animals; Azepines; Carbon Dioxide; Cell Adhesion; Leukocyte Count; Leukopenia; Platelet Activating Factor; Sheep; Thrombin; Thrombocytopenia; Thromboxane B2; Triazoles

For 11 patients with confirmed heparin-induced thrombocytopenia, we used reversible platelet inhibition with iloprost, a stable prostacyclin analogue, to permit safe heparin administration for cardiac (n = 9) or vascular (n = 2) operations. In vitro, iloprost (0.01 mumol/L) prevented both heparin-induced platelet aggregation and 14C-serotonin release in all patients. Therefore, intraoperatively, a continuous infusion of iloprost was started before administration of heparin and was continued until 15 minutes after administration of protamine. For cardiac patients, after heparin administration, the whole blood platelet count did not change (171,000 +/- 29,000/microL versus 174,000 +/- 29,000/microL, mean +/- standard error of the mean); no spontaneous platelet aggregation was observed, and plasma levels of the alpha-granule constituents platelet factor 4 and beta-thromboglobulin increased from 38 +/- 14 and 140 +/- 18 ng/mL to 591 +/- 135 and 235 +/- 48 ng/mL, respectively. Fibrinopeptide A levels actually decreased from 287 +/- 150 to 27 +/- 6 ng/mL. Furthermore, adenosine diphosphate-induced platelet activation was preserved, postoperative bleeding times were unchanged, and no heparin-related deaths occurred. Similar results were obtained in both vascular patients. We conclude that temporary platelet inhibition with iloprost now permits safe heparin administration in all patients with heparin-induced thrombocytopenia who require a cardiac or vascular operation.

Topics: Adult; Aged; Aspirin; beta-Thromboglobulin; Bleeding Time; Epoprostenol; Fibrinopeptide A; Heparin; Humans; Iloprost; Intraoperative Period; Male; Middle Aged; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Count; Platelet Factor 4; Thrombocytopenia; Thromboxane B2

Rabbits were given collagen and arachidonic acid intravenously. Blood pressure, platelet counts, plasma thromboxane-B2 (TXB2) and plasma 6-keto-prostaglandin F1 alpha, (6-keto-PGF1 alpha) were determined. Both thrombogenic agents, upon infusion of a lethal dose, caused thrombocytopenia, indicative of in vivo platelet aggregation and hypotension. These changes were associated with an increase in plasma levels of TXB2 and 6-keto-PGF1 alpha measured by radioimmunoassay (RIA). Pretreatment of rabbits with an aqueous extract of garlic (500 mgkg) provided protection from thrombocytopenia and hypotension. Thromboxane-B2 synthesis was significantly reduced in animals pretreated with garlic and then injected with a lethal dose of either collagen or arachidonic acid. The amount of TXB2 synthesized in these animals was not sufficient to induce thrombocytopenia or hypotension. All animals pretreated with garlic were well protected against the effects of collagen or arachidonate infusion, and no apparent symptoms were observed in these animals. These observations indicate that garlic may be beneficial in the prevention of thrombosis.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acids; Blood Pressure; Collagen; Female; Fibrinolytic Agents; Garlic; Hypertension; Plants, Medicinal; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Count; Rabbits; Thrombocytopenia; Thromboxane B2

HLA alloimmunization is a major problem for thrombocytopenic patients receiving long-term platelet support. It is caused by white cells (WBCs) that are present as contaminants in platelet concentrates (PCs). Recent data have shown that filtration is an effective means to reduce WBC contamination, but it has little effect on the recovery of platelets. The present report evaluates two filters, a cellulose acetate (CA) filter requiring the inactivation of platelets with prostacyclin and a cotton wool (CW) filter requiring no platelet inactivation. The results show that, using fresh pooled PCs from six random donors, both filters reduce WBC contamination below 10(7) per PC, the likely threshold below which alloimmunization does not develop. With platelets stored for 2 to 3 days the efficacy of the CW filter decreases. Neither filter inflicts important damage to the platelets, as there is no considerable platelet activation or cell disruption. Moreover, PCs prepared by both filters show normal survival and effectively reduce the bleeding times. Thus, filtration of PCs results in platelets with optimal responsiveness both in vitro and in vivo.

Topics: Animals; Bleeding Time; Blood Platelets; Blood Transfusion; Cell Separation; Cell Survival; Cellulose; Cytoplasmic Granules; Filtration; Gossypium; Humans; Leukocyte Count; Leukocytes; Platelet Activation; Platelet Count; Platelet Transfusion; Thrombocytopenia; Thromboxane B2; Wool

Platelet-activating factor (PAF) has been demonstrated in the circulation and organs of animals exposed to gram negative endotoxins, whereas PAF antagonists have been shown to exhibit some efficacy in modifying the course of endotoxemia. In this study we evaluated BN 50739, a novel specific PAF antagonist, for its capacity to block PAF or lipopolysaccharide endotoxin (LPS)-mediated effects in rabbits. Pretreatment with BN 50739 (3 and 10 mg/kg i.p.) inhibited PAF (500 pmol/kg i.v.)-induced thrombocytopenia, leukopenia and plasma thromboxane B2 elevation in a dose-dependent manner. The inhibitory effect lasted 3.5 to 4.5 hr. BN 50739 (10 mg/kg) prevented the early phase of LPS (50 micrograms/kg i.v.)-induced thrombocytopenia and thromboxane B2 elevation, and reduced the 24-hr mortality rate from 75 to 22% (P less than .05). Post-treatment with BN 50739 increased the 10-hr survival rate from 33 to 87% (P less than .05); however, it had no effect on the 24-hr mortality. BN 50739 did not affect LPS-induced leukopenia or the elevation in plasma tumor necrosis factor. Our data support possible therapeutic efficacy of PAF antagonists in septic shock despite their inability to prevent the generation of tumor necrosis factor.

Topics: Animals; Azepines; Blood Cell Count; Endotoxins; Leukopenia; Male; Platelet Activating Factor; Rabbits; Thrombocytopenia; Thromboxane B2; Triazoles

The production of thromboxane A2 (TxA2) and prostacyclin (PGI2) was studied in patients with chronic idiopathic thrombocytopenic purpura (10 patients) compared to central thrombocytopenia (five patients) and healthy subjects (10 subjects). This production was monitored by the assay of urinary 2,3-dinor-TxB2 and 2,3-dinor-6-keto-PGF1 alpha as respective breakdown products of TxA2 and PGI2 by stable isotope dilution assays employing negative ion-chemical gas-chromatography-mass-spectrometry. Evidence is presented for the existence of an enhanced PGI2 and TxA2 urinary excretion in the group of idiopathic thrombocytopenic purpura (ITP) patients. Moreover, production of serum TxB2 per platelet was decreased in ITP group. These results provide arguments for an in vivo platelet cyclooxygenase hyperactivity during chronic ITP.

