Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

II. Mechanisms of Hemostasis
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components of the system in close proximity to each other.
The formation of a fibrin clot is not only the physiological
trigger for the activation of the fibrinolytic system, but
it also accelerates the rate of tPA induced conversion of
plasminogen to plasmin up to three orders of magnitude
( Collen, 1999 ). Fibrin essentially acts as a cofactor in plasmin
activation through a two-step process ( Medved and
Nieuwenhuizen, 2003 ). As fibrinogen undergoes polymerization
to fibrin, cryptic tPA and Glu-plasminogen binding
sites are exposed on its D-domains that facilitate the formation
of tertiary complexes between the three molecules.
The Glu-plasmin generated on the fibrin surface begins to
cleave specific arginine and lysine residues in the α , β ,
and γ chains to form a modified fibrin polymer that has
additional exposed C-terminal lysine residues that promote
increased binding of tPA and plasminogen. Thus, this
modified form of the fibrin gel is three-fold more potent
as a cofactor for fibrinolysis than the unmodified form.
Not only does the modified fibrin increase the concentration
of tPA and plasminogen on the fibrin surface, but it
also enhances the plasmin-induced conversion of Gluplasminogen
to Lys-plasminogen ( Nesheim, 2003 ). This
serves as a positive feedback response, because Lysplasminogen
is approximately 20-fold better than Gluplasminogen
as a substrate for tPA catalysis. Plasmin
sequentially digests fibrin with the formation of degradation
products designated as X-, Y-, D-, and E-fragments
( Dobrovolsky and Titaeva, 2002 ). The sites most sensitive
to proteolysis are specific Lys/Arg residues on the α - and
β -chains. The cleavages result in a set of X-fragments
with molecular masses ranging from 330 to 240 kDa.
Degradation of insoluble fibrin clots to the soluble Y-, D-,
and E-fragments occurs after at least one D-fragment has
been cleaved. The presence of fibrin fragments in the circulation
is used diagnostically as evidence of inappropriate
fibrinolysis (see Sections III.B.4.a and b).
4. Inhibitors of Fibrinolysis
The major inhibitor components of fibrinolysis are shown
in Table 10-6 .
a. Plasminogen Activator Inhibitor Type-1 (PAI-1)
PAI-1 is the major endogenous inhibitor of fibrinolysis, or
thrombolysis, as it is effective in blocking the conversion
of plasminogen to plasmin by either tPA or uPA ( Kruithof,
1998 ). PAI-1 is a 52-kDa-glycoprotein synthesized and
secreted by endothelial cells in its active form. In plasma,
PAI-1 is present in two forms: an active form in which the
reactive site is exposed on the surface of the molecule and
a latent form in which the reactive site is concealed within
the protein globule (Dobrovolsky and Titaeva, 2002). The
change of active PAI-1 to its latent form occurs spontaneously
in plasma. Although the physiological mechanisms that
induce the reverse transformation are not fully understood,
it appears that latent PAI-1 can be reactivated by phospholipid
vesicles containing PS or phosphatidylinositol (PI).
This indicates that at sites of vascular injury, activated
platelets may be able to activate latent PAI-1 (see Section
II.B.2.a). Hence, the local concentration of active PAI-1 can
be significantly increased at sites of thrombus formation as
approximately 90% of PAI-1 circulates as one of the constituents
of platelet α -granules. Thus, PAI-1, along with the
action of thrombin activatable fibrinolysis inhibitor (TAFI,
see Section II.D.4.c), is a contributing factor in the stabilization
of the fibrin matrix. It has been shown in people that
circulating PAI-1 levels vary more than any other component
of the fibrinolytic system. This is likely due to the ability of
a wide variety of substances to stimulate PAI-1 production.
These include insulin, TNF α , IL-1, transforming growth
factor β (TGFβ ), and thrombin ( Tsikouris et al ., 2002 ).
Endotoxin and TNF α have been shown to stimulate PAI-1
production in the liver, kidney, lung, and adrenals of mice.
At least in human and murine plasma, PAI-1 exhibits the
characteristics of an acute phase protein. In various disease
states, a companion serine proteinase inhibitor of tPA known
as plasminogen activator inhibitor type-2 (PAI-2) can appear
in the circulation ( Dobrovolsky and Titaeva, 2002 ). PAI-2 is
synthesized by placenta, monocytes, and macrophages and
is an effective inhibitor of t-PA.
b. Antiplasmin (AP)
Antiplasmin (AP, also referred to α 2 -antiplasmin, α 2 AP)
is the principal inhibitor of plasmin in the circulation. The
liver secretes it as a single-chain 70-kDa protein. AP possesses
the conserved core structure of the serpin family of
proteins, and its amino acid sequence is highly conserved
across species (Coughlin, 2005a) . Human AP is 80% identical
with the bovine protein and 74% identical with murine
AP. In people, 30% of AP circulates in the form of Met-AP
and 30% as Asn-AP. The latter is a truncated form that
has lost a 12-amino acid sequence from the N-terminal
end of the protein. Both Met-AP and Asn-AP inhibit plasmin
at similar rates, but Asn-AP has a higher affinity for
fibrin than Met-AP (Dobrovolsky and Titaeva, 2002). The
N-terminal portion of AP is cross-linked to fibrin by a
transglutaminase reaction catalyzed by FXIIIa. The amount
of AP cross-linked to fibrin is a more important determinant
of the rate of clot lysis than is the amount of free plasmin
in the circulation (Coughlin, 2005a) .
c. Thrombin Activatable Fibrinolytic Inhibitor (TAFI)
The function of TAFI is to down-regulate the onset of fibrinolysis
once the fibrin clot has successfully stopped blood
loss. TAFI is a single-chain, 46-kDa plasma protein that is
classified as a zinc-containing metalloproteinase ( Bajzar,
2000 ). It can be cleaved by thrombin, plasmin, and trypsin
to yield an activation peptide and an enzyme, TAFIa, that
exhibits carboxypeptidase activity. Thrombin, by itself,