12 Fibrinogen, Globulins, Albumin and Plasma at Interfaces
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L. VROMAN, A. L. ADAMS, M. KLINGS, and G. FISCHER Veterans Administration Hospital, Brooklyn, New York Changes in plasma protein films preadsorbed and then exposed to plasma or deposited by plasma itself, onto various surfaces were studied using ellipsometry and other techniques to observefilmthickness, antigenicity, and activity of adsorbates. Plasma deposited matter onto 7s gamma-globulins; if intact clotting factor XII was present in film or plasma, some removal followed from oxidized silicon substrate. Fibrinogen films were partially removed and, as did globulin to some extent, lost their antigenicity on exposure to intact plasma even if lacking factor XII. Antigenicity was maintained if the substrate had been non-wettable. Albumin was not adsorbed out of plasma though it competed well against purified proteins. Glass preexposed to proteins adsorbed factor XII out of plasma.
At an air interface, water arranges itself like a hydrophobic film ( 1 ) ; even in very dry air, a metallic surface will obtain such a low energy coat of water (2, 3). Coiled protein molecules also adhere to relatively simple, non-yielding surface (4) to display complex surface properties. Onto a hydrophobic solid they adhere with hydrophilic residues exposed to the aqueous phase while on a hydrophilic one their apolar residues will be exposed (5). However, the forces involved in adhesion to a simple surface such as Lucite may be far from simple (6). On more complex surfaces such as gels and cell membranes (7) and in multilayer adsorption (8), reaction rates will be incomputable. If only 14 residue segments of any protein molecule are involved in adsorption (9), the distortion needed to accomplish such adhesion must have varied and indirect effects (10 11, 12, 13, 14) rendering the adhering shape sometimes more, rather than less, antigenic (15,16) or changing its enzymatic activity which is especially sensitive to orientation (17). Neither a hard 255 In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
256
APPLIED CHEMISTRY AT PROTEIN INTERFACES
surface s u c h as g e r m a n i u m
(18)
nor a hydrophobic
one
need
(19)
destroy t h e g l o b u l a r s h a p e of the a d s o r b e d p r o t e i n m o l e c u l e .
T h u s the
a n t i g e n i c i t y of a d s o r b e d a n t i g e n c a n be r e t a i n e d sufficiently for use i n i m m u n o a s s a y s ( 20, 21)
o n a v a r i e t y of h a r d , h i g h energy, m a t e r i a l s a n d
w i t h a v a r i e t y of t e c h n i q u e s s u c h as a l l o w i n g w a t e r to condense o n glass e l l i p s o m e t r y o n s i l i c o n crystals (23),
(22),
formation on anodized tantalum
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Effects of Adsorption
or interference color p a t t e r n
(24).
on Plasmatic Clotting
Factors
I n b l o o d c l o t t i n g , w h e r e a c t i v i t y r a t h e r t h a n a n t i g e n i c i t y is i m p o r tant, b o t h the adsorbent a n d the adsorbate influence events.
Various
p r o t e o l y t i c enzymes b e c o m e active i n c l o t t i n g i f a l l o w e d to f o r m plexes w i t h the p r o p e r p h o s p h o l i p i d m i c e l l e s (25).
com-
It is the a r r a n g e m e n t
of p h o s p h o l i p i d m o l e c u l e s t h a t i n d u c e s t h e r i g h t p a i r s of
proteinaceous
c l o t t i n g factors, I X w i t h V I I I a n d t h e n X w i t h V , to c o m b i n e forces at the interface.
H e r e the e n z y m e s
(factors I X a n d X ) are h e l d at p o l a r
groups w h i l e t h e i r cofactors ( V I I I a n d V ) are h e l d at a p o l a r ones i n the m i c e l l e (26).
P e r h a p s the i n i t i a t i n g factors X I I a n d X I of this c h a i n c a n
i n t e r a c t at a n y i n t e r f a c e that initiates c l o t t i n g (27),
although purified
f a c t o r X I I , once a c t i v a t e d , activates p u r i f i e d factor X I w i t h o u t f o r m i n g a f u n c t i o n a l c o m p l e x w i t h i t (28).
However, under more physiological
c o n d i t i o n s things m a y b e different.
C o l l a g e n f a i l e d to cover itself w i t h
c l o t - p r o m o t i n g m a t e r i a l w h e n exposed to p l a s m a t h a t l a c k e d either factor X I I or factor X I ; i n a d d i t i o n , the c o l l a g e n m o l e c u l e n e e d e d its n e g a t i v e l y c h a r g e d groups as w e l l as its h e l i c a l structure to activate the X I I and X I
(29).
Ramifications
of Factor XII
Activation
A c t i v a t i o n of factor X I I leads to c o m p l e x s e v e r a l areas of p h y s i o l o g y . times i g n o r e d . X I I (31)
factors
events
(30)
connecting
Interfaces a c c i d e n t a l l y i n t r o d u c e d are some-
F o r e x a m p l e , the surface of b a c t e r i a m a y activate factor
a n d thus be responsible f o r the finding that i m m u n e
complex
activates factor X I I d i r e c t l y . T h e r e is the l a c k of s t r i c t specificity i n the actions of s e v e r a l enzymes.
F o r e x a m p l e , factor X I I is n e e d e d f o r the
c o n v e r s i o n of p r e k a l l i k r e i n ( p e r h a p s i d e n t i c a l w i t h F l e t c h e r f a c t o r ) k a l l i k r e i n b y glass (32).
to
T h e n c o n v e r s i o n of k i n i n o g e n to k i n i n b y k a l l i -
k r e i n f o l l o w s ; b u t s e v e r a l precursors m a y activate themselves as w e l l as others
(33).
T h e p a i n - p r o d u c i n g factor formed b y diluting plasma m a y be i d e n t i c a l w i t h a c t i v a t e d factor X I I ( X l l a ) a n d splits i n t o p r e k a l l i k r e i n a c t i v a t o r w i t h f a c t o r X I I a c t i v i t y a n d some fragments (34).
C o l l a g e n gains k i n i n -
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
12.
VROMAN E T AL.
generating
Protein
( kallikrein-like )
257
Interfaces
activity upon
exposure
to
plasma.
Most
significant h o w e v e r is t h a t m a x i m a l a c t i v i t y is g a i n e d o n shortest exposure (15 sec) to p l a s m a ( 3 5 ) .
Y e t , i n most laboratories the r e m o v a l of k i n i n o -
genase f r o m p l a s m a b y q u a r t z (36)
or b y C e l i t e (37)
is c a r r i e d out
w i t h o u t c o n s i d e r i n g that short r a t h e r t h a n l o n g exposure c a n b e effective.
O n some surfaces n o r m a l p l a s m a deposits
fibrinogen
most
i n two
seconds a n d converts it (renders i t u n a b l e a n y longer to attract m a t t e r out of a n t i - h u m a n
fibrinogen
sera) w i t h i n t w e n t y seconds i f t h e p l a s m a
is i n t a c t ( h a d n o t b e e n exposed
to surface t h a t activates factor X I I ) .
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F a c t o r X I I seems to h a v e n o r o l e i n this c o n v e r t i n g a c t i v i t y , b u t o n l y i n the presence
of i n t a c t factor X I I d i d p l a s m a r e m o v e
converted matter
some of
the
(39).
A n t i b o d y / a n t i g e n c o m p l e x itself m a y not a d s o r b a n d a c t i v a t e factor X I I (31),
b u t its f o r m a t i o n leads to a c t i v a t i o n of c o m p l e m e n t factor C
w h i c h i n t u r n m a y a c t i v a t e c l o t t i n g (40, 41). appear poorly
s o l u b l e a n d m a y offer
6
H e r e too, some p r o d u c t s
p h y s i c a l interfaces
rather than
chemical activity. F o r m a t i o n of the p r o t e o l y t i c p l a s m a e n z y m e , p l a s m i n , out of its precursor,
plasminogen,
r e q u i r e s factor X I I (42) m a y w e l l create
by
adding
k a o l i n or
chloroform
as w e l l as at least one cofactor.
an insoluble protein
film
at the
to
plasma
Chloroform
plasma/chloroform
interface, a g a i n i n t e r p o s i n g m o r e p h y s i c a l events. A t least one H a g e m a n factor cof actor has b e e n p u r i f i e d (43). activating powders
I t is r e m o v e d f r o m p l a s m a b y
s u c h as glass a n d is p e r h a p s
responsible
l y s i n e esterase a c t i v i t y present i n u n a c t i v a t e d p l a s m a (44).
for
the
Thus, par-
t i c u l a r amounts of c e r t a i n interfaces m a y s i m u l t a n e o u s l y a d d a n d r e m o v e e n z y m e activities. Relationships
between Adsorption
of "Plasma Proteins and
Adhesion of Platelets Activation of Clotting and Adhesion of Platelets. p o r t e d (45)
It has b e e n r e -
that a l l u n c h a r g e d h y d r o p h o b i c surfaces a d s o r b the
proteins f r o m p l a s m a i n the same u n d e s t r u c t i v e w a y , b u t the
same
adsorbed
proteins are yet u n i d e n t i f i e d . A n e x p e r i m e n t i n w h i c h a test t u b e first receives h e m o g l o b i n
and
t h e n b l o o d has l e d to the p r o p e r c o n c l u s i o n t h a t h e m o g l o b i n i n h i b i t s surface a c t i v a t i o n of c l o t t i n g (46),
b u t a w i d e r a n g e of other p a r t i a l l y
p o s i t i v e l y c h a r g e d substances w i l l c o m p e t e just as successfully for the n e g a t i v e l y c h a r g e d a c t i v a t i n g sites o n a s o l i d (47)—especially
if given
a chance to get there before f a c t o r X I I does. B l o o d p l a s m a contains some components a b l e to a t t a c h themselves to p o s i t i v e sites o n c o l l a g e n , w h i c h t h e n b e c o m e s u n a b l e to aggregate platelets (48).
