Concept of haemostatis

1. Moderators
Respected :- Ishwar Bihana Sir
Respected :- Joseph Sir
Respected :- S.K.Bose Sir
Presented By
Gaurav Kumar
B.sc. MLT Student Part II
Department of Haematology
Post Graduate Institute of Medical Education And Research, Chandigarh

2. • It is a biological or physiological phenomenon
which is responsible to keep the blood in fluid
state in the circulation as well as to arrest
bleeding followed by an injury to blood vessel.
Defination

3. Aims and Objective
To understand how our vascular system keep our blood in its
fluid state.
To know how bleeding is arrested.
To know the role of haemostasis components.
To know the biology and mechanism of coagulation.
To know how clot is removed from vascular system.

4.  To maintain the blood in fluid state while it remains
circulating within vascular system.
 To maintain the integrity of the vessels wall.
 To arrest bleeding at the site of injury or blood loss by the
formation of haemostatic plug.
 Eventual removal of plug when healing is complete.
Importance of Hemostasis

5. ADPase
TM
Thrombin
Protein CaProtein C
Destroys FVa,FVIIIa
Protein S
HS
AT-III
Inhibit
FXa,FIXa,thrombin
tPA
plasminogen plasmin
fibrinolysis
endothelium
Overview of Hemostasis
Blood coagulation cascade
Vessel injury
Neurogenic
Mayogenic
endothelin
vasoconstriction
Reduce blood flow
Collagen exposure+vWF
Tissue factor
Plt.adhen & activationserotonin
TxA2,ADP
plt.aggregation
Primary hemostatic plug
Stable 2ndry hemostatic plug
healing
fibrinolysis antiplasminogen
Cell migration & proliferation
PF
thrombin
fibrin

6. Blood vessels Platelets
Plasma
coagulation
factors
Inhibitors
Fibrinolytic
system
Components of
Hemostasis

7. Blood vessels
Structure of blood vessels

8. Function of Blood Vessel
Blood vessel with muscular coat help to reduce blood
loss by vasoconstriction.
Blood vessels with a pipe system transport nutrients ,
hormones , gases and other essential factors which are
transported by blood.

9. Endothelial Cell Function
Normal vascular endothelium is a thromboresistant
surface.
Non-thrombogenic- don’t react with plasma or cellular
elements of the blood.
It is antithrombotic. It activates antithrombin III.
When injured (either biologically , chemically or
mechanically) it can profoundly promote hemostasis.

10.  Antiplatelets Effect:
 Inhibition of platelet adhesion, activation and aggregation.
 PGI2 (Prostacyclin)-vasodilator and antiplatelet agent.
 NO-Nitric Oxide/PGI2
 Both bind with the receptors on the platelets and inactivate them. So inhibit platelet
aggregation.
 ADPase:
 Enzymes that break down the ADP (strong proaggregating agent) released by the
platelets and thus inhibit platelet aggregation.
ADPase
TM
Thrombin
Protein CaProtein C
Destroys
FVa,FVIIIa
Protein
S
HS
AT-III
Inhibit
Fxa,FIXa,thrombin
tPA
plasminogen plasmin
fibrinolysis
Endothelial Antithrombotic Mechanism

11. ADPase
TM
Thrombin
Protein CaProtein C
Destroys FVa,FVIIIa
ProteinS
HS
AT-III
Inhibit
Fxa,FIXa,thrombin
tPA
plasminogen plasmin
fibrinolysis
Surface expressed integral membrane protein which binds thrombin & results in loss of the
pro-coagulant properties of thrombin.
Thrombin/thrombomodulin complex is a potent anticoagulant complex.
Since it activates protein C to activated-Protein C.
Activated protein C down regulates coagulation by inactivating important proteins
(FVa, FVIIIa)
Thrombomodulin

12. Heparan sulphate
• Long unbranched polysaccharide expressed at the endothelial
membrane surface.
• Act as a cofactor for the plasma inhibitor antithrombin III.
• Inhibits thrombin and other coagulation factors(FIXa,FXa)
ADPase
TM
Thrombin
Protein CaProtein C
Destroys FVa,FVIIIa
Protein S
HS
AT-III
Inhibit
Fxa,FIXa,thrombin
tPA
plasminogen plasmin
fibrinolysis
Binding and Inhibition of Thrombin

