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J Thorac Cardiovasc Surg 1998;116:1043-1051
© 1998 Mosby, Inc.
CARDIOPULMONARY SUPPORT AND PHYSIOLOGY |
Supported by HL47186 from the National Heart Lung Blood Institute, National Institutes of Health, Bethesda, Md.
Received for publication Feb 12, 1998. Revisions requested April 15, 1998; revisions received July 14, 1998. Accepted for publication July 21, 1998. Address for reprints: L. Henry Edmunds, Jr, MD, Department of Surgery, Hospital of the University of Pennsylvania, Six Silverstein, 3400 Spruce St, Philadelphia, PA 19104.
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Abstract |
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Introduction |
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2 Heparin directly inhibits circulating thrombin and partially inhibits thrombin formation by weakly inhibiting factor Xa and factor IXa 3; it inhibits thrombin activity within blood clots only at very high concentrations. 4 Inhibition of thrombin formation, as opposed to inhibition of thrombin after it is formed, offers the prospect of down-regulating the overall coagulation stimulus during cardiac operations and surmounting disadvantages of unfractionated heparin.
The intrinsic and extrinsic coagulation pathways converge by converting factor X to factor Xa, which initiates formation of the prothrombinase complex and converts prothrombin to thrombin. 2,5 Several peptides, found in leeches and spiders, bind factor Xa in nanomolar concentrations, 6 but recombinant forms of these proteins fail to suppress thrombin formation during in vitro extracorporeal perfusion. 7 Compared with standard heparin, low molecular weight heparins are more potent inhibitors of factor Xa in vivo 8 and better suppressors of thrombin generation in patients with deep venous thrombosis. 9 Low molecular weight heparin has also been used for clinical cardiac operations. 10-12
In a previous study we observed that the low molecular weight heparin, enoxaparin, completely suppressed complement activation and neutrophil elastase release during in vitro, simulated, extracorporeal recirculation of fresh human blood. 7 The same study also showed that thrombin formation and activity, as measured by the generation of the prothrombin fragment F1.2 and fibrinopeptide A, was attenuated by enoxaparin as compared with standard porcine heparin. 7 The present in vivo study tests the hypothesis that anticoagulation with enoxaparin or a combination of enoxaparin and standard heparin attenuates thrombin formation and activity and also reduces complement formation and neutrophil elastase release during CPB in baboons.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In 3 baboons, the dose of enoxaparin was given intravenously (blood volume assumed 0.08 times body weight) to raise the activated clotting time to 625 ± 70 seconds. Sixty minutes later, the activated clotting time was 353 ± 13 seconds. Ten and 60 minutes after protamine (4 mg/100 U enoxaparin), activated clotting times were 195 ± 4 and 147 ± 9 seconds, respectively.The effects of standard porcine intestinal heparin and enoxaparin on complement C1-s and factor Xa inhibition in human and baboon blood were compared. Fresh human (n = 2) and baboon (n = 2) peripheral venous blood was drawn directly into test tubes containing 3 different anticoagulants: porcine heparin (3.75 U/mL), enoxaparin (17.5 U/mL), and 3.8% sodium citrate (10% citrate/90% blood by volume). Blood was centrifuged at 2000g for 20 minutes; the supernatant was further centrifuged at 15,000g for 10 minutes; this supernatant was divided into aliquots and stored at –80°C until studied. For analysis aliquots (12.5 µL) of plasma were incubated with 100 µL of 0.1 U/mL FXa (Enzyme Research Laboratories Inc, Southbend, Ind) in microtitre plate wells and incubated for different time intervals (0 to 10 min). Chromogenic substrate S-2222 (Chromogenix, Franklin, Ohio) was added and absorbance was read at 405 nm after 12 minutes. For C1-s inhibition plasma or phosphate-buffered saline solution (PBS; 0.1 mol/L containing 0.15 NaCl, pH 7.4) and substrate, S2314 (Chromogenix) were preincubated at 37°C for 2 minutes. Activated human C1-s (1.0 µmol/L, final concentration; Enzyme Research Laboratories Inc) was added to yield a total volume of 200 µL and incubated at 37°C in a water bath. The final concentration of S2314 was 0.5 mol/L. The reaction was stopped at 0, 5, 10, and 30 minutes by adding 100 µL 50% (vol/vol) acetic acid. Triplicate samples (80 µL) were placed in a disposable nonsterile assay plate (96 well; Corning, Inc, Corning, NY) and measured at 405 nm by the enzyme-linked immunosorbant assay reader (Multiscan Biochromatic Plate Liter; ICN Pharmaceuticals, Huntsville, Ala).
