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J Thorac Cardiovasc Surg 1994;108:975-983
© 1994 Mosby, Inc.

CARDIOPULMONARY BYPASS,
MYOCARDIAL MANAGEMENT, AND SUPPORT TECHNIQUES

Recombinant platelet factor 4 reversal of heparin in human cardiopulmonary bypass blood

Ronald D. Williams, MD, Michael N. D'Ambra, MD, Theodore E. Maione, PhDa, Karen E. Lynch, BSN, Douglas F. Keene, MD


Boston and Cambridge, Mass.

This study was supported in part by the Repligen Corporation, One Kendall Square, Cambridge, MA 02139, and by the Rowland Foundation, Cambridge, MA.

Received for publication Nov. 23, 1993. Accepted for publication June 14, 1994. Address for reprints: Michael N. D'Ambra, MD, Cardiac Anesthesia Group, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114.

Abstract

The ability of recombinant platelet factor 4, a protein of human origin with high heparin affinity, and the present clinical heparin reversal agent, protamine, to neutralize heparin in human whole blood was studied by means of three standard whole blood coagulation tests: whole blood clotting time, heparin assay, and activated clotting time. Ten subjects were chosen at random among patients undergoing cardiopulmonary bypass operations. Heparinized blood, free of protamine, was obtained from the bypass reservoir for testing. Whole blood aliquots, without reversal agents (controls) or with either protamine (10, 20, 30, or 40µg/ml) or recombinant platelet factor 4 (10, 20, 40, or 80µg/ml), were analyzed. The quantity of each agent required to reverse the ten samples, using 95% upper confidence bounds (t distribution) was determined for each method. Recombinant platelet factor 4 reversed heparin at 40µg/ml and protamine at 20µg/ml, suggesting a reversal ratio for recombinant platelet factor 4/protamine of 2:1 on a milligram basis. Further, currently available methods for testing coagulation should be reliable, without modification, to monitor the restoration of normal coagulation parameters with recombinant platelet factor 4 after cardiopulmonary bypass. (J THORAC CARDIOVASC SURG 1994;108:975-83)

Heparin has been used in cardiac surgery since 1951 when the first extracorporeal bypass procedure was performed to repair an atrial septal defect.Go 1 Protamine is an effective reversal agent and has been in clinical use for many years. The polycationic protamine molecule binds and neutralizes the polyanionic heparin molecule via ionic forces.Go 2 However, protamine has many deleterious effectsGo Go 3-25 and as such must be administered carefully. Rapid infusion of protamine reliably produces hypotension and can also cause thrombocytopenia, leukopenia, pulmonary inflammation, and respiratory distress.Go Go 3,25 Protamine heparin complexes activate the complement cascade in vivo,Go Go 3-12 which may contribute to adverse events after heparin reversal. Also, inappropriate dosing can lead to incomplete heparin reversal or protamine-induced coagulopathy.Go 26

Recombinant platelet factor 4 (rPF4) is a recombinantly produced human protein that has been proposed as an alternative to protamine sulfate for use in heparin reversal. Preliminary animal and human studies have demonstrated that rPF4 safely and effectively reverses heparin anticoagulation.Go Go 27,28 The purpose of this study was to determine an effective heparin neutralization dose of rPF4 versus the standard agent, protamine, in human blood activated through exposure to the cardiopulmonary bypass (CPB) circuit. Three currently available methods for measuring clot formation in whole blood were used to compare the heparin-neutralization responses of rPF4 and protamine. The testing methods were the whole blood clotting time (WBCT), Hepcon heparin assay, and the Hepcon activated clotting time (ACT).

METHODS

Reagents
Porcine intestinal heparin 10 mg/ml (Elkins-Sinn Co., Cherry Hill, N.J.) and a single lot of protamine 10 mg/ml (Eli Lilly Co., Indianapolis, Ind.) were used for the study. rPF4 (Repligen Corporation, Cambridge, Mass.) was provided as a solution containing purified rPF4 in a concentration of 3.56 mg/ml.Go 29 The reversal agents, protamine and rPF4, were diluted with isotonic saline to a final concentration of 1 mg/ml.

