SECONDARY HEMOSTASIS Categories of Coagulation Factors by Hemostatic Function

Substrate Factor I (Fibrination)

Factor V
• Involves the enzymatic activation of series of Cofactors
Factor VIII
plasma proteins in the coagulation system to Serine Protease Ia
form a fibrin meshwork. Factor IXa
Factor Xa
Event Comment Enzymes
Transaminase
Coagulation factors interact Factor XIIIa
on platelet surface to Prekallikrein
Fibrin-Platelet Plug
Step 4 produce fibrin; fibrin-
Formation
platelet plug then form at Classification of Coagulation Factors by Physical Properties
site of vessel injury Factor XI
Fibrin clot must be Factor XII
Step 5 Fibrin Stabilization stabilized by coagulation Contact Group
Prekallikrein
factor XIII HMWF
Factor II
Factors Name Other Name Prothrombin Group / Factor VII
I Fibrinogen Ia – Fibrin Vitamin K Dependent Group Factor IX
II Prothrombin IIa- Thrombin Factor X
III Tissue Factor Tissue Thromboplastin Factor I
IV Calcium Ions Ionic Calcium Factor V
V Proaccelerin Labile Factor Fibrinogen Group
Factor VIII
VII Proconvertin Stable Factor Factor XIII
Antihemophilic Factor A
VIII Antihemophilic Factor (AHF)
Antihemophilic Globulin
Group Contact Prothrombin Fibrinogen
Plasma Thrombopoietin Antihemophilic Factor B
IX XI, XII, PK,
Component (PTC) Christmas Factor Factors II, VII, IX, X I, V, VIII, XIII
X Stuart-Prower Factor Prower/Stuart Factor HMWF
Plasma Thromboplastin Absorbed by
XI Antihemophilic Factor C NO YES NO
Antecedent (PTA) Al3OH / BaSO4
Glass Factor Vit. K
XII Hageman Factor (HF) NO YES NO
Contact Factor Dependent
Laki-Lorand Factor (LLF) Consumed in
XIII Fibrin Stabilizing Factor(FSF) NO NO (except II) YES
Fibrinase Clotting
Prekallikrein Fletcher Factor Found in Serum
Fitzgerald Factor BOTH BOTH PLASMA
High Molecular Weight or Plasma
William Factor
Kininogen
Flaujeac Factor
vonWillebrand Factor Vitamin K Dependent:
• Protein C
Factors Biochemistry Other Notes • Protein S
I Glycoprotein • Protein Z
Precursor of thrombin
II Glycoprotein
Consumed during coagulation
III Lipoprotein Cofactor of Factor VII Pathway of Coagulation
IV Metal Ions Cofactor of Factor X
V Glycoprotein Cofactor of Factor X and II
Main precursor of extrinsic
VII Glycoprotein
pathway
Cofactor for Factor X via intrinsic
VIII Glycoprotein
pathway
IX Glycoprotein
X Glycoprotein
XI  and  globulin
XII Sialoglycoprotein Cofactor of Factor XI and IX
XIII  and  globulin
Prekallikrein Plasma Protein Contact Factors
HMWF Plasma Protein Contact Factors
1) Intrinsic Pathway 3) Common Pathway
- Activation occurs when a vessel is injured, - This pathway starts with the activation of
exposing the subendothelial basement Factor X to factor Xa by either intrinsic or
membrane and collagen. extrinsic pathway.
- This leads to the activation of the “Contact
Factors”, Factor XII together with Factor XI,
HMWF, and Prekallikrein.

