Gihan Gawish.Dr Dr. Gihan Gawish. Gihan Gawish.Dr Coagulation Coagulation is a complex process by...

Gihan Gawish.DrGihan Gawish.Dr Dr. Gihan Gawish

Gihan Gawish.DrGihan Gawish.Dr

CoagulationCoagulation CoagulationCoagulation is a complex process by which is a complex process by which

blood forms clots.blood forms clots.

Coagulation Coagulation begins almost instantly after begins almost instantly after an injury to the blood vessel has damaged an injury to the blood vessel has damaged

the endothelium (lining of the vessel). the endothelium (lining of the vessel).


HemostasisHemostasis Primary hemostasisPrimary hemostasis:: platelets immediately platelets immediately

form a plug at the site of injuryform a plug at the site of injury

Secondary hemostasis:Secondary hemostasis: occurs occurs simultaneously: simultaneously:

1.1. proteins in the blood plasma (proteins in the blood plasma (clotting factorsclotting factors) ) respond in a complex cascade respond in a complex cascade

2.2. to form fibrin strands to form fibrin strands

3.3. which strengthen the platelet plug which strengthen the platelet plug


11 . .Primary hemostasisPrimary hemostasis Platelet activationPlatelet activation

1.1. Damage to blood vessel walls exposes Damage to blood vessel walls exposes sub endothelium proteins, most notably sub endothelium proteins, most notably

collagen, present under the endothelium. collagen, present under the endothelium.

2.2. Circulating platelets bind collagen with Circulating platelets bind collagen with surface collagen-specific glycoprotein surface collagen-specific glycoprotein

Ia/IIa receptors.Ia/IIa receptors.


3. The adhesion is strengthened by 3. The adhesion is strengthened by circulating proteins (vWF) circulating proteins (vWF) Binding intermediaries

von Willebrand factorvon Willebrand factor

BLOOD PLATELETS

ENDOTHELIAL CELL

EXPOSED COLLAGENvWF

vWF

vWF

vWF


4. This adhesion activates the platelets. 4. This adhesion activates the platelets.

5. Activated platelets release the contents of stored 5. Activated platelets release the contents of stored granules into the blood:granules into the blood:

1.1. plasma ADP, plasma ADP,

2.2. serotonin, serotonin,

3.3. platelet activating factor (PAF), platelet activating factor (PAF),

4.4. von Willebrand factor (vWF) , von Willebrand factor (vWF) ,

5.5. platelet factor 4 platelet factor 4

6.6. thromboxane A2 (TXA2)thromboxane A2 (TXA2)


6. The granules' contents activate:6. The granules' contents activate:

Gq-linked protein receptor cascade Gq-linked protein receptor cascade

Increased calcium concentration in the platelets' cytosol. Increased calcium concentration in the platelets' cytosol.

Activates protein kinase C Activates protein kinase C

Activates phospholipase A2 (PLA2). Activates phospholipase A2 (PLA2).

Modifies the integrin membrane glycoproteinModifies the integrin membrane glycoprotein

Increasing its affinity to bind fibrinogen. Increasing its affinity to bind fibrinogen.


7. The activated platelets changed shape from 7. The activated platelets changed shape from spherical to stellate spherical to stellate

8. The fibrinogen cross-links with glycoprotein 8. The fibrinogen cross-links with glycoprotein aid in aggregation of adjacent platelets.aid in aggregation of adjacent platelets.


22 . .Secondary hemostasisSecondary hemostasisThe coagulation cascadeThe coagulation cascade

The coagulation cascade of secondary The coagulation cascade of secondary hemostasis has two pathways:hemostasis has two pathways:

1.1. The contact activation pathway (formerly known The contact activation pathway (formerly known as the intrinsic pathway) as the intrinsic pathway)

2.2. The tissue factor pathway (formerly known as The tissue factor pathway (formerly known as the extrinsic pathway) the extrinsic pathway)

That lead to That lead to fibrinfibrin formation. formation.


The Clotting CascadeThe Clotting Cascade

FibrinogenFibrinmonomers

Fibrinpolymers

Thrombin


The biological of the coagulation The biological of the coagulation factorsfactors

The coagulation factors are generally The coagulation factors are generally serine proteasesserine proteases ( (enzymesenzymes). ).

