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Pharm Ch 22
Physiology of Hemostasis
 Thrombosis – pathologic state in which normal hemostatic processes are activated inappropriately
 Injured blood vessel must induce formation of blood clot to prevent blood loss and allow healing; clot formation
must remain localized to prevent widespread clotting in intact vessels; clot formation occurs in 4 stages
o Localized vasoconstriction occurs as response to reflex neurogenic mechanism and secretion of
endothelium-derived vasoconstrictors such as endothelin
o Primary hemostasis – platelets activated and adhere to exposed subendothelial matrix
 Platelet activation involves change in shape of platelet and release of secretory granule contents
from platelet that recruit other platelets, causing more platelets to adhere to subendothelial
matrix and aggregate with one another at site of vascular injury (primary hemostatic plug
formation)
o Secondary hemostasis (coagulation cascade) – activated endothelium and nearby cells express
membrane-bound procoagulant factor (tissue factor) that complexes with coagulation factor VII to
initiate coagulation cascade (end result is thrombin)
o Thrombin converts soluble fibrinogen to insoluble fibrin polymer that forms matrix of clot and induces
more platelet recruitment and activation
 Fibrin clot formation (secondary hemostasis) overlaps temporally with platelet plug formation
(primary hemostasis), and each process reinforces the other
 Platelet aggregation and fibrin polymerization lead to formation of stable, permanent plug
 Antithrombotic mechanisms restrict permanent plug to site of vessel injury, ensuring permanent
plug does not inappropriately extend to occlude vascular tree
 Vasoconstriction – occurs immediately after vascular injury, mediated by neurogenic mechanism
o Local endothelial secretion of endothelin, a potent vasoconstrictor, potentiates reflex vasoconstriction
o Because vasoconstriction is transient, bleeding would resume if primary hemostasis were not activated
 Primary hemostasis – goal is to form platelet plug that rapidly stabilizes vascular injury
o Platelets – cell fragments that arise by budding from megakaryocytes in bone marrow (contain
cytoplasm but lack nuclei); glycoprotein receptors in platelet PM are primary mediators by which
platelets activated
o Primary hemostasis involves transformation of platelets into hemostatic plug through 3 reactions:
adhesion, granule release reaction, and aggregation and consolidation
 Platelet adhesion – platelets adhere to subendothelial collagen exposed after vascular injury –
von Willebrand factor (vWF) is secreted by both activated platelets and injured endothelium and
binds both to surface receptors (especially glycoprotein Ib [GPIb]) on platelet membrane and to
exposed collagen; bridging action mediates adhesion of platelets to collagen; platelet
glycoprotein VI (GPVI) interacts directly with collagen in exposed vessel wall; both GPIb-vWFcollagen interaction and GPVI-collagen interaction required for initiation of primary hemostasis
 Platelet granule release reaction – initiated by agonist binding to cell-surface receptors, which
activate intracellular protein phosphorylation cascades and ultimately causes release of granule
contents
 Stimulation by ADP, epinephrine, and collagen lead to activation of platelet membrane
phospholipase A2 (PLA2), which cleaves membrane phospholipids and liberates
arachidonic acid, which is converted into a cyclic endoperoxiade by platelet
cyclooxygenase
 Thromboxane synthase converts cyclic endoperoxide into thromboxane A2 (TxA2), which
causes vasoconstriction at site of vascular injury via G protein-coupled receptor,
inducing decrease in cAMP levels in vascular smooth muscle cells – TxA2 also stimulates
granule release reaction within platelets, propagating cascade of platelet activation and
vasoconstriction
 During release reaction, large amounts of ADP, Ca2+, ATP, serotonin, vWF, and platelet
factor 4 actively secreted from platelet granules
 ADP particularly important in mediating platelet aggregation, causing platelets to
become sticky and adhere to one another; although strong agonists (thrombin and

collagen) can trigger granule secretion even when aggregation prevented, ADP can
trigger granule secretion only in presence of platelet aggregation
 Pathway of platelet activation without disruption of endothelium or involvement of vWF
initiated by tissue factor (lipoprotein expressed by activated leukocytes and
microparticles derived from activated leukocytes) – tissue factor forms complex with
factor VIIa, and complex activates factor IX, which leads to proteolytic cascade that
results in generation of thrombin (factor IIa), a muiltifunctional enzyme that plays
critical role in coagulation cascade – in tissue-factor initiated pathway of platelet
activation, thrombin cleaves protease-activated receptor 4 on platelet surface and
thereby causes platelets to release ADP, serotonin, and TxA2; by activating other nearby
platelets, these agonists amplify signal for thrombus formation
 Platelet aggregation and consolidation – TxA2, ADP, and fibrous collagen are potent mediators of
platelet aggregation
 TxA2 promotes platelet aggregation through stimulation of G protein-coupled TxA2
receptor in platelet membrane; binding of TxA2 to platelet TxA2 receptors leads to
activation of phospholipase C (PLC) which hydrolyzes phosphatidylinositol 4,5bisphosphate (PI[4,5]P2) to yield inositol 1,4,5-trisphosphate (IP3) and diacylglycerol
(DAG) – IP3 raises cytosolic Ca2+ concentration, and DAG activates protein kinase C (PKC),
which promotes activation of PLA2, which induces expression of GPIIb-IIIa (membrane
integrin that mediates platelet aggregation)
 ADP triggers platelet activation by binding to G protein-coupled ADP receptors on
platelet surface – 2 subtypes of G protein-coupled platelet ADP receptors: P2Y1
receptors and P2Y(ADP) receptors – activation of ADP receptors mediates platelet shape
change and expression of functional GPIIb-IIIa
o P2Y1 – Gq-coupled receptor that releases intracellular Ca2+ stores through
activation of phospholipase C