Topics: 6-Ketoprostaglandin F1 alpha; Adult; Epoprostenol; Female; Humans; Male; Middle Aged; Platelet Count; Prospective Studies; Purpura, Thrombocytopenic; Thrombocytopenia; Thromboxane A2; Thromboxane B2

Heparin-induced thrombosis is due to an immune-mediated activation of circulating platelets and has significant clinical implications for patients with vascular disease. The purpose of this article was (1) to define the biochemical mechanisms of heparin-induced platelet activation (HIPA) and (2) to determine the relationship between thromboxane A2 (TxA2) synthesis and platelet granule release. In two patients with confirmed HIPA, heparin (3 U/ml) induced extensive platelet aggregation (61.5%), release of 14C-serotonin (81.5% of releasable 14C-serotonin, a dense granule marker) and platelet factor 4 (63.7% of releasable platelet factor 4, an alpha granule marker) and generation of TxB2, a stable metabolite of TxA2 (100% relative to serum control). In one patient heparin did not induce release of n-acetyl-beta-glucosaminadase (N-AC, a lysosomal granule marker), and aspirin (4 mmol/L), which abolished TxA2 synthesis, prevented aggregation and granule release. In the second patient heparin did induce release of N-AC (39.7% of releasable N-AC) and aspirin, despite abolishing TxA2 synthesis, did not prevent aggregation or granule release. In contrast, by elevating intracellular cyclic adenosine monophosphate, iloprost (0.01 mumol/L), a stable prostacyclin analogue, prevented heparin-induced aggregation, granule release, and TxB2 generation in both patients. Thus we show (1) HIPA can proceed independently of TxA2 synthesis; (2) heparin in certain patients can release lysosomal hydrolases, thus mimicking strong platelet agonists such as thrombin; and (3) iloprost but not aspirin prevents HIPA regardless of the biochemical pathways involved.

Topics: Aged; Cytoplasmic Granules; Female; Heparin; Humans; Platelet Aggregation; Thrombocytopenia; Thromboxane A2; Thromboxane B2

Studies were performed to determine the cross-reaction rate of the heparin-dependent antibody with Org 10172, a new low molecular weight heparinoid, and to investigate the effect of Org 10172 on platelet activation induced by the antibody. The plasmas of 17 patients with thrombocytopenia induced by standard heparin were shown, by platelet aggregation studies, to contain the heparin-dependent antibody. Of these 17 patient plasmas, only three cross-reacted with the heparinoid, producing a cross-reaction rate of 18%. When Org 10172 was added to a reaction mixture containing normal platelet-rich plasma, patient plasma, and standard heparin with non-cross-reacting plasmas, it inhibited platelet aggregation and thromboxane B2 production induced by the antibody, provided that the ratio of Org 10172 concentration (anti-Xa U/mL) to standard heparin concentration (IU/mL) exceeded 2.5 to 5.0. This inhibitory effect was observed only with platelet activation mediated by the antibody, but not by collagen (2 micrograms/mL) or ADP (5.0 mumol/L). Additionally, three of 17 patients with serious thrombosis, whose plasma showed no cross-reaction with the heparinoid, received Org 10172 treatment with a good response in each case. These findings suggest that Org 10172 may be a useful drug for the treatment of heparin-induced thrombocytopenia.

Topics: Antigen-Antibody Reactions; Blood Platelets; Chondroitin Sulfates; Cross Reactions; Dermatan Sulfate; Glycosaminoglycans; Heparin; Heparitin Sulfate; Humans; In Vitro Techniques; Molecular Weight; Platelet Aggregation Inhibitors; Thrombocytopenia; Thrombosis; Thromboxane B2

The variations in platelet counts upon intravenous challenge with soluble aggregates of IgG were assessed in normal rats. A time- and dose-dependent thrombocytopenia, followed by recovery to preinfusion values after 30 minutes was observed. Rats injected with immune aggregates showed an increase in plasma levels of immunoreactive thromboxane B2, however, this increase was delayed as compared with the peak level of the thrombocytopenia. Previous treatment of rats with either indomethacin or aspirin, inhibited thromboxane B2 release, but did not affect thrombocytopenia. Pretreatment of the animals with BN 52021, a potent antagonist of platelet-activating factor binding to its receptor, also failed to block thrombocytopenia. Complement depletion by prior treatment with cobra venom factor, caused a significant reduction of the thrombocytopenia, whereas DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, an inhibitor of carboxypeptidase N, potentiated the thrombocytopenia elicited by submaximal doses of either IgG aggregates or a homogeneous preparation of rat anaphylatoxin containing C5a. In addition, rats challenged with doses of IgG aggregates higher than 5 mg/kg showed a massive complement consumption coincident with the onset of thrombocytopenia. "In vitro" aggregation/secretion experiments with rat platelets showed little platelet-stimulating activity either by aggregated IgG through the Fc receptor or through the CR1 receptor. By contrast, a preparation of rat serum anaphylatoxins containing C5a, showed a high platelet-secreting activity. These data suggest that a complement-derived peptide(s), most probably C5a, is one of the effector substances for platelet activation in response to soluble aggregates of IgG.

Topics: Anaphylatoxins; Animals; Antigen-Antibody Complex; Complement C5; Complement C5a; Immunoglobulin G; Peptides; Platelet Count; Rats; Rats, Inbred Strains; Thrombocytopenia; Thromboxane A2; Thromboxane B2

The effects of a highly selective 5-lipoxygenase inhibitor, CGS8515 [methyl 2-[(3,4-dihydro-3,4-dioxo-1-naphthalenyl) amino]benzoate], on endotoxic shock sequelae and eicosanoid synthesis by peritoneal macrophages were evaluated in the rat. Pretreatment of peritoneal macrophages in vitro with CGS8515 significantly inhibited the synthesis (P less than .01) of immunoreactive leukotriene C4/leukotriene D4 stimulated by the calcium ionophore (A23187). Inhibition of 5-lipoxygenase produced significant shunting to immunoreactive thromboxane B2 formation (P less than .05). In rats sedated with ketamine.HCl (82.5 mg/kg) and xylazine. HCl (27.5 mg/kg), i.v. injection of Salmonella enteritidis endotoxin (25 mg/kg i.v.) produced significant decreases at 30 min in mean arterial pressure (from 89 +/- 4 to 44 +/- 8 mm Hg, N = 5, P less than .001); in white blood cell count (from 10.8 +/- 0.6 to 6.5 +/- 0.8 x 10(3)/mm3, N = 5, P less than .01); in platelet count (from 687 +/- 66 to 392 +/- 65 x 10(3)/mm3, N = 5, P less than .01); and produced an increase of hematocrit (from 46 +/- 1.2 to 57.4 +/- 1.8%, N = 5, P less than .03). CGS8515 (5 mg/kg i.v. 30 min before endotoxin injection, N = 6) blunted the endotoxin-induced hypotension by 35% (P less than .001), the leukopenia by 24% (P less than .03), the thrombocytopenia by 45% (P less than .006) and the hemoconcentration by 16% (P less than .03), compared to the shocked control rats 30 min after endotoxin injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arachidonate Lipoxygenases; Blood Pressure; Hematocrit; Leukopenia; Lipoxygenase Inhibitors; Male; Naphthoquinones; ortho-Aminobenzoates; Rats; Shock, Septic; Thrombocytopenia; Thromboxane B2

