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Education  |   May 2002
Recombinant Human Transgenic Antithrombin in Cardiac Surgery: A Dose-finding Study
Author Affiliations & Notes
  • Jerrold H. Levy, M.D.
    *
  • George J. Despotis, M.D.
  • Fania Szlam, M.M.S.
  • Peter Olson, M.D.
    §
  • David Meeker, M.D.
  • Andrew Weisinger, M.D.
    #
  • *Professor, ‡Research Associate, §Assistant Professor, #Clinical Fellow, Department of Anesthesiology, Emory University School of Medicine. †Associate Professor, Departments of Anesthesiology, Pathology, and Immunology, Washington University School of Medicine, St. Louis, Missouri. ∥Sr. Vice President, Therapeutics, Genzyme Corporation, Boston, Massachusetts.
  • Received from the Department of Anesthesiology, Emory University School of Medicine and Division of Cardiothoracic Anesthesiology and Critical Care, Emory Healthcare, Atlanta, Georgia.
Article Information
Education
Education   |   May 2002
Recombinant Human Transgenic Antithrombin in Cardiac Surgery: A Dose-finding Study
Anesthesiology 5 2002, Vol.96, 1095-1102. doi:
Anesthesiology 5 2002, Vol.96, 1095-1102. doi:
HEREDITARY antithrombin III (AT) deficiency is seen in 1 of 2,000–5,000 individuals in the general population. 1 Acquired AT deficiency is a considerably more common disorder associated with a variety of pathologic conditions, including cardiac surgery. During cardiopulmonary bypass (CPB), AT activity frequently decreases to as low as 40–50 U/dl. 2–7 This abnormally low AT activity is observed in patients with hereditary deficiency and implicated in the pathogenesis of thrombotic episodes in these patients. AT deficiency may lessen the anticoagulant response to heparin, resulting in excessive activation of the hemostatic system during CPB 3,5,8 with subsequent consumption of labile coagulation factors and platelets 6 and excessive microvascular bleeding. 9 Excessive microvascular bleeding after cardiac surgery occurs in 5–16% of patients, 10,11 frequently requires transfusion of blood products, increases operative time and 9 need for exploration, 11,12 and overall increases the procedure-related morbidity and mortality. 11,13–16 
Patients undergoing cardiac surgery with CPB are also at increased risk for perioperative thromboembolic complications. 17–27 Despite the use of high doses of heparin during CPB, tissue injury during surgery and exposure of blood to the extracorporeal circuit may initiate excessive activation of the hemostatic system 28–30 and thrombin generation in a setting where the natural circulating anticoagulants such as AT and proteins C and S are commonly reduced. 2–7,31 
Antithrombin III concentrate derived from pooled human plasma is currently approved for patients with hereditary AT deficiency who require conventional heparin therapy. 32 However, clinical use of pasteurized AT concentrates has been limited by availability and concerns regarding the increased risk, albeit low, of infectious agent transmission by pooled products that involve large (i.e.  , 20,000) donor exposures. Although AT concentrate is used in coronary artery bypass grafting procedures, it has not been approved for this and other causes of acquired AT deficiency in the United States. The development of transgenic, recombinant technology has facilitated the production of large quantities of pure proteins for potential use in humans. The potential advantages of transgenically produced recombinant proteins include a negligible risk of infectious agent transmission when compared with plasma-derived proteins, as well as potentially unlimited supply. Based on these considerations, we studied the effects of recombinant AT to determine the doses required to achieve a potential therapeutic concentration (i.e.  , 100% activity) during CPB and to assess biologic activity of incrementally larger doses of AT with respect to enhanced suppression of hemostatic system activation during CPB.
Methods
Patient Enrollment and Recombinant Human Antithrombin III Dosing
Patients were enrolled after approval from the Institutional Human Studies Committee (Emory University Hospital, Atlanta, GA) and written informed consent was obtained. Thirty-six patients who were scheduled to undergo elective, primary coronary artery bypass grafting and who had received heparin for at least 12 h before surgery were eligible for this study. Exclusion criteria were previous coronary artery bypass grafting or valve surgery; unstable angina; preexisting hemostatic disorder, renal disease, or hepatic disease; recent or concurrent treatment with warfarin, streptokinase, tissue plasminogen activator, or abciximab (ReoPro®; Centocor Inc., Malvern, PA); preoperative blood or blood component transfusion; recent or active history of drug or alcohol abuse; known human immunodeficiency virus, hepatitis B, or hepatitis C infections; or known allergy or sensitivity to goat proteins.
This study was of a single-dose, open-label, dose-escalation design. Each of the following dosing cohorts of recombinant human (rh) AT consisted of three patients: 10, 25, 50, 75, 100, 125, 175, or 200 U/kg, respectively. The placebo and 150-U/kg cohorts each consisted of six patients. Patients were enrolled in the next higher-dose cohort after establishment of safety of the previous dose, which was assessed at least 48 h after the last patient within the previous cohort had completed therapy. The decision to advance to the next cohort was reviewed by a safety board composed of the principal investigator from the study site (or a designated representative) and a Medical Affairs representative from the study sponsor (Genzyme Transgenics Corporation, Boston, MA).
Study Procedures
Patients’ medical history was recorded and a physical examination was performed within 30 days before surgery. Study medication was reconstituted from a lyophilized vial preparation of rh-AT (7 U/mg specific activity) provided by Genzyme Transgenics Corp. After induction of general anesthesia and tracheal intubation, patients in each dosing cohort received continuous intravenous infusion of rh-AT over 30 min. Patients then received an initial dose of 300 U/kg of porcine intestinal, unfractionated heparin. During CPB, whole heparin concentrations and the activated clotting times (ACTs) were determined at 30-min intervals using a heparin–protamine titration system (Medtronic Perfusion Systems, Minneapolis, MN). Additional heparin was administered as needed to maintain whole blood heparin concentrations between 2.7 and 3.4 U/ml (there were no target ACTs). Coronary artery bypass grafting surgery was performed during moderate hypothermia to 32°C using either vein or artery grafts. After discontinuation of CPB, heparin was neutralized with protamine at a dose ratio of 1.3 mg protamine per milligram of heparin in plasma. The protamine dose was calculated using whole blood heparin concentration measurements performed at the end of CPB. Subsequently, a heparinase-ACT was measured every 2 h after protamine administration for a total of 8 h to detect possible heparin rebound.
