NORMAL HEMOSTASIS

ModeratorsRespected :- Ishwar Bihana SirRespected :- Joseph SirRespected :- S.K.Bose Sir

Presented By Gaurav KumarB.sc. MLT Student Part II

Department of Haematology Post Graduate Institute of Medical Education And Research, Chandigarh

• It is a biological or physiological phenomenon which is responsible to keep the blood in fluid state in the circulation as well as to arrest bleeding followed by an injury to blood vessel.

Defination

Aims and Objective

To understand how our vascular system keep our blood in its fluid state.

To know how bleeding is arrested.

To know the role of haemostasis components.

To know the biology and mechanism of coagulation.

To know how clot is removed from vascular system.

To maintain the blood in fluid state while it remains circulating within vascular system.

To maintain the integrity of the vessels wall.

To arrest bleeding at the site of injury or blood loss by the formation of haemostatic plug.

Eventual removal of plug when healing is complete.

Importance of Hemostasis

NoPGI2

ADPase

Inhibit platelets

aggregation

TM

Thrombin

Protein CaProtein C

Destroys FVa,FVIIIa

Protein S

HSAT-III

Inhibit FXa,FIXa,thrombin

tPAplasminogen plasmin

fibrinolysis

endothelium

Overview of Hemostasis

Blood coagulation cascade

Vessel injury

NeurogenicMayogenicendothelin

vasoconstriction

Reduce blood flow

Collagen exposure+vWFTissue factor

Plt.adhen & activationserotonin

TxA2,ADP

plt.aggregation

Primary hemostatic plug

Stable 2ndry hemostatic plug

healing

fibrinolysis antiplasminogen

Cell migration & proliferation

PF

thrombin

fibrin

Blood vessels Platelets

Plasma coagulation

factorsInhibitorsFibrinolytic

system

Components of Hemostasis

Blood vesselsStructure of blood

vessels

Function of Blood Vessel Blood vessel with muscular coat help to

reduce blood loss by vasoconstriction.

Blood vessels with a pipe system transport nutrients , hormones , gases and other essential factors which are transported by blood.

Endothelial Cell Function

Normal vascular endothelium is a

thromboresistant surface.

Non-thrombogenic- don’t react with plasma or

cellular elements of the blood.

It is antithrombotic. It activates antithrombin

III.

When injured (either biologically , chemically or

mechanically) it can profoundly promote

hemostasis.

Antiplatelets Effect: Inhibition of platelet adhesion, activation and aggregation. PGI2 (Prostacyclin)-vasodilator and antiplatelet agent. NO-Nitric Oxide/PGI2 Both bind with the receptors on the platelets and inactivate them. So inhibit

platelet aggregation. ADPase: Enzymes that break down the ADP (strong proaggregating agent) released by

the platelets and thus inhibit platelet aggregation.

NoPGI2

ADPase

Inhibit platelets

aggregation

TM

Thrombin

Protein CaProtein C

Destroys FVa,FVIIIa

Protein S

HSAT-III

Inhibit Fxa,FIXa,thrombin

tPAplasminogen plasmin

fibrinolysis

Endothelial Antithrombotic Mechanism

NoPGI2

ADPase

Inhibit platelets

aggregation

TM

Thrombin

Protein CaProtein C

Destroys FVa,FVIIIa

ProteinS

HSAT-III

Inhibit Fxa,FIXa,thrombin

tPAplasminogen plasmin

fibrinolysis

Surface expressed integral membrane protein which binds thrombin & results in loss of the pro-coagulant properties of thrombin.

Thrombin/thrombomodulin complex is a potent anticoagulant complex.

Since it activates protein C to activated-Protein C.

