1 - Pharmaceutic and Pharmacologic Principles of Anticoagulant Drugs and Their Action

What must blood do in the body regarding clotting?

Blood must remain fluid in the vasculature but clot quickly when exposed to subendothelial surfaces → sites of vascular injury

What does coagulation do?

Forms a blood clot

What does fibrinolysis do?

Breaks down a blood clot

What is thrombosis?

Formation of a blood clot in the vasculature that is adhered to the vascular wall

What is an embolus?

A free floating clot

What is a hemorrhage?

Loss of blood from the vasculature

What is hemophilia?

A bleeding disorder caused by a loss or deficiency of a functional coagulation factor

Treatment may involve replacement of the missing factor

What is a thrombi?

Clotted blood

Composed of aggregated platelets, cross-linked fibrin, and trapped red blood cells

Anticoagulants?

Reduce fibrin clot formation by blocking the function of coagulation factors

Antiplatelet agents?

Inhibit platelet activation and or aggregation

Fibrinolytic agents?

Break down fibrin clots after they have formed

Clot buster!

Why do antithrombotic and coagulant drugs have different criteria for clinic use compared to other drugs?

Because their effects → either too much pro or anti thrombotic effect can be deadly!

What activates coagulation factors?

Gamma carboxylation of glutamate

Ca+ Binding

What do activated coagulation factors become?

Proteases → protein cutters

They cleave and activate other coagulation factors

What do endogenous serine protease inhibitors do?

Inactivate coagulation factors

Ex → antithrombin

What is required for an activated coagulation factor to cleave/activate the next factor?

Gamma carboxylation of glutamate and subsequent calcium binding

What enzyme carries out gamma-carboxylation of glutamate?

A vitamin K-dependent carboxylase

What are serpins?

Natural anticoagulant molecules → that inhibit serine proteases involved in coagulation

Which serpin is targeted by heparins?

Antithrombin → a serine protease inhibitor

What is antithrombin?

A serine protease inhibitor → that inhibits coagulation/serine enzymes in the extrinsic, intrinsic, and common pathways

How does heparan sulfate enhance antithrombin activity?

It binds antithrombin and increases its activity ~1,000-fold

Greatly increasing thrombin and factor Xa inactivation

What is Thrombate used for?

Replacement therapy of antithrombin

Made with human antithrombin

IV

What is heparin used for clinically?

Used as an anticoagulant to prevent clot formation

Where does exogenous heparin come from?

Porcine intestinal mucosa

piggys 🐷

How does heparin work, and what determines its function?

The heparin pentasaccharide binds antithrombin

Chain length determines its function

What does unfractionated heparin chains bind/activate?

Pentasaccharide sequence binds → antithrombin and thrombin

Heparin turns ON/activates antithrombin → antithrombin inhibits Factor Xa

How does unfractionated heparin help antithrombin inactivate thrombin?

It binds both antithrombin and thrombin

Bringing them together so antithrombin can → inactivate thrombin much faster (~1,000× faster).

What does low molecular weight heparin (LMWH) bind/activate?

Pentasaccharide sequence → binds/activates antithrombin → inhibiting FXA

Cannot simultaneously bind antithrombin and thrombin

Synthetic pentasaccharide?

The pentasaccharide alone binds/activates → antithrombin

How does chain length affect UFH vs. LMWH?

Length and size of oligosaccharide chain → dictates anticoagulant mechanism and efficacy

How are LMWH made?

Via depolymerization / degradation of UFH

Most of its polysaccharide chains are shorter → average MW around 5000

What do the short chains of LMWH mean?

Too short to simultaneously bind both antithrombin and thrombin → so does NOT promote anticoagulation via the inhibition of thrombin

Only promotes anticoagulation via activation of antithrombin → inhibiting XA

What must remain intact during the depolymerization of UFH to LMWH?

The AT-binding pentasaccharide

How are different LMWHs produced?

Enoxaparin → Benzylation → alkaline hydrolysis

Dalteparin → Nitrous acid depolymerization

Different methods affect cleavage sites, new structures, and % AT-binding pentasaccharides

How does LMWH structure affect its effects?

Size/charge → affects anti-Xa/IIa activity and cell interactions

ATIII/HCII binding → affects anticoagulant effects

Growth factor/signaling effects → may affect other biological effects

Basically different manufacturing methods = different LMWH drugs!!!!!!

What are some major advantages of LMWH over UFH?

Fixed dose administration → and can be used at home via subcutaneous injection

Higher bioavailability and less nonspecific binding

Less variability → routine aPTT monitoring usually unnecessary

Lower risk of HIT → heparin induced thrombocytopenia, immune response to heparin

Fondaparinux / Arixtra ?

Synthetic pentasaccharide

Specific binding and activating antithrombin

Anti-Xa : Anti-IIa activity ratios?

