2. Coagulation(clotting) mechanism
Prothrombin Thrombin `
Thrombin
Fibrinogen Fibrin
(soluble) Calcium (Insoluble)
Thromboplastin released by damaged tissue or platelets converts inactive prothrombin into active thrombin in the presence of calcium. Thrombin converts soluble fibrinogen into insoluble fibrin clot in the presence of calcium.
Thromboplastin
Calcium

3. INITIATION OF BLOOD COAGULATION
Extrinsic Pathway
Intrinsic Pathway
Blood trauma/ contact with collagen
Tissue trauma
Leakage of Tissue Factor
Activation of factor XII, IX, VIII
Ca+2, factor VII
X Xa
X Xa
Ca+2
Ca+2
Prothrombin activator
Prothrombin
activator
Ca+2
Prothrombin Thrombin
(factor II)
Prothrombin Thrombin
(factor II)
Activation of certain factors (VII, II, X and protein C and S) is essential for coagulation. This activation requires vit K (reduced form)

4. Recall ! Intrinsic Pathway Extrinsic Pathway Blood Vessel Injury
Tissue Injury
Tissue Factor
XII
XIIa
Recall !
Thromboplastin XI
XIa IX
IXa
VIIa
VII X Xa X
Prothrombin
Thrombin
Factors affected
By Heparin
Fibrinogen
Fribrin monomer
Fibrin polymer
Vit. K dependent Factors
Affected by Oral Anticoagulants
XIII

5. Function Number and/or name I (fibrinogen) II (prothrombin)
Forms clot (fibrin)
II (prothrombin)
Its active form (IIa) activates I, V, VII, VIII, XI, XIII, protein C, platelets
Tissue factor(formerly known as factor III)
Co-factor of VIIa
Calcium(formerly known as factor IV)
Required for coagulation factors to bind to phospholipid
V (proaccelerin, labile factor)
Co-factor of X with which it forms the prothrombinase complex VI
Unassigned – old name of Factor Va

6. VII (stable factor, proconvertin)
Activates IX, X
VIII (Antihemophilic factor A)
Co-factor of IX with which it forms the tenase complex
IX (Antihemophilic factor B or Christmas factor)
Activates X: forms tenase complex with factor VIII
X (Stuart-Prower factor)
Activates II: forms prothrombinase complex with factor V
XI (plasma thromboplastin antecedent)
Activates IX
XII (Hageman factor)
Activates factor XI, VII and prekallikrein
XIII (fibrin-stabilizing factor)
Crosslinks fibrin

7. COAGULANTS
These are substances which promote coagulation & are indicated in haemorrhagic states

8. Classification Phytonadione (phylloquinone)
1 .Vitamin K
K1(from plants, fat-soluable)
Phytonadione (phylloquinone)
K3(synthetic)
Fat-soluble :menadione,acetomenaphthone
Water soluble :menadione sod.bisulphate menadione sod.diphosphate
2 .Miscellaneous
Fibrinogen (human), Antihaemophilic factor, Desmopressin, Adrenochrome monosemicorbazone, Ruti, Ethamsylate

9. VITAMIN K ACTION: - Acts as cofactor in the synthesis
of coagulation factors II, VII, IX, X
DEFICIENCY:
- Haematuria
- GIT & Nose Bleeding
- Ecchymoses
Deficienry of vitK occurs due to liver disease, obstructive jaundice, malabsorption,long-term antimicrobial therapy which alters intestinal flora. However, deficient diet is rarely responsible. The most important manifestation is bleeding tendency due to lowering of the levels of prothrombin and other clotting factors in blood.Haemafuria is usually first to occur; other conunon sites of bleeding are g.i.t., nose and under the skin-ecchymoses.

10. Use of Vitamin K: Dietary deficiency Prolonged antimicrobial therapy
Obstructive jaundice
Malabsorption syndromes
Liver disease
Premature newborn
To reverse effect of overdose of oral
anticoagulants
Prolonged high dose salicylate therapy

11. Decorboxy prothrombin (or VII, IX, X )
MOA
Decorboxy prothrombin (or VII, IX, X )
Prothrombin (or VII, IX, X )
Vit K epoxide
Vit K hydroquinone
NAD
NADH
BLOCKED BY ORAL ANTICOAGULANTS

12. MISCELLANEOUS Fibrinogen
The fibrinogen fraction of human plasma is employed to control bleeding in haemophilia, antihaemophilic globulin (AHG) deficiency and acute afibrinogenemic states; 0.5 gm i.v infusion

13. Antihaemophilic factor
It concentrated human AHG prepared from pooled human plasma.
Indicated (along with human fibrinogen) in haemophilia and AHG deficiency
Highly effective in control bleeding episodes, but action is short-lasting (1-2 days)
Dose; 5-10 U /kg by i v infu, repeated 6-12 hrly

14. Desmopressin
It releases factor VIII and von Willebrand's factor from vascular endothelium and checks bleeding in haemophilia and von Willebrand's disease

15. Adrenochrome monosemicarbazone
It believed to reduce capillary fragility, control oozing from raw surfaces and prevent microvessel bleeding, ex:epistaxis, haematuria, retinal haemorrhage, secondary haemorrhage from wounds, etc Its efficacy is uncertain
Dose: 1-5 mg oral, i.m.

