These conclusions pertain to Z-Gly-Gly-Arg-AMC at the
usual concentration, i.e. at around twice its Km. They should
not be extrapolated to other substrates or to higher concen-
trations. They certainly should not be interpreted as a laissez
passer for any other substrate. Thrombin generation experi-
ments with high-affinity substrates, e.g. those performed in ,
are very likely to reflect a coagulation mechanism in which
feedback mechanisms are severely disturbed.
Disclosure of Conflict of Interests
The authors state that they have no conflicts of interest.
1 Butenas S, Mann KG. Caution in the interpretation of continuous
thrombin generation assays. J Thromb Haemost 2007; 5: 1084–5.
2 Hemker HC, Giesen P, Al Dieri R, Regnault V, De Smedt E, Wage-
nvoord R, Lecompte T, Beguin S. Calibrated automated thrombin
generation measurement in clotting plasma. Pathophysiol Haemost
Thromb 2003; 33: 4–15.
of thrombin generation in plasma, its use for the determination of the
thrombin potential. Thromb Haemost 1993; 70: 617–24.
via the endogenous thrombin potential. Thromb Haemost 1995; 74:
Dargaud Y, Beguin S, Lienhart A, Al Dieri R, Trzeciak C, Bordet JC,
Hemker HC, Negrier C. Evaluation of thrombin generating capacity
in plasma from patients with haemophilia A and B. Thromb Haemost
2005; 93: 475–80.
Calatzis A. Improved method for the assessment of thrombin for-
mation in blood or plasma. EP Patent 1,367,135, 2003.
Evidence that pre-existent variability in platelet response to
ADP accounts for ?clopidogrel resistance?: a rebuttal
P. GURBEL, K. P. BLIDEN and U. TANTRY
Sinai Center for Thrombosis Research, Baltimore, MD, USA
To cite this article: Gurbel P, Bliden KP, Tantry U. Evidence that pre-existent variability in platelet response to ADP accounts for ?clopidogrel
resistance?: a rebuttal. J Thromb Haemost 2007; 5: 1087–8.
See also Michelson AD, Linden MD, Furman MI, Li Y, Barnard MR, Fox ML, Lau WC, McLaughlin TJ, Frelinger AL. Evidence that pre-existent
variability in platelet response to ADP accounts for ?clopidogrel resistance?. J Thromb Haemost 2007; 5: 75–81; Michelson AD, Furman MI, Lau WC,
McLaughlin TJ, Frelinger III AL. Evidence that pre-existent variability in platelet response to ADP accounts for ?clopidogrel resistance?: reply to a
rebuttal. This issue, pp 1089–90.
We read the article by Michelson et al.  with interest. We
believe that several issues concerning the evaluation of
clopidogrel resistance or response variability should be empha-
sized. Most importantly, in order to assess ?resistance? or
response variability to an antiplatelet agent, it is mandatory to
measure platelet function before and after therapy [2,3].
Therefore, in the study by Michelson et al., the complete
absence of pretreatment and post-treatment measurements in
patients and the absence of an analysis of platelet inhibition in
the healthy control group precludes any conclusions related to
clopidogrel resistance. Moreover, most studies addressing
clopidogrel response variability have enrolled a homogeneous
patient population, as platelet reactivity to adenosine diphos-
phate (ADP) varies with the disease state, and patients were
treated with a uniform dosing regimen. Finally, the lack of
serial prespecified measurements, we believe, is another
important limitation of the study.
In 2001, by serially measuring ADP-induced platelet aggre-
gation and expression of activation-dependent receptors in 100
patients undergoing coronary stenting treated with a 300-mg
clopidogrel loading dose followed by a 75-mg daily mainten-
ance dose, we demonstrated that clopidogrel treatment did not
produce a uniform level of post-treatment platelet reactivity to
ADP . We had also previously demonstrated that there is
variability in ADP-induced platelet aggregation in aspirin-
treated patients not receiving clopidogrel . In 2003, we
reported a study of 96 patients undergoing elective stenting
catheterization laboratory followed by a 75-mg maintenance
dose. ADP-induced platelet aggregation and activation-
dependent platelet surface marker expression [P-selectin and
activated gylcoprotein (GP) IIb/IIIa] were assessed before
clopidogrel therapy and serially for 30 days following treat-
ment for stenting. In the latter study and a subsequent study,
we again demonstrated non-uniform post-treatment platelet
reactivity to ADP that followed a normal distribution [6,7].
Correspondence: Paul A. Gurbel, Sinai Center for Thrombosis
Research, Hoffberger Building, Suite 56, 2401 W. Belvedere Ave,
Baltimore, MD 21215, USA.
Tel.: +1410601 9600; fax:
Received 23 January 2007, accepted 8 February 2007
Letters to the Editor 1087
? 2007 International Society on Thrombosis and Haemostasis
Importantly, we measured the absolute change in aggregation
induced by ADP, and clearly demonstrated that at 24 h after a
300-mg loading dose, ?30% of patients had a £ 10% absolute
change in ADP-induced aggregation. Finally, we hypothesized
that the post-treatment platelet reactivity correlated with the
pretreatment reactivity, and, indeed, the latter was also
demonstrated in our investigation .
Thus, the portion of the report by Michelson et al. [2,3] that
25 normal subjects supports our initial hypothesis that
pretreatment platelet reactivity predicts post-treatment reactiv-
ity. Although the mechanism for thelatter observation remains
unknown, it has been repeatedly suggested that high post-
treatment platelet reactivity is a risk factor for ischemic events.