There are some exceptions. For example, FVIII and FV There are some exceptions. For example, FVIII and FV are are glycoproteinsglycoproteins

Factor XIII is a Factor XIII is a transglutaminasetransglutaminase. .

Serine proteases act by cleaving other proteins at specific Serine proteases act by cleaving other proteins at specific sites. sites.

The coagulation factors circulate as inactive The coagulation factors circulate as inactive zymogenszymogens..

http://en.wikipedia.org/wiki/Serine_protease

http://en.wikipedia.org/wiki/Enzymes

http://en.wikipedia.org/wiki/Transglutaminase

http://en.wikipedia.org/wiki/Zymogens


The pathways are a series of The pathways are a series of reactionsreactions

zymogenzymogen + its + its glycoproteinglycoprotein co-factor are co-factor are activated to become active components that activated to become active components that

then catalyze the next reaction in the then catalyze the next reaction in the cascade, ultimately resulting in cross-linked cascade, ultimately resulting in cross-linked

fibrin. fibrin.

zymogenzymogen is inactive enzyme precursor of a is inactive enzyme precursor of a serine proteaseserine protease

http://en.wikipedia.org/wiki/Zymogen

http://en.wikipedia.org/wiki/Glycoprotein

http://en.wikipedia.org/wiki/Zymogen

http://en.wikipedia.org/wiki/Serine_protease


Coagulation factors are generally indicated Coagulation factors are generally indicated by Roman numerals, with a lowercase by Roman numerals, with a lowercase aa

appended to indicate an active form.appended to indicate an active form.


Generation of ThrombinGeneration of Thrombin The prothrombinThe prothrombin (Factor II) (Factor II) genegene is located is located

on the eleventhon the eleventh chromosomechromosome ((1111p11-q12p11-q12 ( (

Thrombin is produced by the enzymatic Thrombin is produced by the enzymatic cleavage of two sites on prothrombin by cleavage of two sites on prothrombin by

activated activated Factor XFactor X (Xa). (Xa).

The activity of factor Xa is greatly enhanced The activity of factor Xa is greatly enhanced by binding to activated by binding to activated Factor VFactor V (Va), termed (Va), termed

the prothrombinase complex. the prothrombinase complex.

http://en.wikipedia.org/wiki/Gene

http://en.wikipedia.org/wiki/Chromosome

http://en.wikipedia.org/wiki/Factor_X

http://en.wikipedia.org/wiki/Factor_V


Prothrombin is produced in the liver and is Prothrombin is produced in the liver and is post-translationally modified in a post-translationally modified in a vitamin Kvitamin K--

dependent reaction that converts ten dependent reaction that converts ten glutamic acids on prothrombin to glutamic acids on prothrombin to

gamma-carboxyglutamic acidgamma-carboxyglutamic acid (Gla). (Gla).

In the presence of calcium, the Gla residues In the presence of calcium, the Gla residues promote the binding of thrombin to promote the binding of thrombin to

phospholipid bilayersphospholipid bilayers

http://en.wikipedia.org/wiki/Vitamin_K

http://en.wikipedia.org/wiki/Gamma-carboxyglutamic_acid


Deficiency of vitamin K or administration of Deficiency of vitamin K or administration of the anticoagulant the anticoagulant warfarinwarfarin inhibits the inhibits the

production of gamma-carboxyglutamic acid production of gamma-carboxyglutamic acid residues, slowing the activation of the residues, slowing the activation of the

coagulation cascade.coagulation cascade.

In human beings the level of prothrombin in In human beings the level of prothrombin in the blood stream increases after birth and the blood stream increases after birth and typically peaks on the 8th day after which typically peaks on the 8th day after which

the prothrombin level lowers to normal the prothrombin level lowers to normal levels levels

http://en.wikipedia.org/wiki/Warfarin


Action of ThrombinAction of Thrombin Thrombin converts fibrinogen to an active form Thrombin converts fibrinogen to an active form

that assembles into fibrinthat assembles into fibrin. .

Thrombin also activatesThrombin also activates factor XIfactor XI, , factor Vfactor V, , andand factor VIIIfactor VIII. . This positive feedback accelerates the This positive feedback accelerates the

production of thrombinproduction of thrombin..

