Collagen-Induced Platelet Aggregation: -Evidence Against the Essential Role of Platelet Adenosine Diphosphate

1979 ◽  
Vol 42 (04) ◽  
pp. 1193-1206 ◽  
Author(s):  
Barbara Nunn
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Essential Role ◽  
Atp Release ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
High Doses ◽  
Induced Aggregation

SummaryThe hypothesis that platelet ADP is responsible for collagen-induced aggregation has been re-examined. It was found that the concentration of ADP obtaining in human PRP at the onset of aggregation was not sufficient to account for that aggregation. Furthermore, the time-course of collagen-induced release in human PRP was the same as that in sheep PRP where ADP does not cause release. These findings are not consistent with claims that ADP alone perpetuates a collagen-initiated release-aggregation-release sequence. The effects of high doses of collagen, which released 4-5 μM ADP, were not inhibited by 500 pM adenosine, a concentration that greatly reduced the effect of 300 μM ADP. Collagen caused aggregation in ADP-refractory PRP and in platelet suspensions unresponsive to 1 mM ADP. Thus human platelets can aggregate in response to collagen under circumstances in which they cannot respond to ADP. Apyrase inhibited aggregation and ATP release in platelet suspensions but not in human PRP. Evidence is presented that the means currently used to examine the role of ADP in aggregation require investigation.

Opposite Effect of Lysine on Platelet Aggregation Induced by Arachidonate and by Other Aggregants

1982 ◽  
Vol 48 (01) ◽  
pp. 078-083 ◽  
Author(s):  
C Ts'ao ◽  
S J Hart ◽  
D V Krajewski ◽  
P G Sorensen
Keyword(s):  
Platelet Aggregation ◽  
Secondary Phase ◽  
Aminocaproic Acid ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Primary Phase ◽  
High Doses ◽  
The Many ◽  
Induced Aggregation

SummaryEarlier, we found that ε-aminocaproic acid (EACA) inhibited human platelet aggregation induced by adenosine diphosphate (ADP) and collagen, but not aggregation by arachidonic acid (AA). Since EACA is structurally similar to lysine, yet these two agents exhibit vast difference in their antifibrinolytic activities, we chose to study the effect of lysine on platelet aggregation. We used L-lysine-HCl in these studies because of its high solubility in aqueous solutions while causing no change in pH when added to human plasma. With lysine, we repeatedly found inhibition of ADP-, collagen- and ristocetin-induced aggregation, but potentiation of AA-induced aggregation. Both the inhibitory and potentiation effects were dose-dependent. Low doses of lysine inhibited the secondary phase of aggregation; high doses of it also inhibited the primary phase of aggregation. Potentiation of AA-induced aggregation was accompanied by increased release of serotonin and formation of malondialdehyde. These effects were not confined to human platelets; rat platelets were similarly affected. Platelets, exposed to lysine and then washed and resuspended in an artificial medium not containing lysine, remained hypersensitive to AA, but no longer showed decreased aggregation by collagen. Comparing the effects of lysine with equimolar concentrations of sucrose, EACA, and α-amino-n-butyric acid, we attribute the potent inhibitory effect of lysine to either the excess positive charge or H+ and C1− ions. The -NH2 group on the α-carbon on lysine appears to be the determining factor for the potentiation effect; the effect seems to be exerted on the cyclooxygenase level of AA metabolism. Lysine and other chemicals with platelet-affecting properties similar to lysine may be used as a tool for the study of the many aspects of a platelet aggregation reaction.

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.

Functional role of a polymorphism in the Pannexin1 gene in collageninduced platelet aggregation

2015 ◽  
Vol 114 (08) ◽  
pp. 325-336 ◽  
Author(s):  
Filippo Molica ◽  
Jean-François Denis ◽  
Paul Bradfield ◽  
Silvia Penuela ◽  
Anne Zufferey ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Reactivity ◽  
Critical Role ◽  
Atp Release ◽  
Human Platelets ◽  
Immune Synapse ◽  
Cardiovascular Patients ◽  
Exogenous Expression ◽  
First Time

