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Blood coagulation
Dr. Abir Alghanouchi
Biochemistry department
Sciences college
Arrest
of bleeding
Events
preventing excessive blood loss
◦ Vascular spasm: Vasoconstriction of
damaged blood vessels
◦ Platelet plug formation
◦ Coagulation or blood clotting
*DAMAGE TO BLOOD VESSEL leads to:
1. Vascular Constriction:
Immediate constriction of blood vessel
Vessel walls pressed together – become
“sticky”/adherent to each other
Minimize blood loss
2. Platelet Plug formation:
PLATELETS attach to exposed collagen with the presence
of von Willebrand factor (vWF) and Glycoprotein IbIX
Aggregation of platelets causes release of chemical
mediators (ADP, Thromboxane A2)
ADP attracts more platelets
Thromboxane A2 (powerful vasoconstrictor)
* promotes aggregation & more ADP
Leads to formation of platelet plug!
3- Blood Coagulation (clot formation):
Final Step in Hemostasis:
Transformation of blood from liquid to solid
Clot reinforces the plug
Multiple cascade steps in clot formation
Process requires plasma proteins, PLs and calcium.
Thrombin
Soluble fibrinogen
Insoluble fibrin
Stages
◦ Activation of prothrombinase
◦ Conversion of prothrombin to thrombin
◦ Conversion of fibrinogen to fibrin
Pathways
◦ Extrinsic
◦ Intrinsic
Initially independent, then they
converge on common pathway leading
to the formation of a fibrin clot !
PRIMARY
AGGREGATION
Platelet Aggregation Clotting
Hemostatic
clot
Fibrin
SECONDARY
COAGULATION
0 min
Thrombin
5 min
10 min
A cascade is a mechanism
in which enzymes activate
other enzymes
sequentially usually
leading to an amplification
of an initial signal.
Each of these pathways
leads to the conversion of
factor X (inactive) to
factor Xa (active)
The intrinsic and extrinsic coagulation pathways are
a series of reactions involve coagulation factors
known as
1. Enzyme precursors (zymogens)
2. Non-enzymatic (cofactors)
3. Calcium (Ca ++)
4. Phospholipids (PL).
All coagulation factors normally are present in the
plasma, with PL being provided by platelets.
Zymogens:
◦ Factors II, VII, IX, X, XI, XII, and prekallikrein
◦ NO biologic activity until converted by enzymes to
active enzymes called serine proteases
Cofactors
◦ Factors V, VIII, tissue factor, and HMWK
Extrinsic—Release
of biochemicals from
broken blood vessels/damaged tissue.
Intrinsic—No
tissue damage, blood
contacts damaged endothelial layer of
blood vessel walls.
Intrinsic
clotting—all factors are found in
circulating blood.
Extrinsic
clotting—Factor III (tissue
thromboplastin) is found outside of blood.
Activated in vivo by endothelial injury, in vitro by
glass or other contact
A foreign surface such as collagen activates
factor XII (Hageman factor).
Acting as catalysts are high MW Kininogen
(HMWK) and kallikrein in the contact phase.
XI, XII, HMWK, PK
The contact group is adsorbed by contact with
a negatively charged surface such as collagen
or the subendothelium in vivo.
Not Vitamin K dependent
Calcium is involved in three steps: the activation of
FIX, X and prothrombin.
Cofactor VIII interacts in the activation of factor X and
cofactor V reacts with prothrombin.
The platelet phospholipid surface acts as template in
the activation of FX and prothrombin.
Is initiated by the release of tissue thromboplastin
(Factor III) which is exposed to the blood when
there is damage to the blood vessel.
Factor VII which is a circulation coagulation factor,
forms a complex with tissue thromboplastin and
calcium.
This complex rapidly converts Factor X to the
enzyme form Factor Xa.
Factor Xa catalyzes the prothrombin (Factor II) to
thrombin (Factor IIa) reaction which is needed to
convert fibrinogen (Factor I) to fibrin.
XIIIa and Ca++ stabilize fibrin clot
Formation of blood clot causes more clotting to
occur—positive feedback.