In vivo studies
Thirteen juvenile baboons (Papio annubis) weighing 10 to 23 kg were used. The animals were divided into 3 groups: standard procine intestinal heparin (300 U/kg; Elkins-Sinn, Inc, Cherry Hill, NJ) control (group 1, n = 10); enoxaparin (800 U/kg; group 2, n = 8); and a combination of porcine heparin (225 U/kg) and enoxaparin (200 U/kg; group 3, n = 9). Baboons were studied when the mandated rest period of 6 to 8 weeks between studies expired or when new animals completed quarantine; 3 animals were studied thrice; 8 animals, twice; and 2 animals, once. Activated clotting times with the use of kaolin were monitored throughout the study, and all times increased over 500 sec with each bolus dose of anticoagulant. This study was approved by the University of Pennsylvania Committee on Animal Care and Utilization (IACUC).
For each CPB experiment, the baboon was placed in a squeeze cage, sedated with 10 mg/kg ketamine hydrochloride intramuscularly, and induced with 5 mg/kg thiopental sodium intravenously. The animal was intubated; general anesthesia was maintained with inhalational isofluorane. The right or left side of the neck and both groins were prepared and draped appropriately for sterile cutdown and cannulation of vessels. Hemodynamic monitoring was accomplished with an arterial line with a 22-gauge catheter placed in the femoral artery, and a 5F balloon-tipped pulmonary artery catheter placed via a femoral vein. After anticoagulation, a 10F to 14F Bio-Medicus (Medtronic Bio-Medicus, Eden Prairie, Minn) wire-wrapped, polyurethane catheter was introduced into the jugular vein and advanced into the right atrium. A similar, but shorter 8F, arterial catheter for reinfusion was inserted into the femoral artery.
Each bypass circuit was assembled with silicone-rubber tubing (Dow Corning Corporation, Midland, Mich), incorporating a bubble oxygenator (Bentley 5/Pediatric; Baxter Healthcare, Inc, Irvine, Calif), an arterial filter (Intersept Pediatric; Medtronic, Inc, Anaheim, Calif), and a roller pump (model 13400; Sarns/3M, Ann Arbor, Mich). 13 The circuit was primed with approximately 500 mL of Normosol solution (Abbott Laboratories, North Chicago, Ill). Normothermic CPB began at a flow rate of 50 mL/kg/min (approximately one half of the baboon's resting cardiac output), and perfusion was maintained for 60 minutes. Ten minutes after CPB ended, protamine was given for total doses of anticoagulant as follows: 1 mg/100 U heparin; 4 mg/100 U enoxaparin.
Sampling times
Six blood samples (15 to 20 mL each) were obtained at baseline before anticoagulation (time-point labeled BASELINE); after anticoagulation (HEPARIN); 5 minutes after starting CPB (START); 5 minutes before stopping CPB (END); 10 minutes after protamine (3 mg/kg; Elkins-Sinn, Inc, Cherry Hill, NJ) (PROTAMINE); and 60 minutes after time-point PROTAMINE (PROTAMINE-60). Some additional hematologic measurements were made 120 and 180 minutes after protamine and 24 hours later.
Measurements
Heart rate by electrocardiogram, systemic (systolic, diastolic, mean) arterial blood pressures, central venous pressure, pulmonary arterial pressure, and pulmonary capillary wedge pressure were continuously monitored. Intermittent thermodilution cardiac outputs were measured in duplicate before and after CPB. Blood samples were assayed for hematocrit, platelet count, white blood cell count, platelet aggregation to adenosine diphosphate (ADP), ß-thromboglobulin (ßTG) release, release of neutrophil elastase, complement glycoproteins C3b/c and C4b/c, prothrombin fragment F1.2, thrombin-antithrombin complex (TAT), and fibrinopeptide A (FPA). Template bleeding times were measured at 6 time points. Dilution of formed blood elements and plasma proteins were corrected with the use of hematocrit. The total amount of blood withdrawn was limited to less than 10% of body weight (120 to 180 mL) for each experiment, and blood remaining in the perfusion circuit was reinfused at the end of CPB.
