1. Hemostasis & Coagulation Ahmad Sh. Silmi Msc Hematology,FIBMS
Unit 1 : Primary Hemostasis

2. Homeostasis Vessels Coagulation Proteins Platelets
State of fluid equilibrium within the blood vessels
Vessels
Platelets
Fibrinolysis/Inhibitors
Coagulation Proteins

4. Hemostasis Arrest of bleeding Events preventing excessive blood loss
Vascular spasm: Vasoconstriction of damaged blood vessels
Platelet plug formation
Coagulation or blood clotting

5. Haemostasis: Vasoconstriction Platelet activation Haemostatic plug
Coagulation
Stable clot formation
Clot dissolution

6. 35

7. Primary hemostasis Involves: Vasoconstriction Platelet adhesion
Platelet secretion
Platelet aggregation

8. Vascular System Blood Vessels Arteries Veins Capillaries
Carry blood from the heart to capillaries
Thickest walls of the vasculature
Veins
Return blood from capillaries to the heart
Thinnest walls of vasculature
Capillaries
No vessel wall
Do not contribute to hemostasis

9. Vascular System: Blood Vessels
Construction
Endothelium
Single layer of endothelial cells, lining vessels
Coated by glycocalyx
Protects basement membrane
Produces Von Willebrand's factor (vWF), a part of Factor VIII
Secretes prostaglandins, plasminogen activators
Negatively charged, repels circulating proteins and platelets
Subendothelium
Smooth muscle and connective tissue with collagen fibers

10. Vascular System: Blood Vessels
Basement membrane
Collagen material – stimulates platelets
Connective tissue
Elastic fibers- provide support around vessels

12. Vascular System: Blood Vessels
Function
Endothelium
Controls vessel permeability
Controls blood flow rate
Produces and releases substances that inhibit OR stimulate platelets, coagulation and fibrinolysis
Subendothelium
Collagen within is whats exposed upon injury

13. Vascular Endothelium Products: Stimulators
Produces vonWillebrand factor (vWF)
Helps in platelet adhesion to collagen
Carries factor VIII
Tissue factor (TF) activates secondary hemostasis via extrinsic pathway
Tissue plasminogen activator (tPA) is released activating fibrinolysis

14. Vascular Endothelium Products: Inhibitors
Release of tPA activates release of plasminogen activator inhibitor (PAI-1) to inhibit fibrinolysis
Thromomodulin forms a complex with thrombin
Platelet aggregation via prostacyclin production

15. Vascular System: Function Following Injury
Initiate hemostasis
Vasoconstriction of the arterioles
Minimizes blood flow to injured area
Prevents blood loss
Immediate
Short-lived

16. Vasoconstriction Mechanism Neurogenic factors Regulatory substances
Prolong vasoconstriction
Serotonin ( made by platelet activation & endothelium)
Thromboxane A2 ( made by platelet activation & endothelium)
Endothelin-1 (made by damaged endothelial cells)

17. Vasoconstriction Vasodilation Counteracts Vasoconstriction
Endothelial cells
Prostaglandin (PGI2)/ Prostacyclin
Vasodilates to increase blood flow to bring fresh supplies of clotting substances
Inhibits platelet aggregation
Contraction of venules
Causes gaps between them which pushes fluids causing edema or swelling

18. Thought question…
Think about the last time you cut your finger with a piece of paper. Did your finger bleed immediately?
If not, what might have prevented the bleeding?

19. Answer..
No, the finger probably did not bleed immediately, due to vasoconstriction of the blood vessels

20. Discussion
What actions of the endothelial cells prevent clotting from occurring within the blood vessels?

21. Answers..
Since the endothelial lining has a negative charge, it normally repels coagulation proteins and platelets in the circulation. It synthesizes products that help to inhibit fibrin formation.

