Antithrombotics

Prophylaxis and Therapy of Thromboses

Upon vascular injury, the coagulation
system is activated: thrombocytes and
fibrin molecules coalesce into a “plug”
that seals the defect and halts
bleeding (hemostasis). Unnecessary
formation of an intravascular clot – a
thrombosis – can be life-threatening. If
the clot forms on an atheromatous
plaque in a coronary artery, myocardial
infarction is imminent; a thrombus in a
deep leg vein can be dislodged, carried
into a lung artery, and cause complete
or partial interruption of pulmonary
blood flow (pulmonary embolism).
Drugs that decrease the coagulability
of blood, such as coumarins and heparin
(A), are employed for the prophylaxis
of thromboses. In addition, attempts
are directed at inhibiting the aggregation
of blood platelets, which are
prominently involved in intra-arterial
thrombogenesis. For the therapy
of thrombosis, drugs are used that
dissolve the fibrin meshwork!fibrinolytics.
An overview of the coagulation
cascade and sites of action for coumarins
and heparin is shown in A. There are
two ways to initiate the cascade (B): 1)
conversion of factor XII into its active
form (XIIa, intrinsic system) at intravascular
sites denuded of endothelium; 2)
conversion of factor VII into VIIa (extrinsic
system) under the influence of a tissue-
derived lipoprotein (tissue thromboplastin).
Both mechanisms converge
via factor X into a common final pathway.
The clotting factors are protein
molecules. “Activation” mostly means
proteolysis (cleavage of protein fragments)
and, with the exception of fibrin,
conversion into protein-hydrolyzing
enzymes (proteases). Some activated
factors require the presence of phospholipids
(PL) and Ca2+ for their proteolytic
activity. Conceivably, Ca2+ ions
cause the adhesion of factor to a phospholipid
surface, as depicted in C. Phospholipids
are contained in platelet factor
3 (PF3), which is released from aggregated
platelets, and in tissue thromboplastin
(B). The sequential activation
of several enzymes allows the aforementioned
reactions to “snowball”, culminating
in massive production of fibrin.
Progression of the coagulation cascade
can be inhibited as follows:
1) coumarin derivatives decrease
the blood concentrations of inactive factors
II, VII, IX, and X, by inhibiting their
synthesis; 2) the complex consisting of
heparin and antithrombin III neutralizes
the protease activity of activated factors;
3) Ca2+ chelators prevent the enzymatic
activity of Ca2+-dependent factors;
they contain COO-groups that bind
Ca2+ ions (C): citrate and EDTA (ethylenediaminetetraacetic
acid) form soluble
complexes with Ca2+; oxalate precipitates
Ca2+ as insoluble calcium oxalate.
Chelation of Ca2+ cannot be used
for therapeutic purposes because Ca2+
concentrations would have to be lowered
to a level incompatible with life
(hypocalcemic tetany). These compounds
(sodium salts) are, therefore,
used only for rendering blood incoagulable
outside the body.

