Dayle Daniel G.

Sorveto
BMLS- 4A

HEMOSTASIS REVIEWER:
A process that keeps blood in the
circulation and then when theres an
injury occurs, produces a clot to stop the
bleeding.
Are primarily the vascular intima,
extravascular tissue factor (TF) bearing
cells and platelets.

PRIMARY HEMOSTASIS:

Involves the interaction of:
1. Vasoconstriction
2. Platelet aggregation
3. Coagulation enzymes (stop bleeding)

Refers to the whole blood vessels and
platelets in the initial formation of a
platelet plug in responses to a vascular
injury.

*Primary hemostasis activated during:
1. blood vessels injury
2.desquamation of dying/damage endothelial
cells.


Mechanisms:
1. The blood vessel contracts to seal the
wound or reduce the blood flow
(vasoconstriction)
2. Procoagulant released by the damage
endothelial cells
3. Platelets, activated and adhere to
exposed collagen, secret the contents
of their granules.
4. Aggregate w/other platelets to form a
platelet plug.
5. Rapid, short-lived response.

Secondary hemostasis:
Is the activation of a series of plasma
proteins in the coagulation system to
form a fibrin clot.
Plasma transport atleast 16 glycoproteins
mostly tripsinlike serine proteases
Circulate only during process of
coagulation.

1. is triggered by tissue damage that
exposes TF.
Fibroblast
Smooth muscle cells
2. Exposed TF form a factor VII
It activates IX and X
3. Factor IX combine to cofactor, factor
VIII and activate factor X.
4. factor X bind to its cofactor factor V
it converts to prothrombin to
thrombin
*thrombin is an enzyme key for coagulation.
5. Thrombin splits into 2:
i. Fibrinogen- to produce fibrin
ii. Factor XIII- to cross-link and
stabilize the clot.
*activates platelet and feedback to activate
factors V, VIII, XI.
*Fibrin= necessary for control bleeding
*absence of single plasma procoagulant may
doom the individual to lifelong anatomic
hemorrhage.
Fibrinolysis:
Final event of hemostasis
Plasminogen is bound to fibrin
during coagulation
o Activated by tissue plasminogen
activator (TPA) into the serine
protease.
o Plasmin slowly and
systematically degrades the
fibrin degredation products
o Designed X, Y, D, E and D-
Dimer.

VASCULAR INTIMA IN HEMOSTASIS:
1. Endothelial cells
Innermost lining of blood vessels
is a monolayer metabolic active
Forms a smooth, unbroken
surface that promotes the fluid
passage of blood
It prevents turbulence
2. Smooth muscle cells
in the arteries and arterioles
3. fibroblast
occupy the connective tissue
layer and produce collagen.
4. Elastin-rich internal elastic lamina
Supports endothelial cells.
ANTICOAGULANT PROPERTIES OF
INTACT VASCULAR INTIMA
Collagen promotes platelet
activation and adhesion
TF= promotes coagulation and
fibrin formation.
1. Prostacyclin
Platelet inhibitor, is synthesized
through the eicosanoid pathway.
Prevents unnecessary or
undesirable platelet activation in
intact vessels.
2. Nitric oxide
Synthesized in endothelial cells,
vascular SMC, neutrophils and
macrophages
Counteracts vasoconstriction and
maintains healthy arterioles
3. Tissue factor pathway inhibitor
(TFPI)
Controls the TF or extrinsic
coagulation pathway.
4. Thrombomodulin
Membrane protein that activates
protein C pathway.
5. Heparin sulfate
Glycosaminoglycan the retards
coagulation by activating
antithrombin.
Coagulation regulatory protein.
PROCOAGULANT PROPERTIES OF
DAMAGED VASCULAR INTIMA
1. Smooth muscle cells in arterioles and
arteries
Induce vasoconstriction
2. Exposed subendothelial collagen
Binds VWF and platelets
3. Damaged or activated endothelial
cells.
Secrete VWF
Secrete adhesion molecules
a) P-selectin
b) Intra cellular adhesion molecules
(ICAMS)
c) Platelet endothelial adhesion
molecules (PECAMS)
d) Exposed smooth muscle cells and
fibroblast
Tissue factor exposed on cell
membranes.
e) Endothelial cells in inflammation
Tissue factor is induced by
inflammation.
Fibrinolytic properties of vascular intima
During thrombus formation, both
TPA and plasminogen bind to
polymerized fibrin.
TPA activates fibrinolysis by
converting plasminogen to plasmin,
which slowly digest fibrin and
restores blood flow.
Endothelial cells may secrete
plasminogen activator Inhibitor-1
(PAI-1)
Thrombin bound to
thrombomodulin activates thrombin
activatable fibrinolysis inhibitor
(TAFI) w/ inc the tendency for
thrombus formation.
Platelet function:
1. Adhesion:
-platelets roll and cling to nonplatelet
surfaces
2. Aggregation:
-platelet adhere to each other.
3. Secretion:
-platelets discharge the contents of
their granules
Characteristics:
- Irreversible, occurs during aggregation,
platelet contents are secreted, essential to
coagulation.
Platelet granules content:
1. Alpha-granules (-granules)
Large molecules
B-thromboglobulin
Factor V
Factor XI
Protein S
Fibrinogen
VWF
Platelet factor 4
Platelet-derived growth factor
2. Dense bodies
Small molecules
ADP (activates neighboring
platelets)
ATP
Ca
2+

