251
Biol Res 3 1 : 251-262 (1998)
Plasma contact activation: A revised hypothesis
ALVIN H SCHMAIER*
D i v i s i o n of H e m a t o l o g y and O n c o l o g y , D e p a r t m e n t of Internal M e d i c i n e a n d P a t h o l o g y ,
U n i v e r s i t y of M i c h i g a n , A n n A r b o r , M I 4 8 1 0 9 - 0 6 4 0 , U S A
A new hypothesis
for activation
of the contact system of plasma
proteolysis
(i.e., the plasma
kallikrein/kinin
system)
is presented.
Kininogens
have a
multiprotein
receptor on endothelial
cells which consists of at least
cytokeratin
1, urokinase
plasminogen
activator
receptor,
and gClqR.
When
contact
proteins
(high molecular
weight kininogen followed
by prekallikrein)
assemble
on the kininogen
receptor on endothelial
cells, an endothelial
cell
membrane
cysteine
protease
is expressed
to activate
prekallikrein
to kallikrein.
On
endothelial
cells, prekallikrein
activation
is independent
of factor
XHa
activation.
Activation
of prekallikrein
on endothelial
cells results in
kallikrein
cleaving its receptor high molecular
weight kininogen
to liberate
bradykinin.
Bradykinin
liberation
stimulates
release of tissue-type
plasminogen
activator
from
endothelial
cells.
Kallikrein
formation
also results
in
kinetically
favorable
pro-urokinase
activation
on endothelial
cells with
subsequent
plasminogen
activation.
In addition
to stimulating
cellular
fibrinolysis,
kininogens
contribute
to the constitutive
anticoagulant
nature
of the
intravascular
compartment.
Kininogens
block calpain 's participation
in
forming
the heterodimeric
complex of platelet integrin Ot/^jSj. Kininogens
also
block thrombin from binding
to the thrombin
receptor(s)
on platelets.
Last,
kininogens
prevent thrombin from cleaving protease activated
receptor 1 after
arginine .
These combined
data indicate a biologic
system for activation
of
the plasma kallikrein/kinin
system and physiologic
consequences
as result of
this
activation.
4]
Key terms: antithrombin,
XII, fibrinolysis,
kininogen,
bradykinin,
contact
kinins, prekallikrein,
INTRODUCTION
T h e c o n t a c t s y s t e m of p l a s m a p r o t e o l y s i s ,
w h i c h is t h e p l a s m a k a l l i k r e i n / k i n i n
s y s t e m , h a s b e e n v i e w e d as a b i o c h e m i c a l
p a t h w a y w h o s e b i o l o g i c role n e e d s further
c l a r i f i c a t i o n . T w o a s p e c t s of this s y s t e m
h a v e s e r v e d to o b f u s c a t e u n d e r s t a n d i n g and
b r a n d the s y s t e m as u n i m p o r t a n t . T h e first
c o n f o u n d i n g a s p e c t is t h a t a l t h o u g h
d e f i c i e n c i e s of its c o n s t i t u e n t s , factor X I I
activation,
cytokeratin,
thrombin
factor
(FXII), prekallikrein (PK), and high
molecular weight kininogen (HK), give
s t r i k i n g p r o l o n g a t i o n of s u r f a c e - a c t i v a t e d
coagulation assays, patients with these
protein deficiencies do not bleed. The
s e c o n d c o n f o u n d i n g a s p e c t is t h a t the in
vivo a c t i v a t o r ( s ) of this s y s t e m h a s not b e e n
identified. Contact system proteins have
been k n o w n only to a c t i v a t e
when
a s s o c i a t e d w i t h an a r t i f i c i a l , n e g a t i v e l y
charged surface such as glass, kaolin,
Correspondence to: Alvin H Schmaier, M D , Department of Internal Medicine and Pathology, University of Michigan,
5301 M S R B III, 1150 W Medical Center Drive, Ann Arbor, MI 48109-0640, USA. Phone: (1-734) 647-3124. Fax: ( 1 734) 647-5669. E-mail: [email protected]
252
c e l i t e , etc,
hence the name "contact
s y s t e m " . A l t h o u g h a n u m b e r of b i o l o g i c
substances,
acidic
phospholipids,
c h o l e s t e r o l sulfate, s u l f a t i d e s , g o u t c r y s t a l s ,
etc, h a v e b e e n s h o w n t o f u n c t i o n a s
negatively charged surfaces, none are
c o n v i n c i n g to s e r v e as the single in vivo
surface for p h y s i o l o g i c a c t i v a t i o n of this
s y s t e m . T h u s a m o r e c o g e n t m e c h a n i s m for
a c t i v a t i o n of t h i s s y s t e m n e e d s t o b e
discovered.
For the last twenty years
most
i n v e s t i g a t o r s in t h e field h a v e a c c e p t e d the
n o t i o n t h a t t h e initiation of a c t i v a t i o n of the
s y s t e m r e s u l t s f r o m F X I I b i n d i n g to
n e g a t i v e l y c h a r g e d surfaces to a u t o a c t i v a t e
(37, 54). Factor X I I ' s autoactivation leads
to P K a c t i v a t i o n a n d k a l l i k r e i n f o r m a t i o n
amplifies further F X I I a c t i v a t i o n . T h e rate
of i n i t i a t i o n a n d a m p l i f i c a t i o n of t h i s
s y s t e m is a c c e l e r a t e d by H K a n d an
a r t i f i c i a l s u r f a c e . A m p l i f i c a t i o n of t h i s
s y s t e m ' s a c t i v a t i o n by k a l l i k r e i n is at least
1000-fold faster than the autoactivation
p h e n o m e n a ( 4 8 ) . A c t i v a t i o n of t h e
z y m o g e n s F X I I a n d P K r e s u l t s in e n z y m e s
that c o n t r i b u t e to factor XI a c t i v a t i o n
(coagulation), complement activation,
bradykinin liberation, fibrinolysis and
g r a n u l o c y t e a c t i v a t i o n . U n f o r t u n a t e l y , the
p o t e n t i a l i m p o r t a n c e of this s y s t e m to
mediate biologic responses has been
overshadowed by the untenable explanation
for h o w this s y s t e m ' s a c t i v a t i o n is initiated.
Over ten years ago, my laboratory
d e v e l o p e d a w o r k i n g h y p o t h e s i s to s e r v e as
an
alternative
to
the
factor
XII
a u t o a c t i v a t i o n p h e n o m e n a for the initiation
of a c t i v a t i o n of c o n t a c t s y s t e m p r o t e i n s .
W e r e a s o n e d t h a t in vivo it is the a s s e m b l y
of a m u l t i p r o t e i n c o m p l e x of c o n t a c t
s y s t e m p r o t e i n s on cell r e c e p t o r s that a l l o w
f o r l o c a l i z a t i o n a n d a c t i v a t i o n of t h i s
s y s t e m . In o r d e r to p r o v e that h y p o t h e s i s ,
w e s o u g h t to a c c o m p l i s h t h r e e t h i n g s : First,
d e t e r m i n e if t h e r e is a r e c e p t o r for t h e
m a j o r c o f a c t o r of this s y s t e m , H K , on cell
m e m b r a n e s . Second, show how the
a s s e m b l y of c o n t a c t p r o t e i n s on cell
membranes
through
HK results
in
a c t i v a t i o n of t h e z y m o g e n s P K and F X I I .
T h i r d , d e m o n s t r a t e if t h e r e are i m p o r t a n t
biologic activities associated with contact
Biol Res 3 1 : 251 -262 ( 1998)
protein assembly
membranes.
a n d a c t i v a t i o n on
cell
CHARACTERIZATION OF THE MULTIPROTEIN
K I N I N O G E N R E C E P T O R O N E N D O T H E L I A L CELLS
Initial i n v e s t i g a t i o n s to d e t e r m i n e the
kininogen receptor w e r e spent characterizing
the ability of kininogens to bind to various
cells in the i n t r a v a s c u l a r c o m p a r t m e n t . T h e
s t u d y by G r e e n g a r d a n d Griffin first
d e m o n s t r a t e d H K b i n d i n g to activated
platelets required Z n
(10). W e followed
w i t h t h e d e m o n s t r a t i o n t h e H K b i n d s to
u n s t i m u l a t e d p l a t e l e t s in t h e p r e s e n c e of
Z n ( 1 5 ) . A d d i t i o n a l s t u d i e s from m u l t i p l e
laboratories have shown that kininogens
also b i n d to g r a n u l o c y t e s a n d e n d o t h e l i a l
cells in the p r e s e n c e of Z n
(14, 46, 50).
H o w e v e r , the r o l e of zinc ion w a s not j u s t
t o a s s o c i a t e w i t h t h e light c h a i n of H K (9).
