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Anesth Analg 2000;90:808-812
© 2000 International Anesthesia Research Society

CARDIOVASCULAR ANESTHESIA

The Effects of Heparin, Protamine, and Heparinase 1 on Platelets in vitro Using Whole Blood Flow Cytometry

Sibylle A. Kozek-Langenecker, MD*, S. Fazal Mohammad, PhD{dagger},{ddagger}, Takahisa Masaki, PhD§, Craig Kamerath||, and Alfred K. Cheung, , MD§,¶

*Department of General Anesthesiology and Intensive Care B, University of Vienna, School of Medicine, Vienna, Austria; {dagger}Department of Pathology, {ddagger}Artificial Heart Laboratory, and §Division of Nephrology & Hypertension, Department of Medicine, University of Utah School of Medicine; and ||Research and ¶Medical Services, Veterans Affairs Medical Center, Salt Lake City, Utah

Address correspondence and reprint requests to Sibylle A. Kozek-Langenecker, MD, Department of Anesthesiology and General Intensive Care, University of Vienna, Währinger Gürtel 18-20, 1090-Vienna, Austria. Address e-mail to sibylle.kozek-langenecker{at}univie.ac.at


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The effects of heparinization and the reversal of heparin activity on platelet function after cardiopulmonary bypass have not been well defined. Flow cytometry has become a convenient and powerful technique for characterizing platelets. We examined the expression of a secretion marker (P-selectin) and an aggregation marker (activated fibrinogen receptor GP IIb-IIIa) on normal platelets in response to heparin, heparinase 1, and protamine in vitro using whole blood flow cytometry. Unfractionated heparin increased adenosine diphosphate-induced expression of P-selectin and GP IIb-IIIa in a dose-dependent manner. Heparinase 1 alone decreased both markers of platelet activation. Protamine alone increased P-selectin expression but had no effect on GP IIb-IIIa expression. Heparinase 1 antagonized the stimulatory effect of heparin on both markers. In contrast, protamine antagonized the effect of heparin on GP IIb-IIIa expression but potentiated the effect of heparin on P-selectin expression. These in vitro observations suggest that 1) both heparin and its reversal agents affect platelet secretion and aggregation, and 2) heparinase 1 reverses heparin-induced platelet preactivation more effectively than protamine.

Implications: This experimental in vitro study demonstrates that heparin and its reversal agents affect platelet secretion and aggregation.


   Introduction
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 Abstract
 Introduction
Methods
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 Discussion
 References
 
Platelet dysfunctions play a major role in the pathophysiology of thrombosis and hemostasis after cardiopulmonary bypass (CPB) (1). The method of flow cytometry is increasingly used for characterization of cellular abnormalities of platelets during and after CPB (2–12), because this technique permits assessment of platelet activation in a physiological manner (13–16). Flow cytometry also allows monitoring of platelet-directed therapeutic interventions designed to reduce postoperative blood loss in cardiac surgery patients (7–12). However, the effects of platelet-directed interventions on flow cytometric variables may be modified by the concomitant use of drugs during clinical CPB. Concomitant heparin anticoagulation required during CPB has been found to alter P-selectin expression (3–5), a marker for platelet secretion in flow cytometric analyses (17). Ammar and Fisher (18) reported influences of protamine, the drug most widely used for neutralization of heparin anticoagulation, on P-selectin expression. A direct comparison of the effects of heparin and protamine on platelet secretion by using the same controllable test procedure is currently not available. Further, the effects of these drugs on platelet aggregation remain incompletely interrogated. Heparinase 1 (hereinafter referred to as heparinase) is in preclinical trial as an alternative drug for heparin reversal after CPB (19). The effects of heparinase on platelet reactivity have also not been well defined. We attempted to characterize the effects of unfractionated heparin, protamine, and heparinase per se, and the effects of drug combinations on flow cytometric variables of both platelet secretion and aggregation using whole blood assays. Because institutional review board approval could not be secured for an ex vivo study design with iv infusion of protamine or heparinase without previous heparin administration, we assessed platelet reactivity in the absence and presence of these drugs by analyzing platelet response to stimulation with adenosine diphosphate (ADP) in a controllable but artificial in vitro study design.


