Heparin resistance in acute coronary syndromes - Springer Link

J Thromb Thrombolysis (2007) 23:93–100 DOI 10.1007/s11239-006-9049-9

Heparin resistance in acute coronary syndromes Jonathan D. Rich Æ John M. Maraganore Æ Edward Young Æ Rosa-Maria Lidon Æ Burt Adelman Æ Paul Bourdon Æ Supoat Charenkavanich Æ Jack Hirsh Æ Pierre Theroux Æ Christopher P. Cannon

Published online: 13 January 2007 Springer Science+Business Media, LLC 2006

Abstract Background Maintaining a therapeutic level of anticoagulation with unfractionated heparin remains a major challenge for clinicians because of the wide variability of patient responses, which may be explained by variable binding of heparin to plasma proteins. Direct thrombin inhibitors may offer an advantage in more predictable anticoagulation. Methods Plasma samples from normal volunteers, stable coronary artery disease (CAD) patients, unstable angina patients, and acute myocardial infarction patients were obtained. A fixed concentration of heparin (.13 U/ml) or bivalirudin (1.6 lg/ml) was added to plasma from each of the four study groups and measurement of the APTT was performed. In addition, a pool of plasma from patients with acute MI was diluted in pooled normal plasma, and heparin or J. D. Rich C. P. Cannon (&) TIMI Study Group, Cardiovascular Division, Brigham and Women’s Hospital and Harvard Medical School, 75 Francis St, Boston, MA 2115, USA e-mail: [email protected] J. M. Maraganore B. Adelman P. Bourdon S. Charenkavanich Medical Research, Biogen, Inc., Cambridge, MA, USA E. Young Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Canada R.-M. Lidon P. Theroux Division of Cardiology, Montreal Heart Institute, Montreal, Canada J. Hirsh Department of Medicine, McMaster University, Hamilton, Canada

bivalirudin was added to the plasma preparation and APTT measurements performed. Results In heparin-treated plasma samples, mean APTT values were 443 ± 137% baseline for normal volunteers, 347 ± 116% for patients with stable CAD, 290 ± 124% for patients with unstable angina (p < 0.05), and 230 ± 120% for patients with acute MI (p < 0.05). APTT did not differ across the four groups treated with bivalirudin. There was a much higher degree of variability in APTT values in heparin treated controls (272%–671%, SD ~30%) compared to bivalirudin treated controls (284–499%, SD ~12%). When the ‘‘acute MI pool’’ was diluted in pooled normal plasma at fixed concentrations of either bivalirudin (1.6 lg/ml) or heparin (0.13 U/ml), there was a sharp decrease in heparin activity from 407% baseline (at 0% acute MI pool) to values as low as 126% baseline (at 100% acute MI pool). A markedly different pattern was seen in the bivalirudin treated samples, where a trend towards decreased APTT values was seen only at the 100% acute MI pool. Conclusion Both heparin variability and resistance may limit optimal antithrombotic therapy with heparin in patients with ACS and constitutes a potential advantage of direct antithrombin blockade with bivalirudin. Keywords

Anticoagulation Heparin Resistance

Introduction Unfractionated heparin is a heterogeneous glycosaminoglycan which inhibits blood coagulation through the antithrombin-dependent inactivation of numerous coagulation factors including factor Xa and thrombin [1]. Heparin is used widely in the acute coronary

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syndromes (ACS), unstable angina and myocardial infarction (MI), and during percutaneous coronary intervention (PCI). In addition to its well-known bleeding complications, heparin has limitations that are caused by antithrombin-independent binding to positively charged proteins [2]. Thus, binding of heparin to platelet factor 4 neutralizes heparin and causes immune-mediated platelet activation (leading to heparin-induced thrombocytopenia (HIT)) [3, 4], and its binding to positively charged plasma proteins is responsible for the variable anticoagulant response to heparin and to the phenomenon of heparin resistance [2, 5]. Alternative anticoagulant drugs have been developed as replacements for heparin in a number of clinical settings [6–13]. These include low molecular weight heparins (such as enoxaparin), specific anti-Xa inhibitors (such as fondaparinux), and direct thrombin inhibitors. Two of these alternate anticoagulants, low molecular weight heparins and specific anti-Xa inhibitors, exhibit less binding to plasma proteins and platelet factor 4 [14] and therefore have a more stable anticoagulant response than heparin and a lower risk of heparin induced thrombocytopenia [15]. Bivalirudin, a direct thrombin inhibitor, is free of the risk of HIT [3] and, based on its structure should exhibit much less binding to plasma proteins and therefore have a more predictable anticoagulant response than heparin [16]. To investigate the stability of anticoagulant response of bivalirudin, we carried out a head-to-head comparison of bivalirudin and unfractionated heparin across the spectrum of coronary artery disease (CAD) patients including normal controls, stable CAD, unstable angina, and acute MI. We hypothesized that bivalirudin would have a more stable response than heparin, and that unlike heparin, the anticoagulant response would not be reduced in patients with acute myocardial infarction, in whom an acute phase protein response would be expected to result in heparin resistance [17].