Activated protein C down regulates coagulation by inactivating important proteins (FVa, FVIIIa)

Thrombomodulin

Heparan sulphate• Long unbranched polysaccharide expressed at the

endothelial membrane surface.• Act as a cofactor for the plasma inhibitor

antithrombin III.• Inhibits thrombin and other coagulation

factors(FIXa,FXa)

NoPGI2

ADPase

Inhibit platelets

aggregation

TM

Thrombin

Protein CaProtein C

Destroys FVa,FVIIIa

Protein S

HSAT-III

Inhibit Fxa,FIXa,thrombin

tPAplasminogen plasmin

fibrinolysis

Binding and Inhibition of Thrombin

platelet

GPIb receptor

vWF

Endothelialcell

subendothelialcollagen

Subendothelial Cell Function

Subendothelium consist of collagen ,elastic tissues, proteoglycans and non collagenous glycoproteins {fibronectin, vWF}.Exposure of this layer after damage of vessel wall is responsible for platelet adherence.vWF bind with collagen, vWF then undergoes a conformational change and platelets are captured via their surface membrane glycoprotein GpIb binding to vWF.

The mechanism is Damage of vascular endothelium

stimulates endothelial cells

Endothelial cell Synthesize and secrete three

Substances involvedIn haemostatic plug

Formation

vWF(VIII vWF)

It help in adhesion Of platelet to

sub endothelium

PGI2

(synthesized from Arrachidonic acid

And it inhibit platelet Aggregation

PlasminogenIts release is Stimulated by

Vascular damage

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Stages of Hemostasis

Fibrinolysis

Formation of Platelet Plug

Formation of blood clot

Vascular Constriction

Blood Vessels Injury

Vasoconstriction Endothelial cells produce vasoconstrictors such as

angiotensin II and serotonin which help in

vasoconstriction.

Activated platelets produce thromboxane A2 (TXA2)

which is a potent vasoconstrictor.

Function of platelets

Coagulative function

Haemostatic function

Platelets membrane glycoproteins:GPIb-IX:

Constitute active receptor for vWF

Mediates vWF dependent adhesion of platelets to subendothelial

GPIIb/IIIa: On activation serve to bind

fibrinogen Mediates aggregation Also receptor for vWF, fibronectin

and thrombospondinGPIa-IIa:Constitutively active receptor for collagen.Mediates platelets adhesion independent of vWF.

Hemostatic Function

Role of Platelets in Hemostasis

With in 1-2 sec after injury to blood vessel, hemostatic process begins & proceed as out line bellow:

1. platelet adhesion2. platelets activation 3. platelets release reaction4. platelets aggregation

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.

Binding via GpI b initiates activation of platelet.

Platelets Adhesion

Platelet activation:The adhesion of platelets to

the vessel wall activates them.

Platelets undergo a shape change from disc to tiny sphere with projecting pseudopodes.

Activation required for hemostatic plug formation.

Activators released or synthesized at the site of injury

Thrombin Exposed collagen fibres ADP,

Adrenaline,serotonin,TxA2

Immediately after adhesion & activation process of release reaction or secretion begins.

In this process content of platelets organelles are released to the exterior.

Secretion of α granulesa) PF4b) β-Thrombomodulinc) Fibrinogend) Factor Ve) Fibronectinf) Thrombosponding) PDGFh) PAI-1

Platelets Release Reaction

Secretion of dense granules: ADP, GTP, GDP Calcium, serotonin Histamin, epinephrin

ADP released from dense granules promotes platelets aggregation.

PF-4 release from alpha granules neutralize the anticoagulant activity of heparin

PDGF – stimulate proliferation of vascular smooth muscle cell & skin fibroblast & plays a role in cut healing.

TxA2 causes shape change & stimulates release reaction from alpha & dense granules, Also induce aggregation of other platelets & local vasoconstriction

Platelet aggregation

Process by which platelets interact with one another to form a hemostatic plug.Chemical changes cause platelets to

aggregate and stick to one anotherGPIIb-IIIa complex binds vWF,

undergoes Ca++-dependent structural change, then acts as receptor for fibrinogen

Fibrinogen + activated platelets serve as a bridge between two platelets .

April 15, 2023 26

Primary haemostasis involves the binding of platelets to exposed collagen in the sub endothelium of damaged vessels.

Secondary haemostasis is the process of activation of coagulation factors leading to the production of thrombin.