Prothrombin is soluble single chain glycoprotein (72kDa)
synthesized in liver
Thrombin is produced by the enzymatic cleavage of two sites
on prothrombin by activated Factor X (Xa) and generate
active 2 chain thrombin molecule which is then released
from platelet surface
The A and B chains of thrombin are held together by a
dissulfide bond
S
1
A
2
Xa
Fragment 2 -1
S
S
B
Xa
Prethrombin
Prothrombin (72kDa)
1
+
2
Fragment 2 -1
A
S
B
Active thrombin
(34 kDa)
Converts fibrinogen to fibrin
Activation of prothrombin into
thrombin (cont…)
The activation of prothrombin occurs on the surface of
activated platelets and requires assembly of
prothrombinase complex consisting of platelet, anionic
PLs, Ca2+, factor Xa and prothrombin
This complex is termed factor Va which is activated by
traces of thrombin
Factor Va is subsequently inactivated by further action
of thrombin to limit activation of prothrombin to
thrombin
Factor Xa
Thrombin in Hemostasis
Conversion of fibrinogen to fibrin
340kDa (factor I) is soluble plasma
glycoprotein that consists of 3 non
identical pairs of polypeptides chains
(Aα, Bβ, γ)2 covalently linked by
disulfide bonds
The A and B portions of the Aα and
Bβ chains, termed Fibrinopeptide A
(FPA) and Fibrinopeptide B (FPB)
Thrombin
Release of FBs by thrombin
generate fibrin monomer (weak)
Aggregate spontaneously forming
insoluble fibrin polymer (fibrin
clot) (hard, insoluble)
Antithrombin
III
◦ Most important since it can also inhibit the
activities of factors IXa, Xa, XIa and XIIa
◦ Binds to activated factors rendering them inactive
◦ The action of AT-III is enhanced and accelerated by
the presence of Heparin (either naturally released
from basophils or given therapeutically as an
anticoagulant)
Protein
C
◦ Produced by liver; Vitamin K dependent
◦ Inhibits (cleaves) the cofactors VIIIa and Va
◦ Significantly decreases the rate of clot formation
◦ Needs to be activated (by Thrombin (IIa))
◦ Enhancement of Protein C anticoagulant functions
is achieved by Protein S.
Protein
S
◦ Produced by liver
◦ Vitamin K dependent
◦ Acts as a cofactor to Protein C to enhance its
ability to degrade factors V and VIII
Heparin
◦ It is a negatively charged polysaccharide found in
mast cells near the walls of blood vessels and on the
surfaces of endothelial cells
◦ Heparin acts as an anticoagulant by increasing the
rate of formation of irreversible complexes between
antithrombin III and the serine protease clotting
factors
Alpha
2-macroglobulins
◦ Contributes most of the remaining (25%) of
antithrombin activity in plasma
Alpha
1-antitrypsin
◦ Acts as a minor inhibitor under physiological
conditions, which normally inhibits elastase
◦ Alpha 1-Antitrypsin activity normally increases
markedly after injury to counteract excess elastase
arising from stimulated neutrophils
Vitamin K antagonists
1. Coumarin drug:
◦ They are anticoagulant in vivo
◦ They inhibit carboxylation of Glu residues in
prothrombine and factors VII, IX, X
Vitamin
K antagonists
2. Dicumarol
◦ Is naturally occurred (plant origin)
◦ Used only in vivo as anticoagulant to prevent
thrombosis in patients with a tendency to form blood
clot
◦ Slow onset of action 2-3 days but long duration 4-6
days
Heparin
◦ Animal origin (most cells)
◦ Acts both in vivo and in vitro
◦ Rapid onset few minutes
Removal of Ca2+ by citrate oxalate (in vitro)
Defibrination of blood
◦ Break down of fibrin threads once formed by
continuous shaking or by glass rod
Platelets (thrombocytes) have several functions in
blood clotting:
Form platelet plug at the site of injury
Sites of activation of some clotting factors (II, X)
Provide the surface on which certain clotting
factors bind (Va, Xa, II, Ca2+)
Sources of some clotting factors (XIII, PL)
If platelets are not lysed, blood does not clot
Individuals with thrombocytopenia (low
platelets), bleeding for a long time
Platelets deficiency can be due to many agents
(drugs, some infections, ionizing radiation)
Clot is slowly dissolved by the “fibrin
splitting” called Plasmin
Plasmin gets trapped in clot and slowly
dissolves it by breaking down the fibrin
meshwork at various places, leading to the
production of circulating fragments that are
cleaved by other proteases or by the kidney
and liver
Plasminogen is the inactive pre-cursor that is
activated by activators in plasma:
1. Tissue plasminogen activator (t-PA)
2. Urokinase (to lesser extend)
Is
produced as a precursor “prourokinase’’
by epithelial cells
Its main action is probably in the degradation
of extracellular matrix
Inactive t-PA is released from vascular endothelial
cells following injury
It binds to fibrin and is consequently activated
Active t-PA converts plasminogen into plasmin
Dissolves the clot
Inherited bleeding
disorders
◦ Hemophilia A and B
◦ Von Willebrand
disease
◦ Other factor
deficiencies
Acquired bleeding
disorders
◦ Liver disease
◦ Vitamin K deficiency
1- Hemophilia A and B
Are the best-known coagulation factor disorders
Hemophilia A
Coagulation factor deficiency
Inheritance
Incidence
Hemophilia B
Factor VIII
Factor IX
X-linked
recessive
X-linked
recessive
1/10,000 males
1/50,000 males
2- von Willebrand Disease
It is the most common hereditary bleeding disorder
and is characterized as being inherited autosomal
recessive or dominant
In this disease there is a defect in von Willebrand
factor (vWF) which:
1.