Since our initial description, subsequent investigations have
unequivocally demonstrated clopidogrel non-responsiveness,
on the basis of pretreatment and post-treatment analyses using
ADP-induced aggregation, and flow cytometric measurements
of ADP-stimulated P-selectin, active GPIIb/IIIa expression,
and vasodilator-stimulated phosphoprotein (VASP) phos-
phorylation levels [2,3]. It is now well established that the
assessment ofresponsiveness toclopidogreliscriticallydepend-
ent on the time when post-treatment measurements are
conducted, after the load and the dose of the load [2,3]. Higher
estimates of non-responsiveness are present following the 300-
mg loading dose as compared to after the 600-mg loading dose
[2,3]. In the study by Michelson et al., although 635 patients
were included, the study did not assess pretreatment and post-
and nor was there a uniform dose – factors that are critical in
determining clopidogrel response variability. As only post-
treatment platelet reactivity was measured, clopidogrel respon-
siveness could not be determined.
Only 30–50% inhibition of ex vivo ADP-induced platelet
aggregation was demonstrated following a repeated daily dose
of 75 mg in normal volunteers or loading doses of 300 or
600 mg in patients undergoing percutaneous coronary inter-
vention (PCI) . This level of incomplete inhibition, in turn,
correlated with incomplete P2Y12 receptor blockade. The
repeated demonstrations that a high loading dose of 600 mg of
clopidogrel is associated with increased inhibition of ex vivo
ADP-induced platelet aggregation in patients undergoing PCI
and a decreased incidence of non-responders support insuffi-
cient active metabolite generation as a major factor in
clopidogrel resistance [2,3]. This insufficient metabolite genera-
tion may be secondary to limited absorption of clopidogrel
prodrug , or genetic variation in CYP3A4 or drug–drug
interactions with certain statins at the CYP3A4 level . The
pivotal role of limited active metabolite generation in explain-
ing response variability comes from recent studies demonstra-
ting that: (i) unlike prasugrel (a CYP450-dependent P2Y12
receptor inhibitor), clopidogrel is associated with lower plasma
concentrations of active metabolite, lower platelet inhibition,
and wider response variability ; and (ii) administration of
prasugrel to clopidogrel non-responders provides excellent
platelet inhibition . Thus, the above data strongly support
insufficient active metabolite generation as the primary
explanation for response variability rather than pre-existing
platelet function variability.
In summary, clopidogrel response variability was hypothes-
ized in 2001, and was repeatedly and unequivocally demon-
strated by multiple centers employing multiple techniques that
measure ADP-induced responses in platelets before and after
clopidogrel treatment. On the basis of the current findings,
clopidogrel response variability is primarily explained by
insufficient clopidogrel metabolite generation. It also appears
that post-treatment reactivity is a better predictor of ischemic
events than clopidogrel non-responsiveness, as some non-
responders have low pretreatment platelet reactivity .
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.
1 Michelson AD, Linden MD, Furman MI, Li Y, Barnard MR, Fox
ML, Lau WC, McLaughlin TJ, Frelinger AL. Evidence that pre-
existent variability in platelet response to ADP accounts for ?clopi-
dogrel resistance?. J Thromb Haemost 2007; 5: 75–81.
2 Gurbel PA, Tantry US. Clopidogrel resistance? Thromb Res 2007;
3 Gurbel PA, Tantry US. Drug insight: clopidogrel nonresponsiveness.
Nat Clin Pract Cardiovasc Med 2006; 3: 387–95.
4 Gurbel PA, Cummings CC, Alford AB, Meister AF, Serebruany VL;
Plavix Reduction of New Thrombus Occurrence (PRONTO) trial.
Onset and extent of platelet inhibition by loading dose clopidogrel in
patients undergoing elective coronary stenting: the Plavix Reduction
Of New Thrombus Occurrence (PRONTO) trial. Am Heart J 2003;
5 Gurbel PA, Bliden KP. The stratification of platelet reactivity and
activation in patients with stable coronary artery disease on aspirin
therapy. Thromb Res 2003; 112: 9–12.
6 Gurbel PA, Bliden KP, Hiatt BL, O?Connor CM. Clopidogrel for
coronary stenting: response variability, drug resistance, and the effect
of pretreatment platelet reactivity. Circulation 2003; 107: 2908–13.
7 Gurbel PA, Bliden KP, Hayes KM, Yoho JA, Herzog WR, Tantry
US. The relation of dosing to clopidogrel responsiveness and the
incidence of high post-treatment platelet aggregation in patients
undergoing coronary stenting. J Am Coll Cardiol 2005; 45: 1392–6.
A, Schomig A. Absorption, metabolization, and antiplatelet effects of
300-, 600-, and 900-mg loading doses of clopidogrel: results of the
ISAR-CHOICE (intracoronary stenting and antithrombotic regimen:
choose between 3 high oral doses for immediate clopidogrel effect)
Trial. Circulation 2005; 112: 2946–50.
9 Lau WC, Gurbel PA. Antiplatelet drug resistance and drug–drug
interactions: role of cytochrome P450 3A4. Pharm Res 2006; 23: 2691–
10 Brandt JT, Payne CD, Wiviott SD, Weerakkody G, Farid NA, Small
DS, Jakubowski JA, Naganuma H, Winters KJ. A comparison of
prasugrel and clopidogrel loading doses on platelet function: magni-
tudeof plateletinhibitionisrelatedtoactivemetaboliteformation. Am
Heart J 2007; 153(1): 66.
11 Samara WM, Bliden KP, Tantry US, Gurbel PA. The difference be-
tween clopidogrel responsiveness and posttreatment platelet reactivity.
Thromb Res 2005; 115: 89–94.
1088 Letters to the Editor
? 2007 International Society on Thrombosis and Haemostasis