R a t h e r , it w a s e s s e n t i a l for t h e e x p r e s s i o n
of t h e k i n i n o g e n r e c e p t o r b e c a u s e l o w
molecular weight kininogen (LK), which
d o e s n o t h a v e H K ' s light c h a i n , r e q u i r e d
Z n as well for b i n d i n g to p l a t e l e t s ( 3 6 ) .
Investigations also revealed that kininogen
m u s t be b i n d i n g to a p h y s i c o c h e m i c a l
structure. The k i n i n o g e n binding site,
putative receptor, on endothelial cells appears
to be a structure that can b e regulated. First,
treatment of endothelial cells with m e t a b o l i c
i n h i b i t o r s to a e r o b i c a n d a e r o b i c m e t a b o ­
lism and the h e x o s e m o n o p h o s p h a t e shunt
a b o l i s h the ability of H K to b i n d to cells
( 1 8 ) . C y c l o h e x i m i d e has n o effect on H K
b i n d i n g to e n d o t h e l i a l c e l l s . S e c o n d ,
t e m p e r a t u r e or the b r a d y k i n i n s e q u e n c e in
k i n i n o g e n s c o n t r i b u t e s t o t h e l e v e l of
k i n i n o g e n b i n d i n g to e n d o t h e l i a l cells ( 1 8 ,
19, 5 5 ) . T h i r d , b r a d y k i n i n t r e a t m e n t of
e n d o t h e l i a l c e l l s r e s u l t s in i n c r e a s e d H K
a n d L K b i n d i n g a n d t h i s p a t h w a y is
m e d i a t e d by protein k i n a s e C and the
endothelial cell B l bradykinin receptor
(55). Fourth, heavy chain and L K have a
Ca
r e q u i r e m e n t for p h o r b o l 1 2 - m y r i s t a t e
13-acetate 4-0 methyl ether up-regulation
of t h e i r e n d o t h e l i a l cell b i n d i n g s i t e ,
w h e r e a s H K d o e s not (55).
Fifth,
angiotensin-converting e n z y m e inhibitors
p o t e n t i a t e the effect of b r a d y k i n i n on u p 2 +
2 +
2 +
2 +
2 +
253
Biol Res 3 1 : 251-262 (1998)
r e g u l a t i n g the H K b i n d i n g site on
e n d o t h e l i a l cells ( 5 5 ) . L a s t , w h e n H K b i n d s
to e n d o t h e l i a l c e l l s , it initiates a s e r i e s of
e v e n t s that a l l o w for an e n d o t h e l i a l c e l l a s s o c i a t e d e n z y m e to a c t i v a t e P K b o u n d to
H K ( 3 8 ) . T h i s last a c t i o n will b e d i s c u s s e d
in d e t a i l b e l o w . T h u s , b r a d y k i n i n u p regulates kininogen binding on endothelial
cells
and
kininogen
can
influence
bradykinin formation (35). These data
i n d i c a t e t h a t t h i s s y s t e m is t i g h t l y
c o n t r o l l e d in an a u t o c r i n e - l i k e m a n n e r .
The combined information given above
indicated
that
there
must
be
a
p h y s i c o c h e m i c a l k i n i n o g e n r e c e p t o r ( s ) on
e n d o t h e l i a l c e l l s . U s i n g an H K affinity
c o l u m n , the first b i n d i n g p r o t e i n r e p o r t e d
to b e isolated from e n d o t h e l i a l cell l y s a t e s
was a 33 k D a protein which upon aminot e r m i n a l s e q u e n c i n g w a s i d e n t i f i e d as
gClqR,
a known
r e c e p t o r for
the
macromolecular complement protein (23).
This protein only b o u n d H K , not LK.
F u r t h e r , the initial r e p o r t stated that Z n
w a s not r e q u i r e d for b i n d i n g ( 2 3 ) ; a s e c o n d
report stated that Z n
was a requirement
for b i n d i n g ( 2 9 ) . H o w e v e r , t h e r e is s o m e
c o n t r o v e r s y a s t o w h e t h e r g C l q R is a
s u b s t a n t i a l p r o t e i n on e n d o t h e l i a l cell
m e m b r a n e s . It has m o s t l y b e e n d e s c r i b e d as
a m i t o c h o n d r i a l p r o t e i n in e n d o t h e l i a l cells
(8). C D 1 l b / C D 18 on g r a n u l o c y t e s a l s o w a s
p r o p o s e d as b i n d i n g p r o t e i n , p u t a t i v e
receptor,
for
kininogen
because
a
m o n o c l o n a l a n t i b o d y to this s t r u c t u r e
partially blocked kininogen binding (51).
W e found this interpretation untenable
b e c a u s e in o u r o w n s t u d i e s , w e found that
I - H K and
I - L K b o u n d n o r m a l l y to
leukocyte
adhesion
deficiency
g r a n u l o c y t e s , i.e. g r a n u l o c y t e s a b s e n t in
CDllb/CD18
(unpublished).
More
recently, the u r o k i n a s e
plasminogen
activator receptor (uPAR) has been shown
to be a k i n i n o g e n b i n d i n g s i t e on
e n d o t h e l i a l c e l l s ( 6 ) . S i n c e u P A R is linked
to C D l l b / C D 1 8 , it is p o s s i b l e t h a t
a n t i b o d i e s to M a c - 1 that p a r t i a l l y b l o c k e d
H K b i n d i n g c o u l d h a v e b e e n d o i n g so by
interfering w i t h e x p r e s s i o n of u P A R ( 5 2 ) .
H o w e v e r , the fact that u P A R is not p r e s e n t
on p l a t e l e t s i n d i c a t e s t h a t t h i s p r o t e i n
c a n n o t b e a s i n g l e k i n i n o g e n r e c e p t o r on all
2 +
2 +
l 2 S
1 2 5
cells. Additional binding proteins must
exist.
W o r k p e r f o r m e d in o u r o w n l a b o r a t o r y
revealed that the major protein band
purified on a H K affinity c o l u m n from
e n d o t h e l i a l cell lysates w a s 5 4 k D a w h i c h
on a m i n o acid s e q u e n c i n g w a s identified as
cytokeratin 1 (CK1) (20). Cytokeratin 1
a n t i g e n w a s f o u n d on t h e m e m b r a n e of
e n d o t h e l i a l cells by laser s c a n n i n g c o n f o c a l
microscopy, flow cytometry, and direct
a n t i - C K l F ( a b ) ' b i n d i n g . H K specifically
b o u n d to n a t i v e or r e c o m b i n a n t C K 1 only
in the p r e s e n c e of Z n . F u r t h e r , all t h r e e
b i n d i n g d o m a i n s of H K ( d o m a i n s 3 , 4 and
5) b l o c k e d H K b i n d i n g t o c y t o k e r a t i n ( 1 7 ,
19, 2 4 ) . L a s t C K 1 a n t i g e n w a s found on
p l a t e l e t s a n d g r a n u l o c y t e s i n d i c a t i n g that
this protein can serve as a k i n i n o g e n
r e c e p t o r on all of t h e s e c e l l s . H o w e v e r , the
n u m b e r of C K 1 b i n d i n g sites on e a c h of
t h e s e cells is not sufficient to a c c o u n t for
the total n u m b e r of k i n i n o g e n b i n d i n g sites,
i n d i c a t i n g that o t h e r p r o t e i n s , t h o s e a b o v e
as well as p o s s i b l e o t h e r s , m a y s e r v e in a
m u l t i p r o t e i n a s s e m b l y as the k i n i n o g e n
r e c e p t o r . It is q u i t e u n e x p e c t e d to find C K 1
as a k i n i n o g e n b i n d i n g p r o t e i n , p u t a t i v e
r e c e p t o r . R e c e n t s t u d i e s h a v e i n d i c a t e d that
other c y t o k e r a t i n s , C K 8 a n d 18, s e r v e as
b i n d i n g p r o t e i n s for p l a s m i n o g e n and
t h r o m b i n - a n t i t h r o m b i n III c o m p l e x e s ,
respectively (21, 22, 53). Kininogens'
m u l t i p r o t e i n r e c e p t o r c o m p l e x is s h o w n in
F i g u r e 1. Last, b o t h C K 1 a n d u P A R ' s link
to
CDllb/CD18
provide
potential
m e c h a n i s m s for s i g n a l i n g w h e n H K
assembles
on
the
endothelial
cell
m e m b r a n e . T h e s e p o t e n t i a l m e c h a n i s m s for
cell activation may be i m p o r t a n t when
c o n t a c t p r o t e i n s a s s e m b l e on cell s u r f a c e s .