13. platelet
GPIb receptor
vWF
Endothelial
cell
subendothelial
collagen
Subendothelial Cell Function
Subendothelium consist of collagen ,elastic
tissues, proteoglycans and non collagenous
glycoproteins {fibronectin, vWF}.
Exposure of this layer after damage of
vessel wall is responsible for platelet
adherence.
vWF bind with collagen, vWF then
undergoes a conformational change and
platelets are captured via their surface
membrane glycoprotein GpIb binding to
vWF.

14. The mechanism isDamage of vascular
endothelium
stimulates endothelial cells
Endothelial cell
Synthesize and secrete three
Substances involved
In haemostatic plug
Formation
vWF
(VIII vWF)
It help in adhesion
Of platelet to
sub endothelium
PGI2
(synthesized from
Arrachidonic acid
And it inhibit platelet
Aggregation
Plasminogen
Its release is
Stimulated by
Vascular damage

15. Stages of Hemostasis
Fibrinolysis
Formation of Platelet Plug
Formation of blood clot
Vascular Constriction
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16. Blood Vessels Injury

17. Vasoconstriction
Endothelial cells produce vasoconstrictors such as
angiotensin II and serotonin which help in vasoconstriction.
Activated platelets produce thromboxane A2 (TXA2) which is
a potent vasoconstrictor.

18. Function of platelets
Coagulative function
Haemostatic function

19. Platelets membrane glycoproteins:
GPIb-IX:
Constitute active receptor for vWF
Mediates vWF dependent adhesion of
platelets to subendothelial
GPIIb/IIIa:
On activation serve to bind fibrinogen
Mediates aggregation
Also receptor for vWF, fibronectin
and thrombospondin
GPIa-IIa:
Constitutively active receptor for collagen.
Mediates platelets adhesion independent of
vWF.
Hemostatic Function

20. Role of Platelets in
Hemostasis
With in 1-2 sec after injury to blood vessel, hemostatic process begins & proceed as
out line bellow:
1. platelet adhesion
2. platelets activation
3. platelets release reaction
4. platelets aggregation

21.  Platelets attach to non-platelet
surfaces, such as collagen fibers in
the subendothelium.
 Platelets move from the blood vessels
and into the tissues.
 Exposure to surfaces in the tissues
causes them to bind to collagen with
the presence of von Willebrand factor
( vWF) and Glycoprotein IbIX,
making a bridge formation.
 Binding via GpI b initiates activation
of platelet.
Platelets Adhesion

22. Platelet activation:
The adhesion of platelets to
the vessel wall activates them.
 Platelets undergo a shape change
from disc to tiny sphere with
projecting pseudopodes.
 Activation required for hemostatic
plug formation.
 Activators released or
synthesized at the site of injury
 Thrombin
 Exposed collagen fibres
 ADP, Adrenaline,serotonin,TxA2

23.  Immediately after adhesion & activation process of release reaction or
secretion begins.
 In this process content of platelets organelles are released to the exterior.
Secretion of α granules
a) PF4
b) β-Thrombomodulin
c) Fibrinogen
d) Factor V
e) Fibronectin
f) Thrombospondin
g) PDGF
h) PAI-1
Platelets Release Reaction

24. Secretion of dense granules:
 ADP, GTP, GDP
 Calcium, serotonin
 Histamin, epinephrin
 ADP released from dense
granules promotes platelets
aggregation.
PF-4 release from alpha granules
neutralize the anticoagulant
activity of heparin
PDGF – stimulate proliferation
of vascular smooth muscle cell &
skin fibroblast & plays a role in cut
healing.
TxA2 causes shape change &
stimulates release reaction from
alpha & dense granules, Also
induce aggregation of other
platelets & local vasoconstriction

25. Platelet aggregation
Process by which platelets interact with one
another to form a hemostatic plug.
Chemical changes cause platelets to
aggregate and stick to one another
GPIIb-IIIa complex binds vWF, undergoes
Ca++-dependent structural change, then
acts as receptor for fibrinogen
Fibrinogen + activated platelets serve as a
bridge between two platelets .

