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Blood, 15 May 2002, Vol. 99, No. 10, pp. 3868-3870
CORRESPONDENCE
To the editor:
Adenosine diphosphate (ADP) does not induce thromboxane
A2 generation in human platelets
Recently, Jin et al addressed the generally misunderstood
problem of the factors responsible for adenosine diphosphate
(ADP)-induced thromboxane A2 (TxA2) production
by human platelets.1 They used stirred, washed human
platelet suspensions to which fibrinogen but no CaCl2 had
been added; as a consequence, the concentration of external
Ca++ ([Ca++]o) was much lower than
physiological. The main results of their study can be
summarized as follows: (i) the ADP receptor antagonists A2P5P
(anti-P2Y1) or AR-C67085 (anti-P2Y12) inhibited
platelet aggregation and TxA2 production induced by ADP;
(ii) the fibrinogen receptor antagonist SC49992 inhibited platelet
aggregation and TxA2 production induced by ADP; (iii) the
Fab fragment of ligand-induced binding site 6 (LIBS6) antibody, which
induces a fibrinogen binding site on
 IIb 3, caused platelet aggregation and
TxA2 production when added to platelet suspensions with
fibrinogen; (iv) A2P5P (P2Y1 antagonist) or AR-C67085
(P2Y12 antagonist), when added together or alone, inhibited
the secondary wave of aggregation and TxA2 production
induced by LIBS6 plus fibrinogen; and (v) in the presence of
physiological concentrations of Ca++, there was no
production of TxA2 by ADP-aggregated platelets. The authors
concluded that ADP induces TxA2 generation in human platelets, which requires coordinated signaling through the integrin IIb3 and ADP receptors. We do not agree with the authors' interpretation of their own results.
Rather, we think that their results are in accord with well-established
knowledge of the factors involved in ADP-induced TxA2
generation: (1) ADP does not stimulate TxA2 production
directly; (2) it is the close platelet-to-platelet contact that is
brought about by ADP-induced platelet aggregation that triggers the
production of TxA2; and (3) this effect is greatly enhanced
and can be seen in most healthy individuals when [Ca++]o
is decreased to micromolar levels.2-5 Based on this
interpretation, it is not surprising that the experiments performed by
Jin et al showed that antagonists of P2Y1,
P2Y12, or the fibrinogen receptor, which inhibit
ADP-induced platelet aggregation, abolished the TxA2
production. The dependency of TxA2 production and the
ensuing platelet secretion on platelet aggregation is demonstrated by the observation that no TxA2 production or platelet
secretion occurs from normal human platelets that are stimulated by
ADP, even at high concentration, under conditions in which platelet aggregation does not occur: for instance, if the receptor function for
adhesive proteins on IIb3 is inhibited
with inhibitory monoclonal antibodies or
Arg-Gly-Asp-containing peptides, or, more simply, if the
platelet suspension is not stirred. The mechanism by which platelet
aggregation triggers TxA2 formation at low
[Ca++]o is presently unknown. But the mechanism is by no
means selective for ADP-induced platelet aggregation, because similar
effects occur when platelets are aggregated by other weak
agonists,6 and it can be observed also when close
platelet-to-platelet contact is brought about by LIBS6 and
fibrinogen,1 or the agglutinating agents polylysine or
ristocetin.7,8 The platelet-to-platelet contact caused by
weak agonists, agglutinating agents or LIBS6 and fibrinogen causes the
formation of trace amounts of TxA2 and the secretion of
ADP, which supports the secondary full aggregation responsible for the
production of large amounts of TxA2. None of these
responses, including TxA2 production, is observed when the
same agonists or agglutinating agents are added to the same platelet
suspension under nonstirring conditions.6 This observation clearly indicates that a definite distinction should be made between platelet aggregation and fibrinogen receptor
occupancy. When platelets are exposed to ADP under nonstirring
conditions to prevent their aggregation, ADP elicits the inside-out
signaling, which promotes the occupancy of the fibrinogen receptor on
IIb3, which in turn elicits the
outside-in signaling. According to the model proposed by Jin et al,
this should be enough to stimulate the generation of TxA2
by platelets; under these conditions, however, TxA2
production does not occur: it is only when platelets are stirred and,
as a consequence, they are allowed to aggregate that platelet
TxA2 production can be observed. Finally, the fact that platelet secretion and the production of large
amounts of TxA2 occur with platelets from most
healthy individuals only when [Ca++]o is at
micromolar levels demonstrates that the whole process that can be
observed in the turbidometric aggregometer is mostly an in vitro
artifact.2-5 (The widespread use of citrate as an anticoagulant in the preparation of platelet-rich plasma introduces the
same artifact as suspending media without added CaCl2.)