Methods Subjects, blood collection, and plasma preparation Subjects were identified at the participating centers (Brigham and Women’s Hospital, Montreal Heart Institute, and Biogen, Inc.) and informed consent was obtained. This study included a cohort of normal volunteers (n = 19) and patients with stable CAD (n = 26), unstable angina (n = 21), and acute MI (n = 14). Stable CAD patients had a known history of CAD with or

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J Thromb Thrombolysis (2007) 23:93–100

without previous symptoms of exertional angina, but no rest pain during the index hospitalization. Unstable angina patients had a history of chest discomfort or ischemic symptoms at rest lasting at least 5 min with or without ECG changes. Acute MI patients had either non-ST elevation or ST elevation MI. Only unstable angina and acute MI patients who had not received anticoagulant therapy at the time of entry were eligible for the study. Blood was collected by atraumatic venipuncture and collected in either 1/10th volume 3.8% trisodium citrate or (for measurements of platelet factor 4 levels) in a special anticoagulant mixture containing 1/ 10th volume 3.8% trisodium citrate, theophylline (20 mM), and PGE1 (10 lg/ml). Platelet-poor plasma was obtained by centrifugation, and stored at –20C until use. Volunteer/patient characteristics were collected using a questionnaire and via interview or through hospital records. Materials Porcine intestinal heparin was obtained from Sigma (St. Louis, MO). Bivalirudin was then a product of Biogen, Inc., Cambridge, MA. Activated partial thromboplastin time (APTT) assays were performed with a Coag-A-Mate XC instrument (General Diagnostics, Organon Teknica, Durham, NC) with calcium chloride and partial thromboplastin reagents supplied by the manufacturer. Frozen, pooled normal plasma was purchased from George King Biomedical, Overland Park, KS. Assays for platelet factor 4 (PF4) and antithrombin (AT) were performed with Asserachrom PF4 and Liatest AT kits, respectively, obtained from Diagnostica Stago (Parsippany, NJ). Theophylline and PGE1 were purchased from Sigma. Low affinity heparin (LAH) was prepared from unfractionated porcine mucosal heparin (Sigma) by controlled periodate oxidation and borohydride reduction according to a modification of the method of Casu et al. [18]. Identification of heparin and bivalirudin concentrations for study Concentrations of bivalirudin or heparin for the ex vivo study were identified in experiments with pooled, normal plasma. A range of bivalirudin (0 to 4.0 lg/ml) or heparin (0–0.2 U/ml) concentrations was added in 0.025 ml volume to 0.1 ml human plasma. APTT measurements were then performed using a Coag-AMate XC instrument (General Diagnostics/Organon Teknika, Durham, NC) with the manufacturer’s reagents and procedures.


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Table 1 Dose–response for heparin and bivalirudin anticoagulant activity in pooled, normal citrated plasma Heparin, Heparin Bivalirudin, Bivalirudin APTT, % Control lg/ml APTT, % Controla U/ml 0.32 0.80 1.60 4.00

215 263 364 512

0.064 0.096 0.128 0.160 0.192

210 253 334 445 564

a

APTT values are mean, n = 3. ‘‘Control’’ is the APTT value for plasma in the absence of bivalirudin or heparin addition

Measurements of APTT in volunteer and patient plasma A dose response study of bivalirudin and heparin with pooled normal plasma was performed to identify concentrations of each agent, which would prolong APTT to 400% baseline (Table 1). A baseline APTT for volunteer or patient plasma samples was obtained using 0.1 ml plasma diluted with 0.025 ml vehicle. Plasma samples with baseline APTT >65 s were not used further in the study due to the potential errors in blood collection (e.g., traumatic venipuncture) or the potential presence of heparin (e.g., from a ‘‘heparin lock’’ used in an indwelling catheter or from use of heparin anticoagulation in patients). Bivalirudin (1.6 lg/ml) or heparin (0.13 U/ml), corresponding to plasma levels of 5.2 lg/ml and 0.42 U/ml, respectively, was added in 0.025 ml volume to 0.1 ml volunteer or patient plasma for measurement of APTT. These measurements were performed in triplicate, and mean values (±SD) were recorded as % baseline, using the baseline APTT value obtained for each volunteer/ patient plasma sample.

