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(Circulation. 1997;96:2877-2883.)
© 1997 American Heart Association, Inc.
Articles |
Differential Effects of Anticoagulants on the Activation of Platelets Ex Vivo
From the Department of Medicine (D.J.S., B.E.S.) and the Department of Biochemistry (P.B.T., K.G.M.), the University of Vermont College of Medicine, Burlington.
Correspondence to David J. Schneider, MD, Cardiovascular Division, E217 Given Bldg, University of Vermont, Burlington, VT 05405. E-mail djschnei{at}zoo.uvm.edu
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Abstract |
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Background Because activation of platelets and of the coagulation system are interdependent mediators of thrombosis, platelet activation was characterized in whole blood in the presence of anticoagulants used to assess platelet function in vitro or as treatment for patients with occlusive arterial disease.
Methods and Results Blood was anticoagulated alone or in combination with citrate, ethylenediaminetetraacetatic acid, corn trypsin inhibitor (CTI, an inhibitor of activated factor XII), heparin, enoxaparin, recombinant tick anticoagulant peptide (rTAP), or recombinant hirudin. Platelet activation in response to adenosine diphosphate (ADP) or collagen was detected by assay of P-selectin on the platelet surface delineated by flow cytometry. Although minimal activation was seen without ADP, the fraction of platelets expressing P-selectin in response to ADP was greatest in blood anticoagulated with citrate compared with CTI and all other anticoagulants. ADP-induced platelet activation was greater in blood anticoagulated with heparin compared with an equipotent anti-Xa concentration of enoxaparin. More variable results were seen with collagen, but platelet activation in the presence of citrate was greater than that with CTI.
Conclusions Interpretation of assays of inhibition of platelet activation by potentially therapeutic agents in vitro requires consideration of the effects of anticoagulants used. In addition, anticoagulants other than standard heparin may potentiate efficacy of antiplatelet drugs.
Key Words: platelets • occlusion • coagulation • arteriosclerosis
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Introduction |
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Acute coronary syndromes including myocardial infarction and unstable angina are frequently characterized by thrombosis associated with rupture of complex atherosclerotic plaques.1 2 3 Exposure of blood to procoagulant subendothelial surfaces can trigger adhesion of platelets and generation of thrombin that can initiate and accelerate platelet activation and aggregation as well as deposition of fibrin. Treatment of patients with acute coronary syndromes with antithrombotic and antiplatelet agents decreases the incidence of subsequent myocardial infarction and recurrent angina.4 Both antiplatelet and antithrombotic therapy are critical conjunctive approaches in patients treated with thrombolytic drugs.5 6 7
Release of secretory products and aggregation of platelets are increased in blood from patients with acute coronary syndromes8 9 10 11 and are associated with an increased incidence of subsequent cardiac events and mortality.9 12 Increased activation of platelets has been associated also with subsequent coronary events in subjects without acute coronary syndromes.13 14 Despite aspirin therapy, activation of platelets is evident in patients with acute myocardial infarction15 and in those treated with thrombolytic agents.16
Exposure of blood to minute quantities of tissue factor associated with plaque rupture can lead to activated coagulation factor VIIa–tissue factor complexes that initiate activation of factors IX and X, leading to generation of thrombin.17 18 The platelet surface serves as a pivotal site for assembly of the procoagulant intrinsic "tenase" complex (leading to coagulation factor X activation) and prothrombinase.18 19 20 Thus platelet activation can be viewed as a thrombin-generating system that contributes to an explosive increase in local concentrations of thrombin after a lag and initiation phase19 21 as well as to persistence of active thrombin on activated surfaces and hence persistence of the procoagulant state locally.22 Activation may be modified by binding of coagulation factors to the platelet surface.22 23
Relationships between activation of the coagulation system and
activation of platelets imply that effective antithrombotic therapy
will be a necessary conjunct for effective use of antiplatelet
drugs. Conversely, continuing activation of platelets despite use
of antiplatelet drugs in subjects with acute coronary
syndromes may reflect inadequacy of anticoagulation, inadequacy of the
antiplatelet therapy, or both. To explore this possibility we
characterized activation of platelets ex vivo in whole blood drawn
into solutions containing selected concentrations of specific
anticoagulants that were exposed subsequently to ADP. Platelet
activation was recognized on the basis of the appearance of the cell
surface 
-granule protein P-selectin (CD62) and quantified in terms
of the percentage of platelets expressing P-selectin among all
appropriately sized particles expressing surface
glycoprotein IIb/IIIa.
