BIOCHEMISTRY

1.07 Platelet and Coagulation

Dr. Marivic A. Villamor | September 6, 2016
LE1
I. Hemostasis
OUTLINE Figure 1. Stages of Hemostasis
#
 Definition
 3 Phases of Hemostasis
1. Primary
2. Secondary
3. Fibrinolysis
II. Platelets
A. Definition
B. Functions
C. Zones
1. Organelle
2. Peripheral
3. Structural
D. Platelet Plug Formation
1. Adhesion
2. Activation
3. Aggregation
III. Coagulation Cascade Figure 2. Steps Involved in Hemostasis
a. Functions
b. Coagulation B. 3 Phases of Hemostasis
1. Intrinsic
2. Extrinsic 1. Primary
c. Phases  Formation of a loose and temporary platelet
d. Hemostatic Plug
aggregate at the site of injury.
e. Regulatory Proteins
f. Thrombus  Platelets bind to collagen at the site of vessel wall
g. Vitamin K injury, and form thromboxane A2 and release ADP,
h. Thrombin which activate other platelets flowing by the vicinity of
IV. Fibrinolysis the injury.
V. Evaluation Tests for Coagulation Pathway  Thrombin, formed during coagulation at the same site,
a. Prothrombin Time
causes further platelet activation.
b. Activated Partial Thromboplastin Time
 Upon activation, platelets change shape and, in the
presence of fibrinogen, aggregate to form the
OBJECTIVES: hemostatic plug (in hemostasis) or thrombus (in
At the end of the lecture, the student should be able to: thrombosis).
1. Explain the role of the platelets in the hemostasis and 2. Secondary
thrombosis.  Formation of a fibrin mesh that binds to the platelet
2. Discuss the sequential mechanisms in blood coagulation.
aggregate, forming a more stable hemostatic plug or
3. Compare and contrast the proteins involved in the
coagulation cascade. thrombus.
4. Describe the components of the fibrinolytic pathway. 3. Fibrinolysis
 Partial or complete dissolution of the hemostatic plug
I. HEMOSTASIS or thrombus by plasmin.

A. Definition II. PLATELETS

 Coordinated function of blood vessels, platelets,
coagulation factors, and fibrinolytic system. A. Definition
 Cessation of bleeding from a cut or severed vessel  Non-nucleated cells present in the blood (Marks)
(Harper)  Precursor: Megakaryocyte
 There is initial vasoconstriction of the injured vessel,  Extensive actin-myosin system
causing diminished blood flow distal to the injury.  Platelet activation in response to endothelial injury
2+
causes Ca -dependent changes in the contractile
elements
 Contain 3 types of granules:
o Electron-dense granules
o α-granules
o Lysosomal granule

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1.07 Platelet and Coagulation BIOCHEMISTRY 2020C

 Promote the Coagulation Cascade involving plasma D. Platelet Plug Formation

factors to form a blood clot a. Adhesion
1. Platelet–subendothelial interaction:
B. Functions platelets initially adhere to the sites of blood vessel
 Adhesive and cohesive functions that lead to the injury
formation of a hemostatic plug.  collagen, subendothelial matrix-bound vWF,
and other matrix components are exposed
 Aggregate on damaged endothelium and exposed
2. Binding to collagen by GPIa (integrin α2β1)
collagen, release contents of alpha and dense  platelet shape: flat disc  spherical
granules
 spherical cell extrudes long pseudopods 
 Form mechanical plugs at the site of vessel injury promote platelet/platelet interactions.
(Marks) 3. Binding of subendothelial vWF by GPIb
 Secrete regulators of the clotting process and  changes in the platelet membrane: expose
vascular repair (Marks) GPIIb/IIIa (integrin αIIbβ3) binding sites to
fibrinogen and vWF.