Recurrent thrombocytopenia, thrombosis, or sudden death may develop in patients with heparin-induced thrombocytopenia who are reexposed to heparin. Three patients came to us in whom a diagnosis of heparin-induced thrombocytopenia had been made on the basis of clinical and serologic evidence; these patients required reexposure to heparin because of urgent cardiac surgery. Therefore, we evaluated the ability of iloprost (ZK36374), a new analogue of prostacyclin, to prevent heparin-dependent activation of platelets and thereby permit obligatory heparinization for safe extracorporeal circulation. Before operation, we demonstrated that iloprost prevented both heparin-dependent platelet aggregation and tritiated (3H)-serotonin release in vitro. Therefore a continuous infusion of iloprost was begun 1 hour before heparinization and was continued throughout cardiopulmonary bypass and for an additional 15 minutes after protamine administration. The mean platelet count of 130,000/microliters before operation remained stable, and no spontaneous platelet aggregation was observed in samples of platelet-rich plasma obtained before cardiopulmonary bypass but after heparin administration. Similarly, after heparin administration but before bypass, platelet responsiveness to adenosine diphosphate remained unchanged when compared with preoperative values. Plasma levels of platelet factor 4 increased from 26 +/- 1 ng/ml (mean +/- standard error) to 843 +/- 383 ng/ml after heparin administration but actually decreased throughout cardiopulmonary bypass to 52 +/- 25 ng/ml. Beta-thromboglobulin levels increased from 103 +/- 16 to 244 +/- 94 ng/ml with heparinization. The mean bleeding time was 10.5 minutes preoperatively and 13.3 minutes postoperatively. The mean amount of postoperative chest tube drainage (duration: 12 hours) was 432 +/- 67 ml. Thus, despite the confirmed presence of heparin-dependent platelet-activating factor in the plasma of these three patients, iloprost prevented heparin-induced platelet activation during cardiopulmonary bypass while preserving platelet function, as would be desired for postoperative hemostasis.

Topics: Adult; Cardiopulmonary Bypass; Cardiovascular Agents; Epoprostenol; Fibrinopeptide A; Heparin; Humans; Iloprost; Middle Aged; Platelet Aggregation; Platelet Count; Platelet Factor 4; Thrombocytopenia; Thromboxane B2

The hypothesis that thrombocytopenia in liver cirrhosis (LC) could be due to platelet activation was investigated. 18 patients with thrombocytopenia and LC have been studied. Circulating beta-thromboglobulin (beta TG) was normal, but appeared elevated when referred to platelet count. However, this may not accurately reflect alpha-granule release because of reduced liver cell function. Intraplatelet beta TG, on the contrary, should not be affected by liver cell function. It was markedly depressed, thus truly suggesting the existence of an exhausted state caused by platelet activation. Thromboxane B2 production was slightly increased. This could be due to a compensatory mechanism, or simply to platelet size, which was slightly increased, too. Anti-platelet therapy failed to improve thrombocytopenia. A platelet activation state seems therefore to be present in LC, but seems not to be the only cause of thrombocytopenia. Platelet factor 4 approximated zero, regardless of platelet count and therapy. This confirms that elevated values of such a protein represent only a laboratory artifact, due to platelet activation in vitro.

Topics: Aged; beta-Thromboglobulin; Blood Platelets; Dipyridamole; Female; Humans; Isoindoles; Liver Cirrhosis; Male; Middle Aged; Phenylbutyrates; Platelet Aggregation; Platelet Count; Platelet Factor 4; Thrombocytopenia; Thromboxane B2

Changes in the production of proaggregatory (thromboxane A2 and prostaglandin E2) and antiaggregatory (prostacyclin) prostaglandins by blood platelets, macrophages and endothelial cells during acute African swine fever caused by both a highly virulent virus and a less virulent virus were studied. No impairment in thromboxane A2 release by either platelets or macrophages could be detected but prostacyclin production by the endothelium was impaired. There was also a significant increase in prostaglandin E2 release by macrophages at the time when thrombocytopenia was most marked. However, the early event that causes primary aggregation remains obscure.

Topics: African Swine Fever; Animals; Arachidonic Acids; Blood Coagulation; Blood Platelets; Dinoprostone; Endothelium; Hematocrit; Hemorrhage; Macrophages; Platelet Aggregation; Platelet Function Tests; Prostaglandins E; Swine; Swine Diseases; Thrombocytopenia; Thromboxane B2

We found a novel platelet aggregating factor in a patient with steroid-responsive immune thrombocytopenic purpura that is associated with defective collagen-induced platelet functions. The aggregating factor and platelet functions were analyzed. The patient, a 58-year-old female, had purpura and prolonged bleeding time despite adequate platelet counts (greater than 140,000/microL) after steroid therapy. The patient's platelets responded normally to all agonists except collagen. Platelet adhesion to collagen fibrils was decreased. The patient's plasma induced irreversible aggregation and ATP release in normal platelet-rich plasma (PRP). This platelet aggregating factor was found in F(ab')2 fragments of the patient's IgG, which caused thromboxane B2 synthesis, elevation of cytoplasmic Ca2+ levels, and phosphorylation of 40 kDa protein in normal platelets. Platelet aggregation by the patient's IgG was inhibited by prostacyclin, dibutyryl cAMP, diltiazem, disodium ethylenediaminetetraacetate, and antimycin A plus iodoacetate, but ADP scavengers, cyclo-oxygenase inhibitors, and heparin had little or no effect. The aggregating activity of the patient's IgG absorbed to and eluted from normal platelets. The patient's Fab fragments did not induce platelet aggregation in eight of ten normal PRP but specifically inhibited aggregation induced by collagen and by the patient's IgG. The major component of an immunoprecipitate made with the patient's IgG from radiolabeled membrane proteins of normal platelet extract had a 62 kDa mol wt, while no such precipitate appeared in extracts of the patient's platelets. These results indicated that platelet aggregation by the patient's IgG was induced by the reaction of an antibody with a specific antigen on the normal platelet membrane through stimulus-response coupling. This antigen may be a collagen receptor on the platelet, most likely a polypeptide of 62 kDa under reducing condition. The defect of collagen-induced aggregation of the patient's platelets seemed to be due to alteration of the membrane protein related to this putative collagen receptor.

Topics: Autoimmune Diseases; Blood Coagulation Factors; Blood Platelet Disorders; Collagen; Female; Humans; Immunoglobulin G; Middle Aged; Platelet Activating Factor; Platelet Aggregation; Purpura, Thrombocytopenic; Thrombocytopenia; Thromboxane B2

Neonatal and young animals fail to develop antigen-induced, lethal, systemic anaphylactic reactions. Recent evidence has documented that, in the adult rabbit, an unusual phospholipid autacoid, platelet-activating factor, induces almost all of the physiologic events associated with IgE-induced anaphylaxis. Thus, in the present study, the intravascular alterations after intravenous infusion of synthetic platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine; AGEPC) into young rabbits were examined. In comparison to 13-week-old, adult rabbits, the intravascular infusion of greater than 1.0 micrograms/kg AGEPC was not lethal in rabbits of 8 weeks of age or less. In dose-response studies, the amount of AGEPC required to induce a lethal response in 50% of the animals tested (LD50) was found to inversely correlate with age. In contrast, AGEPC-induced platelet aggregation in vitro was not affected by the age of the donor animal. Consistent with age-independent platelet responsiveness in vitro, AGEPC-induced thrombocytopenia and intravascular accumulation of platelet factor 4 and thromboxane B2 were also unaffected by animal age. Neutropenia and basopenia, as well as platelet and neutrophil sequestration in the pulmonary microvasculature after intravenous AGEPC infusion also were similarly unaffected by animal age. Although the mechanisms which modulate the profound and lethal physiologic responses following AGEPC infusion in the adult rabbit remain to be established, the current study clearly documents an age-dependent acquisition of systemic physiologic sensitivity to AGEPC and/or other mediators released as a result of intravascular AGEPC administration.

