Unit 1Unit 1 : Primary : Primary HemostasisHemostasis

State of fluid equilibrium within the blood vesselsVessels

Platelets

Fibrinolysis/Inhibitors

Coagulation Proteins

Hemostasis

Arrest of bleedingEvents preventing excessive blood loss

Vascular spasm: Vasoconstriction of damaged blood vessels

Platelet plug formation Coagulation or blood clotting

Haemostasis:Vasoconstriction Platelet activationHaemostatic plugCoagulationStable clot

formationClot dissolution

Primary hemostasisInvolves:

VasoconstrictionPlatelet adhesionPlatelet secretion Platelet aggregation

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Vascular SystemBlood Vessels

Arteries 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

Vascular System: Blood VesselsConstruction

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

plateletsSubendothelium

Smooth muscle and connective tissue with collagen fibers

Vascular System: Blood Vessels

Basement membrane Collagen material – stimulates platelets

Connective tissue Elastic fibers- provide support around vessels

Vascular System: Blood VesselsFunction

Endothelium Controls vessel permeability Controls blood flow rate Produces and releases substances that inhibit OR

stimulate platelets, coagulation and fibrinolysisSubendothelium

Collagen within is whats exposed upon injury

Vascular Endothelium Products:StimulatorsProduces 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

Vascular Endothelium Products:InhibitorsRelease of tPA activates release of

plasminogen activator inhibitor (PAI-1) to inhibit fibrinolysis

Thromomodulin forms a complex with thrombin

Platelet aggregation via prostacyclin production

Vascular System: Function Following InjuryInitiate hemostasis

Vasoconstriction of the arterioles Minimizes blood flow to injured area Prevents blood loss Immediate Short-lived

VasoconstrictionMechanism

Neurogenic factorsRegulatory substances

Prolong vasoconstriction Serotonin ( made by platelet activation &

endothelium) Thromboxane A2 ( made by platelet activation &

endothelium) Endothelin-1 (made by damaged endothelial cells)

VasoconstrictionVasodilation 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

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?

Answer..No, the finger probably did not bleed

immediately, due to vasoconstriction of the blood vessels

DiscussionWhat actions of the endothelial cells prevent

clotting from occurring within the blood vessels?

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.

All About Platelets…Second major component of the hemostatic

system

PlateletsWhat is a platelet?

Small 2-3 µmAnuclearReddish-purple

granulesFragments of

megakaryocyte cytoplasm

PlateletsLife span

9-10 daysNormal Range

150-440 x 109 /L

Platelet: Side noteSeen 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

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

Anatomy of a Platelet Structural or Sol-Gel zone: Responsible for platelet

retraction/contraction functions and platelet shape Microtubules Cytoskeleton Binding proteinOrganelle zone: Responsible for storage and platelet

release functions Granules

Dense bodies, alpha granules, lysosomal granules and microperoxisomes

Mitochondria Glycogen

Zone

PERIPHERAL

GlycocalyxPhospholipidIntegral Proteins: Ib/IX, IIb/IIIaSodium ATPase

STRUCTURAL

Microtubules: TubulinCytoskeleton: Actin/Myosin

ORGANELLE

Granules: Alpha & DenseEnzymes: Lysosomes & Peroxisomes

MEMBRANE

Canalicular System – derived from DMS of megakaryocyteDense Tubular System – calcium storage

Platelet ReceptorsGPIb/IX – vWF

Required for PLT adhesion

GPIIb/IIIa – FibrinogenRequired for PLT aggregation

Phospholipid (Pl)Bind vitamin K dependent proteins , Ca++

dependentBind Va and VIIIa (called “PF3” in this

context)

Platelet GranulesALPHA 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

DENSE

ATPADPSerotoninCalcium Ions

Diagrammatic Representation of the Platelet

PF4, ß-TG, PDGF, TGF-ß,fibrinogen, VWF, etc.

ADP, ATP, Ca2+, serotonin, etc.

PLATELET STRUCTURE

Production of PlateletsMade in Bone marrowNeed 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-MegCytokines and growth factors such as IL-3 and

GM-CSF influences progenitor stages

Platelet DevelopmentMegakaryoblast

10-15 µm Increased nuclear:

cytoplasmic ratioPromegakaryocyte

80 µmDense alpha and

lysosomal granulesBasophilic megakaryocyteMegakaryocyte

Production of PlateletsPrecursor Cell=

MegakaryocyteProduces about

2000-4000 plateletsPlatelets are

released via sinuses of bone marrow

Production of PlateletsThrombopoietin (TPO)

Regulates platelet developmentInfluences all stages of megakaryocyte

productionProduced in the liver, kidney and spleen

Production of PlateletsHow 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.

Thought question…If a patient had a low platelet count what will

happen?

Answer…TPO increases the number of megakaryocytes

in the bone marrow, increases size and DNA count of megakaryocytes and increases maturation rate

Function of PlateletsSurveillance of blood vessel continuity

Checks endothelial lining for gaps and breaksFill-in small gaps caused by separation of

endothelial cells

Formation of primary hemostatic plugSurface for coagulation factors to make

secondary hemostatic plugAid in healing injured tissue

Formation of Primary Hemostatic PlugOnce the platelets “normal” environment is

changed, they become activated or adhesiveThree stages of plug formation:

AdhesionRelease reaction Aggregation

Stage 1: Platelet Adhesion It is the binding of platelet to non platelet

surface: sub endothelial collageninvolves changes from a disc shape to a slightly

broader, plate like form to increase surface areaa 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.

