Mechanism of Action of Antithrombotic Drugs Rabih R. Azar, MD, MSc, FACC Division of Cardiology Hotel Dieu de France Hospital

Mechanism of Action of Antithrombotic Mechanism of Action of Antithrombotic DrugsDrugs

Rabih R. Azar, MD, MSc, FACCRabih R. Azar, MD, MSc, FACC

Division of CardiologyDivision of Cardiology

Hotel Dieu de France HospitalHotel Dieu de France Hospital


1- The coagulation cascade1- The coagulation cascade

2- Heparin2- Heparin

3- Low molecular weight heparin3- Low molecular weight heparin

4- Direct thrombin inhibitors4- Direct thrombin inhibitors

5- Vitamin K antagonists5- Vitamin K antagonists

6- Platelet antagonists6- Platelet antagonists

7- Thrombolytics7- Thrombolytics

Summary of the coagulation cascadeSummary of the coagulation cascade

Extrinsic pathway:Extrinsic pathway:

VII, Tissue factorVII, Tissue factor

Intrinsic pathway:Intrinsic pathway:

XII, XI, IX, VIIIXII, XI, IX, VIII

Common Pathway:Common Pathway:

V, X, II (prothrombin)V, X, II (prothrombin)

Coagulation Coagulation occurs when occurs when thrombinthrombin is is formed and formed and transform transform fibrinogenfibrinogen into into fibrinfibrin

The Central Role of Thrombin in the Coagulation The Central Role of Thrombin in the Coagulation CascadeCascade

• Thrombin is formed from prothrombin after activation by Thrombin is formed from prothrombin after activation by factor Xafactor Xa

• High concentration of thrombin:High concentration of thrombin:

– activate fibrinogen into fibrinactivate fibrinogen into fibrin

– activate factor XIII leading to fibrin cross-linkingactivate factor XIII leading to fibrin cross-linking

– result in platelets activation and aggregationresult in platelets activation and aggregation

Site of action of drugs

TXA2

ADP

Fibrinogen Fibrin

Thrombin Prothrombin

Intrinsic ExtrinsicCollagen

Clopidogrel

GP IIb/IIIaantagonists

LMWH

Heparin–AT-IIIHirulog

X Xa LMWH

Vascular SurfaceTXA2 = thromboxane A2; ADP = adenosine diphosphate; IIb/IIIa = glycoprotein IIb/IIIa; vWF = von Willebrand factor









Plasma anti-coagulation proteinsPlasma anti-coagulation proteins

Anti-thrombin IIIAnti-thrombin III

XIIa, XIa, IXa, Xa, ThXIIa, XIa, IXa, Xa, Th

Tissue factor Tissue factor inhibitorinhibitor

Still experimentalStill experimental

Protein C/SProtein C/S

VIIIa, VaVIIIa, Va

Vitamin K dependentVitamin K dependent

VII, IX, X, prothrVII, IX, X, prothr

Unfractionated Heparin

Naturally occurring as mixture of linear sulphated mucopolysaccharide chains of various lengths.

Increases activity of the inhibitory factor antithrombin I I I .

Molecular weight = 2,000 - 40,000 Da

Mechanism of Action of Unfractionated HeparinMechanism of Action of Unfractionated Heparin

• Heparin = indirect thrombin inhibitorHeparin = indirect thrombin inhibitor

• Stimulate anti-thrombinStimulate anti-thrombin

• Anti-thrombin binds to thrombin and other activated Anti-thrombin binds to thrombin and other activated coagulation factors and forms inactivation complexescoagulation factors and forms inactivation complexes

• The rate of formation of these inactivating complexes The rate of formation of these inactivating complexes increases by a factor of several thousand in the presence increases by a factor of several thousand in the presence of heparinof heparin

Mechanism of action of heparin on the thrombin anti-thrombin complex

Thrombin and other clotting factors have an active center contained a serine amino acid. This Thrombin and other clotting factors have an active center contained a serine amino acid. This center is inhibited by an arginine on the anti-thrombin (AT) molecule. Heparin binds to a lysine center is inhibited by an arginine on the anti-thrombin (AT) molecule. Heparin binds to a lysine site on the AT molecule and produces a conformational change that converts AT from a slow to site on the AT molecule and produces a conformational change that converts AT from a slow to a very rapid thrombin inhibitor. Heparin then dissociates from the thrombin/AT complex and a very rapid thrombin inhibitor. Heparin then dissociates from the thrombin/AT complex and can be re-utilized.can be re-utilized.

