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(Circulation. 2000;102:2051.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
Novel, Bedside, Tissue Factor–Dependent Clotting Assay Permits Improved Assessment of Combination Antithrombotic and Antiplatelet Therapy
From the Departments of Medicine and Biochemistry (K.G.M.), University of Vermont College of Medicine, Burlington.
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Abstract |
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Background—Because optimal use of combinations of antiplatelet and antithrombotic drugs requires improved methods for assessment of therapeutic efficacy, we developed an assay designed to increase sensitivity that is based on initiation of clotting by tissue factor in minimally altered whole blood.
Methods and Results—Blood samples were obtained from healthy subjects, and the contact pathway of coagulation was inhibited with corn trypsin inhibitor (a specific factor XIIa inhibitor without effect on other coagulation factors). Clotting was initiated with relipidated tissue factor and detected with a Hemochron ACT instrument. Results were reproducible with samples from 25 healthy volunteers (mean time to clot, 125±17 seconds). Blood was also exposed to pharmacological concentrations of antithrombotic and antiplatelet agents in vitro. Heparin (0.25 anti-IIa/Xa U/mL) prolonged the time to clot by 2.4-fold (172 seconds, P<0.05); hirudin (1.0 anti-IIa U/mL), by 3-fold (250 seconds P<0.05); and enoxaparin (0.6 anti-Xa U/mL), by 2 -fold (123 seconds, P<0.05). Additive effects of antiplatelet agents were readily detectable with both heparin and hirudin. Thus, addition of 3 µg/mL abciximab to 1.0 anti-IIa/Xa U/mL heparin and to 1.0 anti-IIa U/mL hirudin further prolonged the times to clot by 140 and 67 seconds, respectively (P<0.05 for each). Addition of abciximab to enoxaparin did not further prolong the time to clot (increment, 13 seconds; P=NS).
Conclusions—The assay developed should facilitate improved dose selection, titration, and monitoring of combination antithrombotic and antiplatelet treatment regimens.
Key Words: coagulation • anticoagulants • platelets
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Introduction |
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Conventional assays used to monitor effects of antithrombotic drugs include activated partial thromboplastin time (aPTT), activated clotting time (ACT), and prothrombin time (PT). Although these assays are used to define risks of thrombosis and bleeding, the assay conditions differ markedly from conditions prevailing in vivo. Vascular injury exposes blood to tissue factor, and thrombosis is initiated by the formation of the extrinsic tenase complex (tissue factor–factor VIIa).1 2 The activities of the extrinsic and intrinsic tenases (factor VIIIa–factor IXa) and the prothrombinase complex are highly dependent on activated platelets and other phospholipid membranes.3 Thus, characterization of the impact of therapeutic agents in vivo is more optimally analyzed by methods in which the contact pathway of activation is attenuated or obviated and clotting is initiated by the tissue factor pathway in the presence of the cellular components of blood.2 4
Clotting is initiated in the aPTT and ACT by the contact pathway despite its equivocal role in vivo in hemostasis.5 6 Conventional aPTT assays are also limited by the requirement of the use of plasma rather than whole blood. Thus, a source of phospholipid must be provided, and the contributions of platelets and other cells or inhibitors of cellular processes cannot be assessed. An advantage of whole-blood clotting assays such as ACT is sensitivity to antiplatelet agents like glycoprotein IIb/IIIa inhibitors.7 8
Assays of PT entail initiation of coagulation by the physiologically critical tissue factor pathway. However, use of the PT assay is limited to plasma. Furthermore, because recalcified plasma in vitro will clot spontaneously owing to contact pathway activation, supraphysiological concentrations of tissue factor are needed to obtain reproducible results with the PT assay. The consequently short time to clot (<15 seconds) eliminates the contribution of the intrinsic tenase (factor IXa–factor VIIIa) and limits the sensitivity of the assay for assessing the impact of agents such as heparin.9
We have previously characterized the sequence of events leading to thrombin generation in contact pathway suppressed, minimally altered whole blood.2 10 11 The present studies were performed to determine the extent to which the approach developed could be incorporated into an integrated point-of-care clotting assay that is sensitive to the relevant plasma and cellular events involved in physiological blood coagulation and its management by pharmacological agents. In the assay developed, clotting is initiated with low concentrations of lipidated tissue factor while the contact pathway is suppressed with corn trypsin inhibitor (CTI, a specific inhibitor of coagulation factor XIIa). The assay detects antithrombotic effects of a variety of agents with considerable sensitivity and facilitates detection of additive and synergistic effects of combinations of antithrombotic and antiplatelet agents.
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Methods |
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Subjects
With the use of a protocol approved by the University of Vermont Institutional Review Board, blood was obtained by peripheral venipuncture from healthy volunteers. All subjects provided written informed consent.
