Transcript ENZYME CASCADES: BLOOD CLOTTING Medical Biochemistry, Lecture 27 Lecture 27, Outline • The extrinsic and intrinsic clotting pathways • Specific reactions in the coagulation pathway:

ENZYME CASCADES:
BLOOD CLOTTING
Medical Biochemistry, Lecture 27
Lecture 27, Outline
• The extrinsic and intrinsic clotting pathways
• Specific reactions in the coagulation
pathway: a. thrombin-fibrinogen-fibrin; b.
Factor XIIIa; c. Hemophilia; d. antiprotease system; e. auto-regulation of
thrombin; f. fibrinolysis
Blood Clotting
• Because of the importance of blood in
regulating pH and the transport of oxygen,
nutrients, carbon dioxide, and wastes,
maintaining the integrity of the process is
crucial to life. When ruptures in the system do
occur, the process of blood clotting is initiated
as an emergency measure to halt the loss of
blood. Biochemically, blood clotting is an
example of signal amplification caused by the
simultaneous activation and inhibition of many
enzymes. A basic overview of the process and
Red blood cells enmeshed in
the insoluble strands of a fibrin
clot
Electron micrograph
of a fibrin fiber
PLEASE REMEMBER
• Do not worry about memorizing the
whole pathway and all of the factors
involved - you will be tested on
specific examples, not the entire
pathway - the focus will be on using
this pathway to illustrate all of the
interactions between proteins,
enzymes and regulatory mechanisms
described in the preceding lectures.
Blood Vessel Damage
• When injury to a blood vessel occurs, three
major events happen to rapidly stop the loss of
blood:
• 1) clumping of blood platelets at the site of
injury to create a physical plug.
• 2) vasoconstriction occurs to reduce blood flow
through the area.
• 3) aggregation of fibrin into an insoluble clot
that covers the rupture and stops loss of blood.
• The clot is dissolved after actual repair of the
blood vessel.
Platelet Aggregation
• The initial phase of platelet aggregation is a
complex formed between the platelets and
underlying collagen fibrils exposed in the
ruptured vessel. An additional circulating
protein, von Willebrand factor (vWF), mediates
binding of platelets to collagen and each other
resulting in activation of the platelets and
release of various activators. The next slide
schematic illustrates the complex biochemical
effects that binding of platelets, collagen and
vWF have on activation of the blood clotting
Blood Vessel Damage (cont)
• In the normal, undamaged vascular
endothelium, platelet aggregation does not
occur since collagen fibrils are not exposed
and other activating factors (like ADP) are
not present in sufficient amounts. Besides
exposure of the collagen fibrils in the underlying
matrix of the vessel, other membrane proteins
in this matrix are exposed to the circulating
blood. It is these matrix and membrane
proteins that serve as receptors for the various
zymogens and protein co-factors that are
released by the activated platelets (or that were
Blood Vessel Damage (cont)
• Ultimately, the final blood clot is formed by
the conversion of fibrinogen to fibrin
eventually resulting in insoluble, crosslinked fibrin polymers. Two activation
pathways (initiated by complexes with
exposed membrane matrix proteins),
historically termed the extrinsic and intrinsic
pathways, supply the protease (Factor Xa)
that activates the thrombin catalyzed
production of fibrin
Intrinsic and Extrinsic
Pathways Schematic
General Characteristics of the
Coagulation Cascade
• The predominant mechanism illustrated in the
activation pathway is that of an enzyme
amplification cascade. One enzyme at the
start of the activation pathway can activate
many molecules of a given substrate as
illustrated:
Amplification Cascade Model
• This preceding model can be interpreted as
follows: If each of 100 individual Factor VIIa
enzymes catalyzed the activation of 100 Factor
Xa enzymes, and each one of these 10,000
Factor Xa enzymes catalyzed 100 thrombin
activations, and so on - there is a resulting
million-fold activation from the start of the
pathway to fibrinogen cleavage. It is this
amplification process that allows a clot to form
rapidly and also illustrates the magnitude that
one defect in these steps could have on the
whole process (an example to be discussed will
General Characteristics (cont)
• Another characteristic of this process is the
regulation of zymogen enzymes. In both
pathways, Factors XII, XI, IX, VII, X and II
are zymogens of serine proteases.
