Proteins, Plasma, and Blood in Narrow Spaces of Clot-Promoting

19 Proteins, Plasma, and Blood in Narrow

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Spaces of Clot-Promoting Surfaces L E O VROMAN, ANN L. ADAMS, G E N A C. FISCHER, PRISCILLA C. M U N O Z , and M I C H A E L STANFORD Veterans Administration Medical Center, Interface Laboratory, Brooklyn, NY 11209

Blood plasma deposits fibrinogen

on many surfaces. On those

able to activate clotting, high molecular weight kininogen and factor XII supplant the adsorbed fibrinogen. only where fibrinogen

Platelets adhere

remains adsorbed. In recent studies we

found that, in spaces of less than about 10 µm wide, plasma contains insufficient high molecular weight kininogen and factor XII per surface area to supplant the fibrinogen; here platelets will adhere. Between a glass convex lens and a glass or anodized tantalum slide, plasma will leave a central disc of fibrinogen;

diluted plasma will leave a larger ring of which the

center—where the thin plasma layer lacks sufficient fibrinogen—may contain a ring of albumin. Whole blood leaves a ring of platelets corresponding

to that of fibrinogen;

in absence of

erythrocytes the ring of platelets deposited is smaller.

W

hen blood plasma comes into contact with a solid, it will deposit proteins at the interface within 1 s. Fibrinogen dominates the proteins

adsorbed onto many materials (I ). O n surfaces such as glass, which is known to activate clotting, plasma will quickly supplant its own fibrinogen deposit by high molecular weight kininogen and by factor XII (2). High molecular weight kininogen carries factor XI and prekallikrein to the surface (3), and the interactions among these factors (4) probably complete the surface activation of the intact plasma clotting system. Where high molecular weight kininogen and factor XII in intact form are limited, for example, in plasma congenitally deficient in this kininogen (5), and in activated plasma, much less fibrinogen is supplanted. Even in normal intact plasma, the concentration of fibrinogen is greater than that of high molecular weight kininogen and factor XII by an order of magnitude. O n the other hand, the affinity of high molecular weight kininogen for glass is very high (5). Consequently, in a thin layer of blood, factor XII and high molecular 0065-2393/82/0199-0265$06.00/0 ©1982 American Chemical Society In Biomaterials: Interfacial Phenomena and Applications; Cooper, S., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

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weight k i n i n o g e n m a y b e d e p l e t e d b y adsorption o n the activating surface before a l l a d s o r b e d f i b r i n o g e n is s u p p l a n t e d . Since platelets o n l y adhere w h e r e they f i n d a d s o r b e d f i b r i n o g e n (6), they s h o u l d adhere i n narrow spaces, rather than o n m o r e o p e n areas w h e r e the activating surface w i l l have supplanted a l l f i b r i n o g e n before the platelets arrive. In the p r e s e n t study w e a l l o w e d n o r m a l intact plasma to fill spaces of


varied thickness o n a n o d i z e d t a n t a l u m - s p u t t e r e d glass (TaO) o r o n glass, a n d then r e n d e r e d the r e s u l t i n g p a t t e r n v i s i b l e b y subsequent exposure to antisera. T h e most suitable configuration o f solid surfaces was a convex lens placed o n a slide: it a l l o w e d us to c o m p u t e a n d correlate l i q u i d f i l m heights w i t h the r a d i i o f c i r c u l a r c o n c e n t r i c patterns r e s u l t i n g f r o m the experiments. F i b r i n o g e n solutions, h e p a r i n i z e d b l o o d , a n d citrated plasma w e r e used i n this a r r a n g e m e n t o f interfaces. O u r m e t h o d s for d e t e c t i n g and i d e n t i f y i n g adsorbed proteins w e r e tested f u r t h e r w i t h k n o w n p r o t e i n s , m a t c h i n g a n d n o n m a t c h i n g antisera i n recent years ( i , 7, 8), a n d p r o v e d to b e specific.

