1. Hemostasis & blood coagulation

2. Learning objectives
Hemostais
Platelets and their role in hemostasis
Clotting : nomenclature of clotting factors
Clotting cascade: extrinsic, intrinsic, common pathways
Fibrin stabilization
Synthesis of clotting factors
Role of vit K in clotting

3. Platelets and their role in hemostasis
MORPHOLOGY
COUNT
THROMBOPOIESIS
LIFE SPAN, ROLE OF SPLEEN
FUNCTIONS
RELATED DISORDERS

4. MORPHOLOGY
SMALL, GRANULATED, LACK NUCLEI, DIAMETER-2-4µ, GLYCOPROTEIN COAT ON MEMBRANE.
EXTENSIVELY INVAGINATED MEMBRANE WITH INTRICATE CANALICULAR SYSTEM IN CONTACT WITH ECF
RING OF MICROTUBULES AROUND THEIR PERIPHERY

5. MEMBRANE RECEPTORS FOR:
COLLAGEN, ADP, VESSEL WALL VON WILLEBRAND FACTOR, FIBRINOGEN.
CYTOPLASMIC CONTENTS:
ACTIN, MYOSIN, GLYCOGEN, LYSOSOMES, ENZYME SYSTEMS FOR SYNTEHESIS OF ATP, ADP, PROSTAGLANDINS, GRANULES.

6. Contents of GRANULES:
Some clotting factors like thrombin
Platelet derived growth factor (PDGF)
Platelet activating factor (PAF)
von Willebrand factor
PROPERTIES: ADHESION, AGGREGATION, AGGLUTINATION

7. THROMBOPOIESIS COUNT: 150000-400000 cells/cubic mm bld Location
adults - red bone marrow
Half life 4 days
Differentiation
hemocytoblasts (stem cells) develop into large megakaryocytes
megakaryocytes have lots of ER & Golgi
megakaryocyte pinches off tiny cytoplasmic packets (4000 plts /megak)

8. Megakaryocyte in bone marrow (note it has a large nucleus)8 8

9. In a stained blood smear platelets are often seen as eosinophilic clumps 9 9

10. REGULATION normally negative feedback homeostasis
primarily the liver and kidney releases thrombopoietin (TPO) to increase platelet production
liver disease leads to abnormally low platelet production from insufficient TPO
WBCs release an interleukin - also weakly stimulates platelet production

11. LIFE SPAN : 9-12 DAYS
ROLE OF SPLEEN:
STORAGE (25-40% sequestered )
DESTRUCTION
SPLENOMEGALY- increased destruction -THROMBOCYTOPENIA
SPLENECTOMY- THROMBOCYTOSIS

12. FUNCTIONS HAEMOSTASIS Definition:
SPONTANEOUS ARREST OF BLEEDING FROM SMALL INJURED VESSELS (arterioles, capillaries, venules)

13. FUNCTIONS A. HEMOSTASIS STEPS OF HEMOSTASIS: 1. VASOCONSTRICTION
2. PLATELET PLUG FORMATION
3. COAGULATION

14. Primary hemostasis : refers to the formation of a temporary hemostatic platelet plug that stops loss of blood
Secondary hemostasis (definitive hemostasis, or clotting): formation of a strong fibrin clot sealing the site of injury

15. VASOCONSTRICTION
DIRECT: LOCAL MYOGENIC SPASM (more the trauma more is the spasm eg: in crush injury)
REFLEX SYMPATHETIC
SEROTONIN, THROMBAXANE A2

16. 16 16

17. Platelet Function: Platelet Activation (is classically described using the following terms)
Process
Brief Description
Platelet activation
Platelets bound to vWF become ‘activated’ and release mediators including ADP, thrombin, serotonin, platelet activating factor, vWF.
They synthesize and release thromboxane (TXA2)
All these mediators promote platelet adhesion to vessel wall, and platelet aggregation (see next slide)
TXA2, thrombin and serotonin also cause vasoconstriction locally
Thrombin also activates the clotting cascade 17 17

