Madam Mwamzi notes on Hemostasis

1. FACILITATOR: MWAMZI IDD
KITURANYA
DATE: 10TH OF APRIL 2023

2. When a body tissue is injured and begins to
bleed, it initiates a sequence of clotting
factor activities leading to the formation of
a blood clot.
Dfn;
Is a complex process in which multiple
components of the blood clotting system
are activated in response to vessel injury to
control bleeding.

3.  Vessel wall
 Platelets
 Plasma coagulation factors and inhibitors
 Fibrinolytic system

4.  VESSEL WALL
Intact vascular endothelium is thromboresistant
and potent regulator of the dynamic hemostatic
system
It inhibits activation of the coagulation cascade to
tightly regulate and maintain blood in the fluid
state through expression and release of various
molecules;

5.  Heparin like sulfates/GAG on its surface,
Thrombomodulin and protein C activation,
Tissue factor pathway inactivator (TFPI),
Secretion of anticoagulants,( nitric oxide,
prostacyclin & ADPase) and T-Pa
 In contrast, subendotheliallayer is highly
thrombogenic and contains collagen, Von
Willebrand factor (vWF) and other proteins
involved in platelet adhesion. Any vascular insult
results in arteriolar vasospasm.

7. • Anucleate, Derived from bone marrow
megakaryocytes, Discoid shaped; change
shape with activation
• Critical role in normal hemostasis is by forming
primary hemostatic plug and providing surface
for coagulation cascade.
• Function dependent on glycoprotein receptors,
cytoplasmic granules and a contractile
cytoskeleton

8.  Dense Granules
– ATP, ADP, Calcium, Serotonin, Mg and epinephrine
 Alpha Granules
– Express the adhesion molecule P-selectin
– Contain and secrete:
• Fibrinogen and vWF
• Factor V and VIII
• Platelet Factor 4 (heparin binding chemokine)
• Platelet derived growth factor (PDGF) and TGF-beta
• Beta thrombospondin and fibronectin (adhesive proteins that
stabilize Plt aggregates)
 Lysozymes and acid hydrolases

9. The coagulation proteins are the core components of the
coagulation system that lead to a complex interplay of
reactions resulting in the conversion of soluble fibrinogen
to insoluble fibrin strands( formation of secondary
hemostatic plug)
 Majority of clotting factors are precursors of proteolytic
enzymes known as zymogens that circulate in an
inactive form.
 Nomenclature of coagulation proteins is rather
complex. The first 4 of the 12 originally identified
factors are referred to by their common names, i.e.,
fibrinogen, prothrombin, tissue factor (TF), and
calcium and are not assigned any Roman
numerals. FVI no longer exists.

11. The more recently discovered clotting factors (e.g.
prekallikrein and high molecular weight kininogen)
have not been assigned Roman numerals. Some
factors have more than one name. Factors V and VIII
are also referred to as the labile factors because their
coagulant activity is not durable in stored blood.
 Von Willebrand factor is a glycoprotein present in
blood plasma and produced constitutively as
ultra-large vWf in endothelium, megakaryocytes,
and subendothelial connective tissue. It mediates
platelet adhesion to subendothelial surface. It also
acts as a carrier protein for coagulant activity of
Factor VIII

12. CFNo: CF NAME FUNCTION
I Fibrinogen Clot formation
II Prothrombin Activation of I, V, VII, VIII, XI, XIII,
protein C, platelets
III Tissue factor Co factor of VIIa
IV Calcium Facilitates coagulation factor binding to
phospholipids
V Labile factor, Proacclerin
VI Unassigned
VII Stable factor, proconvertin
VIII Antihaemophilic factor A Co-factor of IX-tenase complex
IX Antihaemophilic factor B or
Christmas factor
Activates X: Forms tenase complex with
factor VIII
X Stuart-Prower factor Prothrombinase complex with factor V:
Activates factor II
XI Plasma thromboplastin antecedent Activates factor IX
XII Hageman factor Activates factor XI, VII and prekallikrein
XIII Fibrin-stabilising factor Crosslinks fibrin

13. Mechanism of hemostasis has four steps/stages
1. Primary hemostasis
2. Secondary hemostasis( coagulation cascade)
3. Fibrin clot formation and stabilization
4. Inhibition of coagulation( fibrinolysis)
1. PRIMARY HEMOSTASIS
Vasoconstriction occurs at the site of injury to reduce blood
flow.
 The key component of primary hemostasis is the
platelets which aggregates to form platelets plug.
Primary hemostasis is triggered by injury to the vessel
wall, exposing subendothelial collagen. Primary
hemostatic plug is formed in the following steps

14. Platelet adhesion
After vascular injury vWf acts as a bridge between
endothelial collagen and platelet surface receptors GpIb
and promotes platelet adhesion. The platelet glycoprotein
complex I (GP-Ib) is the principal receptor for vWF.
Platelet secretion
After adhesion, degranulation from both types of granules
takes place with the release of various factors. Release of
calcium occurs here. Calcium binds to the phospholipids
that appear secondary to the platelet activation and
provides a surface for assembly of various coagulation
factors(critical for coagulation cascade)

15. Platelet aggregation
Thromboxane A2 produced by activated platelets
provide stimulus for further platelet aggregation.
TxA2 along with ADP enlarge this platelet
aggregate leading to the formation of the platelet
plug, which seals off vascular injury temporarily.
ADP binding also causes a conformational change
in GpIIb/IIIa receptors presents on the platelet
surface causing deposition of fibrinogen on
activated platelets.

