Anesth Analg 2001;93:1446-1452
© 2001 International Anesthesia Research Society
CARDIOVASCULAR ANESTHESIA
A Dose-Determining Trial of Heparinase-I (NeutralaseTM) for Heparin Neutralization in Coronary Artery Surgery
Edward K. Heres, MD*,
Jay Charles Horrow, MD
,
Glenn P. Gravlee, MD*,
Barbara E. Tardiff, MD
,
John Luber, Jr, MD
,
Joel Schneider, MD||||,
Thomas Barragry, MD¶, and
Richard Broughton, BSc#
*Allegheny General Hospital, Pittsburgh, Pennsylvania; MCP-Hahnemann University, Philadelphia, Pennsylvania; Duke University Medical Center, Durham, North Carolina; Albany Medical Center, Albany, New York; ||||St. John’s Hospital, Springfield, Illinois; ¶St. Francis Hospital, Milwaukee, Wisconsin; and #IBEX Technologies, Montreal, Quebec, Canada
Address correspondence to Jay Charles Horrow, MD, MCP-Hahnemann University, 245 N. 15th St., MS 310, Philadelphia, PA 19102. Address e-mail to horrow{at}drexel.edu
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Abstract
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Heparinase-I, a specific heparin-degrading enzyme, may represent an alternative to protamine. We explored the dose of heparinase-I for efficacy and safety in patients undergoing coronary artery surgery. At the conclusion of cardiopulmonary bypass, subjects received 5, 7, or 10 µg/kg of open-label heparinase-I instead of protamine. Activated clotting time (ACT) and its difference from a contemporaneous heparin-free sample (
ACT) at 3 min before and 3, 6, and 9 min after heparinase-I determined reversal efficacy. After surgery, we recorded hourly chest tube drainage. Systemic and pulmonary arterial blood pressure and cardiac output measurements before and immediately after heparinase-I were used to evaluate hemodynamic safety. Coagulation measurements included anti-factor Xa and anti-factor IIa activities. Forty-nine patients from seven institutions participated: 12 received 5 µg/kg, 21 received 7 µg/kg, 4 received two doses of 7 µg/kg, 8 received 10 µg/kg, and 4 received two doses of 10 µg/kg. Treatment groups did not differ demographically. Median ACT 9 min later was 11, 7, and 4 s for the 5, 7, and 10 µg/kg groups, respectively. No adverse hemodynamic changes occurred with heparinase-I administration. The authors conclude that heparinase-I effectively restored the ACT after cardiopulmonary bypass. This effect appeared to be dose dependent.
IMPLICATIONS: Heparinase-I (NeutralaseTM) successfully restored activated coagulation time with no adverse hemodynamic events in patients undergoing coronary artery surgery with cardiopulmonary bypass in an open-label dose-determining trial.
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Introduction
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Patients undergoing surgery with cardiopulmonary bypass (CPB) must receive systemic anticoagulation with intense antithrombin activity to prevent activation of the coagulation system by the artificial surfaces of the CPB apparatus. Heparin provides this effect. After the patient is separated from CPB, heparin’s anticoagulant effect must be neutralized to halt substantial bleeding. Protamine, the only currently approved drug in the United States with antiheparin activity, neutralizes heparin by binding of its polycationic structure to the polyanionic heparin.
Protamine use is associated with significant adverse responses, including systemic hypotension, pulmonary vasoconstriction, and anaphylactic reactions (1). Protamine also inhibits platelet function, and in excess it exerts an anticoagulant effect itself (2,3). Patients who are allergic to protamine and therefore do not receive heparin reversal drug after CPB have experienced life-threatening bleeding (4). Transfusion of platelets and fresh frozen plasma instead of protamine administration after CPB does not prevent substantial hemorrhage (5).
