Coagulation: In medicine, the clotting of blood. The process by which the blood clots to form solid masses, or clots. More than 30 types of cells and substances in blood affect clotting. The process is initiated by blood platelets. Platelets produce a substance that combines with calcium ions in the blood to form thromboplastin, which in turn converts the protein prothrombin into thrombin in a complex series of reactions. Thrombin, a proteolytic enzyme, converts fibrinogen, a protein substance, into fibrin, an insoluble protein that forms an intricate network of minute threadlike structures called fibrils and causes the blood plasma to gel. The blood cells and plasma are enmeshed in the network of fibrils to form the clot.

1. Overview of Primary Hemostasis
Endothelium and Platelets
Lecture 1

2. Hemostasis/Coagulation
•
Hemostasis
•
Coagulation
•
Components
–
Blood vessels
–
Platelets
–
Coagulation proteins
–
Serine protease inhibitors
–
Fibrinolysis
–
Kinin system
–
Complement system
•Hemostasis
–Primary
•Formation of the “platelet plug”
•Initially halts the loss of blood
–Secondary
•Reinforcement of the platelet plug by fibrin formation
Balance between bleeding and clotting
Major components
Minor components 2

3. Hemostasis
•
Primary Hemostasis
–
Platelets interact with damaged endothelium
–
Primary hemostatic plug to arrest bleeding (primary platelet plug)
–
Fragile and easily dislodges
•Secondary Hemostasis
–Formation of insoluble fibrin strands leading to a more stable clot
–Secondary hemostatic plug (secondary platelet plug)
–Allows healing of the damaged tissue
–Involves the coagulation cascade proteins that interact with each other and the platelet plug
•Fibrinolysis
–Once healing has occurred  the clot has to be lysed to prevent occlusion of vessel 3
Fibrin
RBC
Platelet

4. Primary Hemostasis

Primary hemostasis

Defined by platelet adhesion to exposed collagen within the endothelium of the vessel wall

Platelet adhesion is mediated by gpIb and vWF

These reactions result in initial clot formation, however the clot that forms is reversible and unstable 4

5. Secondary Hemostasis

Involves the enzymatic activation of the coagulation proteins to
1.
Generate thrombin
2.
Convert fibrinogen to fibrin

Fibrin cooperates in the formation of a nonreversible, stable clot

The stable clot will then be digested proteolytically via fibrinolysis

Tissue repair and would healing occur as a result of the release of PDGF 5

6. Vascular System 6
•
Endothelial cells play a key role in hemostasis
•
Innermost lining of blood vessels
–
Blood vessels contain
1.
Tunica intima
2.
Tunica media
3.
Tunica adventitia
–
EC’s form a monolayer of metabolically active cells
–
Joined end-to-end providing a smooth surface
1.
Procoagulant role – induce activation of coagulation cascade, platelet adhesion
2.
Anticoagulant role – prevent platelet activation
–
VS contains an elastin-rich internal elastic lamina surrounded by layers of connective tissue
•
Fibroblasts, adipocytes, smooth muscle cells, elastin, collagen
a)
Veins  collagen and fibroblasts
b)
Arteries  collagen, fibroblasts, smooth muscle cells

7. Capillaries 7
The smallest and most numerous of the vessels
1.
Every cell in the body is within 0.13 mm of a capillary
2.
Involved in metabolic exchange between blood and tissues
3.
Lumen of the capillary is surrounded by a single endothelial cell
–
Just large enough for a single RBC or WBC to pass through
McKenzie/Pearson Education 2010

8. Function of Endothelial Cells
When the endothelial lining is disrupted
1.
Vascular system acts to prevent bleeding  promotion of rapid vasoconstriction of the injured vessel and adjacent vessels
2.
Diverts blood flow around the damaged vessels
3.
Enhances contact activation of platelets with coagulation factors
How is this done?
1.Vasoconstriction and reflex stimulation (Vagus nerve)
Diverts flow of blood around the damaged vessel
2.Forms a surface for initiation of contact activation of platelets (collagen)
Subsequent adhesion, release reaction, and aggregation of platelets
3.Platelet plug enhances contact activation of the coagulation proteins
Leads to generation of thrombin and fibrin formation
8