N o t only the polarity
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
258
APPLIED CHEMISTRY AT PROTEIN INTERFACES
of surface groups (49)
b u t also t h e i r a b i l i t y to act specifically as a s u b -
strate f o r a p l a t e l e t e n z y m e m u s t be essential i n b i n d i n g p l a t e l e t surface to n o n - p l a t e l e t surface (50).
T h e r e f o r e the process w o u l d b e affected
by
specific r a t h e r t h a n b y g e n e r a l p r o p e r t i e s of activators a n d i n h i b i t o r s . F o r e x a m p l e , o n l y a specific g r o u p of l i p o p r o t e i n s , p e r h a p s b y m a s k i n g c e r t a i n sites o n platelets, enhances t h e i r a g g r e g a t i o n (51);
certain basic
p o l y m e r s p r o m o t e a g g r e g a t i o n a n d the release of p l a t e l e t m a t e r i a l b u t d o r e q u i r e adenosine
diphosphate
(ADP)
specifically (52).
It seems
l i k e l y t h a t a l l s u r f a c e - r e l a t e d p r o p e r t i e s of p l a t e l e t s : a d h e s i o n , a g g r e g a -
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t i o n , release ( 5 3 ) , a n d clot r e t r a c t i o n (54)
have their o w n requirements.
W h e r e a d h e s i o n or a g g r e g a t i o n of platelets is f o l l o w e d b y release
of
c l o t t i n g f a c t o r m a t e r i a l , n e w p a t h w a y s m a y l e a d to i n i t i a t i o n of c l o t t i n g w h i l e b y p a s s i n g factors X I I a n d X I (55, 56, 57).
W i t h the v i e w to l i n k
c l o t t i n g a n d platelets w i t h t h r o m b o s i s , efforts h a v e b e e n m a d e to s h o w that a c t i v a t i o n of factor X I I leads to p l a t e l e t a d h e s i o n in vivo, b u t e v i d e n c e is s t i l l insufficient (58).
A l s o , o t h e r factors m a y p r e c e d e factor X I I
i n s u c h a c h a i n ; F l e t c h e r factor deficiency is c o r r e c t e d in vitro b y a c t i v a t e d factor X I I (59), the interface. (60),
a n d p e r h a p s this factor precedes factor X I I at
Therefore,
d i a g r a m s of
clotting may
b u t there is always>room at the top.
enzyme
appear
complete
Elsewhere, even a c o m m o n
m a y penetrate the system; for e x a m p l e , t r y p s i n c a n
f a c t o r XI
Adsorption of Other Proteins, and Adhesion vs. Platelets.
activate
(61). Aggregation
of
W e h a v e b e e n interested i n proteins t h a t p l a s m a m a y deposit
a l o n g w i t h factor X I I ( a n d p e r h a p s F l e t c h e r factor a n d factor X I )
on
v a r i o u s surfaces. F i b r i n o g e n seems to p l a y a p e c u l i a r r o l e (38, 39, 62-83). O n t o a glass-like suface, n o r m a l i n t a c t p l a s m a deposits f a c t o r X I I a n d fibrinogen
w i t h i n a f e w seconds at a rate that seems i n d e p e n d e n t of the
p a r t n e r protein's presence i n s o l u t i o n (62).
D o e s either of these
p l a t e l e t a d h e s i o n in vitro or t h r o m b o s i s in vivo?
cause
T h e p r o p e r t i e s of a
surface that d e t e r m i n e its t h r o m b o g e n i c i t y in vivo m a y not be r e l a t e d to its a b i l i t y to m a k e platelets a d h e r e in vitro. T o f o r m a t h r o m b u s , platelets m u s t a d h e r e to platelets (63).
M u c h w o r k has b e e n done to
find
a
specific p r o t e i n t h a t w o u l d act as a g l u e o n either the p l a t e l e t or the s o l i d surface.
Patients l a c k i n g
fibrinogen
c o n g e n i t a l l y (64)
as a r e s u l t of streptokinase injections ( 6 5 )
or p e r h a p s
h a v e platelets w i t h p o o r a d -
h e s i o n to glass, b u t these experiments f a i l e d to d e t e r m i n e w h e t h e r the glue p r o t e i n m u s t b e l o c a t e d o n p l a t e l e t or s o l i d . It m a y b e c o m e active or adhesive u n d e r the i n f l u e n c e of d i s t o r t i n g forces at the i n t e r f a c e 66).
T h e distortions t h a t proteins other t h a n
fibrinogen
may
(19,
undergo
at the site of a w o u n d a p p e a r insufficient to cause p l a t e l e t a d h e s i o n a n d a g g r e g a t i o n : the b l e e d i n g t i m e of a f i b r i n o g e n e m i a patients is p r o l o n g e d a n d c o r r e c t e d b y i n f u s i o n of
fibrinogen
(67).
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
12.
VROMAN E T AL.
Protein
259
Interfaces
It has b e e n suggested t h a t c a t i o n i c proteins—i.e., XII,
fibrinogen,
factor
a n d g a m m a - g l o b u l i n s — c a n a l l restore t h e a g g r e g a t i n g a b i l i t y of
w a s h e d platelets a n d d o so b y r e d u c i n g t h e n e t n e g a t i v e c h a r g e o n t h e platelet membrane
T h a t platelets w i l l also a d h e r e to n e g a t i v e l y
(68).
c h a r g e d surfaces, s u c h as those c o a t e d w i t h h e p a r i n , is e x p l a i n e d b y a s s u m i n g that the h e p a r i n i z e d surface first adsorbs a p r o t e i n f r o m t h e p l a s m a (69).
O t h e r s h a v e suggested it is
needed for platelet aggregation
fibrinogen
e s p e c i a l l y w h i c h is
(70, 71) b u t f o u n d that at least i n a
s o l u t i o n c o n t a i n i n g g e l a t i n glass coated w i t h g l o b u l i n s r a t h e r t h a n w i t h
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fibrinogen nin
c a u s e d platelets n o t o n l y to a d h e r e b u t t h e n to release serotoP o l y s t y r e n e i n t w o configurations a p p e a r e d t o adsorb
(72).
more
g l o b u l i n a n d h e n c e cause m o r e platelet a g g r e g a t i o n i n one c o n f i g u r a t i o n t h a n i n t h e other (73).
S i m i l a r efforts to see i f c o n f o r m a t i o n changes i n
m o r e n a t u r a l substrates, s u c h as c o l l a g e n , fibrin, a n d
fibrin-coated
collagen
(74) h a v e significant consequences a n d h a v e b e e n unsuccessful. Adsorption of Proteins out of Plasma; Relationship of Their Fate Platelets s u s p e n d e d i n s e r u m , the l i q u i d i n
to Adhesion of Platelets.
b l o o d left after c l o t t i n g a n d therefore l a c k i n g fibrinogen, or i n the p l a s m a of a p a t i e n t suffering f r o m a f i b r i n o g e n e m i a a d h e r e d o n l y to glass that had
adsorbed
fibrinogen
on it. T h e
fibrinogen
film
could be deposited
either b y exposure of the glass to fibrinogen s o l u t i o n , or to n o r m a l p l a s m a d u r i n g exposure of less t h a n a b o u t 5 sec (75).
L o n g e r exposure, c a u s i n g
the p l a s m a to convert t h e a d s o r b e d
left a film to w h i c h p l a t e -
lets w o u l d no l o n g e r adhere.
fibrinogen,
I n a range of 0.5 to 10 m g %
v a l u e b e i n g a b o u t 300 m g % ), i n c r e a s e d c o n c e n t r a t i o n of
(a normal
fibrinogen
in
p l a s m a w a s reflected b y i n c r e a s e d n u m b e r s of platelets p e r u n i t surface area of glass f o u n d to adhere (76).
Platelets a d h e r e d m o r e o n c o l l a g e n
i n presence of l o w concentrations of
fibrinogen
t h a n of other p r o t e i n s
tested (77), t h o u g h a l b u m i n at p h y s i o l o g i c a l concentrations times those of necessary f o r for a d h e s i o n
fibrinogen) fibrinogen
(78).
w a s also effective.
( a b o u t 10
W h i l e A D P was found
to cause platelet a g g r e g a t i o n , i t w a s n o t n e e d e d
T h i s is e v i d e n c e t h a t a difference exists
between
platelet-to-platelet a n d p l a t e l e t - t o - s o l i d a d h e s i o n . Could
platelet
surface
a d s o r b e d elsewhere? in vitro.
form
fibrinogen
on the platelet
dimers
fibrinogen;
T h e less dense ones adsorb most
adsorb i n j e c t e d over
fibrinogen
Platelets d o a d s o r b
(79),
with
fibrinogen
b o t h in vivo a n d b u t t h e i r a b i l i t y to
in vivo at a l l w o u l d suggest a r a p i d t u r n -
membrane.
A l s o , t h e amounts
of
fibrinogen
that w i l l a i d a g g r e g a t i o n p r o v o k e d b y other means are v e r y s m a l l 80).