The Gram-negative soil bacterium, Flavobacterium heparinum, synthesizes a family of enzymes that degrade glycosaminoglycans. Heparinase-I (NeutralaseTM; IBEX Technologies, Montreal, Quebec, Canada) lyses heparin at its 
-glycosidic linkages (6), whereas heparinase-III degrades heparan sulfate, a related compound. Animal investigations demonstrated that heparinase-I reverses heparin-prolonged activated clotting time (ACT) without significant hemodynamic changes (7,8). When given in doses up to 30 µg/kg to healthy volunteers, heparinase-I successfully neutralized heparin’s anticoagulant effect in a dose-dependent fashion without significant adverse sequelae (8). This study assessed the heparin-neutralizing activity and safety profile of different doses of heparinase-I in patients undergoing coronary artery surgery.
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Methods
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Seven institutions enrolled patients in this open-label trial after approval of their respective IRBs and written, informed consent from each patient. Patients underwent elective, primary coronary artery surgery with an anticipated on-pump time of <3 h; they were between 40 and 75 yr old and had a left ventricular ejection fraction of at least 30%. Ineligible patients had a history of significant gastrointestinal, pulmonary, renal, endocrine, hematologic, or central nervous system disease or of sensitivity to foreign proteins, had insulin-dependent diabetes mellitus, or had had a transmural myocardial infarction within the previous 2 mo. For this first exposure ever of patients to heparinase-I, the protocol specified additional exclusionary criteria: patients receiving warfarin therapy, those who required more than two inotropic drugs to wean from CPB, and patients who required more than 500 U/kg of heparin to achieve an initial ACT of at least 300 s. The concomitant administration of antifibrinolytic medications was permitted at investigator discretion.
Each institution conducted anesthesia in its usual fashion; all used large-dose opioids with or without volatile anesthetics. Patients received porcine mucosal heparin before CPB according to institutional dosing standards. After separation from CPB and when clinically stable, patients received an IV bolus of heparinase-I over 30 s through a central venous catheter.
Initially the protocol specified a single initial bolus dose of 5 µg/kg of heparinase-I; this dose increased to 7 µg/kg or decreased to 3 µg/kg and, if needed, further increased or decreased to 10 or 1 µg/kg. Each dose was planned for 12 subjects, with safety and efficacy analyses deciding whether to halt, increase to the next dose, or decrease to the next dose. Although the protocol contained provision for protamine neutralization of heparin in case heparinase-I failed to achieve such, it did not provide for neutralization of heparin contained in pump blood administered after neutralization. This omission deviated substantially from clinical practice at many centers. Accordingly, investigators administered protamine for this purpose as they deemed appropriate, thus potentially confounding safety analyses. To remedy this issue, after enrollment of 12 subjects in each of the 5 and 7 µg/kg groups, a protocol amendment permitted a second administration of heparinase-I after the administration of heparinized pump blood under certain circumstances (see below). Thus, four heparinase-I treatment groups emerged from the protocol: 5 µg/kg given once, 7 µg/kg once, 7 µg/kg once or twice, and 10 µg/kg once or twice. For ethical reasons, this protocol did not use a placebo group.
Systolic and diastolic systemic blood pressure mea-surements occurred 3 min before and 5, 15, 30, 45, and 60 min after the first heparinase-I administration. Pulmonary arterial systolic and diastolic pressure and cardiac output measurements occurred 3 min before and 1 min after heparinase-I administration.
The panel of coagulation tests, measured 1 h before surgery, again 3 min before heparinase-I dosing, and 3, 6, 9, 30, and 60 min after heparinase-I dosing, included a standard kaolin ACT, activated partial thromboplastin time, prothrombin time, anti-factor Xa activity, anti-factor IIa activity, and a ACT. This last test consisted of two kaolin-activated ACT channels performed simultaneously, one of which contained excess reagent-grade heparinase-I to degrade all heparin in the sample (HR-HTC; Medtronic, Inc., Parker, CO) (9). The ACT was computed by subtracting the results of that channel from those of the standard kaolin ACT channel. The ACT measured 9 min after the administration of the first dose of heparinase-I constituted the primary efficacy end point.