   Methods
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Results
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Blood samples were obtained from 14 healthy adult male volunteers. All participants denied taking any medication known to alter platelet function within the previous 14 days. The study was approved by the Institutional Review Board of the University of Utah.

Unfractionated porcine heparin, ADP, and paraformaldehyde were obtained from Sigma Chemical Co., St. Louis, MO. Heparinase (specific activity of 526 Sigma units/mg) was purchased from Sigma Chemical Co. Protamine sulfate was obtained from Elkins-Sinn, Cherry Hill, NJ. A fluorescein isothiocyanate (FITC)-conjugated activation-dependent antihuman platelet GP IIb-IIIa monoclonal antibody, PAC-1, was obtained from Becton Dickinson Immunocytometry Systems, San Jose, CA. A FITC-conjugated activation-independent antihuman platelet GP IIIa monoclonal antibody (anti-CD61) was obtained from Biodesign International, Kennebunk, ME. A phycoerythrin (PE)-conjugated monoclonal antibody against human platelet P-selectin (anti-CD62P) was obtained from Pharmingen, San Diego, CA.

Blood was drawn by venipuncture without stasis from the antecubital vein into Vacutainer® tubes (Becton Dickinson, Rutherford, NJ) containing 3.2% trisodium citrate or into plastic tubes containing unfractionated porcine heparin at a final concentration of 2.5 IU/mL (approximately 25 µg/mL). In the experiments comparing the effects of protamine, heparin, and heparinase per se on platelets, anticoagulation was maintained with citrate because other anticoagulants have been shown to alter the physiological behavior and the responses of platelets more than citrate (20). In experiments comparing the effect of heparin reversal agents on heparin-induced platelet alterations, heparin was used as the only anticoagulant because this approach resembles more the in vivo condition of heparin reversal after CPB. Citrated and heparinized whole blood samples were diluted (1:5) in phosphate-buffered saline (PBS, 100 mM sodium phosphate, pH 7.3, and 0.145 M NaCl).

To evaluate the effect of drugs per se on platelets, citrated blood was incubated with heparin (0.5, 2.5, and 5.0 IU/mL), protamine (25 and 32.5 µg/mL), heparinase (0.02, 2 IU/mL), and a complex of heparin with protamine at a ratio of 1:1 by weight prepared at room temperature before incubation with citrated blood. Samples incubated with PBS only served as a control. Incubation was performed at 37°C for 5 min without agitation instead of 2 min at 22°C, used in a previous study (18), because heparinase degradation of heparin has been suggested to be optimal in vitro under these test conditions (19). The dosages of drugs investigated in the present in vitro study represent those used in clinical practice or have been recommended previously. Heparin concentrations of 2.5 IU/mL are commonly used for CPB, although some centers use higher or lower concentrations (21). For reversal of heparin, a ratio of heparin 100 IU:protamine 1–1.3 mg has been recommended (22). Heparinase (0.02 IU/mL ~ 12.5 Sigma units/mL) has been reported to reverse activated clotting times of heparinized samples to baseline values in vitro (18).

To evaluate the effects of heparin reversal agents on heparin-induced platelet alterations, heparinized blood was incubated with protamine (25 and 32.5 µg/mL) or heparinase (0.02 IU/mL) at 37°C for 5 min without agitation. Samples incubated with PBS only served as a control.

To evaluate the extent of platelet reactivity, all samples were activated with ADP (10 µM) before flow cytometric analysis. Expression of activated GP IIb-IIIa complex was analyzed after stimulation with ADP for 10 min at 37°C without agitation. P-selectin analysis was performed after ADP-stimulation for 5 min. Pilot studies confirmed that this duration of stimulation induced maximum response to ADP. Each test sample was divided into two aliquots for fluorescent staining. To determine the expression of activated GP IIb-IIIa complex, one aliquot of each sample was incubated with PAC-1. To determine the percentage of platelets expressing P-selectin, a second aliquot was incubated with anti-CD61, which binds to all platelets, and anti-CD62P, which binds to P-selectin. After 30 min incubation with saturating concentrations of monoclonal antibodies at room temperature in the dark, samples were fixed in 1% paraformaldehyde (pH 7.3) at 4°C. In each experiment, one sample was stained with FITC-conjugated, isotype-matched, nonspecific mouse immunoglobulins as an isotype control. Another sample without addition of conjugated antibody served as an autofluorescence control. Fluorescence was measured with FACScan flow cytometer and analyzed with CellQuest 3.1 software (Becton Dickinson Immunocytometry Systems, San Jose, CA). Quantum fluorescence microbeads (Calibrite Beads; Becton Dickinson Immunocytometry Systems) were used each day for standardization of instrument settings.