Blood specimens were drawn from an indwelling arterial catheter at the following time points: before and 1 min after completion of rh-AT administration; 5 min after heparin bolus administration; 30, 60, and 90 min after initiation of CPB; before protamine administration; and 10 min, 2, 4, 6, 12, and 24 h after protamine administration. AT activity (Coatest Antithrombin; Pharmacia Hepar Inc., Franklin, OH) was measured in all blood specimens. Heparin concentrations (Stachrom Heparin; American Bioproducts, Parsippany, NJ) were measured before rh-AT, after heparin, during CPB every 30 min, and 6 h after protamine. Turbimetric platelet aggregation (platelet aggregometer; Chronolog Corp., Havertown, PA), using 2.4 μm adenosine diphosphate and 2.4 μg/ml collagen (Chronolog Corp.), and bleeding times were measured before rh-AT and 2 h after protamine. The following analyses were performed on all perisurgical samples and at 10 min and 2 and 6 h after protamine: celite ACT using the Hemochron system (International Technidyne, Edison, NJ), kaolin ACT with or without heparinase using the ACT II instrument (Medtronic Perfusion Systems, Minneapolis, MN), heparin concentration (Stockroom Heparin; American Bioproducts), prothrombin fragment 1.2 (Enzygnost F1+2 micro; Behring Diagnostics Inc., Westwood, MA), fibrin monomer (Coatest Fibrin Monomer; Pharmacia Hepar Inc.), D-dimer (Asserachrom D-Di; American Bioproducts), plasmin:antiplasmin complexes, (Enzygnost PAP micro; Behring Diagnostics Inc.), and platelet function and β-thromboglobulin (Asserachrom β-TG; American Bioproducts). Antibody titers were measured before rh-AT administration and again at least 4 weeks after surgery using a qualified indirect enzyme-linked immunosorbent assay developed by Genzyme's Immunology Department and confirmatory radioimmunoprecipitation assay. Based on statistical analysis of the distribution of 200 normal human serum samples, 98% of the population has a final enzyme-linked immunosorbent assay optical density reading less than 0.100. Samples were classified as within normal range or above normal range based on their optical density readings. All serum samples with optical density greater than 0.100 were considered above normal range. A patient with a serum sample within normal range before treatment and above normal range after treatment was considered seroconverted if the above normal range was confirmed as a positive reaction with the confirmatory assay (radioimmunoprecipitation assay).
Hemodynamic variables (e.g.  , systolic and diastolic blood pressure, pulse) were monitored before and after AT administration and 24 h after surgery. A postoperative clinical evaluation was obtained at least 1 month after surgery, during which a blood sample was obtained for rh-AT antibody screen. A 12-lead electrocardiograph was obtained before AT treatment (or before heparin in controls) and within 24–48 after surgery. Chest tube drainage was recorded after protamine administration at 10 min and at 2, 4, 6, 12, and 24 h after arrival in the intensive care unit.
Statistical Analyses
One-way analysis of variance was used to compare continuous variables between treatment cohorts, while the chi-square test was used to compare categoric variables between cohorts. Univariate linear regression was used to evaluate the potential linear relation between any two variables. A P  value ≤ 0.05 was considered significant.
Results
The demographic and operative profiles of patients receiving placebo or rh-AT are compared in table 1. Overall, no clinically important safety concerns were discovered in this study. No differences in hemodynamic variables were found between patients receiving rh-AT or placebo.
Table 1. Demographic and Operative Patient Data
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Table 1. Demographic and Operative Patient Data
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Chest Tube Drainage, Donor Exposures, and Adverse Events
There were no dose-related trends in chest tube drainage or transfusion requirements (table 2). There was no difference (P  = 0.15) for total donor exposures between the placebo (1.08 ± 1.6 U) versus  rh-AT–treated patients (4.8 ± 4.8 U). In addition, there was less transfusion support for the rh-AT-treated patients intraoperatively (1.7 ± 1.8 U) versus  postoperatively (3.1 ± 4.3 U), and there was no significant difference (P  = 0.71) in the cumulative blood loss between the placebo-treated (822 ± 329 ml) and rh-AT-treated (893 ± 440 ml) patients.
Table 2. Adverse Events in Placebo and rh AT Patients
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Table 2. Adverse Events in Placebo and rh AT Patients
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Table 3summarizes the adverse event frequency in the 30 patients treated with rh-AT and 6 patients in the placebo group. No serious adverse events related to rh-AT administration were reported. One rh-AT–treated patient died. This patient was noted at surgery to have severe diffuse atheromatous disease with loose plaques and suffered an atheromatous embolic stroke during surgery.
Table 3. Total Blood Component Donor Exposures and Mediastinal Chest Tube Drainage in Patients Receiving Different Doses of rh AT (n = 30) or Placebo (n = 6)
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Table 3. Total Blood Component Donor Exposures and Mediastinal Chest Tube Drainage in Patients Receiving Different Doses of rh AT (n = 30) or Placebo (n = 6)
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Antibodies to Recombinant Human Antithrombin III
Enzyme-linked immunosorbent assay testing for circulating antibodies to rh-AT was positive in three patients: one patient in each 100-, 125-, and 150-U/kg dose groups. Because no antibodies were detected in any patients in higher dose groups (175 and 200 U/kg), there was no relation between dose and reactivity. Positivity (three patients) of enzyme-linked immunosorbent assay testing was not confirmed on radioimmunoprecipitation assay (negative radioimmunoprecipitation test); thus, none of the three patients seroconverted. One of the three patients showed reactivity (above normal range) before receiving rh-AT.
Antithrombin III Activity
Figure 1shows the dose-dependent increase in plasma antithrombin activity after administration of each dose of rh-AT. With doses that exceeded 50 U/kg, AT plasma concentrations were maintained at greater than 100 U/dl throughout the CPB period. Furthermore, AT concentrations reached 600 U/dl and were maintained at approximately 250 U/dl throughout bypass after 200 U/kg rh-AT, the highest dose administered.
Fig. 1. Plasma antithrombin III (AT) activity concentrations after administration of recombinant human AT (rh-AT) using single doses of 0, 10, 25, 50, 75, 100, 125, 150, 175, and 200 U/kg during cardiac surgery with cardiopulmonary bypass (CPB). rh-AT was administered after intubation. Specimen collection times: Baseline = preadministration, 1 min after rh-AT injection, 5 min after heparin, every 30 min during CPB, before protamine, 10 min and 2, 4, 6, 12, and 24 h after CPB, and protamine reversal. Data are expressed as mean ± SE.
Fig. 1. Plasma antithrombin III (AT) activity concentrations after administration of recombinant human AT (rh-AT) using single doses of 0, 10, 25, 50, 75, 100, 125, 150, 175, and 200 U/kg during cardiac surgery with cardiopulmonary bypass (CPB). rh-AT was administered after intubation. Specimen collection times: Baseline = preadministration, 1 min after rh-AT injection, 5 min after heparin, every 30 min during CPB, before protamine, 10 min and 2, 4, 6, 12, and 24 h after CPB, and protamine reversal. Data are expressed as mean ± SE.
Fig. 1. Plasma antithrombin III (AT) activity concentrations after administration of recombinant human AT (rh-AT) using single doses of 0, 10, 25, 50, 75, 100, 125, 150, 175, and 200 U/kg during cardiac surgery with cardiopulmonary bypass (CPB). rh-AT was administered after intubation. Specimen collection times: Baseline = preadministration, 1 min after rh-AT injection, 5 min after heparin, every 30 min during CPB, before protamine, 10 min and 2, 4, 6, 12, and 24 h after CPB, and protamine reversal. Data are expressed as mean ± SE.