Blood was harvested between 9 AM and noon. Phlebotomies were performed with 19-gauge butterfly needles and a 2-syringe technique with which the first 3 mL blood was discarded. Blood samples were drawn into syringes containing CTI (100 µg/mL) alone or in combination with a pharmacological concentration of heparin (0.1, 0.25, or 0.4 anti-IIa/Xa U/mL), enoxaparin (0.4, 0.6, or 1.0 anti-Xa U/mL; Rhone-Poulenc Rorer), hirudin (0.5, 1.0, or 1.5 anti-IIa U/mL, Sigma), recombinant tick anticoagulant peptide (rTAP; 0.1, 0.2, or 0.4 anti-Xa U/mL, kindly provided by Dr George Vlasuk, Corvas International), or abciximab (3 µg/mL, Eli Lilly). Final volumes were 1 part anticoagulant to 9 parts blood.
Reagents
Recombinant human tissue factor was provided by Dr Roger
Lundblad (Hyland Division, Baxter Healthcare Corp, Duarte, Calif).
Tissue factor was relipidated into small unilamellar vesicles of 25
mol% phosphatidyl serine/75 mol% phosphatidylcholine (10
µmol/L total lipid). Sucrose (60% wt/vol) was subsequently added to
stabilize for long-term storage in the freezer (up to 12 months).
Aliquots of the reagent were stored at -20°C and rehydrated before
each experiment.12 13
CTI was extracted from ground popcorn kernels and concentrated with an Amicon concentrator. Acetone precipitation/purification sequences were then performed, followed by overnight dialysis. The dialyzed concentrate was applied to a DEAE Sephacel column, and fractions were pooled on the basis of prolongation of the aPTT and absorbance.13 CTI was purified to homogeneity so that a single band was apparent by polyacrylamide electrophoresis. The CTI concentration was chosen to completely inhibit initiation of coagulation through the contact pathway determined with a whole-blood assessment of coagulation factor activation.2 CTI is available commercially (Hematologic Technologies).
Clotting Times
A 1-mL aliquot of blood was added to a K-resin ACT tube kindly
provided by International Technidyne without the glass beads
traditionally used to activate the contact pathway. Lipidated
tissue factor (200 pmol) was added to K-resin tubes before the
blood.
The K-resin tubes containing tissue factor and blood were promptly placed in a Hemochron ACT instrument. The time to clot was detected by the displacement of a dependent magnet within the rotating tube when fibrin strands formed. The blood was maintained at 37°C throughout the assay procedure.
Statistical Analysis
Values are mean±SD. Significance of differences was determined
with the use of ANOVA. Differences between treatments were assessed
with the use of the Student-Newman-Keuls tests. Significance was
defined as P<0.05.
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Results |
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Assay Variability
CTI was used to selectively inhibit the contact pathway of coagulation that is otherwise initiated when blood comes into contact with an artificial surface. Blood without CTI or exogenous tissue factor clotted in 360±30 seconds. The addition of CTI led to a concentration-dependent increase in the time to clot. A final concentration of 100 µg/mL CTI was chosen for subsequent experiments. This concentration uniformly prevented clotting for >1200 seconds; most samples (19 of 25) had no evidence of coagulation at 1500 seconds, at which point the ACT instrument displays a fault message. Thus, evaluation of the effects of antithrombotic and antiplatelet agents in a solely tissue factor–initiated system could be accomplished.
The addition of exogenous relipidated tissue factor led to a concentration-dependent decrease in the time to clot. We selected a final concentration of 200 pmol. Clotting occurred in 125±17 seconds in blood samples from 25 subjects. To verify the consistency of the lipidation process, clotting times from 3 separate lots of relipidated tissue factor were assessed on a sample of blood. Variability was similar to interindividual variability within a single lot (127±23 seconds). Five individual ACT instruments were used to ensure that any potential instrument variability was included in the interindividual variability noted.
Intra-assay variability was determined by performing the assay in
triplicate on 12 individual blood samples (4 individuals in whom
phlebotomy was performed on 3 days). Variability was determined with
tissue factor alone and when blood was spiked with heparin (0.25 U/mL)
or enoxaparin (0.6 U/mL). The mean intra-assay coefficient of variation
(percentage error) was 4% for tissue factor alone, 17% for heparin,
and 11% for enoxaparin (Figures 1
and 2). The mean intraindividual coefficients
of variation were 9% for tissue factor alone, 21% for heparin, and
25% for enoxaparin (Figures 1 and 2).
