Bleeding disorders are most common disorders,can be hereditary or acquired

1. NORMAL
HEMOSTASIS
PRESENTED BY-
DR. DHARMENDRA SINGH
JR-I

2. NORMAL HEMOSTASIS
❑ HEMO=BLOOD, STASIS=STANDING
❑ Normal physiological process by which blood clots form
at sites of vascular injury and preventing blood loss
while maintaining blood in a fluid state within vascular
system.

3. NORMAL HEMOSTASIS
Normal blood vessel with linear blood flow
Vessel injury causes constriction of blood vessel around
wound- reduced blood flow to damaged area
Activated platelets stick to injury site and clump together to
form platelet plug
Blood clotting mechanism start to form fibrin which act like a
mesh to stop the bleeding

4. IMPORTANCE OF HEMOSTASIS
➢ To maintain blood in a fluid state while it remains circulating
within the vascular system.
➢ To stop bleeding at the site of vascular injury by formation
of hemostatic plug.
➢ To limit hemostasis process to the site of damaged blood
vessel.
➢ To ensure the eventual removal of the plug while healing is
completed.

5. MAJOR COMPONENTS OF HEMOSTASIS & ITS ROLE
1.Blood vessels
2.Platelets
3.Coagulation factors
4.Coagulation inhibitors
5.Fibrinolytic system
1.Blood vessel
2.Platelets
3.Coagulation factors
4.Coagulation inhibitors
5.Fibrinolytic system

6. SEQUENCE OF HEMOSTASIS
I. Arteriolar vasoconstriction
II. Primary hemostasis : formation of platelet plug
III. Secondary hemostasis : coagulation of blood and
deposition of fibrin clots
IV. Fibrin clots retraction and stabilisation
V. Fibrinolysis

7. ARTERIOLAR VASOCONSTRICTION
Immediately after a blood
vessel injury or cut
Smooth muscle in blood vessel
wall contracts
Reduces the flow of blood
from the ruptured vessel

8. THE SMOOTH MUSCLE CONTRACTION RESULT FROM
1. Reflex neurogenic mechanism- pain nerve impulse that orginate from injured
vessel and nearby tissue.
2. Local myogenic spasm of the blood vessels- due to direct damage to the
vascular wall.
3. Local autocoid factors-
I.Endothelin-Released by injured endothelium
II.Thromboxane A2 & Serotonin-Released by activated platlets.
are potent local vosoconstrictor.
➢ Decreased blood flow and pressure in damaged area lasts for several minutes
to even hours, during which time process of platelet plugging and blood
coagulation can take place.

9. PRIMARY HEMOSTASIS: PLATELET PLUG FORMATION
 4 Steps:
1.Platelet adhesion
2.Platelet activation
3.Platelet aggregation
4.Primary platelet
plug formation
Intact blood vessel
Secretion of
prostacyclin &
nitric oxide

10. PLATELET ADHESION
➢ Endothelial cell injury exposes
subendothelial collagen and
von Willebrand factor.
➢ vWF mediates adhesion of
platelets with collagen of
subendothelium by binding
with Gp1b receptor on
platelets

11. PLATELET ACTIVATION
➢ Platelets activation initiate due to-
o Platelets adhesion to collagen
o PAF release from injured endothelium &
o Thrombin form in coagulation process.
➢ Platelet activation causes-
1.Change in shape of platelets-from disc shape to flat plate
shape with spiky protrusion due to conformation change
in cell surface GpIIb/IIIa.
2.Release reaction-release of secretory granules contents-
serotonin, thromboxane A2, ADP & others
3.Recruitment-ADP and TXA2 release from secondary
granules which activate and attract more platelets.

12. PLATELET AGGREGATION
➢ Platelet activation causes
conformational changes in platelet
GpIIb/IIIa receptor.
➢ Changed GpIIb/IIIa binds with
bivalent plasma polypeptide
fibrinogen that forms bridges
between adjacent platelets leading
to their aggregation.
➢ TXA2 & ADP release during
platelet activation is a potent
inducer of platelets aggregation.

13. PRIMARY PLATELET PLUG FORMATION
➢ More and more platelet
aggregation, cross bridging
between platelets with
fibrinogen and irreversible
platelet contraction by
thrombin, form primary
platelet plug which temporary
closes injured blood vessel.