Cleavage of one zymogen leads to an active
protease that activates another zymogen
protease and so on. These zymogens are
themselves regulated by a series of nonprotease protein co-factors that also must
be activated by proteases (like thrombin
activation of Factor VIII to VIIIa and Factor V
General Characteristics (cont)
• These protease and protein cofactor
interactions lead to large multi-protein
complexes, yet they can be thought of just like
any small molecule substrate to product
enzymatic reaction that we have discussed
previously. It is the specificity of the protein
sequence and conformation in combination with
various metal, lipid or protein co-factors that
determine whether a factor is a substrate for an
activated protease (For instance, Factor XIIa
will not cleave prothrombin to thrombin, only
Intrinsic Pathway and
Protein Complex
Extrinsic Pathway and
Protein Complex
Thrombin-Fibrinogen-Fibrin
• Thrombin is generated from a prothrombinase complex
consisting of prothrombin, factor Va, factor Xa, calcium and
anionic phospholipids. Prothrombin is synthesized in the liver
and contains 10 Gla residues in its amino-terminal domain.
Factor Xa cleaves prothrombin into its activated form
(thrombin). Thrombin acts on fibrinogen, a large fibrous protein
consisting of two tripeptide units. These subunits form three
globular domains - two on the ends and one in the middle
where the two tripeptides associate. Thrombin cleaves four
peptides termed either fibropeptide A (from the a-subunit) or
fibropeptide B (from the b-subunit). These cleaved
fibropeptides are unique in that they contain many negatively
charged Asp and Glu residues, plus unique sulfated tyrosine
residues. Loss of this net negative charge on the fibrin
monomer induces conformational changes in the middle
globular domains that create new sites with high affinities for
Prothombinase Pathway
and Protein Complex
Fibrinogen Activation
Peptides cleaved by thrombin contain many Asp, Glu and sulfated-Tyr
residues, thus very highly negatively charged
•
Factor XIIIa
The "soft clot"(transglutaminase)
of fibrin monomers is stabilized and converted
into the final clot by covalent cross-linkages between specific
glutamine residues on one monomer and lysine residues on
another monomer. This peptide (amide) bond is catalyzed by
activated Factor XIIIa, a transglutaminase:
Factor XIIIa - Fibrin Cross-linking
Roles of Calcium,
Carboxyglutamate, and Vitamin
K
• Some of these factors (thrombin, VII, IX and X)
contain a unique modified glutamate residue, called
carboxyglutamate (Gla). This amino acid is a natural
high affinity binder (or chelator) of calcium ions, hence
the designation of calcium as a co-factor in the
schematic. It is this complex of calcium with the Glafactors that allow specific interactions with acidic
membrane lipids that ultimately lead to the correct
tertiary and quaternary protein stuctures recognized
by other proteins in the pathway. Synthesis of Gla
residues result from post-translational modifications of
the newly synthesized factors in the liver endoplasmic
Vitamin K - Carboxyglutamate
Dicoumarol / Warfarin
• Two analogs of Vitamin K, dicoumarol
(I) and warfarin (II), have been shown
to inhibit formation of the Gla residues
of prothrombin and Factors VII, IX and
X. The lack of Gla residues, and hence
binding of calcium, was shown to inhibit
the participation of these proteins in the
blood coagulation process. This is the
basis for using coumarin drugs clinically
as anti-coagulants.
VITAMIN K
DICOUMAROL
Structures of the
Coumarin Drugs
(Vitamin K analogs)
WARFARIN
Physiological Effect of Inhibiting
Gla Production with Coumarin Drugs
The presence of Gla peptides
released during the clotting
activation cascade signals new
synthesis of Gla-proteases in
the liver. Blocking of the Gla
modification by drugs prevents
secretion of the newly
synthesized proteases and thus
limits their numbers in
circulating blood. In the
absence of drugs, this pathway
replenishes the circulating
pool of zymogen factors
Hemophilia
• Hemophilia is a genetically inherited
disease characterized by an inability to
form proper blood clots. This deficiency
has been attributed to defective production
of Factor VIII (hemophilia A) or Factor IX
(hemophilia B). Hemophilia A is the most
common (80%) form of the disease.
