Section Editor: Kenneth J. Tuman.
Protamine sulfate is administered after cardiopulmonary bypass (CPB) to reverse heparin anticoagulation. Although the administration of a protamine dose based on the measured heparin level results in precise reversal of anticoagulation, this technique is not widely practiced. More often, a fixed dose of protamine is administered based on the total dose of heparin, followed by measurement of an activated clotting time (ACT). A number of investigations have identified potential problems with this practice [1-3]. The commonly used fixed dose of 1.0-1.3 mg of protamine for 100 U of heparin does not account for heparin elimination and can result in excess circulating protamine . Excess protamine has been associated with a prolonged clotting time and weakened clot structure , whereas a smaller protamine dose has been associated with reduced postoperative bleeding . Reliance on the ACT as the sole measure of reversal of heparin effect may be unreliable because it can be affected by many disorders of coagulation . Thus, an increased ACT after protamine administration may be due to excess protamine (rather than inadequate protamine) or coagulation issues unrelated to residual heparin.
The protamine dose or ratio that prolongs an ACT or decreases platelet function is unknown, and alternative drugs that antagonize heparin have not been compared with protamine to evaluate their effects when administered in excess. Therefore, the purpose of this study was to examine the influence of clinically relevant concentrations of protamine and other reversal drugs on heparin antagonism as assessed by ACT. Because platelets influence the ACT and because bleeding after CPB may be related to platelet dysfunction, we also examined the effect of protamine on platelet aggregation.
After institutional approval and informed consent, blood from adult patients was obtained for the analyses. Patients with thrombocytopenia (platelet count <150,000/mL) or those who had received fibrinolytic medications or antiplatelet drugs (ticlopidine or ReoPro-abciximab) within 2 days of surgery were excluded. For studies of heparin reversal after CPB, residual blood was withdrawn from the extracorporeal reservoir at the conclusion of the bypass period. Patients received an initial dose of heparin 400 U/kg and additional heparin doses of 100-500 U to maintain ACT >450 s. For studies of platelet function, blood was obtained from an arterial cannula after the induction of anesthesia but before systemic heparinization.
Before CPB, patients were anticoagulated with 400 U/kg bovine lung heparin. An additional 10,000 U of heparin was present in the pump prime. Celite-based ACTs (Hemochron; International Technidyne, Edison, NJ) were performed during surgery, and additional heparin (100-500 U) was administered as needed to maintain the ACT >450 s. CPB was performed using a membrane oxygenator. At the end of CPB, residual blood was withdrawn from the extracorporeal reservoir into plastic syringes and analyzed for heparin level by heparin-protamine titration (Hepcon[trade mark sign] HMS; Hemotec Inc., Englewood, CO). The precision of this assay is 0.3 U/mL.
Sixty-three patients were studied in this investigation. Blood samples were placed in plastic syringes, then divided into 7 x 0.4 mL aliquots, and baseline kaolin ACTs (Medtronic-Hemotec, Inc.) were performed. Before the addition of blood (0.4 mL), protamine (n = 31 patients), recombinant platelet factor 4 (rPF4; n = 16 patients), or hexadimethrine (n = 16 patients) was added to kaolin ACT cartridges to create concentrations of reversal drugs (reversal drug to heparin ratios) as follows: protamine concentrations of 50 [micro sign]g/mL (1.3:1), 100 [micro sign]g/mL (2.6:1), 150 [micro sign]g/mL (3.9:1), 200 [micro sign]g/mL (5.2:1), 250 [micro sign]g/mL (6.5:1), and 300 [micro sign]g/mL (7.8:1); rPF4 concentrations of 50 [micro sign]g/mL (1:1), 100 [micro sign]g/mL (2:1), 150 [micro sign]g/mL (3:1), 200 [micro sign]g/mL (4:1), 250 [micro sign]g/mL (5:1), 300 [micro sign]g/mL (6:1), and 350 [micro sign]g/mL (7:1); hexadimethrine concentrations of 50 [micro sign]g/mL (1:1), 100 [micro sign]g/mL (2:1), 150 [micro sign]g/mL (3:1), 200 [micro sign]g/mL (4:1), 250 [micro sign]g/mL (5:1), 300 [micro sign]g/mL (6:1), and 350 [micro sign]g/mL (7:1). The volume of reversal drug added was <1.2% of the final volume.
Arterial blood samples were collected from 32 patients into plastic syringes containing 3.8% sodium citrate (9:1 vol/vol). To obtain platelet-rich plasma (PRP), blood samples were centrifuged for 10 min at 1000 rpm and 27[degree sign]C. After the PRP was decanted, the remainder of the sample was centrifuged at 3500 rpm for 15 min at 27[degree sign]C to obtain platelet-poor plasma. The platelet count in the PRP was measured by using a Cell Counter System 9000 (Baker Instruments Inc., Allentown, PA) and adjusted to approximately 150,000/mL by adding the autologous platelet-poor plasma. Bovine lung heparin was added to aliquots of PRP to create a plasma concentration of 4 U/mL. Platelet aggregation was performed by a turbidometric method using a double-channel aggregometer (Model 490 Optical Aggregometer; Chronolog, Havertown, NJ). Incremental doses of protamine (50, 100, 200, 300, and 370 [micro sign]g/mL) were added to heparinized PRP samples, and aggregometry was performed in the presence of adenosine diphosphate (ADP) 2.4 [micro sign]M/L and 2.4 [micro sign]g/mL collagen (concentrations of agonists to produce approximately 50% aggregation of platelets). The protamine concentrations correspond to protamine to heparin ratios of 1.3:1, 2.5:1, 5.0:1, 7.5:1, and 9.3:1.
