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CHAPTER
5 Normal Mechanisms of Vascular
Hemostasis
Elisabeth M. Battinelli, Joseph Loscalzo
Hemostasis occurs in response to vessel injury. The clot is essential for both prevention of blood loss and initiation of the wound
repair process. When there is a lesion present in the blood vessel,
the response is rapid, highly regulated, and localized. If the process is not balanced, abnormal bleeding or nonphysiological
thrombosis can result. In cardiovascular disease, formation of
abnormal thrombus at the area of an atherosclerotic plaque results
in significant morbidity and mortality. This chapter will focus on
normal mechanisms of hemostasis, with specific attention to the
role of the platelet in the process, the coagulation cascade, and
fibrinolytic mechanisms as a basis for understanding how abnormalities in these processes can lead to thrombotic and hemorrhagic disorders.
Endothelial Function and Platelet
Activation
Platelets are anucleate cells produced by megakaryocytes in the
bone marrow. Once they have traversed from the bone marrow to
the general circulation, their lifespan is approximately 10 days. They
function mainly to limit hemorrhage after trauma resulting in vascular injury. Normally in the vasculature, platelets are in a resting state
and only become activated after exposure to a stimulus leads to a
shape change and release reaction that causes the platelet to export
many of its biologically important proteins. Some of the agonists
that can initiate this response include thromboxane A
diphosphate (ADP), thrombin, and serotonin. In areas of vascular
injury, platelets are attracted to the impaired site by collagen through
binding with von Willebrand factor (vWF) via the glycoprotein (GP)
Ib/V/IX complex. This initial binding results in platelet activation,
with a subsequent feedback mechanism in which ADP, thrombin,
and thromboxane A
tional platelets to the area. The complex firmly binds the platelet to
the area of injury so there is no disruption by the high shear forces
of turbulent blood flow that occur with vessel disruption. This amplification of the response is essential to form a hemostatic plug and
represents the first stage in the hemostatic process. When vWF is not
present, hemostatic abnormalities result, with deficiencies leading
to von Willebrand's disease, which can be associated with severe
bleeding. Hemostasis issues also arise when the platelet receptor
complex GPIb/V/IX is mutated, resulting in inability of vWF to bind,
a disorder termed Bernard-Soulier's syndrome.
Additional platelet aggregation occurs through activation of G
protein–coupled receptors (GPCRs), with the final pathway relying on the GP IIb/IIIa complex, the main receptor for platelet aggregation and adhesion.
on different platelets, stabilizing the clot. The integral role of this
receptor is manifest in Glanzmann thrombasthenia, a disorder in
which fibrinogen binding is impaired, leading to spontaneously
occurring mucocutaneous bleeding episodes.
Vascular endothelium is essential to this hemostatic process;
this is the cellular site where regulation and initiation of coagulation begins. Endothelial cells (ECs) modulate vascular tone, generate mediators of inflammation, and provide a resistant surface
that allows for platelets to experience laminar flow with minimal
shear. Endothelial cells regulate hemostasis by releasing a number
of inhibitors of platelets and inflammation. Vascular endothelium is
essential for regulating uncontrolled platelet activity through mechanisms of inhibition including the arachidonic acid– prostacyclin
pathway, l-arginine–nitric oxide pathway, and endothelial
ectoadenosine diphosphatase (ecto-ADPase) pathway6 (Table 5-1).
further activate the platelets and recruit addi-
2
1,2
3,4
Fibrinogen tethers GP IIb/IIIa complexes
5
70
, adenosine
2
Nitric oxide (NO) is produced constitutively by (ECs) via an
endothelial isoform of nitric oxide synthase (eNOS) in a process
dependent on conversion of
is regulated by NO as it controls smooth muscle cell (SMC) contraction. It also inhibits platelets directly, blocking platelet aggregation
through stimulation of guanylyl cyclase and cyclic guanosine
monophosphate (cGMP) and inhibition of platelet phosphoinositol3-kinase (PI-3 kinase). Nitric oxide functions by decreasing the intracellular Ca
change in GP IIb/IIIa suppressing fibrinogen's ability to bind to the
receptor, thereby attenuating platelet aggregation.