14. Platelet Plug formation

15. PLATELETS
➢ Disc shaped anucleated cell fragment.
➢ Produced by cytoplasmic fragmentation of
megakaryocytes in bone marrow.
➢ Size- 1 to 4 micrometer.
➢ Lifespan- 7 to 12 days.
➢ Normal blood count- 1.5 lac to 4.5 lac/ml.
➢ Eliminated by tissue macrophage system in spleen.

16. PLATELETS:ULTRASTRUCTURE
➢ Contractile protein- Actin,
Myosin & Thrombosthenin, that
can causes the platelets to
contract.
➢ ER & Golgi apparatus- synthesis
of various enzymes and store
large quantities of calcium ions.
➢ Mitrochondria- forming ATP &
ADP.
➢ Enzyme systems- synthesis of
prostaglandins

17. ❑ Growth factor-for multiplication & growth of endothelial cells,
vascular smooth muscle cells & fibroblast thus help in repair of
damaged vascular wall.
❑Cell membrane of platelets is coated with a negative charge
glycoprotein glycocalyx that repulses adherence to normal
endothelium.
❑Membrane phospholipid- activate multiple stages in the blood
clotting process and provide surface for it.

18. PLATELETS GRANULES
Histamine

19. PHYSICAL PROPERTIES THAT PREVENT CLOT FORMATION IN NORMAL
BLOOD VESSELS
➢ Lamellar flow of blood in vessel-cellular components flow in center of
stream and away from endothelial surface.
➢ Smoothness of endothelial cell surface.
➢ A layer of glycocalyx on endothelium which repels clotting factors &
platelets.
➢ Surface of endothelium is negatively charged which repels circulating
platelets and proteins which are also negatively charged.
➢ Endothelium shields platelets from collagen, tissue factor & other pro-
aggregatory components of sub-endothelial layer.

20. NORMAL BLOOD FLOW IN BLOOD VESSEL

21. ENDOTHELIAL CELLS
➢ Endothelial cells are central regulators of hemostasis. The balance between
the procoagulants and anticoagulants activities of endothelium determines
wheather thrombus formation, propagation, or dissolution occurs.
➢ Normal endothelial cells express a variety of anticoagulant factors that
inhibit platelet aggregation and coagulation and promote fibrinolysis.
➢ After injury or activation, however, this balance shifts, and endothelial cells
acquire numerous procoagulant activities (activation of platelets and
clotting factor).
➢ Besides trauma, endothelium can be activated by microbial pathogens,
hemodynamic forces, and a number of pro-inflammatory mediators.

22. ANTICOAGULANT ACTIVITIES OF NORMAL ENDOTHELIUM
❑ Prostaglandin I2[PGI2]- produced from endothelium-prevents platelets
aggregation and its activation.
❑ Thrombomodulin- expressed on surface of endothelial cells-binds with
thrombin & modulates function of thrombin- activates Protein C –causes
inactivation of coagulation factor F Va & F VIIIa.
❑ Heparan sulfate- on surface of endothelium-binds & activates
Antithrombin III- inhibits thrombin & coagulation factor IXa, Xa, XIa &
XIIa.

23. ❑ Tissue plasminogen activator[tPA]- released from endothelium-
activates plasminogen to plasmin which breaks down abnormaly
formed fibrin into fibrin degradation product.
❑ Prostacyclin & Nitric oxide-inhibits platelet adhesion & aggregation,
promotes vasodilation.
❑ ATPase & ADPase-ATP & ADP produced by platelets are inactivated.
❑ Tissue factor pathway inhibitors[TFPI]-binds and inhibits TF-F VIIa
complex

25. COAGULATION FACTORS
 Normally coagulation factors are circulating in the blood in an inactive
form.
 All coagulation factors are protein except Tissue factor[thromboplastin] &
Calcium.
 Majority of coagulation factors are serine protease that circulate in blood
as an inactive form.
 Most of coagulation factors are synthesised by liver except Tissue factor
and Calcium.
 Tissue factor synthesised by various tissues and Calcium from bones and
diet.
 F V & F XIII synthesised by platelets in addition to liver.