and PF4 levels were measured using commercially available kits. Assays were performed per the manufacturer’s instructions. Measurements of APTT in pooled MI plasma The final part of the study employed a pool of plasma prepared by mixing plasma (2.0 ml) from five individual subjects with acute MI. Ten separate dilutions (ranging from 0% to 100%) were prepared of the pooled acute MI plasma with pooled normal plasma (George King Biomedical, Overland Park, KS). Subsequently, bivalirudin (1.6 lg/ml) or heparin (0.13 U/ml) was added to the plasma preparations, and APTT measurements were performed. Statistical analyses Differences between bivalirudin or heparin anticoagulation of volunteer or patient plasma samples were analyzed for significance by one-way ANOVA. In order to identify potential predictors of decreased (or increased) APTT values for bivalirudin or heparin treated plasma samples, a best fit model was searched using APTT as a continuous variable and the following variables, obtained through the volunteer/patient questionnaire, as predictors: age, gender, occurrence of recent chest pain at rest, hypertension, diabetes mellitus, high cholesterol levels, smoking, family history of cardiovascular disease, occurrence of previous MI, and use of concomitant medications including aspirin, betablockers, calcium channel antagonists, nitrates, IV nitroglycerin, and ACE inhibitors. Subsequent analysis of any identified predictors employed a Student’s t-test. The potential correlation of APTT values with plasma levels of PF4 or AT was also analyzed.

Measurements of heparin binding to plasma proteins Results Plasma from 13 additional patients with acute MI was obtained for measurement of heparin binding to plasma protein. A fixed amount of heparin (0.13 U/ ml) was added to each plasma sample. The anti-factor Xa activity of the added heparin was measured before and after addition of LAH (45 lg/ml) in each sample. Anti-factor Xa assays were performed according to the method of Teien and Lie [19]. Measurements of AT and PF4 levels Using plasma samples obtained with the citrate/PGE1/ theophylline anticoagulant during blood collection, AT

Patient characteristics and baseline APTT results A summary of characteristics for volunteers and CAD patients is provided in Table 2. Volunteers were significantly younger with a mean age of 38.2 ± 10.1 years as compared with CAD patients who were approximately 61–63 years of age. There were more women in the normal group than the CAD groups. As expected, the occurrence of risk factors for CAD was also found less frequently in volunteers, as was the use of cardiac medications. Mean baseline APTT values for plasma from volunteers and patients

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Table 2 Summary of characteristics of volunteers and patientsa

a Data were obtained from 17/19 normal volunteers, 25/ 26 patients with stable CAD, 21/21 patients with unstable angina, and 13/14 patients with acute MI b ‘‘SCAD’’ is stable coronary artery disease c ‘‘UA’’ is unstable angina

Parameter

Volunteers

SCADb

UAc

Acute MI

Age (mean ± SD) Sex (% F) Recent Pain @ Rest Hypertension diabetes High cholesterol Smoking Family history of CAD Previous MI Aspirin Beta-blockers Calcium antagonists Nitrates IV Nitroglycerin ACE inhibitors

38.2 ± 10.1 65% 0% 0% 0% 0% 6% 18% 0% 0% 0% 0% 0% 0% 0%

60.9 ± 10.0 4% 20% 16% 24% 56% 40% 44% 52% 56% 52% 60% 40% 4% 24%

63.2 ± 13.1 48% 100% 52% 48% 33% 43% 29% 57% 76% 71% 52% 71% 0% 14%

60.7 ± 11% 8% 92% 31% 31% 31% 38% 31% 23% 38% 23% 15% 23% 0% 15%

with stable angina, unstable angina, and acute MI were 33 ± 5 s, 36 ± 7 s, 34 ± 9 s, and 28 ± 4 s, respectively. When bivalirudin (1.6 lg/ml) or heparin (0.13 U/ml) was added to individual volunteer plasma samples, APTT values obtained were 384 ± 43% baseline or 443 ± 137% baseline, respectively. These values were not significantly different, and were in accord with the expected level of anticoagulation at these bivalirudin and heparin concentrations based on experiments with pooled normal plasma (Table 1). A higher degree of variability (with standard deviation at ~30% of the mean) was observed in volunteer plasma samples treated with heparin, where the range of APTT values recorded was from 272% baseline to 671% baseline. In the case of bivalirudin-treated volunteer plasma samples, a lower degree of variability was observed, with the range of APTT values from 284% baseline to 499% baseline (with standard deviation at ~12% of the mean) (Fig. 1). Addition of bivalirudin (1.6 lg/ml) or heparin (0.13 U/ ml) to the patient plasma samples resulted in prolongation of APTT values. In the case of heparin-treated