Coumarin Derivatives (A)
Vitamin K promotes the hepatic !-carboxylation
of glutamate residues on the
precursors of factors II, VII, IX, and X, as
well as that of other proteins, e.g., protein
C, protein S, or osteocalcin. Carboxyl
groups are required for Ca2+-mediated
binding to phospholipid surfaces.
There are several vitamin K derivatives
of different origins: K1 (phytomenadione)
from chlorophyllous
plants; K2 from gut bacteria; and K3
(menadione) synthesized chemically.
All are hydrophobic and require bile acids
for absorption.
Oral anticoagulants. Structurally
related to vitamin K, 4-hydroxycoumarins
act as “false” vitamin K and prevent
regeneration of reduced (active) vitamin
K from vitamin K epoxide, hence
the synthesis of vitamin K-dependent
clotting factors.
Coumarins are well absorbed after
oral administration. Their duration of
action varies considerably. Synthesis of
clotting factors depends on the intrahepatocytic
concentration ratio of coumarins
to vitamin K. The dose required
for an adequate anticoagulant effect
must be determined individually for
each patient (one-stage prothrombin
time). Subsequently, the patient must
avoid changing dietary consumption of
green vegetables (alteration in vitamin
K levels), refrain from taking additional
drugs likely to affect absorption or elimination
of coumarins (alteration in coumarin
levels), and not risk inhibiting
platelet function by ingesting acetylsalicylic
acid.
The most important adverse effect
is bleeding. With coumarins, this
can be counteracted by giving vitamin
K1. Coagulability of blood returns to
normal only after hours or days, when
the liver has resumed synthesis and restored
sufficient blood levels of clotting
factors. In urgent cases, deficient factors
must be replenished directly (e.g., by
transfusion of whole blood or of prothrombin
concentrate).
Heparin (B)
A clotting factor is activated when the
factor that precedes it in the clotting
cascade splits off a protein fragment and
thereby exposes an enzymatic center.
The latter can again be inactivated physiologically
by complexing with antithrombin
III (AT III), a circulating glycoprotein.
Heparin acts to inhibit clotting
by accelerating formation of this
complex more than 1000-fold. Heparin
is present (together with histamine) in
the vesicles of mast cells; its physiological
role is unclear. Therapeutically used
heparin is obtained from porcine gut or
bovine lung. Heparin molecules are
chains of amino sugars bearing -COO–
and -SO4 groups; they contain approx.
10 to 20 of the units depicted in (B);
mean molecular weight, 20,000. Anticoagulant
efficacy varies with chain
length. The potency of a preparation is
standardized in international units of
activity (IU) by bioassay and comparison
with a reference preparation.
The numerous negative charges are
significant in several respects: (1) they
contribute to the poor membrane penetrability—
heparin is ineffective when
applied by the oral route or topically onto
the skin and must be injected; (2) attraction
to positively charged lysine residues
is involved in complex formation
with ATIII; (3) they permit binding of
heparin to its antidote, protamine
(polycationic protein from salmon
sperm).
If protamine is given in heparin-induced
bleeding, the effect of heparin is
immediately reversed.
For effective thromboprophylaxis, a
low dose of 5000 IU is injected s.c. two
to three times daily. With low dosage of
heparin, the risk of bleeding is sufficiently
small to allow the first injection
to be given as early as 2 h prior to surgery.
Higher daily i.v. doses are required
to prevent growth of clots. Besides
bleeding, other potential adverse effects
are: allergic reactions (e.g., thrombocytopenia)
and with chronic administration,
reversible hair loss and osteoporosis.

Low-molecular-weight heparin (average
MW ~5000) has a longer duration
of action and needs to be given only
once daily (e.g., certoparin, dalteparin,
enoxaparin, reviparin, tinzaparin).
Frequent control of coagulability is
not necessary with low molecular
weight heparin and incidence of side effects
(bleeding, heparin-induced thrombocytopenia)
is less frequent than with
unfractionated heparin.
Fibrinolytic Therapy (A)
Fibrin is formed from fibrinogen
through thrombin (factor IIa)-catalyzed
proteolytic removal of two oligopeptide
fragments. Individual fibrin molecules
polymerize into a fibrin mesh that can
be split into fragments and dissolved by
plasmin. Plasmin derives by proteolysis
from an inactive precursor, plasminogen.
Plasminogen activators can be infused
for the purpose of dissolving clots
(e.g., in myocardial infarction). Thrombolysis
is not likely to be successful unless
the activators can be given very soon
after thrombus formation. Urokinase
is an endogenous plasminogen activator
obtained from cultured human kidney
cells. Urokinase is better tolerated than
is streptokinase. By itself, the latter is
enzymatically inactive; only after binding
to a plasminogen molecule does
the complex become effective in converting
plasminogen to plasmin. Streptokinase
is produced by streptococcal
bacteria, which probably accounts for
the frequent adverse reactions. Streptokinase
antibodies may be present as a
result of prior streptococcal infections.
Binding to such antibodies would neutralize
streptokinase molecules.
With alteplase, another endogenous
plasminogen activator (tissue
plasminogen activator, tPA) is available.
With physiological concentrations this
activator preferentially acts on plasminogen
bound to fibrin. In concentrations
needed for therapeutic fibrinolysis this
preference is lost and the risk of bleeding
does not differ with alteplase and
streptokinase. Alteplase is rather shortlived
(inactivation by complexing with
plasminogen activator inhibitor, PAI)
and has to be applied by infusion. Reteplase,
however, containing only the
proteolytic active part of the alteplase
molecule, allows more stabile plasma
levels and can be applied in form of two
injections at an interval of 30 min.
Inactivation of the fibrinolytic
system can be achieved by “plasmin inhibitors,”
such as !-aminocaproic acid,
p-aminomethylbenzoic acid (PAMBA),
tranexamic acid, and aprotinin, which
also inhibits other proteases.
Lowering of blood fibrinogen
concentration. Ancrod is a constituent
of the venom from a Malaysian pit viper.
It enzymatically cleaves a fragment
from fibrinogen, resulting in the formation
of a degradation product that cannot
undergo polymerization. Reduction
in blood fibrinogen level decreases the
coagulability of the blood. Since fibrinogen
(MW ~340 000) contributes to the
viscosity of blood, an improved “fluidity”
of the blood would be expected.
Both effects are felt to be of benefit in
the treatment of certain disorders of
blood flow.