18. Platelet Adhesion: Process Brief Description Platelet adhesion
Platelets adhere to subendothelial collagen;
This process requires von Willebrand factor (vWF);
vWF comes from injured endothelial cells and activates platelets;
Both subendothelial collagen and platelets have receptors that bind vWF; 18 18

19. Platelet Aggregation:
Process
Brief Description
Platelet aggregation
Platelets link up with each other forming a loose mass of platelets;
ADP, thrombin, TXA2, serotonin, platelet activating factor [PAF] from platelets all promote platelet aggregation
Fibrinogen (a clotting factor) cross links platelets
Platelet membranes have fibrinogen receptors
Note – platelet activation, adhesion and aggregation interact and reinforce each other 19 19

20. ADHESION OF PLTS TO COLLAGEN VIA vWF
DAMAGE TO VESSEL WALL
COLLAGEN GETS EXPOSED
ADHESION OF PLTS TO COLLAGEN VIA vWF
ACTIVATION OF PLATELETS
RELEASE OF ADP FROM
SECRETORY VESICLES
SYNTHESIS OF THROMBAXANE A2
FROM ARACHIDONIC ACID ON
PLASMA MEMBRANE
AGGREGATION OF PLATS
LOOSE PLT PLUG

21. Vessel damage, collagen exposed
Platelet activation
Discharge of chemical
mediators
Synthesis of
Thromboxane A2
Serotonin, thromboxane A2,
Thromboxane A2, ADP
Contraction of
Vascular smooth muscle
Platelet aggregation
Vasoconstriction
Platelet plug
Temporary arrest of bleeding

22. PLATELET PLUG attracted to & stick to damaged vessel walls
platelets enlarge & get sticky
platelets release attractant chemicals that attract more platelets
Positive Feedback Cycle
produces a bigger & faster reaction
platelets release chemicals that attract more platelets to site
How limits to injured site ?
ADP stimulates release of prostacyclin & nitric oxides, from intact endothelium prevents aggregation of platelets.

23. FUNCTIONS CONTD SEROTONIN- VASOCONSTRICTION
PLATELET PLUG- TEMPORARY SEALING OF VESSLES
PLATLET FACTOR 3- FOR CLOTTING
ACTIN, MYOSIN- CLOT RETRACTION
PDGF- WOUND HEALING & REPAIR OF ENDOTHELIUM

24. OTHER LESS IMP FUNCTIONS
PHAGOCYTOSIS OF:
DYES, CARBON, MICRO-ORGANISMS, OLD CELLS ETC.
AS ANTIGEN PRESENTING CELL (APC)
SECRETES VARIOUS CYTOKINES:
HELP IN HAEMOPOIESIS, FUNCTIONING OF NK CELLS ETC

26. THROMBOCYTOPENIA
CAUSES – Splenomegaly, infections, toxins, radiation, rare genetic diseases
OUTCOME - increased bruising (purpura) & increased risk of serious hemorrhaging
Thrombaesthenic purpura: due to weak platelets.

27. Consequence of thrombocytopenia
Prolongation of ‘bleeding time’ – excessive bleeding from relatively minor injuries..
However, ‘clotting time’ is normal (details later in Practicals)
Note extensive bruising (purpura) 27 27

28. BLOOD COAGULATION

29. CLOT TRANSFORMATION OF SOLUBLE BLOOD INTO SOLID GEL
CLOT OR THROMBUS CONSISTS OF INSOLUBLE PROTEIN POLYMER OF FIBRIN and TRAPPED BLOOD CELLS

30. WHAT PREVENTS INTRAVASCULAR CLOTTING ?
Physiologic anticlotting or antithrombic mechanisms prevent inappropriate or excess clotting are:
CLOTTING FACTORS ARE INACTIVE
DEGRDATION OF ACTIVATED CLOTTING FACTORS
CONTINUOUS MOTION OF BLOOD AT A NORMAL FLOW RATE

31. SMOOTH INTACT ENDOTHELIUM PGI2 FROM ENDOTHELIAL CELLS.
GLYCOCALYX
THROMOMODULIN RECEPTORS ON ENDOTHELIAL WALL
CIRCULATING ANTICOAGULANTS-Antithrombin III – heparin complex (inhibits IXa, Xa, XIa and XIIa)
FIBRINOLYTIC SYSTEM- Protein C – protein S complex inactivates Va, VIIIa and inactivates inhibitor of tPA