Coagulation Pathways

2) Extrinsic Pathway
- Activated by the release of Factor III (TF,
Tissue Thromboplastin) into the plasma
from injured tissue.
- Fastest
Maintenance of Blood in Liquid State Inside Blood
Vessels
Thrombin formation marks a critical event in the
hemostatic process
Action of Thrombin:
1. Converts fibrinogen to fibrin
2. Activates Factor XIII
3. Enhances the activity of Factor V and VIII cofactor
4. Induces platelet aggregation
Thrombin-Mediated Reactions in Hemostasis
1. Procoagulant
• Induces platelet activation and aggregation
• Activates cofactor VIII to VIIIa
• Converts Factor XIII to XIIIa
• Via autocatalysis converts Prothrombin to
thrombin
2. Coagulation
• Binds antithrombin to inhibit serine
proteases
• Binds to thrombomodulin to activate
Protein C (inhibits Factor Va and VIIIa)
3. Tissue Repair
• Induces cellular chemotaxis
• Stimulates proliferation of smooth muscle
and endothelial cells
Inhibitors of Coagulation (Anticoagulant)
I. Physiologic Inhibitors
1. Protein C
- Glycoprotein that is produced by the liver
and is major inhibitor of blood
coagulation.
- This activates Factors VIII, and Va in the
presence of cofactor Protein S.
2. Protein S
- A vitamin K dependent protein and also
produced by the liver.
- This function to enhance binding of
Protein C to phospholipid surfaces and
increase the rate of Factors Va and VIIIa
inactivation by Protein C.
3. Thrombomodulin– inhibits thrombin and
inactivates the clotting cascade.
4. Antithrombin III – major inhibitor of thrombin
and Factor Xa.
5. Heparin cofactor – inhibits thrombin; activity is
enhanced by heparin.
6. Alpha2 Macroglobulin – forms complex with
thrombin, kallikrein, thus inhibiting their
activities.
7. Extrinsic Pathway Inhibitor – lipoprotein
associated inhibitor (LACI) that inhibits VIIa
which is a tissue complex factor.
8. CI Inhibitor – inactivates Factor XIIa and plasma
kallikrein, Factor XIa and plasmin.
9. Alpha1 Antitrypsin – a slow reacting thrombin
inhibitor which inhibits Factor XIa and Xa.
10. Activated Protein C Inhibitor – inhibits Protein
C. This is more active when heparin is present.
Laboratory Evaluation: Activation of Common Pathway
and Conversion of Prothrombin to Thrombin
Test of Phase II of Coagulation
1. The Coagulation Test
• Tests the composite action of all plasma
acting simultaneously
• Clotting Time is a measure of the ability of
the blood to clot and is not influenced by
the platelet functions other the PF3. It also
measure only the time required for the
formation of the traces thrombin sufficient
to produce a visible clot.
Vacuum tube: Sodium Citrate Tube (Blue Top)
Methods:
a. Micro Methods
▪ Slide or Drop Method
▪ Capillary or Dale and Laidlaw’s Method
b. Macro Methods – superior for there is less
contamination of the plasma with tissue
fluids when blood is drawn from a vein.
▪ Lee-White Method or Whole Blood
Clotting Time
Principle: The whole blood clotting
time is the time required for freshly
collected blood to form a firm clot in
standardized glass tubes at 37OC.
Thus, the whole blood clotting time is
a measure of the integrity of the
intrinsic factorsystem.
Vacuum Tube: Plain Tube (Red Top)
Normal Value: 5-10 minutes
 c. Activated Coagulation Time of whole Blood
Principle: The activated coagulation time of
whole blood is the time necessary for fresh
blood to form a clot when incubated at
37OC in the presence of “surface contact”
(glass beads) activation. This assay, like to
whole blood clotting time, measures overall
activity of the intrinsic clotting system.
Normal Value: 1-2 minutes
d. Plasma Recalcification Time
▪ More sensitive method than the
coagulation time of whole blood
▪ May reveal abnormality which is not
detectable by the determination of
the clotting time of the venous blood
▪ The activated recalcification time
makes use of 0.25 M CaCl2 as
activator
▪ Normal Values: less than 50 seconds
2. Partial Thromboplastin Time
• Simplest test for the INTRINSIC and
COMMON PATHWAY of coagulation
Vacuum tube: Sodium Citrate Tube (Blue Top)
Sample: Citrated Plasma
3. Activated Partial Thromboplastin Time (APTT)
• A test for the deficiencies of factors in the
intrinsic system (Factor VIII, IX, XI, XII)
Normal Value: 25-35 seconds
4. Differential Test of Activated Partial
Thromboplastin Time (DAPTT)
• Used to differentiate factor of deficiency
and disorder of circulating anticoagulants
(Lupus Anticoagulant)
Vacuum tube: Sodium Citrate Tube (Blue Top)
5. Differential Test of Partial Thromboplastin Time
(DPTT)
• Another modification of APTT which is done
by mixing the patient’s plasma with
commercially available correcting reagents,
