Influence of Red Blood Cells and Their Components on Protein

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Influence of Red Blood Cells and Their Components on Protein Adsorption S. UNIYAL, J. L. BRASH, and I. A. D E G T E R E V 1

2

McMaster University, Departments of Chemical Engineering and Pathology, Hamilton, Ontario, Canada

Earlier

observations from this laboratory

showed that red

blood cells have an inhibitory effect on adsorption of albumin and fibrinogen

to polyethylene surfaces. The present work

extends observations of this "red cell effect" to the glass-fibrinogensystem. Adsorption in the presence of red cells was inhibited up to 50%, the inhibition increasing with increasing hematocrit at constant protein concentration in the free fluid volume. Since some hemolysis occurs in these experiments, the effect of deliberately added hemolysate was investigated, but found to be negligible. Adsorption

in the presence of red cell

ghosts was inhibited strongly. These results suggest that the red cell effect is not attributable to leakage of cell contents, but rather is a membrane-related effect. A dsorption of proteins is the primary event upon contact between blood and foreign surfaces (1 ), and subsequent cellular interactions leading to thrombus formation are determined by these adsorbed proteins (2). Much of the early work on the study of adsorption was done in buffered solutions of single proteins or relatively simple mixtures (3-7). More recently, studies have been conducted using more "realistic" media, particularly plasma (8, 9). These studies have shown that the plasma interacts subsequently with the initially adsorbed proteins, causing some unexpected effects. For example, initially adsorbed fibrinogen is desorbed rapidly from several surfaces in the presence of plasma (8). The question might well be asked whether the red cells, another major component of blood, have any effect on protein adsorption. The effect of red cells on platelet sticking has been noted widely, causing an augmentation of the rate of adhesion, probably by a combination of physical and biochemical mechanisms (10). However, studies of protein adsorption in the presence of To whom correspondence should be addressed. Soviet Union-Canada exchange scientist from Institute of Chemical Physics, Moscow.

1 2

0065-2393/82/0199-0277$06.00/0 ©1982 American Chemical Society Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

278

BIOMATERIALS: INTERFACIAL PHENOMENA AND APPLICATIONS

r e d cells are, s u r p r i s i n g l y , almost e n t i r e l y lacking. I n a previous study f r o m this laboratory (11), the a d d i t i o n of r e d b l o o d cells to b u f f e r e d solutions of plasma proteins caused a decrease i n the quantity of p r o t e i n a d s o r b e d f r o m these solutions to a p o l y e t h y l e n e surface. T h e present w o r k extends these observations to the g l a s s - f i b r i n o g e n system, a n d provides n e w i n f o r m a t i o n relevant to the m e c h a n i s m of adsorption i n h i b i t i o n . I n particular, w e w a n t e d to establish w h e t h e r the r e d c e l l effect has intracellular or m e m b r a n e origins. T h e r e f o r e , separate e x p e r i m e n t s w e r e c a r r i e d out i n the presence o f h e m o lysate f r o m r e d cells a n d r e d c e l l ghosts.