- properties

Since heparin is not absorbed by the GI tract, it isusually administered either iv or sc

Half-life of heparin injected iv is 45-90 min

Peak plasma concentration after sc injection isreached after 2 hours

Heparin presentations come either as sodium orcalcium salts, with the sodium salt being absorbedmore rapidly that the calcium ones

Heparin Induced Thrombocytopenia

• Transient mild-moderate thrombocytopenia in 25%

• Severe thrombocytopenia in 5%

• Mild platelet reduction within the first 5 days may result from heparin-induced aggregation that is postulated to be benign and transient

• A smaller subset of pts may develop an antibody-mediated thrombocytopenia that is associated with paradoxical thrombosis. The antibody is directed against the heparin-platelet factor 4 complex

• These antigen-antibody complexes bind to Fc receptors on adjacent platelets, causing aggregation and thromboembolism

Heparin Dosage and Reversal

• Therapeutic concentration: 0.3-0.7 anti-Xa units/mL. This will prolong the aPTT by 2-2.5

• Bolus: 80-100 units/kg; maintenance: 15-22 units/kg/h (aPTT 2-2.5)

• Reversal of heparin: • Protamin combines with heparin as an ion pair to form a stable

complex devoid of anticoagulant activity

• For every 100 units of heparin remaining in the patient, administer 1 mg of protamin sulfate IV

• The rate of infusion should not exceed 50 mg in any 10 min period









Mechanisms of inhibitory action of unfractionated heparin Mechanisms of inhibitory action of unfractionated heparin and low-molecular weight heparin on thrombin and factor Xaand low-molecular weight heparin on thrombin and factor Xa

•Inhibition of thrombin Inhibition of thrombin requires binding of requires binding of heparin through a heparin through a >> 18 18 saccharide residuesaccharide residue

•LMWH do not have that LMWH do not have that residue and are weak residue and are weak thrombin inhibitorsthrombin inhibitors

•Inhibition of Xa requires Inhibition of Xa requires binding to AT only binding to AT only (without formation of a (without formation of a ternary complex).ternary complex).

Mechanism of Action of Low Molecular Weight Mechanism of Action of Low Molecular Weight Heparin (LMWH)Heparin (LMWH)

• Selective Inhibition of factor Xa via Anti-thrombin with a weak effect Selective Inhibition of factor Xa via Anti-thrombin with a weak effect on thrombin on thrombin

• LMWH anti-Xa/thrombin ratio >> 1LMWH anti-Xa/thrombin ratio >> 1

• In contrast, unfract heparin anti-Xa/thrombin ratio = 1In contrast, unfract heparin anti-Xa/thrombin ratio = 1

• Depending on the length of LMWH, the effect on thrombin may vary, Depending on the length of LMWH, the effect on thrombin may vary, and the anti-Xa/thrombin ratio variesand the anti-Xa/thrombin ratio varies

Low-Molecular-Weight HeparinsAnti-Facotr Xa : Anti - Factor IIa Ratios

Agent Trade Xa:IIa Mol Wt (d)

Enosaparin Lovenox 3.8 : 1 4,200

Dalteparin Fragmin 2.7 : 1 6,000

Ardeparin Normiflo 1.9 : 1 6,000

Nadroparin Fraxiparine 3.6 : 1 4,500

Reviparin 3.5 : 1 4,000

Tinzaparin 1.9 : 1 4,500

Why are LMWH considered superior to Why are LMWH considered superior to unfractionated heparinunfractionated heparin

1- 1- Heparin does not inhibit clot bound thrombin or XaHeparin does not inhibit clot bound thrombin or Xa

