PHAR 423: Anticoagulants and Thrombolytic Agents

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PHAR 423 Anticoagulants and Thrombolytic Agents

• Dr Thomas Abraham
• Spring 2004

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Overview of the Coagulation Cascade

• Damage to the vessel wall results in platelet
  adhesion and activation and activation of the
  coagulation cascade.
• DVT, PE, myocardial ischemia, coronary vasospasm,
  atrial arrhythmia
• Adherent platelets release autocoid mediators
  (TXs, ADP, etc.) which lead to aggregation of
  platelets.
• Aspirin inhibits autocoid release
• Thienopyridines decrease aggregation step
• The extrinsic coagulation cascade leads to
  production of thrombin which processes fibrinogen
  to fibrin to form a rigid meshwork.
• Coumarins interfere with coagulation factor
  function while heparins inhibit thrombin activity.

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Overview of the Coagulation Cascade

• Adherence of activated platelets to the fibrin
  strands leads to thrombus formation.
• Thrombin formation has multiple consequences
  but also serves to amplify the extrinsic pathway
  and enhance platelet release of mediators and
  aggregation.
• A formed clot is eventually degraded by the
  fibrinolytic pathway.
• Enhanced by thrombolytic agents e.g TPA, Urokinase

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Overview of the Coagulation Cascade
Extrinsic Coagulation Pathway

• Binding of factor VII to injured blood vessels
  in the presence of tissue factor (TF),
  phospholipid and Ca2 results in activation of
  factor VII.
• Sequential conversion of factors X, V and
  prothrombin ultimately produce thrombin which has
  multiple functions e.g. vasoconstriction,
  mitogenesis of vascular tissue and cleavage of
  fibrinogen.
• Va cofactor for Xa

• Cleavage of fibrinogen to fibrin results in the
  polymerization of fibrin into dense strands that
  form a meshwork onto which platelets adhere.

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Orally active Anticoagulants
Coumarin and Indandione Derivatives

• Bishydroxycoumarin originally identified from
  sweet clover.
• Minimal structural requirements for activity of
  coumarin derivatives are substitutions at
  positions 3, 4.

• Warfarin is weakly acidic, (sodium salt) and
  marketed as racemic mixture of R-(d) and
  S-(l)-forms. S-form has greater potency as
  anticoagulant.
• 4X greater potency of S-isomer
• Major anticoagulant effect due to ability of
  coumarin derivatives to antagonize vitamin
  K-mediated carboxylation of coagulation factors
  II, VII, IX, X, protein C and S.

Wisconsin Alumni Research Foundation-arin
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Orally active Anticoagulants
Mechanism of coumarin action

•    N-terminal glutamate residues are
  dicarboxylated by a carboxylase with vitamin K as
  a cofactor. Dicarboxylation of coagulation
  factors required for calcium binding properties
  of coagulation factors.
• g-carboxylated glutamate residues have the
  divalency required for high affinity Ca2
  binding.
• Cellular reductase regenerates the epoxide to
  the active vitamin K in the presence of NADH.
• Warfarin competes with the epoxide form of
  vitamin K for binding to the reductase.
• This results in build-up of the the epoxide form
  of vitamin K and decreased carboxylation of newly
  synthesized coagulation factors.

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Orally active Anticoagulants
Coumarin derivatives (contd.)

• Since coumarin derivatives only affect the
  g-carboxylation of newly synthesized coagulant
  proteins there is a lag time of several days
  before optimal anticoagulation is achieved.
• More rapidly acting anticoagulants need to be
  coadministered to the patient until warfarins
  effects become more overt.
• Warfarin dosing is adjusted to decrease
  coagulation function by about 75

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Orally active Anticoagulants
Coumarin derivatives (contd.)

• Almost complete bioavailability from oral, IM,
  IV admin. of warfarin. Onset of action requires
  depletion of functional coagulation factors full
  anticoagulant effect may take several days to
  achieve.
• Very high plasma protein binding (mostly to
  albumin) may be a site of major drug
  interaction.
• Caution with oral hypoglycemic agents and cardiac
  glycosides.
• Warfarin will cross the placental barrier and
  cause birth defects and spontaneous abortion.
  These agents should be used with extreme caution
  in any stage of pregnancy.
• Metabolized to inactive metabolites by liver
  (predominant) and kidneys and excreted in stool
  and urine (enterohepatic cycling) half-life of
  elimination 25-60 hours. R-form has more
  prolonged plasma half-life than S-isomer.
• Patients with decreased liver function will have
  decreased coagulant activity increased
  susceptibility to warfarin.