Intra-arterial Thrombus Formation (A)
Activation of platelets, e.g., upon contact
with collagen of the extracellular
matrix after injury to the vascular wall,
constitutes the immediate and decisive
step in initiating the process of primary
hemostasis, i.e., cessation of bleeding.
However, in the absence of vascular injury,
platelets can be activated as a result
of damage to the endothelial cell
lining of blood vessels. Among the multiple
functions of the endothelium, the
production of NO˙ and prostacyclin plays
an important role. Both substances inhibit
the tendency of platelets to adhere
to the endothelial surface (platelet adhesiveness).
Impairment of endothelial
function, e.g., due to chronic hypertension,
cigarette smoking, chronic elevation
of plasma LDL levels or of blood
glucose, increases the probability of
contact between platelets and endothelium.
The adhesion process involves
GPIB/IX, a glycoprotein present in the
platelet cell membrane and von Willebrandt’s
factor, an endothelial membrane
protein. Upon endothelial contact,
the platelet is activated with a resultant
change in shape and affinity to
fibrinogen. Platelets are linked to each
other via fibrinogen bridges: they
undergo aggregation.
Platelet aggregation increases like
an avalanche because, once activated,
platelets can activate other platelets. On
the injured endothelial cell, a platelet
thrombus is formed, which obstructs
blood flow. Ultimately, the vascular lumen
is occluded by the thrombus as the
latter is solidified by a vasoconstriction
produced by the release of serotonin
and thromboxane A2 from the aggregated
platelets. When these events occur in
a larger coronary artery, the consequence
is a myocardial infarction; involvement
of a cerebral artery leads to
stroke.
The von Willebrandt’s factor plays a
key role in thrombogenesis. Lack of this
factor causes thrombasthenia, a pathologically
decreased platelet aggregation.
Relative deficiency of the von Willebrandt’s
factor can be temporarily overcome
by the vasopressin anlogue desmopressin
(p. 164), which increases the
release of available factor from storage
sites.
Formation, Activation, and Aggregation
of Platelets (B)
Platelets originate by budding off from
multinucleate precursor cells, the megakaryocytes.
As the smallest formed
element of blood (dia. 1–4 μm), they can
be activated by various stimuli. Activation
entails an alteration in shape and
secretion of a series of highly active substances,
including serotonin, platelet activating
factor (PAF), ADP, and thromboxane
A2. In turn, all of these can activate
other platelets, which explains the
explosive nature of the process.
The primary consequence of activation
is a conformational change of an integrin
present in the platelet membrane,
namely, GPIIB/IIIA. In its active
conformation, GPIIB/IIIA shows high affinity
for fibrinogen; each platelet contains
up to 50,000 copies. The high plasma
concentration of fibrinogen and the
high density of integrins in the platelet
membrane permit rapid cross-linking of
platelets and formation of a platelet
plug.