Experimental Materials. Anodized tantalum-sputtered glass slides (TaO). Tantalumsputtered glass slides, 1 x 3 and 3 x 4 in., were kindly supplied by N. Schwartz and D. Gerstenberg, Bell Telephone Labs; others were purchased from Millis Research. Slides were anodized at approximately 20 V in 0.01% nitric acid, to obtain a deepbronze, first-order interference color at nearly vertically incident natural light (7). Glass slides (borosilicate glass, 1 x 3 in. or larger) (A.H. Thomas); Kimwipes (Kimberly-Clark Corp.); Sparkleen detergent (Fisher Scientific Co.); antisera to human fibrinogen (Behring Diagnostics, and M . Mosesson, SUNY at Downstate); and Coomassie Brilliant Blue R or G (2), were purchased from the suppliers indicated. Veronal-buffered saline, pH 7.4, was prepared as described in Réf. I. Normal human intact citrated plasma obtained from freshly donated blood by centrifugation (2) at 20°C was stored in aliquots in polystyrene test tubes at - 7 5 ° C . Red cell poor blood was prepared by collecting blood into 100 U of heparin (Lipohepin of Riker Labs, or heparin from K&K Labs. Inc.) per approximately 10 mL of blood, allowing it to settle in polystyrene tubes, and then collecting the red cell poor, but platelet and white cell rich, supernatant. Curved surfaces were provided by several objects: a 1-in. diameter stainless steel ball bearing ball (radius 12.7 mm), and several plano-convex lenses with a diameter of 42 mm and radii of curvature ranging from 44 to 2000 mm. Handling and Cleaning of Test Solids. Slides and curved objects were handled with forceps. Kimwipes wound around applicator sticks were drenched in concentrated Sparkleen, and were used to brush all materials. The materials were then rinsed with large amounts of distilled water, dried in a clean air current, and passed five times across the colorless section of a gas flame. All materials were used within 10 min after cleaning and cooling; all were highly wettable for water. Procedure. All experiments were carried out at room temperature (about 20°C). The lens was placed on a glass or TaO slide. Then, 0.2 to 0.4 mL of intact plasma that had been thawed at 37°C was deposited with an automatic plastic pipet between curved surface and slide so that it spread rapidly between the two surfaces. The preparation was placed in a moist chamber, and after 10 to 30 min (effects of time within a range of 10-60 min having proved minimal), the slide wasfloodedwith large

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


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amounts of Veronal-buffered saline, slightly tilted to allow the lens to float off (a technique that proved not to damage significantly the adsorbed protein film under it), rinsed with more Veronal-buffered saline and then covered with 0.1 mL of an antiserum while wet with the saline. Then, 4 min later, the slides were rinsed with Veronal-buffered saline followed by water. The TaO slides were dried in aflowof clean air, while the glass slides were covered with a solution of one of the Coomassie Blues (250 mg of dye in 50 mL of methanol and 10 mL of glacial acetic acid, diluted to 100 mL with distilled water). Four min later, these slides were flooded and rinsed with water, and then air dried. In other experiments, red cell poor and whole heparinized blood v/ere used. Blood was applied like the plasma and rinsed off with Veronalbuffered saline, after 10 min, but these preparations were then fixed with 2.5% glutaraldehyde in the saline, and stained (if on glass) with Wright's stain or a Coomassie Blue. Preparations on TaO substrates were not stained, but rinsed with water and observed with a vertically illuminating microscope. Experiments with protein solutions were carried out as were those with plasma. When a steel ball was used, it was held in a holder that allowed it to be moved only normal to the plane of the slide, so that it could be lifted off vertically after the first Veronal-buffered saline rinse. Diameters of the resulting circular patterns were measured with calipers, and if small, with a microscope provided with a calibrated grid in the ocular. In previous studies, we avoided creating an air/solution/solid interface, where a protein film on the advancing fluid could otherwise be transferred to the solid. In the present arrangement, prewetting either the lens or slide with Veronal-buffered saline would introduce unpredictable flow and dilution effects when the protein-containing reagent was added. In several tests the following sequence of reagents was applied. Lens and slide were wetted with Veronal-buffered saline, a large amount of plasma was allowed to flow over the lens with its convex side up, and then the plasma-wetted lens was placed rapidly on the buffer-wetted slide. The results of these tests correspond to those of the experiments carried out on the wettable surfaces without prewetting. Data reported here refer to the latter technique.