17. Activation of the clotting cascade to cement the
platelet plug. Platelets have procoagulant activity
and act as assembly points for complexes of the
coagulation cascade
Coagulation cascades
sequential activation of a series of proenzymes/
zymogens(inactive clotting factor) to active
enzymes by proteolysis(active clotting factor)

18. It has divided into three caogulation pathways;
i. Extrinsic pathway
ii. Intrinsic pathway
iii. Common pathway.

20. It is considered as the first step in plasma
mediated haemostasis. It is activated by TF, which
is expressed in the subendothelial tissue. Which
binds with factor VIIa and calcium to promote the
conversion of factor X to Xa.
Under normal physiological conditions, normal
vascular endothelium minimises contact between
TF and plasma procoagulants, but vascular insult
expose TF.

21. It is a parallel pathway for thrombin activation by
factor XII. It begins with factor XII, HMW
kininogen, prekallekerin and factor XI, which
results in activation of factor XI. Activated factor
XI further activates factor IX, which then acts with
its cofactor (factor VIII) to form tenase complex on
a phospholipid surface to activate factor X.

22. Activated factor X along with its cofactor (factor
V), tissue phospholipids, platelet phospholipids
and calcium forms the prothrombinase complex
which converts prothrombin to thrombin. This
thrombin further cleaves circulating fibrinogen to
insoluble fibrin and activates factor XIII, which
covalently crosslinks fibrin polymers
incorporated in the platelet plug. This creates a
fibrin network which stabilises the clot and forms
a definitive secondary haemostatic plug

24. Each reaction in the pathway depends on assembly of
a macromolecular complex consisting of:
 a) enzyme b)cofactor c)substrate e.g
 Enzyme: Factor VIIa
 Cofactor: Tissue Factor
 Substrate(s): Factor IX, X
All macromolecular complexes require calcium and are
assembled on a negatively charged phospholipid
membrane

25. Thrombin converts soluble fibrinogen to insoluble
fibrin monomers which polymerize to form a
soluble clot. Thrombin then activates Factor XIII
to XIIIa which cross-links the fibrin monomers
and stabilizes the clot, hence a stable hemostatic
plug.

26. If the hemostatic response is unchecked, can lead
to thrombosis, inflammation and tissue damage
Clotting is checked by:
 Procoagulant dilution in flowing blood
 Removal of activated factors through the RES
esp in liver
 Control by natural antithrombotic pathways

27. At the same time that a clot is being formed, the clotting process
also starts to shut itself off to limit the extent of the thrombus
formed.
 Thrombin binds to the membrane receptor
thrombomodulin and activates Protein C to Activated
Protein C (APC). APC combines with its cofactor Protein S
which then inhibits Factors Va and VIIIa, slowing down the
coagulation process.
 Thrombin bound to thrombomodulin becomes inactive and
can no longer activate procoagulant factors or platelets.
 The endogenous anticoagulant, antithrombin inhibits the
activity of thrombin as well as several of the other activated
factors, primarily Factor Xa.

28. It is the main inhibitor of thrombin. It is a serine
protease inhibitor, which binds and inactivates
thrombin, factor IXa, Xa, XIa and XIIa.
Naturally occurring anticoagulants in the body
 Antithrombin; heparin sulfate
 Thrombomodullin: Protein C and S
 ADPase (CD39)
 Tissue Factor Pathway inhibitor(inhibiting
TF-VIIa complex)
 Nitric oxide

30. Fibrinolytic system is a parallel system which is activated
along with activation of coagulation cascade and serves to
limit the size of clot. Fibrinolysis is an enzymatic process
that dissolves the fibrin clot into fibrin degradation
products (FDPs).
 Tissue plasminogen activator (t-PA) converts
plasminogen to plasmin which breaks down cross-
linked fibrin to several fibrin degradation products, the
smallest of which is D-dimer.
 Thrombin activatable fibrinolysis inhibitor (TAFI)
prevents the formation of plasmin.
 Anti-plasmin and plasminogen activator inhibitor-1
(PAI-1) inhibit plasmin and t-PA respectively.

32.  Primary Hemostasis involves the processes
that lead to formation of the platelet plug
 Secondary Hemostasis refers to activation of
the coagulation cascade leading to stabilization
of the aforementioned plug/clot
 Fibrinolysis helps control/check the process of
clot formation

33. Depiction of intrinsic and extrinsic pathway is
really an in vitro test view
 Current evidence supports the understanding
that intrinsic pathway is not a parallel pathway
but indeed it augments thrombin generation
primarily initiated by the extrinsic pathway.
 Physiologically and in vivo, it is believed that
the primary initiating clotting event is the
generation/exposure of tissue factor at wound
site and interaction with Factor VIIa

34. Newer model describes coagulation with following
steps;
Initiation
It occurs by expression of TF in damaged vessel
which binds factor VIIa to activate factor IX and
factor X. This activation of factor IX by TF-VIIa
complex serves as the bridge between classical
extrinsic and intrinsic pathways. Factor Xa then binds
to factor II to form thrombin (factor IIa). Thrombin
generation through this reaction is not robust and can
be effectively terminated by TF pathway inhibitor .

