Arachidonate-Induced Fibrinogen Binding To Thrombin-Degranulated Rabbit Platelets Is Independent Of Released ADP

1981 ◽  
Author(s):  
E J Harfenist ◽  
M A Guccione ◽  
M A Packham ◽  
R L Kinlough-Rathbone ◽  
J F Mustar
Keyword(s):  
Binding Sites ◽  
Creatine Phosphokinase ◽  
Synergistic Effects ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Binding Studies ◽  
Fibrinogen Binding ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
Stimulation Of

When human or rabbit platelets are stimulated with ADP, fibrinogen (Fbg) binding sites are revealed, the platelets bind Fbg and aggregate. Since stimulation with other aggregating agents (arachidonate, collagen or ionophores) releases platelet granule contents, including ADP and Fbg, it is difficult to determine whether these agents cause aggregation or Fbg binding that is independent of ADP. Therefore we treated rabbit platelets with thrombin (0.73 U/ml) to release at least 90% of their dense granule contents, as measured by 14C-serotonin, washed and resuspended them, and studied aggregation and Fbg binding upon stimulation with ADP or arachidonate. In the presence of Fbg, thrombin-degranulated platelets (TDP) aggregate in response to ADP or arachidonate at concentrations that aggregate untreated platelets, although TDP aggregate somewhat less extensively. When TDP are aggregated with 50 μM arachidonate, they lose up to 9% of their remaining serotonin, corresponding to a concentration of ADP in the suspending medium of not more than0.06μM, which does not aggregate TDP or cause detectable Fbg binding. When creatine phosphate/creatine phosphokinase (CP/CPK) is added at a concentration that abolishes aggregation in response to 1 μM ADP, it reduces aggregation caused by arachidonate by only 18%. Binding studies with 125I-Fbg show that stimulation of TDP with either ADP or arachidonate results in specific Fbg-binding similar to the binding to ADP-stimulated normal platelets. CP/CPK almost completely inhibits binding induced by ADP but reduces binding induced by arachidonate by only 30%. Aggregation and binding studies with TDP using a combination of arachidonate with low concentrations of ADP failed to show synergistic effects. Thus arachidonate causes aggregation and Fbg binding to TDP that are independent of ADP, although the magnitude of these effects may be increased by released ADP.

scholarly journals Released adenosine diphosphate stabilizes thrombin-induced human platelet aggregates

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1081-1086 ◽  
Author(s):  
M Cattaneo ◽  
MT Canciani ◽  
A Lecchi ◽  
RL Kinlough-Rathbone ◽  
MA Packham ◽  
...  
Keyword(s):  
Binding Sites ◽  
Creatine Phosphokinase ◽  
Adenosine Diphosphate ◽  
Creatine Phosphate ◽  
Human Platelets ◽  
Fibrinogen Binding ◽  
Normal Human ◽  
Platelet Aggregates ◽  
Induced Aggregation ◽  
Selective Impairment

Normal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, chymotrypsin, and prostaglandin E1 (PGE1). In contrast, thrombin-induced aggregates of platelets from patients with delta-storage pool deficiency (delta-SPD), which lack releasable nucleotides, are readily deaggregated by the same combination of inhibitors. The ease with which delta-SPD platelets are deaggregated is caused by the lack of stabilizing effects of released ADP, since: (1) exogenous adenosine diphosphate (ADP) (10 mumol/L), but not serotonin (2 mumol/L), abolishes the ability of these inhibitors to deaggregate delta-SPD platelets; (2) thrombin-induced aggregates of platelets from a patient (V.R.) (whose platelets have a severe, selective impairment of sensitivity to ADP, but normal amounts of releasable nucleotides) can be readily deaggregated, and addition of ADP does not stabilize the platelet aggregates; (3) apyrase or creatine phosphate (CP)/creatine phosphokinase (CPK), added before thrombin, make control platelets more easily deaggregated by hirudin, chymotrypsin, and PGE1, and do not change the deaggregation response of delta-SPD platelets and of V.R.'s platelets. Thrombin-induced aggregation and release of beta- thromboglobulin in control, delta-SPD, and in V.R.'s platelets was similar and not inhibited by apyrase or CP/CPK. The stabilizing effect of ADP on platelet aggregates is specific, since epinephrine in the presence of apyrase to remove traces of released ADP does not stabilize the aggregates of control, delta-SPD, or of V.R.'s platelets. Because epinephrine increases fibrinogen binding to thrombin-stimulated platelets to a greater extent than ADP, but does not stabilize the aggregates, it is unlikely that the additional fibrinogen binding sites induced by ADP have a major role in inhibiting deaggregation by the combination of inhibitors.