Experimental Materials. Fibrinogen (human, Grade L) was obtained from Kabi (Stockholm, Sweden). The lyophilized product was dissolved in distilled water (concentration: 1 g/100 mL) and dialyzed against an appropriate buffer, usually isotonic phosphatebuffered saline (PBS), pH 7.35 (see below). The solution was frozen in 5-mL portions until required. Hemoglobin (human, Type IV, twice crystallized) was purchased from Sigma Chemical Co. and was used as received. The Na I was from New England Nuclear. Glass tubing (0.25 cm in diameter) was Corning code 7740, Pyrex glass, and was washed for 1 h with chromic acid cleaning mixture (Chromerge), then rinsed with copious amounts of distilled water. The surface of this glass previously has been shown to be essentially smooth and featureless from a topographical standpoint by electron microscopy (12). The "normal" adsorption medium was isotonic PBS (0.15M, pH 7.35) containing labeled fibrinogen at a specified concentration. To this medium were added, variously, washed whole red cells, red cell ghosts, or red cell hemolysate in sufficient quantity to give a specified final concentration of hemoglobin. Fibrinogen concentrations for the media containing red cells or ghost cells were based on free fluid volume rather than total volume. Fibrinogen Labeling. Labeling with I was carried out as described previously (12), using a twofold molar excess of iodine monochloride. This procedure gives labeled fibrinogen identical in its adsorption on glass to unlabeled protein (13). Other properties of fibrinogen, including biological properties, also are unaffected by this labeling method (14,15). In general, the fibrinogen solutions used in the present work contained 30% labeled and 70% unlabeled material. Preparation of Washed Red Cells. Freshly drawn human blood, collected into acid citrate dextrose (ACD) anticoagulant, was centrifuged for 15 min at 630 X g, and the plasma and buffy coat were removed. The red cells were washed three times using 3-4 volumes of PBS, centrifuging between each wash at 2000 x g for 10 min. Packed red cells [hematocrit (HCT) about 90%] were added to appropriate volumes of fibrinogen solution (in PBS) to give the required hematocrit. Preparation of Ghost Cells. The method of Steck (16) was modified for use in the present work. Washed, packed red cells prepared as indicated above were lysed in 40 volumes of 5mM sodium phosphate (pH 8.0), then washed three times using 40 volumes of 5mM sodium phosphate. This procedure gives so-called "unsealed" ghost cells (16) from which essentially all the hemoglobin has been removed. At the same time, normal red cell morphology (biconcave disc) and size are maintained as shown by the results of scanning electron microscopy (SEM) (Figure 1). Cells were prepared for SEM by allowing them to settle on Nuclepore polycarbonate membranes. They were then fixed sequentially with glutaraldehyde and osmium tetroxide, and dehydrated with ethanol. 125

125

Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

UNIYAL ET AL.

Influence of Red Blood Cells on Protein Adsorption

Figure 1. SEM of a typical ghost cell. Cells were deposited on a polycarbonate membrane (Nuclepore) whose pores are visible. The small white marker lines represent 1 μτη.


280


In addition to S E M , size analysis of the ghost cells was performed with a Coulter counter, and as shown in Figure 2, the size distribution is similar to that of the washed red cell preparation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of ghost membrane proteins was performed by a modification of the method of Laemmli (17). Samples were reduced with β-mercaptoethanol and run in 11% gels. Molecular weight (MW) standards were obtained from Pharmacia Canada Ltd. Media containing ghost cells for adsorption studies were prepared by mixing appropriate volumes of packed ghost cells and labeled fibrinogen in PBS. Ghost hematocrits were determined by cell counting (Coulter counter), assuming an average ghost cell volume of 75 μιτι . Preparation of Hemolysate. Washed red cells were lysed in distilled water (20% HCT) for 15 min. The stroma was extracted in carbon tetrachloride, and the aqueous layer containing the hemolysate was recovered. This solution was diluted appropriately with PBS to a specified hemoglobin concentration, and labeled fibrin ogen was added. The final solution was adjusted to isotonicity and pH 7.35. Hemo globin concentrations were determined by the cyanmethemoglobin method (18). Adsorption Experiments. Adsorptions were carried out as described pre viously (11). A circuit was set up consisting of several glass tubing segments and a roller pump connected in series. The tubes were positioned vertically, and were connected together using Silastic medical-grade tubing (Dow-Corning) and threeway valves. The total length of the Silastic connectors was about one-third that of the glass test segments. The circuit was primed with PBS, which was then displaced by the test medium (a suspension of red blood cells, ghosts, etc.) in a manner (using the three-way valves to eliminate air bubbles) such that no air-solution-solid interface was created. Experiments were run at a flow rate of 50 mL/min (540 s surface shear rate) at room temperature. To determine the time course of adsorption, tubing segments were disconnected at various times up to 4 h and were rinsed three times with PBS (20 volumes), and the associated radioactivity was determined (Beckman Biogamma system). An aliquot of the labeled fibrinogen solution (of known concen tration) prepared for a given experiment also was counted, and surface concentration Γ ^g/cm ) was calculated from the relation: 3