22. All About Platelets…
Second major component of the hemostatic system

23. Platelets What is a platelet? Small 2-3 µm Anuclear
Reddish-purple granules
Fragments of megakaryocyte cytoplasm

24. Platelets
Life span
9-10 days
Normal Range
x 109 /L

25. Platelet: Side note Seen in conditions with increased need and/or destruction
Giant platelets
Micromegakaryocytes= Dwarf Megs
May Hegglin anomaly, Bernard-Soulier syndrome, pregnancy, malignancy
Seen in malignant disorders such as CML and MDS

26. Anatomy of a Platelet
Peripheral zone: Responsible for platelet adhesion and aggregation
Glycocalyx:
Contains glycoprotein receptors:
GPIb binds von Willebrand’s factor needed for platelet adhesion to collagen
GPIIb/IIIa bind fibrinogen needed for aggregation
Bind ADP and thrombin, promoting aggregation
Factors I, V, VIII on surface, involved in 2o hemostasis
Plasma membrane:
Exposed on platelet activation
Layer called PF3 (platelet factor) surface for interaction of plasma coagulation factors
Initiation of formation of thromboxane A2. This stimulates aggregation and vasoconstriction

27. Anatomy of a Platelet
Structural or Sol-Gel zone: Responsible for platelet retraction/contraction functions and platelet shape
Microtubules
Cytoskeleton
Binding protein
Organelle zone: Responsible for storage and platelet release functions
Granules
Dense bodies, alpha granules, lysosomal granules and microperoxisomes
Mitochondria
Glycogen

28. Zone
PERIPHERAL
Glycocalyx
Phospholipid
Integral Proteins: Ib/IX, IIb/IIIa
Sodium ATPase
STRUCTURAL
Microtubules: Tubulin
Cytoskeleton: Actin/Myosin
ORGANELLE
Granules: Alpha & Dense
Enzymes: Lysosomes & Peroxisomes
MEMBRANE
Canalicular System – derived from DMS of megakaryocyte
Dense Tubular System – calcium storage

29. Platelet Receptors GPIb/IX – vWF GPIIb/IIIa – Fibrinogen
Required for PLT adhesion
GPIIb/IIIa – Fibrinogen
Required for PLT aggregation
Phospholipid (Pl)
Bind vitamin K dependent proteins , Ca++ dependent
Bind Va and VIIIa (called “PF3” in this context)

30. Platelet Granules ALPHA DENSE ATP ADP Serotonin Calcium Ions
Plasma Derived
Fibrinogen – PLT aggrgation Ig
Albumin
Megakaryocyte Derived
vWF – PLT adhesion, VIII carrier
Coagulation Factor V
Plasminogen – source of plasmin
PAI-1 – inhibits activation of fibrinolysis
a2-antiplasmin – plasmin inhibitor
PF4 – inactivates heparin
PLT-derived Growth Factor (PDGF) – tissue repair
Thrombospondin – PLT aggregate stabilizer
Fibronectin – PLT binding
ATP
ADP
Serotonin
Calcium Ions

32. Diagrammatic Representation of the Platelet

33. PLATELET STRUCTURE ADP, ATP, Ca2+, serotonin, etc.
PF4, ß-TG, PDGF, TGF-ß,
fibrinogen, VWF, etc.
ADP, ATP, Ca2+, serotonin, etc.

34. Production of Platelets
Made in Bone marrow
Need dictates the amount of platelets produced.
Stimulus for production is the platelet mass in circulating blood ~ 80 % and megakaryocyte mass in bone marrow
Originate from CFU-GEMM to form CFU-Meg
Cytokines and growth factors such as IL-3 and GM-CSF influences progenitor stages

35. Platelet Development Megakaryoblast 10-15 µm
Increased nuclear: cytoplasmic ratio
Promegakaryocyte
80 µm
Dense alpha and lysosomal granules
Basophilic megakaryocyte
Megakaryocyte

36. Production of Platelets
Precursor Cell= Megakaryocyte
Produces about platelets
Platelets are released via sinuses of bone marrow

37. Production of Platelets
Thrombopoietin (TPO)
Regulates platelet development
Influences all stages of megakaryocyte production
Produced in the liver, kidney and spleen

38. Production of Platelets
How does TPO work?
Maintains a constant number of platelets in peripheral blood by binding Mp1 (platelet receptor). Bound TPO can not stimulate proliferation of bone marrow progenitor cells
The higher the platelet count, the more TPO is bound and stimulation of bone marrow is decreased.