Factor XIIIFactor XIII is also activated by thrombinis also activated by thrombin. . Factor Factor XIIIa is aXIIIa is a transglutaminasetransglutaminase that catalyzes the that catalyzes the

formation of covalent bonds between lysine and formation of covalent bonds between lysine and glutamine residues in fibringlutamine residues in fibrin. . The covalent bonds The covalent bonds

increase the stability of the fibrin clotincrease the stability of the fibrin clot..

http://en.wikipedia.org/wiki/Factor_XI


http://en.wikipedia.org/wiki/Factor_VIII

http://en.wikipedia.org/wiki/Transglutaminase


Action of Thrombin In plateletsAction of Thrombin In platelets

In addition to the thrombin activity in the In addition to the thrombin activity in the coagulation cascades, thrombin also coagulation cascades, thrombin also

promotespromotes plateletplatelet activation, via activation ofactivation, via activation of proteaseprotease--activated receptorsactivated receptors on the plateleton the platelet..

http://en.wikipedia.org/wiki/Platelet

http://en.wikipedia.org/wiki/Protease-activated_receptor




Ca


Electron Micrograph of Fibrin


A fibrin clot is formed by the interplay of the intrinsic, extrinsic, and final common

pathways.

The intrinsic pathway begins with the activation of factor XII (Hageman factor) by

contact with abnormal surfaces produced by injury.


The extrinsic pathway is triggered by trauma, which activates factor VII and releases a

lipoprotein, called tissue factor, from blood vessels. Inactive forms of clotting factors are

shown in red; their activated counterparts (indicated by the subscript "a") are in yellow.

Stimulatory proteins that are not themselves enzymes are shown in blue.

A striking feature of this process is that the activated form of one clotting factor catalyzes the

activation of the next factor. I.


CofactorsCofactors

Various substances are required for the Various substances are required for the proper functioning of the coagulation proper functioning of the coagulation

cascade:cascade:

1.1. Calcium and phospholipidsCalcium and phospholipids (a platelet (a platelet membrane constituent) membrane constituent)

They are required for the tenase and They are required for the tenase and prothrombinase complexes to function. prothrombinase complexes to function.


Calcium mediates the Calcium mediates the binding of the complexes binding of the complexes via the terminal gamma-via the terminal gamma-

carboxyl residues on carboxyl residues on

1.1. FXa FXa 2.2. FIXa FIXa

(to the phospholipids (to the phospholipids surfaces expressed by surfaces expressed by

platelets)platelets)The Calcium-

Binding Region of Prothrombin

Prothrombin binds calcium ions with the modifiedamino acid g-carboxyglutamate (red).


2. 2. Vitamin KVitamin K It It is an essential factor to a hepaticis an essential factor to a hepatic gammagamma--

glutamyl carboxylaseglutamyl carboxylase that adds athat adds a carboxyl group carboxyl group toto glutamic acid residues on:glutamic acid residues on:

Factor II, Factor II, Factor VII, Factor VII, Factor IX Factor IX Factor X, Factor X, Protein SProtein S, , Protein CProtein C

http://en.wikipedia.org/wiki/Vitamin_K


In adding the gammaIn adding the gamma--carboxyl group to carboxyl group to glutamate residues on the immature clotting glutamate residues on the immature clotting

factors Vitamin K is itself oxidizedfactors Vitamin K is itself oxidized. .


Another enzymeAnother enzyme, , Vitamin K Vitamin K epoxide reductase epoxide reductase VKORCVKORC

reduces vitamin K back to its reduces vitamin K back to its active formactive form. .

Vitamin K epoxide reductase Vitamin K epoxide reductase is pharmacologically important is pharmacologically important

as a target for anticoagulant as a target for anticoagulant (antagonists) drugs(antagonists) drugs warfarinwarfarin and relatedand related coumarinscoumarins such such

asas acenocoumarolacenocoumarol, , phenprocoumonphenprocoumon andand

dicumaroldicumarol. .

http://en.wikipedia.org/wiki/Acenocoumarol

http://en.wikipedia.org/wiki/Phenprocoumon

http://en.wikipedia.org/wiki/Dicumarol


These drugs create a deficiency of reduced These drugs create a deficiency of reduced vitamin K by blocking VKORC, thereby inhibiting vitamin K by blocking VKORC, thereby inhibiting

maturation of clotting factorsmaturation of clotting factors. .