9. Functions of Endothelial Cells
•
Procoagulant functions
–
Secrete endothelin
–
Secrete von Willebrand factor (vWF)
–
Allow for the expression of tissue factor
–
Allow for expression of plasminogen activator inhibitor
•
Anticoagulant functions
–
Rhomboid shape
–
Secrete PGI2 (prostacyclin)
–
Release ADPase
–
Secrete NO (nitric oxide)
–
Contain glycosaminoglycans
a.
Chondroitin sulfate
b.
Dermatin sulfate
c.
Heparan sulfate
–
Secrete TFPI (tissue factor pathway inhibitor)
–
Secrete TM (thrombomodulin)
Hemostatic properties Thrombogenic
Hemostatic properties Anti-thrombogenic
Interact and enhance Antithrombin (AT)

10. Functions of Endothelial Cells

Non-hemostatic functions
1.
Selective blood/tissue barrier
•
Keeps cells and macromolecules in vessels
•
Allows nutrient and gas exchange
2.
Processing of blood-borne antigens
•
Contribute to cellular immunity
3.
Synthesis and secretion of connective tissue
•
Basement membrane collagen
–
Provides backup protection for endothelial cells
–
Collagen of the matrix—responsible for platelet adhesion 10

11. Overview of Endothelial Cell Function Hoffman
11

12. Platelets
•
Small, anucleated blood cells
•
Life span 7-10 days
•
Circulate as discoid-shaped structures
•
2.5 - 3 μm diameter, 1 μm thick, MPV 8-10
•
Produced in the bone marrow from megakaryocytes
•
MGKs – polyploid cells, 30-50 μm diameter
–
Repeated rounds of endomitosis — increasing rounds DNA division without cellular proliferation
–
Largest cells in the BM
•
Abundant granular cytoplasm
•
Large multilobated nuclei
•
Polyploid DNA content may reach 32- 64 times normal diploid cell
•
MGK’s descend from pluripotent hematopoietic progenitors—CFU-GEMM

13. Megakaryocytopoiesis

Arise from megakaryocytes in the bone marrow

Megakaryocytes largest cells in bm—30-50 μm diameter— polyploid

Megakaryocytes derived from CFU-GEMM

Differentiation under influence of thrombopoietin (TPO)

3 MGK lineage-committed progenitor stages

BFU-Meg, CFU-Meg, LD-CFU-Meg 13

14. •
Proliferative stage
–
Three megakaryocyte lineage-committed progenitor stages
•
Megakaryocyte-erythrocyte progenitor
•
BFU-Meg
•
CFU-Meg – give rise to terminal differentiation
•
Terminal differentiation stage
–
MK-1  MK-4

15. Growth Factors in Thrombopoiesis
•
TPO – thrombopoietin
–
Binds to a platelet membrane receptor protein called mpl (CD110)
–
Produced in liver, kidney, spleen
–
Influences all stages of megakaryocyte production and regulates platelet development
–
Thrombocytosis  TPO binds to platelets  less TPO free in the plasma  reduces stimulation of MGK precursors
–
Thrombocytopenia  there is more TPO in the circulation  greater stimulation of MGK precursors  increases in platelet release
•
Other growth factors
–
SCF
–
IL-3
–
IL-6
–
IL-11

16. Megakaryocytopoiesis

Megakaryoblast—MK I

10-15 μ diameter

High N:C ratio

Scanty, blue cytoplasm, no granules

Promegakaryocyte—MK II

Enlarges to 80 μ

Granules formed in Golgi (dense, alpha, lysosomal)

Megakaryocyte—MK III

Basophilic megakaryocyte

Distinct granulation

Cytoplasmic lines of demarcation  outlining individual cytoplasmic fragments that will be released as platelets