NORMAL EXPRESSION OF THROMBOSPONDIN RECEPTOR SITES ON PLATELETS OF THREE GLANZMANN THROMBASTHENIC PATIENTS

1987 ◽  
Author(s):  
J L McGregor ◽  
H Boukerche
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Whole Blood ◽  
Binding Sites ◽  
Competitive Binding ◽  
Fibrinogen Binding ◽  
Receptor Sites ◽  
Low Concentrations ◽  
Normal Expression ◽  
Stimulation Of

A well characterised anti-thrombospondin (TSP) monoclonal antibody (Mab) LYP8 was used to investigate the presence of TSP receptors on Glanzmann thrombasthenic (G.T.) platelets. LYP8 inhibited platelet aggregation induced by low concentrations of thrombin (0.05 U/ml) or collagen (0.5 ug/ml). The presence of LYP8 did not affect the number of sites and Kd of 125I-fibrinogen binding to thrombin-stimulated normal platelets. Binding of LYP8 to normal platelets was minimal in whole blood (300 IgG molecules/ olatelet), increased in citrated PRP (1187 ± 209 IgG molecules/ platelet) and washed platelets (2967 ± 1278 IgG molecules/platelet) . Thrombin stimulation of platelets, washed in the presence of 2 mM calcium, increased the number of LYP8 binding sites (14917 ± 42n IgG molecules/platelet). Addition of EDTA (5mM) to thrombin-stimulated platelets did not reduce the number of LYP8 binding sites. The number of LYP8 binding sites on thrombin-stimulated platelets of three Glanzmann thrombasthenic patients (showing an absence of the glycoprotein (GP) lib and IIIa)was similar to normals in the presence of 2 mM calcium or 5 mM EDTA. In competitive binding, Mab LYP18 directed against the GPIIb-IIIa complex did not inhibit the binding of labelled monoclonal antibody LYP8. These results strongly suggest that TSP binds to a membrane receptor different from the GPIIb-IIIa complex in the presence of calcium or EDTA. This unidentified receptor may be GPIV also known as GPIIIb (Asch, A. et al. Clin. Res. 1986, 34:450A).

scholarly journals Released adenosine diphosphate stabilizes thrombin-induced human platelet aggregates

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1081-1086 ◽  
Author(s):  
M Cattaneo ◽  
MT Canciani ◽  
A Lecchi ◽  
RL Kinlough-Rathbone ◽  
MA Packham ◽  
...  
Keyword(s):  
Binding Sites ◽  
Creatine Phosphokinase ◽  
Adenosine Diphosphate ◽  
Creatine Phosphate ◽  
Human Platelets ◽  
Fibrinogen Binding ◽  
Normal Human ◽  
Platelet Aggregates ◽  
Induced Aggregation ◽  
Selective Impairment

Abstract Normal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, chymotrypsin, and prostaglandin E1 (PGE1). In contrast, thrombin-induced aggregates of platelets from patients with delta-storage pool deficiency (delta-SPD), which lack releasable nucleotides, are readily deaggregated by the same combination of inhibitors. The ease with which delta-SPD platelets are deaggregated is caused by the lack of stabilizing effects of released ADP, since: (1) exogenous adenosine diphosphate (ADP) (10 mumol/L), but not serotonin (2 mumol/L), abolishes the ability of these inhibitors to deaggregate delta-SPD platelets; (2) thrombin-induced aggregates of platelets from a patient (V.R.) (whose platelets have a severe, selective impairment of sensitivity to ADP, but normal amounts of releasable nucleotides) can be readily deaggregated, and addition of ADP does not stabilize the platelet aggregates; (3) apyrase or creatine phosphate (CP)/creatine phosphokinase (CPK), added before thrombin, make control platelets more easily deaggregated by hirudin, chymotrypsin, and PGE1, and do not change the deaggregation response of delta-SPD platelets and of V.R.'s platelets. Thrombin-induced aggregation and release of beta- thromboglobulin in control, delta-SPD, and in V.R.'s platelets was similar and not inhibited by apyrase or CP/CPK. The stabilizing effect of ADP on platelet aggregates is specific, since epinephrine in the presence of apyrase to remove traces of released ADP does not stabilize the aggregates of control, delta-SPD, or of V.R.'s platelets. Because epinephrine increases fibrinogen binding to thrombin-stimulated platelets to a greater extent than ADP, but does not stabilize the aggregates, it is unlikely that the additional fibrinogen binding sites induced by ADP have a major role in inhibiting deaggregation by the combination of inhibitors.