Topics: Aging; Anaphylaxis; Animals; Animals, Newborn; Female; Leukopenia; Lung; Male; Neutrophils; Platelet Activating Factor; Platelet Aggregation; Platelet Factor 4; Rabbits; Thrombocytopenia; Thromboxane B2

The interaction between the triazolothienodiazepine WEB 2086 and the in vitro and in vivo bronchopulmonary effects of PAF-acether and active/passive anaphylaxis in the guinea-pig was studied. WEB 2086 (1-100 nM) inhibited PAF-acether (10-100 ng)-induced bronchoconstriction and TXB2 release from isolated and perfused guinea-pig lungs without affecting the response to 100 micrograms arachidonic acid. In addition, 1-10 microM WEB 2086 significantly reduced antigen-induced TXB2 and histamine release from lungs from actively and passively sensitized guinea-pigs. In the presence of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), mepyramine, methysergide, indomethacin and atropine, WEB 2086 (20-50 microM) inhibited by 30-40% the residual contraction of lung parenchyma strips from guinea-pigs actively sensitized by 0.1-10 micrograms antigen. In vivo, WEB 2086 (0.1-1 mg/kg) reversed or abolished the bronchoconstriction, hypotension, thrombocytopenia and leukopenia evoked by perfusion of PAF-acether (3 or 44 ng/kg per min). At 3 mg/kg, WEB 2086 also markedly decreased the bronchoconstriction and leukopenia induced by 100 micrograms/kg antigen in mepyramine (5 micrograms/kg)-treated passively sensitized guinea-pigs. In contrast, WEB 2086 was ineffective against active anaphylaxis in vivo. These results demonstrate that WEB 2086 antagonizes the bronchopulmonary effects due to PAF-acether and to anaphylactic shock in the guinea-pig.

Topics: Anaphylaxis; Animals; Azepines; Female; Guinea Pigs; Histamine; In Vitro Techniques; Indicators and Reagents; Leukopenia; Male; Passive Cutaneous Anaphylaxis; Platelet Activating Factor; Radioimmunoassay; Respiratory System; Spectrometry, Fluorescence; Thrombocytopenia; Thromboxane B2; Triazines; Triazoles

In animals, monocrotaline induces an acute lung injury secondary to capillary endothelial damage. To date, no reports have appeared dealing with the role of prostaglandins in monocrotaline-induced injury. Our studies, in dogs, revealed that monocrotaline (30 mg/kg iv) caused an acute and persistent thrombocytopenia, lung platelet deposition, pulmonary hypertension, and increased extravascular lung water (EVLW). The pulmonary hypertensive response was biphasic. Thromboxane B2 levels were similarly biphasic, peaking at 5 min and 2 h. The levels of 6-keto-PGF1 alpha peaked at 30 min and returned to base line at 3 h. Pulmonary vascular resistance paralleled thromboxane levels. Infusion of prostacyclin (PGI2) at 50 ng X kg-1 X min-1 effectively prevented the thrombocytopenia, lung platelet deposition, pulmonary hypertension, and increased EVLW; and it decreased excess thromboxane production by 79%. These results suggest that platelet activation and lung sequestration play a role in acute lung injury due to monocrotaline, and that the resultant thromboxane production may contribute to the pulmonary hypertension. PGI2 ameliorates monocrotaline-induced injury, perhaps by preventing platelet activation.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Blood Cell Count; Blood Platelets; Body Water; Dogs; Epoprostenol; Hemodynamics; Hypertension, Pulmonary; Lung; Lung Injury; Monocrotaline; Pyrrolizidine Alkaloids; Thrombocytopenia; Thromboxane B2

A patient is described who presented a thrombocytopenia with thrombocytopathy followed by the development of a leukaemia. The disorder was characterized by decreased aggregation in the presence of ADP, and a lack of aggregation in the presence of arachidonic acid, natural endoperoxide or collagen. In parallel, 14C-serotonin release was severely decreased or nil in response to these inducers. Thrombin induced a slightly decreased aggregation and a normal 14C-serotonin release. Thromboxane B2 (T X B2) synthesis was normal after stimulation by arachidonic acid, natural endoperoxide or thrombin showing a normal arachidonate metabolism. In addition, the mepacrine test showed no significant decrease of the number of dense bodies with an average of 4.6 per platelet (versus 5.4 +/- 0.8 sd in controls). Stimulation by ionophore A 23187 failed to induce aggregation, 14C-serotonin release, or T X B2 synthesis. Furthermore, in the presence of EDTA, A 23187 did not provoke activation as reflected by 14C-serotonin release or T X B2 synthesis. Thus, in this case of thrombocytopathy, the hypothesis of abnormal intracellular Ca++ fluxes responsible for the defective platelet release phenomenon, was suggested.

Topics: Adenosine Diphosphate; Blood Platelet Disorders; Blood Platelets; Calcimycin; Calcium; Edetic Acid; Female; Follow-Up Studies; Humans; Leukemia; Middle Aged; Platelet Aggregation; Quinacrine; Serotonin; Thrombocytopenia; Thromboxane B2

The purpose of this study was to compare the effects of dazoxiben (DAZ), UK 38,485 (UK) and aspirin (ASA) in the prevention of thrombotic sudden death in rabbits. In anesthetized male rabbits, sudden death was produced by intravenous administration of 0.75 mg/kg arachidonic acid (AA). AA increased plasma TxB2 levels from 0.20 +/- 0.10 ng/ml to 8.75 +/- 1.79 ng/ml and produced a 42% reduction in the number of circulating platelets. Death occurred in all animals within 5 minutes. Administration of DAZ (8.6 mumole/kg) 15 min before AA prevented the increase in plasma TxB2, the thrombocytopenia and sudden death while pretreatment with DAZ 2 hr before AA did not. The administration of UK (8.6 mumole/kg) 15 min. 4 hrs or 8 hrs before AA resulted in 100%, 67% and 33% survival, respectively. ASA (110 mumole/kg) administered 2 or 24 hrs before AA inhibited the increase in plasma TxB2 and prevented the fall in platelet counts. All animals pretreated with ASA survived. These data demonstrate that DAZ and UK have only a short to moderate duration of action in preventing AA-induced increases in plasma Tx levels and thrombocytopenia.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acid; Arachidonic Acids; Aspirin; Imidazoles; Male; Rabbits; Thrombocytopenia; Thrombosis; Thromboxane B2; Time Factors

