Hemostasis & Coagulation

Ahmad Sh. Silmi

Unit 1 : Primary Hemostasis

Hemostasis

Heme= blood stasis= to halt Process of retaining blood within the

vascular system Repairs injury to blood vessels Stops or prevents blood loss

Balance of Hemostasis

*Balance of bleeding (hemorrhaging) and clotting (thrombosis)

*Imbalance in one direction can lead to:

bleeding : hypocoagulable state OR

thrombosis: hypercoagulable state

Procoagulant Factors

Regulatory Factors

Fibrin

Hemostasis Components

Vascular 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

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

Hemostasis

The hemostatic components remain inert in the presence of intact vascular tissue or endothelium

Following injury, each component must function optimally.

Hemostasis: OverviewConsists 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

http://health.howstuffworks.com/adam-200077.htm

Vascular System

Blood 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 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

Vascular System: Blood Vessels

Basement membrane Collagen material – stimulates platelets

Connective tissue Elastic fibers- provide support around vessels

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

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

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

Vascular System: Function Following Injury Initiate hemostasis

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

Vasoconstriction

MechanismNeurogenic factorsRegulatory substances

Prolong vasoconstriction Serotonin ( made by platelet activation &

endothelium) Thromboxane A2 ( made by platelet activation &

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

Vasoconstriction

Vasodilation Counteracts VasoconstrictionEndothelial 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

Discussion

What 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

Platelets

What is a platelet? Small 2-3 µm Anuclear Reddish-purple

granules Fragments of

megakaryocyte cytoplasm

Platelets

Life span9-10 days

Normal Range150-440 x 109 /L

Platelet: Side noteSeen in conditions with increased need and/or destruction

Giant platelets

May Hegglin anomaly, Bernard-Soulier syndrome, pregnancy, malignancy

Micromegakaryocytes= Dwarf Megs

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 protein Organelle 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 Receptors 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)

Platelet Granules ALPHA 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 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

Platelet Development

Megakaryoblast 10-15 µm Increased nuclear:

cytoplasmic ratio Promegakaryocyte

80 µm Dense alpha and lysosomal

granules Basophilic megakaryocyte Megakaryocyte

Production of Platelets

Precursor Cell= Megakaryocyte

Produces about 2000 platelets

Platelets are released via sinuses of bone marrow

Production of Platelets

Thrombopoietin (TPO)Regulates platelet development Influences all stages of megakaryocyte

productionProduced in the liver, kidney and spleen

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.

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 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

Formation of Primary Hemostatic Plug Once the platelets “normal” environment is

changed, they become activated or adhesive

Three stages of plug formation

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

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

Platelet Shape Change

George Lancet 2000; 355:1531

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

Activated platelet membrane generates TXA2

TXA2 stimulates release

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

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

Steps in platelet plug formation: Extension

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

Steps in platelet plug formation: Perpetuation

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 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.

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

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)

Platelet Function Scout for vascular

damage Form platelet plug of

damaged tissueAdhesionAggregationSecretion

Phospholipid reaction surface for coagulation

Tissue repair Graphic accessed at URL http://www.medicine.mcgill.ca/physio/209A/Blood/blood6a.htm,

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

Agonists

Blood clot

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

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

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