LMWH inhibits clot bound XaLMWH inhibits clot bound Xa

2-2- Heparin binds extensively to plasma proteins including Heparin binds extensively to plasma proteins including acute phase reactants and vascular and blood cellsacute phase reactants and vascular and blood cells

LMWH binds much less to these proteins/cellsLMWH binds much less to these proteins/cells

3-3- LMWH are more resistant to neutralization by platelet LMWH are more resistant to neutralization by platelet factor 4factor 4

4-4- The longer half life and more predictable anticoagulant The longer half life and more predictable anticoagulant response of LMWH allow their administration at a fixed response of LMWH allow their administration at a fixed dose without need for laboratory monitoringdose without need for laboratory monitoring

Dosing of LMWH

• Weight-based dosing of the LMW heparins results in predictable pharmcokinetics and plasma levels in patients with normal renal function

• Levels are determined by anti-Xa units: peak therapeutic levels are 0.5-1 unit/mL for twice daily dosing, determined 4 hours after administration, and 1.5 unit/mL for the once daily dosing

• Neutralization of LMWH by protamin is incomplete. Limited experience suggest that 1 mg of protamin sulfate may be used to partially neutralize 1 mg of enoxaparin. Definite neutralization requires FFP

Fondaparinux

• Is a synthetic pentasachharideIs a synthetic pentasachharide

• Binds antithrombin with high specific activityBinds antithrombin with high specific activity

• Inactivates XaInactivates Xa

• Long half life of 15 hoursLong half life of 15 hours

• Superior to enoxaparin in preventing DVT following orthopedic surgerySuperior to enoxaparin in preventing DVT following orthopedic surgery









Direct Thrombin Inhibitors: Mechanism of ActionDirect Thrombin Inhibitors: Mechanism of Action

Advantages of Direct Thrombin InhibitorsAdvantages of Direct Thrombin Inhibitors

• Do not necessitate anti-thrombin for their action

• Inactivate both free and fibrin-bound thrombin

• They do not bind to plasma proteins

• They have a more predictable anticoagulant response

• Indicated for the treatment of heparin induced thrombocytopenia

Direct thrombin inhibitors

• Hirudin (lepirudin): IV. Short half life

• Bivalirudin: IV. Rapid onset and offset of action.

• Argatropan: IV. Short half life. Elevates INR because of test interference, rendering the transition to warfarin difficult.

• Melgatran: IV but has an oral form: Ximelagatran.

• Ximelagatran: • is a prodrug.

• Predictable pharmacokinetics and bioavailability allowing for fixed dosing and predictable anticoagulant response. No need for routine coagulation monitoring.

• Rapid onset and offset of action allowing for immediate anticoagulation and thus no need for overlap with additional anticoagulant drugs.









Mechanism of action of anti-vitamin K drugsMechanism of action of anti-vitamin K drugs

- Inhibit Vit K epoxide - Inhibit Vit K epoxide reductase and quinone reductase and quinone reductasereductase

-Block the formation of -Block the formation of Vit KH2 which is a Vit KH2 which is a cofactor for the cofactor for the carboxylation of carboxylation of factors factors II, VII, IX, XII, VII, IX, X to to their active formtheir active form

Plasma anti-coagulation proteinsPlasma anti-coagulation proteins

Anti-thrombin IIIAnti-thrombin III

XIIa, XIa, IXa, Xa, ThXIIa, XIa, IXa, Xa, Th

Tissue factor Tissue factor inhibitorinhibitor

Still experimentalStill experimental

Protein C/SProtein C/S

VIIIa, VaVIIIa, Va

Vitamin K dependentVitamin K dependent

VII, IX, X, prothrVII, IX, X, prothr

Clinical Use of Anti-vitamin KClinical Use of Anti-vitamin K

• Vitamin K dependent proteins: VII, IX, X, prothrombin, prot C, S

• Prot C and S have the shortest half life, followed by factor VII

• The initial effect of anti-vitamin K drugs is a hypercoagulability effect secondary to a deficiency in protein C and S

• A large loading dose of anti-vitamin K can decrease factor VII levels without affecting factors IX, X and II. This predisposes to bleeding without protecting against thrombosis because the activity of all 4 factors must be inhibited to achieve a clinically effective anticoagulation









Platelets Activation and Aggregation

Coller. Coller. Heart Disease, Update 4Heart Disease, Update 4. 1995.. 1995.