_1

2

1

CpRf Afi s

where C is the solution concentration of fibrinogen ^g/mL); Rf, the count rate of surface; R , the count rate of solution (per mL); and A, the area of surface (cm ). In experiments where the extent of hemolysis was needed, samples of test fluid were withdrawn from the circuit at various times, and the hemoglobin concentration in the supernatant was measured (18). p

s

2

Results E f f e c t o f W h o l e R e d C e l l s . A s i n d i c a t e d i n F i g u r e 3, w h o l e r e d cells cause a d i m i n u t i o n i n the q u a n t i t y of f i b r i n o g e n adsorbed to a glass surface. T h e extent of this " i n h i b i t i o n " increases w i t h increasing hematocrit, a n d at 4 0 % H C T , the surface c o n c e n t r a t i o n after 4 h was r e d u c e d to about 5 0 % of its value i n the absence of cells. It was shown p r e v i o u s l y (11), that such an effect was not d u e to the d e p l e t i o n of f i b r i n o g e n f r o m the aqueous phase b y adsorption to the c e l l surfaces. A l t h o u g h this may occur to some extent, it is insufficient to cause any detectable alteration i n the concentration o f f i b r i n o -


Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982. 3

3

Figure 2. Distributions of cell volumes of typical red cell and ghost cell preparations, obtained by Coulter counter. Key to mean cell volumes: red cells, 87 μ τ η ; and ghost cells, 76 μ ? η .

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Figure 3. Adsorption of fibrinogen on glass as a function of time at various hematocrits. Conditions: buffer—PBS, pH 7.35; fibrinogen concentration—1.0 mg/mL in free volume; and shear rate at the surface—540 s . Values are the average of at least three experiments; error limits are standard deviations. Key: · , 0% HCT; Q 10% HCT; Q, 20% HCT; and A, 40% HCT.

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ι NIYAL E T A L .


283

gen i n the l i q u i d phase. In a d d i t i o n , for all suspensions u s e d , the c o n c e n tration i n the cell-free v o l u m e was kept constant at 1.0 m g / m L . U n l i k e the results for p o l y e t h y l e n e , p u b l i s h e d p r e v i o u s l y (11 ), the surface concentration does not pass t h r o u g h an early m a x i m u m . Instead, it shows " t y p i c a l " k i n e t i c behavior, a p p r o a c h i n g a steady-state value i n times on the o r d e r of 1-2 h . In the case of the 4 0 % H C T suspension, the surface concentration began to decrease f r o m its steady-state value after 2 - 3 h . Hemolysis D u r i n g Experiments with Whole Red Cells.

A l t h o u g h it

was p r e v i o u s l y c o n c l u d e d (11) that hemolysis i n these experiments is less than 1 % a n d p r o b a b l y of no account,

we w a n t e d to ascertain the exact

concentration of free h e m o g l o b i n a n d to d e t e r m i n e w h e t h e r this concen tration increases w i t h t i m e . H o r b e t t et al. (19) suggested that h e m o g l o b i n is a p r o t e i n of h i g h surface activity, a n d is preferentially adsorbed f r o m plasma relative to f i b r i n o g e n a n d a l b u m i n . T h u s , at least a part of the r e d c e l l - r e l a t e d i n h i b i t i o n of adsorption possibly c o u l d be d u e to preferential adsorption of h e m o g l o b i n . F i g u r e 4 shows that the free h e m o g l o b i n concentration does i n d e e d increase w i t h t i m e i n t y p i c a l adsorption experiments, suggesting that some h e m o l y t i c damage to cells is o c c u r r i n g . T h e effect is m i n i m a l at the lower hematocrit, b u t at 4 0 % H C T , the concentration of free h e m o g l o b i n reaches a value o n the o r d e r of 1.0 m g / m L , comparable to that of f i b r i n o g e n . T h e i n i t i a l concentration of h e m o g l o b i n is finite a n d increases w i t h increasing hematocrit. W i t h the m a n i p u l a t i o n s i n v o l v e d i n p r e p a r i n g the r e d cells, this " r e s i d u a l " free h e m o g l o b i n c o u l d not be e l i m i n a t e d . E v e n at 2 0 % H C T , the initial h e m o g l o b i n concentration was about 0.04 mg/mL. E f f e c t s of H e m o g l o b i n a n d H e m o l y s a t e .