Serotonin(vasoconstriction)
*Aspirin permanently inactivates
cyclooxygenase, blocking thromboxane A
2

production and causing impairment of
platelet function (aspirin effect)

Role of rbc, wbc, TF-bearing cells in
hemostasis:
- Vessel injury exposes blood to the sub-
endothelial TF-bearing cells and leads to
activation of coagulation through factor
VIIa
RBC:
- Add bulk and structural integrity to the
fibrin clot.
Monocyte, Lymphocyte
- Provides surface borne TF that Triggers
coagulation.
WBC
- Have a series of membrane integrins and
selectins that bind adhesion molecule
and help stimulate the production of
inflammatory materials that promote
wound healing process.

Coagulation system:
- 16 procoagulants also called
coagulation factors or clotting factors.
- Synthesized in the liver
o Few are made in monocyte,
endothelial cell and
megakaryocyte.
- 8 are enzymes that circulate in inactive
form called zymogens.
- In 1958 the International Committee for
the Standardization of the nomenclature
of the Blood clotting factors.
o Named by using roman numerals
*factor V used for the complexing the
procoagulant activation of factor VI.

*VonWillebrandFactor(VWF)synthesize
d in megakaryocyte and endothelial
cells.

Vitamin K-dependent Prothrombin
group:
- Prothrombin, factor VII, IX, X
- Protein C, S, Z
o Structural resemblance to
prothrombin
- All 7 proteins have 10-12 glutamic acid
- Vit-K is a quinone found in green leafy
vegetables, fish, liver.
o Produced by intestinal
organisms:
1. Bacteroides fragilis
2. Escherichia coli. (E-coli)
Posttranslational modification of the
prothrombin group:
- - carboxylation of A.A terminal
glutamic acid
- glutamic acid -carboxyglutamic acid
- 2
nd
carboxy grp added to gamma- carbon
- Negative charges w/ enables them to
bind ionic calcium (Ca
2+
)
- Calcium permits Vit-K to bind
negatively charged phospholipids
*phospholipid binding is essential to
coagulation rection.
*Warfarin suppresses Epoxide reductase
In vitamin-K deficiency or intake of
warfarin:
1. Cant produce 2
nd
carboxyl group to
bind gamma- carbon.
2. Called des--carboxyl proteins or
proteins in Vit-K antagonism
(PIVKAs)
VWF:Factor VIII complex:
- VWF is a multemric GP.
- Composed of multiple subunits 240000
D
- Stored in alpha-granules in platelets and
storage sites called Weibel-Palade
Bodies in endothelial cells
- Conc 7-1- mcg/mL
VWF:Factor VIII complex circulates
covalently bound to VWF
- VWF provides 3 active receptor sites.
1. Platelet surface receptor (GP
Ib/IX/V)
2. Platelet integrin (GP IIb/IIIa) during
platelet aggregation
3. Collagen binding site
4. Factor VIII
Factor VIII is produced by Hepatocytes and
other tissues.
Sex linked procoagulants
1. Factor VIII- 260000 D
2. Factor IX- 57,000 D
Free factor VIII is unstable: except
1. During coagulation
2. Cannot be detected in plasma
*Factor VIII depend on VWF stability if
VWF have diminished VWF also
Diminished factor VIII activity levels.

Fibrinogen Structure and Fibrin
formation:
- Fibrinogen is the primary structure of
thrombin
- Synthesized in liver
- Plasma conc of fibrinogen 200-400
mg/dL
o Highest concentration of all
plasma procoagulants
- Fibrinogen is mirror-image dimer.
o Consisting of 3 non-identical
polypeptides
- Designated B and united by disulfide
bonds
- 6 terminal assemble to form bulky
central region called E domain
- Carboxy termini assemble at the 2 ends
of molecule to form 2 D domains.
- Thrombin cleaves fibrinopeptides A and
B
- Cleaved fibrinogen called fibrin
monomer
- Factor XIII catalyzes the formation of
cavalent bonds between carboxy termini
of -chains adjacent to D domains.
- - amino acid of lysine moieties and the
-amide group of glutamine units.
- Fibronectin, a plasma protein involve in
cell adhesion.
-
2
-antiplasmin, rendering the fibrin
mesh resistant to fibrinolysis.
- Plasminogen, the primary serine
protease of fibrinolytic system.