o P2Y(ADP) – Gi-coupled receptor that inhibits adenylyl cyclase – target of
antiplatelet agents ticlopidine, clopidogrel, and prasugrel
 Fibrous collagen binds directly to platelet glycoprotein VI (GPVI) which initiates signaling
cascades that promote granule release reaction and induce conformational changes in
cell-surface integrins (especially GPIIb-IIIa and α2β1) that promote direct or indirect
binding of integrins to collagen – strengthens adhesion of activated platelets to
subendothelial matrix
 Platelets aggregate with one another through bridging molecule (fibrinogen), which has multiple
binding sites for functional GPIIb-IIIa – fibrinogen-GPIIb-IIIa interaction critical for platelet
aggregation ultimately leading to formation of reversible clot (primary hemostatic plug)
 Activation of coagulation cascade leads to generation of fibrin, initially at periphery of primary
hemostatic plug
 Platelet pseudopods attach to fibrin strands at periphery of plug and contract, yielding
compact, solid, irreversible clot (secondary hemostatic plug)
Secondary hemostasis – coagulation cascade; goal is to form stable fibrin clot at site of vascular injury
o Most plasma coagulation factors circulate as inactive proenzymes synthesized by liver; proenzymes
proteolytically cleaved by activated factors that precede them in cascade (activation reaction is catalytic
not stoichiometric, allowing for amplification of product)
o Major activation reactions in cascade occur at sites where phospholipid-based protein-protein complex
has formed (complex composed of membrane surface provided by activated platelets, activated
endothelial cells, and possibly activated leukocyte microparticles; an enzyme (activated coagulation
factor); substrate (proenzymes form of downstream coagulation factor); and cofactor
o Presence of negatively charged phospholipids, especially phosphatidylserine, critical for assembly of
complex – phosphatidylserine, which is normally in inner leaflet of PM, translocates to outer leaflet in
response to agonist stimulation of platelets, endothelial cells, or leukocytes
o Calcium required for enzyme, substrate, and cofactor to adopt proper conformation for proteolytic
cleavage of coagulation factor proenzymes to its activated form
o
Coagulation cascade divided into intrinsic and extrinsic pathways
 Intrinsic pathway activated by factor XII (Hageman factor)
 Extrinsic pathway initiated by tissue factor, activated endothelial cells, subendothelial smooth
muscle cells, and subendothelial fibroblasts at site of vascular injury
 Both pathways merge at activation of factor X, but there is much interconnection between the
pathways
 Because factor VII (activated by extrinsic pathway) can proteolytically activate factor IX (key
factor in intrinsic pathway), extrinsic pathway is regarded as primary pathway for initiation of
coagulation
o Both intrinsic and extrinsic coagulation pathways lead to activation of factor X, which proteolytically
cleaves prothrombin (factor II) to thrombin (factor IIa) [reaction requires factor V]
o Thrombin acts in coagulation cascade to
 Convert soluble plasma protein fibrinogen into fibrin, which forms long, insoluble polymer fibers
 Activate factor XIII, which cross-links fibrin polymers into highly stable meshwork or clot
 Amplify clotting cascade by catalyzing feedback activation of factors VIII and V
 Strongly activate platelets, causing granule release, platelet aggregation, and platelet-derived
microparticle generation
 Modulate coagulation response – thrombin binds to thrombin receptors on intact vascular
endothelial cells adjacent to area of vascular injury and stimulates these cells to release platelet
inhibitors prostacyclin (PGI2) and NO, profibrinolytic protein tissue plasminogen activator (t-PA),
and endogenous t-PA modulator plasminogen activator inhibitor 1 (PAI-1)
o Thrombin receptor (protease-activated G protein-coupled receptor) expressed in PM of platelets,
vascular endothelial cells, smooth muscle cells, and fibroblasts – activation of thrombin receptor
involves proteolytic cleavage of extracellular domain of receptor by thrombin, and a new NH2-terminaltethered ligand binds intramolecularly to a discrete site within receptor and initiates intracellular
signaling
 Activation of thrombin receptor results in G protein-mediated activation of PLC and inhibition of
adenylyl cyclase
o Microparticles – vesicular structures derived from leukocytes, monocytes, platelets, endothelial cells,
and smooth muscle cells that display proteins of the cells from which they were derived (for example,
subpopulation of microparticles is released from monocytes that are activated in context of tissue injury
and inflammation)
 Express both tissue factor and P-selectin glycoprotein ligand-1 (PSGL-1), which binds to Pselectin adhesion receptor expressed on activated platelets
 By recruiting tissue factor-bearing microparticles throughout developing platelet plug (primary
hemostasis), thrombin generation and fibrin clot formation (secondary hemostasis) could be
greatly accelerated within plug itself
 Vessel-wall tissue factor (expressed by activated endothelial cells, subendothelial fibroblasts,
and smooth muscle cells) and microparticle tissue factor important for formation of stable clot
Regulation of Hemostasis
 Hemostasis exquisitely regulated because it must be restricted to local site of vascular injury and size of primary
and secondary hemostatic plugs must be restricted so that vascular lumen remains patent
 After vascular injury, intact endothelium in immediate vicinity of injury becomes activated and presents a set of
procoagulant factors that promote hemostasis at site of injury and anticoagulant factors that restrict
propagation of clot beyond site of injury
 Procoagulant factors, such as tissue factor and phosphatidylserine, tend to be membrane-bound and localized to
site of injury – provide a surface on which coagulation cascade can proceed
 Anticoagulant factors generally secreted by endothelium and are soluble in blood, so activated endothelium
maintains balance of procoagulant and anticoagulant factors to limit hemostasis to site of vascular injury