Results W h e r e p l a s m a h a d r e s i d e d for 10 m i n to several hours, it left little or no i m m u n o l o g i c a l l y d e t e c t a b l e f i b r i n o g e n , except as a tiny disc a r o u n d the p o i n t of contact b e t w e e n c u r v e d a n d planar surface. F i g u r e 1 shows patterns of f i b r i n o g e n left i n a space b e t w e e n a convex lens a n d a glass slide b y i n creasingly d i l u t e d n o r m a l intact p l a s m a (top row) a n d b y f i b r i n o g e n (bottom row). C u r v a t u r e o f the lens was a p p r o x i m a t e l y 95 m m . Results w e r e o b t a i n e d w i t h u n d i l u t e d p l a s m a (top, left), p l a s m a d i l u t e d 1:10 w i t h Veronal-buffered saline (top, center), a n d p l a s m a d i l u t e d 1:50 (top, right). D a r k areas indicate deep staining d u e to the p r e s e n c e of antifibrinogen o n f i b r i n o g e n . L i t t l e f i b r i n o g e n r e m a i n e d w h e r e p l a s m a h a d b e e n injected (upper left corners of each photograph). T h e large circles c o r r e s p o n d to the areas b e t w e e n lens a n d slide that w e r e f i l l e d w i t h p l a s m a ; o n l y the center of the circle left b y u n d i l u t e d p l a s m a contains f i b r i n o g e n . B e y o n d this circle, the plasma deposited some f i b r i n o g e n d u r i n g its b r i e f contact w i t h the surface w h i l e b e i n g r i n s e d off. L i t t l e or no f i b r i n o g e n was d e p o s i t e d b y the d i l u t e d plasma samples i n the narrowest spaces, the p l a s m a that h a d b e e n d i l u t e d 1:50 showing the most n o t a b l e c e n t r a l " e m p t y " hole. A t this d i l u t i o n , the plasma



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Figure 1. Fibrinogen left between convex lens and glass slide by various dilutions of plasma (top row) and of fibrinogen solutions (bottom row) (see text). left f i b r i n o g e n w h e r e v e r it h a d r e s i d e d b e y o n d this " h o l e " , but was apparently too d i l u t e d to leave f i b r i n o g e n d u r i n g its b r i e f contact w h i l e washed b e y o n d the area of p r o l o n g e d residence. Identical e x p e r i m e n t s w e r e p e r f o r m e d w i t h solutions of f i b r i n o g e n ( M . M o s e s s o n , S U N Y at D o w n s t a t e , B r o o k l y n , N Y ; p r o t e i n m o r e than 9 0 % clottable) at concentrations of 6 ( F i g u r e 1, b o t t o m , left), 3 (center), and 0.3 (right) mg/mL of V e r o n a l - b u f f e r e d saline. T h e e m p t y center increased w i t h i n creasing d i l u t i o n . M e a s u r e m e n t s and calculations (see equation below) are given i n Table I. I n several e x p e r i m e n t s , o n l y half of the pattern left by p l a s m a was exposed to a n t i s e r u m (see F i g u r e 2). A lens w i t h a curvature of a p p r o x i mately 147 m m was u s e d o n a T a O surface, and plasma was a p p l i e d as d e s c r i b e d . T h e r i g h t half of the treated, a i r - d r i e d surface was then covered w i t h a n t i s e r u m to h u m a n f i b r i n o g e n , and r i n s e d o f f 5 m i n later w i t h V e r o n a l buffered saline f o l l o w e d b y water. A f t e r air d r y i n g , the interference pattern (purple; darkest i n the p h o t o g r a p h , i n d i c a t i n g thickest p r o t e i n layer) showed a central dark s e m i c i r c l e o n l y o n the right (antiserum) side, i n d i c a t i n g the presence of f i b r i n o g e n . A l s o o n the antiserum-treated side, b e y o n d the large


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circle w h e r e p l a s m a h a d r e s i d e d for 10 m i n , a darker area (right top and bottom o f F i g u r e 2) s h o w e d that, h e r e again, b r i e f contact w i t h plasma d u r i n g r i n s i n g had caused it to deposit b u t not yet to remove f i b r i n o g e n . T h e radius A i of the s m a l l central f i b r i n o g e n disc left b y the plasma d e p e n d e d o n the radius R o f the c u r v e d object that h a d b e e n used (see Table II) i n such a way that the h e i g h t of the p l a s m a layer at the outer edge of the


f i b r i n o g e n disc, b e i n g Ηγ = R — V ( R

2

— Ai ), was m o r e or less constant, that 2

is, about 10 μιη (see Table II a n d F i g u r e 3). S o m e n o r m a l plasma samples y i e l d e d slightly h i g h e r values.