SummaryPannexin1 (Panx1) forms ATP channels that play a critical role in the immune response by reinforcing purinergic signal amplification in the immune synapse. Platelets express Panx1 and given the importance of ATP release in platelets, we investigated Panx1 function in platelet aggregation and the potential impact of genetic polymorphisms on Panx1 channels. We show here that Panx1 forms ATP release channels in human platelets and that inhibiting Panx1 channel function with probenecid, mefloquine or specific 10Panx1 peptides reduces collagen-induced platelet aggregation but not the response induced by arachidonic acid or ADP. These results were confirmed using Panx1-/- platelets. Natural variations have been described in the human Panx1 gene, which are predicted to induce non-conservative amino acid substitutions in its coding sequence. Healthy subjects homozygous for Panx1–400C, display enhanced platelet reactivity in response to collagen compared with those bearing the Panx1–400A allele. Conversely, the frequency of Panx1–400C homozygotes was increased among cardiovascular patients with hyper-reactive platelets compared with patients with hypo-reactive platelets. Exogenous expression of polymorphic Panx1 channels in a Panx-deficient cell line revealed increased basal and stimulated ATP release from cells transfected with Panx1–400C channels compared with Panx1–400A expressing transfectants. In conclusion, we demonstrate a specific role for Panx1 channels in the signalling pathway leading to collagen-induced platelet aggregation. Our study further identifies for the first time an association between a Panx1–400A>C genetic polymorphism and collagen-induced platelet reactivity. The Panx1–400C variant encodes for a gain-of-function channel that may adversely affect atherothrombosis by specifically enhancing collagen-induced ATP release and platelet aggregation.

scholarly journals Electron microscopic observations on platelet aggregation induced by cationized ferritin

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 439-447
Author(s):  
H Yamazaki ◽  
H Suzuki ◽  
N Yamamoto ◽  
K Tanoue
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Atp Release ◽  
Human Platelets ◽  
Cationized Ferritin ◽  
Electron Microscopic ◽  
Neuraminidase Treatment ◽  
Platelet Surface ◽  
Round Form ◽  
Plasma Factors

Washed and gel-filtered human platelets were dose-dependently aggregated by the addition of cationized ferritin (CF). Ca++ and plasma factors were not necessary to induce the aggregation. Immediately after the addition of CF, CF particles were attached to the surface of platelets that showed discoid form, as observed electron microscopically. Some platelets were connected to each other through the CF particles located on their membranes. After the addition of CF, the following was observed: at 15 sec after, platelets showed a round form and were aggregated to each other; at 3 min after, centralization of granules was clearly seen and the aggregates increased their size during the time course; at 3–5 min after, the CF-connected aggregates were found locally. Around the aggregates, other platelets were aggregated, though not through the membrane-located CF. Observing with a lumiaggregometer, the aggregation showed a biphasic curve associated with adenosine triphosphate (ATP) release. The second part of aggregation curve was inhibited by PGI2, PGE1, aspirin, N- ethylmaleimide, and apyrase. The first part of the aggregation curve was inhibited only by heparin. Neuraminidase treatment also inhibited the aggregation dose-dependently. These findings suggest that neutralization of the platelet surface negative charge by a positively charged macromolecule can trigger platelet aggregation, which is followed by the release reaction.

scholarly journals Adenosine Diphosphate (ADP) and ADP Receptor Play a Major Role in Platelet Activation/Aggregation Induced by Sera From Heparin-Induced Thrombocytopenia Patients

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 549-554 ◽  
Author(s):  
János Polgár ◽  
Petra Eichler ◽  
Andreas Greinacher ◽  
Kenneth J. Clemetson
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Atp Release ◽  
Adenosine Diphosphate ◽  
Heparin Therapy ◽  
Dense Granule ◽  
Heparin Induced Thrombocytopenia ◽  
Adp Receptor ◽  
Induced Aggregation ◽  
Granule Release