The correlation between heparin dose and ACT at 9 min by treatment group examined whether each heparinase-I dose yielded sufficient plasma enzyme to degrade the substantial concentration of heparin present for the conduct of CPB. Blood loss, defined as chest tube drainage, was assessed hourly upon arrival to the intensive care unit and continued for 24 h or until removal of the chest tube, whichever came first.
The protocol amendment permitted investigators to administer a second bolus administration of heparinase-I by using a dose identical to that of the initial administration. This second dose could follow administration of heparinized pump blood only if at least 10 min had elapsed from the administration of the initial dose and if the ACT at the time exceeded 20 s or if chest tube drainage exceeded 2.0 mL · kg-1 · h-1, regardless of ACT. A repeat ACT measurement occurred 15 min after the second dose of heparinase-I. If that ACT result still exceeded 20 s or if chest tube drainage exceeded 2.0 mL · kg-1 · h-1, only then could investigators administer protamine as a rescue drug. An automated protamine titration (Hemo-Tec HMS Heparin System; Medtronic) calculated protamine dose in that instance. Protamine administration occurred by IV infusion over 10 min.
Statistical analysis used analysis of variance (ANOVA) and rank ANOVA to compare continuous data among groups, with pairwise comparisons by Fisher’s protected least significant differences test. Regressions for chest tube drainage data, their ranks, and their logarithms at 6, 12, and 24 h underwent residuals analysis (10) and tests for normality (D’Agostino-Pearson omnibus test) to determine the appropriate outcome variable for analysis. Fisher’s exact test compared categorical data. All statistical tests were two tailed at the 0.05 significance level. Where appropriate, P values reflect Bonferroni’s correction for multiple comparisons.
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Results
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Forty-nine subjects participated in the study. Table 1 summarizes the characteristics of the study population samples and their exposures to heparinase-I and protamine. Forty-five subjects (92%) received antifibrinolytic therapy during surgery.
Table 2 presents ACT and ACT test results for the four treatment groups. Heparinase-I returned the ACT to near baseline values in both a temporal and dose-dependent manner. Rank ANOVA disclosed an effect of treatment group on ACT at 3, 9, and 30 min. At 3 min, ACT for the 10 µg/kg group (median 11.5 s) differed significantly from that of the 5 µg/kg group (median, 25.0 s; P < 0.003) but not the 7 µg/kg group (median, 16.5 s; P = 0.066). A similar pattern occurred at the 9-min primary efficacy measurement time: 4.0 s median for the 10 µg/kg group versus 11.0 s for the 5 µg/kg group (P = 0.009) and 7.0 s for the 7 µg/kg group (P = 0.078).
At 30 min, the 7 µg/kg group’s ACT (median 7.0 s) differed significantly from that of the 5 µg/kg group (median, 3.5 s; P = 0.039) but not from that of the 10 µg/kg group (median, 4.0 s; P = 0.183). Unfortunately, potential administration of protamine after heparinized pump blood seriously confounds the 30-min results in the 5 and 7 µg/kg groups.
Figure 1 displays the time course of ACT measurements for the groups, combining the two 7 µg/kg treatment groups at all time points for clarity. Figure 2 displays boxplots for ACT measured 9 min after the administration of 5, 7, or 10 mg/kg of heparinase-I.
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Figure 1. Time course of the change in activated clotting time (ACT) after the administration of heparinase-I. Squares denote the 5 µg/kg group, triangles the 7 µg/kg group, and circles the 10 µg/kg group. Selected error bars were deleted for visual ease. Note discontinuities in the axes.
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To account for a potential effect of heparin dose on ACT, the analysis included a rank ANOVA that used total heparin dose as a covariate. The adjusted ACT for the 10 µg/kg group (5.3 s) differed significantly from that of the 5 µg/kg group (12.1 s, P = 0.003) but not from that of the 7 µg/kg group (8.4 s, P = 0.057). Heparin dose did not correlate well with the 9-min ACT, yielding values of -0.52, 0.05, and 0.34 for the 5, 7, and 10 µg/kg groups, respectively.