Figure 2. Fibrinogen left by intact plasma between convex lens and anodized tantalum-sputtered glass slide. Only right half of pattern was exposed to antiserum to fibrinogen (see text).


270


T h e v e r y s m a l l " b l a n k h o l e " at the c e n t e r o f the f i b r i n o g e n disc, i n creasing i n size w i t h d i l u t i o n o f p l a s m a a n d f i b r i n o g e n solution used, h a d a radius A c o r r e s p o n d i n g to a l i q u i d layer height of H = R - V ( R - A ) (see F i g u r e 3 a n d values i n Tables I a n d II). T h e significance o f H is discussed later. 2

2

2

2

2

O n T a O , rather than o n glass, p l a s m a d i l u t e d w i t h 100 o r m o r e volumes


of V e r o n a l - b u f f e r e d saline left a r i n g o f a l b u m i n w i t h i n the " e m p t y h o l e " that lacked f i b r i n o g e n . A lens w i t h a radius of curvature of approximately 2000 m m was u s e d . T h e e x p e r i m e n t s w e r e c a r r i e d out as d e s c r i b e d , b u t antiserum to h u m a n a l b u m i n rather than to f i b r i n o g e n was used. T h e slide was r i n s e d w i t h V e r o n a l - b u f f e r e d saline 2 m i n after its application, a n d 0.1 m L o f goat antiserum to rabbit g a m m a g l o b u l i n s was a p p l i e d ; 2 m i n later the slide was r i n s e d w i t h the buffered-saline, r i n s e d w i t h water, a n d then air d r i e d . T h e

Figure 3. Top: Diagram showing outer and inner edge of the fibrinogen ring (radii A and A ) left by plasma. Outer vertical lines indicate diameter of area where plasma had resided. Bottom: Pattern of residual fibrinogen created as shown in Figure 1 but on TaO (plasma diluted 1:15). The pattern indicating antibody deposition is created by interference colors (see Figure 2). 7

2


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Table I. Radii of Clear Centers Left by Fibrinogen Solutions of Various Concentrations (Under Lens of R =95 mm) and Computed Heights of Solutions Fibrinogen mg/mL 6 3 0 3 0.06

Radius (A ) 0.7 0.5 1.75 2.75 2

Height (H ) 2.6 1.3 16 40

a

2

2JH .77 1.5 .1 .05

b

C

2

in mm. H in μιτι derived from values A and R. Expected values for mg of fibrinogen/mL on the basis of presumptions are described in the

A

a

b c

text.

2

2

2

Table II. Radii of Areas of Residual Fibrinogen Left by Intact Plasma, and Its Computed Heights 0

Dilution

Lenses

Steel Ball 12.7

R

b

Undiluted

A, H\

1:1

Ai Hi

1:10

A, H\ A H

0.46 8

44

95

147

2095

1.0 11

1.5 12

2.0 14

8.5 17

2.5 21 1.6 101 0.36 5

2

2

5.0 131 0.75 3

Ai and A in mm, Hi and H in μηη derived from values of A and R. R in mm; curvatures of lenses were calculated (a) from diameter and height of edge, (b) from focal length and presumed refractive index of lens, and (c) from Newton ring diameters. a

2

2

h

r e s u l t i n g pattern (see F i g u r e 4) i n d i c a t e d that a l b u m i n h a d b e e n d e p o s i t e d w e l l w i t h i n the area w h e r e no f i b r i n o g e n deposit h a d b e e n f o u n d . T h e d i r e c t i o n o f the t e a r d r o p shape of the d e p o s i t e d a l b u m i n r i n g d e p e n d e d o n the d i r e c t i o n i n w h i c h the p l a s m a h a d b e e n injected ( F i g u r e 4, arrow). E x p e r i m e n t s w i t h b l o o d a n d e r y t h r o c y t e - p o o r b l o o d , as d e s c r i b e d i n the E x p e r i m e n t a l section,