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Platelet count ranged from 162 000 to 302 000 but did not affect the time to clot (r=-0.17, P=0.597). We have previously noted that only platelet counts <10 000 affect coagulation.14 Likewise, hematocrit did not affect the time to clot (r=0.008, P=0.98). The ingestion of aspirin (325 mg at least 2 hours before phlebotomy) did not affect the time to clot (aspirin: n=8, 119±7 seconds; no aspirin: n=17, 128±17 seconds; P=0.276). Furthermore, the ingestion of aspirin did not increase the time to clot in blood samples anticoagulated with heparin, enoxaparin, or hirudin alone or in combination with abciximab (data not shown).
Individual Variability in Response to Addition of
Antithrombotic Agents
Experiments were performed with blood from 25 subjects with tissue
factor alone and in combination with heparin (0.25 U/mL), hirudin (1
U/mL), and enoxaparin (0.6 U/mL), each with and without abciximab (3
µg/mL; Figure 3). Each anticoagulant
led to an increase in the average time to clot. Of note, the
variability in response between samples from different subjects was
marked. Furthermore, an exaggerated response to one anticoagulant in
blood from a given subject did not predict an exaggerated response in
the same blood to another anticoagulant. To delineate differences
between high and low responders, a subject with a marked increase in
time to clot and a subject with a limited increase in the time to clot
were chosen for each anticoagulant. Clotting times obtained under
conditions of 3 concentrations of each anticoagulant for each of these
3 subjects are shown in the Table.
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Effect of Heparin on Clotting Time
The addition of heparin (0.25 anti-IIa/Xa U/mL) prolonged the time
to clot by 2.4-fold (from 125±17 to 297±168 seconds,
P<0.05; Figure 4). Although
the addition of 3 µg/mL abciximab alone did not prolong the time to
clot (131±19 seconds, P=NS), the addition of abciximab to
the heparin prolonged the time to clot further by an average of 140
seconds (from 297±168 to 437±305 seconds, P<0.05; Figure 4). Parallel results were seen when the effect of heparin (0.1,
0.25, and 0.4 anti-IIa/Xa U/mL) was assessed with the clotting time and
aPTT (Figure 5).
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, aPTT values.
Effect of Hirudin on Clotting Time
The addition of hirudin (1.0 anti-IIa U/mL) prolonged the time to
clot by 3.0-fold (from 125±17 to 375±91 seconds, P<0.05).
The addition of abciximab to hirudin prolonged the time to clot further
by 67 seconds (from 375±91 to 442±135 seconds, P<0.05;
Figure 6). When the effect of hirudin
(0.5, 1.0, and 1.5 anti-IIa U/mL) was assessed with the assay developed
and aPTT, increased sensitivity of the tissue factor–initiated time to
clot was seen (Figure 7).
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Effect of Enoxaparin on Clotting Time
The addition of enoxaparin (0.6 anti-Xa U/mL) prolonged
the time to clot by 2-fold (from 125±17 to 249±89 seconds,
P<0.05). The addition of abciximab to enoxaparin did not
further prolong the time to clot (249±89 to 261±141 seconds,
P=NS; Figure 8). To determine
whether the lack of prolongation by abciximab was a function of the
extent of prolongation of the time to clot, a concentration of 1.0 U/mL
enoxaparin with and without abciximab was added to blood from 4
subjects. Blood clotted in 444±43 seconds with enoxaparin alone and
460±43 seconds with enoxaparin and abciximab (increment, 16 seconds;
P=NS). The effect of enoxaparin (0.4, 0.6, and 1.0 anti-Xa
U/mL) was assessed with the clotting time assay developed and aPTT
(Figure 9). Increased sensitivity of the
tissue factor–initiated time to clot was seen with concentrations of
enoxaparin known to provide therapeutic anticoagulation (0.6 to 1.0
U/mL).
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Effect of rTAP on Clotting Time
The effect of rTAP (0.1, 0.2, and 0.4 anti-Xa U/mL) was assessed
with the clotting time assay developed and aPTT. The clotting time
assay developed was more sensitive than the aPTT to a direct
inhibitor of factor Xa (Figure 10).
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Discussion |
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We have developed a simple-to-perform, tissue factor–initiated, contact pathway–suppressed, minimally altered whole-blood clotting assay with high sensitivity. The high sensitivity facilitates determination of the effects of antithrombotic and antiplatelet agents alone and in combination, and the assay can be applied at the point of care. This assay, similar to all in vitro assays of clotting, is limited by assessment of a single captive population of platelets and coagulation factors. Nevertheless, the assay developed more closely simulates thrombosis in vivo than do the other currently available assays. We found an increased sensitivity for direct thrombin inhibitors, upstream inhibitors of coagulation such as factor Xa inhibitors, and potent antiplatelet agents such as glycoprotein IIb/IIIa inhibitors.
