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

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.

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.

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.

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.

scholarly journals Inhibition of platelet phospholipid methylation during platelet secretion

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 537-544
Author(s):  
SJ Shattil ◽  
M McDonough ◽  
JW Burch
Keyword(s):  
Mammalian Cells ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Serotonin Release ◽  
Prostaglandin Synthesis ◽  
Agonist Binding ◽  
Platelet Phospholipid ◽  
Precise Role ◽  
Platelet Secretion

A pathway for the synthesis of membrane phosphatidylcholine involving the N-methylation of phosphatidylethanolamine has been detected in several types of mammalian cells. Furthermore, it has been implicated in the coupling of agonist binding to cell response. We examined whether human platelets exhibit this synthetic pathway and whether platelet agonists influence its activity. When washed platelets were incubated with 0.15 microM L-[methyl-3H]methionine at 37 degrees C, they incorporated methyl-3H into their phospholipids linearly at the rate of 1 pmole/10(9) platelets/hr. When incubated with 20 microM radiolabeled methionine, they incorporated about 15 pmole/10(9) platelets/hr. The radioactivity was found predominantly in phosphatidyl- N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine. Thrombin caused an immediate (within 15 sec) and sustained (up to 30 min) decrease in the rate and extent of N- methylation of platelet phospholipids. This was accounted for by a decrease in synthesis of methylated phospholipids rather than an increase in their degradation. This thrombin effect correlated with serotonin release and could be dissociated from platelet aggregation and prostaglandin synthesis. Thrombin also decreased the synthesis of phosphatidylcholine when choline was used as the radiolabeled substrate. Other agonists such as epinephrine, adenosine diphosphate (ADP), or A23187 also decreased phospholipid methylation under conditions in which they stimulated serotonin release. These data demonstrate that platelets are capable of synthesizing phosphatidylcholine from phosphatidylethanolamine by N-methylation and that agonists perturb this pathway as they induce platelet secretion. The precise role of phospholipid methylation in either resting or stimulated platelets remains to be established.

scholarly journals Inhibition of platelet phospholipid methylation during platelet secretion

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 537-544 ◽  
Author(s):  
SJ Shattil ◽  
M McDonough ◽  
JW Burch
Keyword(s):  
Mammalian Cells ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Serotonin Release ◽  
Prostaglandin Synthesis ◽  
Agonist Binding ◽  
Platelet Phospholipid ◽  
Precise Role ◽  
Platelet Secretion

Abstract A pathway for the synthesis of membrane phosphatidylcholine involving the N-methylation of phosphatidylethanolamine has been detected in several types of mammalian cells. Furthermore, it has been implicated in the coupling of agonist binding to cell response. We examined whether human platelets exhibit this synthetic pathway and whether platelet agonists influence its activity. When washed platelets were incubated with 0.15 microM L-[methyl-3H]methionine at 37 degrees C, they incorporated methyl-3H into their phospholipids linearly at the rate of 1 pmole/10(9) platelets/hr. When incubated with 20 microM radiolabeled methionine, they incorporated about 15 pmole/10(9) platelets/hr. The radioactivity was found predominantly in phosphatidyl- N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine. Thrombin caused an immediate (within 15 sec) and sustained (up to 30 min) decrease in the rate and extent of N- methylation of platelet phospholipids. This was accounted for by a decrease in synthesis of methylated phospholipids rather than an increase in their degradation. This thrombin effect correlated with serotonin release and could be dissociated from platelet aggregation and prostaglandin synthesis. Thrombin also decreased the synthesis of phosphatidylcholine when choline was used as the radiolabeled substrate. Other agonists such as epinephrine, adenosine diphosphate (ADP), or A23187 also decreased phospholipid methylation under conditions in which they stimulated serotonin release. These data demonstrate that platelets are capable of synthesizing phosphatidylcholine from phosphatidylethanolamine by N-methylation and that agonists perturb this pathway as they induce platelet secretion. The precise role of phospholipid methylation in either resting or stimulated platelets remains to be established.