Factor VIII and IX reagents
• If prolonged:
o PTT is corrected with Factor VIII
(hemophilia A)
o PTT is corrected with Factor IX
(hemophilia B)
6. Plasma Prothrombin Time (ProTime)
• Measures the EXTRINSIC and COMMON
PATHWAY of coagulation
• It is used to monitor oral anticoagulant
therapy (warfarin, Coumadin, heparin,
coumarin)
• Can detect prothrombin, fibrinogen,
Factor V, VII, and X deficiencies
Vacuum tube: Sodium Citrate Tube (Blue Top)
Methods:(ProTime)
a. One-Stage Method of Quick (Stanley
Brown Method)
Principle: Tissue thromboplastin and
calcium added to plasma react to fibrinogen
to form a clot. The thromboplastin added to
the plasma takes the place of the juice
formation of extrinsic thromboplastin. The
prothrombin time is therefore prolonged if
there is a deficiency of factors V, VII, or X or
a very severe deficiency of Factor I and II.
Normal Value: 10-15 seconds (depends on
the reagent and machine)
b. Two-Stage Prothrombin and Proconvertin
Test (Owren and Aas)
▪ It offers combined estimation of the
levels of prothrombin and
Proconvertin
Advantages:
 It is more sensitive with Stanley
Brown Method
 Fresh specimens are not
necessary, and the method can
be used for mailed samples for
blood
 The method is not affected by
heparin
c. Owren’sThrombotest Method
▪ For the control of coumarin
anticoagulant therapy, this is
considered as the most sensitive test
d. Fibrometer Method
▪ It is an electromechanical semi-
automated instrument that has been
used extensively in one-stage
prothrombin method of Quick
 e. Micromethod (ProTime)
▪ This is microtechnique employed for
children and the method uses
micropipettes; the principle of the
test is similar with one-stage
prothrombin time or Stypven Time
f. Related Method-Stypven Time (Russell’s
Viper Venom Time)
▪ It is used to distinguish deficiencies
of Factor X and those of Factor VII. It
is also used to detect deficiencies in
prothrombin, fibrinogen, and
Factors V and X. It differs from
prothrombin time in the in that
deficiencies in Factor VII are not
detected
g. Prothrombin Activity or Index
▪ Reported in percentage, with 100%
as the maximum level
ProTime (in seconds of control)
PT Activity(%) = × 100
ProTime (in seconds of patient)
h. International Normalized Ratio
▪ This method of reporting has been
proposed to monitor patients in oral
anticoagulant therapy
▪ It is defined as the prothrombin time
ratio had the test been performed
using international standard
thromboplastin reagent
7. Substitution Test / Mixing Studies
• It is used if PT or APTT is high
• This can be adopted if primarily tests like
PT or APTT are abnormally prolonged
and indicate a factor deficiency. The
patient’s deficient plasma is diluted 1:1
with a plasma or serum substitute and
the APTT or PT is repeated. A correction
if the original prolonged APTT or PT
indicates that the deficient factor has
been added to patient’s plasma by
substitution solutions as follow:
o Aged Plasma – lacks Labile Factor
V and VII but retains normal
activity of all coagulation factors.
(Extract and incubate at 24 hrs.)
o Fresh Absorbed Plasma – lacks
vitamin K dependent factors (II,
VII, IX, X) but retains activity of all
other coagulation factors
o Aged Serum – lack Factors I, II, V,
VII but retains normal activity of
all coagulation factors
(Factor, Circulating Inhibitor-
Lupus anticoagulant, Circulating
Inhibitor)
8. Serum Prothrombin Time or Prothrombin
Consumption Test (PCT)
• Best considered as a test of platelet
phospholipid activity. If the prothrombin
time and the PTT are normal, a short PCT
indicated a deficiency of PF3 due to
thrombocytopenia or thrombopathia.
9. Thromboplastin Generation Test
• Bigg’s and Macfarlane Method
• Hick’s-Pitney Kaolin Modification
Method
Test of Phase III of Coagulation
1. Thrombin Time
• Test for the deficiency or inhibition of
fibrinogen
Principle: Commercially prepared
thrombin reagent is added to citrated
plasma, and the time required for clot
formation is measured.
Normal Value: 10-20 seconds
2. Fibrindex Test
• Commercially available test wherein
upon addition of plasma containing
fibrinogen, thrombin produces clotting
3. Fit-Test (Immunological Test)
• Ba rapid slide test based on the
agglutination of fibrinogen-coated red
blood cells by the latex anti-human
fibrinogen reagent. Normally, presence
of fibrinogen is indicated by
agglutination.
4. Fibrinogen Titer Method
• Serial dilutions of plasma are diluted
with thrombin. The titers is the highest
dilution in which a fibrin clot can be
seen, and is relate to the fibrinogen
concentration and indirectly to the
presence of fibrinogen is indicated by
agglutination.