(77,
F u r t h e r studies are r e q u i r e d to assemble these facts i n t o a coherent
theory. C e r t a i n platelets u n d e r c e r t a i n c o n d i t i o n s w i l l adhere to
fibrino-
gen-coated surfaces w i t h o u t r e l e a s i n g t h e i r a g g r e g a t i n g m a t e r i a l , w h i l e
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
260
APPLIED CHEMISTRY AT PROTEIN INTERFACES
on gamma-globulin-coated f o l l o w (81). fibrinogen
Platelet
surfaces release, a n d t h e n a g g r e g a t i o n
fibrinogen
a n d o c c u r r i n g m o s t l y i n p l a t e l e t granules
(82)
behave unlike the
fibrinogen
will
b e i n g s o m e w h a t different f r o m p l a s m a (83)
may
w e w o r k w i t h o r m a y not b e a v a i l a b l e to
react at a l l . G a m m a - g l o b u l i n s a d s o r b e d onto latex (84)
or glass
(72)
c a u s e d platelet a g g r e g a t i o n , b u t o n glass this was true o n l y i f the platelets were suspended i n an unphysiological m e d i u m . t h e y a d h e r e d , t h o u g h w i t h o u t a g g r e g a t i n g , to
In their o w n plasma,
fibrinogen-coated
surfaces.
S i n c e a l l of these proteins are a v a i l a b l e to the platelets i n t h e i r n a t i v e
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m e d i u m ( the p l a s m a ) itself, s u c h a p r o t e i n ( as w e l l as a n y specific p l a t e let e n z y m e substrate) w o u l d h a v e to c h a n g e c o n s i d e r a b l y to b e c o m e a glue.
P e r h a p s the n o r m a l l y d i s s o l v e d m o l e c u l e s s i m p l y b e c o m e a b n o r -
m a l l y c o n c e n t r a t e d , o r i e n t e d , or p a c k e d at the s o l i d i n t e r f a c e , or t h e y undergo
a change
attractive.
i n conformation
f o r c i n g t h e m to expose s o m e t h i n g
F o r e x a m p l e , as c l o t t i n g f o l l o w s surface a c t i v a t i o n of f a c t o r
X I I , so a c h a i n of reactions c a n f o l l o w the f o r m a t i o n of a c o m p l e x tween
antigen
(such
as a f o r e i g n
protein)
and
antibody.
a d h e r e to a surface that has b e e n exposed to s u c h i m m u n e reactions once complement ponent C
6
component
C
3
h a d joined.
W h e n complement
also j o i n e d , the platelets l y s e d (86).
be-
Platelets (85) com-
O n the other h a n d , no
c o m p l e m e n t was n e e d e d for g a m m a - g l o b u l i n s o n latex to m a k e platelets release t h e i r n u c l e o t i d e s
(87);
t h e g l o b u l i n s h a d m e r e l y to be i n the
a d s o r b e d state. W h e t h e r or n o t t h e c o m p l e m e n t components
themselves
are s t i c k y , i t has seemed that o n l y those surfaces a b l e to a c t i v a t e the c h a i n of c o m p l e m e n t release m a t t e r (88,
reactions
c a n cause
platelets to aggregate
and
89).
T h e p l a t e l e t enzymes i n v o l v e d (90), actions a n d of others (91),
the c o m p l e x i t y of t h e i r i n t e r -
and their relationships, both physiologically
a n d p e r h a p s p h i l o g e n e t i c a l l y , w i t h the reactions of c e r t a i n w h i t e b l o o d cells (90, 9 2 ) h e l p us to see platelets as l i v i n g p a r t i c l e s . T h e i r a g g r e g a t i o n is u s e d as a q u a n t i t a t i v e a i d i n i m m u n o a s s a y (93)
i n a technique
that m a y w e l l b e a p p l i c a b l e to q u a n t i t a t i o n of heat aggregatable g l o b u l i n s (94)
a n d of specific i m m u n o g l o b u l i n G
gamma-
(95).
The Search for Surfaces to Which Platelets Will Not
Adhere
I t is d e s i r a b l e t o find a s i m p l e surface p r o p e r t y that w i l l i n d u c e a n e q u a l l y s i m p l e a n d h e n c e p r e d i c t a b l e b e h a v i o r of p l a s m a a n d its p r o t e i n s a n d t h e n of b l o o d a n d its platelets. A r e s i m p l e g u i d e l i n e s for b u i l d i n g non-thrombogenic (96),
flow
m a t e r i a l s a v a i l a b l e or
(97, 98, 99,100),
even
possible?
Wettability
a n d the effects of a i r / l i q u i d interfaces (101 )
a l l seem to b e r e l a t i v e l y s i m p l e , p h y s i c a l factors w i t h a clear effect o n p l a t e l e t a d h e s i o n . P h y s i c a l , h y d r o p h o b i c b o n d i n g , e.g.,
a force i m p o s e d
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
12.
VROMAN E T AL.
Protein
261
Interfaces
b y t h e c o m p l e x i t i e s of w a t e r , m a y b e i n v o l v e d i n p l a t e l e t a g g r e g a t i o n (102) as w e l l as i n a d h e s i o n . C h a r g e d i s t r i b u t i o n s o n t h e p l a t e l e t surface are p r o b a b l y also r e q u i r e d (103) a n d m a y w e l l b e so specific that t h e y act r a t h e r l i k e c h e m i c a l l y acute a n d d i s c r i m i n a t i n g sense organs w i t h t h e a b i l i t y to r e c e i v e a s h a r p i m a g e o f t h e i r n a t u r a l w o r l d a n d of its abilities to change. F o r platelets n o t to a d h e r e , aggregate, a n d cause t h r o m b o s i s , a n o n - t h r o m b o g e n i c surface m a y h a v e to r e a c t as w e l l as l o o k n a t u r a l H e p a r i n has b e e n t h o u g h t a n a t u r a l a n t i t h r o m b o t i c surface c o n -
(104).
stituent, b u t o n a surface this a n t i c o a g u l a n t m a y cause m o r e r a t h e r t h a n
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less a d s o r p t i o n of proteins a n d a c t i v a t i o n of factor X I I (105), d e p e n d i n g o n t h e substrate c a r r y i n g t h e h e p a r i n (106). Albumin.
T h i s p r o t e i n m a y f o r m a m o r e n o n - t h r o m b o g e n i c surface
t h a n does h e p a r i n . I t is t h e most a b u n d a n t p l a s m a p r o t e i n . A l b u m i n s h a v e b e e n s t u d i e d extensively i n surface c h e m i s t r y laboratories. B o v i n e s e r u m w a s f o u n d to b e a d s o r b e d m o s t s t r o n g l y onto h y d r o p h o b i c surfaces (107) a n d i n t u r n b i n d s a r o m a t i c c o m p o u n d s b o n d i n g (108).
mostly b y hydrophobic
U n d e r a v a r i e t y of c o n d i t i o n s , platelets d o n o t a d h e r e
to a l b u m i n - c o a t e d glass (72, 75), b u t t h e h y d r o p h o b i c n a t u r e per se of the a l b u m i n film f a c i n g t h e platelets m a y n o t b e r e s p o n s i b l e .
Albumin
f o r m i n g a p r o t e c t i v e c o a t i n g o n platelets i n c e r t a i n e x p e r i m e n t s
(109)
m a y d o so b y u s i n g h y d r o p h o b i c b o n d i n g p r e c i s e l y to a d h e r e to t h e platelet m e m b r a n e .
C o n s i d e r i n g the h i g h concentration of a l b u m i n i n
p l a s m a , a n d t h e h i g h a v a i l a b i l i t y o f its h y d r o p h o b i c , t h o u g h p e r h a p s often
occupied,
albumin
even
sites, o u r o b s e r v a t i o n onto
hydrophobic
that plasma never
surfaces
(23,
38,
deposited
110, 111, 112,
113, 114), is s u r p r i s i n g . Present Efforts to Unravel
Interactions Among
Plasma
and Its Proteins at an Interface It is clear t h a t : a ) p l a s m a deposits s e v e r a l proteins onto o n e m a t e r i a l ; b ) i t t h e n affects e a c h d e p o s i t e d p r o t e i n d i f f e r e n t l y ; c ) n e i t h e r c o m p e t i t i o n a m o n g these proteins f o r a surface n o r t h e i r subsequent fate i n t h e p l a s m a / s o l i d i n t e r f a c e is s i m p l y p h y s i c a l . T o separate these o v e r l a p p i n g events, e a c h p u r i f i e d p r o t e i n t y p e w a s p r e - a d s o r b e d s i n g l y onto a v e r y s i m p l e surface a n d t h e n exposed to p l a s m a . T h e results w e r e r e c o r d e d i n terms of thickness a n d a n t i g e n i c i t y . T h e sequences w e r e p u t together to m o d e l t h e b e h a v i o r of w h o l e p l a s m a m e e t i n g this s i m p l e surface. O n e s u c h surface w a s that of a n a c i d - t r e a t e d s i l i c o n c r y s t a l slice.
I t also
s e r v e d to a n s w e r questions s u c h as: does a l b u m i n c o m p e t e as p o o r l y against separate proteins as i t does i n w h o l e p l a s m a ? D o h i g h l y c h a r g e d m o l e c u l e s s u c h as h e p a r i n affect t h e fate of p r e - a d s o r b e d p r o t e i n
films?
A s i n t h e past (22, 23, 24, 38, 75, 106, 111, 112, 113, 114), o u r m e t h o d s
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
262
APPLIED CHEMISTRY AT PROTEIN INTERFACES
i n c l u d e e l l i p s o m e t r y , a n o d i z e d t a n t a l u m interference color
observation,
w a t e r v a p o r c o n d e n s a t i o n p a t t e r n a n d C o o m a s s i e B l u e studies, a d h e s i o n of platelets, a n d the a b i l i t y of p l a s m a deposits to correct t h e c l o t t i n g of f a c t o r X I I deficient p l a s m a . T h e latter t w o m e t h o d s m a y give a m e a s u r e of the significance of a deposit to platelets a n d c l o t t i n g u n d e r o u r e x p e r i mental conditions.
