HEMA 312: Clinical Hematology 2│Lecture

2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

❖ Coagulation Pathways:
Secondary Hemostasis - Extrinsic Pathway
(Preliminary Term, 3rd Topic) - Intrinsic Pathway
Trans Outline: - Common Pathway
I: Clotting Factors - Intrinsic
- Characteristics - Common
o In primary hemostasis, what is formed is the platelet
- Classification III: Coagulation Inhibitors aggregate (plug).
II: Coagulation Pathways o Red clot: incorporation of RBCs to add surface area
- Extrinsic on the clot.
o Because of the negative-charged exposed collagen, it
attracted the clotting factors under intrinsic pathway.
o There are 2 principles to follow when it comes to o For the release of the Tissue Thromboplastin, it will
clotting: further activate the extrinsic pathway.
✓ Plasma-based coagulation: basis for different o Once the aforementioned pathways are activated, and
laboratory testing reached their endpoints, the common pathway will
✓ Cell-based theory: used in the formation of clot then be activated. This will help in the formation of a
o Clotting factors are inactive in nature (as they stable and irreversible clot.
circulate in plasma).

SECONDARY HEMOSTASIS
❖ Activation of a series of coagulation proteins in the
plasma, mostly serine proteases, to form a fibrin clot.
o Interaction of all coagulation proteins called as
clotting factors to form a stable and irreversible clot.
o If more than one of the clotting factors will be missing,
the clot formation will be prolonged. This will lead to
more bleeding.
o Secondary hemostatic process involves enzymatic
activities. Particularly, it involves serine proteases
which helps in stabilizing a clot.
❑ These proteins (coagulation proteins) circulate as
inactive zymogens (proenzymes) that become
activated during the process of coagulation and, in
turn, form complexes that activate other zymogens to
ultimately generate thrombin, an enzyme that converts
fibrinogen to a localized fibrin clot. The final event of
hemostasis is fibrinolysis, the gradual digestion and
removal of the fibrin clot as healing occurs (Rodaks 6th
ed, pg.627).

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2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

especially Factor IV). For Factor III, once released

from the tissues, will activate other factors.
o Factor VII circulates in the plasma inactively. It is
being activated by Factor III (once released from the
tissue, it will bind to Factor VII).
o If the collagen is exposed due to its negative charge,
Factor XII will be attracted to that negative charge,
which will make it to be cleaved or activated.
o The activated coagulation proteins/ clotting factors
must be inhibited later on by regulatory proteins/
control proteins (inhibitors/inactivators).
o Clotting is always localized.

Clotting factors can be classified according to:

o Function:
1. Enzyme (could be a serine protease or
transaminase)
- Serine protease: active sites of CF’s are rich with
serine (amino acid).
- Transaminase: Factor XIII is the only
transaminase which helps in clot stabilization.
2. Co-factor
3. Substrate (most significant is the Factor I)
o Physical Properties:
1. Contact group
- Once they come into contact with a negative
charge, they become activated. In vivo, that
negative charge is the collagen. In vitro, that is
the glass surface (reason why blood coagulates in
a plain red tube). Example of this are Factors XII,
XI, High-molecular-weight kininogen
(HMWK), and Prekallikrein (PK).
❑ These factors are involved in the intrinsic coagulation
pathway. They are moderately stable and are not
consumed during coagulation (Turgeon’s 5th ed, pg.
410).
2. Prothrombin group
- Factors II, VII, IX, X. Factor II is prothrombin,
and the other factors resembles the structure of
prothrombin. This group is a Vitamin-K
dependent group. Vitamin K helps in the
carboxylation of the glutamic acids of the CFs
(involved in the metabolic activities of the amino
o Generally speaking, clotting factors are proteins. If acids). Without this, the factors in this group will
they are proteins, they act as enzymes-that is why they not be able to interact with other factors.
are called as zymogens.
- Protein S, Protein C, and Protein Z are also
o Since they are proteins, they are synthesized by the
Vitamin K-dependent proteins.
liver, except for Factor III (it is on the tissues) and
❑ Vitamin K is available to the body through dietary
Factor IV (Calcium, which will be coming from diet
sources and intestinal bacterial production. This group
and from the bones).
is inhibited by warfarin. This group is considered to be
o Generally, CFs circulate in the plasma inactively,
stable and remains well preserved in stored plasma
except for Factor III and IV (technically active,
(Turgeon’s 5th ed, pg. 410).