UFH → 1:1

LMWH → 2–4:1

Fondaparinux → Xa only

How can UFH cause heparin-induced thrombocytopenia?

UFH will bind to platelet factor 4 → PF4

The heparin-PF4 complex can cause a immunological reaction, causing antibodies to form leading to HIT → low platelet levels with or without thrombosis

What is the HIT risk with LMWH and fondaparinux?

LWMH and fondaparinux → VERY low risk for HIT

Pharmacodynamics of UFH?

MW → 3000-50,000

MOA → Inactivation of FXA and thrombin

Route → IV

Binding → Widespread and high

Monitoring → Tight aPTT monitoring required throughout

Dose → Adjusted based on aPTT

Generics? → Yes

Pharmacodynamics of LMWH?

MW →1000-9000

MOA → Inactivation of FXA

Route → SQ

Monitoring → Only required at start

Dose → Fixed dosage

Generics? → Yes

How does protamine reverse UFH?

Protamine (+) binds heparin (−) → immediate neutralization lasting → 2 hours

1 mg protamine → inactivates 100 units heparin

Give slowly IV to avoid hypotension

How effective is protamine for LMWH and fondaparinux?

LMWH → Partially/less effective

Fondaparinux → Not effective

Why is gamma-carboxylation important?

Gamma-carboxyglutamate binds Ca²⁺ → is essential for their activity and for coagulation factors interacting with the platelet surface

Blocking this via vitamin K antagonists → results in inactive coagulation factors

MOA of Vitamin K antagonists?

Inhibition of formation of vitamin K (active K → KH2), inhibiting the carboxylation of glutamate residues

Factors → II, VII, IX, X and protein C

Ex → Warfarin

How does citrate prevent coagulation?

Citrate binds Ca²⁺ → removes Ca²⁺ needed for coagulation → prevents clotting

Used in blood collection tubes → not as a therapeutic anticoagulant

Why does warfarin have a delayed anticoagulant effect?

Factor Xa and prothrombin have long half-lives → take time to decrease

Peak anticoagulant effect → ~3 days

What is VKORC1 and why is it important for warfarin?

Warfarin inhibits VKORC1 → decreases active vitamin K → decreases clotting factor production

VKORC1 polymorphisms can alter warfarin potency

How does CYP2C9 affect warfarin?

CYP2C9 metabolizes warfarin

CYP2C9 polymorphisms can alter warfarin pharmacokinetics

Why is there >20-fold variation in warfarin dose requirements between patients?

Largely due to genetic differences!

VKORC1 polymorphisms → alter warfarin potency

CYP2C9 polymorphisms → alter warfarin PK/metabolism

Pharmacokinetics of Warfarin?

Delayed onset and offset → 3 days

Mix of R and S enantiomers → S 3-5x more potent

Narrow therapeutic index

Mandatory INR monitoring of coagulation

How can displacement from albumin affect warfarin?

Aspirin, other salicylates, and sulfonamides → displace warfarin from albumin

Increases free warfarin → increase anticoagulant effect!!

What drugs inhibit warfarin major CYPs?

Acetaminophen

Amiodarone

Cimetidine

Azole antifungals

Sulfonamides

Phenytoin

These drugs increase the warfarin effect!!!!

What is the major CYP involved with warfarin?

CYP2C9 is the major enzyme

CYP3A4 and CYP1A2 are minor

How does CYP induction decrease warfarin effect?

CYP induction increases warfarin metabolism, which decreases warfarin levels and its anticoagulant effect!!!

2C9, 3A4, 1A2

What drugs induce warfarin major CYPs?

Carbamazepine

Rifampin

Decrease the anticoagulant effect!!!

How can increased clotting factor synthesis decrease warfarin effect?

Oral contraceptives and vitamin K1 increase clotting factor synthesis

Which decreases warfarin’s anticoagulant effect

What does cholestyramine do to warfarin?

Decrease its absorption

Therefore decreasing its effect

How do antiplatelets and other anticoagulants promote bleeding with warfarin?

They have additive effects with warfarin

Increasing the risk of bleeding.

What drugs have additive effects with warfarin?

Clopidogrel

Prasugrel

Aspirin

Heparin

Promote bleeding !!!

What is vitamin K1 / phytonadione used for with warfarin?

It is a reversal agent and coagulant

Can be used to treat bleeding from warfarin or other vitamin K antagonists

How does vitamin K1 reverse warfarin’s effects?

Promotes gamma-carboxylation of glutamate residues in clotting factors

This promotes coagulation!!

What are important PK facts about oral vitamin K1?

Half-life → 3–5 hours

Can take up to 24 hours to see its effect

Important safety facts about vitamin K1?

Generally safe in many populations → including children and pregnancy!!

Green leafy vegetables contain about 0.05–1 mg/cup → lettuce to kale

What does thrombin do in coagulation?

It cleaves an arginine-glycine bond in fibrinogen

Produces fibrin monomers → clot formation

How does hirudin inhibit thrombin?