26. FACTOR NAME MOLECULE TYPE SOURCE PATHWAY
I Fibrinogen Plasma protein Liver Common
II Prothrombin Plasma protein Liver Common
III Tissue factor/Thromboplastin Lipoprotein mixture Damaged cells & Platelets Extrinsic
IV Calcium ions Inorganic ions Diet, Platelets, Bone matrix Entire process
V Proaccelerin/Labile factor Plasma protein Liver, Platelets Extrinsic & Intrinsic
VI Does not exist
VII Proconvertin/Stable factor Plasma protein Liver Extrinsic
VIII Antihemophilicfactor A/ Antihemophlic globulin Plasma protein Platelets, Endothelial cells Intrinsic
IX Antihemophilic factor B/ Christmas factor Plasma protein Liver Intrinsic
X Stuart prower factor Protein Liver Extrinsic & Intrinsic
XI Antihemophilic factor C/ Plasma thromboplastin
anticedent
Plasma protein Liver Intrinsic
XII Hageman factor Plasma protein Liver Intrinsic
XIII Fibrin stabilizing factor Plasma protein Liver, Platelets Stablizes fibrins
COAGULATION FACTORS

27. SECONDARY HEMOSTASIS:COAGULATION OF BLOOD AND
DEPOSITION OF FIBRIN CLOTS
❑ Coagulation of blood is a series of amplìfying enzymatic reaction that leads
to deposition of an insoluble fibrin clots.
❑ Shape changes in platelets is accompanied by translocation of negatively
charge phospholipid particularily phasphatidylserine to the platelet surface.
These phospholipids bind with calcium and serve as nucleation sites for the
assembly of coagulation factor complexes where coagulation reaction occur.
❑ Each reaction step involves-
▪ An enzyme-an activated coagulation factor
▪ A substrate-an inactive proenzyme form of a coagulation factor
▪ A cofactor-a reaction accelerator

28. COAGULATION CASCADE REACTION ON PLATELETS PHOSPHOLIPID SURFACE
 Initial reaction complex:
Enzyme- F IXa
Substrate- F X
Cofactor- F VIIIa
❑ Second reaction complex:
Enzyme- F Xa
Substrate- F II
Cofactor- F Va
❑ Calcium ions hold the assembled
component together on platelet
phospholipid surface & essential
for the reaction
X Xa
IXa
VIIIa Ca++
II IIa
Va Ca ++
PL
PL

29. INITIATOR OF COAGULATION
▪ Trauma to vessel wall and adjacent tissues.
▪ Trauma to the blood.
▪ Contact of blood with damaged endothelial cells or with
collagen and other tissue elements outside the blood vessel.

30. PROTEIN INVOLVED IN BLOOD COAGULATION
➢ The proteins involved in blood coagulation can be classified into five
types:
1. Zymogens of serine- Dependent proteases, which become activated
during the process of coagulation: Factor II, Factor VII, Factor IX,
Factor X, Factor XI, Factor XII;
2. Cofactors- Factor III, Factor V, Factor VIII.
3. Fibrinogen- Factor I
4. Transglutaminase- Factor XIII- Stabilizes the fibrin clot.
5. Regulatory and other proteins-Protein C, Protein S, Thrombomodulin.

31. COAGULATION COMPLEXES[Tenase(Xase)]
➢ Tenase (Xase) is the final and rate-limiting enzyme
complex.
o Extrinsic tenase complex is made up of Tissue
factor, F VIIa, Phospholipid, and Ca²+ as an
activating ion. Activates F IX and F X.
o Intrinsic tenase complex contains the active factor
IX (IXa), its cofactor factor VIII (VIIIa), Phospholipid
and Ca++ ion. Activates factor X.
o Prothrombinase complex contains F Xa, F Va,
Phospholipid, and Ca++ ion. Activates
Prothrombin.
➢ These vitamin K-dependent procoagulant factors
dock to this surface through their Gla domain with
Ca2+ bridges.

32. PATHWAY OF BLOOD COAGULATION
Three pathway of blood
coagulation:
I. Extrinsic pathway
II. Intrinsic pathway
III. Common pathway

33. PHASES OF BLOOD CAGULATION
 Three phases of blood
coagulation-
1.Formation of prothrombin
activator
I. Extrinsic pathway
II. Intrinsic patway
2. Conversion of prothrombin to
thrombin
3. Conversion fibrinogen to fibrin
Extrinsic & Intrinsic pathway

34. FORMATION OF PROTHROMBIN ACTIVATOR
➢ In response to rupture of the vessel or damage to the blood itself, a complex
cascade of chemical reactions occurs in the blood involving more than a dozen
blood coagulation factors. The net result is formation of a complex of activated
substances collectively called prothrombin activator.
➢ Prothrombin activator is formed by either extrinsic or intrinsic pathway of blood
coagulation which converts prothrombin into thrombin.
➢ Prothrombin activator complex includes-
▪ Factor Xa
▪ Factor Va
▪ Platelet or tissue phospholipid
▪ Calcium ion