A power analysis revealed that a sample size of 10 would provide a power of >80% in detecting a difference in PAC-1 binding of at least 1 SD in heparinized samples incubated with and without protamine. Data were expressed as mean ± SD. Statistical analysis was performed by using analysis of variance for repeated measures. Multiple comparisons within citrated and heparinized blood samples between control and drug exposures were made with Student’s t-test for paired data. The level of significance was adjusted according to the Bonferroni correction. A two-tailed P < 0.05 was considered statistically significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Evaluation of platelet surface GP IIb-IIIa complex is shown in Figure 1. The monoclonal antibody PAC-1 was used to identify activated GP IIb-IIIa receptors on ADP-activated platelets in citrated whole blood. We observed that exposure of platelets to unfractionated porcine heparin led to an increase in GP IIb-IIIa expression on platelets in a dose-dependent manner (by 198% ± 35% at 5.0 IU/mL). Protamine by itself had no significant effect on the expression of GP IIb-IIIa on ADP-activated platelets. Complex binding of heparin with protamine (H/P complex) in a ratio of 1:1 by weight before incubation with whole blood abolished the heparin-induced increase in GP IIb-IIIa expression. Exposure of platelets to heparinase resulted in a decreased expression of GP IIb-IIIa complex on ADP-activated platelets in a dose-dependent manner (by 39% ± 25% at 2 IU/mL).



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  		Figure 1. Evaluation of GP IIb-IIIa complex on platelets in citrated whole blood. After platelets were incubated with phosphate-buffered saline (PBS) or drugs, they were activated with adenosine diphosphate and flow cytometric analysis was performed by using fluorescein isothiocyanate-conjugated activation-dependent monoclonal antibody to platelet surface GP IIb-IIIa (PAC-1). The control sample incubated with PBS was arbitrarily defined as 100%. Expression of activated GP IIb-IIIa was increased on platelets after exposure to heparin (H), not significantly altered after exposure to protamine (P) and H/P complexes, and decreased after exposure to heparinase compared with PBS controls. * P < 0.05 versus PBS (n = 14).

 
Drug-induced effects on the expression of P-selectin were also evaluated (Fig. 2). Platelets were identified as positive for GP IIIa antigen (CD61), and the presence of the PE-labeled antibody against P-selectin (anti-CD62P) was used to determine the percentage of platelets expressing P-selectin. Heparin increased the percentage of platelets expressing P-selectin in a dose-dependent manner (by 173% ± 51% at 5.0 IU/mL). Exposure of platelets to protamine also resulted in an increased expression (by 57% ± 45% at 32.5 µg/mL) while H/P complexes had no effect on the expression of P-selectin. In contrast, exposure to heparinase resulted in a decreased expression of P-selectin on ADP-activated platelets (by 16% ± 7% at 2 IU/mL).



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  		Figure 2. Evaluation of P-selectin expression on platelets in citrated whole blood. Platelets were incubated with phosphate-buffered saline (PBS) or drugs and activated with adenosine diphosphate. Platelets were identified as positive for GP IIIa antigen (CD61), and the presence of the phycoerythrin-labeled antibody against P-selectin (anti-CD62P) was used to determine the percentage of platelets expressing P-selectin. The control sample incubated with PBS was arbitrarily defined as 100%. P-selectin expression was increased on platelets after exposure to heparin (H) and protamine (P), not significantly altered after exposure to H/P complexes. In contrast, P-selectin expression was decreased after exposure to heparinase compared with PBS controls. * P < 0.05 versus PBS (n = 14).

 
Evaluation of the effects of heparin reversal agents on heparin-induced alterations in platelet reactivity is illustrated in Figure 3. There was a decrease in the expression of platelet surface GP IIb-IIIa complex in heparinized samples treated with either protamine (19% ± 9% at 25 µg/mL; P < 0.05) or heparinase (18% ± 8%; P < 0.05) compared with heparinized control samples. Heparinized samples antagonized with heparinase showed a 14% ± 9% decrease in the expression of P-selectin compared with those not treated with heparinase (P < 0.05). In contrast, protamine increased the expression of P-selectin on heparin-activated platelets. At 32.5 µg/mL, protamine increased P-selectin expression by 13% ± 9% (P < 0.05), although a smaller dose of protamine (25 µg/mL) had no effect.