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Methods |
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Consistent with protocols approved by the University of Vermont Institutional Review Board, phlebotomy was performed on normal volunteers after written informed consent had been obtained. All subjects (6 male and 6 female) were free from illness and were not taking aspirin or other nonsteroidal anti-inflammatory medication for at least 10 days before phlebotomy. Age ranged from 18 to 42 years.
Phlebotomy was by peripheral venipuncture with a 19-gauge butterfly needle while subjects were seated and in a nonfasting state. Tourniquets were applied for <1 minute. After discard of the first 2 to 3 mL of blood, the blood was drawn into syringes prefilled with solutions containing specific anticoagulants to yield a selected final concentration.
The anticoagulants included citrate (0.129 mol/L, pH 6.0), EDTA (0.4 mol/L, pH 6.0), heparin (Elkins-Sinn), enoxaparin (Rhone-Poulenc Rorer), recombinant hirudin (Sigma ), and rTAP (kindly provided by Dr George P. Vlasuk, Corvas International, San Diego, Calif). The specific activities of heparin, enoxaparin, and rTAP with respect to inhibition of factor Xa and inhibition of factor IIa were determined with the use of Actichrome Heparin anti-fXa and Actichrome Heparin anti-fIIa kits (American Diagnostica). CTI (Fluka) was used to inhibit the contact pathway of coagulation as previously described18 at a concentration of 32 µg/mL of whole blood. This agent inhibits coagulation factor XIIa.
For flow cytometry procedures, 5-µL aliquots of whole blood were incubated for 15 minutes in HEPES-Tyrode's buffer with both an FITC-conjugated HP11D (Nichols) IgG (directed against glycoprotein IIb/IIIa) and phycoerythrin-conjugated anti–CD62 IgG (Becton Dickinson) in addition to selected concentrations of ADP.11 Blood was aliquoted within 15 minutes of phlebotomy. Conditions of activation in assays performed in triplicate included basal (no ADP), 0.2 µmol/L ADP, 0.8 µmol/L ADP, and 1.5 µmol/L ADP. Activation of platelets in response to collagen was characterized in response to 0.12, 0.23, and 0.35 mg/mL of collagen (Bio/Data). After the 15-minute incubations with the agonist and antibodies, platelets were fixed with Optilyse-C lysing solution (1.5% formaldehyde, Immunotech). Controls used to detect any nonspecific antibody association with platelets (IgG conjugated with phycoerythrin) were included with each sample set. Control tubes contained FITC-conjugated HP11D and nonfractionated IgG conjugated with phycoerythrin.
The association of antibodies with platelets was determined with the use of an FACS (Becton Dickinson). The population of platelets in each sample was characterized on the basis of the forward and 90 degree sidescatter as well as on the expression of glycoprotein IIb/IIIa (HP11D-FITC). The percentage of platelets expressing P-selectin was defined as the fraction exhibiting specific binding (CD62 positive) minus that exhibiting nonspecific binding (percentage defined with the IgG phycoerythrin conjugate).
Analysis of Data
Values are given as mean±SEM. Comparison of the effects of ADP
on P-selectin expression in each anticoagulant (with respect to all
concentrations of ADP) were performed with the use of the
Wilcoxon signed rank test. Differences between anticoagulants
with respect to a specific concentration of ADP were determined with
the use of ANOVA. Significance was defined as P<.05.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Assays for P-selectin expression were performed in triplicate for each concentration of ADP in each anticoagulant. The fraction of platelets expressing P-selectin under baseline conditions was <0.6% with each of the anticoagulants tested, verifying that the methods of phlebotomy and the preparative procedures of the assay did not, per se, activate platelets. The intra-assay coefficient of variation was 3.7% with 1.5 µmol/L ADP, 5.6% with 0.8 µmol/L ADP, and 14.4% with 0.2 µmol/L ADP (n=75 conditions for each).