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3. Fibrinogen group
- Factors I, V, VIII, and XIII.
- These are the CFs consumed during clot
formation because they are part of the clot.
o In coagulation tests, such as for clotting factors, the
most ideal specimen is the plasma (has complete CFs).
o Factor V and VIII are labile factors (easily
deteriorates=tested immediately).
o Factor VIII, to become stable within the circulation,
needs a carrier protein. This is the von Willebrand
factor (VWF). Without vWF, Factor VIII deteriorates.
However, the vWF is unaffected to the absence of
Factor VIII.
o Factor V and Factor VIII makes the activity even
❑ Factors V and VIII are known to decrease during blood
faster.
storage in vitro. These factors are known to increase
o Tissue factor, Factor V, Factor VIII, and HMWK
during pregnancy, in the presence of conditions of
promotes clotting.
inflammation, and subsequent to the use of oral
o Thrombomodulin, Protein S, and Protein Z promotes
contraceptive drugs (Turgeon’s 5th ed, pg. 410).
inhibition of clotting.
CLOTTING FACTORS INVOLVED EACH PHYSICAL CONTA PROTHROM FIBRINOG
PATHWAY PROPERTI CT BIN GROUP EN GROUP
Extrinsic Intrinsic Common ES GROUP
Factor VII Factor IX Factor X Clotting XII, XI, II, VII, IX, X I, V, VIII,
Factor III Factor XI Factor V Factors HMWK XIII
Factor IX Factor II , PK
Factor VIII Factor I Consumed 80% of Factor
HMWK Factor XIII During II is consumed
PK during a clot.
Coagulatio
o Before, in CFs nomenclature, there is Factor VI.
n
However, further studies concluded that Factor VI is
Present in 20% of Factor
the same with Factor V.
Serum II is present.
o Factor IV (Calcium) is present in all pathways. With
Present in All factors are present except for Factor
this, anticoagulants primarily inhibit Calcium as this Stored V and Factor VIII. However, in fresh
is important in clot formation. (aged) plasma, all of them are present.
Plasma
Absorbed
by BaSO4
Once
plasma is
treated
barium
sulfate, it
will absorb
o Fibrinogen is the most significant substrate of all as the
once it is cleaved, it will be the same as fibrin or clot. prothrombi
o Other CFs can also act as a substrate. n group).
o The active CFs will bind to inactive CFs which acts as Present in
a substrate, leading to its activation. adsorbed
o Letter “a” beside a CF number means that it is plasma
activated. (treated
o Activated Factor II: Thrombin (instead of IIa). with
o Activated Factor I: Fibrin or Clot (instead of Ia). barium
sulfate)

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Vit. K o Factor III cleaves a portion of Factor VII, leading to

Dependent Factor VIIa (active).
present o Factor VIIa binds to Factor III with the help of Ca 2+.
absent As they bind, Extrinsic Tenase Complex will be
formed. This is the endpoint (once complex is formed,
PLASMA-BASED COAGULATION CASCADE the common pathway will be activated).
❑ The extrinsic pathway (Fig. 23.9) is initiated by the
entry of tissue thromboplastin into the circulating
blood. Tissue thromboplastin is derived from
phospholipoproteins and organelle membranes from
disrupted tissue cells. These membrane lipoproteins,
termed tissue factors, are normally extrinsic to the
circulation. Platelet phospholipids are not necessary
for activation of the extrinsic pathway because tissue
factor supplies its own phospholipids. Factor VII binds
to these phospholipids in the tissue cell membranes
and is activated to factor VIIa, a potent enzyme
capable of activating factor X to Xa in the presence of
ionized calcium. The activity of the tissue factor–
factor VII complex seems to be largely dependent on
the concentration of tissue thromboplastin. The
proteolytic cleavage of factor VIIa by factor Xa results
in inactivation of factor VIIa. Factor VII participates
only in the extrinsic pathway. Membranes that enter
the circulation also provide a surface for the
attachment and activation of factors II and V. The final
step is the conversion of fibrinogen to fi brin by
thrombin (Turgeon’s 5th ed, pg. 413).

o Green: Extrinsic pathway

o Red: Intrinsic pathway
o Blue: Common pathway

EXTRINSIC PATHWAY
❖ With the help of tissue converts the inactive Factor VII
into an activated Factor VIIa.
▪ Extrinsic Tenase Complex: III+VIIa+Ca++

INTRINSIC PATHWAY
o Under extrinsic pathway, the tissues surrounding the ❖ Negative charge→ Factor XIIa→ Factor XIa→ Factor
vessels during an injury releases Factor III (readily IXa+Calcium,PL, and Factor VIIIa:C
activated).