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Orally active Anticoagulants
Coumarin derivatives (contd.)

• Significant drug interactions are likely with
  many different agents
• Cholestyramine chelation of warfarin in the GI
  tract.
• Increased vitamin K intake decreased ability of
  warfarin to inhibit reductase function
• Amiodarone, sulfinpyrazone, cimetidine,
  metronidazole, grapefruit juice decreased
  warfarin metabolism or increased displacement
  from albumin binding
• Phenytoin, barbiturates, rifampin induction of
  warfarin metabolism.
• Cephalosporins heterocyclic side chains inhibit
  vit. K reductase function to enhance warfarin
  action.
• Adverse effects bleeding (intracranial,
  pericardial, nerve sheath, spinal cord, GI), skin
  necrosis, purple toes syndrome, alopecia,
  dermatitis, fever, NVD are less common.
• Skin lesions, purple toe syndrome may be due to
  coagulation in microvessels due to the loss of
  protein C (natural anticoagulant).
• Continuous serious bleeding can be treated with
  admin. of vitamin K (phytonadione) requirement
  of new functional coagulation factors results in
  delay of antidote effects (up to 24 h).

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Orally active Anticoagulants
Coumarin derivatives (contd.)

• Warfarin and its derivatives may be used in
  cases of venous thromboembolism, arrhythmias,
  cerebral vascular emboli and after prosthetic
  valve placement. Also used in DVT and PE.

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Orally active Anticoagulants

• Interferes with platelet aggregation by
• 1. inhibiting phosphodiesterase in platelets
  which leads to increased cAMP that decreases
  platelet adhesion.
•   Inhibit PDE cAMP metabolism cAMP
  accumulation in platelets
• 2. preventing uptake of adenosine into RBCs
  causing increased A2 receptor stimulation to
  increase cAMP in platelets.

Dipyridamole (Persantine)

• Adverse effects are primarily due to
  vasodilator activity of the agent headache,
  dizziness, GI intolerance, NVD, flushing.
• Used in combination with warfarin for pts with
  prosthetic heart valves, and in pts. with
  thromboembolic disease (usually in combination
  with other agents).

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Orally active Anticoagulants
Aspirin

• Metabolism of arachidonic acid results in
  formation of thromboxane A2 which is a potent
  platelet aggregator and vasoconstrictor.
• Aspirin covalently inactivates cyclooxygenase
  to decrease TBX A2 production in platelets.

• This action occurs at much lower concentrations
  than required for anti-inflammatory and
  anti-pyretic effects.
• 80-160mg taken daily can inhibit platelet
  function.
• More effective in preventing arterial clots than
  venous clots

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Orally active Anticoagulants
Thienopyridines Ticlopidine (Ticlid),
clopidogrel (Plavix)

• Orally active agents. May be prodrugs since
  they cannot prevent platelet aggregation in
  vitro.
• plasma from treated pts. inhibits platelet
  aggregation in vitro.
• Mechanism of action antagonizes adenosine
  diphosphate-mediated binding of activated
  platelets to fibrinogen.
•          ADP via the P2PLC receptor causes the
  activation of phospholipase C and elevation of
  intracellular calcium and change in shape.
•  shape change of platelets allows adhesion of
  fibrinogen
•         ADP via the P2AC receptor inhibits
  adenylate cyclase and decreases VAMP
  phosphorylation.
• Gi inhibits adenylyl cyclase to decrease cAMP
  levels  
• VAMP involved in platelet aggregation and
  adhesion
•          Thienopyridines covalently bind to the
  P2 receptors to prevent ADP-mediated
  intracellular signaling mechanisms that promote
  the stickyness and aggregation of platelets.

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Orally active Anticoagulants
Thienopyridines Ticlopidine (Ticlid),
clopidogrel (Plavix)

• Thienopyridines appear to be activated by
  CYP4503A4 to reactive metabolites that covalently
  interact with the P2 receptors
• Will increase bleeding time and has been found to
  be useful in preventing thrombosis following
  coronary artery stenting. Often used in
  combination with aspirin for synergistic
  therapeutic benefit. May have beneficial effects
  when combined with GP IIb/IIIa inhibitors.
• May be used in pts. who cannot tolerate aspirin,
  in combination with other anticoagulants.
• May decrease metabolism of phenytoin,
  carbamazepine and theophylline to enhance
  toxicity.
• Generally well tolerated compared to aspirin
  with the following adverse effects diarrhea,
  rash, nausea, dyspepsia, neutropenia (rare but
  may be fatal), cholestatic jaundice.
• In general clopidogrel has less toxic profile
  than ticlopidine.

