1. Hemostasis & Coagulation Ahmad Sh. Silmi
Unit 1 : Primary Hemostasis

2. Hemostasis Heme= blood stasis= to halt
Process of retaining blood within the vascular system
Repairs injury to blood vessels
Stops or prevents blood loss

3. Balance of Hemostasis Procoagulant Factors Regulatory Factors Fibrin
Fibrinogen
Procoagulant Factors
Regulatory Factors
Fibrin
Balance of Hemostasis
*Balance of bleeding (hemorrhaging) and clotting (thrombosis)
*Imbalance in one direction can lead to:
bleeding : hypocoagulable state OR
thrombosis: hypercoagulable state

4. Hemostasis Components Vascular System Platelet System
Controls rate of blood flow
Platelet System
Interaction of vasculature and platelets form a temporary plug
Coagulation System
(i.e) fibrin forming
Fibrinolytic System
Fibrin lysing
Coagulation Inhibition System
Natural inhibitors
Control fibrin formation and fibrin lysis

5. Failure or deficiencies in any of these five systems can leads to varying degrees of uncontrolled hemorrhaging or clotting

6. Hemostasis
The hemostatic components remain inert in the presence of intact vascular tissue or endothelium
Following injury, each component must function optimally.

7. Hemostasis: Overview Consists of three stages
Primary Hemostasis
Process of blood clotting in response to injury where blood vessels (vasculature) and platelets are the main “players.”
Primary Hemostatic plug is formed
Platelet plug temporarily arrests bleeding. Insoluble fibrin strands deposit on the initial plug to reinforce and stabilize. The fibrin originates from soluble plasma proteins.
Secondary Hemostasis
Actions of the protein coagulation factors form fibrin in response to injury
At this time, blood has changed into a solid state
Fibrinolysis
Clot is removed following healing of wound

9. 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

10. 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

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

13. 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

14. 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

15. 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

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

17. 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)

18. 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

19. 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?

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

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

22. 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.

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

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

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

26. 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

27. 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

28. 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

29. 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

30. Platelet Receptors Phospholipid (Pl) GPIb/IX – vWF
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)

31. 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

33. Diagrammatic Representation of the Platelet

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

35. 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

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

37. Production of Platelets
Precursor Cell= Megakaryocyte
Produces about 2000 platelets
Platelets are released via sinuses of bone marrow

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

39. 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.

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

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

42. 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

43. Formation of Primary Hemostatic Plug
Once the platelets “normal” environment is changed, they become activated or adhesive
Three stages of plug formation

44. Stage 1: Platelet Adhesion
Platelets attach to non-platelet surfaces, such as collagen fibers in the subendothelium
Platelets move from the blood vessels and into the tissues.
Exposure to surfaces in the tissues causes them to bind to collagen with the presence of von Willebrand factor ( vWF) and Glycoprotein IbIX, making a bridge formation, which triggers a shape change
Reversible
No ADP released

45. 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

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

47. Stage 2: Platelet aggregation
Chemical changes cause platelets to aggregate and stick to one another
Newly arriving platelets become activated by agonists
Exposure of GPIIb/IIIa sites bind fibrinogen
Fibrinogen + activated platelets serves as a bridge between two platelets
Calcium must be present

48. Activated platelet membrane generates TXA2
TXA2 stimulates release

49. 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

50. 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

51. Stage 3: Platelet Secretion & Release
Requires ATP
Platelets release contents of their granules, causing vasoconstriction
Granules trigger a secondary aggregation which is irreversible
Granules consist of
Alpha granules: Factor V, Factor VIII:vWF, Fibrinogen, α2-antiplasmin, platelet factor 4
Dense bodies: ATP, ADP, serotonin, Ca

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

53. 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

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

55. Lets have a close view about the mechanism

56. 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

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

58. 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

59. 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

60. 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)

61. 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

62. 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.

63. 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

64. 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)

65. Platelet Function Scout for vascular damage
Form platelet plug of damaged tissue
Adhesion
Aggregation
Secretion
Phospholipid reaction surface for coagulation
Tissue repair
Graphic accessed at URL

66. 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

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

68. Blood clot

69. Coagulation System
Composed of 14 coagulation factors (serine proteases) which are interdependent (Factors I through XIII – there is no Factor VI – and PK and HMWK)
Inactive form of each is an enzyme precursor which is usually designated by a Roman numeral but also given a name – Ex. Factor I fibrinogen. Numbers correspond to order of discovery NOT order in cascade.
Active forms are usually designated by the letter “a” after the Roman numeral and may also have a different name – Ex. Ia Fibrin
Cofactors are needed for many reactions in the cascade – Ex. Calcium, platelet factor 3 (PF3)
Each molecule must be present in sufficient quantity as well as functioning normally
Final product is fibrin mesh or clot which completely stops bleeding
Secondary hemostasis
Slow contraction and lysis of the clot occurs

70. Fibrinolytic System Plasminogen is converted to plasmin
Plasmin enzymatically attacks the fibrin molecule producing fibrin degradation products (FDPs, sometimes called FSPs) that are cleared from the circulation by macrophages
Fibrin is a product formed during hemostasis, tissue repair or inflammation
Fibrin plays a temporary role
Once injury heals, the fibrin clot is lysed

71. Coagulation Inhibition System
Provides balance and control of clotting mechanisms
Natural inhibitors and anticoagulants circulate in the plasma to:
Prevent clotting when it’s not needed
Limit or localize the clotting that is needed
Examples: Protein C and S, antithrombin III