16. Rutin A plant glycoside claimed to reduce capillary bleeding
Dose 60 mg oral BD-TDS along with vit C which is believed to facilitate its action
Its efficacy is uncertain.

17. Ethamsylate
It reduces capillary bleeding when platelets are adequate; probably exerts antihyaluronidase action
improves capillary wall stability, but does not stabilize fibrin (not an antifibrinolytic)
in the prevention and treatment of capillary bleeding in menorrhagia, after abortion, PPH, epistaxis, malena, hematuria and after tooth extraction, but efficacy is unsubstantiated
S/E:N,H, rash & ↓ BP (only after i.v inj)
Dose: mg TDS oral/i.v
PPH:Post partum hemorrhage

18. DEFINITION
A substance that prevents coagulation or clotting of blood but doesn’t dissolve an already formed clot.
Uses
Storage of blood for blood transfusion or hematological testing
Therapeutic

19. Anticoagulant Purpose of using anticoagulants:
When Blood is collected, it clots after sometime. The anticoagulants are the chemical agents which prevent the clotting of Blood when mixed with Blood in proper proportion.
Purpose of using anticoagulants:
For study of various constituents of Blood components.
Study of coagulation(clotting of Blood).
Preservation of Blood in Blood Bank.
Properties of anticoagulant
It must be soluble in Blood.
It must be keep the blood in fluid condition,
It must not be bring haemolysis of Blood cells.
It must not be change the size of RBC.
It must minimize destruction of leukocytes.
It must minimize aggregation of platelets.

20. Commonly used anticoagulants
EDTA(Ethylene Diamine Tetra Acetic acid)
Double oxalate
Sodium citrate
Sodium Fluoride
Heparin
ACD(Acid Citrate Dextrose)
CPD(Citrate Phosphate Dextrose)
CPDA(Citrate Phosphate Dextrose Adenine)

21. FOR ROUTINE HAEMATOLOGY ACD
EDTA
FOR ROUTINE HAEMATOLOGY
BUFFERED SODIUM CITRATE
FOR COAGULATION STUDIES
POTTASIUM OXALTE OR SODIUM FLOURIDE
GLUCOSE DETERMINATION
NO ADDITIVE
COLLECTION OF SERUM
ACD
PRESERVE RBC FOR BLOOD BANKING &
HLA Typing
HEPARIN
INHIBIT THROMBIN
ACTIVATION

22. Non-calcium chealeter
Anticoagulant
Calcium chealeter
Oxalates
Ammonium oxalate
Potassium oxalate
Double Oxalate
E.D.T.A.
Tri-Sodium citrate
Non-calcium chealeter
Heparin
Warfarin

23. Classification of anticoagulant
1.Calcium chelator
Bind with Calcium
Oxalates
It is following forms:-
-Ammonium Oxalate
-Potassium Oxalate
- Double Oxalate
EDTA (Ethylenediaminetetraacetiacid)
Tri-sodium citrate etc.
2. Non-calcium chelator
Do not bind with Calcium
Heparin
Warfarin etc.
Most of the anticoagulants used in laboratory act by binding with calcium and prevent clotting of blood since calcium ion is essential for many of the steps in coagulation mechanism.

24. CLASSIFICATION USED IN VIVO Parenteral anticoagulants
These are drugs used to reduce the coagulability of blood classified into:
USED IN VIVO
Parenteral anticoagulants
a) Heparins (ITI)
High Molecular Weight Heparin
Unfractionated Heparin (UFH)
Low molecular weight heparin (Enoxaparin, Dalteparin, Tinzaparin, Reviparin, Danaparoid)
b) Heparinoids (DTI)-Heparan sulfate, Hirudin, Lepirudin, Bivalirudin, Argatroban
1. Indirect Thrombin Inhibitors: HMWH, LMWH
2. Direct Thrombin Inhibitors: Hirudin, Lepirudin, Bivalirudin, Argatroban

25. Oral anticoagulants (1)Coumarin derivatives :Bishydroxycoumarin (dicumarol), Warfarin sod, Acenocoumarol (Nicoumalone), Ethylbiscoumacetate (2)Indandione derivative: Phenindione. USED IN VITRO (a)Heparin (b)Sod. citrate :used in blood banks to store the blood (c)Sod. oxalate : (d)Sod. editate :
used as an anticoagulant in laboratory

26. Ammonium oxalate:- Potassium oxalate:- Oxalates
This is used at a concentration of 2 mg for 1 ml of Blood. This anticoagulant causes swelling of the RBC therefore, it is not recommended for use with blood for PCV,ESR and cell morphology.
Potassium oxalate:-
It is used at a concentration of 2 mg for 1 ml of Blood. This anticoagulant is most often used for chemical analysis. It causes shrinkage of RBC therefore it is also is not recommended for the study of PCV,ESR and other cell morphology.
Double oxalate:-
Ammonium oxalate and Potassium oxalate are combined together to balance the swelling effect of Ammonium oxalate and the shrinking effect of Potassium oxalate on the red cells. It is the mixture of 3 parts of Ammonium oxalate and 2 parts of Potassium oxalate which is prepared as follows:-
Ammonium oxalate 2.4 gram
Potassium oxalate 1.6 gram
Distilled water ml