 After vascular injury, endothelium surrounding injured area participates in five separate mechanisms that limit
initiation and propagation of hemostatic process to immediate vicinity of injury
o
Prostacyclin (PGI2) – eicosanoid (metabolite of arachidonic acid) synthesized and secreted by
endothelium; by acting through Gs protein-coupled platelet-surface receptors, PGI2 increases cAMP
levels within platelets and thereby inhibits platelet aggregation and platelet granule release
 PGI2 has potent vasodilatory effects – induces vascular smooth muscle relaxation by increasing
cAMP levels within vascular smooth muscle cells (physiologically antagonistic to TxA2, which
induces platelet activation and vasoconstriction by decreasing intracellular cAMP)
 Prevents platelets from adhering to intact endothelium that surrounds site of vascular injury and
maintains vascular patency around site of injury
o Antithrombin III – inactivates thrombin and other coagulation factors (IXa, Xa, XIa, and XIIa) by forming
stoichiometric complex with coagulation factor enhanced by heparin-like molecule expressed at surface
of intact endothelial cells, ensuring mechanism is operative at all locations in vascular tree except where
endothelium is denuded at site of vascular injury
 Heparin-like molecules on endothelial cells bind to and activate antithrombin III, which is then
primed to complex with and thereby inactivate activated coagulation factors
o Protein C and protein S – vitamin K-dependent proteins that slow coagulation cascade by inactivating
coagulation factors Va and VIIIa; part of feedback control mechanism in which excess thrombin
generation leads to activation of protein C, which, in turn, helps prevent enlarging fibrin clot from
occluding vascular lumen
 Endothelial cell-surface protein thrombomodulin is receptor for both thrombin and protein C in
blood; thrombomodulin binds them in such a way that thrombomodulin-bound thrombin
cleaves protein C to activated protein C (protein Ca); protein Ca then inhibits clotting by cleaving
and thereby inactivating factors Va and VIIIa (requires protein S as cofactor)
o Tissue factor pathway inhibitor (TFPI) limits action of tissue factor – coagulation cascade initiated when
factor VIIa complexes with TF at site of vascular injury and resulting VIIa-TF complex catalyzes activation
of factors IX and X; after limited quantities of factors IXa and Xa are generated, VIIa-TF complex is
feedback inhibited by TFPI by
 TFPI binds to factor Xa and neutralizes its activity in Ca2+-independent reaction
 TFPI-Xa complex interacts with VIIa-TF complex via second domain on TFPI, so that quaternary
Xa-TFPI-VIIa-TF complex formed
 Molecular knots of TFPI molecule hold quaternary complex tightly together and thereby
inactivate VIIa-TF complex, preventing excessive TF-mediated activation of factors IX and X
o Plasmin – exerts anticoagulant effect by proteolytically cleaving fibrin into fibrin degradation products;
generated by proteolytic cleavage of plasminogen (plasma protein synthesized in liver) catalyzed by
tissue plasminogen activator (t-PA), which is synthesized and secreted by endothelium
 Plasmin activity regulated by
 t-PA is most effective when bound to fibrin meshwork
 t-PA activity can be inhibited by plasminogen activator inhibitor (PAI) – when local
concentrations of thrombin and inflammatory cytokines (such as IL-1 and TNF-α) are
high, endothelial cells increase release of PAI, preventing t-PA from activating plasmin,
ensuring stable fibrin clot forms at site of vascular injury
 α2-antiplasmin (plasma protein that neutralizes free plasmin in circulation) prevents
random degradation of plasma fibrinogen
Pathogenesis of Thrombosis
 thrombosis – pathologic extension of hemostasis where coagulation reactions inappropriately regulated so clot
uncontrollably enlarges and occludes lumen of blood vessel (pathologic clot is thrombus)
 Virchow’s triad – three factors that influence one another and predispose to thrombus formation
o Endothelial injury – dominant influence on thrombus formation in heart and arterial circulation;
predisposes vascular lumen to thrombus formation through
 Platelet activators, such as exposed subendothelial collagen, promote platelet adhesion to
injured site
 Exposure of tissue factor on injured endothelium initiates coagulation cascade
 Natural antithrombotics (such as t-PA and PGI2) become depleted at site of vascular injury
because they rely on functioning of intact endothelial cell layer
o
Abnormal blood flow – state of turbulence or stasis rather than laminar flow; turbulent blood flow
causes endothelial injury, forms countercurrents, and creates local pockets of stasis; local stasis can also
result from formation of aneurysm (focal out-pouching of vessel or cardiac chamber) or MI
 Region of infarcted myocardium serves as favored site for stasis
 Cardiac arrhythmias like A fib can also generate areas of local stasis
 Stasis is major cause for formation of venous thrombi
 Disruption of normal blood flow by turbulence or stasis promotes thrombosis by
 Absence of laminar blood flow allows platelets to come into close proximity to vessel
wall
 Stasis inhibits flow of fresh blood into vascular bed so activated coagulation factors in
region not removed or diluted
 Abnormal blood flow promotes endothelial cell activation, which leads to prothrombotic
state
o Hypercoagulability – abnormally heightened coagulation response to vascular injury resulting from
primary (genetic) disorders or secondary (acquired) disorders
 Most prevalent known mutation for hypercoagulability resides in gene for coagulation factor V,
most common of which is Leiden mutation where glutamine is substituted for arginine at
position 506 (part of site in factor Va marked for proteolytic cleavage by activated protein C
 Mutant factor V Leiden protein is resistant to proteolytic cleavage by activated protein
C, so factor Va allowed to accumulate and thereby promote coagulation
 Second common mutation is prothrombin G20210A mutation where adenine is substituted for
quinine in 3’-untranslated region of prothrombin gene; leads to 30% increase in plasma
prothrombin levels