u s i n g the 9 5 - m m radius lens, p r o d u c e d a r i n g of

platelets o n the s l i d e . I n the absence o f significant amounts of erythrocytes, this r i n g was m u c h s m a l l e r than the r i n g d e p o s i t e d i n the presence of e r y t h rocytes (see Table III). B e y o n d a n d i n s i d e these rings, few platelets a d h e r e d to the glass u n d e r these c o n d i t i o n s . G r a n u l o c y t e s a d h e r e d mostly b e y o n d the r i n g of platelets. S o m e e x p e r i m e n t s w e r e c a r r i e d out w i t h n o r m a l intact plasma, b u t i n addition to a n t i s e r u m to f i b r i n o g e n , a n t i s e r u m to kininogens (kindly p r o v i d e d b y R. W . C o l m a n , T e m p l e U n i v e r s i t y School of M e d i c i n e , P h i l a d e l phia, P A ) was a p p l i e d . A lens w i t h an approximately 147-mm

radius of



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Figure 4. Albumin left by intact plasma diluted 1:100 between lens and anodized tantalum- sputtered glass slide (see text).

Table III. Outer Radii of Rings of Platelets Left by Heparinized Blood and by Its Erythrocyte-Poor Supernatant Between Class Side and Lens of J?=95 mm" Whole Blood Sample No. 1 2 3 4 5

Supernatant

A,

Hi

A,

2.22 1.86 5.00 1.90 2.04

26 18 132 19 22

1.32 0.96 2.00 0.96 0.84

Hi 9.2 4.8 21 4.8 3.7

"Radii of platelet rings A i in mm; H i in |xm.


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curvature was u s e d . U n d i l u t e d p l a s m a r e s i d e d b e t w e e n the lens and slide for 10 m i n . T h e two antisera w e r e each a p p l i e d to one-half of the d r i e d surface (see F i g u r e 5, a n t i s e r u m to k i n i n o g e n s o n right half). W h e r e plasma had resided for 10 m i n , l i t t l e or no f i b r i n o g e n r e m a i n e d ( F i g u r e 5, A r e a B), but k i n i n o g e n was present (Area F ) . I n the narrow, central space, f i b r i n o g e n was present (Area C ) , b u t k i n i n o g e n was not (Area G ) . W h e r e contact of the


plasma w i t h the glass h a d o c c u r r e d o n l y b r i e f l y , d u r i n g r i n s i n g , f i b r i n o g e n was d e p o s i t e d ( A r e a A ) , b u t k i n i n o g e n h a d not yet b e e n adsorbed (Area E ) . A faintly dark area was seen i n the c e n t e r b e t w e e n the two antiserum sites (Area D ) , a n d this r e s i d u a l p r o t e i n , p r e s u m a b l y f i b r i n o g e n , was r e m o v e d b y the a n t i s e r u m to k i n i n o g e n s (Area G b e i n g l i g h t e r than A r e a D ) . T h i s removal can be e x p l a i n e d as follows. M o s t sera, a n d even their p u r i f i e d g l o b u l i n s , contain intact surface-activatable c l o t t i n g factors that can displace f i b r i n o g e n . Because f i b r i n o g e n m o l e c u l e s are m u c h larger than k i n i n o g e n or factor X I I molecules, the r e p l a c e m e n t of f i b r i n o g e n causes a decrease i n f i l m thickness (1 ), so that even w i t h o u t exposure to a n t i f i b r i n o g e n , the pattern created b y this process may be d e t e c t e d . W h i l e exposure to an antiserum to f i b r i n o g e n w i l l m e r e l y enhance the f i b r i n o g e n pattern, exposure to any other antiserum may cause its h i g h m o l e c u l a r w e i g h t k i n i n o g e n and factor X I I to remove the residual f i b r i n o g e n a n d thus destroy the pattern.

Discussion T h e contrast b e t w e e n patterns o b t a i n e d w i t h antisera to f i b r i n o g e n and those o b t a i n e d w i t h a n t i s e r u m to k i n i n o g e n s indicates that n o r m a l plasma deposited f i b r i n o g e n , a n d w i t h i n 10 m i n , r e p l a c e d it on these activating substrates, b y h i g h m o l e c u l a r w e i g h t k i n i n o g e n (and p r o b a b l y b y factor X I I ) , except i n spaces of less than about 10 μπι. P r o p o r t i o n a l l y w i d e r spaces were r e q u i r e d for this r e p l a c e m e n t b y p l a s m a that had b e e n d i l u t e d . I n m u c h narrower spaces, even the d e p o s i t i o n of f i b r i n o g e n is restricted, as shown i n patterns created b y f i b r i n o g e n solutions as w e l l as by d i l u t e plasma. A few p r e s u m p t i o n s may suffice to i n t e r p r e t all results qualitatively as w e l l as quantitatively. 1. F o r most proteins, about 1 m g tends to be adsorbed on a substrate area of 1 m (9). 2