27. E.D.T.A.(Ethylenediamine tetra-acetic acid)
It is the most commonly used anticoagulant in Haematology lab because E.D.T.A. is the most powerful calcium chelating (binding) agent we have. It gives the best preservation to the cell morphology therefore E.D.T.A. is the preferred anticoagulant for all cell count and blood smear preparation.
E.D.T.A. is used in two different forms:-
Di-sodium E.D.T.A. salt (Versene)
Di-potassium E.D.T.A. salt (Sequestrene)

28. Tri-sodium citrate It is used as a liquid form. It binds with calcium.
The concentration of Tri- sodium citrate is used as 3.8%.
For PT (Prothrombin Time) a kind of coagulation test, 3.8% Tri-Sodium citrate is used at the ratio of 1:9 i.e. 1 part of anticoagulant and 9 part of Blood.( 200 µl of 3.8% Tri-Sodium citrate and 1.8 ml of Blood is used.)
Fro ESR (Erythrocyte Sedimentation Rate) by Westerngreen method
1 part of 3.8% Tri-Sodium citrate is mixed with 4 part of Blood that is the ratio of 1:4.( 400 µl of 3.8% Tri-Sodium citrate and 1.6 ml of Blood is mixed).
It do not preserve the cell morphology.

29. Heparin
It is a natural anticoagulant and is normally present in the blood in small amount and highly acidic. This is the best anticoagulant for open heart surgery and it causes minimum haemolysis.
It is very expensive.
It produce black back ground in the smear so it is not use for smear preparation.

30. Heparin-MOA
Factors IXa, Xa, XIa, XIIa and IIa (especially IIa,IXa,Xa), need to be neutralized by AT III
Accelerates the interaction of the active clotting factors with AT III.
The negatively charged heparin molecule binds to the positively charged lysine sites on AT III
Causes a conformational change in AT III and exposing its active arginine site
The serine active sites of the active clotting factors bind to the reactive arginine site of AT III
The resulting complex is removed by the RE system
This process is accelerated times by heparin

31. The conformational change of AT exposes its active site for more rapid interaction with the proteases (the activated clotting factors) Heparin functions as a cofactor for the AT protease reaction without being consumed. Once the AT protease complex is formed, heparin is released intact for renewed binding to more AT
HMWHs accelerates the inactivation IIa and Xa.
LMWHs accelerate the inactivation of only Xa

32. Intrinsic Pathway Extrinsic Pathway Vit. K dependent Factors
Blood Vessel Injury
Tissue Injury
Tissue Factor
XII
XIIa
HMW-Kininogen
Thromboplastin XI
XIa
XIIa, Xia, Kallikrein
ca+ IX
IXa
VIIa
VII
ca+
ca+ X Xa X
XIII
ca+
ca+
Thrombin
Prothrombin
Factors affected
By Heparin
XIIIa
Fibrinogen
Fribrin insoluble
Vit. K dependent Factors
Affected by Oral Anticoagulants
Stabilised Fibrin threads

33. Mechanism + Heparin No heparin Active clotting factors
Slow AT III
Inactive clotting factors
Heparin
Active clotting factors
Fast AT III +
Inactive clotting factors

37. ORAL ANTICOAGULANTS

38. Vitamin K-Dependent Clotting Factors
VII
The four Vitamin K dependent clotting factors are synthesized in the liver. IX X II
Synthesis of Functional Coagulation Factors

39. Warfarin Mechanism of Action
Vitamin K
VII
Antagonism of
Vitamin K
Warfarin acts as an anticoagulant by blocking the ability of Vitamin K to carboxylate the Vitamin K dependent clotting factors, thereby reducing their coagulant activity. IX X II
No Synthesis of Functional Coagulation Factors
Warfarin

40. Mechanism of Action
Coumarins block the Gamma Carboxylation of glutamic acid residues of Clotting factors II,VII, IX, X , endogenous anti-coagulants C & S.
This is coupled with oxidative deactivation of Vit K
Coumarins and Indanediones (-) enzyme Vit K epoxide reductase that converts Vit K epoxide to its active hydroquinone (reduced form)
Thus they prevent the activation of Vit K and hence along with it carboxylations of clotting factor residues

41. Why Carboxylation is necessary?
Necessary for ability of clotting factors to bind Ca+ and to get bound to phospholipid surfaces which is necessary for coagulation
Factor VII affected first, then IX, X, and finally Factor II (depends upon half lives of circulating factors)

44. Anticoagulant used in Blood Bank
ACD (Acid Citrate Dextrose)
CPD ( Citrate Phosphate Dextrose )
CPDA (Citrate Phosphate Dextrose Adenine)