Prostacyclin, which is synthesized in the ECs from arachidonic
acid through cyclooxygenase-1 or -2 (COX-1, COX-2)-dependent
pathways, inhibits platelet function by increasing cyclic adenosine monophosphate (cAMP). This is essential for aspirin's ability
to diminish platelet function through acetylation of platelet COX1
at serine 529.
The last pathway important in modulating vascular endothelium's interaction with platelets is the endothelial ecto-ADPase
pathway, which impairs ADP-mediated platelet activation. By
hydrolyzing ADP, this enzyme inhibits the critical state of platelet recruitment to a growing aggregate, thereby limiting thrombus formation. Once the platelet aggregate has been stabilized by
fibrin with red cells to the vessel wall, the next stage of hemostasis
involves activation of the highly regulated coagulation cascade
(
Fig. 5-1).
2+
level through cGMP, which inhibits the conformational
l-arginine to l-citrulline. Vascular tone
7
Coagulation Cascade Leading to Fibrin
Formation
Disruption in the endothelium not only recruits platelets for plug
formation, it also stimulates activation of the coagulation cascade,
which is essential for secondary clot formation through fibrin generation. The coagulation cascade is a dynamic integrated process
in which each step is dependent on another step for activation of
proenzymes or zymogens to their active forms through proteolytic
cleavage. This process is dependent upon calcium and the phospholipid bilayer allowing inactive clotting factors to be converted
to active enzymes through serine protease activity. These coagulation proteins function in a step-by-step fashion to activate downstream members of the cascade, leading to production of the
penultimate clotting factor, thrombin. Thrombin is versatile, playing
a role in many of the essential stages of hemostasis. Not only is it
important for platelet activation, it is also necessary for the crosslinking of fibrin. Recently there have been attempts to limit thrombus formation by directly inhibiting thrombin activity through
anticoagulants such as ximelagatran and the oral medication, dabigatran, which is now available for clinical use.
The clotting cascade is divided into two main pathways, the
intrinsic and extrinsic pathways. The extrinsic pathway begins
with establishment of a complex between tissue factor, found on
the cell surface or on microparticles, and factor VIIa. This complex leads to activation of factor X to Xa, which can then further
the response by looping back and converting factor VII to VIIa
in a feedback mechanism. When factor Xa is present, it binds
to factor Va on the membrane surface and again generates prothrombinase, which converts prothrombin to thrombin and then
generates fibrin as detailed earlier. The activity of factor Xa is accelerated by the presence of factor Va through calcium and formation of a noncovalent association γ-carboxyglutamate residues of
factor Xa and the phospholipid surface of activated platelets.
8
9
Intrinsic Pathway
TABLE 5-1 Factors Involved in Fibrinolysis
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PROHEMOSTATIC ANTIHEMOSTATIC
Circulating
-Antiplasmin Antithrombin III
α
2
Thrombin Protein C
Thrombin-activatable fibrinolysis Protein S
inhibitor (TAFI) Tissue factor pathway inhibitor
Endothelium-Derived
Plasminogen activator inhibitor-1
(PAI-1)
Tissue factor (TF) Heparan sulfate (HS)
von Willebrand factor (vWF) Nitric oxide (NO)
(TFPI)
Ectoadenosine diphosphatase
(Ecto-ADPase)/CD39
Thrombomodulin
Tissue plasminogen activator (tPA)
Urokinase plasminogen activator
(uPA)
The extrinsic pathway is measured by prothrombin time (PT),
which is determined by adding an extrinsic substance such as tissue factor or thromboplastin.
