Overview of Primary Hemostasis

Endothelium and Platelets Lecture 1

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

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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

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Fibrin

RBC Platelet

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

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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

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Vascular System

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• 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

Capillaries

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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

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

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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)

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

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Overview of Endothelial Cell Function Hoffman

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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

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

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• 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

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

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

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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

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

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

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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

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)

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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

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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

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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

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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

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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)

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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

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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

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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

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McKenzie, 2E

GPIb/IX/V

Platelet Adhesion

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

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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

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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

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Resting platelet Activated platelet

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

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McKenzie, 2E

GPIIb/IIIa

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

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

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Platelet Biochemical Pathway

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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

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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

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

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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