plasma samples, mean APTT values were 347 ± 116% baseline for patients with stable CAD, 290 ± 124% baseline for patients with unstable angina (p < 0.05 compared with normal volunteers), and 230 ± 120% baseline for patients with acute MI (p < 0.05 compared with normal volunteers) (Fig. 2). In the case of bivalirudin, mean APTT values were 384 ± 53% baseline in patients with stable CAD, 377 ± 44% baseline in patients with unstable angina, and 354 ± 23% baseline in patients with acute MI (Fig. 2). These values were not different among these three groups. Further analysis of reduced heparin activity in plasma from patients with acute MI employed use of a pool of plasma derived from five individual samples from these patients (Fig. 3). When the ‘‘acute MI pool’’ was diluted in pooled normal plasma at fixed concentrations of either bivalirudin (1.6 lg/ml) or heparin (0.13 U/ml), there was a sharp decrease in heparin activity from 407% baseline (at 0% acute MI pool) to values as low as 126% baseline (at 100% acute MI pool). The reduction of heparin activity was evident upon dilution of the pooled normal plasma with as

800

APTT, % Baseline

700 600 500 400 300 200 100 0

Heparin

Bivalirudin

Fig. 1 Range of APTT values (% baseline) for heparin- or bivalirudin-treated plasma samples from volunteers. Values are mean, high and low range

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Fig. 2 Mean APTT values (% baseline) for heparin- or bivalirudin-treated plasma samples from volunteers or CAD patients. Values are mean + SD


97 Table 3 Recovery of anti-factor Xa activity from AMI plasma Patient no.

1 2 3 4 5 6 7 8 9 10 11 12 13 Mean ± SD

Anti-factor Xa Activity (U/ml) Amount determined

Amount determined after LAH addition

0 0 0 0.07 0.03 0 0 0.18 0.02 0.05 0.14 0.07 0.11 0.05 ± 0.06

0.34 0.34 0.20 0.36 0.38 0.36 0.36 0.36 0.28 0.34 0.34 0.40 0.41 0.34 ± 0.05

a

Fig. 3 Effect of a pool of acute MI patient plasma on the anticoagulant activity of heparin or bivalirudin

little as 10% of acute MI pool plasma. At this dilution, the heparin APTT was ~250% baseline. In the case of bivalirudin, there was a trend toward decreased APTT values at 100% acute MI pool, but a markedly different pattern was seen as compared with heparin. Evidence for plasma protein binding in heparin resistance Plasma samples from additional patients with acute MI were obtained for measurement of APTT responses to bivalirudin or heparin and for determination of heparin binding to plasma proteins. The mean baseline APTT was 29.2 s, and mean APTT values following bivalirudin and heparin addition were 328 ± 26% and 261 ± 70%, respectively. The individual plasma samples were analyzed for the presence of heparin-binding proteins by the methods described previously by Young et al. [2]. After addition of heparin (0.13 U/ml), the mean anti-factor Xa activity recovered was 0.05 ± 0.06 U/ml. Following addition of a large excess of LAH, which lacks antifactor Xa activity, the mean anti-factor Xa activity recovered was 0.34 ± 0.05 U/ml (Table 3). The antifactor Xa activity recovered in the presence of LAH was significantly different from that measured in the absence of LAH (p < 0.0001). PF4 and AT levels Heparin activity can be neutralized by products of the platelet release reaction, such as platelet factor 4

A fixed amount of standard heparin (0.13 U/ml) was added to each plasma sample according to the specific anticoagulant activity supplied by the manufacturer. The anti-factor Xa activity was measured before and after the addition of 45 lg/ml of low affinity heparin (LAH) in each sample to displace active heparin bound non-specifically to plasma proteins

