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.

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.

Ticlopidine Facilitates the Deaggregation of Human Platelets Aggregated by Thrombin

1994 ◽  
Vol 71 (01) ◽  
pp. 091-094 ◽  
Author(s):  
M Cattaneo ◽  
B Akkawat ◽  
R L Kinlough-Rathbone ◽  
M A Packham ◽  
C Cimminiello ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Platelet Aggregation ◽  
Prostaglandin E1 ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Before And After ◽  
Normal Human ◽  
Platelet Aggregates ◽  
Induced Aggregation

SummaryNormal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, prostaglandin E1 (PGE1) and chymotrypsin. Released adenosine diphosphate (ADP) plays an important role in the stabilization of thrombin-induced human platelet aggregates. Since ticlopidine inhibits the platelet responses to ADP, we studied thrombin-induced aggregation and deaggregation of 14C-serotonin-labeled platelets from 12 patients with cardiovascular disease before and 7 days after the oral administration of ticlopidine, 250 mg b.i.d. Before and after ticlopidine, platelets stimulated with 1 U/ml thrombin aggregated, released about 80–90% 14C-serotinin and did not deaggregate spontaneously within 5 min from stimulation. Before ticlopidine, hirudin (5× the activity of thrombin) and PGE1 (10 μmol/1) plus chymotrypsin (10 U/ml) or plasmin (0.06 U/ml), added at the peak of platelet aggregation, caused slight or no platelet deaggregation. After ticlopidine, the extent of platelet deaggregation caused by the same inhibitors was significantly greater than before ticlopidine. The addition of ADP (10 μmol/1) to platelet suspensions 5 s after thrombin did not prevent the deaggregation of ticlopidine-treated platelets. Thus, ticlopidine facilitates the deaggregation of thrombin-induced human platelet aggregates, most probably because it inhibits the effects of ADP on 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 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.

Requirement Of Fibrinogen And Calcium For Inter-Platelet Bridges In Platelet Aggregation: A Hypothesis

1981 ◽  
Author(s):  
Elizabeth Kornecki ◽  
Stefan Niewiarowski
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Proteolytic Enzymes ◽  
Divalent Cations ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Platelet Aggregates ◽  
High Concentrations ◽  
Induced Aggregation ◽  
Aggregation Of Platelets

Fibrinogen and calcium are required for the aggregation of platelets stimulated by ADP or pre-treated with proteolytic enzymes. Specific platelet surface fibrinogen binding sites (receptors) are exposed after platelets are stimulated by ADP or pre-treated with Chymotrypsin or pronase. It has previously been shown in our laboratory that an intact, symmetrical fibrinogen molecule is essential for fibrinogen binding and fibrinogen-induced aggregation of both ADP-stimulated and proteolytically-treated platelets. Here we propose that the mechanism by which fibrinogen and calcium aggregate platelets is by forming inter-platelet bridges linking the fibrinogen receptors of adjacent platelets together. In support of this proposition are the following new lines of evidence: 1) The fibrinogen-induced aggregations of ADP-stfiliulated or proteolytically-treated platelets are inhibited by high concentrations of fibrinogen (Ki=2.6 and 8.5 × 10 5M, respectively). The fibrinogen binding sites on adjacent platelets, at these concentrations, would be saturated by fibrinogen and therefore no inter-platelet fibrinogen bridges could be formed to hold the platelets together. 2) ADP-stimulated or chymotrypsin-treated platelets aggregated by fibrinogen are deaggregated by Chymotrypsin or pronase and this deaggregation coincides with the loss of 125I-fibrinogen from the platelet surface. 3) Preincubation of platelets with EDTA results in inhibition of both platelet aggregation and 125I-fibrinogen binding. Following the aggregations of ADP-stimulated or of chymotrypsin-treated platelets by fibrinogen, the addition of EDTA to the platelet aggregates results in both their deaggregation and their loss of bound 125I-fibrinogen. Thus it appears that divalent cations, especially calcium, are essential for the formation of fibrinogen-linked platelet aggregates.

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.