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Parenteral Anticoagulants
Heparins

• Endogenous heparins are linear polysaccharides
  of 60-100kDa, complexed to a core protein and
  found in mast cells. Acidic characteristic and
  supplied as sodium salt.
• mucopolysaccharide

• Purified from pig intestinal mucosa or bovine
  lung core protein removed. Polysaccharide chain
  degraded during purification to fragments of 2-30
  kDa. Low molecular weight heparins (e.g.
  enoxaparin, less than 7 kDa) are purified from
  this fraction by gel filtration.
• Heparins not absorbed through GI tract and
  given parenterally (i.v. or deep s.c., but not
  i.m.) Subcutaneous has variable bioavailability
  and delayed onset time. Low mol. wt. heparins
  have longer biological t1/2.
• IM injection can result in intramuscular
  bleeding, necrosis.
• Longer t1/2 of the LMWHs allows less frequent
  dosing.

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Parenteral Anticoagulants
Heparins (contd.)
Heparin
Factor Xa or Thrombin
Antithrombin III

• Mechanism of action standard heparins catalyze
  the inhibition of Factor Xa and thrombin by
  antithrombin III.
• Low mol. wt. heparins (enoxaparin, dalteparin)
  predominantly catalyze the inhibition of Xa.
• Used in DVTs, PE, unstable angina, acute MI,
  during/after angioplasty or coronary stent
  placement, cardiac bypass surgery
• Heparins are contraindicated in bleeding
  disorders, hypersensitivity to heparins, severe
  hypertension, renal dysfunction.

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Parenteral Anticoagulants
Heparins (contd.)

• Adverse effects include bleeding,
  thrombocytopenia, hair loss, osteoporosis.
• HIT heparin-induced thrombocytopenia is severe
  form of platelet destruction that can result in
  uncontrolled bleeding or thrombus formation.
• HIT due to antibody formation against complex of
  heparin and platelet factor 4 which results in
  platelet activation, severe coagulation.
• Thrombocytopenia may be less severe with porcine
  heparin than bovine heparins.
• Combined use of standard and LMWHs can lead to
  excessive bleeding and death.
• In case of life-threatening hemorrhage due to
  heparin the anticoagulant effects can be reversed
  by administration of protamine sulfate.
• Ionic interaction between heparin and protamine
  inactivates heparin action.
• The amount of protamine needs to be titrated to
  just neutralize the amount of heparin to prevent
  non-specific effects.

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Parenteral Anticoagulants

• Danaparoid (Orgaran)
• Non-heparin glycosaminoglycans which inhibit
  factor Xa via antithrombin III
• Administered s.c. for DVT prophylaxis and HIT.
• Lepirudin (Refludan)
• Recombinant derivative of hirudin.
• Hirudin originally isolated from the salivary
  gland of medicinal leech
• A direct inhibitor of thrombin activity.
• Primarily used in HIT.

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Parenteral Anticoagulants
Inhibitors of Platelet Glycoprotein IIb/IIIa
Role of Glycoprotein IIb/IIIa in thrombus
formation

• On unstimulated platelets, GP IIb/IIIa is in a
  conformation that has low affinity for soluble
  fibrinogen.
• When platelets are activated, they undergo
  morphologic and physiologic changes, and GP
  IIb/IIIa molecule alters its conformation,
  becoming a high-affinity receptor for fibrinogen.
  

• Each fibrinogen molecule can bind to 2 GP
  IIb/IIIa molecules and therefore cross-link
  receptors on adjacent activated platelets and
  ultimately lead to formation of platelet-rich
  thrombi.

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Parenteral Anticoagulants (GPIIb/IIIa Inhibitors)
Abciximab (ReoPro)

• Is the Fab fragment of a chimeric monoclonal
  antibody. Administered by i.v injection or
  infusion.
• Used in pts. with unstable angina, post-MI and
  following angioplasty
• Binds to platelet glycoprotein IIb/IIIa
  receptors to prevent adhesion of platelets to
  fibrinogen.
• High affinity binding of the mAb to GPIIb/IIIa
  leads to prolonged platelet inhibition.
• Also binds other integrin receptors on vascular
  endothelial cells
•    Adverse effects include bleeding, nausea,
  vomiting, hypotension, atrial fibrillation,
  edema.
• Hypersensitivity reactions due to antibody
  formation

Platelet
Collagen
ADP
RheoPro
Thrombin
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Parenteral Anticoagulants (GPIIb/IIIa Inhibitors)
Eptifibatide (Integrilin)

• A heptapeptide cyclized by a disulfide bridge
  that mimics carpet viper venom toxin in
  preventing coagulation.
• Eptifibatide binds to GP IIb/IIIa with high
  affinity and prevents the interaction with von
  Willebrand factor and fibrinogen to prevent the
  formation of stable platelet aggregates.
• Indicated for pts. suffering from acute MI or
  undergoing PTCA to prevent the formation of new
  clots.
• Used in unstable angina
• Generally used in combination with aspirin and
  heparin
• Due to the relatively specific interaction of
  eptifibatide with GP IIb/IIIa no significant
  adverse effects result apart from potential for
  severe bleeding.

