PT & aPTT Manish Pandey

Hemostasis Is a Balance Between Clot Formation & Clot Dissolution.

Normal Hemostasis Clot formation (Coagulation) Clot dissolution (Fibrinolysis) PT aPTT Thrombin Time Fibrinogen Individual factor tests FDP D-Dimer vWF HMWK Prekallikrein

Thrombosis Abnormal Hemostasis is Thrombosis Formation of Blood Clot ( thrombus) in normal blood vessels Thrombotic occlusion of a vessel after relatively minor injury

Abnormal Hemostasis is Thrombosis

Formation of Blood Clot ( thrombus) in normal blood vessels

Thrombotic occlusion of a vessel after relatively minor injury

Hemostasis involves the interaction of: Vascular Endothelium Platelets Coagulation Factors and Fibrinolytic Proteins

Vascular Endothelium

Platelets

Coagulation Factors and

Fibrinolytic Proteins

Hemostasis has 2 main functions: Induce a rapid & localized hemostatic plug at the site of vascular injury (clot formation) Maintain Blood in a fluid, clot-free state after the injury is healed (clot dissolution)

Hemostasis has 2 main functions:

Induce a rapid & localized hemostatic plug at the site of vascular injury (clot formation)

Maintain Blood in a fluid, clot-free state after the injury is healed (clot dissolution)

Primary Hemostasis Injury Endothelial Cells Exposure of thrombogenic surface (subendothelial extracellular matrix)

Injury

Endothelial Cells

Exposure of thrombogenic surface (subendothelial extracellular matrix)

Platelets adhere and get activated Change shape Release secretory granules (e.g. ADP, TXA2) Attract other platelets and Aggregate Hemostatic plug or Primary Platelet Plug

Platelets adhere and get activated

Change shape

Release secretory granules

(e.g. ADP, TXA2)

Attract other platelets and Aggregate

Hemostatic plug or Primary Platelet Plug

Secondary Hemostasis Fibrin is required to stabilize the primary platelet plug Fibrin is formed by two coagulation pathways i.e. Extrinsic & Intrinsic Extrinsic Pathway is initiated when Tissue Factor (III) present in damaged organ comes in contact with Blood Intrinsic Pathway is initiated when Factor XII binds to a negatively charged “foreign” surface exposed to Blood

Fibrin is required to stabilize the primary platelet plug

Fibrin is formed by two coagulation pathways i.e. Extrinsic & Intrinsic

Extrinsic Pathway is initiated when Tissue Factor (III) present in damaged organ comes in contact with Blood

Intrinsic Pathway is initiated when Factor XII binds to a negatively charged “foreign” surface exposed to Blood

Coagulation Factors Both Calcium IV Pathway Trivial Name Factor Extrinsic Tissue Factor III Both Prothrombin II Both Fibrinogen I Intrinsic Contact activation cofactor HMWK Intrinsic Fletcher factor Prekallikrein

Intrinsic Antihemophilic factor B XI Intrinsic Antihemophilic factor A VIII Extrinsic Proconvertin VII Both Accelerin VI (Va) Both Proaccelerin, Labile factor V

Both Fibrin Stabilizing factor XIII Intrinsic Hageman Factor XII

PT and aPTT testing PT ( Prothrombin Time) test is done for deficiency of factors of extrinsic pathway aPTT ( activated Partial Thromboplastin Time) test is done for deficiency of factors of Intrinsic pathway

PT ( Prothrombin Time) test is done for deficiency of factors of extrinsic pathway

aPTT ( activated Partial Thromboplastin Time) test is done for deficiency of factors of Intrinsic pathway

Effects of Hereditary or Acquired Factor Deficiency on the PT & aPTT aPTT prolonged, PT normal Deficiencies of intrinsic pathway Factor(s) VIII, IX, XI or XII PT prolonged, aPTT normal Deficiency of extrinsic Pathway factor VII Occasionally, mild to moderate deficiency of common pathway factor(s) fibrinogen, II, V or x

aPTT prolonged, PT normal

Deficiencies of intrinsic pathway Factor(s) VIII, IX, XI or XII

PT prolonged, aPTT normal

Deficiency of extrinsic Pathway factor VII

Occasionally, mild to moderate deficiency of common pathway factor(s) fibrinogen, II, V or x

Both PT and aPTT Prolonged Deficiency of common pathway factor(s) fibrinogen, II, V, or X Multiple factor deficiencies

Both PT and aPTT Prolonged

Deficiency of common pathway factor(s) fibrinogen, II, V, or X

Multiple factor deficiencies

Mixing studies in PT PT mixing study (1:1 mix of patient and normal plasma PT normalizes Factor Deficiency; Measure factors I, II, V, VII, X PT initially shortens and then prolongs Factor V inhibitor (rare) aPTT remains prolonged Inhibitor (specific factor inhibitor, rare) Treat specimen with heparinase (degrades heparin) PT normal, prolongation due to heparin PT prolonged

