factors and clotting

1. CLOTTING MECHANISM
1
Done By
A Mahesh Kumar
1st Year Pg student
Dept of Pedodontics
Drs S&N SIDS

2. Contents
2
 Thrombocytes
 Thrombocytopoiesis
 Hemostasis
 Coagulation of blood
 Fibrinolysis
 Bleeding time
 Clotting time
 Plasma
 Plasma proteins

3. THROMBOCYTES :
• Greek : Thrombus : Clot; Cyte: Cell.
• Platelets are not whole cells. ( since they don’t have
nuclear component )
• It posses a cell membrane – covered by fragments of
cytoplasm that are liberated from megakaryocytes.
3

4. • Small disk shaped fragments of granule containing
cytoplasm with diameter 2µm – 3µm.
• The number varies from 1,50,000 – 4,00,000/cumm.
• Spherical or rod shaped - Active form
Oval or disc shaped – Inactive form
4

5. • Life span: 8 to 11 days (median value of 10 days)
• Platelets are destroyed by tissue macrophage system in
spleen
5

6. LM
• Small, irregular with fine cytoplasmic processes
• Cytoplasmic granules are often visible
• Central portion – granulomere (purple colored material)
• Outer region stains a transparent pale blue - hyalomere
6

7. ULTRASTRUCTURE OF PLATELETS
Peripheral zone Exterior coat(glycocalyx), cell
membrane, open canalicular
system
Sol-gel zone Microfilaments, circumferential
microtubules, dense tubular
system
Organelle zone Alpha granules, dense
granules, mitochondria,
lysosomes
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8. 8

9. OPEN CANALICULAR SYSTEM
1. Tubules communicate with the platelet surface.
2. This system is wide and tortuous .
3. Provides a major conduit for the release of number of important
secretory substances to the exterior of the platelet.
DENSE TUBULAR SYSTEM
1. Lumen of the tubules consists of electron dense material and hence
the name.
2. This system is narrow and is derived from the endoplasmic
reticulum.
3. Function is unknown.
4. Has a role in sequester calcium ions that are involved in regulating
the platelet’s internal contractile activities.
9

10. Alpha Delta Lambda
Most numerous.
300-500 nm diameter
Less number &
Smaller size.
similar to lysosomes
Contains:
Fibrinogen
Coagulation factors
Plasminogen
PDGF
Contains
ADP,
ATP,
Serotonin and
histamine
contains
several hydrolytic
enzymes.
Important role
1. initial phase of vessel
repair,
2. blood coagulation &
3. platelet aggregation.
Important Role in :
1. Facilitate platelet
adhesion and
2. Vasoconstriction in
the area of injured
vessel
Important Role in :
1. Clot resorption in
later stages of vessel
repair.
10

11. THROMBOCYTOPOIESIS
• Identifiable stages are
1. Megakaryoblast
2. Promegakaryocyte
3. Megakaryocyte
4. Platelets.
• Unique feature in thrombocytopoiesis is endomitosis.
• Endomitosis : Nuclear division with cytoplasmic
maturation without cell division.
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12. 12

13. As cell matures from Megakaryoblast to Megakaryocyte :
there is
• gradual increase in cell size
• Number of nuclear lobes
• Red-Pink granules
• Gradual decrease in cytoplasmic basophilia
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14. Megakaryocyte
• Most abundant cells of platelet series in marrow
• Large & contain numerous nuclear lobes with dense
nuclear chromatin, small aggregates of granules in
cytoplasm
• On maturation : Megakaryocytes extend pseudopods
through the walls of marrow sinusoids and individual
platelets break off in to the peripheral circulation
• Thrombopoietin controls the maturation of megakaryocytes
14

15. PROPERTIES
Adhesiveness 1. Property of sticking to a rough surface.
2. The factors are: Collagen, thrombin, ADP, Thromboxane
A2, Calcium ions, Von willebrand’s Factors, P selectin
Aggregation 1. Grouping of platelets.
2. The factor responsible are : Platelet activating Factor (
secreted by neutrophills & Monocytes.), ADP,
Thromboxane A2
Agglutination 1. Clumping of platelets.
2. The factor responsible are :
Platelet agglutinins & Platelet Activating Factor.
15

16. FUNCTIONS
Blood clotting Responsible for formation clot by intrinsic &
extrinsic pathway through Prothrombin activator.
Clot retraction Clot contraction & oozing of serum out is called clot
retraction.
It is by contractile proteins like myosin &
thrombosthenin in cytoplasm of platelets.
Role in repair of
ruptured blood
vessel
PDGF formed in cytoplasm of platelets is useful for
the repair of the endothelium.
Role in defense
mechanism
By agglutination property;
Platelets encircle the foreign body and destroy
through phagocytosis.
16