 Associated with significantly increased risk of venous thrombosis and modestly
increased risk of arterial thrombosis
 Other genetic disorders that predispose to thrombosis include mutations in fibrinogen, protein
C, protein S, and antithrombin III genes
 Patients with genetic deficiency of protein C, protein S, or antithrombin III often present
with spontaneous venous thrombosis
 Heparin-induced thrombocytopenia syndrome – acquired hypercoagulability; sometimes
heparin administration stimulates immune system to generate circulating antibodies directed
against complex consisting of heparin and platelet factor 4; because platelet factor 4 present on
platelet and endothelial cell surfaces, antibody binding to heparin-platelet factor 4 complex
results in antibody-mediated removal of platelets from circulation (thrombocytopenia)
 In some patients, antibody binding also causes platelet activation, endothelial injury,
and prothrombotic state (low-molecular-weight heparin associated with lower incidence
of thrombocytopenia than unfractionated heparin)
 Microparticles bearing tissue-factor protein demonstrated in blood of healthy individuals, but no
detectable tissue-factor activity present in normal blood – microparticles contain inactive tissue
factor (in encrypted form) and it is activated only upon recruitment of particles to site of
vascular injury
 In pathologic states, circulating microparticles may contain activated tissue factor, which
could predispose to development of thrombotic events
 High levels of circulating microparticles implicated in thrombosis associated with cancer,
atherothrombosis, and paroxysmal nocturnal hemoglobinuria
Hemorrhagic Disorders
 Disorders involving insufficient levels of functional platelets or coagulation factors can lead to hypocoagulable
state characterized by episodes of uncontrolled hemorrhage
 Can result from disorders of vasculature, vitamin K deficiency, and disorders or deficiencies of platelets,
coagulation factors, and von Willebrand factor
 Hemophilia A – most common genetic disorder of serious bleeding; reduced amount or activity of coagulation
factor VIII; X-linked mode of transmission, and 30% of patients have no family history and presumably represent
spontaneous mutations
o
Severity of disease depends on type of mutation in factor VIII gene: patients with 6-50% normal factor
VIII activity manifest a mild form of disease, those with 2-5% activity manifest moderate disease, and
those with less than 1% develop severe disease
o Patients bruise easy and can develop massive hemorrhage after trauma or surgery
o Spontaneous hemorrhage can occur in body areas normally subjected to minor trauma, including joint
spaces, where spontaneous hemorrhage leads to formation of hemoarthroses
o Petechiae (microhemorrhages involving capillaries and small vessels, especially in mucocutaneous areas)
usually indication of platelet disorders – not present in patients with hemophilia
o Patients treated with infusions of factor VIII that is either recombinant or derived from human plasma –
sometimes complicated in patients who develop antibodies against factor VIII
Pharmacologic Classes and Agents
 Antiplatelet agents – used against MI and stroke caused by thrombosis in coronary and cerebral arteries
o Cyclooxygenase inhibitors – aspirin inhibits synthesis of prostaglandins, inhibiting platelet granule
release reaction and interfering with normal platelet aggregation
 Activation of both platelets and endothelial cells induces phospholipase A2 (PLA2) to cleave
membrane phospholipids and release arachidonic acid, which is then transformed into cyclic
endoperoxide (prostaglandin G2) by enzyme COX
 In platelets, PGG2 converted into TxA2, which causes localized vasoconstriction and is potent
inducer of platelet aggregation and platelet granule release reaction
 In endothelial cells, PGG2 is converted to prostacyclin, which causes localized vasodilation and
inhibits platelet aggregation and platelet granule release reaction
 Aspirin acts by covalently acetylating serine residue near active site of COX enzyme, inhibiting
synthesis of cyclic endoperoxide and various metabolites of cyclic endoperoxide
 In absence of TxA2, there is marked decrease in platelet aggregation and platelet granule
release reaction
 Because platelets do not contain DNA or RNA, they cannot regenerate new COX enzyme
once aspiring has permanently inactivated all available COX enzyme
 Although aspirin inhibits COX enzyme in endothelial cells, its action is not permanent
because they are able to synthesize new COX molecules (endothelial cell production of
prostacyclin relatively unaffected by aspirin at pharmacologically low doses
 Most often used as antiplatelet agent to prevent arterial thrombosis leading to transient
ischemic attack, stroke, and MI – most effective as selective antiplatelet agent when
taken in low doses and/or at infrequent intervals
 Taken at high doses, aspirin can inhibit prostacyclin production without increasing
effectiveness of drug as antiplatelet agent
 Other NSAIDs not as widely used in prevention of arterial thrombosis because inhibitory action
of these drugs on cyclooxygenase not permanent
 COX-1 – predominant COX isoform in platelets; endothelial cells express both COX-1 and COX-2
 Aspirin inhibits both COX-1 and COX-2 , so it serves as effective antiplatelet agent
 Selective COX-2 inhibitors cannot be used as antiplatelet agents because they are poor inhibitors
of COX-1
 Use of selective COX-2 inhibitors associated with increased cardiovascular risk
o Phosphodiesterase inhibitors – in platelets, an increase in concentration of intracellular cAMP leads to
decrease in platelet aggregability; cAMP activates protein kinase A, which decreases availability of
intracellular Ca2+ necessary for platelet aggregation; inhibitors of platelet phosphodiesterase decrease
platelet aggregability by inhibiting cAMP degradation, while activators of platelet adenylyl cyclase
decrease platelet aggregability by increasing cAMP synthesis (no direct adenylyl cyclase activators in
clinical use)
 Dipyridamole – inhibitor of platelet phosphodiesterase that decreases platelet aggregability;