35. EXTRINSIC PATHWAY OF BLOOD COAGULATION
 The term extrinsic came from the observation that there was another factor
extrinsic to circulating blood that facilitate blood cloting identified as factor
III/tissue factor/tissue thromboplastin.
 It is iniatiated at the site of tissue injury with the exposure of tissue factor, a
subendothelial cell surface glycoprotein that bind phospholipid.
 Tissue factor release from injury to tissue, activate factor VII into VIIa and form
complex with it known as tissue factor-F VIIa complex.
 Tissue factor-F VIIa complex in presence of Ca++ activate factor X into Xa.
 Factor Xa, factor Va, form a complex with platlet phospholipid and Ca++ known
as Prothrombin activator.

37. INTRINSIC PATHWAY OF BLOOD COAGULATION
➢ Term intrinsic pathway because that blood clotting would occur
spontaneously when blood was placed in clean glass test tube and all
components for the clotting process were is intrinsic to the circulating
blood.
➢ Intrinsic pathway is initiated when there is contact of blood with exposed
negatively charged surface. Glass contain anionic surfaces that formed
the nucleation points that initiate clotting in the intrinsic pathway.
➢ In mammals, anionic surfaces are exposed upon rupture of the
endothelial lining of the blood vessels and are the binding sites for
specific factors that initiate clooting in the intrinsic pathway.

39. CONVERSION OF PROTHROMBIN TO THROMBIN & FIBRINOGEN TO FIBRIN
COMMON PATHWAY OF BLOOD COAGULATION
1. Prothrombin activator catalyzes conversion of
prothrombin into thrombin.
2. Thrombin act as an enzyme to convert soluble
fibrinogen into insoluble fibrin monomer
which in the presence of calcium ion chage
into fibrin fibers
3.Thrombin activate factor XIII into factor XIIIa
which cross linked fibrin fibers and stablizes
fibrin fibers by forming covalent bond
between monomers that is linked with weak
hydrogen bond.
Prothrombin activator
Complex IX/VIIIa Complex TF/VIIa
Intrinsic pathway Extrinsic pathway

40. ROLE OF LIVER IN BLOOD COAGULATION
▪ Synthesis of plasma clotting factors.
▪ Synthesis of bile salts- which are required for intestinal
absorption of lipid soluble vitamin K.
▪ Most of clotting factors are synthesis by liver, so liver diseases
depress the clotting system.

41. ROLE OF Ca++ IN BLOOD COAGULATION
 Ca++ is required for all steps of blood coagulation except
first two steps of the intrinsic pathway
 Decrease in plasma Ca++ below the threshold level for
clooting decrease blood clotting by both pathways.

42. ROLE OF VITAMIN K IN COAGULATION
 Vitamin K acts as a cofactor of the enzyme Y-glutamyl carboxylase.
 Is required for Y-carboxylation in the liver of factor-
o Factor II, VII, IX, and X
o Protein C and protein S(natural anticoagulants)
❑ Y-carboxylation(introduction of a carboxylic acid group) of certain glutamate
residues in target clotting factors- binding sites for Ca++ and PF3.
❑ Decreased dietary intake of vit K has limited consequences on blood clotting
because vit K is continiously synthesized by the intestinal flora.
❑ Vit K is fat soluble and requires fat for absorption. Lack of the biles decreases fat
digestion and absorption so vit K absorption also decreases.

44. FIBRIN CLOTS RETRACTION & STABILISATION
➢ Within a few minutes after clot formation, it begins to contract
and removes most of the fluid from clot.
➢ Retraction of clot occurs due to contraction of fibrin fibers and
contractile protein- actin, myosin & thrombosthenin of the
platelets. It causes:
▪ Increased clot density
▪ Occlusion of the damaged blood vessel
▪ Bringing the edges of wound together facilitation of wound
healing

45. COAGULATION INHIBITORS
 Tissue factor pathway inhibitor[TFPI]-
o Release from endothelium of blood vessel.
o Inhibit TF-F VIIa complex- inhibit coagulation.
❑ Antithrombin III[AT III]-
o Synthesises in liver.
o Inhibit Thrombin[F IIa], Factor IXa, Xa, XIa, XIIa.
❑ Protein C & S-
o Protein C is activated by thrombin in presence of thrombomodulin on surface
of endothelium causes proteolysis of F Va, F VIIIa.
o Protein S act as a cofactor & inhance activity of protein C.