Primary Hemostasis

First physiological response to vascular injury, which is mediated by platelets, in order to arrest bleedingMechanism:– Activation of platelets via stimulators such as thrombin– Adhesion of platelets to subendothelium via interaction between GPIb and von Willebrand Factor (VWF)– Release of platelet granule products in order to recruit more platelets to the injured site– Aggregation of platelets via interaction between GPIIb/IIIa (aIIb3) and fibrinogen to form the initial plug. Triggers secondary hemostasis (coagulation proteins)

• Process of blood coagulationMechanism: Coagulation proteins work in concern to generate

thrombin Thrombin converts fibrinogen to fibrin Fibrin consolidates the platelet plug made in primary

hemostasis such that a thrombus (secondary hemostatic plug) is formed

• Prevents further blood loss from the injury site

Secondary Hemostasis

April 15, 2023 29

Platelet Aggregation

INTRINSIC PATHWAY

EXTRINSIC PATHWAY

COMMON PATHWAY

Plasma Coagulation System

COAGULATION of blood take place with the help of some proteins called clotting factors present in plasma.

Clotting factor act as zymogens and under the influence of enzyme are themselves converted into active enzymes

Role of Clotting Factors

Coagulation factors in the form of zymogens precursors.• Ca++ as a co-factor and organizing surface,

Provided by platelets in vivo Provided by a phospholipids

emulsion in vitro

The coagulation process is initiated when tissue factor - bearing cells are exposed to blood at a site of injury.

Requirements for Coagulation

FX FXaCa++

PLFVIIIa

Prekallinokerin kallinokerin

FXII FXIIa

FXI FXIa

FIX FIXaCa++

FVIIa

FVIIa

Ca++

Intrinsic pathwayExpose collagen

Tissue factor

FVIIFVIIa

Extrinsic pathway

Prothrombin Thrombin

Fibrinogen Fibrin monomer

Cross linked Fibrin clotFXIII FXIIIa

Ca++

PLFVa

Ca++

FIIa

Common pathway

Fibrinolytic system keep the vascular system free of deposited fibrin clots.

Essential purpose of fibrinolysis is to digest and solublize the fibrin, thus restoring potency to occluded vessel.

Fibrinolytic system

Plasminogen activatorPlasminogen and plasmin Inhibitors of fibrinolysis:

AntiactivatorsAntiplasmins

COMPONENTS OF FIBRINOLYTIC SYSTEM

FIBRINOLYTIC PATHWAY

It involves-conversion of plasminogen to

plasmin

Plasmin break down the fibrinogen or fibrin in their degradation products

Conversion of Plasminogen To Plasmin

Plasminogen

Fibrinogen degradation product

• Plasmin initially attacks alpha chain of the fibrinogen molecule & removes small fragments designed as A,B & C from the C terminal of the Aα chains.

• Followed by degradation of Bβ chains with removal of first 42 amino acids.

• Lead to the formation of a large fragment X that still remains fibrinopeptide A.

• Next cleavage involves all the three chains in an asymmetrical manner with the release of fragment Y & D.

• Fragment Y is rapidly degraded by plasmin liberating two D&E.

Fibrin Degradation ProductDegradation of cross linked fibrin is different from fibrinogen.The fibrin degradation products are different because of the presence of covalant bonding.Thus the characteristic fragments are oligomers of X and Y D-dimer. D-E complex and Y-D complex.Fibrin degradation is slower due to the presence of crosslinkages.Normally the FDPs are cleared from the circulation by macrophages.

INHIBITORSInhibitors of Serine Proteases

Antithrombin

Inhibitor of coagulation having antithrombin activities

Types:

Antithrombin I

Show thrombin adsorbing effect of fibrin

Antithrombin II

Act jointly with heparin act as heparin cofactor

Antithrombin III(alpha 2 glycoprotein)

Antithrombin is a serin protein inhibitor that degrades the serine proteases: thrombin, FIXa, FXa, FXIa, and FXIIa. It is constantly active, but its adhesion to these factors is increased by the presence of heparan sulfate.