Mixing studies in aPTT aPTT mixing study (1:1 mix of patient and normal plasma aPTT normalizes Factor Deficiency; Measure factors VIII, IX, XI, XII aPTT initially shortens and then prolongs Factor VIII inhibitor aPTT remains prolonged Inhibitor, most commonly Lupus Anticoagulant Treat specimen with heparinase (degrades heparin) aPTT normal, prolongation due to heparin aPTT prolonged

Fibrinolytic Mechanism Stable Fibrin Clot Activation of Protein C and Protein S Secretion of t-PA by endothelial cells Plasminogen Plasmin Stabilized fibrin clot X, Y fragments Plasmin Degrades D-E-D fragments E fragment + D dimer

Stable Fibrin Clot

Activation of Protein C and Protein S

Secretion of t-PA by endothelial cells

Plasminogen Plasmin

Stabilized fibrin clot

X, Y fragments

Plasmin Degrades D-E-D fragments

E fragment + D dimer

Fibrinolysis As soon as the injury is healed clot dissolution starts, to restore the normal flow of Blood Plasminogen is converted to the active form Plasmin by 2 distinct Plasminogen Activators (PAs): tissue plasminogen activator (t-PA) from injured endothelial cells Urokinase from Kidney endothelial cells and plasma

As soon as the injury is healed clot dissolution starts, to restore the normal flow of Blood

Plasminogen is converted to the active form Plasmin by 2 distinct Plasminogen Activators (PAs):

tissue plasminogen activator (t-PA) from injured endothelial cells

Urokinase from Kidney endothelial cells and plasma

Fibrinolysis or Kallikrein from Intrinsic Pathway Plasminogen can also be activated by the bacterial product (e.g. Streptokinase) – having significance in certain Bacterial Infections Free Plasmin is neutralized by α 2- plasmin inhibitor (PAI) t-PA activity is also blocked by PAI

or Kallikrein from Intrinsic Pathway

Plasminogen can also be activated by the bacterial product (e.g. Streptokinase) – having significance in certain Bacterial Infections

Free Plasmin is neutralized by α 2- plasmin inhibitor (PAI)

t-PA activity is also blocked by PAI

Endothelial cells modulate the coagulation / anticoagulation balance by releasing PAIs PAIs block fibrinolysis by inhibiting t-PA binding to fibrin as it is most active when bound to fibrin

Endothelial cells modulate the coagulation / anticoagulation balance by releasing PAIs

PAIs block fibrinolysis by inhibiting t-PA binding to fibrin as it is most active when bound to fibrin

Fibrinolysis Three types of Natural Anti-coagulants regulate clotting: antithrombin III – inhibit thrombin activity and Factors IXa, Xa, XIa and XIIa Protein C and Protein S – Vitamin K dependent proteins, inactivate Factors Va and VIIIa Plasmin

Three types of Natural Anti-coagulants regulate clotting:

antithrombin III – inhibit thrombin activity and Factors IXa, Xa, XIa and XIIa

Protein C and Protein S – Vitamin K dependent proteins, inactivate Factors Va and VIIIa

Plasmin

Fibrin Degradation Products or FDP’s include: fragments X and Y – early splits and D and E – late splits D-Dimer is the smallest cross-linked FDP

Fibrin Degradation Products or FDP’s include:

fragments X and Y – early splits and

D and E – late splits

D-Dimer is the smallest cross-linked FDP

D-Dimer are specific FDP formed only by Plasmin activity on fibrin clot and not on intact fibrinogen Thus the presence of D-Dimer indicates that fibrin has been formed and so is a marker for an ongoing in vivo thrombotic condition

D-Dimer are specific FDP formed only by Plasmin activity on fibrin clot and not on intact fibrinogen

Thus the presence of D-Dimer indicates that fibrin has been formed and so is a marker for an ongoing in vivo thrombotic condition

Clinical Significance of Hemostasis Hemophilia A: Caused by the deficiency of Factor VIII Hemophilia B: Caused by the deficiency of Factor IX Vitamin K deficiency (PIVKA’s) P rotein I nduced V itamin K A ntagonism can be used in thrombotic conditions Vitamin K dependent factors are II, V, IX, & X

Hemophilia A: Caused by the deficiency of Factor VIII

Hemophilia B: Caused by the deficiency of Factor IX

Vitamin K deficiency

(PIVKA’s) P rotein I nduced V itamin K A ntagonism can be used in thrombotic conditions