2
2 +
CHARACTERIZATION OF PREKALLIKREIN
ACTIVATION ON THE ENDOTHELIAL CELL
MEMBRANE
It is w e l l a c c e p t e d t h a t t h e m a j o r i t y of
p l a s m a P K a n d f a c t o r X I c i r c u l a t e s in
p l a s m a in c o m p l e x to H K ( 3 4 , 4 9 ) . S i n c e
H K s e r v e s as the cell r e c e p t o r for factor X I ,
w e r e a s o n e d t h a t it a l s o s e r v e s a s t h e
p r e k a l l i k r e i n b i n d i n g site o n e n d o t h e l i a l
254
Biol Res 3 1 : 251-262 (1998)
Fig 1. Multiprotein kininogen receptor on endothelial cell membrane. Circulating plasma PK and factor XI are mostly
bound to plasma HK. The complex between plasma HK and PK or factor XI binds to a multiprotein kininogen receptor on
endothelial cells which consists of cytokeratin 1 (CK1), urokinase plasminogen activator receptor ( u P A R ) , and g C l q R .
Factor XII (XII) also binds to g C l q R .
c e l l s ( 1 1 , 3 8 ) . P K s p e c i f i c a l l y b o u n d to
e n d o t h e l i a l c e l l s o n l y in t h e p r e s e n c e of
Zn
a n d after b e i n g s a t u r a t e d w i t h H K
( 3 8 ) . T h e a p p a r e n t Kd for this i n t e r a c t i o n
w a s 2 3 n M , a v a l u e s i m i l a r to that s e e n
w i t h P K b i n d i n g to H K in s o l u t i o n . P K
b i n d i n g to e n d o t h e l i a l cells in the p r e s e n c e
of a d d e d H K w a s a l m o s t c o m p l e t e l y
i n h i b i t e d by a n t i b o d i e s to the b i n d i n g
d o m a i n s for P K on H K and vice versa ( 3 8 ) .
F u r t h e r studies w e r e p e r f o r m e d to
d e t e r m i n e if w h e n H K a n d P K a s s e m b l e d
on e n d o t h e l i a l c e l l s w o u l d the P K b e c o m e
a c t i v a t e d to k a l l i k r e i n ( 1 1 ) . U s i n g m o d e l s
from the p l a s m a and artificial surface
s y s t e m , P K is o n l y a c t i v a t e d on s u r f a c e s in
the p r e s e n c e of a c t i v a t e d factor X I I (4, 12,
43). Initial studies showed that the
a s s e m b l y of H K , P K , or k a l l i k r e i n a l o n e on
e n d o t h e l i a l c e l l s d i d n o t r e s u l t in a n y
significant a m i d o l y t i c activity (Fig 2 A ) . A s
a control for the a d d i t i o n of a c t i v a t e d f o r m s
of F X I I for the a s s e m b l y of H K and P K , w e
2 +
a d d e d H K f o l l o w e d b y P K a l o n e to
e n d o t h e l i a l c e l l s . T h e a m o u n t of m e a s u r e d
kallikrein formed from the H K and PK
a s s e m b l y a l o n e w a s g r e a t e r than that seen
when zymogen FXII, activated FXII
(FXIIa, a F X I I a ) , or H a g e m a n factor
f r a g m e n t ( F X I I , (3FXIIa) w e r e a d d e d a l o n g
with the P K ( F i g 2 A ) . F u r t h e r , s u b s t i t u t i n g
k a l l i k r e i n at t h e s a m e c o n c e n t r a t i o n t h a n
P K d i d n o t r e s u l t in h i g h e r l e v e l s of
amidolysis (Fig 2A). These
results
s u r p r i s e d u s s i n c e t h e y i n d i c a t e d that on
e n d o t h e l i a l cells t h e r e is a P K a c t i v a t i n g
m e c h a n i s m i n d e p e n d e n t of factor X I I and
its a c t i v a t e d f o r m s . I n f a c t , u n d e r t h e
c o n d i t i o n s of t h e a s s a y , a d d i t i o n of the
e n z y m e k a l l i k r e i n r e s u l t e d in less m e a s u r e d
amidolytic activity.
Further investigations were performed
to
determine
if
there
was
any
c o n t a m i n a t i n g a c t i v a t e d F X I I in t h e
reaction. The ambient tissue culture media
c o n t a i n e d less than 0.0001 U / m l a c t i v a t e d
f
255
Biol Res 3 1 : 251-262 (1998)
H U V E C + H K +K A L H U V E C + H K+ PK*Xllf-jx
H U V E C + HK + PK +
Xlla - §
H U V E C * H K . P K + X I I - :;
HUVEC +H K * P K -|:
H U V E C +K A L H U V E C +P K
H U V E C + H K-
0
KALLIKREIN
FORMED
(pM/min)
B
PK D E F I C I E N T
FXII D E F I C I E N T
|
NHP
H U V E C + H K + P K + Antl-FXII
HUVEC + HK +PK
2B). S o y b e a n t r y p s i n i n h i b i t o r , a k a l l i k r e i n
inhibitor, abolished the measured activity,
b u t c o r n t r y p s i n i n h i b i t o r , an a c t i v a t e d F X I I
inhibitor, d i d n o t abolish t h e generated
kallikrein from P K a s s e m b l e d o n H K on
endothelial cells. Moreover, an antibody to
t h e P K b i n d i n g site on H K a n d a p e p t i d e to
c o m p e t e P K b i n d i n g t o H K a b o l i s h e d the
ability of P K to b e activated on t h e
endothelial cell m e m b r a n e . Last, when
n o r m a l h u m a n p l a s m a o r p l a s m a deficient
in F X I I w e r e i n c u b a t e d o v e r e n d o t h e l i a l
c e l l s , k a l l i k r e i n activity w a s d e m o n s t r a b l e ;
no activity w a s measured when P K
deficient plasma was incubated over
endothelial cells. These combined data
i n d i c a t e d that t h e P K a c t i v a t i o n m e c h a n i s m
was dependent on H K a n d P K a n d
i n d e p e n d e n t of F X I I .
Investigations next were performed to
d e t e r m i n e t h e r o l e , if a n y , f o r F X I I
activation on endothelial cells. Using
optimal Z n
and substrate concentrations,
we
found
no evidence
for FXII
a u t o a c t i v a t i o n o n t h e e n d o t h e l i a l cell
m e m b r a n e ( 4 4 ) . P K a c t i v a t i o n is t h e initial
and central event in contact proteins'
a c t i v a t i o n on e n d o t h e l i u m . F X I I c o n t r i b u t e s
to t h e rate a n d e x t e n t of e n z y m a t i c activity
generated
on the endothelial
cell
m e m b r a n e , b u t n o t t h e initiation of activity.
T h i s p o i n t w a s m a d e by d e t e r m i n i n g t h e
k i n e t i c s of P K a c t i v a t i o n . In t h e a b s e n c e of
FXII, the K (20 ± 8 n M ) and V
(12 ± 3
p M / m i n ) of P K a c t i v a t i o n w a s virtually t h e
s a m e a s that g e n e r a t e d in t h e p r e s e n c e of
F X I I (K = 3 0 + 4 . 2 n M ; V
= 9.2 ± 2.1
pM/min)
( 3 8 ) . These
findings on
e n d o t h e l i a l cells a r e j u s t t h e o p p o s i t e from
that k n o w n t o o c c u r o n artificial surfaces
when activation of F X I I initiates t h e
system. From normal plasma or FXII
deficient p l a s m a , P K b o u n d t o e n d o t h e l i a l
c e l l s b e c o m e s fully a c t i v a t e d w i t h i n 4 - 5
min. Further, using purified contact
p r o t e i n s a t p l a s m a c o n c e n t r a t i o n s , t h e full
e x t e n t of a c t i v a t i o n a l s o o c c u r r e d w i t h i n 5
min. T h e kinetics of activation of this
s y s t e m on e n d o t h e l i a l cells greatly e x c e e d s
F X I I a u t o a c t i v a t i o n w h i c h o n l y r e s u l t s in
l o w l e v e l s of activity e v e n after 1 2 0 m i n of
activation. T h e s e data indicate t h a t P K
a c t i v a t i o n is t h e c r i t i c a l l y i m p o r t a n t
2 +
H U V E C + PK
HUVEC + HK
KALLIKREIN
FORMED
(pM/min)
Fig 2. Activation of P K on H U V E C . Panel A : Endothelial
cell monolayers ( H U V E C ) preincubated with 2 0 0 fjJ of a
solution containing 2 % bovine serum albumin. HK (20
nM) or buffer then added for 1 h at 37°C; unbound protein
removed by washing, and 2 0 nM PK o r 20 nM plasma
kallikrein (Kal) incubated for an additional h. Wells were
then washed and 0.4 m M S 2 3 0 2 added in a b s e n c e o r
presence of 20 n M FXII (XII), 3.4 nM cxFXIIa (Xlla), or
3.4 nM p F X I I a (Xllf), as indicated. Data, means ± SEMs
of 3 experiments. A b s e n c e o f standard error bars in some
columns indicates that variation was too little to portray
visually. Panel B: Endothelial cell monolayers ( H U V E C )
preincubated with 2 0 0 pi of a solution c o n t a i n i n g 2 %
bovine serum albumin. HK ( 2 0 n M ) or buffer then added
for 1 h at 37°C; unbound protein removed by washing, and
20 nM PK incubated for an additional h in absence or
presence of 0.4 m g / m l of an anti-FXII antibody (AntiFXII). In other experiments, H U V E C saturated with HK
(20 n M ) incubated with 5 0 pi of pooled normal plasma
(NHP), FXII-deficient plasma, or PK-deficient plasma for
1 h at 37°C. After w a s h i n g , 0.4 m M S2302 added and
hydrolysis monitored for 1 h. Data, means ± SEMs of 3
e x p e r i m e n t s . A b s e n c e of s t a n d a r d e r r o r bars in s o m e
c o l u m n s i n d i c a t e s that t h e variation w a s t o o little t o
portray visually.