The insensitivity of the aPTT to inhibitors of factor Xa is a result of the nonphysiological mechanism of initiation of coagulation in the assay itself. The initiation of clotting in aPTT leads to the generation of excessive concentrations of factor Xa. Thus, inhibitors of factor Xa have little effect on results obtained with aPTT. In contrast, low concentrations of factor Xa are generated when clotting or thrombosis is initiated with tissue factor.2 As a result, inhibitors of factor Xa are potent inhibitors of coagulation in vivo, and the inhibition is paralleled by effects that are readily apparent in vitro with the use of the assay developed.
The central role of thrombosis in the genesis of acute coronary syndromes has prompted the development and evaluation of novel antithrombotic and antiplatelet agents, including low-molecular-weight heparins, direct thrombin inhibitors, and inhibitors of factor Xa, factor VIIa–tissue factor, and glycoprotein IIb/IIIa.15 16 17 18 19 20 Because conventional assays of coagulation are insensitive to the effects of many of these agents, optimal monitoring is limited. The efficacy of any antithrombotic agent must be defined ultimately with respect to a reduction in clinical events. However, measures of efficacy in vitro are useful for titration of therapy and for evaluation of the potential efficacy of novel pharmacological agents and novel combinations.
Because the assay developed is performed in whole blood, it facilitates tailoring of therapy to the needs of individual patients who are exposed to diverse classes of antithrombotic, antiplatelet, and fibrinolytic drugs. Our results demonstrate marked variation in individual responses to antithrombotic and antiplatelet agents, suggesting the potential utility of monitoring clotting times in patients being treated with these agents. The implications of the clotting times obtained with the current assay need to be delineated on the basis of correlation with clinical end points before target ranges in vitro are established for acceptable levels of anticoagulation.
Compared with unfractionated heparin, both enoxaparin and hirudin reduce the incidence of death and recurrent myocardial infarction in patients with acute coronary syndromes.16 21 22 The aPTT and ACT assay results are only poorly predictive of the antithrombotic effect of hirudin.23 The use of enoxaparin is hampered by the limited availability of assays that effectively monitor its antithrombotic effects. The assay developed demonstrates that therapeutic concentrations of either unfractionated heparin or enoxaparin prolong the clotting time to a similar extent.
The combination of high-dose heparin and glycoprotein IIb/IIIa inhibition used for patients undergoing percutaneous revascularization in the Evaluation of IIb/IIIa Platelet Receptor Antagonist 7E3 in Preventing Ischemic Complications (EPIC) trial resulted in an unacceptably high rate of bleeding complications.24 As a result, lower-dose therapy with heparin was combined with glycoprotein IIb/IIIa inhibition in the Evaluation in Percutaneous Transluminal Coronary Angioplasty to Improve Long-Term Outcome With Abciximab IIb/IIIa Blockade (EPILOG). The lower dose of heparin significantly decreased the incidence of bleeding without precluding clinical benefit.25 Thus, the use of a clotting time assay that can define additive or synergistic effects of antithrombotic and antiplatelet therapeutic regimens should be valuable in defining optimal dose regimens of diverse agents alone and in combination. The ACT assay also can detect additive effects of heparin and abciximab. In the EPIC trial, a 10% increase in ACT was seen in those patients treated with abciximab.7 In vitro studies of abciximab have shown a 10% increase in clotting time when abciximab was added to heparin but no increase with abciximab alone when ACT was used.8 Our assay demonstrates a 50% increase in the time to clot with the addition of abciximab to blood anticoagulated with heparin and a 20% increase with the addition to blood anticoagulated with hirudin.
Unexpectedly, the addition of abciximab to enoxaparin did not further prolong the time to clot. This lack of effect may represent a limitation of the assay to optimally detect the effect of this combination. Alternatively, it may suggest that there is a decreased risk of bleeding with this combination compared with that seen with unfractionated heparin and abciximab.
Implementation of the assay developed in clinical studies and for patient care should be straightforward. Assay tubes can be prepared in advance and stored after addition of the lipidated tissue factor. Thus, blood can be anticoagulated with CTI and added directly to the assay tube. With care taken in the technique used for phlebotomy to prevent introduction of endogenous tissue factor, assay results are highly reproducible.
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Acknowledgments |
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This work was supported by NIH grant PHS HL-46703. We thank Holly Aliesky and Sara Paradis for expert technical assistance.
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Footnotes |
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Reprint requests to Kenneth G. Mann, PhD, Department of Biochemistry, Given Building, University of Vermont College of Medicine, Burlington, VT 05405-0068.
Received March 24, 2000; revision received May 11, 2000; accepted June 8, 2000.
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