The role of thromboxane (Tx) A2 in Streptococcus pneumoniae-induced granulocytopenia, thrombocytopenia, and pulmonary leukostasis is unclear. Rabbits were injected with 0.85% NaCl, nonviable pneumococci, or nonviable pneumococci after pretreatment with TxA2 synthetase inhibition. Blood was obtained immediately before and at times after injection for granulocyte and platelet counts and assays of TxB2 and 6-keto prostaglandin F1 alpha (6-ketoPGF1 alpha). Animals were evaluated for pulmonary leukostasis histologically and biochemically (myeloperoxidase activity). Pneumococcal challenge induced significant granulocytopenia (P less than .001), thrombocytopenia (P less than .001), and elevations in levels of both TxB2 (P less than .05) and 6-ketoPGF1 alpha (P less than .001) as well as pulmonary leukostasis (P less than .001). TxA2 synthetase inhibition blocked the pneumococcus-induced elevation in level of TxB2 without significantly altering levels of circulating granulocytes, platelets, or 6-ketoPGF1 alpha. Pulmonary leukostasis was not blocked. In another group of pneumococcus-challenged animals, no significant transpulmonary gradients of either TxB2 or 6-ketoPGF1 alpha were found.

Topics: 6-Ketoprostaglandin F1 alpha; Agranulocytosis; Animals; Epoprostenol; Lung; Methacrylates; Rabbits; Streptococcal Infections; Thrombocytopenia; Thromboxane B2; Thromboxane-A Synthase; Thromboxanes

We have evaluated several effects of intravenous administration of synthetic platelet-activating factor (PAF) in the non-human primate Cebus apella. Parameters measured were hemoconcentration (monitored by changes in hematocrit), thrombocytopenia (platelet counts), leukopenia (loss of buffy coat), bronchoconstriction (increased airway resistance to fixed airway ventilation), thromboxane A2 production (radioimmunoassay to thromboxane B2) and in vitro aggregation responses of platelets in platelet-rich plasma. Cebus platelets were refractory to PAF-induced aggregation (up to 50 microM) and there was no evidence of thrombocytopenia, elevated thromboxane B2 levels, loss of buffy coat or bronchoconstriction following systemic PAF injection. Animals exhibited reproducible but varying sensitivities to PAF-induced hemoconcentration, where 3.5-30 micrograms/kg PAF (6.6-57 nmol/kg) was required to produce 28-32% increased hematocrit range for the colony. Hemoconcentration induced by PAF in baboons and rhesus occurred at similar doses, suggesting comparable sensitivity. Prior administration of PAF receptor antagonists SRI 63-072 or SRI 63-119 at 3 mg/kg inhibited cebus hemoconcentration responses to 3.5 micrograms/kg PAF by 96% and 100%, respectively. The ED50 values were 0.95 and 0.60 mg/kg, respectively. These results suggest that the cebus exhibits a reproducible hemoconcentration effect to PAF and that these vascular responses can be inhibited by a PAF receptor antagonist.

Topics: Animals; Cebus; Indomethacin; Lung; Macaca mulatta; Male; Papio; Platelet Activating Factor; Platelet Aggregation; Thiazoles; Thrombocytopenia; Thromboxane B2

Topics: Aged; Arginine; Female; Heparin; Humans; Middle Aged; Pipecolic Acids; Platelet Aggregation; Renal Dialysis; Sulfonamides; Thrombin; Thrombocytopenia; Thromboxane B2

CGS 12970 is a potent selective inhibitor of human platelet thromboxane synthetase in vitro (IC50 = 12 nM). It is four orders of magnitude less potent as an inhibitor of sheep seminal vesicle cyclooxygenase, bovine aorta prostacyclin synthetase and human leucocyte 15-lipoxygenase. The compound inhibited collagen-induced thromboxane B2 production by human platelets in vitro without an effect on the accompanying platelet aggregation induced by collagen, ADP, platelet activating factor, thrombin, arachidonic acid or the prostaglandin mimetic, U 46619. Administration of CGS 12970 to rats inhibited collagen-induced thromboxane B2 production but had no effect on platelet aggregation ex vivo. It also had no effect on platelet aggregation induced by thrombin or on plasma clotting times. A single oral dose of 1 or 3 mg kg-1 to rabbits inhibited thromboxane B2 production in clotting blood ex vivo for 12 or 24 h respectively. CGS 12970 inhibited thromboxane B2 production in vivo induced by intravenous administration of collagen to rats or calcium ionophore to guinea-pigs. In both cases there was a concomitant elevation of immunoreactive 6-keto-prostaglandin F1 alpha but no effect on the induced thrombocytopenia. As with other thromboxane synthetase inhibitors, CGS 12970 prolonged tail bleeding time in the rat. However, CGS 12970 was not as potent as other thromboxane synthetase inhibitors in this test. In addition to these usual effects of thromboxane synthetase inhibitors, CGS 12970 inhibited the thrombocytopenia induced by the Forssman reaction or ADP administration. In these tests the effect of the compound was of short duration. 8 CGS 12970 had no effect on the thrombocytopenia associated with the Arthus reaction which distinguishes it from cyclo-oxygenase inhibitors. It also had no effect on thrombus formation on a cotton thread in an arteriovenous shunt in the rat.

Topics: Animals; Arachidonic Acids; Arthus Reaction; Blood Coagulation; Blood Platelets; Collagen; Forssman Antigen; Guinea Pigs; Humans; In Vitro Techniques; Pyridines; Rabbits; Radioimmunoassay; Rats; Rats, Inbred Strains; Thrombocytopenia; Thromboxane B2; Thromboxane-A Synthase; Time Factors

Ten patients for elective cholecystectomy were studied pre-, per-and postoperatively. All had neurolept anesthesia. Plasma concentrations of beta-TG, TXB2 and 5-HT and intraplatelet 5-HT were measured. Aggregation to ADP was recorded. Serum cortisol concentration was used as index of the stress response, showing peroperative increase and postoperative decrease. Closely related to this we observed a significant increase in P--beta-TG and P-TXB2 with postoperative normalization in 6 patients without complications. P--5-HT had a peak peroperatively and remained elevated postoperatively. A negative correlation between P--5-HT and decreasing intraplatelet 5-HT postoperatively was observed. High postoperative levels of P--5-HT seem to be related to low arterial PO2 and pulmonary dysfunction. In 3 patients with complications a second increase in P--beta-TG, P-TXB2 and partly in P--5-HT was found. Platelets were temporarily refractory to ADP immediately following surgery and showed increased aggregability postoperatively. We conclude that platelets are activated in surgical stress.