26.  Primary haemostasis involves the binding
of platelets to exposed collagen in the sub
endothelium of damaged vessels.
 Secondary haemostasis is the process of
activation of coagulation factors leading
to the production of thrombin.
18 July 2015 26

27. Primary Hemostasis
First physiological response to vascular injury, which is mediated by platelets, in
order to arrest bleeding
Mechanism:
– Activation of platelets via stimulators such as thrombin
– Adhesion of platelets to subendothelium via interaction between GPIb and von
Willebrand Factor (VWF)
– Release of platelet granule products in order to recruit more platelets to the
injured site
– Aggregation of platelets via interaction between GPIIb/IIIa (aIIb3) and
fibrinogen to form the initial plug.
 Triggers secondary hemostasis (coagulation proteins)

28. • Process of blood coagulation
Mechanism:
 Coagulation proteins work in concern to generate thrombin
 Thrombin converts fibrinogen to fibrin
 Fibrin consolidates the platelet plug made in primary hemostasis such
that a thrombus (secondary hemostatic plug) is formed
• Prevents further blood loss from the injury site
Secondary Hemostasis

29. Platelet Aggregation
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30. INTRINSIC
PATHWAY
EXTRINSIC
PATHWAY
COMMON PATHWAY
Plasma Coagulation System

31. COAGULATION of blood take place with the help of some proteins called
clotting factors present in plasma.
Clotting factor act as zymogens and under the influence of enzyme are
themselves converted into active enzymes
Role of Clotting Factors

32. Coagulation factors in the form of zymogens
precursors.
• Ca++ as a co-factor and organizing surface,
 Provided by platelets in vivo
 Provided by a phospholipids emulsion in
vitro
The coagulation process is initiated when
tissue factor - bearing cells are exposed to
blood at a site of injury.
Requirements for Coagulation

33. FX FXa
Ca++
PL
FVIIIa
Prekallinokerin kallinokerin
FXII FXIIa
FXI FXIa
FIX FIXa
Ca++
FVIIa
FVIIa
Ca++
Intrinsic pathway
Expose collagen
Tissue factor
FVIIFVIIa
Extrinsic pathway
Prothrombin Thrombin
Fibrinogen Fibrin monomer
Cross linked Fibrin clot
FXIII FXIIIa
Ca++
PL
FVa
Ca++
FIIa
Common pathway

34.  Fibrinolytic system keep the vascular system free of deposited
fibrin clots.
 Essential purpose of fibrinolysis is to digest and solublize the
fibrin, thus restoring potency to occluded vessel.
Fibrinolytic system

35. Plasminogen activator
Plasminogen and plasmin
Inhibitors of fibrinolysis:
Antiactivators
Antiplasmins
COMPONENTS OF FIBRINOLYTIC SYSTEM

36. FIBRINOLYTIC
PATHWAY
It involves-conversion of
plasminogen to plasmin
Plasmin break down the fibrinogen or fibrin in their
degradation products

37. Conversion of Plasminogen To Plasmin
Plasminogen

38. Fibrinogen degradation
product
• Plasmin initially attacks alpha chain of
the fibrinogen molecule & removes
small fragments designed as A,B & C
from the C terminal of the Aα chains.
• Followed by degradation of Bβ chains
with removal of first 42 amino acids.
• Lead to the formation of a large
fragment X that still remains
fibrinopeptide A.
• Next cleavage involves all the three
chains in an asymmetrical manner with
the release of fragment Y & D.
• Fragment Y is rapidly degraded by
plasmin liberating two D&E.

39. Fibrin Degradation Product
Degradation of cross linked fibrin is
different from fibrinogen.
The fibrin degradation products are
different because of the presence of
covalant bonding.
Thus the characteristic fragments are
oligomers of X and Y D-dimer. D-E
complex and Y-D complex.
Fibrin degradation is slower due to
the presence of crosslinkages.
Normally the FDPs are cleared from
the circulation by macrophages.