As expected, all subjects displayed increased prothrombin time and activated partial thromboplastin time values immediately after surgery, with most returning to normal by postoperative Day 7. Anti-factor IIa activity rapidly returned to zero (limit of detection by assay) after the administration of either 3 or 7 µg/kg of heparinase-I (Fig. 3). Unfortunately, a laboratory error invalidated the anti-factor IIa results for samples in the 10 µg/kg group. In contrast, heparinase-I administration neutralized approximately 70% of the anti-factor Xa activity achieved from the large doses of unfractionated heparin given for the conduct of CPB (3.12 U at 3 min before heparinase-I compared with 0.88 U 3 min after the administration of heparinase-I; Fig. 4).
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Figure 3. Time course of anti-factor IIa activity 3 min before and 3, 6, 9, and 30 min after heparinase-I administration. By 6 min, activity measurements occurred within the limits of detection by the assay. Diamonds denote 5 µg/kg, squares 7 µg/kg given at most once, and triangles 7 µg/kg given at most twice.
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Figure 4. Time course of anti-factor Xa activity, similar to Figure 3. Note the 25% residual anti-factor Xa activity (overall mean, 0.79 U/mL at 9 min; 3.1 U/mL before heparinase-I). Diamonds represent 5 µg/kg; squares 7 µg/kg at most once; triangles 7 µg/kg at most twice; and circles 10 µg/kg at most twice. Selected error bars were deleted for visual ease.
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Treatment group did not affect 6-, 12-, or 24-h chest tube drainage (Table 2). Comparison of chest tube drainage data utilized the logarithmically transformed values rather than either the untransformed values or their ranks, based on the failure of the latter two to demonstrate normality at the 12-h measurement.
Table 3 summarizes adverse events that occurred in the conduct of the trial. Adverse event reporting was independent of any opinion regarding causality by study drug. As expected in a trial involving cardiac surgery, 76% experienced at least one adverse event, with fever, bleeding, and atrial fibrillation being the most common. Serum chemistry determinations, including liver and cardiac enzymes, and hematocrit and platelet count measurements also typified a cohort undergoing open heart surgery.
Five (two, two, and one for subjects receiving 5, 7 + 7, and 10 + 10 µg/kg, respectively) possibly serious adverse events relating to increased chest tube drainage occurred. Seven other serious adverse events occurred without dosing-group preference: arthrosis, cerebrovascular accident, dyspnea, pulmonary edema, pericardial effusion, fever, atrial fibrillation, cardiac arrest, hypoxia, noncardiac chest pain, and a vein disorder. Individual investigators designated these event terms. Recovery occurred from all events except arthrosis and vein disorder. Serum chemistry determinations, including liver and cardiac enzymes, and hematocrit and platelet count measurements also typified a cohort undergoing open heart surgery.
Table 4 presents the hemodynamic data associated with the administration of heparinase-I. Clinically relevant changes ( heart rate >15 bpm; systolic blood pressure [SBP] >20 mm Hg; pulmonary artery pressure [PAP] systolic >5 mm Hg) occurred infrequently at the first measurement period after heparinase-I administration. The largest decrease in SBP at 5 min in any patient, from 140 to 100 mm Hg, occurred after 5 µg/kg; the largest increase in systolic PAP at 1 min (32 to 44 mm Hg) followed 7 µg/kg.
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Discussion
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This trial demonstrates that heparinase-I effectively reverses the anticoagulant effect of heparin in patients undergoing coronary artery surgery with CPB. The ACT values measured nine minutes after heparinase-I bolus administration suggest a dose-response relationship, with ACT being the response. This decrease of ACT was sustained, in that ACT remained near baseline levels after surgery and chest tube drainage volumes did not deviate substantially from those expected (11). This clinical trial reaffirmed previous investigations in vitro, in animals, and in humans; these investigations demonstrated the lack of any significant anticoagulant effect of the heparinase-induced cleavage products of heparin (7,12).