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  		Figure 3. Evaluation of the effects of heparin antidotes on ADP-induced expression of GP IIb-IIIa and P-selectin on platelets in heparinized whole blood. After platelets were incubated with phosphate-buffered saline (PBS), protamine or heparinase, they were activated with ADP. Control samples incubated with PBS were arbitrarily defined as 100%. Protamine decreased GP IIb-IIIa expression significantly but increased P-selectin expression in a dose-dependent manner. Heparinase decreased both GP IIb-IIIa and P-selectin expression significantly. * P < 0.05, versus heparin + PBS (n = 14).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The current study shows that both heparin and its reversal agents, protamine and heparinase, affect flow cytometric variables of platelet secretion as well as platelet aggregation. Previous studies identified heparin as a potent activator of platelet secretion as determined by increased binding of monoclonal antibodies to P-selectin (3,4,6). Our study extends these reports. Results presented in Figures 1 and 2 demonstrate that heparin increases not only agonist-induced P-selectin expression, but also the expression of activated GP IIb-IIIa receptors that are competent for binding fibrinogen, as determined by increased binding of an activation-dependent antihuman platelet GP IIb-IIIa monoclonal antibody (PAC-1).

In the present study, protamine facilitated agonist-induced platelet secretion (Fig. 2). This finding is in agreement with previous studies demonstrating increased plasma levels of platelet release products such as platelet factor 4 and ADP on isolated platelets exposed to protamine (23). The mechanism by which protamine increases platelet secretion is unknown, but possibly involves the interaction of the polycationic protamine molecules with the negative surface charge of the platelet membrane. Supporting the hypothesis of a charge-related phenomenon, we found that platelet response to ADP was unaltered on platelets exposed to H/P complexes produced in vitro by incubation of protamine and heparin in a ratio of 1:1 (Figs. 1 and 2).

We showed for the first time that platelets exposed to heparinase in the absence of heparin are less responsive to the pharmacological stimulus ADP (Figs. 1 and 2). The mechanism by which heparinase may impair platelet aggregation and secretion is unknown. The evidence that heparinase may cause platelet inhibition, however, suggests that monitoring platelet function may be useful during the clinical use of heparinase.

For reversal of heparin anticoagulation after completion of CPB, protamine is the only drug available in clinical practice (21). Heparinase is in preclinical trial as an alternative to protamine (19). Interestingly, protamine was not completely effective for reversal of heparin-induced platelet preactivation. Although the expression of GP IIb-IIIa was decreased, P-selectin expression was significantly increased in samples neutralized with protamine at a ratio of 1:1.3 compared with heparinized samples (Fig. 3). Previous investigation has indicated that protamine induces abnormalities of aggregation (24). In contrast to protamine, heparinase antagonized heparin-potentiated expression of GP IIb-IIIa as well as P-selectin. Previous investigations provided indirect evidence that heparinase digestion protects platelets against heparin (25,26). These results are in contrast to the study by Ammar and Fisher (18) who found that expression of P-selectin was decreased by protamine and unchanged by heparinase after heparin reversal. We have no explanation for this discrepancy, but it may reflect the variability in anticoagulants used for blood sampling, in temperature and duration of enzyme incubations, and in the platelet agonists used for assessment of platelet reactivity in vitro.

Our results are limited by the in vitro methodology used for flow cytometric analysis. Nevertheless, our study suggests that concomitant drug-induced effects should be taken into consideration for interpretation of flow cytometric analyses of platelet function during extracorporeal circulation. Additional studies are needed to identify as yet uncharacterized factors such as body temperature, vasopressors, or anesthetics that potentially interfere with flow cytometric assessment of platelet reactivity in cardiac surgery.


   Acknowledgments
 
This work was supported in part by US Department of Veterans Affairs; Dialysis Research Foundation, Ogden, UT; and a grant from the Max Kade Foundation to SK.


   References
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 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication December 28, 1999.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press