Protocol
The protocol was approved by the Massachusetts General Hospital Institutional Review Board in compliance with federal regulations. Written informed consent was obtained from each patient.

Ten patients undergoing cardiovascular surgery involving CPB were randomly selected without regard to sex, coexisting medical problems, or type of operation. Patient data including weight, sex, and preoperative ACT was recorded. Approximately 100 ml of whole blood was extracted from the bypass reservoir at the end of CPB but before any protamine administration. Blood was collected in two 60 ml polypropylene syringes, tightly capped, and placed in a warm water bath (Techne TU-16A) maintained at 37° C. An ACT and heparin assay were immediately performed as described in the Hepcon Operators Manual.Go 30 The heparin assay was repeated with a cartridge of appropriate sensitivity when the initial result was at the limit of the cartridge resolution. The baseline assay was performed on blood extracted directly from the bypass circuit.

Blood in 2 ml aliquots was placed in chemically clean 10 ml glass test tubes after a known quantity of either protamine or rPF4 had been added to each test tube. The specimens were mixed thoroughly and tested immediately for baseline ACT and heparin levels. Protamine concentrations tested were 10, 20, 30, and 40 µg/ml blood and rPF4 concentrations tested were 10, 20, 40, and 80 µg/ml blood. ACTs were recorded in seconds and heparin assay values in milligrams per kilogram body weight.

The WBCT assays were performed promptly, by a method modified to permit simultaneous analysis of nine patient samples. Nine columns of sample tubes labeled 1 through 9 in a three-row matrix were filled with the appropriate quantities of each study drug and placed in the warm water bath at 37° C. All tubes in column 1 contained no drug and served as the control. Columns 2 through 5 contained protamine concentrations of 10, 20, 30, and 40 µg/ml blood and columns 6 through 9 had rPF4 concentrations of 10, 20, 40, and 80 µg/ml blood. All three tubes in each column had the same quantity of drug. Next, 2 ml aliquots of the 37° C whole blood were then added to each tube beginning with row 3. Care was taken not to agitate the mixture or expose the blood to any more glass than was necessary, thereby minimizing contact activation. When the last tube was filled a timer was started. The tubes were quickly capped and placed in the water bath. At 30-second intervals each tube in row 1 was tilted 180 degrees to maximize exposure of the blood to glass/activating surfaces and to mix the blood with the reversal agent. When clotting was noticed the tube was discarded and replaced by the corresponding tube in row 2. When all three tubes in a column had clotted, the time was recorded as the WBCT for that given quantity of reversal agent. All times were rounded off to the nearest half minute. The normal value is considered to be 8.5 to 15.5 minutes with values greater than 17 minutes being grossly abnormal.

Reversal criteria for each test was established before testing as follows: heparin neutralization was considered complete when the ACT returned to baseline + 15 seconds, the heparin assay concentration declined to 0 mg/kg, and the WBCT was less than 17 minutes.

Statistical analysis
Descriptive analysis of all the ACT data is presented as arithmetic mean ± standard error of the mean. The ACT results were analyzed in terms of 90% and 95% upper confidence boundaries based on the (one-sided) t distribution with 9 degrees of freedom. The t variables at the 90% and 95% upper confidence boundaries are 1.38 and 1.83, respectively.

RESULTS

The results of the heparin assay expressed in milligrams per kilogram, with a resolution of 0.5 mg/kg, are presented in GoTable I and demonstrate that protamine neutralized heparin in vitro in nine of ten patients at a concentration of 20 µg/ml as indicated by the Hepcon assay. rPF4 neutralized heparin in vitro in eight of ten patients at a concentration of 40 µg/ml as shown by the Hepcon heparin assay. Patient 8, with a baseline heparin assay value of 2.5 mg/kg, the highest in the study group, required the greatest amount of each agent for reversal. Patient 9, with a baseline heparin assay of 0.5 mg/kg, the lowest in the study group, required the least quantity of each agent. All samples were neutralized when tested at the maximum concentration of both heparin reversal agents.