Topics: Adenosine Diphosphate; Adult; Aged; beta-Thromboglobulin; Blood Platelets; Cholecystectomy; Female; Humans; Hydrocortisone; In Vitro Techniques; Male; Middle Aged; Platelet Aggregation; Postoperative Complications; Serotonin; Stress, Physiological; Thrombocytopenia; Thromboxane B2

The interactions between platelets and dialysis membranes were studied prospectively in 10 patients undergoing long-term stable dialysis. Transient but significant thrombocytopenia and platelet activation were found during dialysis with the commonly used cuprophane membrane. Platelet counts decreased from 231 +/- 21 X 10(3)/mm3 before dialysis to 127 +/- 28 X 10(3)/mm3 at 90 minutes following initiation of dialysis (p less than or equal to 0.007). Thromboxane B2, an index of platelet activation, also increased from a baseline level of 1.06 +/- 0.2 pg/10(6) platelets to 7.3 +/- 3.0 pg/10(6) platelets at 90 minutes (p less than or equal to 0.04). Cuprophane membranes were also shown to induce complement activation with C3a desArg, the stable derivative of C3 activation, showing a threefold increase from baseline 15 minutes after initiation of dialysis. In contrast, during dialysis with a non-complement-activating dialyzer membrane, polymethylmethacrylate, thrombocytopenia and platelet activation were not observed. These data suggest that platelet activation and thrombocytopenia during hemodialysis are associated with complement activation during hemodialysis in a manner similar to dialysis-associated neutropenia.

Topics: 6-Ketoprostaglandin F1 alpha; Blood Platelets; Cellulose; Complement Activation; Epoprostenol; Humans; Membranes, Artificial; Methylmethacrylates; Platelet Count; Prospective Studies; Renal Dialysis; Thrombocytopenia; Thromboxane B2; Time Factors

We have previously demonstrated that platelets obtained from patients with anorexia nervosa or severe peripheral vascular disease are hyperaggregable. Since conventional heparins are known to activate platelets in vitro and occasionally induce thrombosis and consumptive thrombocytopenia in vivo, we have investigated the direct effect of a conventional heparin on platelets obtained from patients with anorexia nervosa or severe peripheral vascular disease. Heparin at therapeutic concentrations was found to induce platelet aggregation of such platelets in vitro. In contrast, a recently developed low molecular weight heparinoid (Org 10172), at therapeutic concentrations, had no effect on these hyperaggregable platelets. We conclude that: heparin may be potentially harmful to patients with hyperaggregable platelets; thrombocytopenia and thrombosis associated with heparin therapy may be mediated through a direct effect of heparin on platelets; it is unlikely that heparin induced thrombocytopenia is always mediated by classical immunological mechanisms, especially in patients with hyperaggregable platelets; and low molecular weight heparinoids may be safer anticoagulants in patients with platelet hyperaggregability.

Topics: Adolescent; Adult; Aged; Anorexia Nervosa; Blood Platelets; Chondroitin Sulfates; Collagen; Dermatan Sulfate; Epinephrine; Female; Glycosaminoglycans; Heparin; Heparitin Sulfate; Humans; Immunoglobulin G; In Vitro Techniques; Male; Middle Aged; Platelet Aggregation; Thrombocytopenia; Thromboembolism; Thromboxane B2; Vascular Diseases

A prospective study was performed on 26 preeclamptic patients and 17 pregnant control subjects relating the platelet count to in vivo platelet function as assessed by the bleeding time and in vitro platelet function as assessed by collagen-stimulated thromboxane B2 biosynthesis. The results of these tests were normal in all control subjects. Nine of the 26 preeclamptic patients (34%) showed thrombocytopenia, and five of these patients had a prolonged bleeding time. Four of the 16 nonthrombocytopenic patients also had a prolonged bleeding time. Eleven patients had impaired thromboxane B2 biosynthesis, and seven of these had a prolonged bleeding time. In all patients, the bleeding time returned to normal, and in most the platelet count returned to normal within five days or after delivery. A significant proportion of patients with preeclampsia develop an acquired defect of platelet function that could contribute to bleeding.

Topics: Bleeding Time; Blood Platelets; Female; Humans; Platelet Count; Platelet Function Tests; Pre-Eclampsia; Pregnancy; Prospective Studies; Thrombocytopenia; Thromboxane B2

A modification of the original thiobarbituric acid (TBA) method for malondialdehyde (MDA) assay is described. The improvement is essentially based on the clearing effect of KClO4 that makes measurements more simple and sensitive. MDA values obtained in normal subjects were almost eightfold higher than those obtainable with Stuart's original assay method, so that after cyclooxygenase block it was possible to assess platelet regeneration time even in thrombocytopenic patients with at least 60,000 platelets/microliter and MDA production early after aspirin intake. To challenge this modification, platelet regeneration time was studied in normal subjects as well as in thrombocytopenic patients, either hypoplastic or idiopathic, and in hypoxemic patients with increased platelet consumption. The initial disappearance kinetics of platelet MDA and thromboxane B2 production after aspirin suggests that TBA-reactive material is synthesized through the lipoxygenase pathway. The reversible block determined by acetylsalicylic acid and salicylate on hydroperoxi-eicosatetraenoic acid peroxidase can be responsible for the initial increase of this TBA-reactive material.

Topics: Adult; Aged; Aspirin; Blood Platelets; Cell Survival; Cyclooxygenase Inhibitors; Humans; Lung Diseases, Obstructive; Malonates; Malondialdehyde; Middle Aged; Prostaglandin-Endoperoxide Synthases; Thrombocytopenia; Thromboxane B2

The arachidonic acid metabolites thromboxane A2, a potent platelet aggregator, and prostacyclin, a potent vasodilator, are released early in endotoxin shock and may contribute to its pathologic sequelae. Plasma levels of thromboxane (Tx) A2 and prostacyclin were measured via radioimmunoassay of their stable metabolites immunoreactive (i) TxB2 and i6-keto-PGF1 alpha in tolerant and nontolerant rats after endotoxin. Long-Evans rats were made tolerant to endotoxin by four daily IV injections of S enteritidis (endotoxin) (0.1, 0.5, 1, and 5 mg/kg). In normal rats (N = 15) given LPS (IV, 15 mg/kg), only 11% survived at 24 h; in contrast, tolerant rats (N = 13) all survived even at a dose of 50 mg/kg. At 1 h, after endotoxin (15 mg/kg) IV, plasma i6-keto-PGF1 alpha in nontolerant rats was 1,005 +/- 149 pg/ml (N = 14) and continued to rise to 4,209 +/- 757 pg/ml (N = 5) (P less than 0.001) after 4 h. In tolerant rats, given endotoxin (15 mg/kg), plasma i6-keto-PGF1 alpha at 1 h was 800 +/- 203 pg/ml (N = 5) and was not significantly different (734 +/- 254 pg/ml) at 4 h. Plasma iTxB2 at both 1 and 4 h was significantly (P less than 0.01) lower in tolerant than nontolerant rats. Both iTxB2 and i6-keto-PGF1 alpha were significantly (P less than 0.01) lower in tolerant rats given 50 mg/kg IV endotoxin than nontolerant rats. Endotoxin-induced elevation in fibrin degradation products was significantly decreased (P less than 0.05) during endotoxin tolerance although there was no difference in the severity of thrombocytopenia. These composite observations demonstrate that endotoxin tolerance in the rat is associated with altered arachidonic acid metabolism.