Table 1. Platelet Functions Figure 4. Surface Molecules Involved in Adhesion

C. Zones b. Activation/Secretion
1. Organelle Zone - responsible for the production of the 1. After initial adherence  some platelets release
needed molecules that will ensure the platelet will function contents of their dense granules and α-granules
effectively. 2. ADP (potent platelet activator) released from
platelets and damaged red blood cells binds to a
2. Peripheral Zone - Glycocalyx, Membrane platelet membrane receptor  further unmasking of
*Glycocalyx = glycoprotein-polysaccharide covering GPIIb/IIIa binding sites.
on a cell surface  Aggregation of platelets can’t take place without
ADP stimulation
3. Structural Zone - includes actin-myosin system which  induces swelling of the activated platelets,
allows activated platelet to form pseudopodia  allow promoting platelet/platelet contact and
platelets to have greater surface area which induce more adherence.
platelet attachment to one another.  Binding of fibrinogen to activated platelets is
necessary for aggregation

Figure 5. Platelet activation and Pseudopodia formation

Figure 3. Platelet Zones

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c. Aggregation

1. Initial adherence of platelets sets off a series of

reactions (platelet activation)  more platelets are
recruited and aggregated at the site of injury.

Figure 6. Platelet Adhesion to the Subendothelial Cell Layer

III. COAGULATION CASCADE

A. Functions Figure 7. A simplified version of the coagulation “cascade.”
a. Substrate – Factor 1 (Fibrinogen) An abnormality in the extrinsic pathway results in a prolonged
b. Cofactors – accelerate enzymatic reaction prothrombin time (PT). An abnormality in the intrinsic pathway
 Factors III, V and VIII results in a prolonged activated partial thromboplastin time
 Factor HMWK (aPTT). An abnormality in the common pathway results in
c. Enzymes prolongation of PT and aPTT.
 Serine Protease – cleave peptide bonds
(Factor II, VII, IX, X, XI and XII) C. Coagulation Phases
 Transamidase – XIII only

Group Contact Prothrombin Fibrinogen Initiation Low amts of active Vasculature

phase coagulant factors are disruption and
Factors XI, XII, II, VII, IX, X I, V, VIII, generated endothelial cells
PK, XIII exposure to the
HMWK bloodstream
Vitamin K NO YES NO Amplification Level of active Accumulating
dependent phase coagulation factors amts of thrombin
Consumed in NO NO (except YES is boosted will further
clotting for FII) activate platelets
Found in BOTH BOTH PLASMA Propagation Coagulation factors Occurs away on
serum or ONLY phase bind to membranes surfaces
plasma of activated platelets containing
Table 2. Coagulation Groups Fibrin clots are procoagulant
formed phospholipids
B. Coagulation
 simultaneous occurrence of two events Table 3. Coagulation Phases
a. reaction of plasma proteins with
subendothelium
b. platelets adhering to subendothelial layer
 two pathways working together (extrinsic and intrinsic)

1. Intrinsic pathway
- inside blood vessel
- direct vascular injury
- ex. Damage of endothelium lining

2. Extrinsic pathway Figure 8. Cross-linked fibrin monomers

- outside blood vessel
- calcium dependent pathway

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 Factor XIII and thrombin activates cross-linking Coagulation Factors

between monomeric units of fibrin
 An amino acid from one fibrin molecule will bind to COAGULATION FACTORS
another amino acid in the next fibrin molecule through
transamidation reaction Factor Descriptive Pathway, Function
name
I Fibrinogen Common pathway,
converted to fibrin
II Prthrombin Common pathway,
converted to
thrombin (converts
fribrinogen to fibrin)
III Tissue Factor Activates extrinsic
pathway
IV Calcium Ions Needed for almost
all stages of
coagulation
process; always
present
Figure 9. Transamidation reaction catalyzed by Factor XIIIa, V Proaccelerin, Common pathway
transglutaminase labile factor
VII Proconvertin Both extrinsic and
 needs Factor XIIIa (active) to catalyze the removal of intrinsic pathways
VII Antihemophilic Intrinsic pathway,
amide group in Gln of a fibrin molecule
Factor deficiency 
 exposed carbonyl group is allowed to bind with Lys of
haemophilia A
another fibrin molecule
IX Plasma Intrinsic pathway,
thromboplastin deficiency results in
D. Hemostatic Plug component haemophilia B
X Stuart Factor Common pathway
XI Plasma Intrinsic pathway,
thromboplastin deficiency results in
antecedent haemophilia C
XII Hageman factor Intrinsic pathway,
activates plasmin,
initiates clotting in
vitro; activation
initiates
inflammation
XIII Fibrin stabilizing Cross-links fibrin,
factor forming a strong,
stable clot
Prekallikrein Serine protease
High-molecular weight kininogen Cofactor

