854
c h a p t e r 36
Biochemistry of Hemostasis
Prothrombin
—► Thrombin
+
Fragment 1-2
Prothrombin
1
143 195
448
T______ I
I________________
J
m m m -
h
h
m
Factor X
F I G U R E 3 6 - 1 0
(Also see color figure.) Activation of prothrombin. Prothrombin is cleaved
by factor Xa at two residues, Arg284 and Arg320. The order of cleavage
produces different products. If the first cleavage is at Arg284, it produces
two products: prothrombin fragment 1-2 (residues 1-284, yellow bar) and
prethrombin 2 (residues 285-579, gray bar), each of which represents
approximately one-half of the prothrombin molecule. When Arg320 is
cleaved first, the intermediate product meizothrombin is formed.
Meizothrombin contains the entire sequence of the prothrombin molecule;
the two halves are held together by a single disulfide bond (CyS283 to
Cys439). In the absence of factor Va, the rate constants for cleavage of the
two bonds are essentially the same. In the presence of factor Va and
phospholipids, Arg320 is cleaved first. Meizothrombin is detectable in
reaction mixtures, but its rapid cleavage produces prothrombin
fragment 1-2 and a-thrombin. In the color version of this figure, motifs and
domains are color coded as follows: Gla domain (blue), kringle domains
(orange), EGF-like domain (magenta), activation peptide (yellow), and
proteinase domain (green). Light chains are indicated in dark gray, heavy
chains in light gray. Regions connecting the motifs are black.
fibrinogen, factor XIII, and protein C. It almost certainly
acts on factor VII to generate factor Vila. In contrast to
the other vitamin K-dependent proteins, thrombin is not
linked to the Gla domain-containing polypeptide and thus
is free to diffuse away from the site of its formation. All
of the other vitamin K-dependent proteins, both in their
precursor and active proteinase forms, retain the Gla do-
main and are able to bind to the phospholipids of the cell
membranes at the injury site. Prothrombin fragment 1
-2 is
measured as a marker of ongoing prothrombin activation
and by inference, an indication of hypercoagulability (an
imbalance of procoagulant subsystem activity over antico-
agulant subsystem activity) when it is present in a higher
than normal concentration.
Although there is an explosive increase in the reaction
rate, only a small fraction (<^
1
%) of the total prothrom-
bin available (~1.5 /xM) is converted to thrombin. The
concentration of thrombin required to convert fibrinogen
to fibrin that is sufficient to form a clot in 15 seconds is
less than 1 nM. When the coagulation “cascade” is viewed
as an amplifier, it must be noted that it is the rates of the
reactions that are being amplified
. 5
An interesting mutation in the gene for prothrombin,
a G-to-A transition in the 3' untranslated region at nu-
cleotide
2 0 2 1 0
, results in an elevated concentration of pro-
thrombin in the circulation (>115% of normal). It is not
known if the mutation causes the elevated prothrombin
levels, but the defect is associated with a twofold increase
in the risk of thrombosis.
36.6 The Extrinsic Pathway
Historically, the reactions of the coagulation system that
are initiated
in vitro
by the addition of an exogenous sub-
stance such as tissue homogenates have been grouped un-
der the title of “extrinsic pathway.” The reaction pathway
that is followed without tissue homogenate being added
was designated the “intrinsic pathway” because it was as-
sumed that all components were present in blood
. 6
The
two pathways are not independent, but the terms continue
to be used both because of their historical roots and be-
cause the commonly performed
in vitro
coagulation tests
can be related to them.
Initiation of the Procoagulant Subsystem and
Activation of Factor VII
The first complex to form is almost certainly the “extrin-
sic” factor X activation complex. Factor Vila binds to tis-
sue factor, an integral membrane protein that is exposed
upon injury of the blood vessel. The phospholipid bilayer
surface is provided by the damaged cell membranes.
In vivo,
Ca2+ is always present in the blood
. 7
Two hypotheses exist for the origin of factor Vila. In
one, factor VII is considered to have some proteolytic ac-
tivity prior to a proteolytic cleavage. In the other view, low
concentrations of thrombin and/or factor Xa are consid-
ered present at all times in the circulating blood. Throm-
bin, factor Xa, and factor Vila can activate factor VII to
5 Amplification in a sequence of enzyme-catalyzed reactions suggests that
more of the product is formed at each stage than in the previous stage. How-
ever, in the coagulation “cascade” the principal amplification is of reaction
rate, not quantity of product formed.
6It is now known that the surface of the glass test tube provides a sur-
face upon which proteins bind and initiate clot formation. See discussion of
contact system (phase) following.
1 In vitro,
Ca2+ is added only because the anticoagulant solutions used for
blood collection employ substances such as citrate ion, EDTA, or oxalate to
bind Ca2+ to prevent activation complex formation. It is worth noting that
factor Xlla can activate factor VII in the absence of Ca2+.