39. Thought question…
If a patient had a low platelet count what will happen?

40. Answer…
TPO increases the number of megakaryocytes in the bone marrow, increases size and DNA count of megakaryocytes and increases maturation rate

41. Function of Platelets Surveillance of blood vessel continuity
Checks endothelial lining for gaps and breaks
Fill-in small gaps caused by separation of endothelial cells
Formation of primary hemostatic plug
Surface for coagulation factors to make secondary hemostatic plug
Aid in healing injured tissue

42. Formation of Primary Hemostatic Plug
Once the platelets “normal” environment is changed, they become activated or adhesive
Three stages of plug formation:
Adhesion
Release reaction
Aggregation

43. Stage 1: Platelet Adhesion
It is the binding of platelet to non platelet surface: sub endothelial collagen
involves changes from a disc shape to a slightly broader, plate like form to increase surface area
a number of plasma proteins are required for normal platelet adhesion.
Thrombin
Fibronectin
vWF
vWF is the largest component of factor VIII and secreted by platelets and by vascular endothelial cells.

44. Adhesion cont’d Increase bond that seal platelet to the vessel wall
Collagen – vWF –Platelet
vWF bridge physical distance between platelate and sub endothelial collagen
Increase bond that seal platelet to the vessel wall
reversible
Adhesion

45. Adhesion is Reversible & No ADP is released.

46. Stage 1: Platelet Activation
Platelets undergo a shape change from disc to spiny sphere with projections
Activation required for 1O hemostatic plug formation
Activation continues until Ca ++ threshold met
Outcome
Activation of GPIIb/IIIa receptors for fibrinogen
Secretion of granules within platelets into tissues

47. Platelet Shape Change
George Lancet 2000; 355:1531

48. Stage 2: Platelet Secretion & Release
Requires ATP
It is release of contents of the granules of platelet
Primarily ADP  stimulates aggregation
Cathecolamine (especially epinephrine) and serotonin enhance vasoconstriction
Granules trigger a secondary aggregation which is irreversible

49. Platelets contain 3 types of secretary granules:
Lysosome containing acid hydrolyses
α-granules containing platelate specific proteins (Plt factor 4, β- thromboglobulin, as well as other proteins such as Platelet derived growth factor and coagulation proteins found in plasma (fibrinogen & von Willebrand’s factor)
-granules containing ATP, ADP, Calcium & serotonin

50. Steps in platelet plug formation: Extension
Figure 1B

51. Granules con’t
Factor V: receptor on platelet surface for factor Xa & prothrombin
PF4: heparin neutralizing factor
ADP: agonist, continues to recruit and stimulate platelets by increasing cytoplasmic calcium

52. Steps in platelet plug formation: Perpetuation
Figure 1C

53. Stage 3: Platelet aggregation
Chemical changes cause platelets to aggregate and stick to one another
Newly arriving platelets become activated by agonists
Aggregation is triggered by:
PLT factor 3,
adenosine diphosphate (ADP),
Thrombin and
Thromboxane A2

54. Exposure of GPIIb/IIIa sites bind fibrinogen
Fibrinogen + activated platelets serves as a bridge between two platelets
Calcium must be present
Activated platelet membrane generates TXA2
TXA2 stimulates release
Irreversible once platelet aggregate they do not disaggregate.