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Thrombin Inhibition
The relation between fibrin monomer and rh-AT dose during and after CPB (n = 226) is shown in figure 2. A significant (P  < 0.0001) inverse relation was observed between fibrin monomer concentrations and either rh-AT dose (fibrin monomer =−0.22 AT dose + 34; r =−0.51) or AT concentration (fibrin monomer =−0.25 AT + 44; r =−0.50) when rh-AT dose was less than 150 U/kg. The relation between AT activity and either mean D-dimer values for each dosing cohort or D-dimer concentrations during CPB (n = 121) is depicted in figure 3. A significant (P  = 0.001) inverse relation was observed between D-dimer concentrations and either AT activity (D-dimer =−5.8 AT + 1,757; r =−0.49;fig. 3, top) or rh-AT dose (D-dimer =−6.5 AT + 1,586, r =−0.51;fig. 3, bottom). There were no significant differences in prothrombin fragment 1.2 concentrations between placebo and rh-AT groups at any time points studied (P  = 0.67, r = 0.02;P  = 0.14, r = 0.06); concentrations of prothrombin fragment 1.2 increased during CPB and were still elevated 6 h after administration of protamine. In addition, there were no significant differences between placebo and AT cohorts in concentrations of plasmin–antiplasmin (P  = 0.33, r = 0.029;P  = 0.74, r = 0.02) and β-thromboglobulin (P  = 0.33, r = 0.03;P  = 0.74, r = 0.02) concentrations measured over the course of CPB, nor at the preprotamine time point.
Fig. 2. Relation between activation of coagulation (fibrin monomer) and recombinant human antithrombin III (rh-AT) dose. Mean plasma concentrations of fibrin monomer for each dosing cohort are shown. The total number of specimens collected during and after cardiopulmonary bypass that were used for the analysis was 226. *P  < 0.05 compared with placebo patients.
Fig. 2. Relation between activation of coagulation (fibrin monomer) and recombinant human antithrombin III (rh-AT) dose. Mean plasma concentrations of fibrin monomer for each dosing cohort are shown. The total number of specimens collected during and after cardiopulmonary bypass that were used for the analysis was 226. *P 
	< 0.05 compared with placebo patients.
Fig. 2. Relation between activation of coagulation (fibrin monomer) and recombinant human antithrombin III (rh-AT) dose. Mean plasma concentrations of fibrin monomer for each dosing cohort are shown. The total number of specimens collected during and after cardiopulmonary bypass that were used for the analysis was 226. *P  < 0.05 compared with placebo patients.
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Fig. 3. Relation between D-dimer as markers of activation of coagulation (fibrin formation) and increased and recombinant human antithrombin III (rh-AT). (Top  ) The relation between individual plasma concentrations of D-dimer and rh-AT during several cardiopulmonary bypass time points is shown; a significant inverse relation was observed between D-dimer concentrations and rh-AT (D-dimer =−5.7 rh-AT + 1,749, r =−0.49, P  = 0.001). (Bottom  ) Mean D-dimer values for each dosing cohort are shown. The total number of specimens collected at 15-min intervals during CPB used for analyses was 124. *P  < 0.05 compared with placebo patients.
Fig. 3. Relation between D-dimer as markers of activation of coagulation (fibrin formation) and increased and recombinant human antithrombin III (rh-AT). (Top 
	) The relation between individual plasma concentrations of D-dimer and rh-AT during several cardiopulmonary bypass time points is shown; a significant inverse relation was observed between D-dimer concentrations and rh-AT (D-dimer =−5.7 rh-AT + 1,749, r =−0.49, P 
	= 0.001). (Bottom 
	) Mean D-dimer values for each dosing cohort are shown. The total number of specimens collected at 15-min intervals during CPB used for analyses was 124. *P 
	< 0.05 compared with placebo patients.
Fig. 3. Relation between D-dimer as markers of activation of coagulation (fibrin formation) and increased and recombinant human antithrombin III (rh-AT). (Top  ) The relation between individual plasma concentrations of D-dimer and rh-AT during several cardiopulmonary bypass time points is shown; a significant inverse relation was observed between D-dimer concentrations and rh-AT (D-dimer =−5.7 rh-AT + 1,749, r =−0.49, P  = 0.001). (Bottom  ) Mean D-dimer values for each dosing cohort are shown. The total number of specimens collected at 15-min intervals during CPB used for analyses was 124. *P  < 0.05 compared with placebo patients.
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Platelet Aggregation and Bleeding Times
In all patients, platelet aggregation values after exposure to adenosine diphosphate and collagen decreased subsequent to protamine administration from the aggregation values observed at baseline. There was apparent effect of supplemental rh-AT (dose) on adenosine diphosphate–induced platelet aggregation (P  = 0.006, r = 0.47) at the 2-h postprotamine time point, but no effect was seen in collagen-induced platelet aggregation.
There was no significant difference in bleeding time between placebo and rh-AT patients with respect to rh-AT concentration (P  = 0.62, r = 0.12) or AT dose (P  = 0.66, r = 0.15). In patients enrolled in the rh-AT dosing cohorts, the increase in median bleeding times from baseline to 2 h after protamine ranged from 0 to 8 min; in patients who received placebo, there was an approximately 4-min increase in the median bleeding time observed after protamine compared with baseline.
Activated Clotting Times, Anti-Xa, and Heparin Concentrations
Increased kaolin ACT values (P  < 0.0001) were observed after systemic heparinization (i.e.  , specimen collection time points 3–8) in specimens (n = 134) obtained from rh-AT–supplemented patients (844 ± 191 s) compared with ACT values obtained using specimens (n = 24) from placebo-treated patients (531 ± 180 s). This effect of rh-AT on ACT values appears to be independent of heparin because heparin concentrations (anti-Xa) measured in rh-AT–supplemented patients (5.0 ± 1.5 U/ml) were similar (P  = 0.14) to those in placebo-treated patients (4.5 ± 1.6 U/ml, n = 24), and the effect of rh-AT on ACT values persisted even when heparin concentrations were included as a covariate in the analysis during CPB. Anti-Xa measurements at baseline, 5 min after heparin, and at CPB were comparable between the placebo and rh-AT groups: 0.38 versus  0.23 IU/ml (range, 0.15–0.41 IU/ml), 6.43 versus  6.67 IU/ml (range, 5.89–9.06 IU/ml), and 3.89 versus  5.07 IU/ml (range, 3.83–5.32 IU/ml), respectively. Likewise, heparin concentrations were 3.4 (placebo group) versus  4.0 IU/ml (rh-AT group) at 5 min after heparin and 2.7 versus  2.98 IU/ml during CPB.