2. T h e " c r i t i c a l h e i g h t " of a p r o t e i n solution spread b e y o n d its capacity of f o r m i n g a c o m p l e t e p r o t e i n m o n o m o l e c u l a r layer on the substrate is the value H or H d e s c r i b e d p r e v i o u s l y . A plasma layer of m o r e than H μπι thick contains e n o u g h h i g h m o l e c u l a r w e i g h t k i n i n o g e n (and factor XII) to displace the f i b r i n o g e n that this plasma layer had d e p o s i t e d . A t h i n n e r layer w i l l contain insufficient h i g h m o l e c u l a r weight k i n i n o g e n (and factor XII) to displace the entire f i b r i n o g e n coating, but w i l l still contain e n o u g h f i b r i n o g e n to create this f i b r i n o g e n coating, unless the plasma layer is less than H μιη thick. x

2

{

2



274


Figure 5. Fibrinogen (left) and kininogen (right) left by normal plasma between lens and glass slide (see text).

T h e n , even the f i b r i n o g e n i n this t h i n plasma layer is insuf ficient to create a "carpet w i t h o u t h o l e s . " H o w e v e r , a l b u m i n , b e i n g m u c h m o r e abundant than f i b r i n o g e n , may then fill these holes. 3. F r o m p r e s u m p t i o n s (1) a n d (2), C m g of a p r o t e i n i n 100 m L of s o l u t i o n can cover C m , so its c r i t i c a l height H, w h e r e a single substrate surface w o u l d be i n v o l v e d , is 100/(10,000 X C ) c m or 100/C μπι. T w i c e as h i g h a layer of solution w i l l be n e e d e d b e t w e e n two surfaces. Because the surface of the c u r v e d objects w i t h i n the n a r r o w ranges of significance to this study is nearly planar, Ηχ a n d H have to be a p p r o x i m a t e l y 200/C μπι, w h e r e C represents the concentration of h i g h m o lecular w e i g h t k i n n i n o g e n ( + factor X I I ) d e r i v e d f r o m Hi, a n d the c o n c e n t r a t i o n of f i b r i n o g e n d e r i v e d f r o m H . 2

2

2

4. Platelets adhere w h e r e they f i n d adsorbed f i b r i n o g e n (6).


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Plasma n o r m a l l y contains about 1 0 - 3 0 m g of c o m b i n e d h i g h m o l e c u l a r weight k i n i n o g e n and factor X I I p e r 100 m L , and about 300 m g of f i b r i n o g e n in the same v o l u m e . F o r u n d i l u t e d plasma, Hi s h o u l d then be about 200/20 μπι, and for plasma d i l u t e d 1:10, H s h o u l d be 200/2 μπι. A c t u a l values w e r e {

about 10 a n d 120 μπι, respectively (see Table II). In u n d i l u t e d plasma, or i n f i b r i n o g e n solutions of physiological concen trations, H (the height b e l o w w h i c h the solution cannot f o r m an u n b r o k e n Downloaded by KTH ROYAL INST OF TECHNOLOGY on September 16, 2015 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch019

2

fibrinogen coating) s h o u l d be about 200/300, or 0.66 μπι, c o r r e s p o n d i n g to a central " e m p t y h o l e " w i t h a d i a m e t e r o f o n l y about 0.4 m m w i t h o u r lenses. A fuzzy circle that small cannot be m e a s u r e d easily, so our data of A and the 2

calculated H vary considerably (see Table I, u p p e r lines) w h e n u n d i l u t e d 2

plasma is used. W h e n d i l u t e d serially, solutions of f i b r i n o g e n y i e l d e d reason able values o f H (see Tables I a n d II): at 30 mg/100 m L , the expected value 2

for H is 200/30, o r about 7 μπι, w h i l e the actual value was 16; at 6 mg/100 2

m L the expected value is 200/6 , o r 33 μπι, w h i l e a value of 40 was f o u n d . H e p a r i n i z e d e r y t h r o c y t e - p o o r b l o o d left a r i n g of platelets between the lens a n d slide that was about as large as the r i n g of f i b r i n o g e n left b y u n d i l u t e d citrated plasma (compare A i n Tables II a n d III). T h e samples of x