(PF4), and is dependent on plasma levels of antithrombin (AT). Accordingly, levels of PF4 and AT were measured in plasma samples from volunteers and CAD patients. PF4 levels (mean ± SD) were 19.5 ± 30.7 U/ml (n = 12) in volunteer samples, 28.2 ± 25.3 U/ml (n = 10) in stable CAD patient samples, 84.9 ± 147.3 U/ml (n = 12) in unstable angina patient samples, and 108.3 ± 142.0 U/ml (n = 4) in acute MI patient samples. While there was a strong trend toward increased PF4 levels in patients with ACS, on average there were no significant differences among groups. The relationship of PF4 levels with APTT values in heparintreated samples showed no evidence for a strong correlation (r2 = 0.085). AT levels (mean ± SD) were 97 ± 12% normal (n = 11) in volunteer samples, 95 ± 13% normal (n = 10) in stable CAD patient samples, 91 ± 11% normal (n = 13) in unstable angina patient samples, and 90 ± 18% normal (n = 4) in acute MI patient samples. On average, there were no significant differences among groups. Further, there was no apparent correlation of AT levels with heparin anticoagulant activity (r2 = 0.022). Predictors of heparin resistance in CAD patients Stepwise linear regression was employed to identify the best fit models of the patient characteristics listed in Table 1 and the APTT (as a continuous response variable). An overall difference (p = 0.014) was observed among plasma samples treated with bivalirudin

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(APTT = 377 ± 5% baseline, mean ± s.e.m., n = 79) as compared with those treated with heparin (APTT = 334 ± 16% baseline, mean ± s.e.m., n = 78). The strongest predictor of reduced APTT in plasma samples treated with heparin was the occurrence of recent chest pain at rest (p < 0.001). Heparin prolonged APTT to 268 ± 19% baseline (mean ± s.e.m., n = 38) in patients with recent chest pain as compared with volunteers or patients without recent chest pain, where APTT was 397 ± 21% baseline (mean ± s.e.m., n = 40). The variable of recent chest pain was, of course, associated with patients with unstable angina or acute MI, where there was a significant reduction in heparin activity as noted above. The only variable associated with differences in the APTT responses for bivalirudin was family history of cardiovascular disease (p = 0.006). Bivalirudin prolonged APTT to 397 ± 10% baseline (mean ± s.e.m., n = 24) in volunteers or patients with a family history as compared to APTT values of 368 ± 5% baseline (mean ± s.e.m., n = 55) in volunteers or patients without a family history.

Discussion Heparin is a heterogeneous glycosaminoglycan preparation which prevents blood coagulation through the antithrombin-dependent inactivation of numerous activated coagulation factors including thrombin and factor Xa [20]. The pharmacokinetics and pharmacodynamics of heparin are complex, and heparin administration for antithrombotic therapy requires careful monitoring of anticoagulant levels for optimal patient management [21]. Interindividual variability of heparin dosing requirements is well-established [21], and has been described in patients requiring heparin for anticoagulant therapy during coronary angioplasty [22]. Moreover, heparin resistance has been documented in patients with deep venous thrombosis, and appears related in part to the presence of non-specific binding with plasma-derived factors (perhaps ‘‘binding proteins’’) [23]. This study was performed to examine the magnitude and mechanism of heparin resistance across the spectrum of ACS and to compare heparin to a direct thrombin inhibitor. Results from this study document that: (i) The level of anticoagulation achieved with a given dose of heparin is less in patients with ACS than stable or no CAD. (ii) the more severe the ACS the greater the heparin resistance. (iii) heparin activity is significantly neutralized (p = 0.006) in plasma samples obtained