Other deficiencies of vitamin K Other deficiencies of vitamin K ((ee..gg. . inin malabsorptionmalabsorption), ), or diseaseor disease ( (hepatocellular hepatocellular

carcinoma impairs the function of the enzyme and carcinoma impairs the function of the enzyme and leads to the formation of PIVKAs leads to the formation of PIVKAs ((proteins formed proteins formed

in vitamin K absencein vitamin K absence) ) this causes partial or non this causes partial or non gamma carboxylation and affects the coagulation gamma carboxylation and affects the coagulation factors ability to bind to expressed phospholipidsfactors ability to bind to expressed phospholipids


The Clotting Process Must Be Precisely Regulated

There is a fine line between hemorrhage and thrombosis. Clots must form rapidly

yet remain confined to the area of injury.

Activated factors are short-lived because they are diluted by blood flow, removed by

the liver, and degraded by proteases.

For example, the stimulatory proteins factors Va and VIIIa are digested by protein

C


Protein C is a protease that is switched on by the action of

thrombin.

Thus, thrombin has a dual function: it catalyzes the formation of fibrin and it initiates the deactivation of

the clotting cascade.


FibrinolysisFibrinolysis


FibrinolysisFibrinolysis is the process wherein a fibrin is the process wherein a fibrin clot is broken down clot is broken down

PlasminPlasmin cuts the fibrin mesh at various cuts the fibrin mesh at various places, leading to the production of places, leading to the production of circulating fragments that are cleared by circulating fragments that are cleared by other other proteasesproteases or by the or by the kidneykidney and and liverliver..

Plasmin is produced in an inactive form, Plasmin is produced in an inactive form, plasminogenplasminogen, in the liver. , in the liver.

http://en.wikipedia.org/wiki/Plasmin

http://en.wikipedia.org/wiki/Plasminogen


Coagulation factors and related Coagulation factors and related substancessubstances

I I fibrinogenfibrinogen Forms clot Forms clot ((fibrinfibrin( (

(II(II ( (prothrombinprothrombin Its active form Its active form ((IIaIIa) ) activates I, V, VII, VIII, activates I, V, VII, VIII, XI, XIII, protein C, plateletsXI, XIII, protein C, platelets

Tissue factorTissue factor Co Co--factor of VIIa factor of VIIa ((formerly known as factor III) formerly known as factor III)

CalciumCalcium Required for coagulation factors to bind to Required for coagulation factors to bind to phospholipid phospholipid ((formerly known as factor IVformerly known as factor IV

))VV ( (proaccelerin, labile factor proaccelerin, labile factor ))CoCo--factor of X with which it factor of X with which it forms theforms the prothrombinase complexprothrombinase complex

http://en.wikipedia.org/wiki/Fibrinogen

http://en.wikipedia.org/wiki/Prothrombin

http://en.wikipedia.org/wiki/Tissue_factor

http://en.wikipedia.org/wiki/Calcium



XIIXII (Hageman factor) Activates factor XI and (Hageman factor) Activates factor XI and prekallikreinprekallikrein

XIIIXIII (fibrin-stabilizing factor) Cross links fibrin (fibrin-stabilizing factor) Cross links fibrin

von Willebrand factorvon Willebrand factor Binds to VIII, mediates Binds to VIII, mediates platelet adhesion platelet adhesion

PrekallikreinPrekallikrein Activates XII and prekallikrein; Activates XII and prekallikrein; cleaves HMWKcleaves HMWK

high molecular weight kininogenhigh molecular weight kininogen (HMWK) (HMWK) Supports reciprocal activation of XII, XI, and Supports reciprocal activation of XII, XI, and prekallikreinprekallikrein

http://en.wikipedia.org/wiki/Factor_XII

http://en.wikipedia.org/wiki/Factor_XIII

http://en.wikipedia.org/wiki/Von_Willebrand_factor

http://en.wikipedia.org/wiki/Prekallikrein

http://en.wikipedia.org/wiki/High_molecular_weight_kininogen


antithrombinantithrombinIIIIII Inhibits IIa, Xa, and other Inhibits IIa, Xa, and other proteasesproteases