55. Platelates shed membranes rich in phospholipid (appearance of PLT factor 3 on the PLT membrane)
this happens during PLT plug formation
serves as a catalytic site for the coagulation proteins
Aggregation is also a response to helps initiation of the coagulation mechanism

56. Aggregation cont’d
platelet aggregation

57. Arachidonic acid Cyclo-oxygenase PLATELET ENDOTHELIAL CELL Thromboxane
Cyclic Endoperoxides
PLATELET
ENDOTHELIAL CELL
Thromboxane
synthetase
Prostacyclin
synthetase
Prostacyclin
(PGI2)
Thromboxane
(TxA2)
Inhibits plt aggregation
Vasodilator
Enhances plt aggregation
Vasoconstrictor

58. Aggregated Platelets Resting Platelet GPIIb/IIIa (inactive) GPIIb/IIIa
Agonist
(activated)
e.g. ADP
TXA2
thrombin
Fibrinogen
high shear: von Willebrand factor substitutes for fibrinogen

59. Fibrinogen binding to Glycoprotein IIb-IIIa on activated platelets
Platelet Aggregation
Fibrinogen binding to Glycoprotein IIb-IIIa on activated platelets

60. Lets have a close view about the mechanism

61. Platelet Activation Pathways (1)
ADP
Adrenaline
COLLAGEN
THROMBIN
ADP
GpIIb/IIIa
Aggregation
GpIIb/IIIa
Aggregation
GpIIb/IIIa
Aggregation
GpIIb/IIIa
Adhesion
Platelet
GpIb
Adrenaline
Adhesion
vWF
Exposed Collagen
Endothelium

62. What happens when you have an injured blood vessel?
Endothelial cells
Coagulation factors…
Platelet
Exposed sub-endothelium
Tissue factor (TF)

63. How does a platelet plug a hole under high shear stress conditions?
-GP Ib binds the exposed vWF on sub-endothelial cells
Platelet
Platelets roll on vWF and become activated…
vWF

64. Platelet activation Thrombin Agonist eg. thrombin Negative charge
(Priming and propagation (Intrinsic pathway) of coagulation)
Agonist
eg. thrombin
Negative charge
Activates the coagulation factors and propagates the production of higher levels of thrombin required to support haemostasis PS PS
Upon platelet activation, phosphatidyl serine (PS)
flips to the outer leaflet of the plasma membrane. PS PS
Activation PS PS
Thrombin
Fibrinogen
Fibrin

65. Platelet activation (continued from previous slide) also results in the release of the constituents of the alpha and dense granules
Activation of aIIbb3 allows
binding to fibrinogen
Alpha granules
P selectin (tm)
-fibrinogen
-PDGF
-VWF
ADP
COX AA
TXA2
Aspirin, inactivates cyclooxygenase (COX)
Activates platelets
Thromboxane A2
Arachidonic acid (AA)

66. Platelet activation (cont.):
- aIIbb3 becomes activated and capable of binding fibrinogen
Fibrinogen
The co-crosslinking
of two or more platelets
by aIIbb3 and fibrinogen
is termed ‘aggregation’
Platelet

67. Side note
Heparin is used on patients who clot excessively. Endothelial cells make heparin-like molecules and expose them on their surface. PF4 binds these substances. Heparin can complex with bound PF4 and heparin will be neutralized.

68. Final Stage : Stabilization of Clot
AKA: primary hemostatic plug formation
Thrombus formation
Platelets release Factor V
Expose factor III, accelerating coagulation cascade
Promote activation of clotting factors

69. Platelet System: Additional Functions
Provides the reaction surface for some coagulation system reactions, as well as platelet factor 3 (PF3) which is platelet phospholipid
Supports and maintains endothelial lining
Defective hemostasis can occur due to
decreased number of platelets (quantitative)
abnormally functioning platelets (qualitative)

70. Factors that activate platelets
ADP
Thrombin
Collagen
5-hydroxytryptamine (serotonin)
Thromboxane A2
Mechanical stimuli
Many stimuli
Several different receptors
Multiple signalling pathways

71. Agonists
“a term used to describe substances that can attach to a platelet membrane receptor and activate platelets causing them to aggregate .”
McKenzie, SB. Clinical Hematology, Glossary, p. 919

72. Agonists agonists produced ADP Collagen Thrombin Epinephrine
Arachidonic acid
Thromboxane A2

73.                                          
                                                                                                                                                                                                                                      
Slide 23 of 79

74. Blood clot