Because substantial concen

trations of h e m o g l o b i n exist i n the adsorption m e d i a , we wanted to k n o w w h e t h e r h e m o g l o b i n p e r se w o u l d exert an i n h i b i t o r y effect on f i b r i n o g e n adsorption. E x p e r i m e n t s u s i n g h e m o g l o b i n f r o m a c o m m e r c i a l source were thus c a r r i e d out, u s i n g concentrations at the l o w and h i g h ends of the range actually e n c o u n t e r e d i n the r e d c e l l suspensions. F i g u r e 5 shows the kinetics of adsorption at h e m o g l o b i n concentrations

of 0.09 and 0.8 mg/mL, and

indicates a strongly i n h i b i t o r y effect such that adsorption of f i b r i n o g e n is all but e l i m i n a t e d at the h i g h e r concentration. T h e shape of the curves suggests that a certain a m o u n t of the f i b r i n o g e n adsorbed at short times is r e m o v e d later. T h e spectacular effect of h e m o g l o b i n shown i n F i g u r e 5 is, i n fact, greater than the r e d c e l l effect itself, suggesting that i f the latter is d u e e n t i r e l y to leakage of c e l l contents, then hemolysate is relatively less i n h i b i t o r y than p u r e h e m o b l o g i n . To investigate further the role of c e l l con tents, adsorption e x p e r i m e n t s w e r e p e r f o r m e d i n the presence of c o n t r o l l e d amounts of hemolysate, such that free h e m o g l o b i n concentrations were again in the range e n c o u n t e r e d i n the r e d c e l l experiments. T h e results of these experiments are s h o w n i n F i g u r e 6; the hemolysate effect was very small, and effectively n e g l i g i b l e u p to a h e m o g l o b i n concentration of 1.0 mg/mL. A slight decrease i n the steady-state l e v e l of adsorption o c c u r r e d at the h i g h e r



to

δ Figure 4. Development of hemoglobin in supernatant as a function of time during red cell experiments at two ζ hematocrits. Values are the average of at least three experiments. Key: Q, 40% HCT and ·, 20% HCT.

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α >

> > ζ

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Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982. LEVEL IN ABSENCE OF HEMOGLOBIN

Figure 5. Adsorption of fibrinogen on glass in the presence of Sigma hemoglobin. Conditions: buffer—PBS, pH 7.35; fibrinogen concentration—1.0 mg/mL; and shear rate at the surface—540 s *. Values are the average of three experiments. Key to hemoglobin concentration: 0> 0.09 mg/mL and ·, 0.8 mg/mL; —, level in absence of hemoglobin.

0.7 r

286


h e m o g l o b i n concentrations. T h e difference b e t w e e n the effects of the c o m m e r c i a l h e m o g l o b i n a n d hemolysate is v e r y p r o n o u n c e d . A m o n g possible explanations is that o t h e r constituents of hemolysate act i n opposition to h e m o g l o b i n . H o w e v e r , F i g u r e 7 suggests another, perhaps m o r e p l a u s i b l e , explanation. F r o m this figure, s h o w i n g v i s i b l e spectra of the hemolysate and S i g m a h e m o g l o b i n , the hemolysate

h e m o g l o b i n is i n the f o r m of oxy

h e m o g l o b i n , whereas the S i g m a h e m o g l o b i n is i n the f o r m of m e t h e m o g l o b i n (20). C o n s e q u e n t l y , m e t h e m o g l o b i n appears to be adsorbed strongly i n pref erence to f i b r i n o g e n , whereas o x y h e m o g l o b i n is not. Effect of Ghost Cells.