heparin cofactor IIheparin cofactor II Inhibits IIa, cofactor for Inhibits IIa, cofactor for heparin heparin

protein Cprotein C Inactivates Va and VIIIa Inactivates Va and VIIIa

protein Sprotein S Cofactor for activated protein C Cofactor for activated protein C

http://en.wikipedia.org/wiki/Antithrombin

http://en.wikipedia.org/wiki/Heparin_cofactor_II

http://en.wikipedia.org/wiki/Protein_C

http://en.wikipedia.org/wiki/Protein_S


Specific inhibitors of clotting factors

1.Antithrombin III is the most important one,

It is a plasma protein that inactivates thrombin by forming an irreversible complex with it.

It resembles alpha 1-antitrypsin except that it inhibits thrombin much more strongly than it

inhibits elastase.

Also, it blocks other serine proteases in the clotting cascade namely, factors XIIa, XIa, IXa,

and Xa.


2.Heparin The inhibitory action of antithrombin III is

enhanced by heparin

It is a negatively charged polysaccharide found in mast cells near the walls of blood vessels and on

the surfaces of endothelial cells

Heparin acts as an anticoagulant by increasing the rate of formation of irreversible complexes

between antithrombin III and the serine protease clotting factors.

Antitrypsin and antithrombin are serpins, a family of serine protease inhibitors.


Electron Micrograph of a Mast Cell. Heparin and other molecules in the dense granules are released into the extracellular space when the

cell is triggered to secrete.


3. Alpha 1-antitrypsin

which normally inhibits elastase

alpha 1-Antitrypsin activity normally increases markedly after injury to counteract excess elastase

arising from stimulated neutrophils.

The mutant a 1-antitrypsin caused the patient's thrombin activity to drop to such a low level that

hemorrhage ensued.


Disease and clinical significance Disease and clinical significance of thrombosisof thrombosis

1. H1. Hemophiliasemophilias are the best are the best--known known coagulation factor disorders. coagulation factor disorders.

hemophilia B (factor IX deficiency or (factor IX deficiency or "Christmas disease") "Christmas disease")

hemophilia C ))factor XI deficiencyfactor XI deficiency , ,

mild bleeding tendencymild bleeding tendency.(.(

hemophilia A ))factor VIII deficiencyfactor VIII deficiency((

The three main forms are:The three main forms are:

http://en.wikipedia.org/wiki/Hemophilia

http://en.wikipedia.org/wiki/Hemophilia_B

http://en.wikipedia.org/wiki/Hemophilia_C

http://en.wikipedia.org/wiki/Hemophilia_A


2. von Willebrand disease2. von Willebrand disease It is the most common hereditary bleeding disorder It is the most common hereditary bleeding disorder

and is characterized as being inherited autosomal and is characterized as being inherited autosomal recessive or dominant. recessive or dominant.

In this disease there is a defect in von Willebrand In this disease there is a defect in von Willebrand factor (vWF) which mediates the binding of factor (vWF) which mediates the binding of glycoprotein Ib (GPIb) to collagen. glycoprotein Ib (GPIb) to collagen.

This binding helps mediate the activation of This binding helps mediate the activation of platelets and formation of primary hemostasis.platelets and formation of primary hemostasis.


3. Deficiency of Vitamin K3. Deficiency of Vitamin K It may also contribute to bleeding disorders It may also contribute to bleeding disorders

because clotting factor maturation depends because clotting factor maturation depends on Vitamin Kon Vitamin K..

44. Liver diseases:. Liver diseases: Some clotting factors; II, IX, VII, X are Some clotting factors; II, IX, VII, X are

synthesized in liversynthesized in liver Liver diseases deficiency of these Liver diseases deficiency of these

factors bleeding disorders. factors bleeding disorders.