All ACT data are expressed mean +/- SD. The Kruskal-Wallis test followed by Dunn procedure was used to compare the ACT for each incremental dose of protamine, rPF4, and hexadimethrine to the maximal reversal. A P value <0.05 was considered significant. All platelet aggregation data are expressed as mean percentage +/- SD. Serial data for each group were evaluated by repeated-measures analysis of variance followed by Bonnferroni/Dunn test. A P value <0.05 was considered significant.
The mean heparin level was 3.3 U/mL (range 2.7-4.1 U/mL). This heparin concentration was used to calculate the reversal drug heparin ratios for the study. Heparin anticoagulation was maximally reversed at a protamine to heparin ratio of 1.3:1. Each increment in protamine concentration (i.e., in excess of a calculated protamine to heparin ratio of 1.3:1) resulted in a larger ACT value that was statistically significant at ratios >2.6:1 (Figure 1). ACT values associated with protamine to heparin ratios of 1.3:1 and 2.6:1 were 143 +/- 13 and 162 +/- 16 s, respectively. In contrast, excess rPF4, even at the highest concentrations did not significantly prolong the ACT (Figure 2). Hexadimethrine only prolonged the ACT at the highest ratios of >or=to5:1 (Figure 3). The precision of this heparin assay is +/- 0.3 U/mL.
Platelet aggregation induced by ADP in the heparinized PRP was significantly reduced in the presence of protamine, even at the lowest ratio studied (1.3:1) (Figure 4). Similar results were observed for collagen-induced aggregation, but this was significant only at a protamine to heparin ratio of 9.3:1 (Figure 4).
We observed that, within a very narrow range, the presence of excess protamine when given for reversal of heparin anticoagulation after CPB can prolong the ACT (Figure 1). This is important because the common practice of administering a fixed dose of protamine based on the total dose of heparin can greatly overestimate the circulating heparin level. Further, if excess protamine is initially administered and the subsequent ACT is prolonged, the administration of additional protamine may contribute to a post-CPB coagulopathy. Thus, when heparin-protamine titrations are not performed, an initial attempt to antagonize the anticoagulant effect should be made with a smaller protamine dose.
The cause of a prolonged ACT in the presence of excess protamine is not fully understood. The anticoagulant effect of protamine may be related to its polycationic structure, by which one active site neutralizes heparin while the other elicits a nonspecific acid-base interaction with various hemostatic elements . Precipitation of fibrinogen and fibrin monomer occurs in the presence of protamine . This may be related to the electrostatic interaction of the positive charge of protamine with the negative charge on the D-domain on fibrinogen . Rapid precipitation of fibrinogen before thrombin activation may mitigate procoagulant substrate levels. Thrombin bound to fibrinogen aggregates lead to a diminished thrombin concentration  in blood from volunteers. A reversible but concentration-dependent interaction between protamine and thrombin may also lessen the procoagulant effect of thrombin .
Platelet dysfunction associated with excess protamine may also prolong the ACT. We observed that ADP-induced platelet aggregation is influenced by the presence of protamine at clinically relevant doses (Figure 4). Protamine and the combination of heparin and protamine have been shown to attenuate human platelet responsiveness to thrombin . Moreover, excess use of protamine may result in an untoward platelet effect further contributing to platelet dysfunction and potential for coagulopathy . Previous studies demonstrated no change in ADP-mediated platelet aggregation in the presence of protamine alone . However, we observed that, in the presence of heparin, protamine diminished ADP-mediated platelet aggregation. Platelet membrane function may be modified by the presence of protamine-heparin complexes. Interaction of the protamine-heparin complex with the platelet membrane may alter ADP-mediated calcium influx and thus hinder platelet activation and aggregation . Curtailed expression of membrane markers of platelet activation (P-selectin) have been observed in response to an agonist challenge when protamine is present . Although we did not measure molecular markers of platelet activation, we observed that clinically relevant protamine reversal was associated with impaired platelet aggregation (Figure 4). This is important because platelet activation is a prerequisite for aggregation to occur during the formation of a clot. Protamine interaction with thrombin may be related to platelet dysfunction . This may be associated with a decrease in thrombin concentration mediated by the aggregation of thrombin with fibrinogen or thrombin with protamine [6,7]. Carr  reported that larger doses of protamine (protamine to heparin ratios of 3.5:1-8.0:1) than we report are required to reverse the antiplatelet effects of heparin in human blood. However, we studied blood at the end of CPB when platelets are dysfunctional.