5. Assay of Plasma Fibrinogen

• Several accurate methods are now
available for the quantitative assay of
plasma fibrinogen. Fibrinogen is usually
converted into fibrin which is quantified
by gravimetric, nephlelometric,
chemical, immunologic and precipitation
methods.
Methods:
a. Ellis and Stransky Method
b. Stirland’s Method
c. Turbidimetric Method of Partfantjevet. Al
d. Ratnoff and Menzie Method
e. Fibrin Clot Method
 FIBRINOLYSIS
• It is a system whereby the temporary fibrin clot
is systematically and gradually dissolves and as
the vessel heals in order to restore normal
blood flow.
Components of Fibrinolytic System
1. Plasminogen activators (half-life: 24-26 hr)
a. Endogenous
• Tissue plasminogen activator
• Single-chain urokinase-like
plasminogen activator
• Two-chain urokinase
b. Exogenous
• Streptokinase
• Acy-plasminogen streptokinase
activator complex (APSAC)
2. Plasminogen (profibrinolysis)
• Comes from the liver
3. Plasmin (Fibrinoysis)
• Proteolytic enzyme
4. Inhibitors of Fibrinolysis
• Neutralize the activity of plasmin
a. Alpha1 Antitrypsin
- The primary inhibitor of plasmin
- Present in the plasma and also in
platelets
b. Alpha2 Macroglobulin
- Inactivates the plasmin that is not
inhibited by Alpha2 Antitrypsin
c. Thrombospondin
- Release by platelets
- Inhibits activation of fibrin-bound
plasminogen
d. Plasminogen activator inhibitor 1 (PAI-
1) and plasminogen activator inhibitor
2 (PAI-2)
- Both are naturally occurring
- They come from endothelial cells
and platelets
Action of Plasmin
1. Destroys fibrinogen and fibrin
2. Produces FDP which increase vascular
permeability and interfere with thrombin-
induced fibrin formation
3. Produces D-Dimer
4. Destroys factor V, VII, IX, XI, and other plasma
proteins
5. Indirectly enhances or amplifies conversion of
factor XII to XIIa
6. Enhances or amplifies conversion of PK to
kallikrein, liberating kinins to kininogen
7. Cleaves C3 fragment
Degradation of Fibrin and Non-cross-linked Fibrin
• Plasma degrades fibrin clot during fibrinolysis
and also native fibrinogen in a process called
fibrinogenolysis.
• The earliest proteolytic activity results in the
still clottable X fragment which is subsequently
degraded to the unclottable Y and D fragments.
The Y fragment, consisting of D plus E portions,
is then itself split into these components. Small
peptides are also produced at a number of the
proteolytic cleavage.
Laboratory Evaluation: Fibrinolysis
1. Whole Blood Clot Lysis Time
• Principle: A clot is dissolved as a result of
plasmin activity. Normally, this does not
occur in less than 72 hours because of the
presence of plasma inhibitors which
inactivate plasmin as it forms.
• Normal Value: Lysis of clot after 24 hours
2. Euglobulin Clot Lysis Time
• Principle: Euglobulin fraction of the plasma
contains fibrinogen, plasminogen and all of
plasminogen activators but only traces of
antiplasmins. The lysis of a fibrin clot formed
by the addition of thrombin is a measure of
the fibribolytic activity.
3. Diluted Blood Clot Lysis Time
• Principle: Plasmin inhibitors loose activity on
dilution. In this method, whole blood is
diluted with a buffer solution and clotted by
the addition of thrombin. Then the clot is
observed for lysis.
• Normal Value: Blood clot should not lyse in
less than 6-10 hours
4. Diluted Plasma Clot Lysis Time
• Principle: Serial dilutions of patient’s
plasma and normal plasma are prepared.
Thrombin is added to each tube and is
then observed later on for the presence
of clot and eventually for lysis. Lysis
within 12 hours means increased
fibrinolysis activity.
5. Quantitative Assay of Fibrin-Fibrinogen
Degradation Products
• The methods for assay of these
fragments are based on red cell
hemagglutination inhibitors,
staphylococcal agglutination and
immunodiffusion
6. Protamine Sulfate Turbidity Test
• Principle: When a dilute solution of
protamine sulphate is added to citrated
plasma incubate at 37oC, a precipitate in
the presence of fibrin monomers or early
degradation products is formed to
produce turbidity (gel-like clots or
paracoagulation)
Laboratory Evaluation: Test Inhibitors of Coagulation
1. Plasma Antithrombin Test
• This involves titration of plasma with
decreasing amounts of thrombin to
detect small amount of anticoagulant
2. Plasma Thrombin Time
• Plasma is clotted by thrombin and the
time taken is dependent on the amount
and quality of fibrinogen and inhibitors
3. Assay for Lupus Anticoagulant (Tissue
Inhibition Test)
• Method: Schleider’s Method
4. Assays of Inhibitors of Other Factors

7. Latex Bead Agglutination Test

• A rapid, semi-quantitative method to
measure fibrin degradation

8. D-Dimer Test
• This test is superior in sensitivity and
specificity as compared with the
conventional FDP assay
• This testis positive in early Disseminated
Intravascular Coagulation (DIC) and is
specific for cross-linked D-Dimer
fragment of fibrin

9. Prothrombin Fragment 1.2 Test (F -1.2)

• A new assay that is sensitive biological
marker of thrombin generation and X
activity because generation of F – 1.2
precedes thrombus formation

10. Tanned Red Cell Hemagglutination Inhibition

Immunoassay
• The FDP present in patient’s serum
neutralizes antifibrinogen antiserum,
thereby preventing the antiserum form
agglutinating fibrinogen-coated
erythrocytes