17. HEMOSTASIS
• Set of process that regulate 2 important functions.
1. They maintain blood in a fluid, clot – free state in
normal vessels.
2. They are poised to induce a rapid and localized
hemostatic plug at a site of vascular injury.
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18. • Physiologic form of blood coagulation is termed as
hemostasis.
• Pathologic counter part of it is Thrombosis ( happens
in unsevered vessel i.e., normal vessel.)
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19. Blood doesn't clot in vascular channel due to
Antithrombin III
Heparin
Protein C & S
Thrombomodulin
Alpha -2 macroglobulin.
These factors inactivate the activated factors.
1. Vascular endothelium prevents activation of clotting factors.
2. High blood flow prevents accumulation of activated clotting factor
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20. VASOCONSTRICTION
20

21. PRIMARY HEMOSTASIS
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22. SECONDARY HEMOSTASIS
22

23. THROMBUS AND ANTITHROMBOTIC EVENTS
23

24. Injury to blood vessel and damage to endothelium
EXPOSURE OF COLLAGEN
Adherence of platelets to
collagen
Activation of platelets
Von willebrand’s Factor
Secretion of serotonin Secretion of ADP &
TXN
Formation of
Prothrombin Activatior
VASOCONSTRICTION Formation of platelet
plug
Blood clotting.
Aggregation of
platelets
PAF
HEMOSTASI
S
24

25. • “Process in which blood looses its fluidity and becomes a
jelly like mass few minutes after it is shed out.”
• Clotting factors are responsible in the process of
coagulation of blood.
COAGULATION OF BLOOD
25

26. • Clotting mechanism occurs in 3 stages:
1. Formation of Prothrombin Activator.
2. Conversion of prothrombin into thrombin.
3. Conversion of fibrinogen into fibrin
26

27. Factor No. Nomenclature
I Fibrinogen
II Prothrombin
III Thromboplastin ( Tissue Factor)
IV Calcium
V Labile factor ( Pro accelerin / Accelerator
globulin.)
VI Presence has not been proved.
VII Stable factor
VIII Anti hemophillic Factor
IX Christmas Factor
X Stuart Prower Factor
XI Plasma thromboplastin Antecedent
XII Hegman factor ( contact Factor)
XIII Fibrin Stabilizing Factor ( fibrinase.)
27

28. Formation of Prothrombin Activator
• Blood clot commences with the formation of
PROTHROMBIN ACTIVATOR.
• Prothrombrin activator formation occurs in 2
pathways
28

29. 29
The two final phases in the haemostatic process, plasma
coagulation with the formation of a fibrin clot, and
fibrinolysis leading to the dissolution of fibrin clots, are
reviewed.
Coagulation may be initiated either by reactions occurring
between components of the blood alone, the intrinsic
pathway, or by reactions which also involve tissue
components, termed the extrinsic pathway.

30. Extrinsic Pathway 1. Formed from the injured tissues.
2. Formation of prothrombin activator
is initiated by the tissue
thromboplastin.
Intrinsic Pathway 1. Formed with in the blood itself.
2. Formation of prothrombin activator
is initiated by platelets.
30

31. Extrinsic pathway
Tissue trauma
Tissue
thromboplastin
Factor VII
Factor X Xa
Ca
Thrombin
FactorV
Prothrombin activator31

32. INTRINSIC PATHWAY
Endothelial damage + Collagen
exposure
XII XII a
Kinogen
Platelets
prekallikrein
XI XI a
Ca
Phospholipid
IX IX a
VIII & Ca
X Xa32

33. Stage 2 Ca
Prothrombin
Thrombin
Stage 3 Fibrinogen a
Fibrinogen
Polymerization
Loose strands of fibrin
XIII, Ca
Prothrombin activator
Fibrin
Tight blood clot
33

34. FIBRINOLYSIS
• Lysis of blood clot inside the blood vessel is
termed as fibrinolysis.
• It requires a substance called plasmin / fibrinolysin.
34

35. 35
The key component in fibrinolysis is plasminogen, which
under the influence of various activators is converted to
plasmin.
Plasmin is a serine protease and its main in vivo target is
fibrin. Alpha 2antiplasmin and a fast acting inhibitor of
tissue plasminogen activator are the most important
inhibitors.