Coagulation Coagulation TestsTests


Coagulation CascadeCoagulation Cascade

PT

PTTVIIIa

Heparin

Hirudin, Argatroban


Coagulation and FibrinolysisCoagulation and Fibrinolysis


Coagulation TestsCoagulation TestsScreen testScreen testBleeding Time (Duke method, Bleeding Time (Duke method, Template method), Thrombin Time, Template method), Thrombin Time, PT, PTTPT, PTT

Antiphospholipid syndromeAntiphospholipid syndromeDilute Russell Viper Venom Time Dilute Russell Viper Venom Time (dRVVT), Anti-Cardiolipin Ab, ACA, (dRVVT), Anti-Cardiolipin Ab, ACA, IgG, Anti-Phospholipid Ab, APA, IgG, IgG, Anti-Phospholipid Ab, APA, IgG, Anti-Cardiolipin Ab, ACA, IgM Anti-Cardiolipin Ab, ACA, IgM

Coagulation factorCoagulation factorFactor I (Fibrinogen), II, V, VII, VWF, Factor I (Fibrinogen), II, V, VII, VWF, VIII, IX, X, Urea solubility test, VIII, IX, X, Urea solubility test,

Other coagulation inhibitor Study, Other coagulation inhibitor Study, VIII, XI, XII VIII, XI, XII DIC profileDIC profileFibrinogen, Fibrinogen, FDP, 3P Test, D-dimerFDP, 3P Test, D-dimer

FibrinolysisFibrinolysisEuglobulin clot lysis time, Euglobulin clot lysis time, Plasminogen activator inhibitor, Plasminogen activator inhibitor, Alpha2-antiplasminAlpha2-antiplasmin

PLT functionPLT functionPlatelet aggregation Platelet aggregation

ThrombosisThrombosisAPC Resistance, Protein S, APC Resistance, Protein S, Antithrombin III, Protein C, Antithrombin III, Protein C, PlasminogenPlasminogen


1 .Bleeding time Done with a template.

Time taken for the blood to stop

Normal range 2-10mts

Prolonged in plt disorders, low plts, severe anemia, Vwf, collagen vascular disease

Great variability in results, unreliable, invasive, insensitive


22 . .Activated Partial Activated Partial Thromboplastin Time (aPTT)Thromboplastin Time (aPTT)

It is a performance indicator measuring the efficancy It is a performance indicator measuring the efficancy of both the "intrinsic" and the common coagulation of both the "intrinsic" and the common coagulation pathways. pathways.

It is also used to monitor the treatment effects with It is also used to monitor the treatment effects with heparin. heparin.

It is used in conjunction with the prothrombin time (PT) It is used in conjunction with the prothrombin time (PT) which measures the which measures the extrinsic pathwayextrinsic pathway. .


Methodology (aPTT)Methodology (aPTT) ContainerContainer:: blue top (3.2% citrate) tube blue top (3.2% citrate) tube

CollectionCollection:: Deliver tubes immediately to the laboratory Deliver tubes immediately to the laboratory

In order to activate the intrinsic pathway, phospholipid, an In order to activate the intrinsic pathway, phospholipid, an activator (such as silica, celite, kaolin, ellagic acid), and activator (such as silica, celite, kaolin, ellagic acid), and calcium (to reverse the anticoagulant effect of the citrate) calcium (to reverse the anticoagulant effect of the citrate) are mixed into the plasma sample . are mixed into the plasma sample .

The time is measured until a thrombus (clot) forms. The time is measured until a thrombus (clot) forms.

The test is termed "partial" due to the absence of tissue The test is termed "partial" due to the absence of tissue factor from the reaction mixture.factor from the reaction mixture.


Performing APTT

At 37 GC Plasma Add kaolin/elgaic acid Phospholipid source Calcium Time the appearance of clot


Activated Partial Thromboplastin Activated Partial Thromboplastin Time (aPTT)Time (aPTT)

Causes for RejectionCauses for Rejection:: Specimen received Specimen received more than 4 hoursmore than 4 hours after collection, tubes not after collection, tubes not filled, clotted specimens, visible hemolysis filled, clotted specimens, visible hemolysis


Activated Partial Thromboplastin Activated Partial Thromboplastin Time (aPTT)Time (aPTT)

Reference IntervalReference Interval:: 2020-25 to 32--25 to 32-3939 seconds. seconds. Prolong in newbornsProlong in newborns

However, newborns and infants do not normally However, newborns and infants do not normally experience bleeding, because a balance between experience bleeding, because a balance between procoagulants and natural anticoagulants is procoagulants and natural anticoagulants is maintained.maintained.