2.
acts as a carrier for factor VIII
mediates the binding of glycoprotein Ib (GPIb)
to collagen
2- von Willebrand Disease
This binding helps the activation of platelets
and formation of primary hemostasis
vWD is characterized by excessive bleeding in
infants because platelets fail to form
hemostatic plug
Source of vitamin K
Green vegetables
Synthesized by intestinal flora
Required for synthesis
Factors II, VII, IX ,X
contribute to bleeding disorders
Causes of deficiency
Malnutrition
Biliary obstruction
Malabsorption
Antibiotic therapy
1. Formation of carboxyglutamate
Vitamin K is essential for the functioning of several
proteins involved in blood clotting (II, VII, IX and X)
These proteins contain a unique modified glutamate
residue, called carboxyglutamate (Gla).
These proteins are synthesized as inactive precursors
that are activated by the vitamin K-dependent
carboxylase which converts glutamate in these
proteins to carboxyglutamate forming mature clotting
factors.
1. Formation of carboxyglutamate (cont…)
(mature)
Dicumarol,
Warfarin
(Gla residue)
2. Interaction of prothombin with platelets
The Gla residue of prothrombin is a natural high affinity
binder (chelator) of positively calcium ions, hence the
designation of calcium as a co-factor (factor IV) in the
schematic.
The prothrombin-calcium complex is then able to bind to
PLs essential for blood clotting on the surface of platelets.
Extrinsic system (tissue damage)
Intrinsic system (surface contact)
XIIa
XII
Tissue factor
XIa
XI
IX
VIIa
IXa
VIII
VII
VIIIa
X
Xa
V
Va
II
Fibrinogen
Vitamin K dependant factors
IIa
(Thrombin)
Fibrin
A number of parameters are used for diagnosis of
clotting disorders including:
1.
Coagulation time (whole blood clotting time)
2.
Bleeding time
3.
Prothrombin time (PT)
4.
Prothrombin consumption test
5.
Activated partial thromboplastin (APTT)
6.
Thrombin clotting time (TT)
1- Coagulation time
Test for intrinsic system
Simple test but takes time and rarely done now
Method:
Venous blood is taken and placed on glass test
tube at 37°C and it observed at time intervals
until clotting occurs
Normal blood takes 5-10min to clot
Longer periods
Coagulation defects
2- Bleeding time
Test for a platelet function
Time taken for the blood to stop:
it is determined by noting time at which blood
coming out a small cut, no longer forms a spot on a
piece of filter paper placed in contact with cut surface
The normal range from 2-4 min
Bleeding time depends on the number and functional
activity of platelets
3- Prothrombin time (PT)
Test for extrinsic system
It involves tissue factor
The level of prothrombin in blood is measured indirectly
by measuring PT
3- Prothrombin time (PT) (cond…)
Method:
An excess of tissue factor and Ca2+ ions are added to
diluted plasma containing citrate (anticoagulant)
and then the time taken for the mixture to clot is
measured
High PT
low level thrombin
Results from liver disease due to
deficiency of prothrombin, fibrinogen,
V, VII and X factors
4- Prothrombin consumption test
This test is used for detection of minor coagulation defects
Method:
Ca2+ is added to both plasma and serum samples
At given time intervals, the mixture is subsampled into tubes
containing fibrinogen and the time taken for fibrinogen to clot
is recorded
The normally plasma will clot fibrinogen very rapidly (12-20s)
because it contains prothrombin but serum takes 2-3 min or
more because it contains normally little prothrombin (it is
removed)
4- Prothrombin consumption test
plasma time x100
Prothrombin consumption time (%) =
Serum time
It is less than 20%
Higher percentage means inefficient hemostasis so
serum contains more thrombin
e.g. 0.5/2.5*100=20%
0.5/1*100=50%>20%
Normal
Disorder
5- Activated partial thromboplastin (APTT)
This test is used for intrinsic system
Method:
A mixture of koalin (provide a maximal stimulus for factor XII)
and PL (acts as platelet substitute) is added to citrate plasma
following by Ca2+ The time taken for mixture to clot is
measured
Prolonged clotting time is almost always due to deficiency of
factors VIII and IX
Normal APTT: 20-25 to 32-39 s;
Critical Values: >100-150 seconds
6- Thrombin clotting time (TT)
TT is a coagulation assay which is usually performed
in order to detect for the therapeutic level of the
anticoagulant Heparin
It is also sensitive in detecting the presence of a
fibrinogen abnormality
6- Thrombin clotting time (TT) (cont…)
Method:
After liberating the plasma from the whole blood by
centrifugation, bovine thrombin is added to the
sample of plasma
The clot is formed and is detected optically or
mechanically by a coagulation instrument
The time between the addition of the thrombin
and the clot formation is recorded as the
thrombin clotting time (generally <21s)
APTT Prolonged, PT Normal: Deficiencies of factor
VIII, IX, XI, and/or XII (intrinsic pathway)
PT Prolonged, APTT Normal: Deficiency of factor VII
(extrinsic pathway), mild-to-moderate deficiencies of
factor II, V, X, and/or fibrinogen (common pathway)
Both PT and APTT Prolonged: Deficiencies of factor II,
V, X, and/or fibrinogen (common pathway), Multiple
factor deficiencies