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A l t h o u g h the d a t a s h o w a r e l a t i o n s h i p b e t w e e n the a b i l i t y of a sur face to a d s o r b
fibrinogen
this
fibrinogen,
a n d the t e n d e n c y of platelets to a d h e r e , i t does n o t c o n
out of p l a s m a , the plasma's i n a b i l i t y to convert
firm
that a d s o r p t i o n of
fibrinogen
m u s t p r e c e d e a d h e s i o n of platelets to
a l l k i n d s of surface, o r t h a t surfaces w h i c h a d s o r b
fibrinogen
u n d e r these
c o n d i t i o n s w i l l be b a d b i o m a t e r i a l s in vivo as h e a r t valves, b l o o d vessels, or c a n u l a e , or ex vivo as a r t i f i c i a l k i d n e y m e m b r a n e s — e v e n
t h o u g h the
latter are most l i k e l y to b e i m p e d e d b y a n y adsorbate. D a t a presented supported
here represent m o s t l y single observations
i n subsequent
modifications
of
each
experiment.
further Because
e l l i p s o m e t r y r e c o r d i n g s are t i m e c o n s u m i n g , a n u m b e r of v a r i a b l e s , s u c h as p H a n d c o m p o s i t i o n of buffer s o l u t i o n , h a v e b e e n k e p t constant. Experimental M a t e r i a l s . T h e m a t e r i a l s u s e d i n these experiments are l i s t e d w i t h t h e i r source a n d p r e p a r a t o r y details. S i l i c o n c r y s t a l slices, η-type, o x i d i z e d ( S i O ) , are u s e d as d e s c r i b e d ( 2 3 ) . 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 ( T a O ) was o b t a i n e d f r o m M i l l i s R e s e a r c h , M i l l i s , M a s s . B u f f e r ( V S ) , isotonic V e r o n a l s o l u t i o n w a s a d j u s t e d to p H 7.4 a n d d i l u t e d 1:4 w i t h 0 . 8 5 % N a C l . 15 m l w e r e u s e d p e r e l l i p s o m e t e r cuvet. A l b u m i n , h u m a n , c r y s t a l l i n e , 1 0 0 % ( M a n n R e search L a b s . , N e w Y o r k , Ν. Y . ) , was p r e p a r e d i n s o l u t i o n of 2 m g / m l V S . 0.2 m l w e r e u s e d p e r e x p e r i m e n t . F i b r i n o g e n , h u m a n , l y o p h i l i z e d , p l a s minogen-free, protein 9 6 % clottable containing 0.4% sodium citrate and 0.9% N a C l (from A l a n J . Johnson: N e w Y o r k U n i v e r s i t y ) was prepared as 3 m g / m l V S . 0.4 m l w a s u s e d p e r e x p e r i m e n t unless o t h e r w i s e i n d i cated. F r a c t i o n I (fibrinogen), h u m a n ( M a n n Research L a b s . ) was pre p a r e d i n s o l u t i o n of 3 m g / m l V S a n d 4 m l w a s a d d e d p e r experiment. 7s gamma-globulins, h u m a n , chromatographically isolated 1 0 0 % ( M a n n R e s e a r c h L a b s . ) i n c o n c e n t r a t i o n of 8 m g / m l V S ; 0.2 m l was a d d e d p e r e x p e r i m e n t . F r a c t i o n I I I - I , h u m a n ( H y l a n d - d i v . of T r a v e n o l , C a l i f . ) i n c o n c e n t r a t i o n of 3 m g / m l V S ; 0.2 m l was a d d e d per experiment., E p s i l o n - A m i n o - n - C a p r o i c A c i d ( E A C A ) , C P . , homogeneous ( M a n n R e s e a r c h L a b s . ) i n c o n c e n t r a t i o n of 0 . 1 M ; 0.4 m l p e r e x p e r i m e n t w a s added. Plasminogen, human, purified ( M a n n Research Labs. ) i n concen t r a t i o n of 1 m g / m l V S (15 R P M I u n i t s / m g ) ; 0.4 m l was a d d e d p e r experiment. A p p r o x i m a t e l y 4 4 - 4 5 u n i t amounts ( d i s s o l v e d i n V S ) of S t r e p t o k i n a s e ( S K ) , 100,000 C h r i s t e n s e n u n i t s / v i a l . 0.1 m g = 400 units ( L e d e r l e Laboratories) was used per experiment. T r y p s i n Inhibitor, S o y b e a n ( S B T I ) , c h r o m a t o g r a p h i c ( c o m p o n e n t V I , salt free, l y o p h . ) , 10,000 B A E E u n i t s of i n h i b i t i o n p e r m g ( M a n n R e s e a r c h L a b s . ) , w a s
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch012
12.
VROMAN ET A L .
Protein
Interfaces
263
p r e p a r e d as 2 m g / m l V S . P l a t e l i n ( G e n e r a l D i a g n o s t i c s D i v . of W a r n e r C h i l c o t t , M o r r i s P l a i n s , N . J . ) was u s e d as d i r e c t e d b y the m a n u f a c t u r e r . Stock s o l u t i o n of C o o m a s s i e B r i l l i a n t B l u e R ( S i g m a C h e m i c a l C o . , St. L o u i s ) w a s p r e p a r e d b y d i s s o l v i n g 2.5 m g i n 50 v o l % m e t h a n o l a n d 10 v o l % acetic a c i d q.s. to 100 cc. F o r s t a i n i n g , the stock w a s d i l u t e d 2 0 - f o l d w i t h a s o l u t i o n of 40 v o l % m e t h a n o l , 5 v o l % acetic a c i d , a n d 2.5 v o l % g l y c e r o l . H e p a r i n , N a , 100,000 i n t e r n a t i o n a l u n i t s / g m ( K & K L a b s . , N e w Y o r k , Ν. Y . ) i n c o n c e n t r a t i o n of 14 m g / m l V S ; 0.1 m l was a d d e d per experiment. P r o t a m i n e ( S a l m i n e sulfate) ( P S ) ( G e n e r a l B i o c h e m i c a l s , C h a g r i n F a l l s , O h i o ) i n c o n c e n t r a t i o n of 2 m g / m l V S ; 0.4 m l was a d d e d p e r experiment. N o r m a l i n t a c t p l a s m a , c o l l e c t e d i n 0.1 v o l . A C D , r e n d e r e d p l a t e l e t p o o r b y c e n t r i f u g a t i o n , a n d stored i n p o l y s t y r e n e tubes ( F a l c o n Plastics D i v . , B e c t o n , D i c k i n s o n , & C o . , O x n a r d , C a l i f . ) at — 4 0 ° C w a s u s e d w i t h i n 4 hrs after t h a w i n g . A c t i v a t e d p l a s m a was p r e p a r e d b y exposing n o r m a l i n t a c t p l a s m a to 60 m g S p e e d e x ( G r e a t L a k e s C a r b o n C o r p . , L o s A n g e l e s ) p e r m l p l a s m a for 10 m i n , c e n t r i f u g i n g , a n d c o l l e c t i n g the supernatant. F a c t o r X I I deficient p l a s m a was c o l l e c t e d i n a 3 % s o d i u m citrate f r o m a p a t i e n t w i t h severe factor X I I deficiency. N o r m a l s e r u m was p o o l e d f r o m n o r m a l donors, stored for several hours at 3 7 ° C , a n d f r o z e n i n glass tubes. R a b b i t antisera to the f o l l o w i n g h u m a n p r o t e i n s : F r o m H y l a n d , antisera to a l b u m i n ( l i s t # 0 7 1 - 1 0 7 ) , fibrinogen (list # 0 7 1 - 1 0 8 ) , h u m a n f r a c t i o n I I I - I ( l i s t # 0 7 1 - 1 0 3 ) , β-lipoproteins ( l i s t # 0 7 1 - 1 1 3 ) , h u m a n s e r u m ( l i s t # 0 7 1 - 1 2 1 ) , F a b ( 0 7 1 - 2 5 8 ) , F c ( 0 7 1 - 2 5 9 ) . F r o m M a n n R e s e a r c h L a b s . , antisera to 7s g a m m a - g l o b u l i n s (cat. # 2 3 1 ) a n d t o t a l g a m m a - g l o b u l i n s (cat. # 8 0 9 0 ) . F r o m H o e c h s t , W o o d b u r y , Ν. Y . : a n t i s e r u m to p r e - a l b u m i n ( c o d e #8506). Techniques. E l l i p s o m e t r y was c a r r i e d out as f o l l o w s : a n S i O c r y s t a l slice c o v e r e d w i t h test m a t e r i a l is p l a c e d i n 15 m l V S w h i c h is s t i r r e d b y v e r t i c a l m o t i o n a n d k e p t at 37 ° C . R e a d i n g s are t a k e n , a n d the r e c o r d i n g was started. O f t e n , 0.1 m l n o r m a l i n t a c t p l a s m a is a d d e d to the V S , a n d the m i x t u r e is r e p l a c e d b y fresh V S t w i c e , after 2 m i n , a n d i n other series after 45 m i n . R e a d i n g s are r e p e a t e d , a n d the differences b e t w e e n first ( b l a n k ) a n d s e c o n d r e a d i n g s of m i n i m u m l i g h t t r a n s m i t t i n g positions of the a n a l y z e r (for the e l l i p s o m e t e r w i t h q u a r t e r w a v e p l a t e p l a c e d after reflecting s u r f a c e ) or p o l a r i z e r (for t h e ellipsometer w i t h q u a r t e r w a v e p l a t e p l a c e d before reflecting s u r f a c e ) is r e p o r t e d . 0.1 m l of a n t i - h u m a n fibrinogen, a n t i - h u m a n 7s g a m m a - g l o b u l i n s or a n t i - h u m a n a l b u m i n s e r u m is a d d e d , f o l l o w e d 45 m i n later b y r e p l a c i n g c u v e t contents w i t h fresh V S , a n d readings are t a k e n . V a r i o u s sequences of the antisera a d d i tions w e r e u s e d since w e f o u n d a n t i g e n i c i t y of a p r o t e i n film m a y be d e s t r o y e d b y e x p o s i n g i t to a n o n - m a t c h i n g a n t i s e r u m . T h e final a n t i s e r u m was one against t o t a l h u m a n s e r u m . T a O interference colors are o b s e r v e d on a 1 X 3- or 2 X 3 - i n c h T a O slide c o v e r e d w i t h test m a t e r i a l . S o m e V S is d r o p p e d o n the slide a n d some n o r m a l i n t a c t p l a s m a is p l a c e d i n t o this d r o p . A f t e r 10 m i n , another d r o p of V S is p l a c e d elsewhere o n the slide a n d p e r m i t t e d to r u n i n t o the V S - p l a s m a m i x t u r e . A s soon as the d r o p s flow together, the entire slide
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
264
APPLIED CHEMISTRY A T PROTEIN INTERFACES
Table I.