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

Eicosapentaenoic Acid Interferes with U46619-Stimulated Formation of Inositol Phosphates in Washed Rabbit Platelets

1989 ◽  
Vol 62 (04) ◽  
pp. 1116-1120 ◽  
Author(s):  
N Chetty ◽  
J D Vickers ◽  
R L Kinlough-Rathbone ◽  
M A Packham ◽  
J F Mustard
Keyword(s):  
Signal Transduction ◽  
Fatty Acid Composition ◽  
Eicosapentaenoic Acid ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Dense Granule ◽  
Detectable Change ◽  
Membrane Phospholipids ◽  
Rabbit Platelets ◽  
Stimulation Of

SummaryEicosapentaenoic acid (EPA) inhibits platelet responsiveness to aggregating agents. To investigate the reactions that are affected by EPA, we examined the effect of preincubating aspirintreated rabbit platelets with EPA on stimulation of inositol phosphate formation in response to the TXA2 analogue U46619. Stimulation of platelets with U46619 (0.5 μM) caused aggregation and slight release of dense granule contents; aggregation and release were inhibited by preincubation of the platelets with EPA (50 μM) for 1 h followed by washing to remove unincorporated EPA. Incubation with EPA (50 μM) for 1 h did not cause a detectable increase in the amount of EPA in the platelet phospholipids. When platelets were prelabelled with [3H]inositol stimulation with U46619 of control platelets that had not been incubated with EPA significantly increased the labelling of mos1tol phosphates. The increases in inositol phosphate labelling due to U46619 at 10 and 60 s were partially inhibited by premcubat10n of the platelets with 50 μM EPA. Since the activity of cyclo-oxygenase was blocked with aspirin, inhibition of inositol phosphate labelling in response to U46619 indicates either that there may be inhibition of signal transduction without a detectable change in the amount of EPA in platelet phospholipids, that changes in signal transduction require only minute changes in the fatty acid composition of membrane phospholipids, or that after a 1 h incubation with EPA, activation of phospholipase C is affected by a mechanism that is not directly related to incorporation of EPA.

COMPARISON OF THE PLATELET AGGREGATION INDUCED BY THREE THROMBIN-LIKE ENZYMES OF SNAKE VENOMS AND THROMBIN

1987 ◽  
Author(s):  
C M Teng ◽  
F N Ko
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Creatine Phosphokinase ◽  
Antithrombin Iii ◽  
Atp Release ◽  
Creatine Phosphate ◽  
Snake Venoms ◽  
Rabbit Platelets ◽  
Bothrops Atrox ◽  
Induced Aggregation

Acutin was isolated from Agkistrodon acutus venom and batroxobin and thrombocytin were isolated from Bothrops atrox venom. These three thrombin-like enzymes had different specificity for platelet activation and fibrinogen clotting. The clotting activities were 700, 170 and 7 μ/mg for batroxobin, acutin and thrombocytin, respectively. They induced aggregation and ATP release of washed rabbit platelets. The aggregating activities were 102, 104 and 105 times less than that of thrombin for thrombocytin, acutin and batroxobin, respectively basing on the clotting unit. The platelet -activating potency was correlated with their effectiveness on the retractility and elasticity of the clots. Platelet aggregation induced by thrombin or thrombocytin could be inhibited by heparin with antithrombin III while that by acutin or batroxobin could not. The thrombin-like enzymes did not induce aggregation of thrombin-degranulated platelets even fibrinogen was added. Indomethacin showed weak inhibition on the aggregation while the ADP - scavenging system, creatine phosphate/creatine phosphokinase, or apyrase inhibited the aggregation induced by the three thrombin-like enzymes but not that by thrombin. In the presence of EGTA, only thrombin could induce ATP release from platelets. It is concluded that the aggregation induced by thrombin-like enzymes is different from that of thrombin and mainly due to ADP released from platelets.