w h o l e h e p a r i n i z e d b l o o d left a larger r i n g of platelets than d i d their m a t c h i n g samples of e r y t h r o c y t e - p o o r b l o o d . T h i s result suggests that the erythrocytes acted as a d i l u e n t o f the plasma, r e d u c i n g the amount o f h i g h m o l e c u l a r weight k i n i n o g e n (and factor XII) available to displace adsorbed f i b r i n o g e n i n areas of less than about 20 μπι, so that platelets c o u l d f i n d a w i d e r r i n g of fibrinogen to adhere to i n the presence of, rather than i n the absence of erythrocytes. T h i s explanation contrasts those p r o p o s e d b y others i n the past; namely, that erythrocytes leak a substance such as A D P w h i c h stimulates platelet adhesion (10), o r that erythrocytes u n d e r conditions of flow usually associated w i t h tests for platelet retention i n glass w o o l wicks (11) or glass bead c o l u m n s (12), mechanically force platelets into contact w i t h the glass surface (13). U n d e r the conditions o f o u r present experiments, flow was m i n i m a l , a n d p r e l i m i n a r y tests i n d i c a t e d that the f i b r i n o g e n d e p o s i t e d b y plasma i n narrow spaces may r e m a i n for several hours, suggesting that dif fusion o f h i g h m o l e c u l a r weight k i n i n o g e n into these areas has not o c c u r r e d to a measurable extent. W h e t h e r the r e m o v a l o f f i b r i n o g e n b y intact plasma represents physical or e n z y m a t i c d i s p l a c e m e n t b y h i g h m o l e c u l a r weight k i n i n o g e n a n d factor X I I , or an i n d i r e c t result of activation of p l a s m i n o g e n to the fibrino(gen)olytic e n z y m e p l a s m i n v i a t h e activation o f factor X I I (14), remains to be s t u d i e d .

Acknowledgment T h i s w o r k was s u p p o r t e d i n part b y the N a t i o n a l H e a r t , L u n g , and B l o o d Institute G r a n t N o . 1 R 0 1 H L 2 3 8 9 9 0 1 .


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Literature Cited 1. Vroman. L.; Adams, A. L.; Klings, M . ; Fischer, G. C. In "Applied Chemistry at Protein Interfaces," Advances in Chemistry Series, No. 145; ACS: Washing ton, D . C . , 1975; p. 255-289. 2. Vroman, L.; Adams, A. L . ; Fischer, G . C.; Munoz, P. C. Blood 1980, 55, 156-159. 3. Wiggins, R. C.; Bouma, B. N.; Cochrane, C. G . ; Griffin, J. H . Proc. Natl. Acad. Sci. USA 1977, 74, 4636-4640. 4. Ratnoff, O. D . ; Davie, E. W.; Mallet, D. L. J. Clin. Invest. 1961, 40, 803-819. 5. Schmaier, A. H . ; Colman, R. W.; Adams, A. L . ; Fischer, G. C. Munoz, P. C.; Vroman, L. Circulation 1980, 62, III-57. 6. Zucker, Μ .B.; Vroman, L. Proc. Soc. Exp. Biol. Med. 1969, 131, 318-320. 7. Vroman, L. Thromb. Diath. Haemorrh. 1964, 10, 455-493. 8. Adams, A. L . ; Klings, M . ; Fischer, G. C.; Vroman, L. J. Immunol. Methods 1973, 3, 227-232. 9. Langmuir, I.; Waugh, D. F. J. Am. Chem. Soc. 1940, 62, 2771-2793. 10. Hellem, A. J. Scand. J. Clin. Lab. Invest. Suppl. 1960. 11. Moolten, S. E.; Vroman, L. Am. J. Clin. Path. 1949, 19, 701-709. 12. Salzman, E. W. J. Lab. Clin. Med. 1963, 62, 724-735. 13. Goldsmith, H . L. Fed. Proc. 1971, 30, 1578-1588. 14. Goldsmith, G. H . , Jr. J. Lab. Clin. Med. 1980, 96, 222-231. R E C E I V E D for review January 16, 1981. A C C E P T E D March 3, 1981.


Proteins, Plasma, and Blood in Narrow Spaces of Clot-Promoting

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