35. Amplification
Since the amount of thrombin generated is not
sufficient, therefore numerous positive feedback
loops are present that bind thrombin with
platelets. Thrombin that is generated in the
initiation phase further activates factor V and
factor VIII, which serves as a cofactor in
prothrombinase complex and accelerates the
activation of Factor II by F Xa and of F Xa by F
IXa, respectively.

36. Propagation
The accumulated enzyme complexes (tenase
complex and prothrombinase complex) on
platelet surface support robust amounts of
thrombin generation and platelet activation. This
ensures continuous generation of thrombin and
subsequently fibrin to form a sufficiently large
clot.

37. Stabilization
Thrombin generation leads to activation of factor
XIII (fibrin stabilizing factor) which covalently
links fibrin polymers and provides strength and
stability to fibrin incorporated in platelet plug. In
addition, thrombin activates thrombin activatable
fibrinolysis inhibitor (TAFI) that protects the clot
from fibrinolysis.

40. Initiation of coagulation
Tissue factor (TF) is released from injured tissue cells,
endothelial cells and monocytes. TF and Factor VIIa
form the TF / Factor VIIa complex. TF / Factor VIIa
activates a small amount of Factor IX and X to
generate a small amount of thrombin. Factor XII (and
other “contact” factors) play a minor role in the
activation of Factor XI.
Amplification phase
Thrombin activates Factor V to Va, Factor VIII to
VIIIa and activates more platelets. Thrombin also
activates FXI to FXIa.

41. Propagation phase
Additional Factor Xa is produced when TF /
Factor VIIa complex activates Factor IX. The
resultant Factor IXa along with Factor VIIIa forms
the tenase complex which then converts more
Factor X to Xa. Factor Xa and Va along with
calcium and a phospholipid (PL) surface
(activated platelets) form the prothrombinase
complex which converts prothrombin (Factor II)
to large amounts of thrombin (Factor IIa).

42. The classical theory of blood coagulation is
particularly useful for understanding the In vitro
coagulation tests, but fails to incorporate the central
role of cell-based surfaces in In vivo coagulation
process. Interestingly contact activation critical for In
vivo haemostasis does not get support from following
observations
Deficiencies of FVIII and FIX (both intrinsic pathway
factors) lead to haemophilia A and B, respectively,
however the classic description of two pathways of
coagulation leave it unclear as to why either type of
haemophiliac cannot not simply clot blood via the
unaffected pathway.

43. Factors II, VII, IX, and X (also protein C and S) are
dependent to vitamin K
For the coagulation proteases to work, they need
 Post translational modification to produce
gamma carboxy glutamic acid (Gla) residue,
which is a vitamin K dependent process
 𝛄 carboxylation of Gla allows for Ca binding
and complex formation
 Phospholipid surface for the proteases to bind
along with their cofactors

45.  Is the most common of the hereditary clotting factor
deficiencies.
 Caused by deficiency of factor VIII
 The inheritance is sex‐linked recessive
 Commom in males than females
 Soft tissue bleeding and excessive bruising when they
start to be active.
 Recurrent painful haemarthroses and muscle
haematoma.
 Prolonged bleeding occurs post trauma, after dental
extractions, surgical procedures.

46.  The following tests are abnormal:
 Activated partial thromboplastin time (APTT).
 Factor VIII clotting assay.
 Mixing study-Normalize in deficiency and low in
inhibitors.
 The platelet function analysis‐100 (PFA‐100)
and prothrombin time (PT) are normal.

47.  The inheritance and clinical features of factor IX
deficiency (Christmas disease, haemophilia B) are
identical to those of haemophilia A.
 Indeed, the two disorders can only be
distinguished by specific coagulation factor assays.
 Abnormal
 APTT
 Factor IX clotting assay.
 Mixing study
 As in haemophilia A, the PFA‐100 and PT tests are
normal

48.  In this disorder there is either a reduced level or
abnormal function of von Willebrand factor (VWF)
resulting from a wide variety of mainly missense
mutations in different parts of the gene.
 VWF is produced in endothelial cells and
megakaryocytes.
 It has two roles
 It promotes platelet adhesion to sub endothelium
and each other at high shear rates
 It is the carrier molecule for factor VIII, protecting it
from premature destruction.

49. Usually, the inheritance is autosomal dominant.
 The severity of the bleeding is highly variable,
depending on mutation type, Women are more badly
affected than men at a given VWF level.
Laboratory findings
 The PFA‐100 test is abnormal. Factor VIII levels are
often low.
 The APTT may be prolonged.
 VWF levels are usually low.
 There is defective platelet aggregation by patient
 The platelet count is normal except for type 2B disease
(where it is low)