Comparison of Calcium Chelators With Other Anticoagulants
Trisodium citrate (0.129 mol/L, pH 6.0) is used
conventionally in assays of the coagulation system and of platelet
aggregation. Although no P-selectin expression was observed under basal
conditions (without ADP), ADP-induced P-selectin expression was
uniformly increased by citrate (n=12 subjects) compared with CTI (32
µg/mL) (n=8 subjects) and with the average of results with all
other anticoagulants studied (heparin, enoxaparin, rTAP, and hirudin)
(Fig 1
, P<.05 for each
comparison). Despite
recalcification of the blood in Tyrode's buffer, activation of
platelets in response to 0.8 µmol/L ADP was 38±5%
in citrate, 21±5% in CTI, and 21±1% (average) in the other
anticoagulants (P<.01 for citrate compared with each other
condition). Although variability between subjects was seen, the
difference between citrate and all the other anticoagulants studied was
consistent.
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To determine whether the effect of citrate was related to its ability
to chelate calcium, blood was anticoagulated with either citrate or
EDTA (0.4 mol/L, pH 6.0). When blood was processed within 5
minutes of phlebotomy, the percent of platelets activated
in response to ADP was greater with citrate than with EDTA (activation
of platelets in response to 0.8 µmol/L ADP for
platelets in citrate was 40±6%; for platelets in EDTA,
26±5%, Fig 2). If blood was
exposed to EDTA for 10 to 15 minutes, the difference between citrate
and EDTA was no longer observed (activation of platelets in
response to 0.8 µmol/L ADP for platelets in citrate,
42±8%; for platelets in EDTA, 48±4%). Accordingly, the effects
of citrate and EDTA on activation of platelets by ADP appear to be
related to chelation of calcium and vary in relation to time.
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Comparisons for Anticoagulants Used In Vivo
To compare anticoagulants in terms of functional activity,
inhibition of activated coagulation factor X (Xa) and thrombin
(IIa) was determined in pooled plasma containing antithrombin III.
Because activity of enoxaparin for clinical use is defined on the basis
of the WHO standard for inhibition of Xa, enoxaparin was used to
standardize the anti-Xa assay in our study. Units are anti-Xa units for
heparin, enoxaparin, and rTAP. Activity of hirudin is expressed in NIH
antithrombin units. As described in the product information, the
quantity of heparin exhibiting 1 U/mL of anti-Xa activity exhibited 1
U/mL of anti-IIa activity as well. For the enoxaparin used in these
experiments, the amount exhibiting 1 U/mL of anti-Xa activity exhibited
0.29 U/mL of anti-IIa activity. The relative anti-Xa and anti-IIa
activities for enoxaparin observed are in accordance with values
described in product information.
ADP-induced expression of P-selectin was determined for platelets
suspended in solution with one of two concentrations of heparin (1 U/mL
or 0.1 U/mL) selected to be in the range (steady state and trough
concentrations) of those seen in vivo with pharmacological doses given
to patients with acute coronary syndromes. ADP-induced
platelet activation in whole blood anticoagulated with 1 U/mL of
heparin was greater than that seen in blood anticoagulated with 0.1
U/mL (P<.05 for all concentrations of ADP, n=12 subjects
for each). A directionally similar difference was seen when results
with 1 U/mL of heparin were compared with those with 1 U/mL of
enoxaparin. Thus with each concentration of ADP, activation of
platelets was greater with 1 U/mL of heparin compared with 1 U/mL
of enoxaparin (n=12 subjects for each). By contrast, ADP-induced
expression of P-selectin in blood anticoagulated with 0.1 U/mL of
enoxaparin was similar to that seen with 1 U/mL of enoxaparin. A
nonsignificant trend toward increased activation of platelets in
response to ADP with 0.1 U/mL of enoxaparin was observed (Fig 2
).
The increased activation of platelets with heparin in response to
ADP was greater with lower concentrations of ADP (Fig 3). The expression of
P-selectin in response to 0.2 µmol/L ADP in blood from
the 12 subjects was 2.8±0.5% in blood with 1 U/mL of heparin,
1.9±0.3% in blood with 0.1 U/mL of heparin, and 2.0±0.3% in blood
with 1 U/mL of enoxaparin (P<.05 for 1 U/mL heparin
compared with 0.1 U/mL and with enoxaparin at 1 U/mL).
