Nov 21st, 2011

Jaehyung (Gus) Cho, Ph.D.

Assistant ProfessorDepartment of Pharmacology

Tel: 312-355-5923 (Office: 5095 CoMRB)Email: thromres@uic.edu

Medical PharmacologyDrug Treatment of Thrombosis

Anti-thrombotic agents

I. Anti-coagulant agents

II. Fibrinolytic agents

III. Anti-platelet agents

* Thrombosis - Pathological conditions in which thrombi form and/or occlude blood vessels in organs.

Thrombus formation (two sides of a coin)

* Hemostasis - Host defense mechanisms to arrest blood loss from injured blood vessels.

* Two major factors: 1) Platelet aggregation 2) Fibrin generation

Blood Coagulation Cas-cade

Thrombus formation (platelet accumula-tion &

fibrin generation) at the site of vessel in-jury

Initiation (adhesion and activation)Extension (adhesion and secretion)

Stabilization (platelet aggregation and fibrin formation)

1. Coagulation factors are plasma glycoproteins produced by hepatocytes in a Vit K-dependent way. Zymogens active form

Blood Coagulation and Platelets

2. In the coagulation cascade, an active coagulation factor cleaves and activates its substrate (a coagulation factor).

3. The key product in the coagulation cascade is thrombin which activates platelets to aggregate and produces insoluble fibrin.

4. Platelets contribute to clotting in many ways: 1) by providing the surface upon which clotting reactions occur 2) by secreting procoagulant substances such as factor V/Va 3) by synthesizing tissue factor 4) by stimulating tissue factor synthesis from monocytes through released substances.5. Coagulation and platelet function are intricately linked. Hemostasis requires the coordinate function of both.

* Formation of occluding thrombi under pathological conditions (platelet aggregation and fibrin generation)

Thrombosis

* Major culprit of heart attack in the U.S. - Heart: myocardial infarction, Brain: stroke

* High incidence in patients over 40 year-old especially after hip or knee surgery. * High incidence in genetic groups

* In veins, red thrombi due to trapped red cells. In arteries, white thrombi with no trapped red cells.

Anti-thrombotic agents

Therapeutic aims of thrombosis

* Anti-coagulant and anti-platelet therapy:

- Prevent propagation if already present - Prevent thrombus formation in high risk groups

* Fibrinolytic (thrombolytic) therapy:

- Lysis of existing thrombi

I. Anti-coagulant agents

1) Parenteral anti-coagulant:

a) Indirect inhibitors: heparin & LMWH, fondaparinux, and idraparinux b) Direct thrombin inhibitors: hirudin, bavalirudin, and argatroban

2) Oral anti-coagulant: Warfarin (Coumarin)

Heparin1. Heparin is a heterogeneous group of anionic mucopolysaccharide called glycosaminoglycans with molecular weight ranging from 3- to 100-kDa (average 15-kDa). Low molecular weight heparin (LMWH) ranges from 1- to 10-kDa.

2. Activate anti-thrombin III (AT III).

AT IIIAction mechanisms of heparin and AT III

Pentasaccharide

* Once the coagulation pathways are activated, factor Xa and thrombin need to be neutralized by AT III.

Heparin

* The negatively charged heparin molecule binds to the positively charged Lysine residues on AT III, thereby causing a conformational change on AT III and accelerating the rate at which AT III inhibits clotting enzymes.

1. Absorption and clearance:

1) Heparin is highly charged, crosses membranes poorly, given parenterally. 2) Low dose: sc (LMWH only) / high dose: iv or sc 3) Monitored by aPTT (2x increase) 4) Heparin does not cross placenta, given during pregnancy: 5) Metabolized by the liver. 6) Half-life (0.5 - 2.5 hr) depends on dosage. 7) Patients with high titer of active clotting factors require a high dose given by a continuous or intermittent injection.