scholarly journals Participation of ADP in the binding of fibrinogen to thrombin- stimulated platelets

Blood ◽  
1980 ◽  
Vol 56 (3) ◽  
pp. 553-555 ◽  
Author(s):  
EF Plow ◽  
GA Marguerie
Keyword(s):  
Creatine Phosphokinase ◽  
Adenosine Diphosphate ◽  
Creatine Phosphate ◽  
Human Platelets ◽  
Serotonin Release ◽  
Fibrinogen Binding ◽  
Platelet Secretion

Abstract Thrombin and adenosine diphosphate (ADP) supported the binding of 125I- fibrinogen to washed human platelets with similar kinetics and affinity. Platelet secretion, as measured by 14C-serotonin release, and fibrinogen binding exhibited an identical dependence on thrombin concentration. Enzymatic removal of ADP with apyrase or creatine phosphate/creatine phosphokinase (CP/CPK) from thrombin-stimulated platelets markedly inhibited 125I-fibrinogen binding, but pretreatment of platelets with CP/CPK prior to thrombin stimulation was without effect. Thus, ADP, released from the platelet, participates in the binding of fibrinogen to thrombin-stimulated platelets.

Studies on the Binding of 3H-SR121566, an Inhibitor of Gp IIb-IIIa Activation

2001 ◽  
Vol 85 (04) ◽  
pp. 702-709 ◽  
Author(s):  
P. Savi ◽  
G. Zamboni ◽  
O. Rescanières ◽  
J. M. Herbert
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelets ◽  
Gamma Chain ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Direct Competition ◽  
Stimulation Of

SummarySR121566 is a new synthetic agent which inhibits the binding of fibrinogen to activated platelets, and platelet aggregation. 3H-SR121566 bound with nanomolar affinity (KD ranging from 45 to 72 nM) to Gp IIb-IIIa expressing cells only. On activated human platelets, this ligand allowed the detection of a maximal number of 100-140,000 binding sites. The binding of SR121566 to platelets, was displaced by several agents including RGD-containing peptides and synthetic RGD mimetics, but not by ReoPro®, a humanised monoclonal antibody which inhibits the binding of fibrinogen to the Gp IIb-IIIa complex. Neither the fibrinogen dodecapeptide nor fibrinogen itself were able to compete with SR121566 whether platelets were activated or not.Flow cytometry studies indicated that SR121566 which did not activate Gp IIb-IIIa by itself, dose-dependently prevented the detection of activation-induced binding sites on TRAP-stimulated platelets in the presence or absence of exogenous fibrinogen, indicating a direct effect on the activation state of the Gp IIb-IIIa complex. Moreover, SR121566 was able to reverse the activation of Gp IIb-IIIa and to displace the binding of fibrinogen when added up to 5 min after TRAP stimulation of platelets. When added at later times (15 to 30 min), SR121566 failed to displace fibrinogen binding, even if SR121566 binding sites were still accessible and the Gp IIb-IIIa complex not activated.In conclusion, our study is in accordance with the finding that fibrinogen is recognised by the activated Gp IIb-IIIa complex through the dodecapeptide sequence present on its gamma chain, and that this interaction is inhibited by SR121566 by preventing and reversing the activated conformation of Gp IIb-IIIa and not by direct competition with fibrinogen.