Thrombin Interaction with Human Platelets Potentiation of Thrombin-induced Aggregation and Release by Inactivated Thrombin

1974 ◽  
Vol 32 (01) ◽  
pp. 207-215 ◽  
Author(s):  
David R. Phillips
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Induce Platelet Aggregation ◽  
Release Reaction ◽  
Proteolytic Site ◽  
Induced Aggregation ◽  
Inhibited Enzyme

SummaryThe possibility that thrombin acts on platelets by a mechanism other than proteolysis was investigated. The proteolytic site of thrombin was modified with phenylmethylsulfonyl fluoride (PMSF). This modified enzyme did not induce platelet aggregation or the platelet release reaction. Platelets were then incubated with the inactivated enzyme (PMS-thrombin) and later with active thrombin. In this sequence of incubation, PMS-thrombin enhanced not only platelet aggregation induced by active thrombin but also the thrombin-induced release reaction. Preincubation with PMS-thrombin was essential for this enhancement as the inhibited enzyme did not affect aggregation if added after active thrombin. The effect of PMS-thrombin was limited to thrombin-induced reactions of the platelet. The inhibited enzyme had no effect on aggregation induced by adenosine diphosphate or collagen, or on thrombininduced coagulation of fibrinogen. These results suggest (1) that both proteolytic and binding sites for thrombin are present on the human platelet plasma membrane ; and (2) that interaction of thrombin with the binding site potentiates the activity of the proteolytic site.

Relationship Between Aggregation and Binding of 125I-Fibrinogen and 45Calcium to Human Platelets

1979 ◽  
Author(s):  
E.I. Peerschke ◽  
R.A. Grant ◽  
M.B. Zucker
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Silicone Oil ◽  
Human Platelets ◽  
Mechanisms Of Action ◽  
Ionized Calcium ◽  
Clot Retraction ◽  
Fibrinogen Binding ◽  
Induced Aggregation ◽  
Stimulation Of

Since calcium and fibrinogen are essential cofactors for ADP-induced aggregation, their mechanisms of action were investigated. Aspirin-treated platelets were filtered through Sepharose 2B equilibrated with cation-poor Tyrode’s solution. After adding the radioactive compounds at 22, platelets were centrifuged through silicone oil. Trapping was assessed in separate samples with 14C-sorbitoi. Calcium binding was maximal at 1 hr and with 200 uH CaCl2. Two binding sites could be demonstrated on normal and thrombasthenic platelets and on platelets which had lost their ability to aggregate (but not to change shape or promote clot retraction) after treatment with EDTA (8 min, 37°, pH 7.8). ADP did not alter calcium binding in the presence or absence of fibrinogen. Fibrinogen, however, bound to normal gel filtered platelets in the presence of ADP and ionized calcium “ but not to thrombasthenic or EDTA-treated platelets or to normal platelets in the presence of EDTA or at pH 6.5. Binding of fibrinogen increased with concentration but saturation was not observed even at physiologic levels. Fibrinogen binding was similar in gel filtered platelets and citrated piatelet-rich plasma. These studies indicate that stimulation of platelets with ADP under conditions favorable to aggregation is associated with binding of fibrinogen but not of additional calcium.

Reactions of Polylysine with Human Platelets in Plasma and in Suspensions of Washed Platelets

1976 ◽  
Vol 36 (02) ◽  
pp. 360-375 ◽  
Author(s):  
M. A Guccione ◽  
M. A Packham ◽  
R. L Kinlough-Rathbone ◽  
D. W Perry ◽  
J. F Mustard
Keyword(s):  
Acetylsalicylic Acid ◽  
Prostaglandin E1 ◽  
Creatine Phosphokinase ◽  
Platelet Rich Plasma ◽  
Creatine Phosphate ◽  
Human Platelets ◽  
Lag Phase ◽  
Tyrode Solution ◽  
Two Phases ◽  
Induced Aggregation

SummaryThe effects of polylysine on human platelets have been examined in citrated platelet-rich plasma (PRP) and in suspensions of washed platelets in various media. In PRP, polylysine caused aggregation after a lag phase. Heparin inhibited this completely. At certain concentrations of polylysine, two phases of aggregation occurred, the second being associated with release of 14C-serotonin from prelabelled platelets; this phase was inhibitable with prostaglandin E1, acetylsalicylic acid, sulphinpyrazone, adenosine, apyrase, or creatine phos- phate/creatine phosphokinase. Polylysine-induced release also occurred in PRP with EDTA or hirudin as anticoagulant. In suspensions of washed platelets in Tyrode solution containing 0.35% or 4% albumin, or 1% gelatin, polylysine caused immediate platelet-to-platelet adherence and very little release of 14C-serotonin or platelet lysis. Heparin inhibited aggregation, but acetylsalicylic acid, prostaglandin E1, adenosine, apyrase, creatine phosphate/creatine phosphokinase or EDTA did not. In a modified Tyrode-albumin medium containing 1 mM magnesium but no calcium, polylysine-induced aggregation was associated with the release of 14C-serotonin which could be inhibited by acetylsalicylic acid or indomethacin; this is similar to the effect of ADP in this medium. In Tyrode solution without albumin or gelatin, polylysine-induced platelet aggregation was associated with release of a large percentage of 14C-serotonin, together with as much as 18% lysis; indomethacin inhibited this release reaction.

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