weak antiplatelet effects, so usually administered in combination with warfarin or aspirin
 Combination of dipyridamole and warfarin can be used to inhibit thrombus formation
on prosthetic heart valves

o
o
Combination of dipyridamole and aspirin used to reduce likelihood of thrombosis in
patients with thrombotic diathesis
 Dipyridamole has vasodilatory properties
 May induce angina in patients with coronary artery disease by causing coronary steal
phenomenon, which involves intense dilation of coronary arterioles
ADP receptor pathway inhibitors – derivatives of thienopyridine that irreversibly inhibit ADP-dependent
pathway of platelet activation; covalently modify and inactivate platelet P2Y(ADP) receptor (P2Y12),
which is physiologically coupled to inhibition of adenylyl cyclase
 Ticlopidine – first-generation thienopyridine; prodrug that requires conversion to active thiol
metabolites in liver; maximal platelet inhibition observed 8-11 days after initiating therapy with
drug (4-7 days if used in combination with aspirin); administration of loading dose can produce
more rapid antiplatelet response
 Used for secondary prevention of thrombotic strokes in patients intolerant of aspirin or
in combination with aspirin to prevent stent thrombosis after placement of coronary
artery stents
 Use occasionally associated with neutropenia, thrombocytopenia, and thrombotic
thrombocytopenic purpura (TTP)
 Largely been replaced by clopidogrel because of more favorable adverse-effect profile
and more rapid onset of action
 Clopidogrel – second-generation thienopyridine closely related to ticlopidine; used in
combination with aspirin for improved platelet inhibition during and after percutaneous
coronary intervention
 Prodrug that must undergo oxidation by hepatic P450 enzymes to active drug form, so it
may interact with statins, proton pump inhibitors, and other drugs metabolized by P450
isoforms
 Used for secondary prevention in patients with recent MI, stroke, or peripheral vascular
disease; can also be used for acute coronary syndromes treated with either
percutaneous coronary intervention or coronary artery bypass grafting
 Requires loading dose to achieve maximal antiplatelet effect rapidly
 GI effects of clopidogrel similar to those of aspirin
 Lacks significant bone marrow toxicity associated with ticlopidine
 Prasugrel – third-generation thienopyridine; irreversible antagonist of P2Y12 ADP receptor; used
for acute coronary syndromes treated with percutaneous coronary intervention
 Prodrug used in combination with aspirin
 More efficiently metabolized than clopidogrel, resulting in higher concentrations of
active drug and more complete platelet inhibition, it may increase risk of bleeding
relative to clopidogrel, especially in patients over 75 years of age or weighing less than
60 kg
 Main limitations of this class of drug attributed to irreversible antiplatelet effect of drugs and
variability of platelet inhibition among individuals
GPIIb-IIIa antagonists – GPIIb-IIIa receptors constitute final common pathway of platelet aggregation,
serving to bind fibrinogen molecules that bridge platelets to one another; antagonists of GPIIb-IIIa
prevent fibrinogen binding to GPIIb-IIIa receptor and thus serve as inhibitors of platelet aggregation
 Eptifibatide – highly efficacious inhibitor of platelet aggregation; synthetic peptide that
antagonizes platelet GPIIb-IIIa receptor with high affinity; used to reduce ischemic events in
patients undergoing percutaneous coronary intervention and treat unstable angina and non-ST
elevation MI
 Abciximab – chimeric mouse-human monoclonal antibody directed against human GPIIb-IIIa
receptor; occupation of 50% of platelet GPIIb-IIIa receptors by abciximab significantly reduces
platelet aggregation; binding is irreversible with dissociation half time of 18-24 hours
 Adding abciximab to conventional antithrombotic therapy reduces long-term and shortterm ischemic events in patients undergoing high-risk percutaneous coronary
intervention


Tirofiban – nonpeptide tyrosine analogue that reversibly antagonizes fibrinogen binding to
platelet GPIIb-IIIa receptor; inhibits platelet aggregation; used for acute coronary syndromes
 Any GPIIb-IIIa antagonist can cause bleeding as adverse effect because of antiplatelet agents
 Reversibility differs with different drugs of this class
o Abciximab is irreversible inhibitor of platelet function, and all abciximab
previously infused is already bound to platelets, so infusion of fresh platelets
after drug has been stopped can reverse antiplatelet effect
o Because eptifibatide and tirofiban bind receptor reversibly and are infused in
great stoichiometric excess of receptor number, infusion of fresh platelets offers
new sites to which drug can bind and it is not practical to deliver sufficient
number of platelets to overwhelm vast excess of drug present, so best way to
reverse is to stop treatment and let platelets recover as drug is eliminated
Anticoagulants – used to prevent and treat thrombotic disease; 4 classes (warfarin, unfractionated and lowmolecular-weight heparins, selective factor Xa inhibitors, and direct thrombin inhibitors); target various factors
in coagulation cascade, interrupting cascade and preventing formation of stable fibrin meshwork; bleeding is
adverse effect – recombinant activated protein C has anticoagulant activity, but clinical indication is severe
sepsis
o Warfarin – act by affecting vitamin K-dependent reactions
 Vitamin K required for normal hepatic synthesis of coag factors II, VII, IX, and X, protein C, and
protein S, which are biologically inactive as unmodified polypeptides following protein synthesis
on ribosomes and gain biological activity by post-translational carboxylation of amino-terminal
glutamic acid residues
 γ-carboxylated glutamate residues (not unmodified glutamate residues) capable of
binding Ca2+ ions, which induces conformational change required for efficient binding of
proteins to phospholipid surfaces
 Binding of Ca2+ to γ-carboxylated molecules increases enzymatic activity of coag factors
IIa, VIIa, IXa, Xa, and protein Ca by about 1000x (also necessary for cofactor function of
protein S