PARTIAL PLATELET FUNCTION DEFECT IN A VARIANT OF GLANZMANN'S THROMBASTHENIA WITH INTERMEDIATE LEVELS OF GP IIb/IIIa

1987 ◽  
Author(s):  
R M Hardisty ◽  
A Pannocchia ◽  
N Mahmood ◽  
T J C Nokes ◽  
D Pidard ◽  
...  
Keyword(s):  
Platelet Function ◽  
Binding Sites ◽  
Adenine Nucleotide ◽  
Bleeding Tendency ◽  
Binding Studies ◽  
Platelet Factor ◽  
Glanzmann’S Thrombasthenia ◽  
Fibrinogen Binding ◽  
Glanzmann's Thrombasthenia ◽  
Sds Page

A 17-year-old Italian boy has had a lifelong bleeding tendency, with frequent epistaxes and gum bleeding. The bleeding time is prolonged and the platelet count low normal. Electron microscopy showed a wide diversity of platelet size with many giant forms. In citrated PRP, ADP and other agonists induce slow and incomplete aggregation. The response of washed platelets varied with the agonist but ranged from subnormal to almost normal. Fibrinogen binding to washed platelets occurred slowly in response to ADP but eventually approached normal levels. No significant abnormality was observed of 5HT uptake, adenine nucleotide content, platelet factor-3 availability, β-thromboglobulin content or release, or malonyldialdehyde production. Clot retraction was normal. SDS-PAGE showed reduced amounts of GPIIb and GPU Ia. Crossed immunoelectrophoresis of Triton X-100 extracts of washed platelets showed the presence of GPIIb/IIIa complexes at 25-50% of normal levels. SDS-PAGE combined with an immunoblot procedure confirmed unchanged mobilities of GPIIb and GPIIIa and a normal proportion of GPIIb to GPIIIa. However, binding studies with radiolabelled monoclonal antibodies showed that intact washed platelets expressed only 12-20% of the normal binding sites for M148, AP-2 and Tab. These antibodies recognize different epitopes on GPIIb/lIIa complexes. Similar levels of these glycoproteins were detected by autoradiography after SDS-PAGE of radio-iodinated patient's platelets. GP lb was normally present. A possible defect in the exposure of fibrinogen binding sites might contribute to the altered platelet function. Meanwhile, the patient appears to be a unique variant of Glanzmann's thrombasthenia with GP IIb/IIIa complexes at the borderline of those able to support platelet aggregation.

scholarly journals Platelet-activating factor (PAF-acether) induces high- and low-affinity binding of fibrinogen to human platelets via independent mechanisms

1986 ◽  
Vol 240 (2) ◽  
pp. 403-412 ◽  
Author(s):  
E Kloprogge ◽  
J W Akkerman
Keyword(s):  
Platelet Activation ◽  
Binding Sites ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Affinity Binding ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
A Minor

When human platelets are incubated with 500 nM-PAF-acether (platelet-activating factor. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) under equilibrium conditions (60 min, 22 degrees C, non-stirred suspensions), two classes of fibrinogen binding sites are exposed: one class with a high affinity [Kd (7.2 +/- 2.1) X 10(-8) M, 2367 +/- 485 sites/platelet, n = 9] and one class with a low affinity [Kd (5.9 +/- 2.4) X 10(-7) M, 26972 +/- 8267 sites/platelet]. Preincubation with inhibitors of cyclo-oxygenase (acetylsalicylic acid, indomethacin) or thromboxane synthetase (UK 38.485) completely abolishes high-affinity binding, leaving low-affinity binding unchanged. In contrast, ADP scavengers (phosphocreatine/creatine kinase or phosphoenol pyruvate/pyruvate kinase) completely prevent low-affinity binding, leaving high-affinity binding unaltered. Initial binding studies (2-10 min incubation) confirm these findings with a major part of the binding being sensitive to ADP scavengers, a minor part sensitive to indomethacin and complete blockade with both inhibitors. Increasing the temperature to 37 degrees C decreases the number of low affinity-binding sites 6-fold without changing high-affinity binding. Aggregation, measured as the rate of single platelet disappearance, then depends on high-affinity binding at 10 nM-fibrinogen or less, whereas at 100 nM-fibrinogen or more low-affinity binding becomes predominant. These findings point at considerable platelet activation during binding experiments. However, arachidonate metabolism [(3H]arachidonate mobilization and thromboxane synthesis) and secretion [(14C]serotonin and beta-thromboglobulin) are about 10% or less of the amounts found under optimal conditions (5 units of thrombin/ml 37 degrees C, stirring). We conclude that PAF-acether induces little platelet activation under binding conditions. The amounts of thromboxane A2 and secreted ADP, however, are sufficient for initiating high- and low-affinity fibrinogen binding via mutually independent mechanisms.

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