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  		Table I. Heparin assay data in milligrams per kilogram (n = 10)
 
Protamine returned the ACT of whole blood to less than 130 seconds (normal = 80 to 130 seconds) in vitro in nine of ten samples, and ten of ten were reversed to within 15 seconds of baseline (Fig. 1, A). However, at higher doses, protamine often decreased the ACT to below the preoperative baseline sample. In fact, protamine decreased the ACT to less than 120 seconds in vitro in the blood of six of ten patients, and no preoperative values were less than 120 seconds. Under equivalent conditions, rPF4 decreased only four of ten ACTs to less than 130 seconds, the upper range of normal, and the fully reversed ACTs of all ten patients were reduced to within 15 seconds of the preoperative value (Fig. 1, B). The dependence of mean ACT (±standard error of the mean) on drug concentration as shown in Fig. 2, A confirms the average reversal dose of rPF4 to be 40 µg/ml. The reversal dose for protamine is 20 µg/ml, suggesting a 2:1 (weight/weight) reversal ratio for rPF4 and protamine (Fig. 2, B).




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  		Fig. 1. Individual ACT results for whole blood samples from ten patients, which were titrated in vitro with protamine (A) and rPF4 (B). Baseline values were Hemochron assay results recorded from the patients' anesthetic records. All other data were performed with the Hepcon ACT assay.

 



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  		Fig. 2. Mean ACT for blood samples from ten patients having CPB, which were titrated in vitro with rPF4 (A) and protamine (B). Error bars represent standard error of the mean. **Values within the 90% confidence limits of the baseline values. *Titrated sample values that were within the 95% confidence limits of the baseline values.

 
The reversal dose for each agent was determined to be the first concentration that exceeded the 90% upper confidence boundary. Both agents exceeded the criteria for reversal at the 95% upper confidence boundary within the study concentrations chosen.

The 40 µg/ml concentration of rPF4 produced ACTs that exceeded the 90% upper confidence boundary and were within 0.6 second of the 95% upper confidence boundary. In contrast, both of the next lower dilutions, rPF4 at 20 µg/ml and protamine at 10 µg/ml, were more than 60 seconds from reversal criteria.

The WBCT results for both protamine (Fig. 3, A) and rPF4 (Fig. 3, B) demonstrated adequacy of heparin reversal despite the lack of specificity of this assay. All specimens were monitored for 1 hour. In the specimens that had not clotted within 60 minutes, there was no evidence of clot formation. In contrast to the evidence of reversal by both the ACT and heparin assay, the WBCT results for patient 3 were abnormal (>17 minutes) for reversal with both agents; however, the patient did not require therapy for post-CPB coagulopathy. Overall, protamine failed to fully reverse the heparin anticoagulation when tested with WBCT in two patients at any concentration. Of the remainder, heparin was reversed with a protamine concentration of 20 µg/ml in all but the patient with the highest heparin level. rPF4 failed to fully reverse only one aliquot, and the majority were reversed with 40 µg/ml. At the maximum concentration (80 µg/ml), rPF4 successfully reversed nine of ten samples whereas protamine reversed eight of ten by WBCT.




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  		Fig. 3. Individual WBCT responses to protamine (A) and rPF4 (B) titration in vitro of ten blood samples from patients having CPB. Unclotted samples at 1 hour were assigned a value of 60 minutes forthese calculations. The horizontal line at 17 minutes indicates the upper limit of normal. Preoperative baseline values were not available for this assay.

