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Anesth Analg 1999;89:573
© 1999 International Anesthesia Research Society

CARDIOVASCULAR ANESTHESIA

The Effects of Heparin Coating of Oxygenator Fibers on Platelet Adhesion and Protein Adsorption

Yoshinari Niimi, MD*, Fumito Ichinose, MD*, Yoshiki Ishiguro, MD*, Katsuo Terui, MD*, Shoichi Uezono, MD*, Shigeho Morita, MD*, and Shingo Yamane, PhD{dagger}

*Department of Anesthesiology, Teikyo University School of Medicine, Ichihara Hospital; and {dagger}Tokatsu Clinic Hospital, Chiba, Japan

Address correspondence and reprint requests to Yoshinari Niimi, MD, PhD, Department of Anesthesiology, Teikyo University Ichihara Hospital, 3426-3, Anesaki, Ichihara, Chiba 299-0111, Japan.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Platelet adhesion on the cardiopulmonary bypass oxygenator membrane is associated with impaired hemostasis. We investigated the effects of heparin coating of the oxygenator membrane on protein adsorption and platelet adhesion on the surface. Noncoated and heparin-coated polypropylene membranes were incubated in whole blood with small- (1 U/mL) or large-dose (5 U/mL) heparin as an anticoagulant for 3 h at 37°C. The amount of platelets adhering on each fiber was assessed by using enzyme immunoassays using monoclonal antibodies directed against CD42b (GP Ib) and CD61 (GP IIb/IIIa). Platelet activation was assessed by measuring plasma guanosine monophosphate 140 levels. The amount and composition of the adsorbed proteins on the surface were analyzed by using a bicinchoninic acid protein assay and by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting technique. The heparin coating of the fibers significantly reduced platelet adhesion on the surface. However, platelet activation was reduced by heparin coating only with small-dose heparinization. The adsorption of platelet adhesive proteins such as fibrinogen and von Willebrand factor was not altered, whereas that of fibronectin was increased by heparin coating. We conclude that heparin coating of the oxygenator fibers can decrease platelet adhesion without affecting adsorption of major adhesive proteins. Surface heparin coating is associated with an increased fibronectin adsorption on the fibers.

Implications: Heparin coating can reduce platelet adhesion and activation in the presence of small-dose heparinization, potentially reducing the inflammatory response and activation of thrombosis and fibrinolysis.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Cardiopulmonary bypass (CPB) for cardiac surgery generates a systemic inflammatory response (1–3) and impairment of hemostasis (4,5). Progressive loss of platelet function during CPB is a major cause of postbypass bleeding. Heparin coating of the extracorporeal circuit attenuates an inflammatory response by reducing activation of complement and granulocytes (6–8). However, the effects of heparin coating on platelets are unknown. Some investigators reported the reduction of platelet activation or platelet loss (6,9), but others did not (10,11). The conflicting results are mainly due to the influence of biomaterial-independent factors on platelets during CPB, including using chamber venting and cardiotomy suction, return of shed mediastinal blood, change in blood temperature, and exposure to high shear stress. Thus, clinical studies cannot easily isolate the effects of heparin coating on platelet adhesion and activation.

Within seconds of blood contact with a synthetic surface, plasma proteins are adsorbed on the surface. These adsorbed proteins control the subsequent platelet adhesion through interactions with two different platelet adhesive receptors, glycoprotein (GP) IIb/ IIIa and GP Ib. Surface-bound fibrinogen and von Willebrand factor (vWF) are proteins containing RGD sequence (arginine-glycine-aspartic acid), which have a high affinity for GP IIb/IIIa. Fibronectin is also a plasma protein-containing RGD sequence, and it supports platelet adhesion via the GPIIb/IIIa receptor. The platelet receptor GP Ib induces platelet adhesion and activation by binding to surface-bound vWF, especially under in vivo high shear conditions. In contrast, surface-bound albumin decreases platelet adhesion because of the absence of the albumin-specific binding receptor on the platelet membrane. Adsorption of proteins on the synthetic surface varies depending on surface characteristics, such as surface roughness, free energy, and chemical composition. Heparin coating may alter the adsorption of the major surface-adhesive proteins because of its highly charged character. However, little is known about the composition of adsorbed proteins on heparin-coated surfaces (12).