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▪ Intrinsic Tenase Complex: Ca++ + VIIIa + IXa + the presence of factor V, factor Xa activates
PF3 prothrombin (factor II) to thrombin, which in turn
converts fibrinogen to fi brin. Strong negatively
charged solids that can participate in the activation of
factor XII include glass and kaolin in vitro as well as
elastin, collagen, platelet surfaces, kallikrein, plasmin,
and high–molecular-weight kininogen in vivo.
Collagen exposed by blood vessel injury greatly
influences the rate of reaction. Factor XIIa interacts in
a feedback loop to convert prekallikrein to additional
kallikrein. This reaction is facilitated by the action of
HMWK. In the absence of prekallikrein, factor XIIa is
o Under intrinsic pathway, it will then be because of generated more slowly. Ionized calcium plays an
collagen. important role in the activation of certain coagulation
o It will begin upon the activation of Factor XII (Factor factors in the intrinsic pathway. Calcium is not
XIIa). It is activated by the negative charge of the required for the activation of factor XII, prekallikrein,
collagen. or factor XI but is necessary for the activation of factor
o However, collagen results to partial activation only of IX by factor Xia (Turgeon’s 5th ed, pg. 413-414).
Factor XII. Thus, to activate more Factor XII, PK and
HMWK is needed.
o Once XIIa binds to HMWK, it converts PK to
Kallikrein (K).
o Once K is formed, it will bind to HMWK for it to
activate more Factor XII.
o The binding of K and HMWK will further release
Brady kinins (BK). Kinins help in the inflammatory
response of the body. Inflammatory responses help in
clearing out the fragments of the clots formed.
o Once there are lots of Factors XIIa, K, and XIa, it will
activate plasminogen to become plasmin (lysing
clots; formed already even if there is no clot yet in this
stage).

o Factor XIIa will activate Factor XI, and Factor XIa COMMON PATHWAY
will activate IX. This Factor IXa will bind to Factor ❖ Starts from the conversion of Factor X to Xa which is
VIIIa, Ca2+, and PF3 (Platelet Factor 3; Platelet initiated by both the Extrinsic and Intrinsic Pathways.
phospholipid). ❖ Xa→IIa (Ca++, PL, Factor Va)
o Once Intrinsic Tenase Complex (Ca++ + VIIIa + IXa ❖ PROTHROMBINASE COMPLEX
+ PF3) is formed, it will activate the common pathway. ❖ II→Ia = soluble fibrin clot
❑ The intrinsic pathway (Fig. 23.10) involves the contact ❖ Factor XIIIa – stabilizes the fibrin clot
activation factors prekallikrein, HMWK, factor XII,
and factor XI. These factors interact on a surface to
activate factor IX to IXa. Factor IXa reacts with factor
VIII, PF 3, and calcium to activate factor X to Xa. In

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o The job of thrombin is to cleave the helix (protein) and

will form fibrin monomers.

o Common pathway will begin upon the activation of

Factor X=Factor Xa (activated by either the Extrinsic
or Intrinsic Tenase Complex).
o Question 1: Is it possible to activate common pathway
with just one complex (intrinsic or extrinsic only)? Yes
o Question 2: What will be the major effect is there is
only a single complex activating the common
pathway? The formation of clot will be slower o These fibrin monomers are still connected with each
prolonged. other because the E domain is positively-charged and
o Factor Xa binds to Factor Va. It will then bind to Ca2+ D domain is negatively -charged.
and PF3. This is then called as the Prothrombinase o As more thrombin cleaves fibrinogen, there will be
complex (Xa + Va + Ca++ + PF3). This complex will continuous formation of fibrin monomers. Until, fibrin
activate Prothrombin into Thrombin. Of all the CFs, monomers become a polymer.
thrombin (with the help of Ca2+) is the only one that o However, these polymers are interconnected with
can cleave Fibrinogen into Fibrin or Clot. hydrogen bonds (positive and negative charge), which
❑ Once factor X is activated to Xa, the extrinsic and are weak bonds.
intrinsic pathways enter a common pathway. Factor II, o At this point, there is already a fibrin or clot, but it is
prothrombin, is activated to thrombin (factor IIa), still unstable.
which normally circulates in the blood as an inactive o Factor XIIIa (activated by thrombin and Ca++) will
factor. Following the activation of factor Xa, it result to transfer of groups, polymerizing fibrin
remains platelet bound and activates factor V. The polymers. It will then strengthen the bonds of the fibrin
complex of factors Xa and Va on the platelet surface polymers. With this, Factor XIII is also known as
is formed near platelet-bound factor II molecules. In “Fibrin Stabilizing Factor”.
turn, the platelet-bound Xa/Va complex cleaves factor
II into thrombin, factor IIa. The stage is accelerated by
factor V and ionized calcium (Turgeon’s 5th ed, pg.
413-414).
FIBRINOGEN

o Most critical step in coagulation: Formation of

thrombin (only one capable of cleaving fibrinogen to
fibrin).
o Any factors deficient from any pathway will lead to
thrombin depletion. Less thrombin=longer time to
form a clot (more bleeding).