 
DISCUSSION

Human PF4
Human PF4 is a protein synthesized by megakaryocytes and partitioned into {alpha}-granules during platelet maturation. Platelet-activating events cause the release of PF4, as well as other {alpha}-granule proteins, leading to locally elevated levels of this protein and transient increases in circulating levels.Go Go 31,32 Although the physiologic role of this protein remains to be definitively established, the heparin-binding capacity of PF4 has been clearly defined.Go 33 Other biologic activities of rPF4 administration including inhibition of endothelial cell growth and angiogenesis,Go 34 chemotactic attraction of several cell types,Go 35 inhibition of bone resorption, Go 36megakaryocyte maturation,Go Go 37,38 and immune modulationGo Go 39,40 have also been reported.

Although rPF4 has not demonstrated significant toxicity when used as a heparin neutralizing agent in ratsGo 27 or in preliminary human studies,Go 28 its absolute safety cannot yet be defined. PF4 is a member of a family of proteins sometimes referred to as chemokines, or intercrines, many of which possess the potential for neutrophil or monocyte activation.Go 41 Although earlier reports suggested that PF4 was chemotactic for neutrophils and monocytes in vitro,Go 35 recent studies have indicated that this activity is minimal in vitro, and neutropenia has not been reported as a sequale of PF4 use in vivo.Go Go Go 27,28,42 Interestingly, rPF4 produced a transient inflammatory response when injected into mouse foot pads.Go 43 However, this effect could be blocked by systemic indomethacin and had no long-term effects. The angiostatic activityGo 34 of PF4 could also be of concern after invasive operations; however, this effect has been shown to be abrogated by heparin,Go 34 and no adverse effects on wound healing have been reported to date. Protamine is also a well-characterized angiogenesis inhibitor that does not show notable effect on angiogenesis in the presence of heparinGo 44 but is associated with vascular toxicity when administered as a sole agent.Go 45

Unlike protamine, PF4 exists as a tetramerGo 46 possessing distinct heparin-binding domains. Although the precise structural features of heparin-PF4 interaction have not been defined, the orientation of complementary lysine-rich helices on opposite sides of the tetramerGo 47 present a surface well suited for multiple protein-sulfated carbohydrate interactions without the requirement for significant conformational changes. The neutralization of heparin by protamine has been interpreted to be the result of direct charge neutralization necessitating minimal structural specificity and leading to large molecular weight aggregatesGo 48 not observed with the smaller molecular weight heparin-rPF4 interaction. Periodic adverse effects of protamine have been widely reportedGo Go 2-25 and may be the result of complement activation or other events initiated by these heterogeneous protamine/heparin complexes.

Two other agents are known to reverse heparin anticoagulation: the polymer polybrene (a quaternary ammonium salt) and toluidine blue (an aniline dye compound). Polybrene causes fatal renal failure when administered in doses in excess of 5 mg/kg and its availability for heparin reversal was discontinued in 1962.Go Go 49-51 Toluidine blue causes methemoglobinemia and hypoxemia and has never been considered as a routine heparin reversal agent.Go 51

The ability of PF4 to neutralize the anticoagulant properties of heparin have been clearly established in vitro.Go Go 52-54 Early studies, however, illustrated the efficacy of PF4 for restoration of the activity of specific coagulation enzymes (factor Xa, thrombin) inhibited by heparin/antithrombin III or heparinized plasma but not whole blood, plasma from human beings, or blood activated by the CPB circuit. The present study definitively demonstrates the ability of rPF4 to restore normal coagulation properties in heparinized human blood extracted from the CPB circuit.

The use of rPF4 as a postoperative heparin-neutralizing agent has been proposed based on a lack of protamine-like adverse side effects in animal studies,Go 27 and the results of the present study provide support that rPF4 will be effective for the restoration of normal hemostasis.