Despite this, Jin et al suggest that ADP-induced TxA2
production may have physiological relevance in vivo, because
[Ca++]o might be "drastically
lower" 1(p196) at sites of thrombus formation.
We do not know of any support for this hypothesis, and we believe that,
due to the secretion of Ca++ from platelet granules,
[Ca++]o might even increase, rather than decrease.
Needless to say, we agree with the authors' statement that the results
of clinical trials with aspirin indicate that TxA2 plays an
important role in thrombus formation in vivo.9
But this is not a demonstration that, in vivo, ADP causes platelet
TxA2 formation. Platelet thrombus formation in vivo depends
on the synergistic interaction of several agonists, some of which, such
as collagen and thrombin, directly cause the formation of
TxA2. The recent trials combining drugs such as clopidogrel
with aspirin10 add evidence to the fact that separate
pathways contribute to thrombosis and that combined therapies are more
effective than aspirin alone or clopidogrel alone.
Marco Cattaneo, Christian Gachet, Jeanne-Pierre Cazenave, and Marian A. Packham
Correspondence: Marco Cattaneo, Department of Medicine, Surgery
and Dentistry, Ospedale San Paolo via Di Rudini, 8, University of
Milano, Milano, Italy
References
1.
Jin J, Quinton TM, Zhang J, Rittenhouse SE, Kunapuli SP.
Adenosine diphosphate (ADP)-induced thromboxane A2 generation in human platelets requires coordinated signaling through integrin alphaIIbbeta3 and ADP receptors.
Blood.
2002;99:193-198[Abstract/Free Full Text].
2.
Mustard JF, Perry DW, Kinlough-Rathbone RL, Packham MA.
Factors responsible for ADP-induced release reaction of human platelets.
Am J Physiol.
1975;228:1757-1765[Abstract/Free Full Text].
3.
Packham MA, Bryant NL, Guccione MA, Kinlough-Rathbone RL, Mustard JF.
Effect of the concentration of Ca2+ in the suspending medium on the responses of human and rabbit platelets to aggregating agents.
Thromb Haemost.
1989;62:968-976[Medline]
[Order article via Infotrieve].
4.
Falcon CR, Cattaneo M, Ghidoni A, Mannucci PM.
The in vitro production of thromboxane B2 by platelets of diabetic patients is normal at physiological concentrations of ionized calcium.
Thromb Haemost.
1993;70:389-392[Medline]
[Order article via Infotrieve].
5.
Cattaneo M, Gachet C.
ADP receptors and clinical bleeding disorders.
Arterioscler Thromb Vasc Biol.
1999;19:2281-2285[Abstract/Free Full Text].
6.
Charo IF, Feinman RD, Detwiler TC.
Interrelations of platelet aggregation and secretion.
J Clin Invest.
1977;60:866-873.
7.
Guccione MA, Packham MA, Kinlough-Rathbone RL, Perry DW, Mustard JF.
Reactions of polylysine with human platelets in plasma and in suspensions of washed platelets.
Thromb Haemost.
1976;36:360-375[Medline]
[Order article via Infotrieve].
8.
Cattaneo M, Chiodini M, Mannucci PM.
Ristocetin and platelet aggregation.
Br J Haematol.
1989;71:301-302[Medline]
[Order article via Infotrieve].
9.
Collaborative overview of randomised trials of antiplatelet therapy, I: prevention of death, myocardial infarction, and stroke by prolonged antiplatelet therapy in various categories of patients. Antiplatelet Trialists' Collaboration.
Br Med J.
1994;308:81-106[Abstract/Free Full Text].
10.
Yusuf S, Zhao F, Mehta SR, et al.
Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation.
N Engl J Med.
2001;345:494-502[Abstract/Free Full Text].