Adhesion cont’d Collagen – vWF –PlateletvWF bridge physical distance between

platelate and sub endothelial collagenIncrease bond that seal platelet to the

vessel wallreversible

Adhesion is Reversible & No ADP is released.

Stage 1: Platelet Activation Platelets undergo a shape change from disc to

spiny sphere with projectionsActivation required for 1O hemostatic plug

formationActivation continues until Ca ++ threshold metOutcome

Activation of GPIIb/IIIa receptors for fibrinogenSecretion of granules within platelets into

tissues

Platelet Shape Change

George Lancet 2000; 355:1531

Stage 2: Platelet Secretion & Release

Requires ATPIt is release of contents of the granules of

plateletPrimarily ADP stimulates aggregationCathecolamine (especially epinephrine)

and serotonin enhance vasoconstrictionGranules trigger a secondary aggregation which is

irreversible

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

Steps in platelet plug formation: Extension

Granules con’tFactor V: receptor on platelet surface for

factor Xa & prothrombinPF4: heparin neutralizing factorADP: agonist, continues to recruit and

stimulate platelets by increasing cytoplasmic calcium

Steps in platelet plug formation: Perpetuation

Stage 3: Platelet aggregationChemical changes cause platelets to

aggregate and stick to one anotherNewly arriving platelets become activated

by agonists Aggregation is triggered by:

PLT factor 3, adenosine diphosphate (ADP), Thrombin and Thromboxane A2

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.

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

Aggregation cont’d

platelet aggregation

Arachidonic acid

ENDOTHELIAL CELLPLATELET

Thromboxane synthetase

Prostacyclinsynthetase

Cyclo-oxygenase

Prostacyclin (PGI2)

Thromboxane(TxA2)

Inhibits plt aggregationVasodilator

Enhances plt aggregationVasoconstrictor

Cyclic Endoperoxides

GPIIb/IIIa(inactive)

Agonist

e.g. ADPTXA2thrombin

Fibrinogen

Aggregated PlateletsResting Platelet

GPIIb/IIIa(activated)

high shear: von Willebrand factor substitutes for fibrinogen

Platelet AggregationFibrinogen binding to Glycoprotein IIb-IIIa on

activated platelets

Lets have a close view about the mechanism

Adhesion

GpIIb/IIIa

Platelet Activation Pathways (1)Platelet Activation Pathways (1)

GpIIb/IIIaGpIIb/IIIa Aggregation

ADP

Adrenaline Platelet GpIb

Exposed Collagen

Endothelium

vWF

COLLAGEN

GpIIb/IIIaGpIIb/IIIa AggregationGpIIb/IIIaGpIIb/IIIa Aggregation

AdhesionAdhesion

ADP

Adrenaline

THROMBINTHROMBIN

Platelet

Coagulation factors…

What happens when you have an injured blood vessel?

Exposed sub-endothelium

Endothelial cells

Tissue factor (TF)

Platelet

vWF

How does a platelet plug a hole under high shear stress conditions?

Platelets roll on vWF and become activated…

-GP Ib binds the exposed vWF on sub-endothelial cells

Platelet activation(Priming and propagation (Intrinsic pathway) of coagulation)

Upon platelet activation, phosphatidyl serine (PS)flips to the outer leaflet of the plasma

membrane.

Agonist eg. thrombin

PS

PS

PSPS

Activation

PS

PS

Negative charge

Activates the coagulation factors and propagates the production of higher levels of thrombin required to support haemostasis

Thrombin

Fibrinogen Fibrin

Platelet activation (continued from previous slide) also results in the release of the constituents of the alpha and dense granules

Alpha granules P selectin (tm) -fibrinogen -PDGF -VWF

Activation of IIb3 allows binding to fibrinogen

ADP

Thromboxane A2

Aspirin, inactivates cyclooxygenase (COX)

AA

Activates platelets

TXA2

COX

Arachidonic acid (AA)

Platelet activation (cont.):

- IIb3 becomes activated and capable of binding fibrinogen

PlateletFibrinogen The co-crosslinking

of two or more plateletsby IIb3 and fibrinogenis termed ‘aggregation’

Side noteHeparin 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.

Final Stage : Stabilization of Clot

AKA: primary hemostatic plug formationThrombus formationPlatelets release Factor VExpose factor III, accelerating coagulation

cascadePromote activation of clotting factors

Platelet System: Additional FunctionsProvides the reaction surface for some coagulation

system reactions, as well as platelet factor 3 (PF3) which is platelet phospholipid

Supports and maintains endothelial liningDefective hemostasis can occur due to

decreased number of platelets (quantitative)abnormally functioning platelets (qualitative)

Factors that activate platelets

ADPThrombinCollagen

5-hydroxytryptamine (serotonin)Thromboxane A2

Mechanical stimuli

Many stimuliSeveral different receptors

Multiple signalling pathways

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

agonists produced ADP Collagen Thrombin Epinephrine Arachidonic acid Thromboxane A2

                       

                 

                                                  

                                                                                                                                        

                                             Slide 23 of 79

Blood clot