E. Regulatory Proteins
Figure 10. Plug Formation
1. Thrombin
 Platelets adhere to collagen material of blood vessel  Antithrombotic effects from its binding to an
endothelial cell receptor called
 further stabilized and attract more platelets
thrombomodulin
 Activation of clotting factors and secretion of thrombin
 Thrombin activates fibrinogen into fibrin to form fibrin 2. Thrombomodulin
clot  abolishes the clotting function of thrombin
 allows thrombin to activate protein C, which
has anticoagulant effects.

3. Proteins C and S
 Activated protein C forms a complex with
protein S
 Protein S anchors the activated protein C
complex (APC) to platelet phospholipid

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 APC destroys Factors VIIIa and Va by o Done in the liver

proteolytic cleavage  decreasing the o Vitamin K-dependent coagulation/regulatory
production of thrombin. factors require activation in a lipid-rich
 APC stimulates endothelial cells to increase environment
secretion of prostaglandin PGI2  reduces
o Vitamin K = lipid-soluble
platelet aggregation
 Main source: large intestines, where gut bacteria
4. Serpins synthesize Vitamin K
 No bacteria = No Vitamin K = No Activation of
 serine protease inhibitors Coagulating and Regulatory Factors = Hemorrhage
 naturally occurring 
o in plasma at high concentrations (~10%
of the plasma proteins)

F. Thrombus

Figure 12. Vitamin K Cycle

Figure 11: Thrombus and Embolus
Guide to the diagram:

1. Thrombus Formation - mediated by complex a. Begins with the reduced Vitamin K (KH2)
interaction between proteins (enzymes or cofactors)  Functional form
b. Vitamin KH2 becomes a cofactor for γ-glutamyl
2. Thrombus vs. Embolus carboxylase in the prozymogens (inactive) 
a. Thrombus carboxylated zymogens
 blood clot = more stable and made of covalently  Carboxyl group added on glu residues at
bonded fibrin amino terminal region
 outcome of the coagulation cascade  Active form = γ-glutamyl carboxyls

2+
o blood-borne fibrin stick together  seal the Have Ca binding property
inside of the wound c. Vitamin KH2 is oxidized  Vitamin K Epoxide.
 remains attached where it was initiated/formed d. Vitamin K Epoxide is reduced  Vitamin KH2
 By Vitamin K Reductase (VCORK1)
b. Embolus  NADH = cofactor (donates the H atoms)
 blood clot that breaks loose from the area
where it was initiated/formed and travels; adapts Note: Warfarin (Coumadin) is a drug that inhibits activity of
a migratory system Vitamin K reductase, preventing γ-carboxylation of glu
 danger: may travel to a blood vessel that is residues in factors II, VII, IX, X; and proteins C and S.
smaller in diameter  clog  possible stroke
Result: Non-carboxylated anti-coagulation factors remain
G. Vitamin K inactive and increase in concentration in both the blood and
plasma, but unable to promote blood coagulation because they
cannot bind calcium and thus cannot bind to their phospholipid
 Needed in the synthesis of several proteins that sites of activation
mediate BOTH coagulation and anticoagulation
 Cofactor in the activation of Vitamin K-dependent
proteins (coagulation and regulatory factors)

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H. Thrombin (Factor IIa)

Thrombin - activated form of Factor II (Prothrombin) which is a

serine protease that converts fibrinogen to fibrin.