Topics: Acid Phosphatase; Animals; Arachidonic Acid; Arachidonic Acids; Aspartate Aminotransferases; Blood Platelets; Disease Susceptibility; Fibrin Fibrinogen Degradation Products; Glucuronidase; Macrophages; Male; Prostaglandins F; Rats; Shock, Septic; Thrombocytopenia; Thromboxane B2

Although cyclo-oxygenase products have been detected at inflammatory sites the tissue of origin remains uncertain. Inflammatory exudates were collected from rats 4, 6, 8, 12 or 24 h after subcutaneous implantation of carrageenin-impregnated sponges. Concentrations of the cyclo-oxygenase products prostaglandin E2 (PGE2), 6-oxo-PGF1 alpha and thromboxane B2 (TXB2) in inflammatory exudates and serum (obtained from blood clotted at 37 degrees C) were measured by specific radioimmunoassays. TXB2 concentrations in exudates increased to about 100 ng ml-1 at 8 h but decreased to less than 20 ng ml-1 after 24 h. PGE2 concentrations increased from 4-12 h and remained between 80 and 120 ng ml-1 from 12-24 h. 6-oxo-PGF1 alpha had the same time course as that of PGE2 but concentrations were approximately one third of PGE2 values. TXB2 concentrations in serum from thrombocytopaenic rats were less than 5% of control values. Thrombocytopaenia did not affect TXB2, PGE2 or 6-oxo-PGF1 alpha concentrations or total leukocyte numbers in inflammatory exudates. Methotrexate-induced neutropaenia did not affect serum TXB2 concentrations but cyclo-oxygenase products (including TXB2) in 6 h inflammatory exudates were reduced by 60-95%. Colchicine (1.0 mg kg-1 s.c.) prevented leukocyte accumulation in sponge exudates and this was accompanied by a reduction in TXB2, PGE2 and 6-oxo-PGF1 alpha concentrations at 6 h. These results indicate that platelets are the source of TXB2 in clotting blood but do not contribute to cyclo-oxygenase activity in experimental inflammation. The results also suggest that migrating leukocytes are the major source of thromboxane and to a lesser degree prostaglandins in acute 6 h inflammatory exudates.

Topics: Animals; Blood Platelets; Colchicine; Exudates and Transudates; Inflammation; Leukocyte Count; Male; Neutropenia; Prostaglandins; Radioimmunoassay; Rats; Thrombocytopenia; Thromboxane B2; Thromboxanes

Bleeding time, platelet thromboxane B2 production and megakaryocyte nuclear DNA concentration were measured in rabbits recovering from thrombocytopenia caused by a single injection of anti-platelet serum. Similar measurements were made on rabbits in a steady state of normal platelet production. The effects of a sustained state of thrombocytopenia on megakaryocyte DNA concentration were investigated by repeated daily injections of anti-platelet serum. It is shown that bleeding time depends on both platelet count and mean platelet volume. Furthermore changes in mean platelet volume appear to play a more important role in haemostasis than changes in platelet count. The mean megakaryocyte nuclear DNA concentration is significantly increased after 24 hours of thrombocytopenia and continues to increase as thrombocytopenia is sustained. Thromboxane B2 production/unit volume of platelet is increased in platelets produced after 24 hours of thrombocytopenia compared with platelets produced in normal steady state function. As a consequence platelets produced in response to thrombocytopenia not only have a larger mean platelet volume but are also more reactive. Mean platelet volume, as well as platelet count, should be considered as an index of haemostasis and its dysfunction, thrombosis.

Topics: Animals; Bleeding Time; Blood Platelets; Bone Marrow Cells; Cytoplasm; DNA; Hematopoiesis; Immune Sera; Male; Megakaryocytes; Platelet Count; Rabbits; Thrombocytopenia; Thromboxane B2; Thromboxanes

Infusion into sheep of plasma containing zymosan-activated complement produces leukopenia, pulmonary leukostasis, and pulmonary artery hypertension. We previously demonstrated a close relationship between the pulmonary vascular response and elevations of plasma thromboxane. We have investigated the source of thromboxane synthesis in this model. Plasma containing zymosan-activated complement added to whole blood did not stimulate thromboxane synthesis. This observation suggested that leukocytes do not synthesize thromboxane directly in response to complement added to whole blood did not stimulate thromboxane synthesis. This observation suggested that leukocytes do not synthesize thromboxane directly in response to complement. Sheep rendered severely thrombocytopenic by the administration of antiplatelet serum responded to complement infusion in the usual way. Pretreatment with aspirin (10 mg/kg) protected sheep against the pulmonary vascular response and completely blocked thromboxane synthesis. Transfusion of functional platelets did not restore these responses. Twenty-four hours after aspirin treatment, in vivo thromboxane synthesis was significantly greater than platelet thromboxane synthesis in vitro. Thromboxane is synthesized by a tissue which recovers cyclooxygenase enzyme activity at a rate that is more rapid than platelet turnover. Sheep lung synthesizes thromboxane actively in vitro. It is postulated that leukocytes exposed to activated complement components damage pulmonary vascular endothelial cells and stimulate synthesis of thromboxane A2 which causes pulmonary vasoconstriction.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Aspirin; Blood Platelets; Complement Activation; Female; Hypertension, Pulmonary; In Vitro Techniques; Leukocytes; Lung; Male; Pulmonary Artery; Sheep; Thrombocytopenia; Thromboxane B2; Thromboxanes

N(7-Carboxyheptyl) imidazole, 4-[2-(1H-imidazol-1-yl) ethoxy] benzoic acid (dazoxiben) and imidazo [1,5-alpha] pyridine-5-hexanoic acid (CGS 13080) are potent selective inhibitors of platelet thromboxane synthetase that have little or no effect on the cyclooxygenase activity. Oral doses of the substances given to rats inhibited platelet thromboxane B2 production induced by intra-venous administration of collagen (100 micrograms/kg). Plasma concentrations of immunoreactive 6-keto PGF1 alpha in treated animals were increased above corresponding concentrations in untreated animals. There were small effects on the thrombocytopenia with CGS 13080 and carboxyheptylimidazole but not with dazoxiben. However these results did not always achieve statistical significance. Confirmation that the immunoreactive prostaglandin measured was actually 6-keto PGF1 alpha was obtained by the facts that indomethacin abolished its appearance in plasma and that the other prostaglandins were not present in sufficient quantities to cross-react with the antiserum to 6-keto PGF1 alpha. Two different antisera to 6-keto PGF1 alpha detected the same changes. Administration of thromboxane synthetase inhibitors to rats causes redirection of prostaglandin production from thromboxane to prostacyclin when platelets are stimulated with collagen in vivo.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Blood Platelets; Collagen; Imidazoles; Indoles; Indomethacin; Male; Oxidoreductases; Pyridines; Radioimmunoassay; Rats; Rats, Inbred Strains; Thrombocytopenia; Thromboxane B2; Thromboxane-A Synthase; Thromboxanes

The intravenous injection of the mast cell degranulator C 48/80 (1 mg/kg) in rats did not produce thrombocytopenia nor circulating platelet aggregates but sensitized the platelets to aggregate upon turbulence challenge. Such turbulence-induced platelet aggregation was not accompanied by formation of thromboxane B2. Electron microscopy revealed absence of platelet degranulation. Turbulence-induced platelet aggregation was completely prevented by pre-treatment of the rats with cyproheptadine, dipyridamole and VK 774, partially with ketanserin (5HT2-receptor antagonist), but not with methysergide (antiserotonergic drug), pyrilamine (antihistaminic drug), suprofen, aspirin (cyclo-oxygenase inhibitors), phentolamine, propranolol, flunarizine, lidoflazine, oxycoumarin or Trasylol. Combined treatment with the anti-histaminic drug pyrilamine and the 5HT2-receptor antagonist ketanserin resulted in a dose-related inhibition for ketanserin of the turbulence-induced platelet aggregation. These experiments point to an interaction between histamine and 5-hydroxytryptamine in the platelet activation by mast cell released mediators.

