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Chapter 10
Thrombin
Nancy Swords Jenny
Roger L. Lundblad
Kenneth G. Mann
The enzyme thrombin has long been recognized for its multiple
functions in blood coagulation as well as for its more recently
defined roles in tissue repair, development, and pathogenic
processes (1,2,3,4,5,6). Thrombin originates from prothrombin, a
circulating zymogen precursor protein. Prothrombin and thrombin
are members of the family of vitamin Kdependent blood clotting
proteins characterized by an NH 2 -terminal Gla domain, which
contains several -carboxyglutamic acid residues. Prothrombin,
M r = 72,000, is the most abundant of these proteins, circulating at
a plasma concentration of 1 to 2 M (7,8). The human prothrombin
molecule (see Fig. 10-1) is synthesized primarily in the liver. Low
levels of prothrombin expression have been reported in brain,
diaphragm, stomach, kidney, spleen, intestine, and in uterine,
placental, and adrenal tissues (9,10). The 21-kb-long prothrombin
gene, located on chromosome 11p11-q12, has been extensively
characterized (11,12,13,14). Of the 26,929 base pairs (bp) of
continuous sequence that has been determined, 6,544 bp are
upstream from the methionine initiator, 20,241 bp span the site of
initiation of transcription to the site of polyadenylation, and 145 bp
constitute the 3 flanking region (14). The gene is organized into
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Human
prothrombin
numbering
Chymotrypsinogen
numbering
271284
1 U1 I
Notes
Deleted in stable
form of human
-thrombin
285292
1 H1 A
307319
14 A14 M
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NH 2 -end; the
COOH-terminal end of
this loop appears to
be flexible
343
37 A
Contributes to
hirudin binding
367375
60 A60 I
Contains
carbohydrate
implicated in
chemotactic activity;
peptide has growth
factor activity; this
loop, the B loop,
caps the active site
of thrombin
393
77 A
Contributes to
hirudin binding
414
97 A
Protrudes from
molecule
447449
129 A129 C
470474
149 A149 E
Site of extensive
proteolysis, flexible;
the loop or C
loop
510
184 A
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plentiful in this
region of structure
513516
186 A186 D
The E loop
535536
204 A204 B
Large 16 turn
549550
218
Loop similar to
trypsin
553
221 A
578579
245 A245 B
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Designation
Chymotrypsin
Thrombin
Histidine loop
Cys42Cys58
Cys348Cys364
Methionine loop
Cys168Cys182
Cys493Cys507
Serine loop
Cys191Cys220
Cys521Cys551
Cys1Cys122
Cys293Cys439
Cys136Cys201
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equivalent position.
Although the primary binding sites of -thrombin and trypsin are
similar, the nature of the specificity of -thrombin is more complex
because of the active site region and the exosites. In
chymotrypsin, the NH 2 -terminal segment of the substrate peptide
bond P 1 to P 3 forms an antiparallel sheet with the S 1 to S 3
sequence of the enzyme. After the S 3 position, the chymotrypsin
structure bends sharply away from the substrate and does not
interact with the substrate beyond the substrate P 3 position. In
-thrombin, however, the binding site is far more extensive. The
-thrombin cleavage sites on various macromolecular substrates,
presented in Table 10-3, often occur after the sequence X-Pro-Arg.
In three cleavage sites on human and bovine prothrombins, the
residues Gly-Ser are found at subsites P 3 and P 4 (Table 10-3). In
addition, phenylalanine at the P 9 position in the fibrinogen A
chain is highly conserved among various species and is required
for specificity in the fibrinogen-thrombin interaction (73,74).
The fibrinogen-binding site extends from the P 9 to at least the P 3
site, and perhaps as far as the P 8 to at least the P 3 site (75).
There may also be multiple secondary binding sites for fibrinogen
(76). -Thrombin binding interactions with substrates are far
larger in scope than equivalent interactions between trypsin and its
substrates.
The specificity of -thrombin toward the arginylglycyl bonds of
the A and B chains of fibrinogen is most likely a result of the
primary, rather than the tertiary, structure of fibrinogen (101).