Discussion
In this first study of a transgenic recombinant human protein in patients, rh-AT was shown to increase plasma AT concentrations without any serious adverse effects even when supraphysiologic doses were administered to supplement native AT in cardiac surgical patients. Furthermore, administration of rh-AT to 30 patients enrolled in current study resulted in no apparent differences in hemodynamic parameters, chest tube drainage, or transfusion requirements as compared with placebo-treated cohorts (table 2). Because pasteurized AT concentrates have been limited by availability, a new transgenic AT has been developed by modification of goat genome through transgenic technology.
Recombinant human AT is produced in milk of transgenic female goats during parturition. 33,34 The initial step in the development of a transgenic goat was creation of an expression vector that contained the sequence for the desired protein linked to the promoter sequence of a specific milk protein gene. This promoter sequence directs the production of the recombinant protein in the goat mammary gland on lactation. Specifically, the human AT cDNA coding for human AT was linked to the caprine β casein gene promoter. 35 The β casein gene is regulated in both a tissue and temporal fashion with maximal mammary gland expression occurring after parturition. The goat β casein gene was cloned from a Saanen goat genomic library and characterized in transgenic mice. 36 The caprine β casein gene was cloned as an 18.5-kilobase (kb) fragment in a λ EMBL3 vector. The human AT cDNA was obtained in plasmid pβAT6. 37 The sequence of cDNA is the same as that published by Bock et al.  , 38 with the exception of the silent nucleotide changes at base pair 1050 (T-C), 1317 (C-T), and 1371 (A-G). The cDNA was engineered with an Xhol site at the 5′ end by site-directed mutagenesis to allow excision of the cDNA as a 1.45-kb Xhol to Sa1l fragment. 35,36,38–40 The 6.2-kb β casein gene promoter was fused to human AT cDNA and 7.1-kb 3′ flanking region of the β casein gene was added to the 3′ end of the AT cDNA to help to stabilize expression levels. 33,35,38,39 The 14.75-kb transgene was excited from bacterial sequences and microinjected into preimplantation goat embryos, which were then transferred to the oviducts of surrogate mothers and carried to term. 33,34 A founder transgenic goat, a male, was identified for the presence of the transgene by analyzing genomic DNA from both a sample of ear tissue and blood by polymerase chain reaction 41 and Southern blot analysis. 38 This founder goat was bred to nontransgenic females and produced transgenic progeny. Once established in the first generation of transgenic animals, the transgene is transmitted as other genetic traits to future generations through traditional breeding, thus generating a production herd that supplies the raw rh-AT ready for purification. 34,42 A purification process that involves isolation of rh-AT from goat milk has been described with a yield greater than 50% and purity greater than 99.99%. 34,42 It involves clarification through a 500-kd tangential flow membrane filtration unit, heparin affinity, anion exchange, and hydrophobic interaction chromatography. This process removes endogenous goat AT present in the milk and other contaminating proteins. 42 Structurally, the recombinant human AT purified from goat milk is indistinguishable from plasma-derived AT with the exception of the glycosylation and carbohydrates. 34,42 Recombinant AT shows fourfold higher affinity for heparin than plasma-derived AT, which is attributed to the difference in glycosylation between recombinant and plasma-derived AT. 42 Recombinant and plasma-derived AT were shown to have equivalent activity in factor Xa and in vitro  thrombin inhibition assays. 42 Lu et al.  43 investigated pharmacokinetics of a single intravenous dose of recombinant transgenic AT in healthy volunteers (table 4). The concentrations of AT after initial doses (50–200 U/kg) given intravenously over 30 min were best described by a weight-normalized two-compartment model. 43 Additional information regarding transgenically produced human AT has been previously reported. 44–46 
Table 4. Pharmacokinetic Variables in Healthy Volunteers after a Single Intravenous Dose of rh AT
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Table 4. Pharmacokinetic Variables in Healthy Volunteers after a Single Intravenous Dose of rh AT
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Although hereditary AT deficiency is rare, 1 acquired AT deficiency is common after noncardiac 47 and cardiac surgery. 2–7,48 Low AT concentrations during cardiac surgery are likely to develop because of preoperative use of heparin, 3–5 the effects of hemodilution, 3–5 and CPB-associated excessive hemostatic system activation. 6 AT concentrations commonly decrease to 40–50 U/dl activity 32 during CPB. 2–7 Native AT concentrations as low as 20–30 U/dl were observed during CPB in the current study in patients not receiving supplemental AT. Supraphysiologic AT concentrations were achieved at higher doses of rh-AT, reaching 600 U/dl immediately after the initial loading dose. Concentrations then declined secondary to hemodilution that occurs during initiation of CPB. Doses of rh-AT 75 U/kg normalized AT activity to greater than 100 U/dl during CPB, and AT concentrations remained elevated close to 100 U/dl on arrival in the intensive care unit in patients who received higher doses (i.e.  , 150 U/kg). Thus, a single dose of AT maintains normal AT concentrations to cover the clinically important period of CPB during which excessive hemostatic system activation may occur and can lead to bleeding or thrombotic complications.
Anticoagulation is used during cardiac surgery to prevent thrombosis of the extracorporeal circuit and to minimize excessive CPB-related activation of the hemostatic system. Unfractionated heparin is routinely used because it is immediately effective, rapidly reversible, generally well tolerated, and inexpensive. 49 The heparin anticoagulant effect is predominately mediated by binding of heparin to AT, requiring a specific pentasaccharide sequence, 50 and subsequent irreversible complex formation and inactivation by AT of several activated coagulation factors, in particular, factors IIa and Xa. 51 Decreased heparin anticoagulant response or heparin resistance is associated with AT deficiency. 52 This has led to the use of either fresh frozen plasma 53 or human plasma-derived AT concentrates (AT concentrates are not approved for this indication in the United States) 5,54 in patients that show appreciable resistance to heparin before initiation of CPB. 53 Administration of rh-AT in the current study improved heparin anticoagulant effect, as reflected by increased ACTs during heparin therapy when compared with placebo patients.
Detection and treatment of acquired AT deficiency may also be important with respect to potential reduction in perioperative bleeding and thromboembolic complications. CPB-related activation of the hemostatic system 28–30 can consume labile coagulation factors and platelets, which may lead to excessive bleeding (i.e.  , in 5–16% of patients) 10,11 or reexploration (3–4%) 11,12 after cardiac surgery. Acquired AT deficiency may render heparin ineffective in suppressing thrombin generation–activity during CPB. 3,5,8 This is important because recent evidence indicates that inadequate heparin anticoagulation can result in excessive bleeding and transfusion requirements 55 secondary to consumption of coagulation factors and platelets. 6 Therefore, detection and treatment of AT deficiency may be important with respect to preservation of labile coagulation factors and platelets, which may reduce blood loss and need for transfusion. The importance of AT in suppressing activation of the hemostatic system is supported by data from several studies. 3,52 Hashimoto et al.  3 showed that AT supplementation more effectively suppresses thrombin activity in pediatric patients who received AT concentrates. Despotis et al.  52 demonstrated that appreciable increases in markers of platelet activation and coagulation activation were only seen when AT concentration decreased to less than 60 U/dl.