Topics: Animals; Blood Platelets; Cytoplasmic Granules; Histamine; Male; Mast Cells; p-Methoxy-N-methylphenethylamine; Platelet Aggregation; Rats; Rats, Inbred Strains; Serotonin; Thrombocytopenia; Thromboxane B2

Eleven patients in whom thrombocytopenia developed during heparin therapy were studied. Six patient (group 1) had severe thrombocytopenia with delayed onset and five of these patients had thromboembolic complications. A serum factor which induced heparin-dependent thromboxane B2 synthesis, 14C-serotonin release, and platelet aggregation was found in all patients in group 1. The serum factor was shown to be IgG. These findings suggest that the mechanism of the severe thrombocytopenia secondary to heparin therapy is immunological and the associated thromboembolic complications may be attributed to in-vivo activation of the platelet prostaglandin pathway and platelet aggregation induced by the heparin-dependent antibody. The five patients in group 2 had mild symptomless thrombocytopenia with early onset. In this group, the heparin-dependent antibody was not found and the mechanism of the thrombocytopenia is probably a direct action of heparin on platelets.

Topics: Adult; Aged; Female; Heparin; Humans; Immunoglobulin G; Male; Middle Aged; Platelet Aggregation; Serotonin; Thrombocytopenia; Thromboxane A2; Thromboxane B2; Thromboxanes; Time Factors

The intravenous infusion of 1-O-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) in baboons (28 micrograms per kg.) induced acute, but reversible, thrombocytopenia and neutropenia and the intravascular release of platelet factor 4 and thromboxane B2. Maximal depression of circulating platelets and neutrophils occurred within 30 seconds after AGEPC infusion and was accompanied by significant elevations in plasma platelet factor 4 and thromboxane B2 levels (p less than 0.02). Hematocrit values increased after AGEPC infusion, but this increase was delayed relative to the other intravascular alterations, i.e., maximal hematocrit values occurred at 10 to 20 minutes after AGEPC infusion.. The thrombocytopenia induced by AGEPC was reversed within 2 to 3 minutes; in contrast, circulating neutrophils did not return to preinfusion levels until 30 minutes after AGEPC infusion. Plasma platelet factor 4 and thromboxane B2 elevations gradually decreased and returned to preinfusion levels within 30 to 60 minutes. The deacetylated derivative of AGEPC, lyso-glyceryl ether phosphorylcholine, had no effect when similarly infused into baboons. These studies demonstrate that the intravenous administration of AGEPC into baboons initiated significant but reversible intravascular alterations; thus, this unusual acetylated alkyl phosphoglyceride may be an important mediator of inflammation in primates, including man.

Topics: Animals; Blood Cell Count; Blood Coagulation Factors; Blood Proteins; Hematocrit; Infusions, Parenteral; Lysophosphatidylcholines; Neutropenia; Papio; Platelet Activating Factor; Platelet Factor 4; Species Specificity; Thrombocytopenia; Thromboxane B2; Thromboxanes

The plasma of two patients with heparin-induced thrombocytopenia has been shown to cause platelet aggregation in the presence of heparin. The platelet aggregating factor was isolated in the IgG reaction of the patients' sera suggesting that it was an antibody. This heparin anti-platelet antibody (HAP-Ab) induced platelet aggregation and release but did not cause platelet lysis, although it fixed complement. Platelet aggregation was inhibited by EDTA and by inactivation of complement. There was a significant production of malondialdehyde (MDA) and thromboxane B2 (TXB2) implying a role of the prostaglandin synthesis pathway in HAP-Ab induced aggregation. ADP-release also appeared to be involved as apyrase blocked aggregation while hirudin, a thrombin inhibitor, had no effect. The thrombotic complications that have recently been reported in patients with heparin-induced thrombocytopenia may be explained by some effects of HAP-Ab on platelets, namely: the antibody mediated platelet factor 3 release, prostaglandin endoperoxides and thromboxane A2 (TXA2) production and platelet aggregation in vivo. These HAP-Ab mediated effects could be inhibited by anti-platelet drugs such as aspirin, indomethacin and dipyridamole and thus may have therapeutic implications.

Topics: Adenosine Diphosphate; Adult; Antibodies; Aspirin; Blood Platelets; Complement Fixation Tests; Dipyridamole; Female; Heparin; Humans; Indomethacin; Male; Malondialdehyde; Middle Aged; Platelet Aggregation; Platelet Factor 3; Thrombocytopenia; Thromboxane B2

Topics: Animals; Blood Pressure; Cardiopulmonary Bypass; Pulmonary Circulation; Sheep; Thrombocytopenia; Thromboxane B2; Thromboxanes; Vascular Resistance

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Fibrin Fibrinogen Degradation Products; Imidazoles; Lysosomes; Male; Oxidoreductases; Prostaglandins E; Rats; Shock, Septic; Thrombocytopenia; Thromboxane B2; Thromboxane-A Synthase

The in vivo inhibitory effect of aspirin on platelet cyclo-oxygenase is irreversible and lasts for the entire platelet life-span. Reappearance of cyclo-oxygenase activity in blood after aspirin has been proposed as a measure of the formation of new platelets and as an indirect indicator of platelet survival. A delay of 24--72 h in recovery, however, has been observed and it has been suggested that aspirin might also inhibit megakaryocyte cyclo-oxygenase. To test this possibility, aspirin (100 mg/kg) or saline were administered i.p. to rats made thrombocytopenic 2 h later (platelet count less than 5% of basal value) by a specific antiplatelet antiserum. Malondialdehyde (MDA) and thromboxane B2 (TxB2) production by platelets was measured by spectrophotometry and radioimmunoassay respectively, during the period of platelet count restoration. By 24 h after thrombocytopenia was induced, platelet count was about 15% of basal values in control and aspirin-treated rats. However, while in controls MDA and TxB2 production was restored to about 20% of basal values, in aspirin-treated rats less than 5% return of activity was detected. A marked difference between the two groups was still found 96 h after induction of thrombocytopenia, when platelet count restoration was similar. Since aspirin disappeared very rapidly from the circulation, the delay in recovery of cyclo-oxygenase activity supports the hypothesis of a megakaryocyte effect of this drug.

Topics: Animals; Aspirin; Blood Platelets; Male; Malondialdehyde; Platelet Count; Rats; Thrombocytopenia; Thromboxane B2; Thromboxanes; Time Factors