40. INHIBITORS
Inhibitors of Serine Proteases
Antithrombin
Inhibitor of coagulation having antithrombin activities
Types:
 Antithrombin I
Show thrombin adsorbing effect of fibrin
 Antithrombin II
Act jointly with heparin act as heparin cofactor
 Antithrombin III(alpha 2 glycoprotein)
Antithrombin is a serin protein inhibitor that degrades the serine proteases:
thrombin, FIXa, FXa, FXIa, and FXIIa. It is constantly active, but its adhesion
to these factors is increased by the presence of heparan sulfate.

41. Other Inhibitors of Coagulation
 α2 macroglobulin – It act as antithrombin, antiplasmin and
inactivate kallikrein.
 α2 antitrypsin ( alpha globulin) – Inhibitor of factor XIa and
antiplasmin and is weakly antithrombin.
 α2 antiplasmin – Inactivate plasmin.
 Heparin – inhibit the action of thrombin.
delay the interaction of thrombin and fibrinogen.

42. Protein C:
• It is a vitamin K dependent glycoprotein synthesized in the liver.
• Protein circulate as an inert zymogen and is activated by thrombin in
the presence of thrombomodulin on the surface of vascular
endothelial cell.
• Protein C proteolytic destruction of activated FV & FVIII.
Protein S :
• It is also vitamin k dependent protein.
• Act as a cofactor in protein C reaction and enhance the action of
protein C
Tissue Factor Pathway Inhibitor(TFPI):
• (TFPI) limits the action of tissue factor (TF). It also inhibits excessive
TF-mediated activation of FIX and FX.
INHIBITION OF COAGULATION CO FACTORS

43. • Endothelial cell produced Nitric oxide, PGI2 and ADPase.
• They act as a antiplatelets agents (inactivate the platelets receptor by
binding with them)
• ADP produced by platelets is favour to bind with endothelial cell but
ADPase inactivate or digest ADP.
• Healthy endothelium does not tolerate the presence of activated
coagulation factor.
• When anti thrombin III binds with heparan sulfate. AT-III is activated
& cut down the thromin molecules & some activated coagulation
factors ( FIXa, Fxa)
SUMMARY
How blood is kept in fluid state in circulation ????

44.  Normally thrombin helps in coagulation but when binds with
thrombomodulin (TM) it modulate the function of thrombin which
activate the protein C.
 Activated protein C digest the activated FVa & FVIIIa.
 Endothelial cells not only prevent the platelets aggregation but also
inhibit the proteins that involve coagulation.
 Endothelial cells also produce tissue plasminogen activator(tPA)
which activate the plasminogen to plasmin
 Plasmin cleaves fibrin strand in to the FDPs.
 FDPs are removed from the circulation by macrophages and some
eosinophill.
 By this entire process blood is kept in fluid state .

45. • When the blood vessels are injured mechanically, chemically & physically the
first step is vasoconstriction.
• After the vasoconstriction platelets are adhere to the endothelial cell
membrane. And under goes activation, release reaction and aggregation.
• Which forms the primary hemostatic plug and stop the blood leakage from
injured site.
• Simultaneously activate the coagulation cascade.
(extrinsic pathway, intrinsic pathway and common pathway) and forms the
fibrin mesh in the clot which establish the plug and form secondary
hemostasic plug
Which is stable and irreversible. So that bleeding is arrested permanently.
How bleeding is arrested???

46. Essential haematology: A. V. Hoffbrand, P. A. H. Moss, J. E. Pettit - 2006 - 380 pages
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pages
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Hemostasis and thrombosis protocols: Volume 489 - Page 3 David J. Perry, K. John Pasi - 1999
- 368 pages
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35
Beckman Coulter Webinars: Fundamentals of Hemostasis at URL
http://www.beckmancoulter.com/LARS/personnel/webinars.asp
Hemostasis Basics: Programmed Learner Part I (Provided by Siemens Healthcare Diagnostics
Hemostasis Technical Services at URL
http://www.dadebehring.com/education/hemostasis/tutorial.htm
References