 Carboxylation reaction requires precursor form of target protein with its 9-12 aminoterminal glutamic acid residues, CO2, molecular oxygen, and reduced vitamin K
 During carboxylation reaction, vitamin K is oxidized to inactive 2,3-epoxide – vitamin K
epoxide reductase (VKORC1) required to convert inactive 2,3-epoxide into active,
reduced form of vitamin K
 Regeneration of reduced vitamin K essential for sustained synthesis of biologically
functional clotting factors II, VII, IX, and X
 Warfarin acts on carboxylation pathway by blocking epoxide reductase that mediates
regeneration of reduced vitamin K; because depletion of reduced vitamin K in liver prevents γcarboxylation reaction required for synthesis of biologically active coagulation factors, onset of
action of oral anticoagulants parallels half-life of coagulation factors in circulation
 Pharmacologic effect of single dose of warfarin not manifested for 18-24 hours (3-4
factor VII half-lives because factor VII has shortest half-life)
 Delayed action distinguishes warfarin class of anticoagulants from all other classes of
anticoagulants
 Small population of patients genetically resistant to warfarin because of mutations n
epoxide reductase gene – require 10-20x usual dose to achieve desired effect
 Genetic variation of VKORC1 gene associated with 25-30% of variance in warfarin
maintenance dose in patients
 Often administered to complete course of anticoagulation that has been initiated with
heparin and to prevent thrombosis in predisposed patients
 Orally administered warfarin is nearly 100% bioavailable, and levels peak 0.5-4 hours
after administration
 99% of racemic warfarin bound to plasma protein (albumin)


o
Relatively long elimination half-life (~36 hours)
Hydroxylated by cytochrome P450 system in liver to inactive metabolites that are
subsequently eliminated in urine
 Drug-drug interactions must be carefully considered because warfarin is highly albuminbound drug (other albumin-bound drugs can increase free plasma concentrations of
both drugs)
 Because warfarin is metabolized by P450 enzymes in liver, coadministration of other
drugs that induce and/or compete for P450 metabolism can affect plasma
concentrations of both drugs
 For severe hemorrhage, patients should receive fresh frozen plasma, which contains
biologically functional clotting factors II, VII, IX, and X
 Warfarin should never be administered to pregnant women because it can cross
placenta and cause hemorrhagic disorder in the fetus
 Newborns exposed to warfarin in utero may have serious congenital defects
characterized by abnormal bone formation (certain bone matrix proteins γcarboxylated)
 Rarely causes skin necrosis as result of widespread thrombosis in microvasculature
 Since warfarin prevents synthesis of biologically active proteins C and S (anticoagulants),
patients genetically deficient in protein C or protein S (most commonly, patients
heterozygous for protein C deficiency) have an imbalance between warfarin’s effects on
coagulation factors and its effects on proteins C and S may lead to microvascular
thrombosis and skin necrosis
 Monitored by PT (test of extrinsic and common pathways of coagulation); warfarin
prolongs PT mainly because it decreases amount of biologically functional factor VII in
plasma (INR)
Heparin – sulfated mucopolysaccharides stored in secretory granules of mast cells; highly sulfated
polymer of alternating uronic acid and D-glucosamine; strongest organic acid in human body
 Unfractionated heparin – often prepared from bovine lung and porcine intestinal mucosa (5-30
kDa)
 LMW heparins prepared from standard heparin by gel-filtration chromatography (1-5 kDa)
 Mechanism of action depends on presence of specific plasma protease inhibitor (antithrombin
III), which inactivates thrombin and serine proteases, including factors IXa, Xa, Xia, and XIIa
 Antithrombin III can be considered as stoichiometric suicide trap for serine proteases
 When one protease encounters antithrombin III molecule, serine residue at active site
of protease attacks specific Arg-Ser peptide bond in reactive site of antithrombin,
resulting in formation of covalent ester bond between serine residue on protease and
arginine residue on antithrombin III – results in stable 1:1 complex between protease
and antithrombin molecules, which prevents protease from further participation in
coagulation cascade
 In absence of heparin, binding reaction between proteases and antithrombin III
proceeds slowly; heparin acts as a cofactor and accelerates the reaction 1000x
 Heparin serves as catalytic surface to which both antithrombin III and serine proteases
bind and induces conformational change in antithrombin III that makes reactive site of
molecule more accessible to attacking protease
 Binding sequence/pathway of heparin/antithrombin III reaction
o Negatively charged heparin binds to lysine-rich region (region of positive
charge) on antithrombin III
o During conjugation reaction between protease and antithrombin, heparin may
be released from antithrombin III and become available to catalyze additional
protease-antithrombin III interactions, but in practice, heparin’s highly negative
charge often causes it to remain electrostatically bound to protease,
antithrombin, or another nearby molecule in vicinity of thrombus
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Heparins of different molecular weights have divergent anticoagulant activities because
they derive from the differential requirements for heparin binding exhibited by
inactivation of thrombin and factor Xa by antithrombin III; to catalyze most efficiently,
single molecule of heparin must bind simultaneously to both thrombin and
antithrombin III (scaffolding function required in addition to heparin-induced
conformational change in antithrombin III that renders antithrombin susceptible to
conjugation with thrombin; to catalyze inactivation of factor Xa by antithrombin III,
heparin molecule must bind only to antithrombin because conformational change in
antithrombin induced by heparin binding is sufficient to render antithrombin susceptible
to conjugation with factor Xa