Mechanism of Potentiation by Manganese Ion of Aggregation of Porcine Pancreatic Elastase-Treated Human Platelets

1989 ◽  
Vol 62 (03) ◽  
pp. 984-988 ◽  
Author(s):  
Hiroyuki Azuma ◽  
Toshio Shigekiyo ◽  
Shinji Miura ◽  
Yuka Uno ◽  
Shiro Saito
Keyword(s):  
Binding Sites ◽  
Creatine Phosphokinase ◽  
Adenosine Diphosphate ◽  
Creatine Phosphate ◽  
Human Platelets ◽  
Extracellular Adenosine ◽  
Pancreatic Elastase ◽  
Manganese Ion ◽  
Porcine Pancreatic Elastase ◽  
Induced Aggregation

SummaryThe effect of manganese ion (Mn2+) on the aggregation of porcine pancreatic elastase-treated platelets (ETP) induced by fibrinogen (Fbg) was studied. Mn2+ enhanced the aggregation of ETP on addition of Fbg specifically and dose-dependently. This effect of Mn2+ was not associated with the formation of thromboxane A2, and was not affected by pretreatment of ETP with acetylsalicylic acid in the presence of Mn2+. Moreover, it was not dependent on extracellular adenosine diphosphate, as shown by removal of extracellular adenosine diphosphate by pretreatment of ETP with creatine phosphate/creatine phosphokinase. Studies on the binding of 125I-Fbg to ETP showed that Mn2+ increased the Kd value of binding but did not affect the number of Fbg binding sites. These results indicate that Mn2+ specifically and dose-dependently potentiated Fbg-induced aggregation of ETP and that this effect of Mn2+ may be due to an increase in the affinity of binding of Fbg to the glycoprotein IIb-IIIa complex on the membranes of ETP.

Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2648-2656 ◽  
Author(s):  
Juan A. Rosado ◽  
Else M. Y. Meijer ◽  
Karly Hamulyak ◽  
Irena Novakova ◽  
Johan W. M. Heemskerk ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding

Abstract Effects of the occupation of integrin αIIbβ3 by fibrinogen on Ca++signaling in fura-2–loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca++] concentrations ([Ca++]i) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca++ but not in the absence of external Ca++ or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca++entry. Fibrinogen also inhibited store-mediated Ca++ entry (SMCE) activated after Ca++ store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to αIIbβ3 was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca++ chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60src to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin αIIbβ3 inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60src. This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected to low-level stimuli in vivo.

Platelet Secretion Induced by Calcium Ionophores: Effect of Anti-Inflammatory Drugs

1975 ◽  
Author(s):  
E. H. Mürer ◽  
K. Davenport ◽  
H. J. Day
Keyword(s):  
Incubation Time ◽  
Surrounding Medium ◽  
Human Platelets ◽  
Serotonin Release ◽  
Time Dependent ◽  
Metabolic Parameters ◽  
Platelet Serotonin ◽  
Inflammatory Drugs ◽  
Anti Inflammatory ◽  
Platelet Secretion

Washed human platelets prelabeled with 3 H-serotonin and 14 C-adenine were incubated at 37° C with ionophores A23187 and X537A. 0.1 μM A23187 released the serotonin store of preincubated platelets after 1 min at 37° C, but was less effective when added in the cold. An increase in incubation time at 37° C did not result in increased release. Platelets preincubated with indomethacin showed reduction of up to 85% in released serotonin, while the metabolic parameters 14 C-ATP and 14 C-IMP were not significantly altered. The platelets from some donors did not show reduced release after treatment with indomethacin. This may indicate a variation in sensitivity to the release inducer similar to that described for Sr++-induced release (Biochim. Biophys. Acta 53-59, 362, 1974), or to the effect of indomethacin. 1 μM X537A caused a time-dependent serotonin release which increased from 2% at 1 min to 58% at 10 min incubation at 37° C. There was little change in 14 C-ATP following release and none in intra- or extracellular 14 C-IMP. 10 μM X537A caused release of 75-80% of the platelet serotonin after 1 min incubation. Longer incubation resulted in 14 C-IMP accumulation and leakage of 14 C-IMP to the surrounding medium. The results do not support the view that X537A and A23187 cause release from platelets by different mechanisms.(Supported by grant HL 14217 from NHLI. A23187 was a gift from Dr. R. L. Hamill, Eli Lilly and Co., Indianapolis, Ind., and X537A from Drs. Zane Gaut and W. E. Scott, Hoffmann-LaRoche, Inc., Nutley, N. J.)