Coagulation pathway:
Tissue Factor Pathway:
- TF is a membrane receptor for factor
VIIa.
- Coagulation is triggered by the exposure
of TF to plasma proteins on injury.
- Factor VIIa binds to TF in the presence
of phospholipid and calcium and triggers
the activation of both factor IX and
factor X.
- Factor IXa binds factor VIIIa on platelet
phospholipid surfaces
o Thus complex activates factor X
- Factor Xa, activated by both TF:VIIa
and IX:VIIIa, binds factor Va on
phospholipid surfaces
- Xa:Va complex activates prothrombin
o Multistep hydrolytic process that
release peptide fragment from
prothrombin called prothrombin
1 and 2
- Activated prothrombin is termed
thrombin, thrombin cleaves
fibrinopeptides A and B from plasma
fibrinogen.
- Tissue factor pathway is characterized
by three multimolecular complex
formation
First complex:
Composed TF, factor VIIa,
phospholipid and Ca
2+
on the TF-
bearing cell complex IX and X.
Second Complex:
Factor IXa, Factor VIIIa,
phospholipid and Ca
2+
called
Tenase
Also activates factor X.
Third complex:
Composed of factor Xa, Factor Va,
phospholipid and Ca
2+

Often called prothrombinase
This complex convert prothrombin to
thrombin.
Contact factors
Composed of Factor XIIa,HMWK,
Pre-K, activates Factor XI.
Factor XIIa transform pre-K, a GP
the circulates bound to HMWK, into
its active form.
Kallikrein
- Cleaves HMWK to form bradykinin.
- Deficiencies of factor XII,HMWK, or
pre-K does not cause clinical bleeding
disorders.
- Factor XII is activated in vitro by
contact w/ negative charged surfaces
such as non-siliconed glass or ellagic
acid in PTT reagent.
Factor XI activation pathway
- Factor XI activated by thrombin
- Also a contact factors
- Factor Xia activates factor IX
- Associated with Rosenthal syndrome
o Deficiencies of factor XI
o Mild bleeding disorders
Thrombin
- Primary function of thrombin is to
cleave fibrinopeptides a and b from the
alpha and beta chains of fibrinogen
molecule triggering fibrin
polymerization
- Thrombin amplifies the coagulation
mechanism by activating cofactors V
and VIII and factor XIII
- Thrombin also factor XIII, the fibrin-
stabilizing factor.
- Factor XIIIa covalent bonds between the
D-domains of the fibrin monomer to
cross-link and stabilize the fibrin clot.

*thrombin initiates aggregation of
platelets.
- thrombin bound to thrombomodulin
activates the protein C pathway to suppress
coagulation
- activates TAFI to suppress fibrinolysis.

Plays role in coagulation= Fibrin
Platelet activation, in coagulation
control= Protein C
Fibrinolysis= TAFI
- Thrombin is a chief protease of the
coagulation pathway.
Extrinsic, Intrinsic and Common
Pathway
- Most common coagulation experts
identified the activation of factor XII as
the primary step of coagulation
- It is found in blood
- TF is not found in blood
Intrinsic pathway:
- Order of reactions:
1. XII 8. V
2. Pre-K 9. prthrombin
3. HMWK 10. Fibrinogen
4. XI
5. IX
6. VIII
7. X
Laboratory test for screening thes
factors:
*APTT

Extrinsic pathway;
- Order of reaction
1. Factor VII
2. Factor X
3. Factor V
4. Prothrombin
5. fibrinogen