F X I I c o a g u l a n t a c t i v i t y . I n t h e p r e s e n c e of
a n e u t r a l i z i n g c o n c e n t r a t i o n of an a n t i b o d y
to F X I I a , P K a c t i v a t i o n w a s t o t h e s a m e
extent when n o antibody was present (Fig
m
m
max
max
256
Biol Res 3 1 : 251-262 (1998)
m e c h a n i s m for a c t i v a t i o n of this s y s t e m in
a p h y s i o l o g i c m a n n e r on e n d o t h e l i a l cells.
F u r t h e r s t u d i e s w e r e p e r f o r m e d to
d e t e r m i n e the m e c h a n i s m by w h i c h P K
b e c a m e a c t i v a t e d on e n d o t h e l i a l c e l l s .
Initial i n v e s t i g a t i o n s e x a m i n e d the r o l e of
H K . In the a b s e n c e of a d d e d H K , t h e r e is
little c h a n g e in the s t r u c t u r e of 85 and 88
k D a P K bound to endothelial cells when
i n c u b a t e d for 2 h. In the p r e s e n c e of H K ,
b o u n d P K is r a p i d l y c l e a v e d w i t h i n 1 to 2
m i n to s h o w its h e a v y c h a i n (51 k D a ) and
its l i g h t c h a i n s at 3 7 a n d 3 4 k D a ( 3 8 ) .
T h e s e d a t a i n d i c a t e that the p r e s e n c e of H K
and P K a s s e m b l y w i t h H K are critical for
the a c t i v a t i o n of P K . I n v e s t i g a t i o n s n e x t
p r o c e e d e d to d e t e r m i n e if the a c t i v a t i o n of
P K w h e n b o u n d to H K was an e n z y m a t i c
process. Initial studies determined if serine
protease inhibitors w o u l d block P K activation
on endothelial cell m e m b r a n e s . Neutralizing
a n t i b o d y to F X I I a , b e n z a m i d i n e , P M S F ,
soybean trypsin inhibitor, and P r o - P h e - A r g c h l o r o m e t h y l k e t o n e did not inhibit the
c h a n g e in s t r u c t u r e of z y m o g e n P K to
kallikrein as s h o w n on S D S - P A G E . T h e s e
d a t a i n d i c a t e d t h a t t h e a c t i v a t i o n of P K
b o u n d to H K w a s n o t d u e to F X I I a , P K
a u t o a c t i v a t i o n , or a s e r i n e p r o t e a s e ( 3 8 ) .
Metal chelators like E D T A , E G T A ,
o r t h o p h e n a n t h r o l i n e , etc, b l o c k e d P K
activation p r e s u m a b l y by b l o c k i n g H K
b i n d i n g to e n d o t h e l i a l c e l l s . A l t e r n a t i v e l y ,
a n t i p a i n ( 1 0 0 pJVI), c y s t e i n e , H g C l , D T T ,
2 - m e r c a p t o e t h a n o l all fully b l o c k e d P K
a c t i v a t i o n . I n t e r e s t i n g l y , c y s t a t i n , ne t h y l m a l e i m i d e , i o d o a c e t a m i d e did not
inhibit this PK activating enzyme(s).
Further metalloprotease inhibitors, T I M P - 1 ,
T I M P - 2 , and B B 9 4 were not inhibitory.
These c o m b i n e d data indicate that a
p e c u l i a r c y s t e i n e p r o t e a s e a s s o c i a t e d with
t h e m e m b r a n e of e n d o t h e l i a l c e l l s is
r e s p o n s i b l e for t h e e n z y m a t i c c o n v e r s i o n of
P K to k a l l i k r e i n o n l y w h e n b o u n d to H K .
T h e s e d a t a i n d i c a t e that e n d o t h e l i a l cells
h a v e a m e c h a n i s m by w h i c h c o n t a c t p r o t e i n
a s s e m b l y a l l o w s for P K a c t i v a t i o n . W e m a y
have discovered a fundamental mechanism
in c e l l b i o l o g y . W e h a v e
already
demonstrated that HK-regulated
PK
activation by a c y s t e i n e p r o t e a s e actually
can
occur
on rat
3T3
fibroblasts
2
( u n p u b l i s h e d ) . F i g u r e 3 s h o w s a c a r t o o n of
the m u l t i p r o t e i n a s s e m b l y of P K on H K on
its r e c e p t o r c o m p l e x on e n d o t h e l i a l cells
a n d the role of P K in F X I I a c t i v a t i o n .
DETERMINATION OF BIOLOGICALLY
IMPORTANT ACTIVITIES ASSOCIATED WITH
PREKALLIKREIN ACTIVATION
Fibrinolysis.
T h e c o n s e q u e n c e s of P K a c t i v a t i o n o n
e n d o t h e l i a l cells n e e d s to b e e n u m e r a t e d .
First and f o r e m o s t after very rapid PK
a c t i v a t i o n on H K on e n d o t h e l i a l c e l l s ,
k a l l i k r e i n c l e a v e s i t s r e c e p t o r , H K , to
l i b e r a t e b r a d y k i n i n ( 3 8 ) . T h e local b i o l o g i c
e f f e c t s of b r a d y k i n i n o n b l o o d p r e s s u r e
regulation, prostaglandin formation, N O
formation, superoxide formation, stimulation
of s m o o t h m u s c l e hyperpolarization factor,
and tissue p l a s m i n o g e n a c t i v a t o r liberation
are i m p o r t a n t a l o n e ( 3 , 7, 2 5 , 2 6 , 3 9 , 4 0 ,
4 7 ) . B r a d y k i n i n h a s b e e n d e m o n s t r a t e d to
b e the m o s t p o t e n t s t i m u l a t o r of t i s s u e - t y p e
p l a s m i n o g e n r e l e a s e a c t i v a t o r in vivo in
r a b b i t s a n d m a n ( 3 , 4 7 ) . T h i s fact a l o n e is
an i m p o r t a n t c o n t r i b u t i o n to f i b r i n o l y s i s .
However, the kallikrein/kinin system has
b e e n s h o w n t o h a v e o t h e r r o l e s in
f i b r i n o l y s i s . P l a s m a k a l l i k r e i n is k n o w n to
b e the m o s t f a v o r a b l e k i n e t i c a c t i v a t o r of
p r o - u r o k i n a s e ( s i n g l e c h a i n u r o k i n a s e ) in
vitro ( 2 7 ) . G u r e w i c h a n d h i s c o w o r k e r s
( 1 3 , 3 1 , 33) p e r f o r m e d an i m p o r t a n t series
of e x p e r i m e n t s w h e r e they s h o w e d that H K
a n d P K a s s e m b l y on p l a t e l e t s or e n d o t h e l i a l
cells, after the a d d i t i o n of a c t i v a t e d F X I I ,
led to k i n e t i c a l l y f a v o r a b l e p r o - u r o k i n a s e
a c t i v a t i o n . T h i s m e c h a n i s m of s i n g l e c h a i n
urokinase activation was kallikrein, HK,
and cell-dependent (platelet,endothelial
cell) after t h e a d d i t i o n of a P K a c t i v a t o r ,
i.e. F X I I a .