Critical ValuesCritical Values:: >100-150 seconds >100-150 seconds


PTT in clinical states

PTT prolonged in

1. Congenital or acquired def of intrinsic pathway factors

2. Heparin

3. Lupus AC (antiphospholipid antibody)

PTT shortened in

1. Pregnancy

2. In conditions causing activation of factors


33 . .Prothrombin Time (PT)Prothrombin Time (PT)

Clotting time from factor VII to fibrin clotClotting time from factor VII to fibrin clot

PTPT↑: ↑: fibrinogen or factors II, V, VII, or X fibrinogen or factors II, V, VII, or X deficiency, therapeutic anticoagulants (heparin, deficiency, therapeutic anticoagulants (heparin, hirudin, or argatroban) hirudin, or argatroban)

ContainerContainer:: Blue top (3.2% sodium citrate) tube Blue top (3.2% sodium citrate) tube


Prothrombin Time (PT)Prothrombin Time (PT)

heparin prolongs the PT to a lesser extent heparin prolongs the PT to a lesser extent than PTT. Hirudin and argatroban prolong than PTT. Hirudin and argatroban prolong the PT and PTT. the PT and PTT.

► ► CollectionCollection:: directly from a peripheral vein directly from a peripheral vein

Causes for RejectionCauses for Rejection:: Specimen received Specimen received more than 24 hoursmore than 24 hours after collection, tube not after collection, tube not filled, clotted specimen, visible hemolysis filled, clotted specimen, visible hemolysis



Reference IntervalReference Interval:: 1010-12 to 12--12 to 12-1414 seconds. seconds. Prolong in newborns.Prolong in newborns. Up to 16 Up to 16 seconds at birth, and gradually shortens into seconds at birth, and gradually shortens into the adult normal range by the age of 6 the adult normal range by the age of 6 months. months.

Critical ValuesCritical Values:: >30 seconds >30 seconds



MethodologyMethodology:: Reagent called Reagent called thromboplastinthromboplastin ((phospholipidphospholipid with with tissue factortissue factor and and calciumcalcium) ) added, measure clot formation time. added, measure clot formation time.

Vitamin K trialVitamin K trial may be performed with an may be performed with an unexplained PT prolongation. If vitamin K unexplained PT prolongation. If vitamin K deficiency, the PT becomes normal or significantly deficiency, the PT becomes normal or significantly shorter within 12-24 hours after vitamin K shorter within 12-24 hours after vitamin K administration. administration.



Monitoring warfarinMonitoring warfarin:: international normalized international normalized ratio (INR), therapeutic goal is an INR of 2-3. ratio (INR), therapeutic goal is an INR of 2-3.

► ► INR = [patient PT/normal PT]ISI INR = [patient PT/normal PT]ISI

► ► international sensitivity index (ISI), varies in international sensitivity index (ISI), varies in reagentsreagents

The ISI is usually between 1.0 and 1.4. The ISI is usually between 1.0 and 1.4.


Effects of Factor Deficiencies on Effects of Factor Deficiencies on PT and PTTPT and PTT

PTT Prolonged, PT NormalPTT Prolonged, PT Normal: : Deficiencies of Deficiencies of factor VIII, IX, XI, and/or XII (intrinsic pathway) factor VIII, IX, XI, and/or XII (intrinsic pathway)

PT Prolonged, PTT NormalPT Prolonged, PTT Normal: : Deficiency of factor Deficiency of factor VII (extrinsic pathway), mild-to-moderate VII (extrinsic pathway), mild-to-moderate deficiencies of factor II, V, X, and/or fibrinogen deficiencies of factor II, V, X, and/or fibrinogen (common pathway) (common pathway)

Both PT and PTT ProlongedBoth PT and PTT Prolonged: : Deficiencies of Deficiencies of factor II, V, X, and/or fibrinogen (common factor II, V, X, and/or fibrinogen (common pathway), Multiple factor deficiencies pathway), Multiple factor deficiencies


44 . .Thrombin clotting timeThrombin clotting time

TTTT is a coagulation assay which is usually is a coagulation assay which is usually performed in order to detect for the performed in order to detect for the therapeutic level of the anticoagulant therapeutic level of the anticoagulant Heparin. Heparin.

It is also sensitive in detecting the presence It is also sensitive in detecting the presence of a fibrinogen abnormality. of a fibrinogen abnormality.