CELL-BASED PHYSIOLOGIC COAGULATION

o On the fibrinogen, there are two domains which are o Platelets are highlighted here.
called as D domain and E domain. These domains are o It is called as cell-based as it is dependent on tissue-
interconnected by a helix. bearing cells (extravascular) and platelets

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(intravascular). Because of these extra- and
intravascular activities, the clotting is localized.
2 Phases:
❖ Initiation
▪ tissue factor-expressing cells and produces 3% to
5% of the total thrombin generated.
o It is the formation of Extrinsic Tenase Complex.
o Only little amount of thrombin is formed. But this
small amount other CFs and platelets (COAT
platelets).
❖ Propagation
▪ occurring on platelets, which produces 95% or
more of the total thrombin.
o Involves the activity of platelets that will give the
majority of thrombin needed.
o It will begin whenever the damaged vessel has
released the Tissue Factor.
o Factor VII will be activated (VIIa). With the help of
Calcium, the Extrinsic Tenase Complex will be
formed.
o The said complex will activate Factor X binding with
Factor V, forming the prothrombinase complex later
on.
o With this, thrombin will be formed. This formed
thrombin will activate the Factor VIII. It will also
propagate Factor V, XI, and platelets.
o To further facilitate the thrombin formation, The
Extrinsic Tenase Complex can activate Factor IX
(unlike in plasma-based where Factor XIa activated
Factor IX).
o Factor IXa will bind to Factor VIIIa (activated by
thrombin).
o Once IXa, VIIIa, and Ca++ will bind to platelet
because it needs the phospholipids (PF3) under
intrinsic pathway, this will activate Factor X.
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o The prothrombinase complex will also bind to platelet
because it needs phospholipids. With this complex,
more thrombin will be generated.
o Summarily, the small amount of thrombin will be the
initiation phase. Once platelets are active, it will then
proceed to propagation phase because activated
platelets will further expose its phospholipids,
allowing surface receptors for clotting factor. Without
these phospholipids, there will be no thrombin
generation. More thrombin is needed in clotting to
easily cleave fibrinogen into fibrin.
o To differentiate plasma-based and cell-based, what is
added on cell-based is the platelet activation.
THROMBIN
1. Procoagulant – fibrinogen→fibrin
❖ Activation of Factors V, VIII, XI, XIII
❖ Induces plt. Activation and aggregation
2. Coagulation Inhibitor – binds with anti-thrombin III
❖ Forms complex with thrombomodulin
❖ High levels = inhibits Factor V and VIII
❖ **Lysis: activates TAFI
3. Tissue Repair:
❖ Release of PDGF
❖ Chemotaxis and phagocytic cells
COAGULATION INHIBITORS
A. TFPI
❖ Principal regulator of the TF pathway (to
eliminate the extrinsic pathway)
❖ Inactivates Xa and inhibits TF: VIIa complex
B. Protein C
❖ Inactivates Va and VIIIa (as well as Protein S)
❖ ECPR (EC protein C receptor) to facilitate the
binding of thrombin and thrombomodulin
❖ Free Protein S
C. Anti-thrombin
❖ binds and neutralizes serine proteases, including
thrombin and factors IXa, Xa, XIa, XIIa,
prekallikrein, and plasmin
o Heparin: makes the anti-thrombin to be stronger
and more potent (amplify its activity).
D. ZPI
❖ Inhibitor of Xa and XIa
❖ Cofactor is protein Z (Vit-K dependent protein
and increases the ability of ZPI to inhibit the
aforementioned CFs)
E. Protein C inhibitor
❖ Inhibits APC, thrombin, factor Xa, factor XIa, and
urokinase
F. F. Alpha 2 macroglobulin
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❖ Inhibits thrombin and kallikrein

G. Alpha 1 antitrypsin
❖ Inhibits XIa, Xa and thrombin
H. C1 esterase inhibitor
❖ Inhibits XIIa, FXIa, kallikrein
I. Alpha 2 antiplasmin
❖ Inhibits FXI, FII, FX
J. Primary inhibitor of plasmin

Book reading suggestions:

Rodak’s Hematology, 6th edition
- Chapter 35: Normal Hemostasis, pg. 626
Turgeon’s Hematology, 5th edition
- Chapter 23: Principles of Hemostasis and Thrombosis,
pg. 410

REFERENCES:

Antonio Pascua Jr., RMT, MSMT (2023). Hematology

Lecture. Our Lady of Fatima University. Valenzuela City.

Rodak’s Hematology: Clinical Principles and

Applications, 5th Edition

Clinical Hematology: Theory and Procedures, 5 th Edition,

Turgeon, M.L.

Steininger, Cheryl et al. Clinical Hematology: Principles,

Procedures and Correlations.

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