W e a s k e d t h e q u e s t i o n if o u r F X I I i n d e p e n d e n t , P K a c t i v a t i o n s y s t e m on
e n d o t h e l i a l c e l l s a l s o c o u l d r e s u l t in
kinetically favorable pro-urokinase and,
s u b s e q u e n t l y , p l a s m i n o g e n a c t i v a t i o n (Fig
4 ) . T h e ability of H K , P K , p r o - u r o k i n a s e ,
or H K + P K a l o n e t o g e n e r a t e e n z y m a t i c
activity to c l e a v e a c h r o m o g e n i c substrate
257
Biol Res 3 1 : 251-262 (1998)
./•WW
be
txxx
Vi
v
f W W f r f r f r f r r r r f f f f
M 1/lfVUW
(
-ffTHTr r f f f'f"f(TW ( T T r T T f f f
f T f r f f r F r f
Fig 3 . Mechanism of FXII activation on endothelial cells. Plasma complex of HK and PK binds to endothelial cell
multiprotein kininogen receptor. Binding of PK t o HK on this receptor allows for expression of an endothelial cell
membrane cysteine protease (MP) that activates PK to kallikrein (K). FXII (XII) bound t o the multiprotein kininogen
receptor is then activated by kallikrein to activated FXII (Xlla). Kallikrein cleaves its receptor, HK, to liberate bradykinin
(BK) and release itself from the m e m b r a n e .
for t w o chain u r o k i n a s e w a s little (Fig 4 A ) .
A s p r e v i o u s l y s h o w n , the addition of p r o u r o k i n a s e t o e n d o t h e l i a l cells r e s u l t e d in
more enzymatic activity which was n o t
potentiated b y P K alone (1) (Fig 4 A ) .
H o w e v e r , the a s s e m b l y of H K and P K along
with p r o - u r o k i n a s e o n endothelial cells
resulted in i n c r e a s e d t w o c h a i n u r o k i n a s e
a c t i v i t y ( F i g 4 A ) . F u r t h e r , this i n c r e a s e d
activity w a s not b l o c k e d by a n antibody to
factor X l l a ( 3 8 ) . A d d i t i o n a l studies s h o w e d
that the K (135 + 81 n M ) and V
(14.5 ±
8 n M / m i n ) of p r o - u r o k i n a s e activation o n
e n d o t h e l i a l cells in the p r e s e n c e of H K , P K
and F X I I w a s the s a m e a s that seen in the
a b s e n c e of F X I I (K = 6 4 + 5 n M ; V
= 10
± 0 . 1 nM/min) (38).
S i n c e the c h r o m o g e n i c s u b s t r a t e for t w o
chain urokinase h a s a high K , w e
performed studies with two chain urokinase
and its b i o l o g i c s u b s t r a t e , p l a s m i n o g e n (Fig
4B) (38). HK, PK, plasminogen, or prom
max
m
max
m
u r o k i n a s e result in little h y d r o l y s i s of the
chromogenic substrate for plasminogen
(Fig 4 B ) . F u r t h e r , the a s s e m b l y of H K +
PK, plasminogen alone, plasminogen +
pro-urokinase, or PK, plasminogen, + prou r o k i n a s e on e n d o t h e l i a l cells also has little
activity on the p l a s m i n s u b s t r a t e . H o w e v e r ,
if w e a s s e m b l e H K and P K on e n d o t h e l i a l
cells followed by p l a s m i n o g e n and prou r o k i n a s e is a d d e d at the t i m e of the a d d i n g
the c h r o m o g e n i c s u b s t r a t e , there is a
m a r k e d i n c r e a s e in t h e a m o u n t of p l a s m i n
f o r m e d ( F i g 4 B ) . T h e g e n e r a t i o n of this
p l a s m i n is not i n h i b i t e d b y a n a n t i b o d y to
F X I I . T h e s e d a t a i n d i c a t e a m e c h a n i s m for
fibrinolysis independent of FXII, tissuet y p e p l a s m i n o g e n a c t i v a t o r , and fibrin. T h i s
w o r k has a l r e a d y b e e n c o n f i r m e d by s t u d i e s
from another laboratory (32). Figure 5
represents a cartoon o n how prekallikrein
a s s e m b l e s o n e n d o t h e l i a l c e l l s t o result in
kinetically favorable pro-urokinase and
258
Biol Res 3 1 : 251-262 (1998)
Thrombin
ProUK-
inhibition.
HUVEC + HKHUVEC + PK- i
HUVEC + HK + PK- '
HUVEC
ProUK-
HUVEC . PK . ProUKHUVEC * HK . PK * ProUK- ;
HUVEC + HK v f K + Anti-FXII + ProUK-
TcuPA FORMED (nM/min)
HK
PK PLG
ProUK
A s e c o n d b i o l o g i c activity of k i n i n o g e n s is
t h e i r a b i l i t y to f u n c t i o n as i n h i b i t o r s of
t h r o m b i n a c t i v a t i o n of p l a t e l e t s . It a p p e a r s
that there are m u l t i p l e m e c h a n i s m s by
w h i c h k i n i n o g e n s f u n c t i o n as i n h i b i t o r s to
t h r o m b i n a c t i v a t i o n of p l a t e l e t s ( F i g 6 ) .
T h e first m e c h a n i s m to b e d e s c r i b e d w a s
that kininogens inhibit calpain-induced
platelet aggregation (45). W h e n platelets
a r e a c t i v a t e d w i t h t h r o m b i n , c a l p a i n is
m e m b r a n e - e x p r e s s e d and its p r e s e n c e
allows
for
the
formation
of
the
h e t e r o d i m e r i c c o m p l e x of o c p i n t e g r i n
(platelet g l y c o p r o t e i n I l b / I I I a ) to b e f o r m e d
to s u p p o r t f i b r i n o g e n b i n d i n g a n d platelet
a g g r e g a t i o n ( 4 1 , 4 5 ) . T h i s m e c h a n i s m can
fully a c c o u n t for k i n i n o g e n i n h i b i t i o n of
thrombin-induced platelet aggregation.
However,
when
thrombin
activates
platelets, platelets secrete their contents
before they aggregate and kininogens
inhibit b o t h p r o c e s s e s ( 3 6 ) . T h u s a n o t h e r
m e c h a n i s m ( s ) of k i n i n o g e n i n h i b i t i o n of
t h r o m b i n a c t i v a t i o n of p l a t e l e t s n e e d e d to
be sought.
B o t h H K a n d L K h a v e b e e n s h o w n to
inhibit oc-thrombin b i n d i n g to p l a t e l e t s and
e n d o t h e l i a l cells ( 1 6 , 17, 3 6 ) . T h e actual
l o c a t i o n on p l a t e l e t s that t h r o m b i n b i n d s to
is n o t c o m p l e t e l y k n o w n . T h r o m b i n h a s
b e e n p o s t u l a t e d to b i n d to at least t w o sites
o n t h e p l a t e l e t s u r f a c e . I n d a t a to b e
p r e s e n t e d b e l o w , it i s r e a s o n a b l e t o
c o n s i d e r that k i n i n o g e n s b i n d to the first
cloned
thrombin
receptor,
protease
a c t i v a t e d r e c e p t o r 1 ( P A R I ) to b l o c k a t h r o m b i n f r o m b i n d i n g to t h i s site on
platelets (16). O t h e r data suggest that
kininogens may interact with platelet
g l y c o p r o t e i n I b - I X - V c o m p l e x or c o m p e t e
w i t h t h r o m b i n ' s a b i l i t y t o b i n d to t h a t
complex (2, 28). Preliminary studies
s u g g e s t that g l y c o p r o t e i n l b m a y b e a zinc
d e p e n d e n t b i n d i n g site for H K (28).
H o w e v e r , o u r o w n s t u d i e s h a v e s h o w n that
H K binds n o r m a l l y to B e r n a r d - S o u l i e r
p l a t e l e t s , i.e. p l a t e l e t s d e f i c i e n t in p l a t e l e t
glycoprotein
lb
(unpublished).
A l t e r n a t i v e l y , it h a s b e e n p r o p o s e d t h a t
k i n i n o g e n w i t h its d o m a i n 3 s e q u e n c e
LNAENNA
m a y s e r v e as a c o m p e t i n g
I I b
HUVEC . HK . PK HUVEC + PLG
HUVEC • PLG + ProUK
HUVEC + PLG + ProUK + PK
HUVEC + PLG + ProUK t HK + PK
HUVEC + PLG + ProUK + HK • PK + Anti-FXII
PLASMIN
FORMED
(nM/min)
Fig 4. Influence of HK and PK on pro-urokinase and
plasminogen activation. Panel A : Pro-urokinase activation.
E m p t y m i c r o t i t e r p l a t e w e l l s or w e l l s c o a t e d with a
monolayer of endothelial cells ( H U V E C ) incubated with
HK (20 nM) or buffer for 1 h. Unbound HK removed and
cells incubated with PK (20 nM) for another h and washed.