32. V Proaccelerin, labile factor VII Proconvertin
CLOTTING FACTORS
I Fibrinogen
II Prothrombin
III Tissue Factor
IV Calcium
V Proaccelerin, labile factor
VII Proconvertin

33. VIII Antihemophiliac factor A, antihemophilic globulin (AHG)
IX Christmas Factor, Antihemophilic factor B
X Stuart-Prower Factor
XI Plasma thromboplastin antecedent (PTA)
XII Hageman Factor
XIII fibrin stabilizing factor

34. OTHER FACTORS von Willebrand factor Protein C Protein S Thrombomodulin
Antithrombin III
Prekallikrein
High mol wt kininogens

35. SOME IMP FACTS ABOUT COAGULATION
inactive clotting factors present in blood at all times
most factors produced by liver or platelets
cascading effect
1 enzyme used to activate next enzyme in series --> greatly amplifies final step
Ca++ - used as an inorganic catalyst for many of these coagulation steps
2 PATHWAYS INITATE CLOTTING: Initiator is different for extrinsic & intrinsic pathway
Most clotting factors are produced in the liver
Vitamin K is required for the synthesis of clotting factors II, VII, IX and X

36. Mechanism of clotting:
inactive prothrombin converted into thrombin (active enzyme) by E & I pathway
Common pathway:
thrombin converts soluble plasma protein fibrinogen into insoluble fibrin
Positive Feedback
thrombin stimulates part of its own production pathway

37. INTRINSIC: ACTIVATION BY CELLULAR ELEMENTS WITHIN BLOOD
INITIATOR: EXPOSED COLLAGEN, CONTACT FACTOR, ELECTRONEGATIVE SURFACE (test tube or capillary tube)
EXTRINSIC: ACTIVATION BY CELLULAR ELEMENT OUTSIDE THE BLOOD (from tissue injury)
INITIATOR: TISSUE FACTOR

38. ‘a’ denotes activated form of the clotting factor38 38

39. The Clotting Cascade
Note details not contained in the previous slide; TPL – tissue thromboplastin; TFI – tissue factor inhibitor; ‘a’ denotes activated form of the factor. 39 39

40. INTRINSIC PATHWAY EXTRINSIC PATHWAY
COMMON PATHWAY 40 40

41. Intrinsic and Extrinsic Pathways compared
Intrinsic Pathway
Extrinsic Pathway
Stimulus for activation
Factor XIIa comes into contact with subendothelial collagen;
Electronegative surface if collected in glass tube
Blood vessel injury; tissue thromboplastin from tissues enters bloodstream and activates Factor VII
Why this name?
The entire cascade is intrinsic to the circulatory system and the vessel wall – thus this name
Called “extrinsic” because it is activated by a factor extrinsic to plasma (i.e., tissue thromboplastin)
Inhibitor
Thrombomodulin-thrombin complex and activated protein C
Tissue factor pathway inhibitor (TFPI) forms a complex with tissue thromboplastin and Factor VIIa and Factor Xa
Time
Longer of the two cascades;
aPTT is seconds
Shorter of the two cascades;
PT is seconds 41 41

43. CLOT RETRACTION Def - consolidation or tightening of clot
Method - fibrin threads contract/shrink
Benefits - pull damaged edges of vessel wall together, repair is faster; tighter, stronger clot
Timing - retraction occurs within min.

46. Learning outcomes
At the end of this class student should be able to
Define hemostasis.
Know the difference between primary hemostasis & definitive hemostasis.
Describe the structure & function of platelets
Normal count, factors regulating thrombopoiesis

47. Common causes of thrombocytoenia & thrombocytosis
Role of platelets in hemostasis
Clotting factors with their roman numerals & common names, & where they are produced from ?
Cascade of events in extrinsic & intrinsic mechanism of clotting, and initiating factors.
Role of vitamin K in clotting.