Hemophilia - Mechanistic Defect
• It may not be readily apparent why defects in
Factor VIII and Factor IX lead to poorly formed
clots. Both of these factors are in the intrinsic
pathway. In hemophiliacs, the extrinsic
pathway still functions normally. As will be
discussed, the whole clotting pathway is tightly
regulated and interdependent. When an injury
occurs in a patient with hemophilia, a
disproportionate clotting factor response to the
wound occurs (i.e., defective intrinsic pathway
activation), and regulation of the entire process
Anti-protease system
• Critical features of the blood coagulation
pathway are the mechanisms of inactivation. If
activated thrombin was not regulated, then the
fibrin clot could continue to form and eventually
block circulation. Also present in the blood are
protein protease inhibitors that specifically bind
to the different activated serine proteases. In
normal blood circulation scenarios, the
antiproteases proteins are present in sufficient
concentrations to act as security against
random thrombin activation (or other proteases)
and the resulting fibrin clots.
Anti-protease system (cont)
• An example is antithrombin III, which
tightly binds and inactivates thrombin this complex is later cleared from
circulation in the liver. The clinical
administration of heparin promotes the
association between antithrombin III and
thrombin. This is the basis for use of
heparin as an anti-coagulant.
Auto-regulation of thrombin
• Besides cleaving fibrinogen, thrombin
has forms complexes with an
endothelial cell protein receptor termed
thrombomodulin. Thrombomodulin and
calcium act as co-factors of thrombin
activation of Protein C, a Gla containing
protease. When activated, Protein C in
conjunction with another protein cofactor, Protein S, will proteolyze and
inactivate Factors Va and VIIIa.
Regulation of Thrombin - Summary
• Activation of the blood coagulation process
involves the site-specific, rapid amplification
and activation of factors that quickly form the
fibrin clot. Since this activation is localized to
the site of injury, formation of fibrin clots should
not be occuring throughout the entire circulatory
system. The combination of the antiproteases
and Protein C ensures this. Since thrombin is
near the end of the amplification cascade, there
will be a high localized concentration of
thrombin at the site of injury. This high level of
thrombin will correspondingly activate a high
Thrombin Summary (cont)
• Protein C inactivation of Factor Va leads to an
inhibition of Factor Xa activation of
prothrombin (and thus more thrombin) while
inactivation of Factor VIIIa effectively inhibits
formation of additional stable fibrin clots.
Circulating anti-proteases further downregulate the activated clotting factors. Thus
the activation-inactivation systems of blood
coagulation exist in a dynamic equilibrium
that can be rapidly mobilized and just as
quickly turned off.
Mechanism of Clotting Summary
INITIATE AMPLIFY
INHIBIT
Dissolving of Blood Clots:
Fibrinolysis
• Similar mechanisms of activation and
inactivation described for clot formation apply to
this pathway, too. Plasmin, which exists in
circulating blood as plasminogen, is the
protease responsible for degrading the fibrin
clots. The activator of plasminogen, another
protease termed tissue plasminogen activator
(tPA), binds with high affinity to the fibrin clot
along with plasminogen. The tPA-plasminogenfibrin complex results in proteolytic activation of
plasminogen to plasmin, which then begins
Clot Dissolving Pathway
Fibrinolysis (cont)
• To keep one plasmin molecule from
degrading the whole clot, after plasmin
degrades a region of the clot, the resulting
digested peptides dissociate from the clot
and take the plasmin-tPA complex with
them. Again similar to the antiprotease
factors described above, anti-plasmin and
anti-tPA proteins have been described that
perform the same function as the anticoagulation proteins
Coagulation Complexes