Pro-UK (20 nM) and 0.6 m M S2444 added to empty wells
or wells coated with H U V E C , and hydrolysis monitored
c o n t i n u o u s l y o v e r 7 5 m i n at 3 7 ° C . In o n e set of
experiments, 0.4 mg/ml of a neutralizing antibody to FXII
a d d e d a l o n g w i t h t h e P K . F o r m a t i o n of t w o c h a i n
urokinase plasminogen activator (TcuPA) determined by
c o m p a r i n g s u b s t r a t e h y d r o l y s i s on cells w i t h k n o w n
concentrations of soluble TcuPA. Data, means ± SEMs of
3 experiments. Panel B : Plasminogen activation. Empty
microtiter plate wells or wells coated with a monolayer of
H U V E C incubated for 1 h with 1 |iM plasminogen (PLG)
before 0.3 m M S 2 2 5 1 w a s a d d e d e i t h e r a l o n e or in
presence of 2 nM Pro-UK. In other experiments, H U V E C coated wells incubated for 1 h with 20 nM HK. After
r e m o v a l of H K , wells i n c u b a t e d with 20 nM PK for
another h. After removal of excess PK, cells incubated
with 1 p.M plasminogen (PLG) for a third h. As indicated,
in one case, 0.4 mg/ml of a neutralizing antibody to FXII
was added a l o n g with the PK. Hydrolysis of substrate
m e a s u r e d o v e r 2 1 0 min at 3 7 ° C . P l a s m i n f o r m a t i o n
determined using a standard curve made by adding known
amounts of purified plasmin to S 2 2 5 1 . Data, means ±
SEMs of 4 independent experiments. Absence of standard
error bars in some columns indicates that variation was too
little to portray visually.
plasminogen
activation.
Thus,
PK
a c t i v a t i o n o n e n d o t h e l i a l cells results in a
m e c h a n i s m for c e l l u l a r f i b r i n o l y s i s . T h i s
p a t h w a y c o u l d result in t h e initial levels of
p l a s m i n w h i c h c o u l d s u b s e q u e n t l y amplify
pro-urokinase activation.
3
Biol Res 3 1 : 251-262 (1998)
259
HK
HKa
5c*
HKa
Fig 5. Mechanism of kallikrein-mediated cellular fibrinolysis. PK bound to HK becomes activated by an endothelial cell
membrane cysteine protease (MP). Kallikrein (K) bound to HK proteolyzes HK to liberate bradykinin (BK). Bradykinin
stimulates the release of endothelial cell tissue-type plasminogen activator (tPA). Kallikrein also activates endothelial cellbound single chain urokinase (SuPA) bound to the urokinase plasminogen activator receptor (uPAR) to form two chain
urokinase plasminogen activator (TcuPA). Two chain urokinase plasminogen activator initiates the conversion of zymogen
plasminogen (PLG) to plasmin (P) bound to its endothelial cell receptor.
b i n d i n g s i t e for o c - t h r o m b i n b i n d i n g t o
glycoprotein-Ib-IX-V
complex
(2).
Glycoprotein lb contains the sequence
NAEN at r e s i d u e s 2 2 3 - 2 2 6 w h i c h is w i t h i n
2 4 a m i n o a c i d s of a s e q u e n c e w h i c h is
k n o w n to i n h i b i t t h r o m b i n - i n d u c e d p l a t e l e t
a g g r e g a t i o n ( 3 0 ) . T h u s , it is p o s s i b l e that
k i n i n o g e n s e r v e s as a p s e u d o g l y c o p r o t e i n
lb, binding available thrombin and keeping
it f r o m a s s o c i a t i n g w i t h t h i s p l a t e l e t
g l y c o p r o t e i n . In a d d i t i o n , it is a l s o p o s s i b l e
that k i n i n o g e n s c o u l d b i n d to g l y c o p r o t e i n
I b - I X - V at a n o t h e r r e g i o n and t h u s interfere
w i t h t h r o m b i n b i n d i n g to p l a t e l e t s . B o t h
m e c h a n i s m s c o u l d b e o p e r a t i v e , but at this
t i m e t h e r e is insufficient i n f o r m a t i o n to say
w h i c h is a c t u a l l y o c c u r r i n g .
A third m e c h a n i s m by w h i c h k i n i n o g e n s
or a d i s c r e e t p e p t i d e f r o m d o m a i n 4 ,
RPPGF, interfere w i t h t h r o m b i n a c t i v a t i o n
of p l a t e l e t s h a s b e e n d e s c r i b e d ( 1 6 ) .
RPPGF
p r e s e r v e s t h e e p i t o p e of t h e
c l e a v a g e site on P A R I after t h r o m b i n
a c t i v a t i o n . It d o e s so b y p r e v e n t i n g octhrombin from cleaving P A R I
after
a r g i n i n e , a critical site for t h r o m b i n
a c t i v a t i o n o f t h i s r e c e p t o r ( 1 6 ) . It is
i n c r e d i b l y i n t e r e s t i n g that the a n g i o t e n s i n
c o n v e r t i n g e n z y m e b r e a k d o w n p r o d u c t of
b r a d y k i n i n , RPPGF,
has
additional
biologic activity to prevent t h r o m b i n ' s
a c t i o n s . In w o r k w h i c h is i n - p r o g r e s s ,
RPPGF
d i r e c t l y b i n d s to p l a t e l e t s to
p r e v e n t t h r o m b i n ' s a c t i v a t i o n of t h i s
receptor.
T h u s , k i n i n o g e n s ' interactions with
t h r o m b i n a p p e a r to b e m u l t i f a c e t e d . O n e
m a y a r g u e that k i n i n o g e n s m a y c o n t r i b u t e
to the c o n s t i t u t i v e a n t i c o a g u l a n t n a t u r e of
the
intravascular
compartment.
In
kininogen deficient platelet-rich plasma,
2.5 t i m e s l e s s y - t h r o m b i n is n e e d e d to
4 1
260
Biol Res 3 1 : 251-262 (1998)
ft
Fig 6. Mechanisms of k i n i n o g e n s ' inhibition of thrombin. One mechanism interferes with calpain stimulation of formation
of heterodimeric complex between integrin cc I I b P 3 . HK or LK blocks calpain which prevents formation of this integrin
complex. A second mechanism of kininogen inhibition of thrombin activation of platelets is that HK blocks thrombin (I la)
from binding to thrombin receptor. A third mechanism of inhibition of thrombin-induced platelet activation is that HK and
fragments of its domain 4 prevent thrombin (Ila) from cleaving P A R I , the seven transmembrane thrombin receptor, near its
amino-terminus.
a g g r e g a t e p l a t e l e t s t h a n t h a t n e e d e d for
normal plasma (42). Further, preliminary
s t u d i e s r e v e a l that the t i m e to t h r o m b o s i s in
a m i n i m a l injury m o d e l of the left iliac
a r t e r y w a s m u c h s h o r t e r in k i n i n o g e n
deficient rats than in n o r m a l rats ( 6 ) .
SUMMARY
In c o n c l u s i o n , t h e r e h a s b e e n a m a j o r
e v o l u t i o n in u n d e r s t a n d i n g t h e p l a s m a
c o n t a c t s y s t e m of p r o t e o l y s i s . K i n i n o g e n s '
a s s e m b l y on their m u l t i p r o t e i n r e c e p t o r
a l l o w s for r e g u l a t e d P K a c t i v a t i o n in a
biologic
environment.
Obviously,
r e g u l a t i o n of t h e r e c e p t o r a n d k i n i n o g e n
and P K b i n d i n g will modify the a s s o c i a t i o n
of t h e s e p r o t e i n s a n d a c t i v a t i o n of t h i s
system. On endothelial cells, FXII
a c t i v a t i o n is s e c o n d a r y a n d a m p l i f i e s P K
a c t i v a t i o n . T h e i m m e d i a t e c o n s e q u e n c e of
a c t i v a t i o n of t h e p l a s m a k a l l i k r e i n / k i n i n
s y s t e m is the l i b e r a t i o n of b r a d y k i n i n with
its a t t e n d a n t a c t i v i t i e s . F u r t h e r , c o n t a c t
p r o t e i n s p r o b a b l y c o n t r i b u t e to c e l l u l a r
fibrinolysis
and
the
constituent
a n t i c o a g u l a n t n a t u r e of t h e i n t r a v a s c u l a r
c o m p a r t m e n t . It is a p a r a d o x that p r o t e i n s
t h o u g h t t o c o n t r i b u t e t o h e m o s t a s i s in
r e a l i t y c o n t r i b u t e to p r e v e n t i o n
of
t h r o m b o s i s . In c o n c l u s i o n , I w a n t to c o n v e y
to you that I b e l i e v e that w e are at the d a w n
of a n e w u n d e r s t a n d i n g of the p h y s i o l o g i c
m e c h a n i s m of a c t i v a t i o n a n d i m p o r t a n c e of
the p l a s m a k a l l i k r e i n / k i n i n s y s t e m . M a n y
d i s c o v e r i e s on the p h y s i o l o g i c r o l e of this
s y s t e m still await o u r r e c o g n i t i o n .