Vitamin K dependent factors are

II, V, IX, & X

Liver Dysfunction Fibrinogen and Factor XIII deficiency Factor XI and Contact Activation Antithrombin Deficiency leads to DVT and PE von Willebrand Disease: Deficiency of von Willebrand Factor Clinical Significance of Hemostasis

Liver Dysfunction

Fibrinogen and Factor XIII deficiency

Factor XI and Contact Activation

Antithrombin Deficiency leads to DVT and PE

von Willebrand Disease: Deficiency of von Willebrand Factor

DIC (Disseminated Intravascular Coagulation) Massive Injury or Sepsis Massive release of Tissue Factor III Excessive Activation of Thrombin Coagulation becomes systemic

Massive Injury or Sepsis

Massive release of Tissue Factor III

Excessive Activation of Thrombin

Coagulation becomes systemic

High consumption of Platelets, coagulation factors Over production of fibrin clot Fibrin clot “disseminates” or spreads throughout the microcirculation Obstructing the blood flow to capillaries, smaller vessels

High consumption of Platelets, coagulation factors

Over production of fibrin clot

Fibrin clot “disseminates” or spreads throughout the microcirculation

Obstructing the blood flow to capillaries, smaller vessels

Lack of blood supply leads to tissue injury (decreased oxygenation, organ infarction & necrosis) Once again release of Tissue Factor Second time coagulation activation More consumption of coagulation factors and platelets

Lack of blood supply leads to tissue injury (decreased oxygenation, organ infarction & necrosis)

Once again release of Tissue Factor

Second time coagulation activation

More consumption of coagulation factors and platelets

Continuous thrombi formation Production capacity of Bone Marrow and Liver reaches its maximum level Activation of fibrinolysis at first site High consumption of Plasmin, antithrombin, Protein C and Protein S

Continuous thrombi formation

Production capacity of Bone Marrow and Liver reaches its maximum level

Activation of fibrinolysis at first site

High consumption of Plasmin, antithrombin, Protein C and Protein S

Generation of Thrombin & Plasmin at the same time Plasmin acts on Fibrin Clots and produces FDPs and D-Dimer FDPs interfere with platelet function and impair fibrin clot formation Further bleeding results

Generation of Thrombin & Plasmin at the same time

Plasmin acts on Fibrin Clots and produces FDPs and D-Dimer

FDPs interfere with platelet function and impair fibrin clot formation

Further bleeding results

Thus an initial thrombotic disorder gets converted into a serious bleeding disorder Whenever there is a widespread activation of Thrombin the chances are that it may lead to DIC

Thus an initial thrombotic disorder gets converted into a serious bleeding disorder

Whenever there is a widespread activation of Thrombin the chances are that it may lead to DIC

Pharmacological Intervention Most commonly patients are on OAC (oral anti-coagulant) therapy: warfarin – over dose can lead to Vit. K deficiency heparin Fibrinolysis: Aspirin Streptokinase, urokinase injections t-PA injections

Most commonly patients are on OAC (oral anti-coagulant) therapy:

warfarin – over dose can lead to Vit. K deficiency

heparin

Fibrinolysis:

Aspirin

Streptokinase, urokinase injections

t-PA injections

INR & ISI values All PT reagents are calibrated against WHO IRP (International Reference Preparation) International Normalisation Ratio is intended to make comparison similar irrespective of the type of PT reagent used worldwide International Sensitivity Index is a measure of the sensitivity of particular PT reagent

All PT reagents are calibrated against WHO IRP (International Reference Preparation)

International Normalisation Ratio is intended to make comparison similar irrespective of the type of PT reagent used worldwide

International Sensitivity Index is a measure of the sensitivity of particular PT reagent

Insensitive PT reagents will give prolonged results only when factor levels are very low Sensitive PT reagent give prolonged results even when there is a mild change in factor level Insensitive PT reagents have higher ISI values Sensitive PT reagents have ISI value as close to 1.0 as possible

Insensitive PT reagents will give prolonged results only when factor levels are very low

Sensitive PT reagent give prolonged results even when there is a mild change in factor level

Insensitive PT reagents have higher ISI values

Sensitive PT reagents have ISI value as close to 1.0 as possible

Calculation of PT results INR = (Ratio) ISI Patient PT Time (secs) Mean Normal PT (MNPT) MNPT = Mean PT Time of at least 20 known normal samples INR = ISI

INR = (Ratio) ISI

Patient PT Time (secs)

Mean Normal PT (MNPT)

MNPT = Mean PT Time of at least 20 known normal samples

Thanks Please send your feedbacks to: Priyank Dubey Ph: 09999990845 [email_address] Application Specialist

Please send your feedbacks to:

Priyank Dubey

Ph: 09999990845

[email_address]

Application Specialist