Heparin

2. Side effect: 1) Minor side effects: allergy, alopecia, osteoporosis 2) Major side effects: hemorrhage, thrombocytopenia (HIT), hypersensitivity 3) Antidote: protamine sulfate (a highly basic peptide)

Heparin Dosage

1. For acute coronary syndrome, an iv bolus injection of 5,000 units, followed by an infusion of 12-15 U/kg/hr.

2. For venous thromboembolism, an iv bolus injection of 5,000 units, followed by an infusion of 18 U/kg/hr.

* Patients with venous thromboembolism appear to require higher doses of heparin to achieve a therapeutic aPTT than do patients with acute coronary syndrome.

LMWH (Enoxaparin & Dalteparin) vs.

heparinAdvantage Consequence

Better bioavailabilityLonger half-life

s.c. injectiononce or twice per day

Predictable anti-coagulanteffect

No monitoring required inmost patients

Lower risk of heparin-inducedthrombocytopenia (HIT) and osteoporosis

Safer than heparin forshort- and long-termtreatment

* LMWH has more anti-FXa activity than standard heparin.

Fondaparinux and Idraparinux(Synthetic heparin analogs)

Fondaparinux and Idraparinux

(hr)

Parenteral direct thrombin inhibitors

Property Hirudin Bivalirudin Argatroban (Lepirudin)

Molecular weight 7000 1980 527 (recombinant) (synthetic) (small compound)

Renal clearance Yes No No

Hapatic metabolism No No Yes

Plasma half-life (min) 60 25 45

Advantage Can be used for treatment with patients with HIT

I. Anti-coagulant agents

1) Parenteral anti-coagulant:

a) Indirect inhibitors: heparin & LMWH, fondaparinux, and idraparinux b) Direct thrombin inhibitors: hirudin, bavalirudin, and argatroban

2) Oral anti-coagulant: Warfarin (Coumarin)

Warfarin (orally-available Vit K antagonist)

Often prescribed in North America Vit K

hydroquinone

Vit Kepoxide

Coagulation factors (FII, FVII, FIX and FX) and anti-clotting proteins in blood (protein C and S) contain γ-carboxylglutamic acid (Gla) which is formed by post-translational modification of glutamic acid involving a Vit K-dependent reaction.

Action mechanism of warfarin

The Gla residues on these clotting factors interact with Ca2+ to promote the formation of coagulation complexes on phospholipid (PL)surfaces. Activated platelets provide the PL surfaces.

Activation of FX by FIXa in the presence of Ca2+, PL and FVIII, and the activation of prothrombin to generate thrombin by FX in the presence of Ca2+, PL and FV are greatly accelerated.

Warfarin1. Given orally. rapidly and completely absorbed. Tmax = within 1 hr

3. Food decreases the rate of absorption and affects the effect.

2. After drug ingestion, there is a lag phase depending on the turnover of existing clotting factors in blood. SLOW ONSET

4. In plasma, 99% is bound to albumin. Only the free form is active. Drugs (e.g., phenylbutazone) that compete with warfarin binding to albumin are dangerous because a transient increase of free warfarin may occur.

5. Warfarin is metabolized by the liver with a half-life of 36 hr, but there are large variations among individuals.

6. Side effect: hemorrhage - treated with Vit K and plasma infusion. Usually not given with anti-platelet drugs.

7. Monitoring warfarin Therapy: INR (International Normalized Ratio)1) The results were reported as a ratio of the two PT values. 2) Highly variable: depending on the reagent and instrument.

II. Fibrinolytic (thrombolytic) agents

Plasminogenactivator

Plasminogen Plasmin

Fibrin Fibrin degradation

Fibrinolysis

- The breakdown and removal of fibrin clots by plasmin converted from plasminogen.

XPlasminogen

activator inhibitor

X a2-antiplasmin

1. tPA (tissue-type plasminogen activator):

- A serine protease of 68-kDa. - Synthesized by endothelial cells - It exists in both single-chain and two-chain forms. In the presence of fibrin, both forms have similar activities. - It binds to fibrin specifically and activates plasminogen at the fibrin surface more efficiently than in the circulation. - It is inactivated by plasminogen activator inhibitor.