Hematocrit and platelet count assays were performed on whole blood. Platelets were counted by phase microscopy or by Coulter Counter (model STKR; Coulter Electronics Inc, Hileah, Fla) in triplicate. After correction for hemodilution, platelet counts were expressed as a percentage of BASELINE values. Platelet aggregation to ADP was studied with a Payton aggregometer (model 440; Chrono-Log, Inc, Havertown, Pa). Platelet-rich plasma and platelet-poor plasma were prepared from citrated blood (10 mL with 10% by volume of 3.8% citrate) by differential centrifugation at 150g for 10 minutes and 13,600g for 5 minutes, respectively. Before studies of aggregation, the platelet count of platelet-rich plasma was adjusted to 150,000/µL by dilution with platelet-poor plasma. The concentration of ADP required to produce complete second-wave aggregation was measured; complete second-wave aggregation was assumed when light transmission was 62.5% or greater within 5 minutes. 14 Platelet aggregation is reported as a percentage and normalized to the concentration of ADP required to obtain full aggregation of the baseline sample. The percent aggregation observed at that ADP concentration in subsequent samples is recorded.
Template bleeding times were measured in duplicate on the forearm at the same time points as blood samples, except during CPB, and were measured 120 and 180 minutes and 24 hours after PROTAMINE (time points labeled PROTAMINE-120, PROTAMINE-180, and 24 hours) with a blood-pressure cuff inflated to 40 mm Hg. The Simplate II (Organon Teknika Corporation, Durham, NC) lancet was used to create reproducible skin incisions for determinations of bleeding times.
For plasma ß-TG analysis blood was withdrawn into centrifugation tubes containing 10% (by volume) of 3.8% acid citrate dextrose and prostaglandin E1 solution at 0°C. ß-TG was measured by radioimmunoassay. 15
Measurements of complement activation in baboon blood is difficult because of poor cross-reactivity with human antibodies. 16,17 Assays for C3b/c and C4b/c have been developed and used successfully in studies of sepsis. 16,17 C3b/c levels were assessed by radioimmunoassay with a monoclonal anti-C3-28 antibody and a polyclonal125I-anti-C3c antibody. 16 C4b/c was measured by a radioimmunoassay similar to that used for the determination of C3b/c, except that a monoclonal anti-C4-1 antibody and a polyclonal125I-anti-C4 antibody were used. 17 Both C3b/c and C4b/c results were expressed as a percentage of the amount of C3b/c or C4b/c present in normal baboon serum.
Elastase-
1 proteinase inhibitor levels were measured by 2 different enzyme-linked immunosorbent assay (ELISA) methods. The first assay (elastase-1 proteinase inhibitor) was performed with commercial kits that used a polyclonal antibody to elastase as a capture antibody and conjugation with a antibody to 1-proteinase inhibitor complexed with alkaline phosphatase (Merck Immunoassay; Merck Inc, West Point, Pa). The second assay (elastase-1-antitrypsin) used 2 monoclonal antibodies; one against elastase complexed with sephadex beads and another against 1-antitrypsin labeled by125I. 18
Plasma levels of F1.2 (Behring Diagnostics, Inc, Westwood, Mass), 19 FPA (American Bioproducts, Parsipanny, NJ), 20 TAT (Behring Diagnostics, Inc) 21 were measured by ELISA with commercial assay kits.
Statistics
Data points represent the mean ± SEM of measurements. Three-way analysis of variance (MANOVA) for repeated measures with the Bonferroni adjustment (SPSS for Windows 7.5; SPSS Inc, Chicago, Ill) was used for statistical analysis of time and group effects. When group effects were significant (P < .05), 2-way ANOVA between control and both enoxaparin groups (separately) was used to establish significant differences (P < .05) between the control group and each experimental group. The unpaired t statistic was used for specific comparisons between groups when the 2-way ANOVA group effect was significant. The paired Student t test with Bonferroni correction was used for analysis of differences within groups when the time effect was significant by 3-way MANOVA and 1-way ANOVA. Differences were considered statistically significant at the P < .05 level.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In vivo studies
There were no significant differences between groups for arterial blood pressure, central venous pressure, pulmonary arterial and capillary wedge pressures, or cardiac output at any time point. During CPB, activated clotting times remained higher than 400 seconds for all animals (Table I). For 3 hours after protamine, activated clotting times remained significantly elevated in both the enoxaparin and combination groups as compared with control animals but were similar at 24 hours (Table I).
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). In the enoxaparin group, however, bleeding times were still prolonged at 24 hours despite full protamine neutralization. 22,23 No wound hematomas or postprotamine wound bleeding occurred in either the heparin or combination groups. However, hematomas developed at cutdown sites in 6 of 8 baboons in the enoxaparin group, 2 of which required evacuation.