Heparin can be attached to synthetic surfaces via two different processes, ionic attachment or covalent binding. The Carmeda BioActive Surface (CBAS; Medtronic Cardiopulmonary, Anaheim, CA) (13) and the Duraflo II surface (Baxter Health-Care Corp., Irvine, CA) (14) are two different heparin-coated surfaces available for clinical use. The CBAS covalently binds end point-attached degraded heparin, whereas the Duraflo II surface binds heparin partially ionically. The CBAS is thought to be more stable than the Duraflo II surface because ionic attachment of the Duraflo II surface may be associated with leaching of heparin to the circulation (15). Accordingly, in this study, we used the CBAS to evaluate a heparin-coated oxygenator surface.

Our objectives were to determine whether heparin coating of the oxygenator membrane alters protein adsorption and decreases platelet adhesion and activation on the surface of the oxygenator fibers. The hypothesis that heparin coating reduces platelet adhesion and activation by changing the conformation of the surface adhesive proteins was tested under in vitro static conditions. We used isolated oxygenator fibers to assess platelet adhesion by using enzyme immunoassays using monoclonal antibodies against platelet receptor proteins CD42b (GP Ib) and CD61 (GP IIb/IIIa), and we assessed platelet activation by measuring plasma guanosine monophosphate (GMP)-140 levels (16).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Polypropylene hollow fibers (3 cm) were removed from heparin-coated oxygenators (Maxima Plus; Medtronic Inc., Anaheim, CA) and noncoated control oxygenators (Maxima 1380; Medtronic Inc.). The surface area was 50 cm2 for analysis of adsorbed proteins and was 12.5 cm2 for platelet adhesion. Both ends of the fibers were mechanically occluded to prevent blood from entering the inner lumen. These fibers were placed in polypropylene tubes containing approximately 120 fibers for analysis of adsorbed proteins, and approximately 30 fibers for platelet adhesion and activation analyses.

Fresh whole human blood samples were collected from healthy consenting volunteers. Blood was anticoagulated with two different concentrations of heparin, 1 or 5 IU in 1 mL of whole blood. Both the heparin-coated and noncoated groups were divided into two subgroups according to the heparin concentration administered as an anticoagulant.

The fibers were incubated in 2 mL of heparinized whole blood (test sample) or in 1.5 mL of plasma (control) for 3 h at 37°C, followed by washing six times with 10 mM phosphate-buffered saline (PBS). For evaluation of platelet adhesion, the mouse immunoglobin G (IgG) anti-human monoclonal CD42b antibody (anti-GP Ib; Dako, Glostrup, Denmark) and the mouse IgG monoclonal CD61 antibody (anti-GPIIIa; Dako) were prepared by diluting them 200 times with 1% bovine serum albumin (BSA) dissolved in PBS. After 45 min incubation at room temperature, the fibers were washed five times with 10 mM PBS. Two milliliters of peroxidase-conjugated anti-mouse immunoglobulin antiserum (Dako) diluted 2000 times with 1% BSA in 10 mM PBS was added. The fibers were incubated 45 min and washed five times with 10 mM PBS, then 1 mL of tetramethylbenzidine (TMB; Dako) was added for chromogenic substrate assay. After a further 10 min incubation, 2 mL of 2N-sulfaric acid was added as a "stop solution." The optical density was determined spectrophotometrically at a wavelength of 450 nm.

For the evaluation of platelet activation, the GMP-140 level was determined for the plasma obtained by centrifugation of blood incubated with fibers (test sample). The GMP-140 level was also determined for blood incubated without fibers (sham control). An enzyme-linked immunosorbent assay was conducted with a commercially available kit (Research & Diagnostic Systems, Minneapolis, MN).