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Parenteral Anticoagulants (GPIIb/IIIa Inhibitors)
Tirofiban (Aggrastat)

• Synthetic, nonpeptide antagonist of the
  GPIIb/IIIa receptor that prevents fibrinogen
  binding to platelets.

• Poor oral bioavailability requires parenteral
  administration of tirofiban but other agents in
  this class are being devloped for oral
  adminstration e.g. sibrafiban, xemilofiban and
  orbofiban.
• Major adverse effects observed are potential
  for bleeding and thrombocytopenia especially when
  administered in combination with heparin.
• Anticoagulant effect is more reversible after
  withdrawal than that seen with RheoPro.

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Thrombolytic Agents

• Physiological Fibrinolysis
• Fibrinolytic system dissolves intravascular
  clots as a result of plasmin activation.
• Targets pathological rather than physiological
  clots
• Plasmin cleaves fibrin strands and coagulation
  factors to dissolve clots.

t-PA
Fibrin
Plasminogen

• Regulation of plasmin activation
• removes unwanted fibrin thrombi but fibrin in
  wounds is maintained.
• endothelial cells release tissue plasminogen
  activator (t-PA) in response to hemostasis.
• t-PA rapidly inactivated by plasminogen
  activator inhibitor 1 and 2 (unless t-PA is bound
  to fibrin) to prevent systemic activation of
  plasminogen to plasmin.
• t-PA binds to fibrin and converts plasminogen,
  that is also bound to fibrin, to plasmin.
• activated plasmin bound to fibrin is protected
  from the negative actions of a2-antiplasmin
• pharmacological application of plasminogen
  activators overwhelms the inhibitory controls on
  fibrinolysis.

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Thrombolytic Agents

• Streptokinase (Kabbikinase, Streptase)
• 47 kDa protein produced by b-hemolytic
  streptococci not an enzyme but forms stable 11
  complex with plasminogen.
• Alters the configuration of plasminogen to
  expose the proteolytic catalytic site which
  autocatalyzes its own activation to free plasmin.
  
• Will activate plasminogen that is not bound to
  fibrin less specific than t-PA
• High loading doses are required to overcome
  antibody inhibition has half-life of 40-80 min
  streptokinaseplasminogen complex not inhibited
  by anti-plasminogen inhibitors.
• Adverse rxns. include bleeding, allergic rxns.,
  anaphylaxis and fever (rare).
• Allergic rxns. in pts. who have had b-hemolytic
  streptococcal infections
• Anistraplase (Eminase) is a plasminogen-strepto
  kinase complex that is more fibrin specific than
  streptokinase alone.

Streptokinase
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Thrombolytic Agents
Tissue plasminogen activator (Alteplase,
Activase)

• Serine protease of 500kDa, produced by
  recombinant DNA technology. Metabolized by liver
  with half-life of 5-10 min.
• Requires the presence of fibrin to convert
  plasminogen to plasmin. Effective in lysing
  clots during acute MI, pulmonary embolism and
  DVT.
• Reteplase is modified by the removal of several
  amino acids from the t-PA sequence.

• Urokinase (Abbokinase)
• A serine protease isolated from cultured human
  kidney cells .
• Used in the clearing of clots from IV lines.
• Metabolized by liver with half-life of 15-20
  min.
• Is not fibrin specific and can cause systemic
  lytic state.
• Current supply is inconsistent due to production
  problems

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Thrombolytic Agents

• Adverse effects of thrombolytic agents
• Hemorrhagic toxicity is the main adverse effect
  in thrombolytic therapy and is due to
• Lysis of fibrin in physiological thrombi
• Systemic lytic state
• After inhibitory protein have been overwhelmed
  and destruction of coagulant proteins results
• Can lead to intracranial hemorrhage
• Excessive fibrinolysis due to t-PA can be limited
  with aminocaproic acid
• Contraindications GI ulcers, bleeding
  disorders, hypertension, recent surgery, aortic
  dissection.