Behavior of N o r m a l Intact Plasma at 7s
Normal
Intact
Plasma
Top
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Exp. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 α 6 c
Globulin 2.30 2.85 2.91 2.93 2.81 2.25 2.04 2.48 2.23 3.09 2.33 2.39 2.94 2.46 1.83
time min 10 15 20 10 10 20
Bottom meas. 0.97 1.18 1.10 1.60 2.28 0.87* 1.21* 1.44 1.67 1.13 0.38 1.24 1.36 1.43' 0.26
—
25 60 10 45 10 20 20 2
c
c
d
time min
meas.
— — —
— — —
180 210 40 150
-1.86 -1.93 -1.02 -0.73
120 220
-0.39 -1.24
20 140 180
-0.09 -1.08 -0.89
—
—
—
—
—
—
e
EACA
SK
— — — — — — — — — — —
— — — — — — — — — —
-0.54
— — —
0.15
— — — —
Plasma premixed 1:1 with S K . Plasma premixed 1:2 with E A C A . Plasma premixed 1:1 with PS (1 mg/ml VS).
30
60
90
MINUTES
Figure 1. Tracing of ellipsometer recording, starting when 0.1 ml normal intact plasma had been added to the 15 ml buffer containing a slice of oxidized silicon on which 7s gamma-globulins had been adsorbed. Curve shows adsorption of about 60 A , followed by desorption. Between R and R, curve was reversed by reversing analyzer deviation from minimal light transmitting position.
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
12.
VROMAN E T AL.
Protein
265
Interfaces
G a m m a - G l o b u l i n - C o a t e d O x i d i z e d Silicon Surfaces Normal
Intact
Top time min
Plasma
Antiserum
meas.
time min
meas.
SK
Globulin
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To
Bottom Fr. llli
Fibrinogen
3.92 2.81
-0.29 10 10 10 12 10
0.50 1.01 0.62 1.04 0.25
840 35 840 100 60
-0.66 -0.09 -0.84 -0.07 -0.41
— -1.66
1.31 —
-0.07
—
Plasminogen
—
0.21 d e /
Plasma premixed with antiplasminogen. Cuvet contents changed 10 min after the start of removal. Plasma premixed 1:1 with soybean trypsin inhibitor (2 mg/ml VS).
is r i n s e d w i t h m o r e V S a n d t h e n w i t h d i s t i l l e d w a t e r after w h i c h i t is a l l o w e d to d r i p d r y . T h e s l i d e thus consists of 2 areas, one area w h e r e p l a s m a o n l y r e s i d e d a f e w seconds or less a n d a n o t h e r w h e r e i t r e s i d e d for 10 m i n . O n b o t h areas, d r o p s of v a r i o u s antisera are p l a c e d , r i n s e d off w i t h V S , a n d t h e n w i t h w a t e r after 1 or 2 m i n . O n b a r e T a O surfaces, the interference colors of n a t u r a l , v e r t i c a l l y ( n o r m a l ) reflected l i g h t a r e : b r o n z e for the u n t r e a t e d surface, r e d d i s h p u r p l e for s u r f a c e exposed to p l a s m a or p r o t e i n , a n d d e e p v i o l e t for sites w h e r e m a t c h i n g antisera h a d r e s i d e d . O n T a O surfaces p r e t r e a t e d w i t h a b i o m a t e r i a l w h i c h itself causes a shift i n color, a shift t o w a r d b l u e w i l l b e n o t e d i n subsequent deposits. G l a s s surfaces w e r e u s e d i n the same m a n n e r as w e r e T a O surfaces. T h e s e surfaces w e r e o b s e r v e d for a n t i g e n / a n t i b o d y r e a c t i o n sites either b y v a p o r p a t t e r n t e c h n i q u e or b y s t a i n i n g . W a t e r v a p o r p a t t e r n a l l o w s d e t e c t i o n of w e t t a b l e r e a c t i o n sites, as r e p o r t e d ( 2 2 ) . F o r s t a i n i n g , slides are c o v e r e d w i t h C o o m a s s i e B r i l l i a n t B l u e R s o l u t i o n w h i l e w e t a n d r i n s e d w i t h w a t e r 5 m i n later. D r i e d surfaces w e r e p l a c e d face d o w n o n y e l l o w p a p e r for o b s e r v a t i o n . Results and
Discussion
Ellipsometry Data.
M o s t experiments w e r e c a r r i e d out i n 15 m l of
V S at 3 7 ° C o n S i O surfaces. V a l u e s i n the tables u s u a l l y represent single e x p e r i m e n t s ; exceptions are n o t e d . I n a l l tables values are g i v e n i n c h r o n o l o g i c a l s e q u e n c e of a d d i t i v e s f r o m left to r i g h t . T h e r e a d i n g s represent
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
266
APPLIED CHEMISTRY AT PROTEIN INTERFACES
Table II.
Behavior of Factor XII Deficient and of Activated
Normal Intact Plasma
XII-Deficient
Top
Top
Bottom
Globulin
time min
meas.
time min
meas.
time min
meas.
1 2 3 4
2.83 2.90 2.60 2.36
10 5
0.84 1.12
10
0.06
220
-0.81
—
—
10 10 10
1.38 1.42* 2.39
—
120 110 110
-0.17 -2.80 -0.15
5 6
2.34 2.93
— —
— —
— —
Exp. No.
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Plasma
—
— — — — —
— — — — —
— —
6
—
° Streptokinase (44-45 u) added to the plasma without rinsing after the curve leveled off. a p e a k or e q u i l i b r i u m p o i n t .
W h e n recordings showed a
down-slope,
suggesting d e s o r p t i o n , the p o l a r i z e r or a n a l y z e r ( d e p e n d i n g o n the e l l i p someter u s e d ) was t u r n e d b e y o n d its m i n i m u m l i g h t t r a n s m i t t i n g p o s i t i o n to a p o i n t w h e r e the r e c o r d e r p e n h a d b e e n i m m e d i a t e l y before. T h e angle of the e l e m e n t w a s n o t e d a l l o w i n g a d d i t i o n a l checks of o p t i c a l thickness change as w e l l as r e v e r s a l of the c u r v e i f t r u e d e s o r p t i o n w a s t a k i n g p l a c e . C u r v e s are not r e v e r s e d b y this m a n i p u l a t i o n b u t c o n t i n u e to d r o p i f a n a i r b u b b l e or a n increase i n t u r b i d i t y of the s o l u t i o n w e r e t h e cause. T y p e s of antisera w e r e a d d e d i n different sequences f o l l o w i n g otherw i s e i d e n t i c a l e x p e r i m e n t s , e.g. i n T a b l e V I , E x p . N o . 1, a n t i - f i b r i n o g e n p r e c e d e d a n t i - g l o b u l i n s e r u m , a n d i n E x p . N o . 2 the sequence w a s r e versed. (110),
O n the basis of p r e v i o u s c a l i b r a t i o n s w i t h step-coated
surfaces
the entries expressed i n degrees c a n be m u l t i p l i e d b y a b o u t 30 to
o b t a i n thickness i n angstroms for a r e f r a c t i v e i n d e x of about 1.60. B E H A V I O R O F P L A S M A A T 7s G A M M A - G L O B U L I N - C O A T E D O X I D I Z E D S I L I -
C O N . F i l m s of 7s g a m m a - g l o b u l i n s w e r e p r e p a r e d i n the e l l i p s o m e t e r c u v e t w h i l e r e c o r d i n g , b y a d d i t i o n of 0.4 m l of 8 m g g l o b u l i n p e r m l V S to the 15 m l V S i n the c u v e t c o n t a i n i n g the S i O slice. A f t e r 90 m i n t h e s o l u t i o n was r e p l a c e d three times b y fresh V S , a n d r e a d i n g s w e r e d o n e w h e n the t e m p e r a t u r e ( 3 7 ° C ) h a d c o m e to e q u i l i b r i u m . F i l m s w e r e a b o u t 2 ° to 3 ° t h i c k (see
T a b l e s I - I V ) . O n t o these, 0.1 m l n o r m a l i n t a c t p l a s m a depos-
i t e d a b o u t 1° to 2 ° of m a t t e r w i t h i n a b o u t 10 m i n after i t h a d b e e n a d d e d to the V S ( T a b l e I , E x p . N o . 1 - 7 ) ,
and then removed nearly all
a n d sometimes m o r e t h a n i t h a d d e p o s i t e d ( E x p . N o . 4 - 7 ) . a n a c t u a l r e c o r d i n g is s h o w n ( F i g u r e 1 ) .