Other Inhibitors of Coagulation

α2 macroglobulin – It act as antithrombin, antiplasmin

and inactivate kallikrein.

α2 antitrypsin ( alpha globulin) – Inhibitor of factor

XIa and antiplasmin and is weakly antithrombin.

α2 antiplasmin – Inactivate plasmin.

Heparin – inhibit the action of thrombin.

delay the interaction of thrombin and fibrinogen.

Protein C:• It is a vitamin K dependent glycoprotein synthesized in the liver.• Protein circulate as an inert zymogen and is activated by

thrombin in the presence of thrombomodulin on the surface of vascular endothelial cell.

• Protein C proteolytic destruction of activated FV & FVIII.Protein S :• It is also vitamin k dependent protein.• Act as a cofactor in protein C reaction and enhance the action of

protein CTissue Factor Pathway Inhibitor(TFPI):• (TFPI) limits the action of tissue factor (TF). It also inhibits

excessive TF-mediated activation of FIX and FX.

INHIBITION OF COAGULATION CO FACTORS

• Endothelial cell produced Nitric oxide, PGI2 and ADPase.

• They act as a antiplatelets agents (inactivate the platelets receptor by binding with them)

• ADP produced by platelets is favour to bind with endothelial cell but ADPase inactivate or digest ADP.

• Healthy endothelium does not tolerate the presence of activated coagulation factor.

• When anti thrombin III binds with heparan sulfate. AT-III is activated & cut down the thromin molecules & some activated coagulation factors ( FIXa, Fxa)

SUMMARY

How blood is kept in fluid state in circulation ????

Normally thrombin helps in coagulation but when binds with thrombomodulin (TM) it modulate the function of thrombin which activate the protein C.

Activated protein C digest the activated FVa & FVIIIa. Endothelial cells not only prevent the platelets

aggregation but also inhibit the proteins that involve coagulation.

Endothelial cells also produce tissue plasminogen activator(tPA) which activate the plasminogen to plasmin

Plasmin cleaves fibrin strand in to the FDPs. FDPs are removed from the circulation by macrophages

and some eosinophill. By this entire process blood is kept in fluid state .

• When the blood vessels are injured mechanically, chemically & physically the first step is vasoconstriction.

• After the vasoconstriction platelets are adhere to the endothelial cell membrane. And under goes activation, release reaction and aggregation.

• Which forms the primary hemostatic plug and stop the blood leakage from injured site.

• Simultaneously activate the coagulation cascade. (extrinsic pathway, intrinsic pathway and common pathway) and

forms the fibrin mesh in the clot which establish the plug and form secondary hemostasic plug

Which is stable and irreversible. So that bleeding is arrested permanently.

How bleeding is arrested???

Essential haematology: A. V. Hoffbrand, P. A. H. Moss, J. E. Pettit - 2006 - 380 pages Hemostasis and thrombosis: basic principles and clinical practice - Page 1569 Robert W. Colman - 2006 - 1827 pagesClinical laboratory medicine: Volume 2001 - Page 987 Kenneth D. McClatchey - 2002 - 1693 pagesConsultative hemostasis and thrombosis - Page xxi Craig S. Kitchens, Barbara M. Alving, Craig M. Kessler - 2002 - 617 pageHemostasis and thrombosis protocols: Volume 489 - Page 3 David J. Perry, K. John Pasi - 1999 - 368 pagesRodak, BF, Fritsma, GA & Doig, K (2008). Hematology Clinical Principles & Applications. Saunders Elsevier. Chapters 40 & 45.McKenzie, Shirlyn B (2004). Clinical Laboratory Hematology. Pearson Prentice Hall. Chapter 35Beckman Coulter Webinars: Fundamentals of Hemostasis at URL http://www.beckmancoulter.com/LARS/personnel/webinars.asp Hemostasis Basics: Programmed Learner Part I (Provided by Siemens Healthcare Diagnostics Hemostasis Technical Services at URL http://www.dadebehring.com/education/hemostasis/tutorial.htm

References