o LMW heparins efficiently catalyze inactivation of factor Xa by antithrombin III
but less efficiently catalyze inactivation of thrombin by antithrombin III
o Unfractionated heparin is of sufficient length to bind simultaneously to
thrombin and antithrombin III and thus efficiently catalyzes inactivation of both
thrombin and factor Xa by antithrombin III
 LMW heparin has 3x higher ratio of anti-Xa to anti-thrombin activity than unfractionated
heparin, so it is more selective therapeutic agent
 Both LMW heparin and unfractionated heparin use pentasaccharide structure of high
negative charge to bind antithrombin III and induce conformational change in
antithrombin required for conjugation reactions
 Used for highly selective inhibitor of factor Xa (fondaparinus)
Heparins used for prophylaxis and treatment of thromboembolic diseases – both unfractionated
and LMW heparins used to prevent propagation of established thromboembolic disease such as
deep vein thrombosis and pulmonary embolism
For prophylaxis against thrombosis, heparins used at much lower doses than for treatment of
established thromboembolic disease
Because enzymatic coagulation cascade functions as amplification system, administration of
relatively small amounts of circulating heparin at first generation of factor Xa highly effective
Heparin cannot cross epithelial layer of GI tract because of high negative charge, so it must be
administered parenterally, usually via IV or subcutaneous routes
Unfractionated heparin often used in combination with antiplatelet agents in treatment of acute
coronary syndromes
 Monitoring unfractionated heparin therapy important for maintaining anticoagulant
effect within therapeutic range because excessive heparin administration significantly
increases risk of bleeding
 Usually monitored with aPTT (activated partial thromboplastin time), which tests
intrinsic and common pathways of coagulation
Small fraction of patients taking heparin develop heparin-induced thrombocytopenia (HIT),
where patients develop antibodies to hapten created when heparin molecules bind to platelet
surface
 In HIT type 1, antibody-coated platelets targeted for removal from circulation, and
platelet count decreases 50-75% about 5 days after heparin therapy begins;
thrombocytopenia is transient and rapidly reversible upon heparin withdrawal
 HIT type 2 – heparin-induced antibodies target platelets for destruction and act as
agonists to activate platelets, leading to platelet aggregation, endothelial injury, and
potentially fatal thrombosis
 Higher incidence of HIT in patients receiving unfractionated heparin than in those
receiving LMW heparin
LMW heparins enoxaparin, dalteparin, and tinzaparin – relatively selective for anti-Xa compared
to anti-IIa; all approved for use in prevention and treatment of deep vein thrombosis
 Have higher therapeutic index than unfractionated heparin, escpecially when used for
prophylaxis, so genearlyy not necessary to monitor blood activity levels of LMW
heparins as closely
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Because LMW heparins excreted via kidneys, care should be taken to avoid excessive
anticoagulation in patient with renal insufficiency
Selective factor Xa inhibitors – fondaparinux (synthetic pentasaccharide that contains sequence of five
essential carbohydrates necessary for binding to antithrombin III and inducing conformational change in
antithrombin required for conjugation to factor Xa
 Fondaparinux specific, indirect inhibitor of Xa with negligible anti-IIa activity; approved for
prevention and treatment of DVT
 Factor Xa inhibitors currently include idraparinux, rivaroxaban, and apixaban
 Idraparinux – indirect inhibitor of factor Xa, hypermethylated derivative of fondaparinux
that binds antithrombin with high affinity; administered subcutaneously and produces
predictable anticoagulant response
 Riveroxaban and apixaban – directly inhibit factor Xa by binding to active site and
competitively inhibiting enzyme; both bind factor Xa that is complexed with factor Va
and Ca2+ on phospholipid surfaces; both administered orally
Direct thrombin inhibitors – currently approved ones include lepirudin, desirudin, bivalirudin,
argatroban, and dabigatran; specific inhibitors of thrombin with negligible anti-factor Xa activity
 Lepirudin – derived from medicinal leech protein hirudin; used to prevent thrombosis in fine
vessels of reattached digits; binds with high affinity to 2 sites on thrombin molecule (enzymatic
active site and exosite (region of thrombin protein that orients substrate proteins))
 Highly effective anticoagulant because it can inhibit both free and fibrin-bound
thrombin in developing clots; is essentially irreversible; approved for use in treatment of
heparin-induced thrombocytopenia
 Has short half-life is available parenterally, and is renally excreted
 Can be administered with relative safety to patients with hepatic insufficiency
 Clotting times must be monitored closely
 Small percentage of patients may develop anti-hirudin antibodies, limiting long-term
effectiveness of lepirudin as anticoagulant
 Desirudin – also recombinant form of hirudin; approved for prophylaxis against DVT in patients
undergoing hip replacement
 Bivalirudin – synthetic peptide that binds to both active site and exosite of thrombin and
thereby inhibits thrombin activity
 Thrombin slowly cleaves arginine-proline bond in bivalirudin, leading to reactivation of
thrombin
 Approved for patients undergoing coronary angiography and angioplasty and may
reduce rates of bleeding relative to heparin for this indication
 Excreted renally and has short half-life (25 minutes)
 Argatroban – small-molecule inhibitor of thrombin approved for treatment of patients with
heparin-induced thrombocytopenia; binds only to active site of thrombin (does not interact with
exosite); excreted by biliary secretion and can therefore be administered with relative safety to
patients with renal insufficiency; short half-life and administered by continuous IV infusion
 Dabigatran – oral drug approved for prevention of thromboembolism in patients with nonvalvular A fib (not related to mitral stenosis or prosthetic heart valve); prodrug that is