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To determine whether the increased activation of platelets in
response to ADP in the presence of 1 U/mL of heparin was related to
limited efficacy of anticoagulation, blood was drawn into citrate
containing 1 or 0.1 U/mL of heparin and into citrate containing 1 or
0.1 U/mL of enoxaparin (Fig 4). The fraction of
platelets expressing P-selectin in response to ADP was greatest in
the blood with citrate and 1 U/mL heparin, intermediate in blood with
citrate and 0.1 U/mL heparin, and lowest in blood with either
concentration of enoxaparin (P<.05 for all concentrations
of ADP in 1 U/mL heparin compared with 0.1 U/mL of heparin, 1 U/mL of
enoxaparin, and 0.1 U/mL of enoxaparin). For each of these conditions,
ADP-induced activation of platelets was greater when citrate was
present in combination with each concentration of heparin or
enoxaparin compared with activation seen with either agent alone
(P<.05 for all concentrations of ADP for the combination
compared with each agent alone).
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To determine whether the particular mechanism through which
anticoagulation is conferred (that is, the activated
coagulation factor that is inhibited by the anticoagulant) accounts for
differences in the extent of ADP-induced activation of platelets,
ADP-induced platelet activation was determined in blood from 5
normal subjects anticoagulated in vitro with rTAP (a specific Xa
inhibitor) or hirudin (a specific IIa
inhibitor). To simulate the functional activities induced
by heparin and enoxaparin, blood was anticoagulated with either 1 or
0.1 U/mL of rTAP and hirudin. Units were anti-Xa units for rTAP and
anti-IIa units for hirudin. No difference in the fraction of
platelets expressing P-selectin in response to ADP was seen with
the two anticoagulants that target different procoagulant proteins (Fig 5). Furthermore, the
ADP-induced activation of platelets was similar in blood
anticoagulated with enoxaparin, CTI, rTAP, and hirudin. Thus the
augmentation of ADP-induced activation of platelets by 1 U/mL
heparin does not appear to depend on the particular mechanism of
anticoagulation by heparin or on limited efficacy of anticoagulation
contingent on a difference in thrombin inhibition (as is evident also
from the lack of increased P-selectin expression with 0.1 U/mL compared
with 1 U/mL of heparin).
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The degranulation (activation) of platelets in response to collagen
was determined in blood from 9 subjects that was anticoagulated (in
vitro) with citrate, CTI, heparin (1 U/mL), and enoxaparin (1 U/mL). In
contrast to results with ADP, the activation induced by collagen was
quite variable. The intra-assay coefficient of variation was 71%
for 0.12 mg/mL of collagen, 35% for 0.23 mg/mL of
collagen, and 20% for 0.35 mg/mL of collagen. As can be seen in
Fig 6, the variability between individuals was also quite large, with
the coefficient of variation
>100%. Despite this
variability, the activation induced by collagen was
consistently greater in blood anticoagulated with citrate
compared with blood anticoagulated with CTI (P=.01 for all
concentrations of collagen, Fig 6). No difference was observed when
results with blood anticoagulated with heparin were compared with
results with blood anticoagulated with enoxaparin.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Platelets are pivotal in thrombosis and hemostasis. Activation of platelets can be initiated by their adherence to damaged vessel surfaces and is associated with the release of factors such as ADP, serotonin, thromboxane, growth factors, and coagulation factors from
-granules.19 21 22 This
activation leads to the release of platelet factor 4 and the
surface expression of P-selectin. In addition, the lipoprotein surface
of an activated platelet can serve as a catalytic moiety
for the assembly of the tenase and prothrombinase
complexes.18 19 20 Cross-linking of activated
glycoprotein IIb/IIIa by fibrinogen on the surface of
activated platelets contributes to the thrombotic mesh and
the consolidated mass of platelets.24 The
cross-linking of platelets is enabled by the conformational change
in glycoprotein IIb/IIIa exposing binding sites for
fibrinogen. Exocytosis of -granules accounts for release of proteins
and for expression of P-selectin. This expression of P-selectin
facilitates binding of platelets to cells.25 26 In our studies, platelet activation was determined in whole blood. ADP and collagen were used to characterize thresholds for activation under specific conditions. Results with collagen were highly variable in this assay system. One factor potentially underlying the variability may be that the blood cannot be stirred efficiently during exposure to collagen under the conditions that we used. A strength of the assay procedure with the use of ADP as agonist was the intra-assay reproducibility, reflected by coefficient of variation of 14% with the lowest concentration of ADP, 6% with the middle concentration, and 4% with the highest.