The finding of a prolonged ACT after protamine administration is a potential source of confusion after CPB. Our results demonstrate that protamine in excess of circulating heparin will prolong the ACT situation that is likely to occur when a fixed dose of protamine based on the initial heparin dose is administered. The reappearance of a prolonged ACT after protamine reversal of heparin is often attributed to reappearance of heparin in the circulation (i.e., heparin rebound). However, this phenomenon, when superimposed on the hemostatic perturbation caused by CPB and/or the overdose of protamine, makes the etiology and treatment of postoperative bleeding less clear. Thus, the decision to administer more protamine based on the visual evaluation of the operative field and ACT alone should be avoided.
Other tests to evaluate coagulation should be considered when bleeding is observed in the postbypass period. If the ACT is prolonged, then thrombin time, heparinase-ACT, and low-level heparin-protamine titration are methods that help to determine whether excess heparin is present. The thromboelastogram (or other tests of the viscoelastic properties of the clot as it forms), platelet function tests, fibrinogen levels, and platelet counts are useful diagnostic tests for guiding therapeutic interventions for the patient with persistent bleeding.
rPF4 and hexadimethrine were less likely to prolong the ACT compared with protamine. rPF4, a naturally occurring heparin-binding protein stored in the alpha granules of platelets, is released during platelet aggregation and neutralizes heparin in humans . Recombinant technology has made this a potential new alternative for antagonism of heparin. When administered with the rPF4 to heparin ratio >or=to3:1, the ACT was not prolonged (Figure 2).
Alternatively, hexadimethrine, a basic compound, also neutralizes heparin . Only the hexadimethrine to heparin ratio >or=to5:1, which is far in excess of the clinical dose, significantly prolonged the ACT (Figure 3). Thus, compared with protamine, neither rPF4 nor hexadimethrine significantly prolonged the ACT even when supratherapeutic doses were administered. Although both of these drugs may be potential alternatives for reversing the heparin effect, they are not currently available for clinical use.
Recent data suggest that coagulation systems are better preserved and that perioperative blood loss is reduced when larger than usual doses of heparin are given and when protamine administration is based on the measured level of circulating heparin . Administration of reduced or optimal doses of protamine based on in vitro estimates can also result in reduced blood loss and transfusion . Avoiding excess protamine administration may represent an important mechanism of this better preservation of the coagulation status and reduction in bleeding.
1. DeLaria GA, Tyner JJ, Hayes CL, Armstrong BW. Heparin-protamine mismatch: a controllable factor in bleeding after open heart surgery. Arch Surg 1994;129:944-50.
2. Jobes DR, Aitken GL, Shaffer GW. Increased accuracy and precision of heparin and protamine dosing reduces blood loss and transfusion in patients undergoing primary cardiac operations. J Thorac Cardiovasc Surg 1995;110:36-45.
3. Despotis GJ, Joist JH, Hogue CW, et al. The impact of heparin concentration and activated clotting time monitoring on blood conservation: a prospective, randomized evaluation in patients undergoing cardiac operation. J Thorac Cardiovasc Surg 1995;110:46-54.
4. Carr ME, Carr SL. At high heparin concentrations, protamine concentrations which reverse heparin anticoagulant effects are insufficient to reverse heparin anti-platelet effects. Thromb Res 1994;75:617-30.
5. Guffin AV, Dunbar RW, Kaplan JA, Bland JW Jr. Successful use of a reduced dose of protamine after cardiopulmonary bypass. Anesth Analg 1976;55:110-3.
6. Moorehead MT, Westengard JC, Bull BS. Platelet involvement in the activated coagulation time of heparinized blood. Anesth Analg 1984;63:394-8.
7. Horrow JC. Protamine: a review of its toxicity. Anesth Analg 1985;64:348-61.
8. Okano K, Saito Y, Matsushima A, Inada Y. Protamine interacts with the D-domains of fibrinogen. Biochim Biophys Acta 1981;671:164-7.
9. Cobel-Geard RJ, Hassouna HI. Interaction of protamine sulfate with thrombin. Am J Hematol 1983;14:227-33.
10. Lindblad B, Wakefield TW, Whitehouse WM Jr, Stanley JC. The effect of protamine sulfate on platelet function. Scand J Thorac Cardiovasc Surg 1988;22:55-9.
11. Mills DC. ADP receptors on platelets. Thromb Haemost 1996;76:835-56.
12. Fisher C, Ammar T. The effects of heparinase 1 and protamine on platelet reactivity. Anesthesiology 1997;86:1382-6.
13. Ereth MH, Klindworth JT, Campbell SC, et al. Protamine attenuates agonist-induced platelet signaling/adhesion molecule expression [abstract]. Anesth Analg 1996;82(Suppl):5.
14. Levy JH, Cormack JG, Morales A. Heparin neutralization by recombinant platelet factor 4 and protamine. Anesth Analg 1995;81:35-7.
15. Kikura M, Lee MK, Levy JH. Heparin neutralization with methylene blue, hexadimethrine, or vancomycin after cardiopulmonary bypass. Anesth Analg 1996;83:223-7.