initiation of coagulation by:
TF:VIIa
o the principal mechanism in vivo for
generating thrombin.
Test used for Extrinsic pathway:
- prothrombin time (PT)
*factor IXa:VIIIa complex is activation of
factor X (tenase)
Hemophilia A= deficient factor VIII
Hemophilia B= deficient factor IX
- can cause severe hemorrhage.
Common pathway:
- Both extrinsic and intrinsic have the
some common factors
1. Factor X
2. Factor V
3. Prothrombin
4. Fibrinogen
Cell-based (In Vivo) coagulation
- A spectacular combination of cellular
and biochemical function in harmony to
keep blood liquid within the veins and
arteries
3 phases:
1. Initiation:
- Continuous low-level activation of the
TF pathway occurs on TF-bearin cells
outside the circulation
- 1% or less of factor VII is present
normally in activated form.
1. Factor VIIa bind to TF
2. TF:VIIa complex activates small amount of
IX and X
3. Xa:Va complexes to form prothrombinase
and generates small amount of thrombin
4. Xa:Va bound to the cell is protected for
inactivation by control proteins
- If it dissolves from the cell, it is rapidly
inactivated by the protease inhibitors
TFPI, antithrombin and protein Z-
dependent protease inhibitor (ZPI)
- Xa:Va in the initiation phase does not
produce fibrin clot.
2. Amplification:
- Starts when theres injury and bleeding
occur.
- VIII:VWF spill into extravascular space
coat platelets
- Partially activated by collagen and
thrombin
- Have higher levels of procoagulant
activity and platelet exposed to collagen
alone.
- In thrombin,
o Factor V release from a-arginine
o Factor VIII dissociates it from
VWF
o It activates XI.
3. Propagation
- Reaction occurs on the surface of the
activated platelet
- Factor IXa from initiation phase binds to
VIIIa on the platelet.
- For IX:VIIIa complex (tenase)
o It activates Xa
- Factor Xa is complex to Va it will
activate prothrombin and generate a
burst of thrombin which cleaves
fibrinogen into fibrin clot.
- Thrombin activates also factor XIII to
stabilize clot
o It binds to thrombomodulin to
activate protein C control
pathway and activates TAFI to
inhibit fibrinolysis.
Coagulation Regulatory Mechanisms
- Serine proteases and cofactor in
coagulation system are regulated by
inhibitors, cofactor and feedback loops
to maintain complex and delicate
balance between thrombosis and
abnormal bleeding.
- Principal regulator
1. TFPI
2. Antithrombin
3. Protein C pathway
- They prevent excessive clot formation or
thrombosis.

Tissue factor Pathway inhibitor
(TFPI)
- TFPI is synthesized by endothelial cells
- 80% bound to the vessel wall
- 20% circulates in the plasma
- 2% is the full-length free form that
effective inhibitor of tissue factor
pathway.
TFPI inhibit coagulation in 2 step process:
1. Inactivating Xa
2. Binding TF:VIIa to prevent it from
inactivating additional Xa.
- TFPI provides feedback inhibition
o Its not functional until X is
activated.
Protein S
- Cofactor of activated protein C (APC)
- Also cofactor of TFPI
- Stimulates Xa inhibition by TFPI
- It synthesized in the liver.
- It circulates in the plasma in 2 forms:
1. Free Protein S
2. Covalently bound to the complement
control protein C4b-binding protein
(C4bBP)
- Only free plasma protein S can serve
as APC cofactor.
- Protein S-C4bBP binding is particular
interest in inflammatory conditions
Protein C regulatory system
-thrombin binds to EC membrane protein
thrombomodulin and trigger an essential
coagulation regulatory system called Protein
C system.
- thrombin-thrombomodulin complex
activates zymogen protein C to serine
protease.
- APC binds its cofactor protein S
o Stabilized APC-protein S
complex.
- The APC-protein S complex hydrolyzes
and inactivates factor Va and VIIIa
o It slowing or blocking
coagulation.

Thromboembolic disease:
- Chronic, acquired or inherited:
o Protein C deficiency
o Protein S deficiency
- Or mutations of Protein C and S and
Factor V
o It compromised down
regulation of Va and VIIIa.
Serine protease inhibitors (serpins)
- Antithrombin the first of the
coagulation regulatory proteins
- The first to be assayed routinely in the
clinical hemostasis laboratory
Other members of the serpin family:
Heparin cofactor II
ZPI
Protein C inhibitor
A
1
-protease inhibitor (alpha-antitrypsin)
Alpha2-macroglobulin
Alpha2-antiplasmin
PAI-1
Antithrombin
- It binds and neutralize all serine
proteases except factor VIIa.
- Neutralize and bind also the plasmin.
Heparin cofactor II
- Inactivates primarily thrombin
- Both antithrombin and heparin cofactor
II require heparin for effective
anticoagulant activity.
Unfractionated heparin:
- A heterogenous glycosaminoglycan
composed of 15-30 saccharide unit
- It increase the ability of antithrombin to
neutralize thrombin and factor IX, Xa,
Xia, and XIIa 1000-fold.
Firbrinolysis
- Final stage of coagulation
- Plasminogen, plasmin, TPA and PAI-1
are incorporated into the fibrin clot by
binding to lysine through kringle
loops
- Systematic, accelerating hydrolysis of
fibrin by bound plasmin cleaves peptide
bond at arginine and lysine moieties in
fibrins D and E domains.
- TPA and Urokinase activate fibrin-
bound plasminogen several hours after
thrombus formation.