Figure 14. Fibrinolysis

1. Plasminogen, a circulating serum protein and

prozymogen, is cleaved by 1 or 2 activators to form
the active fibrin-degrading serine protease plasmin.
2. Plasmin then hydrolyzes peptide bonds of fibrin into
soluble degradation products → clot dissolution.
Figure 13. Thrombin as Anticoagulant
V. EVALUATION TESTS FOR COAGULAION PATHWAY
Two Purposes:
A. Prothrombin Time (Extrinsic)
a. Prothrombic Regulatory Role (Feedback Amplification)
 As a procoagulant  measures the amount of time it takes for plasma to
 Stimulates its own formation: activate factors V, VIII, clot
and XI  tests [prothrombin]
o accelerates the rate of clot formation to  specifically evaluates the presence of factors VII, V, X
lessen blood loss  used to monitor warfarin (Coumadin)
 normal PT time: 11-14sec
 Activation of platelet aggregation: stimulate release of
Low INR= high risk of clotting
Factor VIII from vWF, cleaves factor XIII  XIIIa. High INR= high risk of bleeding
b. Antithrombotic Regulatory Role (Feedback Inhibition)
 As an anticoagulant
 Binds to thrombomodulin.
 Thrombin-thrombomodulin complex  activation of
Protein C which has anticoagulant effects
o inhibition of factors VIIIa and Va

IV. FIBRINOLYSIS

A. Definition
 Involves the degradation of fibrin in a clot by plasmin.
(Mark’s, p.857)
 Primary fibrinolysis refers to the normal breakdown
Figure 15. Prothrombin Time
of clots.
 Secondary fibrinolysis is the breakdown of blood
clots due to a medical disorder, medicine, or other B. Activated Partial Thromboplastin Time (Intrinsic)
cause.  measures the integrity of the intrinsic system and
o may cause severe bleeding. common clotting pathways
 also measures time to form clot
 investigates bleeding and clotting disorders
 monitor patients taking anti-coagulant drug like
Heparin
 normal aPTT: 25-30 s (longer than PT)

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4. What amino acids are present in the cross-linked fibrin?

a. Lysine and Arginine
b. Glutamine and Lysine
c. Glysine and Leucine
d. Isoleucine and Phenylalanine

5. What enzyme is required for Fibrinolysis?

a. Plasmin
b. Warfarin
c. Thrombin
d. None of the above

Answer Key: C,C,C,B,A

REFERENCES:
Figure 16. Activated Partial Thromboplastin Time
 Lecture Notes
SUMMARY  Ma’am Villamor’s PPT
 Marks’ Basic Medical Biochemistry
 Many coagulation factors are zymogens of serine 
th
Harper’s Illustrated Biochemstriy 30 Edition (2015).
proteases, becoming activated, then inactivated during
Mc GrawHill Edu.
the overall process
 Platelets are involved in both primary and secondary  Borlongan, R., Garcia, J., Gatbonton, B., Hou, M.,
hemostasis to form a thrombus or a blood clot Lim, L., Obillos, J., Villalon, J., Yoingco, L. (2014).
 The clotting cascade consists of a series of protease 2019B Transcription: Platelets and Coagulation.
activation steps leading to the activation of thrombin,
which converts fibrinogen to fibrin
 Thrombin also activates transglutaminase which cross-
links the fibrins and leads to hard clot formation
 Factors II, VII, IX and X and proteins C and S require
vitamin K-dependent γ-carboxylation process of certain
glutamate residues to function in coagulation. This
carboxylation process can be inhibited by the
anticoagulant warfarin.
 Serpins are serine protease inhibitors, the serpin anti-
thrombin III (AT III) aids in regulating blood coagulation
by modulating thrombin activity
 Heparin enhances the interaction of thrombin with AT III
 Plasmin, the active product of plasminogen, is the only
protease that can dissolve fibrin clots

SHORT QUIZ
1. What platelet function is reversible?
a. Aggregation
b. Secretion
c. Adhesion
d. Intrinsic Pathway

2. The end-product of both the extrinsic and intrinsic

pathways of blood coagulation is:
a. Activation of clotting factors
b. Activation of Plasminogen
c. Fibrin clot formation
d. Fibrin clot dissolution

3. The following are Vitamin K dependent except for

a. Factor X
b. Factor IX
c. Factor VI
d. Factor II

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