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