Although no subject in the current trial received only protamine, a critique of these results for heparinase-I necessarily requires parallels to protamine. Unlike protamine dosing, which is based on estimated circulating heparin, only the patient’s body weight determines the dose of heparinase-I. This distinction arises from the disparate mechanisms by which these drugs affect neutralization: stoichiometric polycation-polyanion complex formation for protamine versus enzymatic degradation for heparinase-I. In this trial, the magnitude of total heparin dose did not substantially affect the ACT measured nine minutes after heparinase-I dosed by body weight for either 7 or 10 µg/kg. However, 5 µg/kg of heparinase-I displayed ACTs that increased with larger total heparin doses, suggesting that this dose yields too little enzyme in plasma.
The adverse hemodynamic consequences of protamine are in part related to its rate of administration (1). Thus, clinicians typically slowly infuse protamine over 10 or more minutes to moderate these effects. Heparinase-I, administered as a bolus, does not alter systemic or pulmonary pressures. In this study, amid a background of the expected post-CPB cardiovascular variability and continuing surgical maneuvers, substantial alterations of SBP (>20 mm Hg decrease) occurred in only four subjects (8.2%). Likewise, PAP increased by more than 5 mm Hg in only four (different) subjects. Cardiac output and systemic vascular resistance remained stable in all patients.
Protamine returns heparin-induced anti-factor IIa and anti-factor Xa activities to undetectable levels (13,14). Heparinase-I, however, neutralizes only approximately 70% of the anti-factor Xa activity while also returning anti-factor IIa activity to zero. The clinical effects of the residual anti-factor Xa activity after heparinase-I administration remain unknown, because this trial was not designed to probe the possible beneficial (e.g., antithrombotic) (15) or deleterious (e.g., hemorrhagic) effects of residual anti-factor Xa activity.
Protamine may contribute to postoperative bleeding because of its inhibition of platelets (2). Although not designated the primary efficacy variable for this dose-finding trial, blood loss should contribute to the assessment of any protamine alternative. Chest tube drainage volumes in this trial seemed consistent with those generally experienced after protamine reversal of heparin for CPB (11). However, this trial contains too few subjects for a valid assessment of the effect of heparinase-I on blood loss. A prospective, randomized, double-blinded comparison between heparinase-I and protamine may clarify this issue.
The ACT represents the standard for monitoring the anticoagulant effect of heparin and its reversal for CPB. Celite and kaolin serve as activators for most ACT devices. They yield similar results, although the use of aprotinin requires kaolin activators to avoid underdosing heparin (16). This study used kaolin ACTs for all patients to standardize the activator and permit appropriate interpretation of ACT values with aprotinin use.
This trial chose ACT as the primary end point to evaluate the efficacy of heparinase-I. The ACT allowed contemporaneous evaluation of residual heparin effect. The in vitroheparinase-treated channel removes all heparin from the sample, thus individualizing the baseline for each ACT measurement (9). On the basis of expert opinion, this trial required a ACT value 
20 seconds to administer a second dose of a neutralizing drug. However, no previous clinical data validate that a ACT of 20 seconds separates complete from incomplete heparin neutralization.
Heparinase-I, 7 or 10 µg/kg, effectively restores the ACT after unfractionated heparin given to patients undergoing CPB for coronary artery surgery. Heparinase-I caused no clinically significant hemodynamic or other adverse responses in this cohort of 49 patients. The dose of 5 µg/kg seems too little for neutralization of heparin doses administered for the conduct of CPB. Further studies should compare heparinase-I and protamine in double-blinded, randomized fashion by using clinically relevant outcome variables such as blood loss.
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Acknowledgments
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Supported by IBEX Technologies, Montreal, Quebec, Canada.
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Footnotes
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Address reprint requests to IBEX Technologies, 5485 Pare, Montreal, Quebec, Canada H4P 1P7. Address e-mail to Neutralase@ibexpharma.com.
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Accepted for publication August 1, 2001.
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Abstract
Introduction