Fibers were exposed to the whole blood with slow rotations for 3 h at 37°C. After incubation, fibers were washed six times with saline, and adsorbed proteins were eluted with 2 mL of 1% sodium dodecyl sulfate (SDS) 20 mM Tris-HCL (pH 8.0) buffer for 16 h. Elutes were stored at -80°C until assayed. The amount of adsorbed proteins was measured by using a bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, IL).

Adsorbed proteins on the fibers were analyzed by using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The samples were diluted with 1% SDS 20 mM Tris-HCL (pH 8.0) buffer to adjust the protein concentration and treated with the Leammli buffer (Dai-ichi Kagaku Co., Tokyo, Japan) for 5 min at 99°C. The volume ratio of the sample to Leammli buffer was 1:1. The samples were stored at -80°C until electrophoresis was performed using 6.0% and 12.5% polyacrylamide gels. The gels were stained with silver (Dai-ichi Kagaku Co.) after electrophoresis.

To identify the antibody-combined proteins, the electrophoresed samples were then electrically transferred to a polyvinylidene difluoride transmembrane (Schleicher-Schull, Dassel, Germany). A constant current of 200 mA was used for 4 h with a buffer composition of 25 mM Tris, 192 mM glycine, 0.1% SDS, 20% methanol. The transmembrane was blocked with 1% blocking reagent (Boehringer Mannheim, Indianapolis, IN) in 0.1 M maleic acid and 0.15 M NaCl for detection of albumin. For the detection of fibrinogen, fibronectin and vWF, 3% BSA in 0.1% Tween-20 in 20 mM Tris buffer, pH 8.0, 0.15 M NaCl (TBST), was used as the blocking reagent. The membrane was incubated with rabbit anti-human serum proteins polyclonal antibody at a dilution of 1:1000 for 1 h at room temperature. Monospecific primary antisera directed against the following proteins were used: albumin (Dako), fibrinogen (Dako), fibronectin (Dako), vWF (Dako). After washing three times with the washing buffer (0.1% Tween-20 in 0.1 M maleic acid for albumin detection and TBST for proteins other than albumin), the membrane was soaked in horseradish peroxidase-conjugated protein A (Sigma, St. Louis, MO) at a dilution of 1:1000 for 1 h at room temperature. The membrane was rinsed three times, and a color reaction was finally detected by using enhanced chemiluminescence (RPN 2109; Amersham Life Science Inc., Arlington Heights, IL).

All data are expressed as means ± SD. To obtain the actual optical density, a blank control sample was subtracted from each test sample. The plasma GMP-140 level of the sham control (incubated without fibers) was subtracted from that of the test sample incubated with fibers. We performed a nonparametric test because of small sample sizes and inability to determine whether the samples were normally distributed. A comparison of the amount of platelet adhesion on the heparin-coated and noncoated membranes within the same subject was performed by using the paired Wilcoxon test. Total variation of plasma GMP-140 levels among the groups was analyzed by using Kruskal-Wallis analysis of variance. When a significant difference was found, the control group was compared with the heparin-coated or noncoated group by using the Mann-Whitney U-test. A P value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
We used two different doses of heparin as an anticoagulant. Activated clotting times with large-dose heparin (5 U/mL) and small-dose heparin (1 U/mL) were 514 ± 39 and 236 ± 38 s, respectively. The optimal dilution for the monoclonal antibodies was determined to be 1:200 for both CD42b and CD61 in the preliminary studies. Platelet adhesion, as represented by the optical density at a wavelength of 450 nm for CD42b and CD61, was reduced in the heparin-coated group, independent of the doses of heparin administered as an anticoagulant (Table 1). No difference in platelet adhesion could be demonstrated between small-dose and large-dose heparinization. In the small-dose subgroup, platelet activation, as assessed by plasma GMP-140 level, increased significantly in the plasma incubated with noncoated fibers compared with that incubated without fibers (sham control) (Table 2). In contrast, the plasma GMP-140 level was not significantly different between heparin-coated membranes and sham control. In the large-dose subgroups, plasma GMP-140 levels were increased significantly in the plasma incubated with fibers, regardless of the presence or absence of heparin coating.