metabolized to active species that binds competitively to active site of thrombin; may cause
significant bleeding; do not need to monitor plasma levels
Recombinant activated protein C (r-APC) – endogenously activated protein C (APC) exerts anticoagulant
effect by proteolytically cleaving factors Va and VIIIa; APC also reduces amount of circulating
plasminogent activator inhibitor 1, enhancing fibrinolysis; APC reduces inflammation by inhibiting
release of tumor necrosis factor α (TNF-α) by monocytes
 Enhanced coagulability and inflammation both hallmarks of septic shock
 r-APC significantly reduces mortality in patients at high risk of death from septic shock; also
approved for treatment of patients with severe sepsis who demonstrate evidence of acute
organ dysfunction, shock, oliguria, acidosis, and hypoxemia
 Not indicated for treatment of patients with severe sepsis and lower risk of death
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Increases risk of bleeding, so contraindicated in patients who have recently undergone surgery
and in those with chronic liver failure, kidney failure, or thrombocytopenia
Thrombolytic agents – used to lyse already-formed clots and thereby restore patency of obstructed vessel
before distal tissue necrosis occurs; act by converting inactive zymogen plasminogen to active protease plasmin
(relatively nonspecific protease that digests fibrin to fibrin degradation products); has potential to dissolve
physiologically appropriate fibrin clots as well as pathological thrombi, so use can lead to hemorrhage
o Streptokinase – produced by β-hemolytic streptococci as component of tissue-destroying machinery
 Complexation reaction – streptokinase forms stable, noncovalent 1:1 complex with
plasminogen; produces conformational change in plasminogen that exposes proteolytically
active site
 Streptokinase-complexed plasminogen with active site exposed can proteolytically cleav other
plasminogen molecules to plasmin
 Thermodynamically stable streptokinase-plasminogen complex most catalytically efficient
plasminogen activator
 Limited because it is a foreign protein capable of eliciting antigenic responses upon repeated
adminsitrations and because its thrombolytic actions are relatively nonspecific and can result in
systemic fibrinolysis
 Approved for treatment of ST elevation myocardial infarction and for life-threatening pulmonary
embolism
o Recombinant tissue plasminogen activator (t-PA) – serine protease produced by endothelial cells and
binds to newly formed thrombi with high affinity, causing fibrinolysis at site of thrombus; once bound to
fresh thrombus, it undergoes conformational change that renders it potent activator of plasminogen
 t-PA is poor activator of plasminogen in absence of fibrin-binding
 Recombinant t-PA (alteplase) – effective at recanalizing occluded coronary arteries, limiting
cardiac dysfunction, and reducing mortality following ST elevation myocardial infarction
 At pharmacological doses, it can generate systemic lytic state and cause unwanted bleeding,
including cerebral hemorrhage
 Use is contraindicated in patients who have recent hemorrhagic stroke
 Approved for use in treatment of patients with ST elevation myocardial infarction or lifethreatening pulmonary embolism as well as acute ischemic stroke
o Tenecteplase – genetically engineered variant of t-PA with molecular modifications to increase fibrin
specificity and make it more resistant to plasminogen activator inhibitor 1
 Longer half-life than t-PA and similar to decreased risk of bleeding
 Can be administered as single weight-based bolus
o Reteplase – genetically engineered variant of t-PA with longer half-life and increased specificity for fibrin
 Efficacy and adverse-effect profile similar to those of streptokinase and t-PA
 Longer half-life allows it to be administered as double bolus (2 pills 30 minutes apart)
Inhibitors of anticoagulation and fibrinolysis
o Protamine – low molecular weight polycationic protein; chemical antagonist of heparin
 Rapidly forms stable complex with negatively charged heparin molecule through multiple
electrostatic interactions
 Administered IV to reverse effects of heparin in situations of life-threatening hemorrhage or
great heparin excess (i.e., at conclusion of coronary artery bypass graft surgery)
 Most active against large heparin molecules in unfractionated heparin and can partially reverse
anticoagulant effects of LMW heparins, but is inactive against fondaparinux
o Serine-protease inhibitors – aprotinin is naturally occurring polypeptide and inhibits serine proteases
(plasmin, t-PA, and thrombin)
 By inhibiting fibrinolysis, aprotinin promotes clot stabilization and may promote platelet activity
by preventing platelet hyperstimulation
 At higher doses, it may inhibit kallikrein and thereby inhibit coagulation cascade
 Decreased perioperative bleeding and erythrocyte transfusion requirement in patients treated
with aprotinin during cardiac surgery
 May increase risk of postoperative acute renal failure
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Lysine analogues – aminocaproic acid and tranexamic acid; bind to and inhibit plasminogen and plasmin
 Used to reduce perioperative bleeding during coronary artery bypass grafting
 May not increase risk of post-operative acute renal failure
Conclusion and Future Directions
 Future development of new antiplatelet, anticoagulant, and thrombolytic agents will be forced to contend with
2 major constraints
o Highly effective, orally bioavailable, and inexpensive therapeutic agents already available
o Virtually every antithrombotic and thrombolytic agent is associated with mechanism-based toxicity of
bleeding, and this adverse effect is likely to continue to new drugs
 Likely that pharmacogenomics techniques will be capable of identifying individuals in population who carry
elevated genetic risk of thrombosis and such individuals may benefit from long-term anti-thrombotic treatment
 There remains a need for new agents that can achieve rapid, noninvasive, convenient, and selective lysis of
acute thrombosis associated with life-threatening emergencies such as ST elevation MI and stroke