Because maintenance of normal or elevated plasma AT concentrations during CPB can enhance suppression of the hemostatic system, the current study investigated the efficacy of increasing doses of transgenic rh-AT to suppress markers of hemostatic activation. The findings of the current study indicate that normalizing AT concentrations with rh-AT during CPB can not only reduce thrombin activity (fibrin monomer), but can also reduce fibrinolytic activity (D-dimer) and reduce impairment of platelet function after CPB, which is thought to be the most important hemostatic defect after CPB. 56,57 Although this study did not show any reductions in excessive microvascular or perioperative thrombotic complications in patients receiving rh-AT, this could be because of the small sample size and low-risk population studied. Because we studied a small number of patients in each group, this study was not powered to prevent significant type II error; therefore, additional studies involving more patients will be necessary in the future to fully assess benefits of supplemental administration of rh-AT to cardiac surgical patients. Nevertheless, our data support the concept that rh-AT administration enhances heparin responsiveness and modulates activation of the hemostatic system.
Perioperative thrombotic complications such as deep venous thrombosis, pulmonary embolism, stroke (1.6–5.6%), 23–25 and perioperative myocardial infarction–injury (5–82%) 26,27 occur with cardiac surgery. Coronary, 17,18,22 pulmonary, 19,21 and intracardiac 20 thrombosis have also been described intraoperatively. Surgery and CPB result in excessive activation of the hemostatic system via  both the intrinsic 28 and extrinsic 29,30 pathways, which leads to substantial thrombin generation and a prothrombotic state. The vascular endothelium and multiple circulating factors, including endothelium-derived relaxing factor, prostacyclin, tissue plasminogen activator, endothelium heparan sulfate, proteins C and S, and circulating AT normally inhibit intravascular thrombosis. However, normal circulating anticoagulants such as AT and proteins C and S are commonly reduced secondary to CPB-related consumption and hemodilution, 2–7,31 which may lead to hypercoagulability in the perioperative cardiac surgical period and thrombotic complications. Because perioperative bleeding and thrombotic complications after cardiac surgery may be related to inadequate suppression of the hemostatic system during and after cardiac surgery, detection and treatment of acquired AT deficiency may have important clinical consequences.
This is the first reported use of a transgenic recombinant protein for therapeutic application. Reductions in AT concentrations that occur during heparin therapy and extracorporeal circulation represent an important clinical application for rh-AT. Our data indicate that administration of transgenic rh-AT to patients and volunteers shows no adverse effects, can improve heparin responsiveness at doses used for CPB, and potentially preserve the hemostatic system via  enhanced anticoagulation during CPB. Because our study included only a small number of patients in each dosing cohort, further studies with larger number of patients are needed to determine the clinical usefulness of rh-AT with respect to management of heparin resistance, prevention of excessive bleeding, and blood conservation, as well as prevention of thromboembolic sequelae. Other potential therapeutic applications that warrant investigation include administration of rh-AT to patients with unstable angina on heparin therapy or in patients undergoing angioplasty or other arterial manipulations.
References
Rosenberg RD: Actions and interactions of antithrombin and heparin. N Engl J Med 1975; 292: 146–51Rosenberg, RD
Wolk LA, Wilson RF, Burdick M, Selik N, Brown J, Starricco A, Mammen EF: Changes in antithrombin, antiplasmin, and plasminogen during and after cardiopulmonary bypass. Am Surg 1985; 51: 309–13Wolk, LA Wilson, RF Burdick, M Selik, N Brown, J Starricco, A Mammen, EF
Hashimoto K, Yamagishi M, Sasaki T, Nakano M, Kurosawa H: Heparin and antithrombin III levels during cardiopulmonary bypass: Correlation with subclinical plasma coagulation. Ann Thorac Surg 1995; 58: 799–805Hashimoto, K Yamagishi, M Sasaki, T Nakano, M Kurosawa, H
Zaidan JR, Johnson S, Brynes R, Monroe S, Guffin AV: Rate of protamine administration: Its effect on heparin reversal and antithrombin recovery after coronary artery surgery. Anesth Analg 1986; 65: 377–80Zaidan, JR Johnson, S Brynes, R Monroe, S Guffin, AV
Irani MS: Antithrombin concentrates in heparin-resistant cardiopulmonary bypass patients. Clin Appl Thromb Haemost 1996; 2: 103–6Irani, MS
Despotis GJ, Joist JH, Hogue CW, Alsoufiev A, Joiner-Maier D, Santoro SA, Spitznagel E, Weitz J, Goodnough LT: More effective suppression of hemostatic system activation in patients undergoing cardiac surgery by heparin dosing based on heparin blood concentrations rather than ACT. Thromb Hemost 1996; 76: 902–8Despotis, GJ Joist, JH Hogue, CW Alsoufiev, A Joiner-Maier, D Santoro, SA Spitznagel, E Weitz, J Goodnough, LT
Dietrich W, Spannagl M, Schramm W, Vogt W, Barankay A, Richter JA: The influence of preoperative anticoagulation on heparin response during cardiopulmonary bypass. J Thorac Cardiovasc Surg 1991; 102: 505–14Dietrich, W Spannagl, M Schramm, W Vogt, W Barankay, A Richter, JA
Despotis GJ, Levine V, Joist HJ, Santoro SA, Mendeloff E: Multiple episodes of thrombosis with biventricular support devices with inadequate anticoagulation and evidence of accelerated intravascular coagulation. J Thorac Cardiovasc Surg 1996; 112: 538–40Despotis, GJ Levine, V Joist, HJ Santoro, SA Mendeloff, E
Despotis GJ, Santoro SA, Spitznagel E, Kater KM, Cox JL, Barnes P, Lappas DG: Prospective evaluation and clinical utility of on-site monitoring of coagulation in patients undergoing cardiac operation. J Thorac Cardiovasc Surg 1994; 107: 271–9Despotis, GJ Santoro, SA Spitznagel, E Kater, KM Cox, JL Barnes, P Lappas, DG
Despotis GJ, Filos KS, Zoys TN, Hogue CW Jr, Spitznagel E, Lappas DG: Factors associated with excessive postoperative blood loss and hemostatic transfusion requirements: A multivariate analysis in cardiac surgical patients. Anesth Analg 1996; 82: 13–21Despotis, GJ Filos, KS Zoys, TN Hogue, CW