36. Damaged tissue and endothelium
Lysosomal enzymes
Thrombomodulin +
Thrombin
Thrombomodulin – thrombin complex
Protein C Activated Protein C
Protein S :: Cofactor
Inactivates V and VII
Inactivation of t- PA inhibitor
Activation of t- PA
Thrombin urokinase PA
Plasminogen Plasmin
Lysis of clot
36

37. ANTICOAGULANTS
1. Heparin
2. Coumarin derivatives – dicoumoral and warfarin
3. EDTA
4. Oxalate compounds
5. Citrates
37

38. PROCOAGULANTS
1. Thrombin
2. Some snake venom
3. Extracts of lungs and thymus
4. Sodium calcium alginate
5. Oxidized cellulose
38

39. LAB INVESTIGATIONS
Bleeding time
Clotting time
Prothrombin time
Activated Partial Thromboplastin time
Thrombin Test
Capillary Resistance test by Hess
Platelet count
Clot retraction Time.
39

40. BLEEDING TIME
DUKE’S METHOD IVY’S METHOD
Ear lobe is pricked and
escaping blood is dried every
30 seconds on the edge of a
circle.
The forearm is punctured after
the application of BP cuff over
the upper arm and BP raised
to 40mm Hg.
Here the arrest of bleeding is from platelet plug formation
Normal value is 2-6 minutes. Normal value is 2-6 minutes.
40

41. 41

42. Prolonged bleeding time
• Thrombocytopenia
• Von-Willebrand’s disease
• Purpura
• Antiplatelet medication – Aspirin
• Uraemia
• Myeloproliferative disorders
• Leukaemias
• DIC
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43. CLOTTING TIME
Glass Capillary tube method
(Wright’s)
Lee & White Method
Finger is pricked and blood is
allowed to flow in capillary tube of
15 cm length.
3-5ml of blood is taken in 3 test
tubes kept in a bath at 37ºc.
A small bit of tube is carefully
broken till the thread of clotted
blood appears
Test tube is tilted at every 30
seconds and checked for the
solidification of blood.
Normal value by this method is 3-7
minutes.
Normal value by this method is 4-
11 minutes at 37oc.
Prolonged in Hemophilia
43

44. PROTHROMBIN TIME
Quick’s One stage Method:
In a test tube 0.2ml of Commerical Thromboplastin containing
calcium is kept at 37°c.
After 30 seconds, 0.1ml of plasma is added from a pippete and
stop watch is started.
The test tube kept in water bath and shaken constantly.
The test tube is tilted to find out the appearance of clot
The normal value is 11-16 seconds.
This is test for extrinsic pathway.
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45. Prolonged prothrombin time
• Hypoprothrombinaemia
• Afibrinogenaemia
• Vit K Deficiency.
• VII, X, V, II, I deficiency
• Dicoumarol therapy
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46. ACTIVATED PARTIAL THROMBOPLASTIN TIME
PROCEDURE
0.1ml of plasma kept at 37ºc is mixed with 0.2ml of
thromboplastin and Kaolin
Kaolin activate XII.
And is kept in a bath for 37ºc for about 10 minutes.
& at the end point 0.025ml of CaCl2 is added.
Normal value 30-40 seconds.
This test used to find out the deficiency of VIII and IX.
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47. Prolonged APTT is seen in
• Deficiency of VIII, IX
• DIC
• Heparin
• Liver disease
• Vit K deficiency
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48. THROMBIN TIME
This test is test for conversion of fibrinogen to fibrin
Add 0.1ml of thrombin + CaCl2 mixture to 0.1ml of
citrated platelet poor plasma.
Normal value 8-12 seconds.
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49. Prolonged thrombin time is seen in
• Disorders of fibrinogen – afibrinogenaemia,
hypofibrinogenaemia, dysfibrinogenaemia
• Presence of heparin in plasma
• Chronic liver disease
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50. CAPILLARY RESISTANCE TEST OF HESS
This test is done to check for resistance of capillary endothelium
or capillary fragility test.
BP cuff is applied on the arm & pressure is raised to 80mm of Hg
for about 5 minutes.
Capillaries leak the blood leading to appearance of minute
hemmorhagic spots called petechia.
When there is more than 10 such spots – indicates diminished
resistance of capillary endothelium.
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51. Defective capillary fragility is seen in
• Thrombocytopenia
• Defective platelet function
• Vascular purpura
• Thrombasthenia
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52. THROMBOCYTOPENIA
• Splenomegaly
• X-ray Radiation
• Aplastic Anemia
• Pernicious anemia
• Allergy
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53. THROMBOCYTOSIS
• >4,00,000/mm3
• Leukemia
• Hodgkin’s disease
• Polycythemia Vera
• Splenectomy
• Stress
• Adrenaline injection
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54. PLASMA
• Water 91-92%
• Solids 8-9%
Solids
Organic Inorganic
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55. Organic substances
• Proteins : Albumin, Globulin, Fibrinogen
• Carbohydrates : Glucose
• Fats : Neutral fats, Phospholipids, Cholesterol
• Non protein nitrogenous substances : ammonia,
creatine, creatinine, xanthine, hypoxanthine, urea,
uric acid
• Hormones & antibodies
• Enzymes : amylase, carbonic anhydrase, alkaline
phosphatase, acid phosphatase, lipase, esterase,
protease, transaminase
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56. Inorganic substances
Na, Ca, K, Mg, Cl, I, Fe, PO4, Cu
Gases
O2, CO2
Serum = Plasma - Fibrinogen
57