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  		Table 1. Effects of Heparin-Coated Oxygenator Fibers on Platelet Adhesion
 

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  		Table 2. Effects of Heparin-Coated Oxygenator Fibers on Platelet Activation
 
The amount of total adsorbed protein on the membrane surface was not different between the heparin-coated and noncoated membranes (1.64 ± 0.38 vs 1.65 ± 0.63 mg/cm2). The composition of the adsorbed proteins as assessed by using SDS-PAGE analysis was similar among the membranes (Fig. 1). Proteins adsorbed on the surface of the oxygenator were further analyzed by using Western blot analysis with antibodies specific for albumin, fibrinogen, vWF, and fibronectin. Western blot analysis showed no significant difference in the pattern of adsorption for albumin, fibrinogen, and vWf among the fibers (Fig. 2). For fibronectin, two bands were shown for heparin-coated surfaces, whereas only a single band was observed for noncoated surfaces. The lower band at 230 kD in heparin-coated membranes was identical with the band observed in noncoated membranes, which corresponds to surface adsorbed fibronectin. The elutes from the heparin-coated membrane exhibited an additional band at 250–260 kD, which is probably the fibronectin binding to 20–30 kD heparin molecules.



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  		Figure 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of elutes from heparin-coated (HC-PP) and noncoated (PP) polypropylene oxygenator membranes. The pattern of adsorbed proteins was similar in both membranes. Molecular weight standards are indicated on the left lane.

 


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  		Figure 2. Western blot analysis of elutes from heparin-coated (HC-PP) and noncoated (PP) polypropylene oxygenator membranes. Blots were incubated with antibody to albumin, fibrinogen, von Willebrand factor, and fibronectin. Note two bands appearing with antibody to fibronectin only on elutes from heparin-coated membranes. The band at 250–260 Kd (arrowhead) corresponds to the binding product of surface heparin molecules and plasma fibronectin.

 

   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The alterations in platelet adhesion and activation we observed are due to heparin-coated oxygenator fibers, not an oxygenator in general. Several observations were made: 1) heparin coating of the fibers reduced platelet adhesion independent of the doses of the heparin administered as an anticoagulant; 2) a mild but significant increase in plasma GMP-140 level observed on the noncoated membrane was attenuated only in the small-dose heparin-coated group; 3) heparin coating did not alter surface adsorption of the major platelet adhesive proteins, such as fibrinogen and vWF; and 4) the heparin-coated surface was associated with increased adsorption of fibronectin.

The most important mechanism contributing to the thrombo-resistance of heparin-coated membrane is considered to be thrombin inhibition via binding to antithrombin (AT) (17–19). Plasma AT is one of the serine protease inhibitors, which has a much higher affinity for heparin than other plasma heparin-binding proteins, including fibronectin, vitronectin, and complement. The formation of an AT-heparin complex inhibits heparin-induced platelet aggregation. However, only approximately one fourth of surface-immobilized heparin has a high affinity for AT (20). Molecules of heparin can bind plasma heparin-binding proteins other than AT and directly stimulate platelets (21,22) or indirectly augment platelet activation induced by agonists, such as ADP or thrombin (23–25). The role of these plasma-adhesive proteins on platelet adhesion should not be neglected because their concentrations are much higher than that of AT. In this study, the net effect of heparin coating on platelets was a modest but significant decrease in adhesion.