T h e results of the experiments i n the presence

of hemolysate suggest that c e l l contents, p e r se, do not influence f i b r i n o g e n adsorption, thus i m p l i c a t i n g the c e l l m e m b r a n e s d i r e c t l y , or alternatively, the particulate character of the cells, as b e i n g responsible for the effect. To investigate this p o s s i b i l i t y , e x p e r i m e n t s w i t h ghost cells were c o n d u c t e d . T h e preparative t e c h n i q u e r e s u l t e d i n ghost cells of n o r m a l shape and size. T h u s , S E M results such as those p r e s e n t e d i n F i g u r e 1 show retention of bioconcave shape a n d a c e l l d i a m e t e r b e t w e e n 6 and 7 μπι. C o u l t e r counter analysis ( F i g u r e 2) s h o w e d the d i s t r i b u t i o n of c e l l volumes to be substantially m a i n t a i n e d , a l t h o u g h the m e a n c e l l v o l u m e was somewhat less than for the n o r m a l cells. T w o concentrations of ghost cells, namely, 15% and 4 5 % b y v o l u m e , w e r e u s e d i n these e x p e r i m e n t s . T h e results, p r e s e n t e d i n F i g u r e 8, show a m a r k e d d i m i n u t i o n i n quantity adsorbed (on the o r d e r of 5 0 % and 8 0 % at 15% a n d 4 5 % b y v o l u m e of ghost cells, respectively). A g a i n , as w i t h

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HEMOGLOBIN CONC IN HEMOLYSATE (mg ml ) 1

Figure 6. Adsorption of fibrinogen on glass in the presence of red cell hemolysate. Conditions: buffer—PBS, pH 7.35; fibrinogen concentration—1.0 mg/mL; and surface shear rate—540 s . Values are the average of three experiments. _ i


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UNIYAL E T A L .

287


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WAVELENGTH (nm) Figure 7. Visible spectra of hemolysate preparation and Sigma hemoglobin in the wavelength range 460-700 nm. Key: —, hemolysate and —, Sigma hemoglobin. the kinetics i n the p r e s e n c e of m e t h e m o g l o b i n , the ghost cells appear to be able to remove i n i t i a l l y a d s o r b e d f i b r i n o g e n . S D S - P A G E of Adsorption M e d i a . S D S - P A G E of various m e d i a used in this study was u n d e r t a k e n to obtain a d d i t i o n a l information on their p r o t e i n compositions. T y p i c a l gels are p r e s e n t e d i n F i g u r e 9. G e l 3 corresponds to the proteins f r o m ghost c e l l m e m b r a n e s and shows a complex pattern of bands that agrees w e l l w i t h p r e v i o u s l y p u b l i s h e d results, for example, those of Fairbanks et a l . (21). C o m p a r i s o n of G e l s 2 a n d 4, c o r r e s p o n d i n g to K a b i f i b r i n o g e n and f i b r i n o g e n plus supernatant f r o m a ghost c e l l suspension, respectively, shows the presence of an a d d i t i o n a l b a n d i n G e l 4 at a m o l e c u l a r weight of about 37,000. T h i s p r o t e i n p r e s u m a b l y o r i g i n a t e d i n the ghost membranes, and



0.6 r LEVEL IN ABSENCE OF GHOSTS

Figure 8. Adsorption of fibrinogen on glass in the presence of ghost cells. Conditions: buffer—PBS, pH 7.35; and fibrinogen concentration—1.0 mg/mL in free volume. Values are the average of three experiments. Key: O , ghosts at 15% HCT; φ, ghosts at 45% HCT; and —, level in absence of ghosts.

ÎT

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Figure 9. SDS-PAGE of various materials. Numbers on left are molecular weights of standards shown in Gel 1. Key to gels: 1, molecular weight standards; 2, human fibrinogen (Kabi); 3, ghost cell membrane polypeptides; 4, fibrinogen with supernatant from ghost cell preparation; 5, ghost cell supernatant alone; and 6, hemolysate from red cells.

appears to c o r r e s p o n d to " B a n d 6" i n the n o m e n c l a t u r e of Fairbanks et al. (21 ). B a n d 6 has b e e n i d e n t i f i e d w i t h the m o n o m e r i c f o r m of glyceraldehyde3-phosphate d e h y d r o g e n a s e (22), w h i c h is b e l i e v e d to be an " e x t r i n s i c " p r o tein, loosely b o u n d to the c y t o p l a s m i c surface of the m e m b r a n e . G e l 5 represents a sample of h e m o l y s a t e , a n d shows essentially a single b a n d c o r r e s p o n d i n g to the h e m o g l o b i n s u b u n i t , thus c o n f i r m i n g that the h e m o lysate does not contain any m e m b r a n e or plasma p r o t e i n contaminants.