-Thrombin can cleave fibrinopeptides isolated from the A and B
chains, and the sequence of fibrinopeptide A is highly conserved
among different species. Synthetic peptide analogs of
fibrinopeptides A and B also act as inhibitors of -thrombin. The
first 51 amino acids of the A chain of fibrinogen appear to be
responsible for the interactions with the binding sites on
-thrombin (102). The primary binding site appears to be located
in the NH 2 -terminal sequence of the A between residues 1 and 23,
with secondary binding interactions mediated by residues 34 to 44
and 45 to 51.
In addition to the exosites and active site region implicated in
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MEIZOTHROMBIN
Figure 10-4 depicts the prothrombin molecule and the proteolytic
processing required to obtain the various forms of thrombin. In
addition to the -, -, and -thrombins, there are two larger active
species: meizothrombin and meizothrombin des-fragment 1 (117).
Meizothrombin was first identified as a product of Echis carinatus
venom protease cleavage of human
P.199
prothrombin at Arg320 (118). Meizothrombin is autolytically
cleaved at Arg156, releasing prothrombin fragment 1 (the Gla
domain and kringle 1 region) from the molecule, generating
meizothrombin des-fragment 1 (117). Subsequent autolytic
cleavage at Arg283 gives rise to the stable form of human
-thrombin (Fig. 10-4). Meizothrombin is now recognized as an
obligate intermediate in normal prothrombin activation and the
first product produced by the prothrombinase complex during the
activation of prothrombin (119,120).
Subsite
Protein
P9P8P7P6P5P4P3P2P1
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Fibrinogen (A),
Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg
human
(16)
Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg
Gly
Val
(19)
Fibrinogen (B),
Asn-Glu-Glu-Gly-Phe-Phe-Ser-Ala-Arg
human
(14)
Thr-Val-Glu-Leu-Glu-Gly-Val-Pro-Arg
Gly
Gly
(36)
Factor XIII, bovine
Val-Glu-Leu-Gln-Gly-Leu-Val-Pro-Arg
Gly
(37)
Factor VIII, human
Asn-Ser-Pro-Ser-Phe-Ile-Gln-Ile-Arg
Ser
(372)
Factor VIII
Leu-Ser-Asn-Asn-Ala-Ile-Gly-Pro-Arg
Ser
(740)
Factor VIII
Gln-Asn-Phe-Val-The-Gln-Ser-Lys-Arg
Ala
(1,313)
Factor VIII
Asp-Glu-Asp-Glu-Asn-Gln-Ser-Pro-Arg
Ser
(1,689)
Factor V, human
Arg-Leu-Ala-Ala-Ala-Leu-Gly-Ile-Arg
Ser
(709)
Factor V
Thr-His-His-Ala-Pro-Leu-Ser-Pro-Arg
Thr
(1,018)
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Factor V
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Asp-Asn-Ile-Ala-Ala-Trp-Tyr-Leu-Arg
Ser
(1,545)
Factor VII, human
Arg-Asn-Ala-Ser-Lys-Pro-Gln-Gly-Arg
Ile-
(152)
Factor VII, bovine
Arg
Gly
Prothrombin, human
Gln-Val-Thr-Val-Met-Val-Thr-Pro-Arg
Ser
(155)
Arg-Pro-Leu-Thr-Phe-Phe-Asn-Pro-Arg
Thr
(286)
Prothrombin, bovine
Arg-Val-Thr-Val-Glu-Val-Thr-Pro-Arg
Ser
(156)
Protein C, human
Asp-Gln-Gly-Asp-Gln-Val-Asp-Pro-Arg
Leu
(169)
Protein C, bovine
Asp-Gln-Lys-Asp-Gln-Leu-Asp-Pro-Arg
Ile-
(171)
Protein S, human
Pro-Asp-Leu-Arg-Ser-Cys-Val-Asn-Arg
Ile-
(74)
Secretin, porcine
Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg
Asp
(14)
Chorionic -subunit,
NH 2 -Ser-Lys-Glu-Pro-Leu-Arg-Pro-Arg
Cys
(8)
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human
Leu-Pro-Gln-Val-Val-Cys-Asn-Tyr-Arg
Asp
(60)
Ser-Ile-Arg-Leu-Pro-Gly-Cys-Pro-Arg
Gly
(74)
Leu-Ser-Cys-Gln-Cys-Ala-Leu-Cys-Arg