The parallel dose-response relationship of protamine and rPF4 in these assays is consistent with approximate molar equivalence in their effects on heparin-induced anticoagulation. Based on the ACT results presented here, approximately 20 µg of protamine (molecular weight 4400 daltons) and 40 µg of rPF4 (MW 7800 daltons) were needed to effectively neutralize residual heparin in 1 ml of CPB blood. These values convert to 4.55 nmol protamine and 5.13 nmol rPF4 per milliliter of blood as equally effective doses. Similar equimolar efficacy was observed when comparing protamine and rPF4 in other in vitro assays of heparin-modulated coagulation. These results are also consistent with direct binding studies of rPF4 and protamine with tritiated heparin, which demonstrate a distribution coefficient of approximately 56 to 81 nmol/L for both proteins.

The ultimate use of rPF4 for clinical heparin neutralization is expected to depend on the characteristics of the specific clinical setting where reversal of anticoagulation would be desirable. In some applications, such as heparin neutralization after kidney dialysis or plasmapheresis, the need for protamine, and therefore less toxic rPF4, may not be great. If less toxic than protamine, however, its predictable neutralizing capacity may prove to have significant value in cardiovascular surgery applications where post-CPB coagulopathies of unknown origin remain common. If rPF4 can be infused more rapidly than currently recommended for protamine after operations, without adverse consequences, as suggested by animal studies,Go 27 it is possible that the use of this agent may ultimately provide an improved pharmacoeconomic profile for specific classes of invasive cardiovascular procedures.

One apparent difference between rPF4 and protamine was the tendency of protamine to restore coagulation times to levels below normal baseline values. This effect seemed to be less pronounced with rPF4. Whether this represents a protamine-induced procoagulant effect or an assay artifact has not been determined, but it does lead to ambiguity in interpreting the efficacy of heparin neutralization by protamine, as well as difficulty in prospectively selecting the appropriate neutralizing dose based on these assay results. Perhaps more important, in two patients (Nos. 3 and 6), protamine failed to normalize coagulation based on the WBCT assay, which may be more physiologically relevant than the ACT data. In one additional patient (No. 5), excess protamine elongated the WBCT beyond the normal range. In only the most problematic patient (No. 3), a patient with end-stage renal disease, rPF4 also failed to fully normalize the coagulation time but was able to restore the WBCT to a greater extent than protamine. Interestingly, the highest dose of rPF4 also produced a slight elongation of the WBCT of blood from patient 5, although the time did remain within the normal range at this level of rPF4. Clearly, rPF4 administration was not associated with any notable in vitro coagulopathies.

The conventions for choosing the appropriate dose of protamine to neutralize heparin at the conclusion of CPB varies significantly between institutions. We administer heparin at 3.0 mg/kg and neutralize with a dose of 1 mg protamine per 1 mg residual heparin. Residual heparin is estimated on the basis of duration of CPB and hemodilution for any particular patient. The technology currently exists to quickly monitor heparin levels more precisely and to accurately define the dose of protamine needed in each specific case. The approach, however, is not yet used very widely and a standard formula for prescribing protamine is still generally used. The present study shows that the effects of rPF4 on coagulation parameters parallel those of protamine in a quantitative relationship. A simple doubling of the number of milligrams of protamine traditionally administered provides a simple formula to calculate the correlating dose of rPF4. The results of this study suggest that if previous animal studiesGo 27 and preliminary human studiesGo 28 predict future human experience, rPF4 may represent a clinically important alternative to protamine for postoperative heparin neutralization.

Acknowledgments

We thank Repligen Corporation for contributing materials for this study. In addition, we thank Alan Zaslavsky and Ralph D'Agostino for their help with the statistical analysis.

Footnotes

From the Department of Anesthesia, Massachusetts General Hospital, Harvard Medical School, Boston, Mass., and the Repligen Corporation, a Cambridge, Mass. Back

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ANN THORAC SURG ASIAN CARDIOVASC THORAC ANN EUR J CARDIOTHORAC SURG
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