A t r a c i n g of
A n o t h e r a d d i t i o n of p l a s m a
r e c r e a t e d to some degree this a d s o r p t i o n a n d d e s o r p t i o n sequence
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
(Exp.
12.
VROMAN ET AL.
Protein
267
Interfaces
P l a s m a a t 7s G a m m a - G l o b u l i n - C o a t e d O x i d i z e d S i l i c o n S u r f a c e s Normal
Activated Plasma
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Τον
Normal
Bottom
Τον
time min
meas.
time min
meas.
time min
10 — — — 120 10
0.61 — — — 1.42 1.57
180 — — — — 120
-0.94 — — — 0.00 -0.25
— 10 — — — 15
b
Intact
Plasma Bottom
meas. — 0.44 — 0.00 — 0.44
time min
meas.
Anti-7s GammaGlobulin
— 840 — — — 120
— -1.93 — — — -0.77
— — 0.27 — — 1.97
Plasma premixed 1:1 with S K .
Figure 2. Tracing of ellipsometer recording; uneventful sections omitted. To buffer containing oxidized silicon slice on which 7s gamma-globulins had been adsorbed, 0.1 ml of normal activated plasma was added at A, causing adsorption. After 135 min, removal still being minimal, cuvet contents were replaced twice by fresh buffer, and at B, 0.1 ml intact plasma was added, resulting in removal. R: reversal (see Figure 1).
No. 10-14). event (exp.
N e i t h e r streptokinase nor 2 A C A a p p e a r e d no. 6 a n d 7 ) ;
therefore,
to affect this
i t seems n e i t h e r a c t i v a t i o n
i n h i b i t i o n of p l a s m i n i n f l u e n c e d this d e s o r p t i o n .
nor
P l a s m i n per se m a y n o t
be i n v o l v e d i n i t at a l l . P r o t a m i n e sulfate d i d i n h i b i t i t ( E x p . N o . 9; also, see b e l o w a n d T a b l e I V ). Factor XII-deficient plasma, l i k e normal plasma, d i d deposit matter onto the g a m m a - g l o b u l i n films b u t was u n a b l e to remoVe i t ( T a b l e I I ,
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
268
APPLIED CHEMISTRY AT PROTEIN INTERFACES
Table III.
Behavior of Variously Anticoagulated Plasmas at Normal
Intact
Plasma
Citrated Bottom
Top
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Exp. No.
Globulin
1 2 3 4 δ 6
2.75 2.94 3.09 2.81 3.12 26.3
Exp. No. 3).
time min
meas.
time min
20 10
1.63 1.36 1.13
240 220
— — —
meas.
—
— — —
-0.98 -1.08 -1.24
— — —
— — —
H o w e v e r , i t d i d r e m o v e m a t t e r left b y n o r m a l p l a s m a o n
b r i e f c o n t a c t w i t h a g l o b u l i n film ( T a b l e I I , E x p . N o . 1 ) .
I n this aspect,
n o r m a l a c t i v a t e d p l a s m a b e h a v e d l i k e factor X I I deficient p l a s m a ( E x p . N o . 5 a n d 6 vs. E x p . N o . 2 ) (see
Figure 2).
Streptokinase premixed w i t h
f a c t o r ΧΙΙ-deficient p l a s m a c a u s e d i t to r e m o v e a v e r y large a m o u n t of m a t t e r after d e p o s i t i n g some ( E x p . N o . 4 ) , p r o b a b l y b y a c t i v a t i n g p l a s m i n o g e n i n the p l a s m a . T h e a n t i g e n i c i t y of the u n d e r l y i n g g l o b u l i n was d e s t r o y e d b y this a c t i v i t y ( c o m p a r e E x p . N o . 4 a n d 6 ) . T h u s i t appears t h a t s o m e t h i n g i n t a c t is n e e d e d either w h i l e p l a s m a is f o r m i n g its d e p o s i t or later w h i l e the d e p o s i t is e x p o s e d to p l a s m a , to allow
removal
other
than by
plasminogen
of a c t i v a t e d p l a s m a to r e m o v e its o w n
activation.
The
inability
deposit on the g l o b u l i n
films
suggests that p r o t e o l y t i c a c t i v i t y , c r e a t e d b y the a c t i v a t i o n , was not the d e s o r b i n g agent.
It also suggests t h a t a cofactor of factor X I I m a y h a v e
b e e n r e m o v e d b y a c t i v a t i o n a n d is at least c o - r e s p o n s i b l e for the d e s o r b i n g a c t i v i t y of i n t a c t p l a s m a
Table IV.
Exp. No. 1 2 3 4 5 6 7
(42).
Effects of Protamine Sulfate and of Heparin
Globulin
PS
2.77 2.34 2.65 2.41 2.43 2.85 2.68
0.07 0.14 0.04 0.14 0.21 0.10 0.06
Heparin
— — — — —
-0.25 0.02
Globulin
— —
0.76 0.66 0.91 0.54 0.25
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
12.
Protein
VROMAN ET A L .
269
Interfaces
7s Gamma-Globulin-Coated Oxidized Silicon Surfaces Normal
Intact
Plasma EDTA
Heparinized
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Top
Bottom
time min
meas.
time min
10
1.72
240
Bottom
Top
meas.
time min
meas.
time min
meas.
1.18 — —
— 2-10 10
0.32 0.44
120 60
-0.17 -0.77
N e i t h e r the presence of h e p a r i n nor t h a t of E D T A i n s o l u t i o n i n hibited desorption b y intact plasma ( T a b l e I I I ) .
O n the other h a n d ,
w h e n a p r e f o r m e d g l o b u l i n film w a s first exposed to p r o t a m i n e sulfate, t h o u g h this exposure affected film thickness v e r y l i t t l e ( T a b l e I V ) , n o r m a l i n t a c t p l a s m a c o u l d no l o n g e r r e m o v e
m a t t e r b u t o n l y deposit i t
(Table IV, Exp. No. 2). T h e m a t t e r d e p o s i t e d b y n o r m a l p l a s m a onto g l o b u l i n films that h a d ( T a b l e I V , E x p . N o . 1 ) or h a d not b e e n p r e - e x p o s e d to p r o t a m i n e sulfate ( n o t l i s t e d ) d i d not adsorb m a t t e r out of a n t i - h u m a n a n d therefore was p r o b a b l y not
fibrinogen
serum
fibrinogen.
W h e n exposure of a g l o b u l i n film to p r o t a m i n e sulfate w a s f o l l o w e d b y r e n e w e d exposure to g l o b u l i n , m o r e of the latter w a s d e p o s i t e d after w h i c h n o r m a l i n t a c t p l a s m a w o u l d deposit m o r e matter b u t r e m o v e l i t t l e (Table IV, Exp. No. 4 and 5).
If, h o w e v e r , exposure of the g l o b u l i n film
to p r o t a m i n e sulfate w a s f o l l o w e d b y exposure to h e p a r i n ( E x p . N o . 6 a n d 7 ) a g a i n c a u s i n g m i n i m a l changes i n film thickness, the r e s u l t i n g surface
on 7s Gamma Globulin and its Interaction with Plasma Normal
Intact
Plasma
Top time min
Antiserum
Bottom
to
meas.
time min
meas.
2 10
0.15 1.84
—
—
0.20
-0.09
—
2.30
180
10 10 10 10
1.64 1.83 1.28 1.56
90 180 130 840
-0.23 -0.29 -1.49 -1.34
— — — —
— — — —
Fibrinogen
Globulin
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
—
270
APPLIED CHEMISTRY AT PROTEIN
INTERFACES
w o u l d a d s o r b o n l y l i t t l e a d d i t i o n a l g l o b u l i n . I n contrast, n o r m a l p l a s m a w a s n o w a b l e to d e p o s i t a n d t h e n to r e m o v e l a r g e a m o u n t s of m a t t e r , suggesting t h a t the i n h i b i t o r y a c t i o n of t h e p r o t a m i n e sulfate o n this a c t i v i t y h a d b e e n n e u t r a l i z e d b y h e p a r i n at the interface. I n most e x p e r i ments successive
a d d i t i v e s d o n o t meet i n s o l u t i o n ; after e a c h
added
a n t i s e r u m c u v e t contents w e r e r e p l a c e d b y f r e s h V S once, a n d after e a c h of the other a d d i t i v e s , at least t w i c e .
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Table V . Effects of Substrate and of D r y i n g on Conversion of Pre-adsorbed Fibrinogen and on Desorption by Plasma Fibrinogen Wettable
Exp. No.