- Alteplase (recombinant for of single-chain tPA) Tenecteplase (a genetically engineered variant of tPA) Reteplase (a recombinant tPA derivative)

II. Fibrinolytic (thrombolytic) agents

2. Urokinase:

- A serine protease of 54 kDa. - Synthesized by the kidney and excreted in the urine. - In plasma, it exists as a zymogen (pro-urokinase or single-chain urokinase-type plasminogen activator (scu-PA)). - In plasma, pro-urokinase forms a complex with an inhibitor (inactive). - In the presence of fibrin which dissociates the inhibitor, pro-urokinase has about 5% of plasminogen activating activity as compared to urokinase.

3. Streptokinase (SK):

- A 47 kDa protein produced by β-hemolytic streptococci. Most people have anti-SK antibodies in blood. - SK forms a complex with plasminogen, altering its conformation and exposing its active site. The SK-plasminogen complex becomes a potent plasminogen activator. - SK activates plasminogen in plasma leading to non-specific degradation of fibrinogen and fibrin in the circulation. Therefore, SK is NOT a fibrin-specific plasminogen activator. - Anistreplase (Apsac): Anisoylated plasminogen streptokinase activator complex. When injected, Apsac is catalytically inactive. It binds to fibrin via the lysine-binding sites of plasminogen and deacylated on the fibrin surface to yield the active enzyme complex.

Inhibitors of plasmin(ogen)

(Treatment of bleeding)1. α2-antiplasmin: present in plasma and rapidly inactivates plasmin by forming a 1:1 complex.

2. Aprotinin: a serine protease inhibitor.

3. ε-aminocaproic acid: competes with Lys residues on fibrin(ogen) for the lysine binding site in plasmin(ogen). An antidote for overdoses of plasminogen activator.

Platelet signaling during thrombus formation

III. Anti-platelet agents

1. Cyclooxygenase inhibitor: Aspirin

III. Anti-platelet agents

2. ADP receptor (P2Y12) antagonists:

Clopidogrel (Plavix) and Ticlopidine

4. GPIIb-IIIa (fibrinogen receptor) antagonists:Abciximab (ReoPro), Eptifibatide, and Tirofiban

3. Agents that increase cAMP level: Dipyridamole

1. Aspirin

* Mechanism of action:- In activated platelets, arachidonic acid is released and

metabolized by cyclooxygenases to the potent platelet agonists PGH2 and TXA2

- Aspirin acetylates cyclooxygenases, rendering them inactive.

* Complications: - Gastrointestinal bleeding - It also inhibits cyclooxygenases on endothelial cells to block the formation of PGI2, a natural platelet inhibitor.

* Recommended uses: Low doses (160 mg/day)

Aspirinx

2. ADP Receptor (P2Y12) Antagonists(Clopidogel (Plavix) and Ticlopidine)

* Mechanism of action:- A prodrug- A thienopyridine structure- Blocks the P2Y12, an ADP receptor on platelets irreversibly.

* Complications: - Nausea, dyspepsia, diarrhea, hemorrhage, severe neutropenia, anemia

* Recommended uses: - In combination with low dose aspirin - In aspirin-intolerant and aspirin-resistant patients Active metabolite of

clopidogrel

3. Dipyridamole

* Mechanism of action:- An increase in cAMP in platelets inhibits platelet function

by sequestering Ca2+ into its platelet storage sites- Dipyridamole inhibits cAMP phosphodiesterases and

increases platelet cAMP by preventing its breakdown

* Complications: - Not-specific and not effective

* Recommended uses: - In combination with warfarin to prevent thromboembolism in patients with artificial heart valves

4. GPIIb-IIIa Antagonists(Abciximab (ReoPro), Eptifibatide,

&Tirofiban)

* Complications: - Bleeding, thrombocytopenia (1% of patients receiving abciximab) - Not orally-available

* Recommended uses: - Monitoring is required.

* Mechanism of action: - Blocks fibrinogen binding to GPIIb/IIIa receptor, thereby inhibiting platelet aggregation.

Platelet aggregation

Platelet activation Activation of coagulation

Vascular injury

Platelet coagulation

Thrombin generation

Fibrin formation

Thrombus formation

Anti-platelet agents

X

Fibrinolytic agents

Anti-coagulant agents

X

Summary of anti-thrombotic agents