Methodology of TTMethodology of TT

After liberating the plasma from the whole blood After liberating the plasma from the whole blood byby centrifugationcentrifugation, , bovinebovine ThrombinThrombin is added to the is added to the sample of plasmasample of plasma. .

The clot is formed and is detected optically or The clot is formed and is detected optically or mechanically by a coagulation instrumentmechanically by a coagulation instrument..

The time between the addition of the thrombin The time between the addition of the thrombin and the clot formation is recorded as the thrombin and the clot formation is recorded as the thrombin clotting timeclotting time


Reference Interval TTReference Interval TT

The reference interval of the Thrombin The reference interval of the Thrombin Clotting time is generally <21 seconds, Clotting time is generally <21 seconds, depending on the method and the endemic depending on the method and the endemic patient population. patient population.

Results outside of reference interval indicate Results outside of reference interval indicate heparin therapy, Hypofibrinogenemia, heparin therapy, Hypofibrinogenemia, hyperfibrinogenemia fibrinogen abnormality, hyperfibrinogenemia fibrinogen abnormality, or Lupus anticoagulant.or Lupus anticoagulant.


55 . .D-Dimers and Fibrin D-Dimers and Fibrin Degradation Products (FDP)Degradation Products (FDP)

Plasmin Plasmin degrades fibrin clots degrades fibrin clots into D-dimers and fibrin into D-dimers and fibrin degradation products (FDP). degradation products (FDP).

Limitations:Limitations: elevate whenever elevate whenever the coagulation and fibrinolytic the coagulation and fibrinolytic systems are activated. systems are activated.

High High rheumatoid factor (RF)rheumatoid factor (RF) levels may cause false-positive levels may cause false-positive result. result.


D-Dimers and Fibrin Degradation D-Dimers and Fibrin Degradation Products (FDP)Products (FDP)

MethodologyMethodology:: semi quantitative or quantitative semi quantitative or quantitative immunoassaysimmunoassays

D-dimer is a specific FDP formed only by plasmin D-dimer is a specific FDP formed only by plasmin degradation of fibrin, not of intact fibrinogen. degradation of fibrin, not of intact fibrinogen.

D-Dimer and FDP(+): thrombosis, liver disease, D-Dimer and FDP(+): thrombosis, liver disease, postoperatively, significant bleeding, hemodialysis, postoperatively, significant bleeding, hemodialysis, eclampsia, sickle cell crisis, cancer, pregnancy eclampsia, sickle cell crisis, cancer, pregnancy


66 . .Disseminated Intravascular Disseminated Intravascular Coagulation (DIC) ScreenCoagulation (DIC) Screen

D-dimer or fibrin degradation products D-dimer or fibrin degradation products (FDP), prothrombin time (PT), activated (FDP), prothrombin time (PT), activated partial thromboplastin time (PTT), platelet partial thromboplastin time (PTT), platelet count, and fibrinogen. count, and fibrinogen. These tests are not These tests are not specific for DIC.specific for DIC.

SpecimenSpecimen:: Plasma (and whole blood for Plasma (and whole blood for platelet count and peripheral blood smear)platelet count and peripheral blood smear)


Disseminated Intravascular Disseminated Intravascular Coagulation (DIC) ScreenCoagulation (DIC) Screen

DIC is a common acquired coagulation DIC is a common acquired coagulation disorder resulting from disorder resulting from excessive activationexcessive activation of the coagulation system, usually due to of the coagulation system, usually due to massive tissue injurymassive tissue injury, , sepsissepsis, or certain , or certain pregnancy complications. pregnancy complications.


Disseminated Intravascular Disseminated Intravascular Coagulation (DIC) ScreenCoagulation (DIC) Screen

disseminated micro vascular thrombi disseminated micro vascular thrombi →consumes platelets, coagulation factors, →consumes platelets, coagulation factors, and natural anticoagulants →PT, PTT and natural anticoagulants →PT, PTT prolongations, bleeding prolongations, bleeding

Reference valueReference value:: FDP< 5.0 ug/ml, D- FDP< 5.0 ug/ml, D-Dimer<324 ug/LDimer<324 ug/L

Gihan Gawish.Dr Dr. Gihan Gawish. Gihan Gawish.Dr Coagulation Coagulation is a complex process by...

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