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Discussion T h e three p r i n c i p a l conclusions e m e r g i n g f r o m this study are that: (1) the " r e d c e l l effect" can n o w be f u r t h e r g e n e r a l i z e d to i n c l u d e glass surface; (2) a c o n s i d e r a b l e difference i n c o m p e t i t i v e adsorption exists b e t w e e n oxyh e m o g l o b i n a n d m e t h e m o g l o b i n ; and (3) i n h i b i t i o n of adsorption b y r e d cells appears to be a c e l l m e m b r a n e - r e l a t e d effect. W i t h regard to the first p o i n t , i n i t i a l w o r k w i t h p o l y e t h y l e n e (11) postulated that the m e c h a n i s m of a d s o r p t i o n i n h i b i t i o n i n v o l v e d collision of r e d cells w i t h the surface, a n d transfer of some material f r o m the c e l l surface to the tube w a l l . S u c h an effect m i g h t be c o n s i d e r e d to be m o r e probable i f it i n v o l v e d h y d r o p h o b i c interactions, w h i c h w o u l d be more likely for polyethylene than for glass. H o w e v e r , as is shown by the present study, the extent of i n h i b i t i o n is about the same for b o t h surfaces, so that whatever the m e c h a n i s m or m e c h a n i s m s , the i n h i b i t i o n appears equally likely for h y d r o p h o b i c and h y d r o p h i l i c surfaces. T h e effect may be a general one that w o u l d occur for any surface. V r o m a n et al. (9) o b s e r v e d transient adsorption (i.e., adsorption followed by r a p i d desorption) of f i b r i n o g e n o n glass i n the presence of plasma, and a t t r i b u t e d this result to r e p l a c e m e n t of f i b r i n o g e n w i t h h i g h molecular weight k i n i n o g e n . T h e present results c o u l d possibly be explained b y this plasma effect r e s u l t i n g f r o m carry over of plasma w i t h the r e d cells. Since the cells are w a s h e d v e r y extensively, we do not believe such an effect w o u l d be important. In a d d i t i o n , w e have o b s e r v e d a r e d c e l l effect for other proteins i n c l u d i n g a l b u m i n (11) a n d I g G (23). T h e d r a m a t i c difference b e t w e e n the effects of hemolysate and S i g m a h e m o g l o b i n is perhaps somewhat s u r p r i s i n g , and must r e m a i n , for the m o ment, w i t h o u t explanation. C e r t a i n l y , however, o n l y the o x y h e m o g l o b i n situation is relevant to b l o o d , since m e t h e m o g l o b i n constitutes o n l y 0 . 5 - 3 % of total h e m o g l o b i n (24). T h e observations of H o r b e t t et al. (19), suggesting that h e m o g l o b i n a d s o r p t i o n m i g h t be i m p o r t a n t i n extracorporeal c i r c u lations w h e r e h e m o l y s i s is m o r e l i k e l y to occur, w e r e based on w o r k w i t h m e t h e m o g l o b i n (25). T h e r e f o r e , the q u e s t i o n of the possible role of h e m o g l o b i n i n b l o o d - s u r f a c e p h e n o m e n a needs to be r e - e x a m i n e d . F r o m a more fundamental standpoint, h o w such differences i n adsorption behavior m i g h t arise s h o u l d be c o n s i d e r e d based o n subtle structural differences b e t w e e n the various h e m o g l o b i n types. A p p a r e n t l y , the oxidation state of the iron affects a d s o r p t i o n p r o p e r t i e s , thus suggesting that the h e m e groups may be i n v o l v e d . C l e a r l y , m o r e systematic studies relevant to this q u e s t i o n are r e q u i r e d to resolve the various issues. T h e results w i t h ghost cells suggest that the m e m b r a n e s play a key role i n the " r e d c e l l effect." T h i s p o i n t of v i e w is i n accord w i t h conclusions f r o m


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UNIYAL ET AL.