System of microtubules, canals, and cytoplasmic granules

Glycogen stores  help sustain platelets for 9-11 days

Megakaryocyte—MK IV

Final stage of maturation

Releases cytoplasmic fragments (platelets) through marrow sinusoid fenestrations  budding or shedding of platelets

Once platelets are released the naked nuclei are phagocytized by marrow histiocytes 16

17. Platelet Ultrastructure
•
Platelet ultrastructure divided in 4 arbitrary zone
a.
Peripheral zone
b.
Structural zone
c.
Organelle zone
d.
Membranous zone 17
McKenzie/Pearson Education 2010

18. Peripheral Zone
•
Outer-most layer consisting of
–
Glycocalyx
•
Amorphous, spongy exterior coat
•
Adsorbed plasma proteins, glycolipids, glycoproteins, proteoglycans
–
Protein membrane
•
Bilipid layer composed of lipids, glycoproteins, and proteoglycans
•
Maintain cytoplasmic integrity and mediates platelet function
•
Adsorbed plasma proteins, glycolipids, glycoproteins, proteoglycans
–
Open canalicular system (OCS)
•
System of invaginations of the platelet membrane
•Function
1.Transports stored products from interior of the platelet to the exterior upon activation
2.Provides increased surface area for absorption and storage of proteins (coagulation factors) from the plasma

19. The Peripheral Zone

Membrane—phospholipid bilayer

Phosphatidylserine (PS)

Phosphatidylethanolamine (PE)

Phosphatidylinositol (PI)

Phosphatidylcholine (PC)

Sphingomyelin

Phosphatidylinositol is precursor of arachidonic acid

Arachidonic Acid—unsaturated fatty acid –major component of the phospholipid membrane—precursor of Thromboxane A2 (TXA2)
During platelet activation  there is a translocation of polar phospholipids (phosphatidylserine) from the inner to the outer surface of the plasma membrane
Provides a surface for binding for coagulation complexes and formation of fibrin
NEGATIVE Charge—inner leaf
NEUTRAL Charge—outer leaf 19

20. Structural Zone
•
Organized network of protein filaments
–
Maintain shape of resting platelet—discoid
–
Shape change –> platelet activation—spherical
•
Submembraneous cytoskeleton
•
3 principle types of filaments
A.
Microtubules
–
Located beneath the cell membrane of resting platelet
–
Maintain discoid shape
B.
Microfilaments
–
Mediate contractile events
C.
Intermediate filaments

21. Organelle Zone

Responsible for metabolic activity of platelets

Mitochondria are present

Lacks nucleus, Golgi apparatus, and RER

Contain 3 types of granules

Alpha granules—20-200/plt, most numerous

Dense Bodies—10-20/plt

Lysosomal Granules

Dense Tubular System—DTS
1.
Sequesters calcium
–
Triggers platelet contraction
–
Subsequent internal activation of platelets
–
Calcium functions in the coagulation protein reactions
2.
Site of thromboxane synthesis (TXA2) 21

22. Platelet Granules

Dense bodies—2 to 10/platelet

ADP

Promotes platelet aggregation

ATP

Energy source

GTP—signal transduction

GDP—signal transduction

Calcium

Primary and secondary messenger regulating platelet activation and aggregation

Serotonin – vasoconstrictor
–Appear more dense under electron microscopy than the surrounding structures
–ADP released from dense bodies perpetuates the aggregation process by attracting additional platelets 22

23. Alpha Granules

Alpha granules—20 to 200/platelet

PF4 (platelet factor 4)

Neutralized heparin

β-TG (β -thromboglobulin)

Promotes smooth muscle growth for vessel repair

PDGF (platelet derived growth factor)