Citrate is used as an anticoagulant in most conventional assays of coagulation and platelet function. Citrate did not activate platelets under basal conditions when no exogenous ADP was present. However, compared with the other anticoagulants studied, citrate and EDTA potentiated ADP-induced activation of platelets. No ideal "gold standard" exists against which the effect of citrate can be measured. However, CTI was selected for this purpose because it is an effective inhibitor of factor XIIa devoid of inhibition of other coagulation factors.18 Activation of factor XII occurs in vitro when blood is exposed to foreign surfaces, such as glass or plastic. It does not appear to underlie initiation of coagulation in vivo under physiological conditions.27 28 29 30 By contrast, initiation of coagulation in vivo appears to depend on formation of tissue factor–VIIa complexes.17 18 Accordingly, activation of platelets in blood supplemented with CTI is likely to simulate conditions in vivo in the absence of vascular injury that exposes blood to tissue factor.
The mechanism by which citrate increases the activation of platelets by ADP in this study probably involves chelation of calcium and consequent changes in intracellular concentrations of calcium in specific compartments that influence activation.31 The increased activity of platelets in response to ADP that occurs after exposure to EDTA for 10 to 15 minutes is consistent with the critical role of intraplatelet calcium stores in platelet activation. Extracellular calcium is necessary for the association of platelets with cells such as monocytes or endothelium.25 26 The association can interfere with detection of platelet surface proteins by flow cytometry.26 Thus a calcium chelator would be expected to facilitate detection of platelets expressing P-selectin. Although this may explain in part the increased activation observed with citrate and EDTA, it is unlikely to account for the differences observed because the assay mixture is recalcified (Tyrode's buffer).
Activation of coagulation factors on platelet surfaces is likely to modulate activation of platelets in response to agonists such as ADP.22 23 However, in the present study activation of coagulation was inhibited. Furthermore, the combination of citrate with heparin or enoxaparin led to a greater fraction of platelets becoming activated in response to ADP compared with that seen with citrate or either other agent alone. Thus the combination of citrate with other anticoagulants expected to maximally suppress generation of thrombin led to a paradoxically greater increase in the fraction of platelets expressing P-selectin in response to ADP.
Increased activation of platelets in response to ADP occurred in blood anticoagulated with heparin compared with a low-molecular-weight heparin, enoxaparin, as well as in blood anticoagulated with a specific inhibitor of factor IIa (hirudin) or factor Xa (rTAP). In contrast to heparin, inhibition of Xa predominates with enoxaparin, with an anti-Xa to anti-IIa inhibitory ratio of approximately 3.3:1.32 33
Potential factors underlying differences in ADP-induced activation of platelets in blood containing heparin as opposed to enoxaparin include efficacy and mechanisms of anticoagulation, chelation of cations such as calcium by the negatively charged heparin, and binding of the standard or low-molecular-weight heparin to platelets.33 In our studies, concentrations of heparin and enoxaparin were selected on the basis of the efficacy of each agent in inhibiting activated factor X. Thus it is unlikely that differences in ADP-induced platelet activation were a result of differences in the efficacy of anticoagulation induced. When both anti–factor Xa and anti–factor IIa activity are considered, heparin would be anticipated to exert more rather than less of an overall anticoagulant effect. Furthermore, increased activation of platelets induced by ADP was observed with 1 U/mL of heparin compared with 0.1 U/mL. In addition, the combination of heparin or enoxaparin with citrate showed that heparin, at both concentrations, led to greater ADP-induced activation of platelets than did either concentration of enoxaparin.
Mechanisms of anticoagulation induced by standard and low-molecular-weight heparin differ somewhat. Antithrombin III–mediated inhibition of factors IIa and Xa is equivalent with standard heparin. By contrast, predominant antithrombin III–dependent inhibition of factor Xa is induced by enoxaparin. To determine whether the specific site of anticoagulant activity (factors Xa or IIa) accounts for differences in activation of platelets in whole blood in response to ADP, we studied effects of inhibitors specific to each. No difference in the ADP-induced activation of platelets was observed with rTAP (anti-Xa) or hirudin (anti-IIa). Thus in a system in which blood is not exposed to substantial quantities of tissue factor, the particular site of inhibition of the coagulation cascade does not appear to influence the extent to which activation of platelets is induced by ADP.