Platelet GP IIb/IIIa Receptor in Vascular Injury: AggregationPlatelet GP IIb/IIIa Receptor in Vascular Injury: Aggregation

`̀

GP IIb/IIIaGP IIb/IIIaGP IIb/IIIaGP IIb/IIIa

Fibrinogen Fibrinogen (or von Willebrand factor)(or von Willebrand factor)

Fibrinogen Fibrinogen (or von Willebrand factor)(or von Willebrand factor)

AggregationAggregationAggregationAggregation

Platelet Activation

Oral Antiplatelet AgentsOral Antiplatelet Agents

Mechanism of ActionMechanism of Action

CollagenCollagenThrombinThrombin

TXATXA22

ADPADP

(Fibrinogen(FibrinogenReceptor)Receptor)

ADP = adenosine diphosphate, TXAADP = adenosine diphosphate, TXA22 = thromboxane A = thromboxane A22, COX = cyclooxygenase., COX = cyclooxygenase.

Schafer AISchafer AI. Am J Med. Am J Med.. 1996;101:199–209.1996;101:199–209.

clopidogrel bisulfateclopidogrel bisulfate

TXATXA22

ADPADP

dipyridamoledipyridamole

phosphodiesterasephosphodiesterase

ADPADP

Gp IIb/IIIaGp IIb/IIIa ActivationActivation

COXCOX

ticlopidine HClticlopidine HCl

aspirinaspirin

Effects of aspirin

ADP= adenosine diphosphate; GP= glycoproteinThickness of line indicates strength of activator

Adapted from Ohman EM et al Eur Heart J 1995;16(suppl L):50-55.

ADPCollagen

ThrombinEpinephrine

Thromboxane A2

Serotonin

Platelet-activating factor Fibrinogen

TiclopidineClopidogrel

ASPIRIN

GP llb/llla

Ticlopidine (ticlid) and clopidogrel (plavix)

TiclopidineTiclopidine ClopidogrelClopidogrel

twice / day once / day

Neutropenia No neutropenia

CBC monitoring No need for CBC monitoring

Skin reactions (rash) No skin reactions

Delayed onset of effect Rapid onset of effect

Platelet-fibrinogen interaction

Platelet aggregation

Blood vessel wall

Platelet

Platelet

IIb/IIIa

Fibrinogen

Ib receptor

Site of injury Platelet adhesion

IIb/IIIa receptorblockers

Ib Ib Ib Ib

GP IIb/IIIa Inhibitors: Chemical StructuresGP IIb/IIIa Inhibitors: Chemical Structures

AbciximabAbciximabAbciximabAbciximab

Topol et al. Topol et al. LancetLancet. 1999;353:227-231.. 1999;353:227-231.

EptifibatideEptifibatideEptifibatideEptifibatideTirofibanTirofibanTirofibanTirofiban

OO

OO

OOOO

OO

OOOO

OOOHOH

HNHNHNHN

SSSS

NNHH

NNHH

NN

NNHH

HHNN

NHNH

NHNH

HH22NN

HH22NN

HHN–SON–SO22–C–C44HH99

OO

CCOOOOHH

HHNN

Chimeric Monoclonal Chimeric Monoclonal AntibodyAntibody

MW MW 50,000 D 50,000 D

Chimeric Monoclonal Chimeric Monoclonal AntibodyAntibody

MW MW 50,000 D 50,000 D

Nonpeptide Tyrosine Nonpeptide Tyrosine DerivativeDerivative

MW MW 500 D 500 D

Nonpeptide Tyrosine Nonpeptide Tyrosine DerivativeDerivative

MW MW 500 D 500 D

Cyclic Cyclic HeptapeptideHeptapeptide

MW MW 800 D 800 D

Cyclic Cyclic HeptapeptideHeptapeptide

MW MW 800 D 800 D

Type of Type of Chimeric monoclonal Chimeric monoclonal NonpeptideNonpeptide CyclicCyclicmoleculemolecule antibodyantibody tyrosine derivativetyrosine derivative heptapeptide heptapeptide