Dry
Slide Wet
Nonwettable
Slide Wet
Dry
Normal Intact Plasma
AF
S i l i c o n slide 2.70
1
1.64
2
1.60
3
—
1.99
4
—
1.83
5
—
2.98
6
—
2.18
7
—
1.80
8
—
2.85
2.29
0.29
3.36 0.17
0.04
3.56 2.09
-0.43
3.47 3.89
-0.04
O x i d i z e d t a n t a l u m slide 9
1.62
10
1.77
1.40
11
1.46
12
1.47
1.34
-0.53
0.56
1.58
13
1.67
14
1.60
1.40 1.76
-0.64
15
1.74
16
1.76
BEHAVIOR OF P L A S M A
-0.45
1.48 -0.90
1.19
AT FIBRINOGEN-COATED OXIDIZED A N D U N O X I -
D I Z E D S I L I C O N . I n a large n u m b e r of e x p e r i m e n t s ( 2 2 , 2 3 , 2 4 , 3 8 , 3 9 ) o g e n p r e a d s o r b e d onto S i O , as w e l l as
fibrinogen
fibrin-
deposited b y plasma
itself is affected b y i n t a c t a n d f a c t o r - X I I - d e f i c i e n t p l a s m a i n s u c h a w a y t h a t its a b i l i t y to c o m b i n e w i t h a n t i - f i b r i n o g e n is lost. T h i s c o n v e r s i o n is slower on non-wettable
surfaces a n d also o n e i t h e r w e t t a b l e or
w e t t a b l e a n o d i z e d t a n t a l u m surfaces. it is f o l l o w e d b y some d e s o r p t i o n .
non-
I n presence of i n t a c t f a c t o r X I I ,
T o o b t a i n some i m p r e s s i o n of
effects that c o n f o r m a t i o n changes i m p o s e d o n the a d s o r b e d
the
fibrinogen
c o u l d h a v e o n t h e a b i l i t y of p l a s m a to c o n v e r t a n d desorb i t , the f o l l o w i n g e x p e r i m e n t s w e r e done.
A s o l u t i o n of 3 m g
fibrinogen
per m l V S was
a p p l i e d d i r e c t l y to d r y surfaces of t h e f o l l o w i n g : u n o x i d i z e d , n o n w e t t a b l e
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
12.
VROMAN
Protein
ET AL.
s i l i c o n (23,112);
271
Interfaces
oxidized, wettable silicon; anodized tantalum-sputtered
glass ( T a O ) ; a n d the same r u b b e d w i t h f e r r i c stéarate ( T a N ) to r e n d e r it n o n w e t t a b l e . A s t u d y to be p u b l i s h e d elsewhere suggests t h a t a
fibrino-
g e n s o l u t i o n , or e v e n p l a s m a itself, forms a fibrinogen film at t h e l i q u i d / a i r interface w h i c h , w h e n c a r r i e d b y a d r o p r o l l i n g over a h y d r o p h o b i c surface, is t r a n s f e r r e d at the a i r / s o l i d / l i q u i d interface i n a f o r m t h a t cannot be converted b y intact plasma. exposure to the
fibrinogen
I n the present series, after 1 m i n . of
s o l u t i o n , the slides a n d slices w e r e r i n s e d w i t h
V S a n d either p l a c e d i n to the 15 m l V S i n the e l l i p s o m e t e r or first rinsed Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch012
w i t h w a t e r , a i r d r i e d , a n d t h e n p l a c e d i n V S i n the ellipsometer.
After
r e a d i n g s w e r e p e r f o r m e d o n the s i l i c o n set, 0.1 m l n o r m a l i n t a c t p l a s m a was a d d e d ; 45 m i n l a t e r the c u v e t contents w e r e r e p l a c e d b y f r e s h V S and
readings were repeated.
T h e 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 slides,
a l l o w i n g slower or less c o n v e r s i o n of p r e a d s o r b e d
fibrinogen,
were
ex-
p o s e d to 0.2 m l r a t h e r t h a n to 0.1 m l p l a s m a for 90 m i n r a t h e r t h a n for 45 m i n . I n a l l of these tests, 0.1 m l of a n t i - h u m a n
fibrinogen
serum was
a d d e d next, a n d r e a d i n g s w e r e p e r f o r m e d 45 m i n later. T h e results o n s i l i c o n substrates as l i s t e d ( T a b l e V ) are averages of 2 tests for
each
e x p e r i m e n t , w h i l e those o n a n o d i z e d t a n t a l u m substrates represent single experiments.
T h e data indicate that d r i e d
fibrinogen
was n o t c o n v e r t e d
w e l l o n a n y surface ( E x p . N o . 2, 6, 10, a n d 1 4 ) , a n d less c o n v e r s i o n t o o k p l a c e o n n o n w e t t a b l e t h a n o n w e t t a b l e surfaces ( c o m p a r e E x p . N o . 4 a n d 8 w i t h E x p . N o . 12 a n d 1 6 ) , p e r h a p s because of the air interfaces a l l o w e d during deposition.
C o n v e r s i o n o n w e t t a b l e a n o d i z e d t a n t a l u m was less
t h a n o n o x i d i z e d s i l i c o n , e v e n t h o u g h r e m o v a l w a s greater ( C o l u m n 5 ) i n this p r o l o n g e d exposure to p l a s m a , t h a n i t was o n S i O . T h e s e d a t a fit the q u a l i t a t i v e observations of interference colors r e s u l t i n g f r o m exposure of fibrinogen
a d s o r b e d onto T a O slides a n d exposed w h i l e w e t w i t h V S to
u n d i l u t e d c i t r a t e d or h e p a r i n i z e d p l a s m a . F i b r i n o g e n - c o a t e d areas t h a t had
b e e n exposed to p l a s m a are p a l e r ( t h i n n e r ) b u t t u r n p u r p l e w h e n
exposed to a n t i - f i b r i n o g e n sera as do the areas not exposed to p l a s m a . T h u s , the d a t a o b t a i n e d on T a O i n d i c a t e that r e m o v a l of t h e a d s o r b e d film
b y p l a s m a n e e d not be p r e c e d e d
r e m a i n e d that
fibrinogen
b y conversion.
T h e possibility
itself c o u l d be v a r i a b l y a n t i g e n i c d e p e n d i n g o n
the w e t t a b i l i t y of its substrate a n d o n its o w n state of d r y i n g .
However,
i n a series of six tests ( not l i s t e d ), no significant differences w e r e caused b y these v a r i a b l e s ,
fibrinogen
films
a l l c a u s i n g a deposit of about
2.7°-
3.5° b y a n t i - f i b r i n o g e n s e r u m . THE
B E H A V I O R O F P L A S M A TOWARDS ITS O W N DEPOSITS O F F I B R I N O G E N
A N D 7s G A M M A - G L O B U L I N S .
T h e v a r i o u s fates of p r e a d s o r b e d
o n exposure to p l a s m a a n d of
fibrinogen
fibrinogen
a n d 7s g a m m a - g l o b u l i n s
de-
p o s i t e d b y p l a s m a itself d e p e n d e d o n the state of a c t i v a t i o n i n the p l a s m a . B o t h a n t i - f i b r i n o g e n a n d anti-7s g a m m a - g l o b u l i n sera d e p o s i t e d
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
much
272
APPLIED CHEMISTRY
Table V I .
AT
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Fibrinogen
— — — — — — — — — 2.19 2.28 1.83 1.77
— —
c
INTERFACES
Effects of Activation of the Ability of Plasma to Convert Intact Plasma
Exp. No.
PROTEIN
Activated
Plasma
90 sec
45 min
90 sec
45 min
1.61 1.53
— —
1.54 1.32 1.34
— — — — — —
— — — — — —
— — — — — — —
—
— — — — — — —
1.61
1.37
— — — — — — —
-0.56
1.38
—
— — — —
2.61 2.40
—
-0.14 0.01 0.26 0.49
—
1.72
0.38
b
° A n t i s e r u m to fibrinogen a d d e d after a n t i s e r u m to g l o b u l i n . 14 m i n exposure, instead of 45 m i n . 6
m a t t e r onto films left b y n o r m a l i n t a c t p l a s m a i n IV2 m i n of contact w i t h o x i d i z e d s i l i c o n ( T a b l e V I , E x p . N o . 1 a n d 2 ) b u t n o t onto films left b y the p l a s m a after 45 m i n of contact ( E x p . N o . 3 ) .
This conversion was
o b t a i n e d w i t h 0.1 m l i n t a c t p l a s m a b e i n g a d d e d to the 15 m l V S . A n e a r l i e r s t u d y ( 3 9 ) s h o w e d that p l a s m a converts its o w n
fibrinogen
deposit
w i t h i n 30 sec i f u n d i l u t e d , e v e n at r o o m t e m p e r a t u r e . I n a p r e l i m i n a r y set of single experiments, w e f o u n d n o c l e a r e v i d e n c e of o r i e n t a t i o n i n the g l o b u l i n films left b y p l a s m a o n IV2 m i n of contact w i t h S i O . R e a c t i v i t y w i t h antisera to g l o b u l i n fragments
was
s l i g h t a n d u n i f o r m . C o n v e r s i o n of g l o b u l i n s d i d not a p p e a r as c o m p l e t e as t h a t of
fibrinogen.
N o . 8 and 9)
A g a i n w e could show that activated plasma ( E x p .
w a s less a b l e to c o n v e r t
fibrinogen,
b u t the
fibrinogen
deposited b y intact plasma could be converted b y activated plasma ( E x p . N o . 14) a n d vice versa ( E x p . N o . 1 5 ) . C o n v e r s i o n of p r e a d s o r b e d
fibrino-
g e n o n S i O b y i n t a c t p l a s m a ( E x p . N o . 10) w a s a l w a y s less c o m p l e t e t h a n t h a t of the
fibrinogen
d e p o s i t e d b y p l a s m a itself ( E x p . N o . 3 ) ,
though
i n b o t h situations t h e r e m a i n i n g film adsorbs m u c h less t h a n the 2 . 7 ° - 3 ° a d s o r b e d out of a n t i - f i b r i n o g e n s e r u m b y
fibrinogen
films
that h a d not
b e e n exposed to i n t a c t p l a s m a . T H E BEHAVIOR OF A L B U M I N IN P L A S M A AT T H E OXIDIZED SILICON SUR-
F A C E . A c h a n c e f o r a l b u m i n to b e a d s o r b e d out of p l a s m a , not yet e n c o u n t e r e d o n a n y of the m a t e r i a l s tested, c o u l d b e p r o v i d e d b y r e m o v i n g
fibrin-
o g e n a n d some g l o b u l i n s f r o m c o m p e t i t i o n . S o m e n o r m a l i n t a c t p l a s m a w a s
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
12.