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from patients with acute MI. (iv) that, as compared with heparin, bivalirudin demonstrates a more consistent anticoagulant activity when added to plasma samples from volunteers or ACS patients. Optimal heparin therapy requires careful monitoring of anticoagulant responses with APTT or activated clotting time measures and adjustment of the administered dose [1, 21] with titration protocols for heparin in venous thromboembolism [23] and ACS [24, 25]. Heparin monitoring is required due to the wide variability of patient responses, which appears to occur as a consequence of variable clearance rates [26], quantitative or qualitative reductions in antithrombin [27], and levels of certain heparin-binding proteins [28]. Young and coworkers have described heparin resistance in patients with venous thromboembolism and have attributed reduced anticoagulant responses to binding of heparin to plasma proteins. The presence of heparin resistance in patients undergoing cardiac surgery requiring cardiopulmonary bypass has been described as well [29–31]; the administration of platelet factor 4 in these patients can inhibit the anticoagulation produced by heparin. In the use of heparin for ACS and during PCI in patients with stable angina and unstable angina, several studies have documented variability in anticoagulant responses. Kroon et al. showed excessive interindividual and intraindividual variations in the anticoagulant activity of heparin administered subcutaneously in patients with acute MI [26]. McGarry and coworkers demonstrated a substantial variability in anticoagulant responses in patients receiving heparin during PCI, and a relationship of increased risk for coronary ischemic events with inadequate anticoagulant responses [22]. In the GUSTO study, where intravenous heparin therapy was aided by the use of a nomogram [32], more than 50% of patients who received heparin therapy were outside the therapeutic range of anticoagulation at twelve hours. Becker et al. showed that despite the use of combined weight-adjusted heparin dose titration and point-of-care coagulation monitoring, a target level of anticoagulation was maintained only approximately 30% of the time over the 48 h study period [24]. In a substudy from PARAGON A [33], Newby and colleagues found that despite utilizing a bedside APTT testing and computerized infusion adjustment device, only 42% of subsequent APTT measurements were within target range despite initially achieving a target APTT value; there was also a trend towards worse adverse outcomes associated with such delays in achieving adequate anticoagulation [34]. Thus, despite aggressive monitoring and titration protocols, under and over anticoagulation remains a problem.


In this study, we observed a high degree of variability in the anticoagulant response with heparin, with the standard deviation at ~30% of the mean. (Fig. 1). Heparin-treated plasma samples showed a mean (±SD) prolongation of APTT of 334 ± 143% baseline. In all 79 bivalirudin-treated plasma samples, the standard deviation for anticoagulant activity was ~12%. When added to plasma samples from volunteers or patients with ACS, bivalirudin showed consistent anticoagulant activity. The degree of APTT prolongation by bivalirudin was not statistically different in plasma samples obtained from volunteers or patients with CAD. Variability of heparin activity may be caused by intrinsic heterogeneity of the glycosaminoglycan preparation, quantitative or qualitative differences in the levels of its obligate cofactor antithrombin, and/or presence of neutralizing heparin-binding proteins [27, 28]. None of these factors would affect the pharmacologic effect of bivalirudin, which may explain the consistent level of anticoagulation seen. In addition to observations of heparin variability, the present study also identified a resistance to heparin activity in plasma samples from patients with acute MI using both APTT and reversible heparin binding. We used a chemically modified heparin as a probe to determine the amount of anticoagulantly active heparin bound to plasma proteins other than antithrombin. When a fixed amount of heparin was added to plasma samples from acute MI patients, the amount of heparin (measured as anti-factor Xa activity) was much lower than expected indicating heparin resistance. The addition of an excess of LAH significantly increased the heparin levels. We attribute this increase to its displacement from plasma proteins that compete with antithrombin for heparin binding. Heparin activity in these samples was significantly reduced as compared with plasma samples from volunteers or patients with stable CAD. The reduction in heparin activity was not correlated with levels of antithrombin nor with levels of PF4. There was no significant inhibition of bivalirudin activity in plasma samples from patients with ACS. Additional examination of heparin resistance in patients with acute phases of CAD employed a pool of plasma from patients with acute MI. At a fixed concentration of heparin, with dilution of the acute MI pool in pooled normal plasma, there was a potent neutralization of heparin activity. A small reduction in the activity of bivalirudin was also observed, but this was within the range of intrinsic variability for APTT responses in bivalirudin-treated samples. The potent neutralization of heparin activity indicates the presence of high-affinity heparin-binding proteins in plasma from patients with ACS.

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Limitations There were several limitations of this study. First, the patient population studied was of a limited sample size. However, the variability of APTT response in ACS patients has been previously seen in multiple clinical trials [22, 24–26, 32–34] adding support to our findings. Another potential limitation is that the baseline APTT appeared slightly different in the four study groups. This may be a reflection of a baseline prothrombotic state in ACS, but further analyses in larger numbers of patients are needed.

Conclusions A reduction in the level of anticoagulation achieved with unfractionated heparin was seen in patients with ACS compared with normal volunteers or stable CAD. This suggests the presence of ‘‘heparin resistance’’ in ACS. In contrast, bivalirudin showed consistent anticoagulant activity across all patient groups. The factor(s) accounting for heparin resistance in ACS remain to be identified. Both heparin variability and resistance may limit optimal antithrombotic therapy with heparin in patients with ACS, and constitutes a potential advantage of direct antithrombin blockade with bivalirudin.

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