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Heparin Neutralization by Recombinant Platelet Factor 4 and Protamine

Levy, Jerrold H. MD; Cormack, James G. MD; Morales, Antonio MD

Cardiovascular Anesthesia
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Protamine is the only available drug to reverse heparin-induced anticoagulation. Platelet factor 4 (PF4) is a basic polypeptide stored in platelets that reverses heparin. To investigate its potential as a reversal drug, we studied recombinant PF4 on anticoagulated blood obtained during cardiac surgery. Blood was obtained from 33 different venous reservoirs, and activated clotting time (ACT), heparin concentrations, and heparinase-ACT were determined. Anticoagulation was reversed by adding incremental PF4:heparin and protamine:heparin ratios to the heparinized blood, and the ACTs were determined (n = 21). Viscoelastic analysis of anticoagulation reversal was performed by adding protamine or PF4 at reversal ratios of 1.3:1 protamine:heparin, and 3.2:1 PF4:heparin using thromboelastography (n = 12). PF4 reversal ratios of 3:1 and 3.5:1 and protamine reversal ratios of 1:1, 1.5:1, 2:1 were not statistically different from heparinase-ACT values. There were no significant differences in viscoelastic measurements of clot formation between protamine and PF4. Recombinant PF4 at a 3.0:1 ratio reverses heparin-induced anticoagulation after cardiopulmonary bypass, and represents a potential alternative, especially for the protamine allergic patient.

(Anesth Analg 1995;81:35-7)

Department of Anesthesiology, Emory University School of Medicine, Division of Cardiothoracic Anesthesia and Critical Care, The Emory Clinic, Atlanta, Georgia (Levy, Morales), and Department of Anaesthesia, University of Alberta, Edmonton, Alberta, Canada (Cormack).

Accepted for publication February 17, 1995.

Address correspondence and reprint requests to Jerrold H. Levy, MD, Department of Anesthesiology, Emory University Hospital, 1364 Clifton Rd. NE, Atlanta, GA 30322.

Protamine, a basic polypeptide derived from salmon sperm, reverses heparin, an acidic glycosaminoglycan derived from bovine or porcine tissues by nonspecific acid-base interactions to form heparin-protamine complexes [1]. A spectrum of adverse reactions has been described to protamine administered to reverse heparin's anticoagulant activity [2-4]. The life-threatening reactions to protamine appear to represent anaphylaxis due to prior sensitization [4]. Unfortunately, there are no available alternatives to protamine if the patient develops a reaction or is known to be allergic.

Platelet factor 4 (PF4) is a heparin binding protein stored in the alpha granules of platelets that is released during platelet aggregation. In an animal model, PF4 reverses heparin anticoagulation without the adverse effects of protamine on complement activation or on the cardiovascular system [5]. Because PF4 is a naturally occurring polypeptide in humans, it would also have the advantage of being less antigenic. Therefore, we compared the dose-response relationship between recombinant human PF4 and heparin to the dose-response relationship between protamine and heparin to determine the optimal dose of PF4 for complete heparin reversal. Because little is known about the effects of heparin reversal with PF4 on platelet-fibrinogen interaction that forms the final hemostatic plug, we also studied protamine and human recombinant PF4 reversal of heparin using thromboelastography.

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Methods

After institutional approval, 10 mL of residual blood remaining in the extracorporeal reservoir was obtained from 33 different reservoirs at the end of cardiopulmonary bypass. Heparin concentration was measured by heparin-protamine titration using the Hepcon HMS system. Activated clotting time (ACT) was measured by Medtronic-Hemotec high range heparinase test cartridges resulting in a baseline ACT with and without heparin. Heparin levels were determined by heparin-protamine titration (Medtronic-Hemotec HMS Registered Trademark; Parker, CO). Microliter quantities of recombinant PF4 (Repligen, 5 mg/mL) or protamine sulfate (5 mg/mL) were added to Medtronic ACT cartridges that contain kaolin as the activator in PF4: heparin ratios of 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, and 3.5:1 (n = 21). Protamine:heparin reversal ratios were 0.5:1, 1:1, 1.5:1, and 2:1. Blood, 0.37 mL, was then added to the ACT cartridges and the times for clotting to occur were measured. Data are reported as mean +/- SD and were compared by analysis of variance and a Scheffe's test for multiple comparisons; P < 0.05 was considered significant.

Viscoelastic analysis of blood reversal was performed using a nonactivated thromboelastograph by mixing microliter quantities of protamine or PF4 to plastic cartridges using the reversal ratios of 1.3:1 protamine:heparin and 3.2:1 PF4:heparin (n = 12). The blood was then immediately pipetted into a Haemoscope TEG coagulation analyzer Registered Trademark (Skokie, IL). Reaction time, clot formation rate, coagulation time, maximum amplitude, and amplitude 60 min after maximum amplitude are reported as mean +/- SD and were compared by paired Student's t-testing with P < 0.05 considered significant.