Promotes smooth muscle growth

Involved in atherosclerosis and lipid metabolism

Thrombospondin

Promotes platelet-to-platelet interaction

Mediates cell-to-cell interaction 23

24. Platelet Granules
•
Lysosomes
–
Contain enzymes known as hydrolases (glycosidases, proteases)
•
Neutral proteases, acid hydrolases, antibacterial enzymes
–
Act to digest materials brought into the platelet by endocytosis
•
Mitochondria
–
Few mitochondria are present in the platelet
–
Power house of the platelet 24

25. Membrane System
•
Open canalicular system (OCS)
–
Surface connected canalicular system (SCCS)
–
Invagination of platelet plasma membrane
–
System of channels
•
Network throughout the entire cytoplasm
•
Functions
1.
Secretion of platelet granule content
»
Content of granules fuse with OCS during platelet activation
2.
Uptake of plasma proteins
3.
Contributes extra membrane material to surface membrane during platelet activation 25

26. Membrane System
•
Dense tubular system
–
Originates from MK ER
–
Composed of channels located near OCS
–
Site of
•
Ca2+ storage
–
Important for triggering contraction of actinomysin
•
Several enzymes, ATPases, cyclooxygenase (PG synthesis)
26

27. Function of Platelets
Role of Platelets in the Circulation
1.
Surveillance of blood vessel continuity
–
Checks endothelial lining for gaps and breaks
–
Fill-in small gaps caused by separation of endothelial cells
2.
Formation of primary hemostatic plug
3.
Surface for coagulation factors to make secondary hemostatic plug
4.
Aid in healing injured tissue
Platelet Function
1.
Adhesion
2.
Activation and shape change
3.
Secretion or release
4.
Aggregation 27

28. What is von Willebrand Factor (vWF)
•
Large multimeric protein synthesized in the endothelial cells and megakaryocytes
•
It is constitutively synthesized by the endothelial cells
•
It is present in the:

Subendothelium

Plasma

Alpha granules of platelets

Weibel-Palade bodies
•
The vWF present in plasma is of endothelial origin
•
Function of vWF

Mediate platelet adhesion to the collagen in the subendothelium

Bind Factor VIII to protect it from proteolysis in the plasma 28

29. Platelet Adhesion
•
Adhesion
–
Initial response to vessel wall injury
–
Independent of platelet activation
–
Passive (no energy required)
•
Does not require Ca2+
•
Mechanism of platelet adhesion
1.
Exposure of subendothelium
2.
vWF:GPIb/IX interaction mediates binding to collagen at high shear rates
3.
Gp Ia/IIa and GPVI mediate direct binding to collagen at low shear rates
4.
vWF induces signaling events that include:
a.
↑ cytosolic Ca2+
b.
Activation of protein kinase C (PKC)
c.
Phosphorylation events 29
McKenzie, 2E
GPIb/IX/V

30. Platelet Adhesion

31. Multistep Platelet Adhesion to Collagen Hoffman
1.
Injured vascular wall  vWF adheres to subendothelial collagen 
2.
vWF binds to GPIb/V/IX 
3.
Rapidly formed bonds are quickly broken and reestablished 
4.
Causes the platelet to roll along the injured vessel
5.
Rolling process slows down the platelet
6.
Allows platelet signaling receptor GPVI to bind to collagen
7.
Leads to a cascade of signals  activation of GPIIb/IIIa  platelet aggregation
31

32. Platelet Shape Change
•
Platelet shape change occurs simultaneously with platelet activation
1.
Flattened disc  spiny sphere with long pseudopod projections
2.
Reorganization of proteins in structural zone
•
Microtubules contract

Concentration of organelles in center of cell close to OCS

Increases platelet surface area
•
Biochemical reactions during shape change 
–
Membrane GP alteration
•
GPIb/IX ↓
•
GPIIb/IIIa ↑
–
Granule secretion
–
Converts platelet from “adhesive” state to an “aggregation” state
32
McKenzie, 2E