261
Bio] Res 3 1 : 251-262 (1998)
ACKNOWLEDGEMENTS
11
I w o u l d like to t h a n k D r A h m e d A K H a s a n
for his i n s i g h t s a n d efforts in d e t e r m i n i n g
the k i n i n o g e n r e c e p t o r a n d p e r f o r m i n g the
critical initial a s s a y s to r e c o g n i z e a n o v e l
PK activation system and
thrombin
i n h i b i t i o n m e c h a n i s m . F u r t h e r , I w a n t to
thank Drs G u a c y a r a d a M o t t a and R a s m u s
Rojkjaer for their efforts in c h a r a c t e r i z i n g
the P K activation m e c h a n i s m . I also w a n t to
thank M s F a k h r i M a h d i and D r Z i a ShariatM a d a r for t h e i r w o r k c h a r a c t e r i z i n g t h e
kininogen receptor. This work
was
s u p p o r t e d by N I H g r a n t s H L 3 5 5 5 3 ,
H L 5 2 7 9 9 and H L 5 6 4 1 5 .
12
REFERENCES
1
2
3
4
5
6
7
8
9
10
B A R N A T H A N E S , K U O A, R O S E N F E L D L,
K A R I K O K, L E S K I M, ROBBIAT1 F, CINES DB
( 1 9 9 0 ) I n t e r a c t i o n of s i n g l e - c h a i n u r o k i n a s e - t y p e
plasminogen activator with human endothelial cells. J
Biol C h e m 2 6 5 : 2865-2872
BRADFORD
HN,
DE
LA
CADENA
RA,
K U N A P U L I SP, D O N G J-F, L O P E Z J, C O L M A N
RW ( 1 9 9 7 ) H u m a n k i n i n o g e n s r e g u l a t e t h r o m b i n
binding to platelets through the glycoprotein Ib-IX-V
complex. Blood 90: 1508-1515
B R O W N NJ, N A D E A U JH, V A U G H A N DE (1997)
S e l e c t i v e s t i m u l a t i o n of t i s s u e - t y p e p l a s m i n o g e n
activator (t-PA) in vivo by infusion of bradykinin.
Thromb Haemost 7 7 : 522-525
C O C H R A N E C G , R E V A K SD, W U E P P E R KD
(1973) Activation of Hageman factor in solid and
fluid phases: a critical role of kallikrein. J Exp Med
138: 1564-1583
C O L M A N RW, P I X L E Y RA, N A J A M U N N I S A S,
YAN W - Y , W A N G J, M A Z A R A, M c C R A E KR
(1997) Binding of high molecular weight kininogen
to human endothelial cells is mediated via a site
within domains 2 and 3 of the urokinase receptor. J
Clin Invest 100: 1481-1487
C O L M A N R W , S C O V E L L S, S T A D N I C K I A,
S A R T O R R B , W H I T E JV (1997) K i n i n o g e n s are
antithrombotic proteins in vivo. Blood 90 (suppl 1): 29a
C R U T C H L E Y DJ, RYAN JW, RYAN US, FISHER GH
(1983) Bradykinin-induced release of prostacyclin and
Ihromboxanes from bovine pulmonary artery endothelial
c e l l s . S t u d i e s w i t h l o w e r h o m o l o g s and c a l c i u m
antagonists. Biochim Biophys Acta 7 5 1 : 99-107
DEDIO J. M U E L L E R - E S T E R L W (1996) Kininogen
b i n d i n g p r o t e i n p 3 3 / g C l q R is l o c a l i z e d in the
vesicular fraction of endothelial cells. F E B S Lett 399:
255-258
DE LA C A D E N A RA, C O L M A N RA (1992) The
s e q u e n c e HGLGHGHEQQHGLGHGH
in the light
chain of high molecular weight kininogen serves as a
primary structural feature for zinc-dependent binding
to an anionic surface. Protein Sei 1: 151-160
G R E E N G A R D JS, G R I F F I N JH (1984) Receptors for
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 on s t i m u l a t e d
washed human platelets. Biochemistry 2 3 : 6863-6869
13
14
15
16
17
18
19
20
21
22
23
24
25
26
G R E E N G A R D JS, HEEB MJ, E R S D A L E, W A L S H
PN, GRIFFIN JH (1986) Binding of coagulation factor
XI to washed platelets. Biochemistry 25: 3884-3890
GRIFFIN JH (1978) Role of surface in the surfaced e p e n d e n t a c t i v a t i o n of H a g e m a n f a c t o r ( b l o o d
coagulation factor XII). Proc Natl Acad Sci USA 75:
1998-2002
G U R E W I C H V, J O H N S T O N E M , L O Z A J - P ,
P A N N E L L R (1993) Prouorkinase and prekallikrein
are both associated with platelets: Implications tor
t h e i n t r i n s i c p a t h w a y s of f i b r i n o l y s i s and for
therapeutic thrombolysis. F E B S Lett 318: 317-321
G U S T A F S O N EG, SCHMA1ER AH, W A C H T F O G E L
YT, K A U F M A N N, K U C I C H U, C O L M A N RW
(1989) Human n e u t r o p h i l s c o n t a i n and b i n d high
molecular weight kininogen. J Clin Invest 84: 28-35
G U S T A F S O N E G , S C H U T S K Y D , K N I G H T L,
S C H M A I E R AH ( 1 9 8 6 ) 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 b i n d s to u n s t i m u l a t e d p l a t e l e t s . J Clin
Invest 78: 310-318
H A S A N AAK, A M E N T A S, S C H M A I E R AH (1996)
Bradykinin and its metabolite, Arg-Pro-Pro-Gly-Phe,
a r e s e l e c t i v e i n h i b i t o r s of a - t h r o m b i n - i n d u c e d
platelet activation. Circulation 94: 1465-1473
HASAN
AAK, CINES
DB, H E R W A L D
H.
SCHMAIER AH, M U E L L E R - E S T E R L W (1995)
Mapping the cell binding site on high molecular weight
kininogen's domain 5. J Biol Chem 270: 19256-19261
HASAN AAK, CINES DB, NGAIZA JR, J A F F E E,
SCHMAIER AH (1995) High molecular weight
k i n i n o g e n is e x c l u s i v e l y m e m b r a n e b o u n d on
endothelial cells to influence activation of vascular
endothelium. Blood 85: 3134-3143
H A S A N AAK, C I N E S DB, Z H A N G J, S C H M A I E R
AH (1994) The carboxyl terminus of bradykinin and
a m i n o t e r m i n u s of the light c h a i n of k i n i n o g e n s
comprise an endothelial cell binding domain. J Biol
Chem 269: 31822-31830
H A S A N AAK, Z I S M A N T. S C H M A I E R AH (1998)
Identification of c y t o k e r a t i n 1 as a b i n d i n g protein
a n d p r e s e n t a t i o n r e c e p t o r f o r k i n i n o g e n s on
e n d o t h e l i a l c e l l s . Proc Natl A c a d Sci U S A 9 5 :
3615-3620
H E M B R O U G H TA, LI L, G O N I A S SL (1996) Cellsurface cytokeratin 8 is the major plasminogen recep­
tor on breast cancer cells and is required for the
accelerated activation of cell-associated plasminogen
by tissue-type plasminogen activator. J Biol Chem
271: 25684-25691
H E M B R O U G H T A , V A S U D E V A N J, A L L I E T T A
M M , G L A S S W F I I , G O N I A S SL ( 1 9 9 5 ) A
cytokeratin 8-like protein with plasminogen-binding
a c t i v i t y is p r e s e n t on t h e e x t e r n a l s u r f a c e of
hepatocytes, HepG2 cells, and breast carcinoma cell
line. J Cell Sci 108: 1071-1082
H E R W A L D H, D E D I O J, K E L L N E R R, L O O S M,
M U E L L E R - E S T E R L W ( 1 9 9 6 ) I s o l a t i o n and
characterization of the kininogen-binding protein p33
from endothelial cells: Identity with the g C l q recep­
tor. J Biol Chem 2 7 1 : 13040-13047
HERWALD
H, H A S A N A A K ,
GODOVACZ I M M E R M A N J, S C H M A I E R A H , M U E L L E R E S T E R L W (1995) Identification of the endothelial
cell binding site on domain D 3 . J Biol Chem 270:
14634-14642
H O L L A N D JA, P R I T C H A R D KA, PAPPOLLA MA,
WOLIN M S , ROGERS NJ, S T E M E R M A N MB (1990)
Bradykinin induces superoxide anion release from
human endothelial cells. J C e l l Physiol 143: 21-25
HONG SL (1980) Effect of bradykinin and thrombin
on prostacyclin synthesis in endothelial cells from
262
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Biol Res 3 1 : 251-262 (1998)
calf and pig aorta and human umbilical cord vein.