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Results

The data from PF4 assays and protamine assays are shown in Figure 1 and Figure 2, respectively. Heparin levels were 3.4 +/- 0.3 U/mL (range, 2.7-4.1 U/mL). PF4 reversal ratios of 3.0:1 and 3.5:1 and protamine reversal ratios of 1:1, 1.5:1, and 2.0:1 were not statistically different from heparinase-ACT determinations. The optimal reversal ratio of PF4 appears to be 3.0:1 to 3.5:1. The thromboelastograph data from 12 patients are shown in Table 1. There were no significant differences in viscoelastic measurements of clot formation between protamine and PF4.

Figure 1

Figure 1

Figure 2

Figure 2

Table 1

Table 1

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Discussion

We determined that a recombinant PF4 reversal ratio of 3.0:1 was the minimum dose required for heparin reversal. This finding is different than the ratio of 2.0:1 previously reported [6]. We also used kaolin ACT cartridges with smaller volumes (0.37 mL) compared to celite-activated ACT tubes that use a volume of 2 mL. The different activators and volume may have accounted for the differences. However, when thromboelastography was attempted with ratios less than 3.0:1, coagulation did not occur. The use of kaolin or celite to enhance contact activation in the ACT systems may also enhance the release of PF4 from platelets in the blood sample assayed. We also found no interference by PF4 on platelet-fibrinogen interaction or differences in clot lysis compared to protamine when reversing heparin as determined by thromboelastography.

In the rat model, heparin anticoagulation was effectively reversed by protamine and PF4 but the platelet count, white blood cell count, complement titer, and mean arterial blood pressure all decreased significantly in those rats receiving protamine but not in those receiving PF4 [5]. The lung interstitium in animals receiving PF4 appeared normal whereas animals receiving protamine demonstrated interstitial edema and hemorrhage [5]. PF4 has been shown to form heparin-PF4 complexes that are different from those formed by heparin and protamine [7]. In contrast to heparin-protamine complexes, heparin-PF4 complexes did not activate antithrombin III in vitro [7]. Incubation of PF4-neutralized heparinized plasma at 37 degrees C for 5 h did not result in liberation of heparin and prolongation of thrombin clotting time, as found with protamine neutralized plasma [7]. This lack of heparin liberation with PF4 reversal suggests less heparin rebound in the PF4-reversed patient [7]. Excess protamine is required to maintain stable protamine-heparin complexes. If excess heparin or antithrombin III is introduced then antithrombin-III reactivation may occur resulting in heparin rebound [8]. The large protamine-heparin complexes could be the cause of the acute pulmonary hypertensive reactions that can occasionally occur with protamine [8]. Recombinant PF4 has been administered safely to patients to reverse heparin after cardiac catheterization without changes in blood pressure, right atrial pressure, pulmonary artery pressure, pulmonary artery occlusion pressure, or cardiac output [9].

We determined that recombinant PF4 at a ratio of 3.0:1 offers a potential alternative to protamine for heparin reversal after cardiopulmonary bypass, especially for the protamine-allergic patient. PF4-reversed patients may be less likely to develop heparin rebound and have fewer side effects than protamine-reversed patients. Further clinical studies are required to determine which patients would be best suited to heparin reversal with recombinant PF4.

The authors would like to acknowledge the support of Theodore E. Maione, PhD, in this project and for kindly providing us with recombinant PF4 for evaluation.

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REFERENCES

1. Levy JH. Anaphylactic reactions in anesthesia and intensive care, 2nd ed. Boston: Butterworth-Heinemann, 1992.
2. Levy JH, Zaidan JR, Faraj BA. Prospective evaluation of risk of protamine reactions in NPH insulin-dependent diabetics. Anesth Analg 1986;65:739-42.
3. Levy JH, Schwieger IM, Zaidan JR, et al. Evaluation of patients at risk for protamine reactions. J Thorac Cardiovasc Surg 1989;98:200-4.
4. Weiss ME, Nyhan D, Peng Z, et al. Association of protamine IgE and IgE antibodies with life-threatening reactions to intravenous protamine. N Engl J Med 1989;320:886-92.
5. Cook JJ, Niewiarowski S, Yan Z, Schaffer L. Platelet Factor 4 reverses heparin anticoagulation in the rat without adverse effects of heparin-protamine complexes. Circulation 1992;85:1102-9.
6. Williams RD, Maione TE, Lynch KE, et al. Recombinant platelet factor 4 reversal of heparin in human cardiopulmonary bypass blood. J Thorac Cardiovasc Surg 1994;108:975-83.
7. Shanberge JN, Quattrociocchi-Longe TM, Martens MH. Interrelationships of protamine and platelet factor 4 in the neutralization of heparin. Thromb Res 1987;46:89-100.
8. Shanberge JN, Murato M, Quattrociocchi-Longe TM, et al. Heparin-protamine complexes in the production of heparin rebound and other complications of extracorporeal bypass procedures. Am J Clin Path 1987;87:210-7.
9. Dehmer GJ, Tate DA. Reversal of heparin anticoagulation by recombinant platelet factor 4. Circulation 1993;88;I-418.
© 1995 International Anesthesia Research Society