In this study, two different heparin doses were used as an anticoagulant. The ACTs in the large- and small-dose heparin groups corresponded to those reported in clinical extracorporeal circulation under standard and reduced heparinization (26). Although interactions between heparin and platelets may take place in the fluid phase as well as on the synthetic surface, no difference in platelet adhesion could be demonstrated between the small- and large-dose groups. There are two possible explanations for the different concentrations of the fluid-phase heparin not altering platelet adhesion. First, molecular heterogeneity in heparin may result in differential biologic actions of heparin (27). Accordingly, the effects of heparin may not correlate with the heparin dose. Second, heparin has bidirectional effects on platelet function, resulting in both inhibition and stimulation, as previously mentioned. Both inhibitory and proaggregating effects of heparin on platelet function could be augmented with large-dose heparin, resulting in no significant alterations in platelet adhesion.

Heparin coating of the surface tended to attenuate platelet activation mildly, as represented by the release of soluble GMP-140. This attenuation was observed only with small-dose heparinization, whereas plasma GMP-140 levels increased similarly in heparin-coated and noncoated groups with large-dose heparinization. Large-dose heparin may trigger pronounced platelet activation and mask the attenuation of platelet activation by the heparin-coated surface (28). The mechanism of decrease in platelet adhesion by heparin coating could not be attributed to the attenuation of platelet activation because a reduction in platelet adhesion on the heparin-coated fibers was observed even with large-dose heparinization.

Heparin coating may influence platelet adhesion by altering the adsorption of the surface-adhesive proteins (12). In this study, however, heparin coating did not change the adsorption pattern of major platelet-adhesive proteins, such as fibrinogen and vWF. The early study by Larsson et al. (29) showed a marked reduction in fibrinogen adsorption on the heparinized surface by using radioimmunoassay of fibrinopeptide A (FPA). However, FPA is the product of fibrinogen-fibrin conversion and cannot be used as an index of surface fibrinogen concentration. Gorman et al. (30) used a radioimmunoassay to detect surface-adsorbed proteins and found that AT adsorption was increased by heparin coating of the surface and that adsorption of other proteins, including fibrinogen, factor XII, and vWF, was unchanged. Plasma AT-mediated inhibition of thrombin by surface heparin and a resultant inhibition of fibrinogen-fibrin conversion likely explain the decrease in FPA concentration in the study by Larsson et al. (29). Indeed, the reduction in platelet adhesion on the heparin-coated surface was not attributable to the altered adsorption of fibrinogen or vWF.

However, fibronectin adsorption on the surface was increased by the surface heparin coating. Fibronectin is a high molecular weight glycoprotein that is present in plasma and is secreted by a variety of cells. Western blotting analysis using monospecific antisera directed against fibronectin showed two bands for the elutes from heparin-coated surfaces but only one band for those from noncoated surfaces. The mechanism responsible for the additional band detected in the heparin-coated group is unknown. We speculated that an additional band at 250–260 kD may correspond to fibronectin (230 kD) binding to 20–30 kD heparin molecules separated from the surface, because the only difference between the two groups was the presence or absence of surface-immobilized heparin. Heparin is a heterogeneous mixture of sulfated polysaccharides with molecular weights of 5–30 kD (average 12–15 kD). The molecular weight of covalently bonded heparin is approximately 8 kD (20). However, human plasma fibronectin has several different domains that bind to heparin, and the high-affinity heparin-binding domain (Hep-2 domain) has two binding sites for heparin (31).

Fibronectin supports platelet adhesion under static conditions (32) and under shear conditions (33). However, when platelet activation is inhibited under static conditions, fibrinogen plays a major role in platelet adhesion on the synthetic surface, whereas the role of fibronectin may be minimal (34). The extent of adhesion of unstimulated platelets to fibronectin is much smaller than that to fibrinogen (35). Nagai et al. (34) demonstrated that polyclonal antibodies against fibronectin have no effect on platelet adhesion, whereas those against fibrinogen inhibited adhesion by 70%.