Affinity for Affinity for circulating platelets circulating platelets HighHigh Moderate Moderate UnknownUnknown

KKD D (nmol/L)(nmol/L) 55 1515 120120

Molecules of drug Molecules of drug per receptor per receptor 1.5 1.5 >100>100 240-440240-440

Drug clearanceDrug clearance tt1/21/2 = 12-24 h = 12-24 h tt1/21/2 = 2 h = 2 h tt1/21/2 = 2.5 h = 2.5 h

GP IIb/IIIaGP IIb/IIIa NonspecificNonspecific SpecificSpecific SpecificSpecificreceptor specificityreceptor specificity (RGD)(RGD) (KGD)(KGD)

Type of Type of Chimeric monoclonal Chimeric monoclonal NonpeptideNonpeptide CyclicCyclicmoleculemolecule antibodyantibody tyrosine derivativetyrosine derivative heptapeptide heptapeptide

Affinity for Affinity for circulating platelets circulating platelets HighHigh Moderate Moderate UnknownUnknown

KKD D (nmol/L)(nmol/L) 55 1515 120120

Molecules of drug Molecules of drug per receptor per receptor 1.5 1.5 >100>100 240-440240-440

Drug clearanceDrug clearance tt1/21/2 = 12-24 h = 12-24 h tt1/21/2 = 2 h = 2 h tt1/21/2 = 2.5 h = 2.5 h

GP IIb/IIIaGP IIb/IIIa NonspecificNonspecific SpecificSpecific SpecificSpecificreceptor specificityreceptor specificity (RGD)(RGD) (KGD)(KGD)

AbciximabAbciximab TirofibanTirofiban EptifibatideEptifibatideAbciximabAbciximab TirofibanTirofiban EptifibatideEptifibatide

Scarborough et al. Scarborough et al. CirculationCirculation. 1999;10:437-444; Tcheng. . 1999;10:437-444; Tcheng. Am J CardiolAm J Cardiol. 1999;83:7E-11E.. 1999;83:7E-11E.

Comparative Properties of GP IIb/IIIa InhibitorsComparative Properties of GP IIb/IIIa Inhibitors









The Fibrinolytic System

• Plasmin is the key protease enzyme of the coagulation system

• 2 major activators of plasminogen: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA)

• Both t-PA and plasminogen possess specific affinity for fibrin and thereby bind selectively to clots. In the absence of fibrin, t-PA activates plasminogen to plasmin relatively slowly

Thrombolytic Drugs

• Because indiscriminate plasmin lysis of both fibrin and fibrinogen can produce a sysemic state of fibrin(ogen)olysis, which might cause a serious systemic bleeding tendency, attempts have been made to develop thrombolytic agents that generate plasmin preferentially at the fibrin surface in preformed thrombus

• Streptokinase and urokinase induce a systemic lytic state

• In contrast, t-PA activate plasminogen preferentially at the fibrin surface.

• The risk of hemorrhage is however not reduced, because of the inability of plasmin to discriminate between fibrin in pathological thrombi and fibrin in physiological hemostatic plugs

Streptokinase

• Isolated from hemolytic streptococci

• Streptokinase itself possesses no enzymatic activity

• Streptokinase forms a complex with plasminogen and it is the strepto-plasminogen complex that actually possesses enzymatic activity

• Streptokinase is anti-genic. Most individuals have preexisting antibodies from previous streptococcal infection

• It can cause allergic reactions: transient hypotension, serum sickness-type syndrome

tPA

• Produced by recombinant DNA technology

• Fibrin specific

• It activates plasminogen directly

• The efficiency of plasminogen activation by t-PA is enhanced in the presence of fibrin

• Short half life: 40- 50 minutes

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Mechanism of Action of Antithrombotic Drugs Rabih R. Azar, MD, MSc, FACC Division of Cardiology Hotel Dieu de France Hospital