T h r o m b Res 18: 7 8 7 - 7 9 5
ICHINOSE A, FUJIKAWA K, SUYAMA T (1986) The
activation of pro-urokinase by plasma kallikrein and its
inactivation by thrombin. J Biol Chem 261: 3486-3489
JOSEPH K, BAHOU W, KAPLAN AP (1997) Evidence
that the Zinc-dependent platelet-binding protein for fac­
tor XII and high m o l e c u l a r w e i g h t k i n i n o g e n is
glycoprotein lb. J Invest Med 45: 267A (abstract)
J O S E P H K, G H E B R E H I W E T B, P E E R S C H K E EI,
R E I D K B M , K A P L A N A P (1996) Identification of
the z i n c - d e p e n d e n t endothelial cell binding protein
for high m o l e c u l a r weight kininogen and factor XII:
Identity with the receptor that binds to the globular
" h e a d s " of C l q ( g C l q R ) . Proc Natl Acad Sci USA
93: 8552-8557
K A T A G I R I Y , H A Y A S H I Y, Y A M A M O T A K,
T A N O U E K, K O S A K I G, Y A M A Z A K I H (1990)
Localization of von Willebrand factor and thrombininteractive domains on human platelet glycoprotein
lb. T h r o m b Haemost 6 3 : 122-126
L E N I C H C, P A N N E L L R, G U R E W I C H V (1995)
Assembly and activation of the intrinsic fibrinolytic
pathway on the surface of human endothelial cells in
culture. Thromb Haemost 74: 698-703
LIN Y, HARRIS RB, YAN W, M c C R A E KR, ZHANG
H, C O L M A N R W ( 1 9 9 7 ) High m o l e c u l a r weight
kininogen peptides inhibit the formation of kallikrein
on endothelial cell surfaces and subsequent urokinasedependent plasmin formation. Blood 90: 690-697
L O Z A J - P , G U R E W I C H V, J O H N S T O N E M,
P A N N E L L R (1994) Platelet-bound prekallikrein
promotes pro-urokinase-induced clot lysis: a mechanism
for targeting the factor XII dependent intrinsic pathway
of fibrinolysis. Thromb Haemost 7 1 : 347-352
M A N D L E R Jr, C O L M A N RW, K A P L A N AP (1976)
Identification of prekallikrein and high molecular
weight kininogen as a complex in human plasma.
Proc Natl Acad Sci USA 7 3 : 4 1 7 9 - 4 1 8 4
M E L O N I FJ, G U S T A F S O N E G , S C H M A I E R AH
(1992) High m o l e c u l a r weight kininogen binds to
platelets by its heavy and light chains and when
b o u n d h a s a l t e r e d s u s c e p t i b i l i t y to k a l l i k r e i n
cleavage. Blood 7 9 : 1233-1244
M E L O N I FJ, S C H M A I E R AH (1991) Low molecular
w e i g h t k i n i n o g e n b i n d s to p l a t e l e t s to m o d u l a t e
t h r o m b i n - i n d u c e d platelet activation. J Biol C h e m
266: 6 7 8 6 - 6 7 9 4
MILLER G, S I L V E R B E R G M, K A P L A N AP (1980)
Autoactivability of human Hageman factor. Biochem
Biophys Res C o m m u n 9 2 : 803-810
M O T T A G, R O J K J A E R R, H A S A N AAK, C I N E S
D B , S C H M A I E R AH (1998) High molecular weight
k i n i n o g e n r e g u l a t e s p r e k a l l i k r e i n a s s e m b l y and
activation on endothelial cells: A novel mechanism
for contact activation. Blood 9 1 : 516-528
N A K A S H I M A M, M O M B O U L I JV, T A Y L O R AA,
V A N H O U T T E PM ( 1 9 9 3 ) E n d o t h e l i u m - d e p e n d e n t
h y p e r p o l a r i z a t i o n caused by b r a d y k i n i n in h u m a n
coronary arteries. J Clin Invest 92: 2867-2871
P A L M E R RMJ, F E R R I G E AG, M O N C A D A S (1987)
Nitric oxide release account for the biologic activity
of endothelium-derived relaxing factor. Nature 327:
524-526
41
P U R I R N , M A T S U E D A R, U M E Y A M A
H,
B R A D F O R D HN, C O L M A N R W (1993) Modulation
of t h r o m b i n - i n d u c e d p l a t e l e t a g g r e g a t i o n by
inhibition of calpain by a synthetic peptide from the
thiol-protease inhibitory sequence of kininogens and
S-(3-nitro-2-pyridinesulfenyl)-cysteine.
Eur J
Biochem 214: 233-241
42
PURI RN, Z H O U F, HU C-J, C O L M A N RF, C O L ­
MAN RW (1991) High molecular weight kininogen
inhibits t h r o m b i n - i n d u c e d platelet aggregation and
c l e a v a g e of a g g r e g i n by i n h i b i t i n g b i n d i n g of
thrombin to platelets. Blood 7 7 : 500-507
REVAK SD, C O C H R A N E C G , GRIFFIN JH (1977)
The binding and cleavage characteristics of human
Hageman factor during contact activation. A
comparison of normal plasma with plasma deficient
in factor XI, prekallikrein or high molecular weight
kininogen. J Clin Invest 59: 1167-1175
ROJKJAER
R, M O T T A G , H A S A N
AAK,
S C H O U S B O E I, S C H M A I E R AH (1998) Factor XII
d o e s n o t i n i t i a t e p r e k a l l i k r e i n a c t i v a t i o n on
endothelial cells. T h r o m b Haemost (in press)
S C H M A I E R AH, B R A D F O R D HN, L U N D B E R G D,
F A R B E R A, C O L M A N R W ( 1 9 9 0 ) M e m b r a n e
expression of platelet calpain. Blood 7 5 : 1273-128 1
S C H M A I E R A H , K U O A, L U N D B E R G
D,
M U R R A Y S, C I N E S DB (1988) The expression of
high molecular weight kininogen on human umbilical
vein endothelial cells. J Biol C h e m 2 6 3 : 16327-16333
SMITH D, G I L B E R T M, O W E N WG (1983) Tissue
plasminogen activator release in vivo in response to
vasoactive agents. Blood 6 6 : 835-839
T A N K E R S L E Y D L , F I N L A Y S O N JS ( 1 9 8 4 )
Kinetics of activation and autoactivation of human
factor XII. Biochemistry 2 3 : 273-279
THOMPSON RE, MANDLE R Jr, KAPLAN AP (1977)
Association of factor XI and high molecular weight
kininogen in human plasma. J Clin Invest 60: 1376-1380
VAN I W A A R D E N F, D E G R O O T PG, B O U M A BN
( 1 9 8 8 ) T h e b i n d i n g of h i g h m o l e c u l a r w e i g h t
kininogen to cultured human endothelial cells. J Biol
Chem 2 6 3 : 4 6 9 8 - 4 7 0 3
W A C H T F O G E L YT, DE LA C A D E N A RA,
KUNAPULI SP, RICK L, MILLER M, S C H U L T Z E
RL, ALTIERI DC, E D G I N G T O N T S , C O L M A N RW
(1994) High molecular weight kininogen binds to Mac1 on neutrophils by its heavy chain (domain 3) and its
light chain (domain 5). J Biol Chem 269: 19307-19312
WEI Y, L U K A S H E V M, S I M O N DI, B O D A R Y SC.
R O S E N B E R G S, D O Y L E M V , C H A P M A N HA
( 1 9 9 6 ) R e g u l a t i o n of i n t e g r i n f u n c t i o n by t h e
urokinase receptor. Science 2 7 3 : 1551-1555
W E L L S M J , H A T T O N M W C , H E W L E T T B,
P O D O R TJ, S H E F F I E L D W P , B L A J C H M A N MA
(1997) Cytokeratin 18 is expressed on the hepatocyte
plasma m e m b r a n e surface and interacts with
thrombin-antithrombin complexes. J Biol Chem 272:
28574-28581
W I G G I N S RC, C O C H R A N E CC (1979) The
autoactivation of rabbit Hageman factor. J Exp Med
150: 1122-1133
ZINI JM, S C H M A I E R AH, C I N E S DB (1993)
B r a d y k i n i n r e g u l a t e s the e x p r e s s i o n of kininogen
binding sites on endothelial cells. Blood 8 1 : 2936-2946
43
44
45
46
47
48
49
50
51
52
53
54
55