Both heparin and enoxaparin are mixtures of negatively charged glycosaminoglycans and thus could be expected to bind cations such as calcium. The binding of platelet factor 4 to heparin (observed almost exclusively with unfractionated heparin) would be expected to neutralize the negative charge.33 The affinity of the low-molecular-weight fraction of heparin for proteins such as platelet factor 4 and fibronectin is lower.32 33 Compared with preparations of low-molecular-weight fractions of heparin, different preparations of unfractionated heparin exhibit more variable affinity for proteins. Accordingly, if the charge associated with heparin is responsible for the increased activation of platelets in response to ADP, more variability of platelet reactivity would be anticipated with unfractionated heparin compared with low-molecular-weight heparin.
Increased activation of platelets in response to ADP with a
relatively high concentration of heparin suggests that binding of
heparin to platelets may play a role. Heparin binds to
thrombospondin, a platelet -granule adhesive protein. The
association potentiates platelet aggregation.34 In
addition, heparin binds to platelet factor 4, another -granule
constituent. The combination of heparin, platelet factor 4, and
antibodies binding to the heparin/platelet factor 4 complex appears
to mediate heparin-induced thrombocytopenia and heparin-induced
thrombosis.35 36 However, it is unlikely that the antibody
binding to heparin/platelet factor 4 complexes mediates the
increased platelet activation observed in our study because
antibodies to the complex are absent from normal plasma.36
Nevertheless, association of heparin with platelet factor 4 may
potentiate platelet activation. Another membrane-associated protein
that may contribute to interactions between heparin and platelets
that augments activation of platelets induced by ADP is
multimeric vitronectin, which is present in
-granules.37
Some Potential Clinical Implications
The extrapolation of results from studies in vitro to those in
vivo must be performed with caution. However, if the increased
activation of platelets in blood anticoagulated with heparin that
we observed ex vivo occurs in patients with acute coronary
syndromes who are treated with heparin, beneficial effects conferred by
anticoagulation may be counterbalanced, to some extent, by increased
responsivity of platelets to weak agonists such as ADP. The
recently reported results in the ESSENCE study are consistent
with this possibility. Patients with unstable angina and non–Q-wave
myocardial infarctions treated with enoxaparin had a 16% lower
incidence of cardiac events (triple end point of death, myocardial
infarction, and recurrent angina) compared with the incidence in those
treated with standard unfractionated heparin.38 In
addition, association of platelets with fibrin clots was increased
in plasma containing heparin compared with that containing
enoxaparin.39 Although these results alone certainly do
not justify a change in current therapy, delineation of the specific
mechanism(s) underlying increased activation of platelets in blood
anticoagulated with heparin compared with the other anticoagulants may
identify additional biochemical targets for pharmacological enhancement
of anticoagulation and effective therapy with antiplatelet drugs,
thereby improving the care of patients with acute coronary
syndromes.
Assessment of platelet activation in whole blood ex vivo is an attractive approach for delineation of the reactivity of platelets to agonists in the presence or absence of potential antiplatelet agents under specific conditions. Our results indicate that apparent effects of such agents are likely to be influenced by the anticoagulant used. Trisodium citrate, used conventionally for assays of the coagulation system and aggregation of platelets, potentiates ADP-induced activation of platelets without increasing their activation under basal conditions. Heparin, compared with enoxaparin, potentiates ADP-induced activation of platelets apparently directly by interacting with platelets rather than indirectly through anticoagulation per se. The increased activation of platelets in blood containing heparin compared with enoxaparin may explain in part the reduced incidence of cardiac events seen previously in patients with acute coronary syndromes who were treated with enoxaparin compared with heparin.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by a grant from the National Institutes of Health (H2-P01-46703).The authors thank Mary C. Stahle and Colette Charland for expert technical assistance.
Received March 3, 1997; revision received June 3, 1997; accepted June 5, 1997.
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