VROMAN E T AL.
Protein
273
Interfaces
Fibrinogen and 7s Gamma-Globulins at the Oxidized Silicon Surface Antiserum Fab Frag.
Fc Frag.
IgG H&Lch
To Fibrinogen 1.25 0.80 0.12
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0.41 — —
— 0.93 —
Human Serum
Globulin
— —
1.50 1.28 0.32
a
— — 0.80
— —
1.40 0.64 0.42 0.76
0.95* 0.40 0.42* 0.73 2.81 2.01 1.90 0.27 0.04
— —
1.13
—
—
1.92 2.06
1.96 2.59 0.54
— —
—
* Activated plasma was added before intact plasma. h e a t e d to 5 6 ° C , a l l o w e d to c o o l to r o o m t e m p e r a t u r e ( a b o u t 2 5 ° C ) after 1 h r , a n d c e n t r i f u g e d to r e m o v e the p r e c i p i t a t e d t r e a t e d d i d not deposit detectable
fibrinogen
fibrinogen.
P l a s m a thus
onto o x i d i z e d s i l i c o n ( T a b l e
V I I , E x p . N o . 1) b u t d i d d e p o s i t some g l o b u l i n s w h i c h o n
prolonged
exposure p e r h a p s d i d u n d e r g o some c o n v e r s i o n ( c o m p a r e E x p . N o . 1 a n d 3).
S o m e other, single experiments suggest t h a t
fibrinogen
preadsorbed
out of its s o l u t i o n ( E x p . N o . 4 a n d 5) or out of n o r m a l p l a s m a ( E x p . N o . 6) w a s at least p a r t i a l l y c o n v e r t e d b y h e a t e d p l a s m a . T h e a b i l i t y of h e a t e d p l a s m a to d e p o s i t a l b u m i n ( T a b l e V I I I ) onto S i O was e n h a n c e d Table VII.
Effects of Heating on Intact Plasma Intact Unheated
Exp. No. 1 2 3 4 5 6
Fibrinogen
— — — 2.19 2.38
—
and
Plasma Antiserum
Heated
a
90 sec
45 min
90 sec
45 min
— — — — —
— — —
1.26 1.31
— —
0.86
-0.56
— —
— — — —
2.40
—
-0.04 1.21
Fibrinogen 0.32 -0.02 0.00 0.73 0.94 0.02
° Plasma maintained at 56°C for 1 hr and centrifuged. Antiserum to globulin added before antiserum to fibrinogen. 6
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
to Globulin 1.05 1.19* 0.58 0.76 0.73 0.48
274
APPLIED CHEMISTRY AT PROTEIN INTERFACES
Table VIII.
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Exp. No.
Effects of Heating on Subsequent Behavior of Plasma A l b u m i n on Oxidized Silicon
Intact 90 sec
Heated Plasma 1 1.73 2 1.61 3 — 4 — 5 — 6 — 7 — 8 — Unheated plasma 9 1.46 10 —
Antiserum
Activated
45 min
90 sec
45 min
— —
— — — —
— — — — — —
2.56 2.77
Albumin
Globulin 1.12 1.34 1.07 0.77
2.65 2.82* 2.54 2.03* 3.20
— — — —
1.41 1.40
— —
1.99 1.99
2.74
—
— —
— —
0.54 0.11
1.60
to
—
—
1.32
—
1.18
—
— —
° A n t i s e r u m to a l b u m i n a d d e d after a n t i s e r u m to g l o b u l i n .
r e m a i n e d so e v e n w i t h a d d i t i o n of
fibrinogen
to the h e a t e d p l a s m a . I n 9
tests, one v o l u m e of n o r m a l a c t i v a t e d or n o r m a l i n t a c t p l a s m a w a s d i l u t e d w i t h a n e q u a l v o l u m e of V S , k e p t at 65 ° C for 2 hrs, a n d c e n t r i f u g e d . one v o l u m e of the s u p e r n a t a n t , a n e q u a l v o l u m e of 3 m g / m l V S Table IX.
Exp. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 α
% wt/wt in Albumin 100» 100 100 98.2 93.0 93.0 92.8 88.9 80.0 80.0 57.1 57.1 40.0 25.0 10.0
— —
Soin
Fibrinogen
— — — 1.8 7.0 7.0 7.2 11.1 20.0 20.0 42.9 42.9 60.0 75.0 90.0 100 100
Film Meas. 1.09 0.69 0.94 1.15 1.13 1.49 0.83 0.89 1.00 1.08 1.07 1.43 1.53 1.94 2.05 1.71 1.67
To
fibrinogen
Competition between Antiserum Fibrinogen 0.16 0.66 0.77" 0.52 1.41 0.93 " 1.69 1.73 1.59 0.94 2.15 1.26 2.23" 3.16 3.28 3.18 1.51 6
6
6
To: Albumin 3.41 1.39 3.85 3.32 2.65 2.10 2.26 2.55 1.74 0.78 1.86 0.51 0.26 1.35 0.77 0.29 -0.17
I n this case o n l y , 40 m g a l b u m i n per m l V S was used.
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
12.
VROMAN E T AL.
Protein
275
Interfaces
was a d d e d a n d t h e n 0.1 m l of this m i x t u r e to t h e 15 m l V S at 37 ° C i n t h e ellipsometer.
Measurements, followed
c a r r i e d o u t as d e s c r i b e d . mixtures of
fibrinogen
b y exposure
to antisera, w e r e
T h e y s h o w e d that t h e films d e p o s i t e d b y t h e
a n d h e a t e d p l a s m a w e r e 1.30°-2.27° t h i c k , a d -
s o r b e d 0 . 1 4 ° - 1 . 3 5 ° ( a v e r a g i n g 0 . 6 1 ° ) o u t of a n t i - f i b r i n o g e n s e r u m , a n d t h e n 1.27°-2.75° ( a v e r a g i n g 2 . 1 2 ° ) o u t of a n t i - a l b u m i n s e r u m . H u m a n s e r u m , another m i x t u r e that lacks t h e c o m p e t i n g
fibrinogen,
d e p o s i t e d films u n d e r t h e same e x p e r i m e n t a l c o n d i t i o n s . T h e films w e r e less a b l e to a d s o r b m a t t e r o u t of a n t i - a l b u m i n s e r u m t h a n w a s h e a t e d Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch012
plasma; neither heated convert
fibrinogen
nor unheated
C O M P E T I T I O N B E T W E E N PROTEINS.
globulins a n d
serum showed
clear a b i l i t y to
(data not listed).
fibrinogen,
C o m p e t i t i o n b e t w e e n 7s
albumin and
fibrinogen,
gamma-
a n d a l b u m i n a n d 7s
g a m m a - g l o b u l i n s f o r t h e S i O surface w a s o b s e r v e d b y p l a c i n g 0.4 m l of a m i x t u r e i n t o t h e 15 m l V S of t h e e l l i p s o m e t e r at 37 ° C a l l o w i n g a d s o r p t i o n f o r 9 0 m i n , r e p l a c i n g t h e s o l u t i o n 3 times w i t h fresh V S , a n d e x p o s i n g to successive antisera as u s u a l , e a c h for 4 5 m i n . 60 tests w e r e m a d e ; i n m a n y , t h e s e q u e n c e of a d d i t i o n of antisera w a s r e v e r s e d . O n e set of d a t a is g i v e n here as a n e x a m p l e ( T a b l e I X ) . G r a p h s are p r e s e n t e d
(Figures
3 a n d 4 ) i n w h i c h t h e p e r c e n t of m a t t e r a d s o r b e d o u t of t h e first a n t i s e r u m a d d e d p e r t o t a l a m o u n t a d s o r b e d o u t of b o t h antisera, is p l o t t e d against r e l a t i v e c o n c e n t r a t i o n , w t / w t , of t h e t w o constituents i n s o l u t i o n . A l b u m i n and Fibrinogen
Total Antisera 3.57 2.05 4.62 3.84 4.06 3.03 3.95 4.28 3.33 1.72 4.01 1.77 2.49 4.51 4.05 3.47 1.34 6
1 Antiserum, % of Total Sera Fibrinogen 4.5 30.7 16.7" 13.5 34.7 30.7 42.8 40.4 47.7 54.6» 53.6 71.9" 89.2» 70.0 89.0 91.6 100 b
b
Albumin 95.5 69.3 83.3 86.5 65.3 69.3 57.2 59.6 52.3 45.4 46.4 28.1 10.8 30.0 11.0 8.4 0.0
Antiserum to fibrinogen added after antiserum to albumin.
In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.
276
APPLIED CHEMISTRY I
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Figure 3. Amount (degrees, ellipsom eter readings) adsorbed out of antialbumin serum, in % of total adsorbed out of it and out of anti-7s gamma globulin serum (curve G) or anti-fibrin ogen serum (curve F), plotted against % of albumin (wt/wt) in 7s gamma globulin (curve G) or fibrinogen solu tion (curve F). Intercept of curve G with Y axis shows some matter was adsorbed out of anti-albumin serum onto albumin-free globulin film.
00
A T PROTEIN
INTERFACES
r