Moulton MJ, Creswell LL, Mackey ME, Cox JL, Rosenbloom M: Reexploration for bleeding is a risk factor for adverse outcomes after cardiac operations. J Thorac Cardiovasc Surg 1996; 111: 1037–46Moulton, MJ Creswell, LL Mackey, ME Cox, JL Rosenbloom, M
Dacey LJ, Munoz JJ, Baribeau YR, Johnson ER, Lahey SJ, Leavitt BJ, Quinn RD, Nugent WC, Birkmeyer JD, O'Connor GT: Reexploration for hemorrhage following coronary artery bypass grafting: Incidence and risk factors. Northern New England Cardiovascular Disease Study Group. Arch Surg 1998; 133: 442–7Dacey, LJ Munoz, JJ Baribeau, YR Johnson, ER Lahey, SJ Leavitt, BJ Quinn, RD Nugent, WC Birkmeyer, JD O'Connor, GT
Gravlee GP, Haddon WS, Rothberger HK, Mills SA, Rogers AT, Bean VE, Buss DH, Prough DS, Cordell AR: Heparin dosing and monitoring for cardiopulmonary bypass: A comparison of techniques with measurement of subclinical plasma coagulation. J Thorac Cardiovasc Surg 1990; 99: 518–27Gravlee, GP Haddon, WS Rothberger, HK Mills, SA Rogers, AT Bean, VE Buss, DH Prough, DS Cordell, AR
Paone G, Spencer T, Silverman NA: Blood conservation in coronary artery surgery. Surgery 1994; 116: 672–7Paone, G Spencer, T Silverman, NA
van de Watering LM, Hermans J, Houbiers JG, van den Broek PJ, Bouter H, Boer F, Harvey MS, Huysmans HA, Brand A: Beneficial effects of leukocyte depletion of transfused blood on postoperative complications in patients undergoing cardiac surgery: A randomized clinical trial. Circulation 1998; 97: 562–8van de Watering, LM Hermans, J Houbiers, JG van den Broek, PJ Bouter, H Boer, F Harvey, MS Huysmans, HA Brand, A
Goodnough LT, Brecher MG, Kanter MH, AuBuchon JP: Medical Progress: Transfusion medicine—Blood transfusion. N Engl J Med 1999; 340: 438–47Goodnough, LT Brecher, MG Kanter, MH AuBuchon, JP
Loubser PG, McCarthy JJ, Attar M: Coronary thrombosis associated with antithrombin-III deficiency. J Cardiothorac Vasc Anesth 1997; 11: 756–9Loubser, PG McCarthy, JJ Attar, M
Umbrain V, Christiaens F, Camu F: Intraoperative coronary thrombosis: Can aprotinin and protamine be incriminated? J Cardiothorac Vasc Anesth 1994; 8: 198–201Umbrain, V Christiaens, F Camu, F
Alvarez JM, Goldstein J, Mezzatesta J, Flanagan B, Dodd M: Fatal intraoperative pulmonary thrombosis after graft replacement of an aneurysm of the arch and descending aorta in association with deep hypothermic circulatory arrest and aprotinin therapy. J Thorac Cardiovasc Surg 1998; 115: 723–4Alvarez, JM Goldstein, J Mezzatesta, J Flanagan, B Dodd, M
Cheung AT, Levin SK, Weiss SJ, Acker MA, Stenach N: Intracardiac thrombus: A risk of incomplete anticoagulation for cardiac operations. Ann Thorac Surg 1994; 58: 541–2Cheung, AT Levin, SK Weiss, SJ Acker, MA Stenach, N
Bohrer H, Fleischer F, Lang J, Vahl C: Early formation of thrombi on pulmonary artery catheters in cardiac surgical patients receiving high-dose aprotinin. J Cardiothorac Anesth 1990; 4: 222–5Bohrer, H Fleischer, F Lang, J Vahl, C
Cosgrove DM, Heric B, Lytle BW, Taylor PC, Novoa R, Golding LA, Stewart RW, McCarthy PM, Loop FD: Aprotinin therapy for reoperative myocardial revascularization: A placebo-controlled study. Ann Thorac Surg 1992; 54: 1031–8Cosgrove, DM Heric, B Lytle, BW Taylor, PC Novoa, R Golding, LA Stewart, RW McCarthy, PM Loop, FD
Hogue CW Jr, Barzilai B, Pieper KS, Coombs LP, DeLong ER, Kouchoukos NT, Davila-Roman VG: Sex differences in neurological outcomes and mortality after cardiac surgery: A society of thoracic surgery national database report. Circulation 2001; 103: 2133–7Hogue, CW Barzilai, B Pieper, KS Coombs, LP DeLong, ER Kouchoukos, NT Davila-Roman, VG
Newman MF, Wolman R, Kanchuger M, Marschall K, Mora Mangano C, Roach G, Smith LR, Aggarwal A, Nussmeier N, Herskowitz A, Mangano DT: Multicenter preoperative stroke risk index for patients undergoing coronary artery bypass graft surgery: Multicenter Study of Perioperative Ischemia (McSPI) Research Group. Circulation 1996; 94: II74–80Newman, MF Wolman, R Kanchuger, M Marschall, K Mora Mangano, C Roach, G Smith, LR Aggarwal, A Nussmeier, N Herskowitz, A Mangano, DT
Roach GW, Kanchuger M, Mangano CM, Newman M, Nussmeier N, Wolman R, Aggarwal A, Marschall K, Graham SH, Ley C: Adverse cerebral outcomes after coronary bypass surgery: Multicenter Study of Perioperative Ischemia Research Group and the Ischemia Research and Education Foundation Investigators. N Engl J Med 1996; 335: 1857–63Roach, GW Kanchuger, M Mangano, CM Newman, M Nussmeier, N Wolman, R Aggarwal, A Marschall, K Graham, SH Ley, C
van Vlies B, van Royen EA, Visser CA, Meyne NG, van Buul MM, Peters RJ, Dunning AJ: Frequency of myocardial indium-111 antimyosin uptake after uncomplicated coronary artery bypass grafting. Am J Cardiol 1990; 66: 1191–5van Vlies, B van Royen, EA Visser, CA Meyne, NG van Buul, MM Peters, RJ Dunning, AJ
Burns RJ, Gladstone PJ, Tremblay PC, Feindel CM, Salter DR, Lipton IH, Ogilvie RR, David TE: Myocardial infarction determined by technetium-99m pyrophosphate single-photon tomography complicating elective coronary artery bypass grafting for angina pectoris. Am J Cardiol 1989; 63: 1429–34Burns, RJ Gladstone, PJ Tremblay, PC Feindel, CM Salter, DR Lipton, IH Ogilvie, RR David, TE
Heimark RL, Kurachi K, Fujikawa K, Davie EW: Surface activation of blood coagulation, fibrinolysis and kinin formation. Nature 1980; 286: 456–60Heimark, RL Kurachi, K Fujikawa, K Davie, EW
Boisclair MD, Lane DA, Philippou H, Esnouf MP, Sheikh S, Hunt B, Smith KJ: Mechanisms of thrombin generation during surgery and cardiopulmonary bypass. Blood 1993; 82: 3350–7Boisclair, MD Lane, DA Philippou, H Esnouf, MP Sheikh, S Hunt, B Smith, KJ
de Haan J, Boonstra PW, Monnink SH, Ebels T, van Oeveren W: Retransfusion of suctioned blood during cardiopulmonary bypass impairs hemostasis. Ann Thorac Surg 1995; 59: 901–7de Haan, J Boonstra, PW Monnink, SH Ebels, T van Oeveren, W
Menges T, Wagner RM, Welters I, Ruwoldt R, Boldt J, Hempelmann G: The role of the protein C-thrombomodulin system and fibrinolysis during cardiovascular surgery: Influence of acute preoperative plasmapheresis. J Cardiothorac Vasc Anesth 1996; 10: 482–9Menges, T Wagner, RM Welters, I Ruwoldt, R Boldt, J Hempelmann, G