Arg
(94)
Chymotrypsinogen,
Gln-Pro-Val-Leu-Ser-Gly-Leu-Ser-Arg
bovine
(15)
Gly-Thr-Asp-Val-Gln-Ala-Trp-Ile-Arg
Ile-
Gly
(125)
Cholecystokinin,
NH 2 -Lys-Ala-Pro-Ser-Gly-Arg (6)
Val
Growth hormone,
Gly-Arg-Leu-Glu-Asp-Gly-Ser-Pro-Arg
Thr
human
(133)
Growth hormone,
Arg-Glu-Leu-Glu-Asp-Gly-Thr-Pro-Arg
bovine
(131)
Growth hormone,
Arg-Glu-Leu-Glu-Asp-Val-Thr-Pro-Arg
ovine
(52)
Gly-Phe-Ala-Gly-Asp-Asp-Ala-Pro-Arg
pancreozymin,
porcine
Ala
Ala
Ala
(28)
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muscle
Phe-Pro-Ser-Ile-Val-Gly-Arg-Pro-Arg
His
(39)
Leu-Thr-Glu-Ala-Pro-Leu-Asn-Pro-Lys
Ala
(113)
Apolipoprotein-C-III-1
Ser-Gln-Gln-Val-Ala-Ala-Gln-Gln-Arg
Gly
(40)
Troponin C
Ala-Gly-Glu-Leu-Ala-Glu-Ile-Phe-Arg
Ala
(120)
Thrombin receptor,
Ala-Thr-Asn-Ala-Thr-Leu-Asp-Pro-Arg
PAR-1
(41)
Thrombin receptor,
Trp-Val-Leu-Ala-Thr-Gln-Ala-Pro-Arg
PAR-4
(47)
TAFI
Ile-Ser-Asn-Asp-Thr-Val-Ser-Pro-Arg
Ser
Leu
Ala
(92)
PAR, protease-activated receptor; TAFI, thrombin-activatable fibrinoly
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P.200
Meizothrombin activity toward fibrinogen and platelets, however, is
greatly reduced compared to that of -thrombin (117,123).
Meizothrombin does have the ability to cleave other -thrombin
substrates. Meizothrombin and -thrombin have comparable
activity toward protein C, factor V, factor XI, and small peptide
substrates (117,123,124,125,126,127). Recombinant
meizothrombin has been reported to produce factor Va at a slightly
faster rate than -thrombin indicating that meizothrombin likely
contributes to factor V activation in vivo (126).
Meizothrombin-catalyzed activation of factor XI may likewise
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diatheses (142).
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k ca t /K m
Km
Enzyme
Substrate
(M)
k ca t /min
M/min
VIIa
IX
NA
NA
VIIa/TF/PCPS/Ca 2+
IX
0.243
15.6
6.42
Ef
10 7
VIIa
4.87
0.024
4.93
10 3
VIIa/TF/PCPS/Ca 2+
0.45
69.0
1.53
10 8
IXa
299.0
0.002
6.69
IXa/VIIIa/PCPS/Ca 2+
0.063
500.0
7.94
10 9
Xa
II
131.0
0.6
4.58
10 3
Xa/Va/PCPS/Ca 2+
II
1.0
1800.0
1.80
10
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P.204
The roles of the intrinsic and extrinsic tenase complexes in factor
Xa generation have been well characterized. Studies of the
extrinsic tenase demonstrate that in the presence of both factor X
and factor IX, factor IX appears to be the preferred substrate and
the rate of factor Xa generation is decreased (229,248). The
extrinsic tenase is therefore proposed to act as a trigger of blood
coagulation and initiate procoagulant events by providing low
levels of factor Xa and essential levels of factor IXa. Factor IXa
forms the intrinsic tenase with factor VIIIa on a membrane surface
and produces factor Xa at a rate that is 50-fold greater than the
extrinsic tenase. The extrinsic tenase complex is also subject to
inhibition by tissue factor pathway inhibitor (TFPI) that binds the
tissue factorfactor VIIafactor Xa ternary complex and blocks
production of both factor Xa and factor IXa (see Fig. 10-5B) (249).
The increased rate of factor Xa generation by the factor
VIIIafactor IXa complex should overcome circulating inhibitors of
factor Xa (i.e., AT-III and TFPI) and the dynamic APC inhibition
system, permitting the formation of the prothrombinase complex.