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our earlier w o r k (11 ) that the r e d cell-surface c o l l i s i o n results i n transfer o f material f r o m the c e l l to the surface. K e l l e r a n d Y u m (26) also p r o v i d e d evidence that such a transfer o f material may occur. T w o possible m e c h anisms p r o b a b l y are operative i n this c o n n e c t i o n : (1) the deposition of material f r o m the c e l l as j u s t n o t e d , p r o v i d i n g a n e w surface that is less adsorptive toward proteins than t h e o r i g i n a l substrate, a n d (2) the ability of the r e d c e l l to " s t r i p " the surface o f p r e v i o u s l y adsorbed p r o t e i n , as suggested b y the shape o f the curves i n F i g u r e s 5 a n d 8. I n the case of p o l y e t h y l e n e (11 ), r e d cells c i r c u l a t e d over a p r e v i o u s l y adsorbed p r o t e i n layer d i d not remove the layer. S u c h e x p e r i m e n t s w e r e n o t p e r f o r m e d i n the present w o r k , but p r o b a bly s h o u l d be c a r r i e d o u t to test the p o s s i b i l i t y that s t r i p p i n g can occur. T h e relevance o f these results to b l o o d - b i o m a t e r i a l interactions has several facets. If r e d c e l l material is d e p o s i t e d o n the surface, then this becomes an i n t e r a c t i o n o f p o t e n t i a l i m p o r t a n c e that has not b e e n r e c o g n i z e d p r e v i o u s l y . Previous observations o f early events i n b l o o d - m a t e r i a l i n t e r actions (1,3) have e m p h a s i z e d the r a p i d d e p o s i t i o n of a p r o t e i n layer, generally a s s u m i n g it to consist o f proteins o r i g i n a t i n g f r o m the plasma. H o w e v e r , red c e l l interactions also may p o s s i b l y c o n t r i b u t e to this layer. Identification of material d e p o s i t e d f r o m the r e d c e l l a n d the acquisition of knowledge of h o w its p r e s e n c e o n t h e surface m i g h t influence subsequent interactions such as platelet adhesion, are clearly tasks of some significance. I n a d d i t i o n , w h e t h e r the q u a n t i t y a n d c o m p o s i t i o n o f the deposit d e p e n d o n the specific b i o m a t e r i a l surface s h o u l d be investigated. R e d c e l l - s u r f a c e interactions may play a role i n the dynamics of p r o t e i n adsorption. W e have b e e n investigating the turnover o f p r o t e i n b e t w e e n solution a n d surface for several years (27-29), a n d have established

that

turnover occurs o n a variety o f surfaces. T h e rate a n d extent o f turnover d e p e n d strongly o n t h e surface character, w i t h h y d r o p h i l i c materials, for example, s h o w i n g m u c h m o r e r a p i d turnover than h y d r o p h o b i c materials. I f r e d cells have the a b i l i t y to strip p r o t e i n off a b i o m a t e r i a l surface, then clearly this effect c o u l d i n f l u e n c e the characteristics o f the turnover process, particularly from a rate p o i n t o f v i e w . T h i s process, i n t u r n , c o u l d affect the d e v e l o p m e n t of the p r o t e i n layer over a p e r i o d of t i m e . F i n a l l y , based o n results f r o m the present w o r k , i f studies o f p r o t e i n adsorption are to b e m e a n i n g f u l i n terms o f b l o o d - b i o m a t e r i a l interactions, then they s h o u l d b e c a r r i e d o u t i n the presence o f r e d b l o o d cells.

Acknowledgments F i n a n c i a l support o f this w o r k b y the M e d i c a l Research C o u n c i l o f Canada, t h e O n t a r i o H e a r t F o u n d a t i o n , a n d the C a n a d a - S o v i e t U n i o n scientific exchange p r o g r a m is gratefully a c k n o w l e d g e d .


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Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

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R E C E I V E D for review January 16, 1981. A C C E P T E D September 16,


1981.

Influence of Red Blood Cells and Their Components on Protein

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