Schwartz RS, Bauer KA, Rosenberg RD, Kavanaugh EJ, Davies DC, Bogdanoff DA: Clinical experience with antithrombin III concentrate in treatment of congenital and acquired deficiency of antithrombin. The Antithrombin III Study Group. Am J Med 1989; 87: 53S–60SSchwartz, RS Bauer, KA Rosenberg, RD Kavanaugh, EJ Davies, DC Bogdanoff, DA
Glick B, Pasternak JJ: Development and Use of Transgenic Animals in Molecular Biotechnology: Principles of and Applications of Recombinant DNA. Washington DC, ASM Press, 1994, pp 359–80
Levy JH, Weisinger A, Ziomek CA, Echelard Y. Recombinant antithrombin: Production and role in cardiovascular disorder. Semin Thromb Hemost 2001; 27: 405–16Levy, JH Weisinger, A Ziomek, CA Echelard, Y
Ebert KM, DiTullio P, Barry CA, Schindler JE, Ayres SL, Smith TE, Pellerin LJ, Meade HM, Denman J, Roberts B: Induction of tissue plasminogen activator in the mammary gland of transgenic goats. Bio Technol 1994; 12: 699–702Ebert, KM DiTullio, P Barry, CA Schindler, JE Ayres, SL Smith, TE Pellerin, LJ Meade, HM Denman, J Roberts, B
Roberts B, DiTullio P, Vitale J, Hehir K, Gordon K: Cloning of the β-casein-encoding gene and expression in transgenic mice. Gene 1992; 121: 255–62Roberts, B DiTullio, P Vitale, J Hehir, K Gordon, K
Broker M, Ragg H, Karges HE: Expression of human antithrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pompe. Biochim Biophys Acta 1987; 908: 203–13Broker, M Ragg, H Karges, HE
Bock SC, Wion KL, Vehar GA, Lawn RM: Cloning and expression of the cDNA for human antithrombin III. Nucl Acids Res 1982; 10: 8113–25Bock, SC Wion, KL Vehar, GA Lawn, RM
Varki A: Biological roles of oligosaccharides: All of the theories are correct. Glycobiology 1993; 3: 97–130Varki, A
DiTullio P, Cheng SH, Marshal J, Gregory RJ, Ebert KM, Meade HM, Smith AE: Production of cystic fibrosis transmembrane regulator in the milk of transgenic mice. Bio Technol 1992; 10: 74–7DiTullio, P Cheng, SH Marshal, J Gregory, RJ Ebert, KM Meade, HM Smith, AE
Gould SJ, Subramani S, Scheffler IE: Use of the DNA polumerase chain reaction for homology probing: Isolation of partial cDNA or genomic clones encoding the iron-sulfur protein succinate dehydrogenase from several species. Proc Natl Acad Sci U S A 1989; 86: 1934–8Gould, SJ Subramani, S Scheffler, IE
Edmunds T, Van Patten SM, Pollock J, Hanson E, Bernasconi R, Higgins E, Manavalan P, Ziomek C, Meade H, McPherson JM, Cole ES: Transgenically produced human antithrombin: Structural and functional comparison to human plasma-derived antithrombin. Blood 1998; 91: 4561–71Edmunds, T Van Patten, SM Pollock, J Hanson, E Bernasconi, R Higgins, E Manavalan, P Ziomek, C Meade, H McPherson, JM Cole, ES
Lu W, Mant TGK, Levy JH, Bailey JM: Pharmacokinetics of recombinant transgenic antithrombin in volunteers. Anesth Analg 2000; 90: 531–4Lu, W Mant, TGK Levy, JH Bailey, JM
Olson ST, Frances-Chmura AM, Swanson R, Bjork I, Zettlmeissl G: Effect of individual carbohydrate chains of recombinant antithrombin on heparin affinity and on generation of glycoforms differing in heparin affinity. Arch Biochem Biophys 1997; 341: 212–21Olson, ST Frances-Chmura, AM Swanson, R Bjork, I Zettlmeissl, G
Garone L, Edmunds T, Hanson E, Ernasconi R, Huntington JA, Meagher JL, Fan B, Gettins PGW: Antithrombin–heparin affinity reduced by fucosylation of carbohydrate at asparagine 155. Biochemistry 1996; 35: 8881–9Garone, L Edmunds, T Hanson, E Ernasconi, R Huntington, JA Meagher, JL Fan, B Gettins, PGW
Pollock DP, Kutzko JP, Birck-Wilson E, Williams JL, Echelard Y, Meade HM: Transgenic milk as a method for the production of recombinant antibodies. J Immunol Meth 1999; 231: 147–57Pollock, DP Kutzko, JP Birck-Wilson, E Williams, JL Echelard, Y Meade, HM
Buller HR, Ten Cate JW: Acquired antithrombin III deficiency: Laboratory diagnosis, incidence, clinical implications, and treatment with antithrombin III concentrate. Am J Med 1989; 87: 44S–8SBuller, HR Ten Cate, JW
Esposito RA, Culliford AT, Colvin SB, Thomas SJ, Lackner H, Spencer FC: Heparin resistance during cardiopulmonary bypass: The role of heparin pretreatment. J Thorac Cardiovasc Surg 1983; 85: 346–53Esposito, RA Culliford, AT Colvin, SB Thomas, SJ Lackner, H Spencer, FC
Despotis GJ, Gravlee GP, Filos KS, Levy JH: Anticoagulation monitoring during cardiac surgery: A review of current and emerging techniques. A nesthesiology 1999; 91: 1122–51Despotis, GJ Gravlee, GP Filos, KS Levy, JH
Choay J, Petitou M, Lormeau JC, Sinay P, Casu B, Gatti G: Structure-activity relationship in heparin: A synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity. Biochem Biophys Res Commun 1983; 116: 492–9Choay, J Petitou, M Lormeau, JC Sinay, P Casu, B Gatti, G
Hirsh J, Raschke R, Warkentin TE, Dalen JE, Deykin D, Poller L: Heparin: Mechanism of action, pharmacokinetics, dosing considerations, monitoring, efficacy, and safety. Chest 1995; 108: 258S–75SHirsh, J Raschke, R Warkentin, TE Dalen, JE Deykin, D Poller, L
Despotis GJ, Levine V, Joist JH, Joiner-Maier D, Spitznagel E: Antithrombin III during cardiac surgery: Effect on response of activated clotting time to heparin and relationship to markers of hemostatic activation. Anesth Analg 1997; 85: 498–506Despotis, GJ Levine, V Joist, JH Joiner-Maier, D Spitznagel, E
Sabbagh AH, Chung GK, Shuttleworth P, Applegate BJ, Gabrhel W: Fresh frozen plasma: A solution to heparin resistance during cardiopulmonary bypass. Ann Thorac Surg 1984; 37: 466–8Sabbagh, AH Chung, GK Shuttleworth, P Applegate, BJ Gabrhel, W
Van Norman GA, Gernsheimer T, Chandler WL, Cochran RP, Spiess BD: Indicators of fibrinolysis during cardiopulmonary bypass after exogenous antithrombin-III administration for acquired antithrombin III deficiency. J Cardiothorac Vasc Anesth 1997; 11: 760–3Van Norman, GA Gernsheimer, T Chandler, WL Cochran, RP Spiess, BD
Despotis GJ, Joist JH, Hogue CW, Alsoufiev A, Kater K, Goodnough LT, Santoro SA, Spitznagel E, Rosenblum M, Lappas DG: The impact of heparin concentration and activated clotting time monitoring on blood conservation: A prospective, randomized evaluation in patients undergoing cardiac operations. J Thorac Cardiovasc Surg 1995; 110: 46–54Despotis, GJ Joist, JH Hogue, CW Alsoufiev, A Kater, K Goodnough, LT Santoro, SA Spitznagel, E Rosenblum, M Lappas, DG