The molecular basis for heparin-induced thrombocytopenia (HIT), a relatively common complication of heparin therapy, is not yet fully understood. We found that pretreatment of platelets with AR-C66096 (formerly FPL 66096), a specific platelet adenosine diphosphate (ADP) receptor antagonist, at a concentration of 100 to 200 nmol/L that blocked ADP-dependent platelet aggregation, resulted in complete loss of platelet aggregation responses to HIT sera. AR-C66096 also totally inhibited HIT serum-induced dense granule release, as judged by measurement of adenosine triphosphate (ATP) release. Apyrase, added to platelets at a concentration that had only minor effects on thrombin- or arachidonic acid-induced aggregation, also blocked completely HIT serum-induced platelet aggregation. Furthermore, AR-C66096 inhibited platelet aggregation and ATP release induced by cross-linking FcγRIIA with specific antibodies. These data show that released ADP and the platelet ADP receptor play a pivotal role in HIT serum-induced platelet activation/aggregation. The thromboxane receptor inhibitor, Daltroban, had no effect on HIT serum-induced platelet activation whereas GPIIb-IIIa antagonists blocked platelet aggregation but had only a moderate effect on HIT serum-induced dense granule release. Pretreatment of platelets with chondroitinases but not with heparinases resulted in concentration dependent inhibition of HIT serum-induced platelet aggregation. These novel data relating to the mechanism of platelet activation induced by HIT sera suggest that the possibility should be examined that ADP receptor antagonists or compounds that inhibit ADP release may be effective as therapeutic agents for the prevention or treatment of complications associated with heparin therapy.

scholarly journals Electron microscopic observations on platelet aggregation induced by cationized ferritin

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 439-447 ◽  
Author(s):  
H Yamazaki ◽  
H Suzuki ◽  
N Yamamoto ◽  
K Tanoue
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Atp Release ◽  
Human Platelets ◽  
Cationized Ferritin ◽  
Electron Microscopic ◽  
Neuraminidase Treatment ◽  
Platelet Surface ◽  
Round Form ◽  
Plasma Factors

Abstract Washed and gel-filtered human platelets were dose-dependently aggregated by the addition of cationized ferritin (CF). Ca++ and plasma factors were not necessary to induce the aggregation. Immediately after the addition of CF, CF particles were attached to the surface of platelets that showed discoid form, as observed electron microscopically. Some platelets were connected to each other through the CF particles located on their membranes. After the addition of CF, the following was observed: at 15 sec after, platelets showed a round form and were aggregated to each other; at 3 min after, centralization of granules was clearly seen and the aggregates increased their size during the time course; at 3–5 min after, the CF-connected aggregates were found locally. Around the aggregates, other platelets were aggregated, though not through the membrane-located CF. Observing with a lumiaggregometer, the aggregation showed a biphasic curve associated with adenosine triphosphate (ATP) release. The second part of aggregation curve was inhibited by PGI2, PGE1, aspirin, N- ethylmaleimide, and apyrase. The first part of the aggregation curve was inhibited only by heparin. Neuraminidase treatment also inhibited the aggregation dose-dependently. These findings suggest that neutralization of the platelet surface negative charge by a positively charged macromolecule can trigger platelet aggregation, which is followed by the release reaction.

scholarly journals Deficiency of intact thrombospondin and membrane glycoprotein Ia in platelets with defective collagen-induced aggregation and spontaneous loss of disorder

Blood ◽  
1988 ◽  
Vol 71 (4) ◽  
pp. 1074-1078 ◽  
Author(s):  
B Kehrel ◽  
L Balleisen ◽  
R Kokott ◽  
R Mesters ◽  
W Stenzinger ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Type I Collagen ◽  
Adenosine Diphosphate ◽  
Membrane Glycoprotein ◽  
Type I ◽  
Bleeding Tendency ◽  
Clot Retraction ◽  
Spontaneous Disappearance ◽  
High Doses ◽  
Induced Aggregation

Platelets from a patient with a severe lifelong bleeding tendency, which later spontaneously disappeared, lacked intact thrombospondin and glycoprotein (GP) Ia. Before disappearance of the bleeding disorder, results of coagulation studies and platelet aggregation in response to adenosine diphosphate (ADP), arachidonic acid, thrombin, A23187, epinephrine, and ristocetin were normal. In contrast, aggregation only occurred in the presence of collagen or wheat germ agglutinin at unusually high doses of these agonists. The platelets adhered normally to purified bovine and human type I collagen, and they did not spread in the presence of methylated type I collagen. No collagen-induced clot retraction was observed. Two-dimensional gel electrophoretic analyses of platelet proteins and immunologic studies showed that intact thrombospondin and GP Ia were absent. Aggregation in response to collagen could be restored by adding thrombospondin. Disappearance of the bleeding tendency occurred at the onset of menopause; subsequent analyses revealed that thrombospondin and GP Ia were present in platelets and that collagen-induced platelet aggregation was normal. These results suggest that both thrombospondin and GP Ia are essential in collagen-induced platelet aggregation. The spontaneous disappearance of the bleeding tendency may have been related to hormonal influences.