33. Platelet Secretion
•
Platelet secretion
1.
ATP required for centralization of organelles
2.
Release of granule contents into OCS 
•
Recruitment and activation of additional platelets from the circulation
•
Released products promote secondary hemostasis and repair
–
ADP, serotonin → platelet activation
–
Ca2+ → activation, assembly of plasma coagulation factors
–
Growth factors & mitogens → stimulate vessel wall repair 33
Resting platelet
Activated platelet

34. Platelet Aggregation
•
Platelet aggregation is absolutely dependent on activation of GPIIb/IIIa receptor complex
•
Primary functions of GPIIb/IIIa
1.
Receptor for fibrinogen (low shear)
2.
vWF (high shear)
–
Requires Ca2+ + ATP
–
Activation  up-regulation of GPIIb/IIIa receptors on to the platelet membrane
–
Triggered by a number of agonists
•
ADP, TXA2, Collagen, Epinephrine, Thrombin, Arachidonic Acid 34
McKenzie, 2E
GPIIb/IIIa

35. Platelet Aggregation
•
Agonists
–
Collagen, thrombin, arachidonic acid 
–
Stimulate release of arachidonic acid from membrane phospholipids by PLA2
•
PC/PE + PLA2  AA
•
AA + COX (cyclo-oxygenase)  PGG2 (PGH2)
•
(Platelets)
•
PGG2 + thromboxane synthetase  TXA2 (thromboxane A2)
•
TXA2  platelet aggregation
•
TXA2  TXB2
•
(Endothelial cells)
•
PGG2 + PGI2 (prostacyclin synthetase)
–
PGI2 inhibits platelet aggregation
Platelets
Endothelial cells

36. The Cyclooxygenase (Eicosanoid) Pathway in the Endothelial Cell
•
TXA2 stimulates platelet aggregation by decreasing cAMP  calcium is released from the DTS  stimulating platelet aggregation and secondary hemostasis
•
PGI2 inhibits platelet aggregation by increasing cAMP  movement of calcium into DTS

This inhibits platelet activation
Platelets vs. Endothelial Cells
36

37. Platelet Biochemical Pathway 37

38. Platelet Signal Transduction
•
Agonists bind to receptors on the platelet surface
1.
Platelets become activated
2.
Initiates signaling events within the platelets
•
Lead to reorganization of the platelet cytoskeleton  granule secretion and aggregation
•
G-proteins – αβγ-heterotrimers that bind guanosine diphosphate (GDP) when inactive
–
Membrane receptor-ligand (agonists) binding promotes GDP release and its replacement with GTP
•
Major player in
–
Platelet activation
–
Signal transduction
–
Platelet aggregation
38
Platelet Adhesion
↓
Activating signals
(GPΙα/ΙΙα, GPVI)
↓
Platelet shape change
Release ADP, TXA2
↓
Recruit additional platelets
↓
Platelet aggregation
(GPIIb/IIIa:fibrinogen)
↓
Platelet plug stabilized by fibrin

39. Mechanism of Signal Transduction in Platelet Activation
1.
Thrombin binds to specific receptors on platelet surface (PARs)
2.
PAR receptors coupled to G-proteins which activate phospholipase-C
3.
PLC hydrolyzes (PIP2) [phosphatidy-inositol-4,5- bisphosphate]  formation of
A.
(IP3) [inositol-1,4,5-triphosphate]

Induces the release of intracellular Ca2+ 

Activation of PLA2 (phospholipase A2) 

Releases of AA
B.
(DAG) (diacylglycerol)

Activates PKC (protein kinase C)  phosphorylates proteins
39

40. Steps in Vascular Damage and Clotting Responses
1.
Vascular Spasm
•
Contraction of smooth muscle in arteriole walls to reduce blood flow
2.
Platelet plug formation
•
Platelet adhesion
o
Platelets contact and stick to free collagen fibers of the damaged blood vessel
3.
Platelet release reaction
o
Activated platelets extend many projections that enable them to contact and interact with other platelets and liberate their granule content
o
Liberated ADP and TXA2 help activate other platelets
o
Serotonin and TXA2 function as vasoconstrictors helping to decrease blood flow