Neutrophil elastase increased significantly during CPB in all groups, and although 3-way MANOVA indicated significant differences between groups, comparisons of each test group with the control group by 2-way ANOVA were not significant (Table II). Similarly, complement C3b/c also increased significantly during CPB in all groups but did not increase further with protamine. Group differences were significant by 3-way ANOVA but were not significantly different from the control group by 2-way ANOVA. C4b/c, a marker for the classic pathway, did not significantly change during CPB in any group (Table II).
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). Enoxaparin significantly inhibited the rise in prothrombin fragment F1.2, TAT, and FPA during CPB as compared with control animals (Figs. 3, 4, and 5; Table II). The combination of enoxaparin and heparin also attenuated the rise in these markers, but only TAT concentrations were significantly less than those measured in heparinized baboons. After protamine, FPA increased nearly 19-fold in animals that were anticoagulated with enoxaparin, but neither TAT or F1.2 increased. This transitory rise in FPA was not observed in other groups and did not persist in the enoxaparin group.
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Discussion |
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7 As shown in the present study, suppression of complement activation in human blood by enoxaparin is not due to inhibition of C1s but may occur later in the cascade.
Complete suppression of thrombin generation during cardiac operations offers prospects of significantly reducing the thromboembolic and bleeding complications associated with these operations. Thrombin plays a central role in the regulation of coagulation and also in cell growth and migration. 2 Thrombin directly activates platelets, forms fibrin, and activates key coagulation proteins (factors V, VIII, and XIII) but also activates protein C and fibrinolysis. 24 Although standard heparin remains a prerequisite for CPB and cardiac surgery, thrombin is progressively generated in both wound 25 and perfusion circuit and circulates during extracorporeal perfusion. 1 Several alternatives to heparin have been investigated. These include coating perfusion circuits with ionic or covalently bound heparin, 26,27 r-hirudin, 28 heparinoids, 29 and inhibitors of factors IXa 30 and Xa. 7 None of these alternatives at the concentrations used reduce the generation or activity of thrombin. In contrast, enoxaparin attenuates thrombin formation and activity in the baboon model of extracorporeal circulation and in humans with deep vein thrombosis 9; these observations encourage efforts to reduce circulating thrombin during cardiac operations.
Because platelets express a thrombin receptor on their external plasma membrane 31 and are directly activated by thrombin, we expected to observe reduced platelet adhesion and alpha granule release of ß-TG in animals anticoagulated with enoxaparin. This attenuation of platelet activation did not occur and probably reflects the fact that only small amounts of circulating thrombin are sufficient to activate platelets or that other agonists, such as ADP, contribute. Because the baboon model does not produce increases in D-dimer and plasmin-antiplasmin, 13 fibrinolytic activity cannot be assessed in this model.
Protamine does not fully reverse the anticoagulant effect of enoxaparin and other low molecular weight heparins, 8,10,22,23 and further studies are needed to elucidate the mechanism of this deficiency. In vitro 1:1 doses of protamine completely neutralize thrombin, 22,23 but residual anti-factor Xa activity persists 22 and is not fully reversed by higher doses. 23 In this in vivo study with CPB, however, immediately after protamine, FPA and ß-TG significantly increase and platelet count decreases only in animals anticoagulated with enoxaparin. Sixty minutes after protamine, plasma FPA concentrations are not significantly different from other groups. By 24 hours, however, activated clotting times do not differ from control animals, but a functional deficiency of platelets remains and bleeding times are prolonged.
The increase in FPA levels with an accompanying slight rise in plasma TAT or F1.2 concentrations suggests a substantial increase in thrombin action with marginally enhanced thrombin generation after protamine. One explanation for this is that protamine preferentially neutralizes the inhibitory effect of enoxaparin on thrombin but not factor Xa. 22 The increase in plasma ß-TG and the additional decline in platelet counts immediately afterwards is consistent with platelet activation by thrombin but does not explain the elevated bleeding times 24 hours later. Presence of thrombin-activated partially degranulated platelets in circulation may provide a partial explanation, but no continuing stimulus for thrombin formation exists early after CPB ends in the baboon model. Alternatively, direct inhibition of platelet function by enoxaparin or activation of platelets by enoxaparin-protamine complexes that are slowly cleared may interfere with platelet function and prolong bleeding times. Our data do not explain the mechanism of prolonged bleeding times 24 hours after protamine.
The failure to restore normal bleeding times after CPB and late wound bleeding observed with enoxaparin is consistent with clinical experience1012 and negates use of enoxaparin for cardiac operations. Nevertheless, this study shows that a short-acting, quickly cleared, or metabolized factor Xa inhibitor offers the prospect of reducing thrombin formation and activity during open cardiac surgery.
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Acknowledgments |
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