Factor Xa requires an additional protein species, factor Va, for
efficient activation of prothrombin (Table 10-4). Factor Va is the
cofactor form of the circulating procofactor factor V. Single-chain
factor V can be proteolyzed by a variety of enzymes including
-thrombin factor Xa, and plasmin (170,250,251,252,253,254).
The active factor Va produced by -thrombin is the best
characterized species and is composed of two polypeptide chains.
The M r = 94,000 heavy chain is obtained from the NH 2 -terminus of
factor V, and the M r = 74,000 light chain is derived from the
COOH-terminus of the factor V molecule. The heavy and light
chains of factor Va are noncovalently associated through divalent
calcium ion bridging between the chains. A large portion of factor
V, corresponding to nearly 50% of the total mass of factor V, is
excised from the middle of the molecule upon activation. No
function has yet been established for the activation peptide, or
B-domain, of factor V.
The functions of the heavy and light chains of factor Va have been
determined from studies of the isolated chains and by binding and
immunochemical techniques. The heavy chain appears to mediate
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P.205
-Thrombin also participates in inactivation of the prothrombinase
complex. The -thrombinthrombomodulin complex activates
protein C to APC, which competes with factor Xa for binding to
membrane-associated factor Va (Fig. 10-6D). Bound factor Xa
blocks the binding of APC to factor Va. However, if factor Xa
dissociates from the prothrombinase complex, APC can bind. Once
bound to factor Va on the membrane surface, APC cleaves and
inactivates factor Va (Fig. 10-6E). APC cleaves both the heavy and
light chains of factor Va, although the cleavages associated with
the heavy chain play the dominant role in inactivation of the
molecule (261,262).
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P.206
Two pathways for prothrombin activation are illustrated in Figure
10-7. Prothrombin can be activated by factor Xa alone at a very
slow rate. In the presence of factor Xa and calcium ions, the initial
cleavage (step 1) occurs at Arg271, giving rise to prothrombin
fragment 1.2 and prethrombin 2. Cleavage of prethrombin 2 at
Arg320 (step 2) gives rise to prothrombin fragment 1.2 and
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SUMMARY
The process of blood coagulation was first described as a
sequential series of proteolytic actions (290,291). However, the
nature of hemostasis is perhaps better described as
threshold-limited, complex, intertwined processes that include
physical, cellular, and biochemical events. -Thrombin plays a
central role in the overall maintenance of blood fluidity and
contributes to reactions at all levels (see Fig. 10-8).
-Thrombin has direct effects on coagulation through activation of
platelets, formation of fibrin clots, and activation of various
zymogens and cofactors in the coagulation cascade. -Thrombin
activity extends from the coagulation process to anticoagulation
and stimulation of fibrinolytic reactions. -thrombin's roles
continue into the tissue repair and remodeling phase necessary to
regenerate damaged vascular tissue. -Thrombin is a potent
mitogen (292,293,294,295,296,297) and stimulates cell division in
smooth muscle cells (205,293,298), macrophages (6), and
endothelial cells (6,299). The proliferative effects of -thrombin
result from direct activation of mitogenic pathways (297) and/or
-thrombin-mediated secretion of a variety of growth factors. The
combination of -thrombin and growth factors often demonstrates
a synergistic effect on cellular proliferation
(300,301,302,303,304). -Thrombin also induces the release of
cytokines (305,306,307,308,309), vasoactive compounds
(310,311,312,313), and chemoattractants (313) from cells in the
vicinity of tissue damage. The processes involved in development
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ACKNOWLEDGMENTS
We would like to acknowledge the intellectual contributions of Dr.
Ruth Ann Henriksen to elements of this chapter dealing with
thrombin. We would also like to thank Drs. Wolfram Bode and
Alexander Tulinsky for making x-ray coordinates available for
direct inspection and Drs. Tim Rydel and Alexander Tulinsky for
providing Figures 10-2 and 10-3. We would like to thank Drs.
Matthew Rand and Jeffrey Lawson for their assistance in clarifying
nomenclatures for thrombin amino acid residues. Finally, Dr. Mann
would like to acknowledge the research support of the National
Heart, Lung, and Blood Institute for the last 36 years.
P.208
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