Is Phospholipase A2 (PLA2) Involved In PAF-Acether Formation By Platelets

1981 ◽  
Author(s):  
M Chignard ◽  
B B Vargaftig ◽  
J P Le Couedic ◽  
J Benveniste
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
Chelating Agents ◽  
Creatine Phosphokinase ◽  
Platelet Activating Factor ◽  
Calcium Ionophore ◽  
Adenosine Diphosphate ◽  
Creatine Phosphate ◽  
Dibutyryl Cyclic Amp ◽  
A 23187

PAF-acether (platelet-activating factor) has been recently identified as l-O-alkyl-2-acetyl-sn-glyceryl-phosphorylcholine, and later chemically synthetized. Platelets form PAF-acether upon stimulation with the calcium ionophore A 23187 or with more physiological stimuli such as thrombin or collagen. By contrast, arachidonic acid (AA) and adenosine diphosphate (ADP) do not trigger formation of PAF-acether. Since 1) PAF-acether is a phospholipid derivative and 2) aggregating agents which trigger PAF-acether formation are potent platelet PLA2 stimulators, we speculated that PLA2 could be implicated in its formation.Rabbit washed platelets were incubated at 37°C in the presence of thrombin (2.5 U/ml) or of ionophore A 23187 (2.5 uM) for 7 min and ethanol (80 % final) was added. After centrifugation, the supernatant was evaporated and concentrated. The extract was tested for its aggregating property on rabbit washed platelets preincubated with a cyclo-oxygenase inhibitor (aspirin) and an ADP scavenging system (creatine phosphate and creatine phosphokinase).In the presence of calcium chelating agents such as EDTA (5 mM) and EGTA (5 mM) most of the synthesis of PAF-acether was suppressed (93 % and 100 % of inhibition respectively). Dibutyryl cyclic AMP (5 mM) also suppressed PAF-acether formation from platelets challenged by thrombin or by the ionophore A 23187 (100 % and 62 % inhibition respectively). Bromophenacyl bromide (0.1 mM) and compound CB 874 (0.1 mM) proved also to be very potent inhibitors of PAF-acether synthesis (100 % inhibition both). All these drugs are well-known platelet PLA2 inhibitors. Upon stimulation platelets also form a deacetylated PAF-acether (lyso- PAF-acether) which could be the direct precursor of PAF-acether. The release of lyso-PAF-acether and the blockade of PAF-acether formation by various molecules having in common a PLA- inhibitory activity lead us to conclude that a PLA2 may be implicated in PAF-acether formation from platelets. Alternative explanations include the possibility that the various inhibitors act on other membrane-related sites.

Inhibition and potentiation of platelet function by lysolecithin

Blood ◽  
1977 ◽  
Vol 49 (1) ◽  
pp. 101-112 ◽  
Author(s):  
JH Joist ◽  
G Dolezel ◽  
MP Cucuianu ◽  
EE Nishizawa ◽  
JF Mustard
Keyword(s):  
Platelet Function ◽  
Platelet Rich Plasma ◽  
Membrane Damage ◽  
Adenosine Diphosphate ◽  
Lactic Dehydrogenase ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Serotonin Release ◽  
Prolonged Incubation ◽  
Induced Aggregation

Abstract The effects of lysolecithin (LPC) on aggregation, serotonin release, shape, and lysis of rabbit, pig, or human platelets in platelet-rich plasma (PRP) or Tyrode albumin solution were examined during prolonged incubation. LPC added to citrated or heparinized PRP from humans or rabbits at a final concentration above 100 muM caused instantaneous inhibition of platelet aggregation induced by adenosine diphosphate (ADP), epinephrine (human PRP only), collagen, or thrombin. The inhibitory effect of LPC was found to be partially reversible over a period of 60–90 min. LPC at final concentrations above 30 muM also caused inhibition of ADP-, collagen-, and thrombin-induced aggregation and collagen- and thrombin-induced release of serotonin in suspensions of rabbit, pig, or human platelets. With washed platelets, the inhibitory effect not only rapidly disappeared but was followed by transient potentiation of aggregation and serotonin release. This potentiating effect of LPC was most pronounced when thrombin was used as stimulus. Both inhibition and potentiation were observed at concentrations of LPC that did not cause a significant change in platelet shape or loss from platelets of lactic dehydrogenase. Inhibition and potentiation were also observed when platelets were added to suspending medium containing LPC, although considerably higher concentrations of LPC were required under these conditions. Potentiation was not observed when LPC was added to citrated or heparinized rabbit or human PRP or to washed rabbit platelets suspended in a medium containing 4% bovine serum albumin. It seemed likely that some or all of the observed effects of LPC on platelet function were due to structural modification of the platelet membrane insufficient to result in gross membrane damage or platelet lysis. In addition, the results of experiments using 14C-LPC seemed to indicate that the observed potentiating effect of LPC on platelet function may be related to its rapid uptake and metabolism by the platelets.

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