Moreover, heparin molecules may inhibit platelet-fibronectin interaction. Heparin has a high affinity to fibronectin and can inhibit platelet adhesion to fibronectin by tightly binding to heparin-binding domain of fibronectin. Beumer et al. (33) showed that heparin with an average molecular weight of up to 18 kD reduced platelet adhesion by 70%, and that of up to 21 kD blocked adhesion almost completely. The authors suggested that a high-affinity heparin-binding domain is close to the RGD site, and that heparin binding to this site can shield the RGD site. Fibronectin has also been reported to inhibit heparin-induced platelet aggregation and AT (25). Thus, the formation of a heparin-fibronectin complex on the heparin-coated surface may inhibit platelet adhesion.

We used enzyme immunoassays using monoclonal antibodies directed against platelet receptor glycoproteins to determine the amount of adhering platelets. Groth et al. (36) demonstrated a qualitative relationship of adhering platelets as assessed by CD42b and scanning electromicroscopic findings and concluded that the enzyme immunoassay using monoclonal antibody against CD42b provides a useful estimate of the adhering platelets on the synthetic surface. However, previous studies have demonstrated that a down-regulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by agonists (37,38), whereas down-regulation does not occur in the GPIIb-IIIa complex. Thus, we used CD61 in addition to CD42b. In this study, both CD42b and CD61 showed almost the same tendencies, reflecting only mild platelet activation even on the polypropylene surface.

These experimental data cannot be immediately applied clinically because the present experiments were conducted under nonshear conditions to evaluate the direct effects of heparin coating of the surface on platelet function. Platelet function during extracorporeal circulation is influenced by multiple factors, including platelet-surface interactions, shear stress, temperature, and use of the chamber venting and cardiotomy suction. Moreover, levels of shear stress on the oxygenator fibers during clinical CPB vary depending on the oxygenator design, synthetic surface area, total pressure drop of bypass system, and extracorporeal circulation technique. Thus, our study was performed under static conditions at 37°C using oxygenator fibers to eliminate the many uncontrolled variables (other than platelet-surface interaction) associated with CPB. In fact, platelet adhesion in vivo is a shear stress-dependent process that requires different platelet receptors and adhesive proteins depending on the levels of shear stress. Under low-shear conditions, platelet adhesion may depend on fibrinogen and GPIIbIIIa, whereas, under high-shear conditions, platelet adhesion largely depends on vWF and GPIb (34,39,40). Effects of heparin coating on interactions between platelet receptors and adhesive proteins under various shear conditions remain to be elucidated in further studies. The lack of endothelium in the current in vitro study may be another important consideration in assessing platelet-surface interaction. The presence of intact endothelium inhibits platelet activation by the release of vasoactive products, such as prostacyclin (41) or nitric oxide (42).

In conclusion, heparin coating of the membrane surface inhibits platelet adhesion on the oxygenator fibers. Heparin coating did not alter adsorption of major adhesive proteins, such as fibrinogen and vWF, but was associated with increased fibronectin adsorption. Fibronectin binding to surface heparin may partly contribute to the inhibition of platelet adhesion.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication May 12, 1999.




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D. A Palanzo, D. L Zarro, N. J Manley, R. M Montesano, M. Quinn, B.-A. Elmore, P. A Gustafson, and J. M Castagna
Effect of Carmeda(R) BioActive Surface coating versus TrilliumTM Biopassive Surface coating of the oxygenator on circulating platelet count drop during cardiopulmonary bypass
Perfusion, July 1, 2001; 16(4): 279 - 283.
[Abstract] [PDF]

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H. Suhara, Y. Sawa, M. Nishimura, H. Oshiyama, K. Yokoyama, N. Saito, and H. Matsuda
Efficacy of a new coating material, PMEA, for cardiopulmonary bypass circuits in a porcine model
Ann. Thorac. Surg., May 1, 2001; 71(5): 1603 - 1608.
[Abstract] [Full Text] [PDF]

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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 1999 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press