Harker LA: Bleeding after cardiopulmonary bypass. N Engl J Med 1986; 314: 1446–8Harker, LA
George JN, Shattil SJ: The clinical importance of acquired abnormalities of platelet function. N Engl J Med 1991; 324: 27–39George, JN Shattil, SJ
Fig. 1. Plasma antithrombin III (AT) activity concentrations after administration of recombinant human AT (rh-AT) using single doses of 0, 10, 25, 50, 75, 100, 125, 150, 175, and 200 U/kg during cardiac surgery with cardiopulmonary bypass (CPB). rh-AT was administered after intubation. Specimen collection times: Baseline = preadministration, 1 min after rh-AT injection, 5 min after heparin, every 30 min during CPB, before protamine, 10 min and 2, 4, 6, 12, and 24 h after CPB, and protamine reversal. Data are expressed as mean ± SE.
Fig. 1. Plasma antithrombin III (AT) activity concentrations after administration of recombinant human AT (rh-AT) using single doses of 0, 10, 25, 50, 75, 100, 125, 150, 175, and 200 U/kg during cardiac surgery with cardiopulmonary bypass (CPB). rh-AT was administered after intubation. Specimen collection times: Baseline = preadministration, 1 min after rh-AT injection, 5 min after heparin, every 30 min during CPB, before protamine, 10 min and 2, 4, 6, 12, and 24 h after CPB, and protamine reversal. Data are expressed as mean ± SE.
Fig. 1. Plasma antithrombin III (AT) activity concentrations after administration of recombinant human AT (rh-AT) using single doses of 0, 10, 25, 50, 75, 100, 125, 150, 175, and 200 U/kg during cardiac surgery with cardiopulmonary bypass (CPB). rh-AT was administered after intubation. Specimen collection times: Baseline = preadministration, 1 min after rh-AT injection, 5 min after heparin, every 30 min during CPB, before protamine, 10 min and 2, 4, 6, 12, and 24 h after CPB, and protamine reversal. Data are expressed as mean ± SE.
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Fig. 2. Relation between activation of coagulation (fibrin monomer) and recombinant human antithrombin III (rh-AT) dose. Mean plasma concentrations of fibrin monomer for each dosing cohort are shown. The total number of specimens collected during and after cardiopulmonary bypass that were used for the analysis was 226. *P  < 0.05 compared with placebo patients.
Fig. 2. Relation between activation of coagulation (fibrin monomer) and recombinant human antithrombin III (rh-AT) dose. Mean plasma concentrations of fibrin monomer for each dosing cohort are shown. The total number of specimens collected during and after cardiopulmonary bypass that were used for the analysis was 226. *P 
	< 0.05 compared with placebo patients.
Fig. 2. Relation between activation of coagulation (fibrin monomer) and recombinant human antithrombin III (rh-AT) dose. Mean plasma concentrations of fibrin monomer for each dosing cohort are shown. The total number of specimens collected during and after cardiopulmonary bypass that were used for the analysis was 226. *P  < 0.05 compared with placebo patients.
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Fig. 3. Relation between D-dimer as markers of activation of coagulation (fibrin formation) and increased and recombinant human antithrombin III (rh-AT). (Top  ) The relation between individual plasma concentrations of D-dimer and rh-AT during several cardiopulmonary bypass time points is shown; a significant inverse relation was observed between D-dimer concentrations and rh-AT (D-dimer =−5.7 rh-AT + 1,749, r =−0.49, P  = 0.001). (Bottom  ) Mean D-dimer values for each dosing cohort are shown. The total number of specimens collected at 15-min intervals during CPB used for analyses was 124. *P  < 0.05 compared with placebo patients.
Fig. 3. Relation between D-dimer as markers of activation of coagulation (fibrin formation) and increased and recombinant human antithrombin III (rh-AT). (Top 
	) The relation between individual plasma concentrations of D-dimer and rh-AT during several cardiopulmonary bypass time points is shown; a significant inverse relation was observed between D-dimer concentrations and rh-AT (D-dimer =−5.7 rh-AT + 1,749, r =−0.49, P 
	= 0.001). (Bottom 
	) Mean D-dimer values for each dosing cohort are shown. The total number of specimens collected at 15-min intervals during CPB used for analyses was 124. *P 
	< 0.05 compared with placebo patients.
Fig. 3. Relation between D-dimer as markers of activation of coagulation (fibrin formation) and increased and recombinant human antithrombin III (rh-AT). (Top  ) The relation between individual plasma concentrations of D-dimer and rh-AT during several cardiopulmonary bypass time points is shown; a significant inverse relation was observed between D-dimer concentrations and rh-AT (D-dimer =−5.7 rh-AT + 1,749, r =−0.49, P  = 0.001). (Bottom  ) Mean D-dimer values for each dosing cohort are shown. The total number of specimens collected at 15-min intervals during CPB used for analyses was 124. *P  < 0.05 compared with placebo patients.
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Table 1. Demographic and Operative Patient Data
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Table 1. Demographic and Operative Patient Data
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Table 2. Adverse Events in Placebo and rh AT Patients
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Table 2. Adverse Events in Placebo and rh AT Patients
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Table 3. Total Blood Component Donor Exposures and Mediastinal Chest Tube Drainage in Patients Receiving Different Doses of rh AT (n = 30) or Placebo (n = 6)
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Table 3. Total Blood Component Donor Exposures and Mediastinal Chest Tube Drainage in Patients Receiving Different Doses of rh AT (n = 30) or Placebo (n = 6)
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Table 4. Pharmacokinetic Variables in Healthy Volunteers after a Single Intravenous Dose of rh AT
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Table 4. Pharmacokinetic Variables in Healthy Volunteers after a Single Intravenous Dose of rh AT
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