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.

Plasmin Inhibition of Thrombin-induced Platelet Aggregation

1975 ◽  
Vol 33 (02) ◽  
pp. 286-309 ◽  
Author(s):  
Jonathan L Miller ◽  
Alfred J Katz ◽  
Maurice B Feinstein
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Calcium Concentration ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Platelet Protein ◽  
Platelet Receptor ◽  
Induced Aggregation ◽  
Plasmin Inhibitor ◽  
Maximal Block

SummaryThe effects of plasmin treatment upon washed human platelets were studied in an attempt to elucidate the mechanisms underlying thrombin-induced platelet aggregation. At calcium concentrations of 10–20 μM, plasmin (0.2 CTA U/ml) inhibited thrombin-induced aggregation almost completely, but did not diminish the thrombin-induced release of adenine nucleotides, 5-hydroxytryptamine, or calcium. Increasing the calcium concentration partially antagonized plasmin’s inhibition of aggregation.Studies utilizing calcium chelators and the Kunitz soybean trypsin inhibitor (SBTI) as a plasmin inhibitor indicated that in order to achieve maximal block of aggregation, plasmin must act upon a substrate made fully available only after an initial thrombin-platelet interaction has taken place. Moreover, the time course of this inhibition parallels the time course of the thrombin-induced release reaction.Plasmin inhibition of aggregation could not be mimicked by exposing the platelets to proteolytic digests of fibrinogen at concentrations as high as 17% total platelet protein. Nor could inhibitory activity be recovered from supernatants of plasmin -treated platelets, upon centrifugation and treatment with SBTI.With the use of a “cold initiation” technique, the release by thrombin of 46.7 ± 6-7 (mean ± SEM) μg of fibrinogen immunological equivalents per mg platelet protein could be demonstrated. Platelets in which thrombin-induced aggregation was abolished by plasmin treatment (and the plasmin subsequently inactivated by SBTI) aggregated normally upon addition of as little as 10 μg human plasma fibrinogen per mg platelet protein.It is concluded that plasmin inhibition of aggregation most likely results from its attack upon a protein that is released or becomes fully available subsequent to interaction of thrombin with a platelet receptor mediating release. The results of this study are consistent with a cofactor role for fibrinogen in the aggregation of human platelets by thrombin.

Aggregation, Release and Desensitization Induced in Platelets from Five Species by Platelet Activating Factor (PAF)

1983 ◽  
Vol 49 (03) ◽  
pp. 204-207 ◽  
Author(s):  
D I Cargill ◽  
D S Cohen ◽  
R G Van Valen ◽  
J J Klimek ◽  
R P Levin
Keyword(s):  
Guinea Pig ◽  
Platelet Rich Plasma ◽  
Platelet Activating Factor ◽  
Atp Release ◽  
Human Platelets ◽  
Threshold Concentration ◽  
Dense Granule ◽  
Second Phase ◽  
High Doses ◽  
Induced Aggregation

SummaryPAF-induced aggregation and dense granule release (ATP release) were investigated in human, primate, guinea pig, rabbit and rat platelet-rich plasma (PRP). Guinea pig PRP was most sensitive to PAF, followed by rabbit and then human. PRP from rats and primates did not aggregate or release when exposed to PAF.In guinea pig and rabbit PRP, release was independent of aggregatory response. In human PRP high doses of PAF (5 × 10-7M) caused maximum aggregation and a biphasic ATP release, whilst lower concentrations (3×10-8M) induced biphasic aggregation and one phase of ATP release concomitant with the second phase of aggregation.Aggregation and ATP release induced by PAF in guinea pig and rabbit PRP is dependent upon Ca++ levels, whilst the response of human platelets is relatively independent of the Ca++ level above a certain threshold concentration.The PRP from the three species which aggregated with PAF also demonstrate the phenomenon of desensitization.

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