Plasminogen and plasmin
- Plasminogen is a 92,000 D plasma
zymogen produced in liver
- Plasminogen is a single chain protein
possessing five glycosylated loops
termed kringles.
- Kringles enables plasminogen to bind
fibrin lysine residue during
polymerization.
- Plasminogen binds plasma alpha2-
antiplasmin the major fibrinolysis
control protein.
- Fibrin-bound plasminogen converted
into 2-chain active plasmin.
o When cleaved between
arginine at position 561 and
valine position 562 by
neighboring bound TPA or
Urokinase
Plasmin:
- Serine protease that systematically
digest fibrin polymer by hydrolysis of
arginine and lysine peptide bonds.
- Fibrin becomes digested the lysine
residue, bind additional plasmin.
- Plasmin is capable of digesting fluid-
phase fibrinogen and factor V and VIII
- Plasma alpha2-antiplasmin rapidly
binds and inactivates free plasmin.
Plasminogen activation and Control:
1. Tissue Plasminogen Activator
(TPA)
- Secreted by endothelial cells
- Hydrolyzes fibrin-bound plasminogen
and initiates fibrinolysis.
- Have 2 glycosylated kringle regions
form covalent lysine bonds w/fibrin
during polymerization
- Segregate the surface of the thrombus
w/ plasminogen.
- Free TPA circulates bound to inhibitor
such as PAI-1 and is cleaved from
plasma.
Urokinase
- Urinary tract epithelial cells, monocytes
and macrophages
- Circulates in plasma at a concentration
2-4mg/mL
- Incorporated into the fibrin-bound
plasminogen and TPA at the time of
thrombus formation
- It has only one kringle region
- Does not bind to fibrin
- Purified urokinase preparation are
used to dissolve thrombi in myocardial
infarction, stroke and deep thrombosis.
- It activates plasminogen

Plasminogen Activator Inhibitor 1(PAI-1)
- Inhibits TPA and urokinase from
activating fluid-phase plasminogen.
- Sources: Endothelium, adipocytes,
megakaryocytes and hepatocytes.
- PAI-1 is present in excess of TPA conc.
- Circulating TPA normally bound to
PAI-1
- PAI-1 deficiency associated w/chronic
mild bleeding due to increase
fibrinolysis
- Excess PAI-1 may be associated with
thrombosis due to reduction in
fibrinolysis.
Alpha2-antiplasmin
- Synthesized in liver
- Primary inhibitor of plasma
- Bound plasmin digest clots and restores
blood vessel patency
- Free plasmin in the circulation digests
plasma fibrinogen, factor V, factor VIII
and fibronectin causing potentially fatal
primary fibrinolysis.
- A2-antiplasmin is a plasma protein that
rapidly and irreversibly binds free
plasmin.
- Also become cross-linked through
lysine bonds to fibrin kringles during
polymerization
- It slows fibrin digestion by plasmin.
Thrombin-Activatable Fibrinolysis
Inhibitor (TAFI)
- A plasma procarboxypeptide that
become activated by the thrombin-
thrombomodulin complex.
- It inhibit fibrinolysis.
o By cleaving exposed
carboxy-terminal lysine
residue from partially
degraded fibrin that prevents
binding of TPA and
plasminogen
o Blocking formation of
plasmin.
*decrease thrombin production may
reduce the activation of TAFI, resulting
increase the fibrinolysis.
*inc thrombin, Inc activation of TAFI it
will decrease fibrinolysis that may
contribute further to thrombosis.

- TAFI play a role in regulating
inflammation and wound healing.
Fibrin degradation products and D-
dimer.
- Fibrinolysis produces a series of
identifiable fibrin fragments X, Y, D, E
and D-dimer.
Fibrin X
- Described as central E domain w/2
Domains.
- Some peptide cleaved by plasmin
Fragment Y
- E domain after cleavage of one D
domain (D-E)
- Further digested to individual D and E
domains.
D-D fragment
- Also called D-dimer
- Two D domains from separate fibrin
molecules cross-linked by the action of
factor XIIIa
- Fragment X,Y, D, E are produced by
digestion of either fibrin or fibrinogen
by plasmin, but D-dimer is a product of
digestion of fibrin.
- D-dimer immunoassay is used to
identify chronic and acute DIC and to
rule out venous thromboembolism is
suspected cases of deep venous
thrombosis of pulmonary embolism