Response:
Signaling events regulating thromboxane A2
generation in platelets
Cattaneo et al argue that the results of our recently published
paper are in accord with well-established knowledge of the factors
involved in adenosine diphosphate (ADP)-induced thromboxane A2 generation, but they do not agree with the
interpretation of our results. They state that (1) ADP does not
stimulate thromboxane A2 production directly; (2) it is the
close platelet-to-platelet contact that is brought about by ADP-induced
platelet aggregation that triggers the production of thromboxane
A2; and (3) this effect is greatly enhanced and can be seen
in most healthy individuals when extracellular calcium is decreased to
micromolar levels. Our results have demonstrated that blockade of the P2Y1
receptor by A2P5P, of the P2Y12 receptor by AR-C67085, or of fibrinogen binding to its receptor by SC49992 inhibits both platelet aggregation and thromboxane A2 generation.1 These results
are consistent with the argument of Cattaneo et al that ADP-induced
thromboxane A2 generation is dependent on ADP-induced
platelet aggregation. We addressed this issue and went further to
investigate whether A2P5P and AR-C67085 block thromboxane
A2 generation simply by inhibiting ADP-induced platelet
aggregation, or whether ADP contributes beyond that to signaling events
in thromboxane A2 generation. ADP under stirring conditions
causes platelet aggregation, which results in close cell-to-cell
contact. Signaling under these conditions is termed as "outside-in"
signaling, which Shattil et al defined as "initiated at localized
regions of cell matrix and cell-cell contact." 2(p2651)
This outside-in signaling translates into several
intracellular events including tyrosine phosphorylation of platelet
proteins.2 Blockade of fibrinogen binding to activated
integrin IIb3 by Arg-Gly-Asp-Ser, EDTA
(ethylenediaminetetraacetic acid), or mAb A2A9 abrogated
tyrosine phosphorylations of platelet proteins, indicating the
important role of fibrinogen binding.3 Our
results1 with SC49992 are consistent with the importance of
fibrinogen binding in outside-in signaling events. When LIBS6 Fab
fragments and fibrinogen were used to induce platelet aggregation
without adding any other agonist, thromboxane A2
was generated.1 But under these conditions, the
ADP-receptor antagonists A2P5P or AR-C67085 inhibited thromboxane
A2 generation.1 Furthermore, a combination of
both A2P5P and AR-C67085 nearly abolished thromboxane A2
generation induced by LIBS6 and fibrinogen, without affecting primary
aggregation, although full irreversible aggregation was impaired.1 But inhibition of full, irreversible
aggregation by wortmannin did not impair thromboxane A2
accumulation in response to LIBS6 plus fibrinogen. These data suggest
that the close platelet-to-platelet contact that is brought about by
platelet aggregation (primary or full) is not sufficient to generate
thromboxane A2 and depends on signaling from the ADP
receptors. Hence we disagree with the interpretation of Cattaneo et al
of the previously published results from several laboratories. We stand
by our interpretation of our results that outside-in signaling from
both the integrin IIb3 and the P2 receptors is required for
ADP-induced thromboxane A2 generation in platelets. We
agree with Cattaneo et al that ADP-induced thromboxane generation
is greatly enhanced and can be seen in most healthy individuals when
extracellular calcium is decreased to micromolar levels.
Jianguo Jin, Todd M. Quinton, Jin Zhang, Susan E. Rittenhouse, and Satya P. Kunapuli
Correspondence: Satya P. Kunapuli, Department of Physiology,
Temple University Medical School, Philadelphia, PA 19140
References
1.
Jin J, Quinton TM, Zhang J, Rittenhouse SE, Kunapuli SP.
Adenosine diphosphate (ADP)-induced thromboxane A2 generation in human platelets requires coordinated signaling through integrin IIb3 and ADP receptors.
Blood.
2002;99:193-198.
2.
Shattil SJ, Kashiwagi H, Pampori N.
Integrin signaling: the platelet paradigm.
Blood.
1998;91:2645-2657[Free Full Text].
3.
Golden A, Brugge JS, Shattil SJ.
Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins.
J Cell Biol.
1990;111:3117-3127[Abstract/Free Full Text].
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Adenosine diphosphate (ADP)-induced thromboxane A2 generation in human platelets requires coordinated signaling through integrin IIb3 and ADP receptors
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