46. NATURAL COAGULATION INHIBITORS AND THEIR ACTIONS
Tissue factor pathway inhibitor
Antithrombin III
Protein C
Protein S

47. THROMBIN/THROMBO-MODULIN/PROTEIN C PATHWAY

49. FIBRINOLYTIC SYSTEM
Fibrinolysis is the the process of dissolution of blood clot
to limit the size of clot and to ensure free flow of blood
in vascular system.
➢ After the wound has healed, fibrin is degraded into
fibrin degradation product by plasmin.

50. FIBRINOLYTIC SYSTEM:ACTIVATOR
❑ Tissue plasminogen activator[tPA]-
➢ Secreted by endothelial cells.
➢ Activates Plasminogen to Plasmin.
 Urokinase-
➢ Produced by kidney.
➢ Activates plasminogen to plasmin.
 Plasmin-
➢ Degraded fibrinogen/fibrin into fibrinogen/fibrin degration
product[FDPs].

51. FIBRINOLYTIC SYSTEM:INHIBITORS
❑ Plasminogen activator inhibitor type 1& 2-
o Inhibit tPA.
❑ Alpha 2 antiplasmin, alpha 2 macroglobulin-
o Bind with free plasmin in plasma so inhibit action of plasmin
on fibrin/fibrinogen.
❑ Thrombin activable fibrinolysis inhibitors-
o Inhibit degradation of fibrin to fibrin degration product

52. Tissue plasminogen
activator(tPA)
Plasminogen activator
inhibitor 1& 2
Urokinase
Plasminogen
Plasmin
Fibrin Fibrin degradation products
Factor XIa,
XIIa
Kallikrein
Alpha 2 antiplasmin
Alpha 2 macroglobulin
Thrombin Thrombin activatable
Fibrinolysis inhibitors
Fibrinogen
MECHANISM OF FIBRINOLYSIS
a
Activation
Inhibition

54. BLEEDING TIME (BT)
➢ Assesses primary hemostasis& is dependenton adequate fuctioning of platelets & blood
vessels
➢ Normal range of bleeding time- 2-7 minutes
➢ Causes of prolongation of bleedingtime:
➢Thrombocytopenia
➢Disorders of platelet function
➢Von Willebrand disease
➢Disorders of blood vessels

55. EVALUATION OF PROLONGED BLEEDING TIME

56. CLOTTING TIME (CT)
➢ Clotting time measures the time required for the blood to clot
in a glass test tube kept at 37 degree C temp.
➢ Prolongation of clotting time only occurs in severe deficiency
of clotting factor and normal in mild or moderate deficiency

57. PROTHROMBIN TIME (PT)
▪ Assesses coagulation factors in extrinsic pathway &
common pathway
▪ Normal range of PT- 11-16 seconds
❑ Casuses of prolongation of prothrombin time-
➢ Tt with oral anticoagulants
➢ Liver disease
➢ Vit K deficiency
➢ DIC
➢ Extrinsic & common pathway coagulation factor
deficiency

58. USES OF PROTHROMBIN TIME
➢ To monitor patient on oral anticoagulant therapy-oralanticoagulants warfarin inhibit
carboxylation of vit K dependent factors-F II,VII,IX, X. PT should be reported in INR
. PT-INR =PT of patient/PT of cntrol.
. Normal PT-INR is < 1.1
. INR range of 2.0 to 3.0 is generally an effective therapeutic range for people taking warfarin
➢ To assess liver function
➢ Detection of vit K deficiency
➢ To screen hereditary deficiency of coagulation factors

59. ACTIVATED PARTIAL THROMBOPLASTIN TIME(APTT)
▪Assesses coagulation factor in intrinsic pathway &
common patway
▪Normal APTT- 30-40 seconds
❑Causes of prolongation of APTT:
➢Hemophilia A or B
➢Intrinsic & common pathways coagulation factors
deficiency
➢Presence of coagulation inhibitors
➢Heparin therapy
➢Disseminated intravascular coagulation
➢Liver disease

60. USES OF APTT
➢ Screening for hereditary disorders of coagulation.
➢ To monitor heparin therapy-heparin potentiates the action of natural anticoagulant AT III
which is an inhibitor of thrombin and activated factors F IXa,Xa and XIa. Maintain the dose
the therapeutic range- 1.5 to 2.5 times the upper reference limit of APTT.
➢ Screening for circulating inhibitors of coagulation

61. EVALUATION OF PROLONG APTT

62. EVALUATION OF SUSPECTED BLEEDING DISORDERS

63. THANK YOU