Inhibitors of Platelet Aggregation
Platelets can be activated by mechanical
and diverse chemical stimuli, some of
which, e.g., thromboxane A2, thrombin,
serotonin, and PAF, act via receptors on
the platelet membrane. These receptors
are coupled to Gq proteins that mediate
activation of phospholipase C and hence
a rise in cytosolic Ca2+ concentration.
Among other responses, this rise in Ca2+
triggers a conformational change in
GPIIB/IIIA, which is thereby converted
to its fbrinogen-binding form. In contrast,
ADP activates platelets by inhibiting
adenylyl cyclase, thus causing internal
cAMP levels to decrease. High cAMP
levels would stabilize the platelet in its
inactive state. Formally, the two messenger
substances, Ca2+ and cAMP, can
be considered functional antagonists.
Platelet aggregation can be inhibited
by acetylsalicylic acid (ASA), which
blocks thromboxane synthase, or by recombinant
hirudin (originally harvested
from leech salivary gland), which
binds and inactivates thrombin. As yet,
no drugs are available for blocking aggregation
induced by serotonin or PAF.
ADP-induced aggregation can be prevented
by ticlopidine and clopidogrel;
these agents are not classic receptor antagonists.
ADP-induced aggregation is
inhibited only in vivo but not in vitro in
stored blood; moreover, once induced,
inhibition is irreversible. A possible explanation
is that both agents already
interfere with elements of ADP receptor
signal transduction at the megakaryocytic
stage. The ensuing functional defect
would then be transmitted to newly
formed platelets, which would be incapable
of reversing it.
The intra-platelet levels of cAMP
can be stabilized by prostacyclin or its
analogues (e.g., iloprost) or by dipyridamole.
The former activates adenyl cyclase
via a G-protein-coupled receptor;
the latter inhibits a phosphodiesterase
that breaks down cAMP.
The integrin (GPIIB/IIIA)-antagonists
prevent cross-linking of platelets.
Their action is independent of the aggregation-
inducing stimulus. Abciximab
is a chimeric human-murine monoclonal
antibody directed against GPIIb/IIIa
that blocks the fibrinogen-binding site
and thus prevents attachment of fibrinogen.
The peptide derivatives, eptifibatide
and tirofiban block GPIIB/IIIA
competitively, more selectively and have
a shorter effect than does abciximab.
Presystemic Effect of Acetylsalicylic Acid

Inhibition of platelet aggregation by
ASA is due to a selective blockade of
platelet cyclooxygenase (B). Selectivity
of this action results from acetylation of
this enzyme during the initial passage of
the platelets through splanchnic blood
vessels. Acetylation of the enzyme is irreversible.
ASA present in the systemic
circulation does not play a role in platelet
inhibition. Since ASA undergoes extensive
presystemic elimination, cyclooxygenases
outside platelets, e.g., in endothelial
cells, remain largely unaffected.
With regular intake, selectivity is enhanced
further because the anuclear
platelets are unable to resynthesize new
enzyme and the inhibitory effects of
consecutive doses are added to each
other. However, in the endothelial cells,
de novo synthesis of the enzyme permits
restoration of prostacyclin production.
Adverse Effects of Antiplatelet Drugs
All antiplatelet drugs increase the risk of
bleeding. Even at the low ASA doses
used to inhibit platelet function (100
mg/d), ulcerogenic and bronchoconstrictor
(aspirin asthma) effects may occur.
Ticlopidine frequently causes diarrhea
and, more rarely, leukopenia, necessitating
cessation of treatment. Clopidogrel
reportedly does not cause hematological
problems.
As peptides, hirudin and abciximab
need to be injected; therefore their use
is restricted to intensive-care settings.