Hirudin binds 1:1 to thrombin

Blocks thrombin’s protease active site → this part catalyzes the cleave of fibrinogen to fibrin

Prevents fibrinogen → fibrin

What is the difference between natural hirudin and lepirudin?

Natural hirudin → Found in certain leeches.

Lepirudin / Refludan → Recombinant hirudin produced using yeast cells and used clinically

Direct Factor Xa inhibitors?

Rivaroxaban

Apixaban

Edoxaban

Direct Thrombin inhibitors?

Dabigatran

Hirudins

Argatroban

What is Bivalirudin / Angiomax?

hirudin-like direct thrombin inhibitor

It is a 20-amino-acid peptide designed from hirudin

How does bivalirudin inhibit thrombin?

Binds to both the catalytic site and anion-binding exosite of thrombin

Inhibits both circulating and clot-bound thrombin

How does bivalirudin interact with thrombin?

Inhibits thrombin reversibly and competitively.

Thrombin slowly cleaves bivalirudin → but the part remains bound and continues inhibiting thrombin

Does not last long!!

What is Argatroban / Acova?

A small-molecule direct thrombin inhibitor

Given IV

It is a peptidomimetic of the Phe-Pro-Arg sequence in fibrinogen

How does argatroban inhibit thrombin?

Competitively and reversibly inhibits thrombin’s catalytic site

Inhibits both free and clot-bound thrombin

What effects are blocked by direct thrombin inhibitors?

Block fibrinogen → fibrin

Block thrombin activation of other coagulation factors

Block platelet aggregation and protein C activation

Examples of direct oral anticoagulants (DOACs)?

Dabigatran / Pradaxa

Rivaroxaban / Xarelto

Apixaban / Eliquis

Edoxaban / Savaysa

Dabigatran / Pradaxa?

Direct thrombin, IIA inhibitor

Oral capsules → very sensitive to moisture!!

Pro-drug

Major P-gp DDI

Rivaroxaban / Xarelto?

Factor Xa inhibitor

Oral tablets → 15/20 mg doses must be taken with a large meal

Major P-gp and 3A4 DDIs

Apixaban / Eliquis?

Factor Xa inhibitor

Oral tablets → do not need to take with food

Major P-gp and 3A4 DDIs

Edoxaban / Savaysa?

Factor Xa inhibitor

Oral tablets → do not need to take with food

Major contraindication for → CrCl <15 or >95

What are the properties of dabigatran?

Very polar and water-soluble

Poor oral absorption

Low tissue distribution

Why is dabigatran given as dabigatran etexilate?

Dabigatran etexilate → inactive prodrug form

More lipophilic → better oral absorption

Prodrug groups are cleaved by esterases

Why is tartaric acid included in dabigatran capsules?

Dabigatran etexilate mesylate has very low water solubility, slightly soluble in ethanol

Tartaric acid maintains an acidic capsule core → helps with drug dissolution/absorption

This helps with drug dissolution and absorption!

How should dabigatran capsules be stored?

Protect from air and moisture using a gas-impermeant seal with desiccant

Shelf-life → ~4 months once opened

What is the reversal agent for dabigatran?

Idarucizumab / Praxbind

Idarucizumab / Praxbind ?

Developed to bind dabigatran and reverse/block the anti-coagulant activity

IV dosed mAB

DOES NOT CAUSE BLOOD TO CLOT

Average wholesale price for 5g → $3,000

What is P-gP and why is it important with anticoagulants?

P-gP (MDR1/ABC1) → is an ATP-dependent efflux transporter

It pumps drugs out of cells, affecting their absorption and drug levels

Common drugs that inhibit P-gP and what effect does that have on DOACs?

Amiodarone

Dronedarone

Diltiazem
Felodipine

Nifedipine

Verapamil

INCREASE DOAC EFFECT

Common drugs that induce P-gP and what affect does that have on DOACs?

Rifampin

Phenytoin

St. Johns wort

DECREASE DOAC EFFECT

How do direct FXa inhibitors work?

Competitively bind and block the active site of Factor Xa

This decreases thrombin generation

How are direct FXa inhibitors different from thrombin inhibitors?

They do not directly inhibit thrombin

Therefore, they do not directly affect platelets → since thrombin activates platelets

How are direct FXa inhibitors similar to LMWHs?

Both primarily target Factor Xa inhibition

Direct FXa inhibitors are very different from LMWHs in how they inhibit Xa

What is the reversal agent for FXa inhibitors?

Andexanet alfa

Andexanet alfa?

A modified recombinant Factor Xa that acts as a decoy receptor.

It binds and sequesters Xa inhibitors so they can no longer inhibit the body's Factor Xa

What is unique about rivaroxaban absorption/PK?

Its lipophilic with limited aqueous solubility.

15 mg and 20 mg doses must be taken with food to improve absorption