57. Cholesterol up to 200mg/dl
Plasma proteins 6.4 to 8.3 gm/dl
Bilirubin 0.5 to 1.5 mg/dl
Iron 50 to 150 micro gram/dl
Copper 100 to 200mg/dl
Calcium 9 to 11 mg/dl
Sodium 135 to 145mg /dl
Potassium 3.5 to 5.0 mg/dl
Magnesium 1.5 to 2.0 mg/dl
Chloride 100 to 110 mg/dl
Bicarbonate 22 to 26 mg/dl
58

58. Plasma proteins :
Total proteins 7.3 gm% (6.4-8.3)
Serum albumin 4.7gm%
Serum globulin 2.3 gm%
Fibrinogen 0.3 gm%
59

59. Origin of Plasma Proteins :
In embryo : mesenchymal cells
First albumin is synthesized
In adults : mostly from reticuloendothelial cells of liver
Also synthesized by spleen, bone marrow, disintegrating
blood cells
ϒglobulin – B lymphocytes
60

60. FUNCTIONS OF PLASMA PROTEINS :
1. Role in coagulation of blood
2. Role in defense mechanism of body
3. Role in transport mechanism
4. Role in maintenance of osmotic pressure of blood
5. Role in acid base regulation
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61. 6. Role in viscosity of blood
7. Role in ESR
8. Role in suspension stability of RBC
9. Role as reserve proteins
10. Role in production of trephone substances
62

62. Separation of Plasma Proteins
1. Precipitation method
2. Salting out method
3. Electrophoretic method
4. Cohn’s fractional precipitation method
5. Ultra centrifugation method
6. Immunoelectrophoretic method
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63. Plasmapheresis :
• Experimental procedure done in animals to
demonstrate the importance of plasma proteins
• Demonstrate the synthesis of plasma proteins by
the liver
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64. Plasma proteins Condition when it
increases
Condition when it
decreases
Albumin Dehydration Malnutrition ,
Cirrhoses of liver ,
Burns ,
Hypothyroid disease ,
Nephrosis
Globulin Cirrhosis of liver,
Chronic infections ,
Nephrosis,
Rheumatoid arthritis
Emphysema ,
Acute hemolytic
anemia.
Fibrinogen Acute infections , Stroke ,
Trauma.
Liver dysfunction ,
steroids ,
use of phenol
a/g ratio Hypothyroidism ,
excess of glucocorticoids ,
hypogamma globunemia
Liver dysfunction
65

65. REFERENCES :
1. Ham’s Histology – 9th edition – David H Cormack
2. Text book of Medical Physiology- 10th edition- Guyton & Hall
3. Concise Medical Physiology- 4th edition- Chaudhuri
4. Essentials of Hematology; Shirish M Kawthalkar
5. Essentials of Medical physiology-3rd edition-Sembulingam
6. Scand J Gastroenterol Suppl. Coagulation and fibrinolysis
1987;137:118.
7. Narla Mohandas and Patrick G. Gallagher. Red cell membrane:
past, present, and future. Blood 2008; 112: 3939-48
66

66. 8. Michael Fo¨ller1, Stephan M. Huber2 and Florian Lang.
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668
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endothelial nitric oxide synthase. Blood 2006; 107: 2943-2951
10.Veronique Witko-Sarsat et al. Neutrophils: Molecules, Functions
and Pathophysiological Aspects. Laboratory investigations 2000;
80(5): 617-653
11.SM Rashmi et al. Neutrophils in health and disease: An overview.
J Oral Maxillofac Pathol 2006; 10: 3-8
12. Per venge. What is the role of eosinophil?. Thorax 1990; 45: 161-
163
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67. THANK YOU
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