10
The extrinsic pathway, which is dependent on tissue factor,
appears to be the main pathway responsible for hemostasis, with
the intrinsic pathway playing a supporting role. Tissue factor is a
membrane-bound GP that is constitutively expressed by SMCs and
fibroblasts but selectively expressed by ECs when there is vessel
wall injury. The “encrypted” activated form of factor VIIa is made
functional through a conformational change that occurs at cysteines 186 and 209, leading to disulfide bond formation upon vessel wall injury. Protein disulfide isomerase, glutathione, and NO all
may have a role in these allosteric changes; however, recent studies have questioned the importance of “de-encryption” in this pro-
11–14
cess.
Tissue factor functions through activation of factors X
and IX after interactions with factor VII as a complex. Factor VII,
although at low levels in an active state (factor VIIa) in the circulation, only becomes biologically important after it is bound to tissue
factor in complex with factors X and IX. This complex formation is
essential for activation of thrombin.
9
The role of tissue factor has recently been expanded. It circulates in the blood in association with microvesicles that are derived
from cellular membranes produced from lipid rafts on monocytes
and macrophages.
15
These tissue factor–bearing microvesicles can
directly initiate the coagulation cascade on activated platelets in a
process that may be important for understanding the hypercoagulable state.
16,17
Once thrombin is activated in the tissue factor X/IX/VIIa complex, it initiates further activation within the coagulation cascade.
In addition to activating platelets and factor V, it also activates factor VIII, which exists in the circulation in association with vWF.
Activated factor VIII (factor VIIIa) works in a feedback loop with
factor IXa to activate further factor X to Xa and thereby yield
more thrombin to accelerate its own activation. Factors VIII and
IX are essential in coagulation, as is evident in patients who suffer deficiencies of these factors leading to hemophilia A and B,
respectively. These disorders lead to severe bleeding due to loss of
activation of factor X, leading to decreased thrombin formation.
Another deficiency that is seen occurs when factor XI is mutated,
resulting in a disorder associated with delayed bleeding in the
postoperative setting. The importance of this coagulation cascade
is highlighted by the severity of this disorder, suggesting that the
feedback mechanism by which thrombin activates factor XI, with
subsequent activation of IX and then further generation of thrombin, is an essential stage of amplification necessary for hemostasis.
71
CH
5
NoRmAl mECHANisms of VAsCulAR HEmosTAsis
Contact with vascular injury
Factor XII Factor XIIa
Factor XIa Factor XI
Factor IX
Factor X Factor XFactor Xa
Ca
Ca, Factor VIII,
Platelet phospholipid
Prothrombin
(Factor II)
Factor IXa
Ca
, Factor VI,
Platelet phospholipid
Ca
Factor Va,
Factor VIIIa
Thrombin
Ca
, Phospholipid
Extrinsic Pathway
Tissue damage
Tissue factor
Ca
Factor VIIa Factor VII
Fibrinogen
(Factor I)
Fibrin
Factor XIII Factor XIIIa
FIGURE 5-1 Coagulation cascade.
72
The extrinsic pathway, described earlier, joins up with the
intrinsic pathway through factor X to form the common pathway.
The intrinsic pathway is initiated by contact and results in activation of factor IXa, which then goes on to activate factor X as
described. It is generally accepted that the intrinsic pathway is of
less importance in coagulation than the tissue factor–mediated
CH
extrinsic pathway, although it plays an essential role in inflam-
5
mation and fibrinolysis. The intrinsic pathway is based on exposure of blood to a negatively charged surface, and is classically
initiated by activation of factor XIIa by kallikrein, which is facilitated by kininogen. Kallikrein is generated from prekallikrein
through proteolytic cleavage by activated factor XII in a reaction
dependent on the presence of high-molecular-weight kininogen
(HMWK). When kallikrein has been generated, it also functions
to cleave HMWK to bradykinin, which functions as an inflammatory mediator to potentiate vasodilation and vascular permeability, thereby expanding the role of factor XIIa to inflammation,
regulation of vascular tone, and fibrinolysis.
catalyzes conversion of factor XI to the active enzyme form, factor XIa. When calcium is present, factor XIa next functions to convert IX to IXa, which then binds to VIIIa on membrane surfaces,
converting X to its active form, factor Xa. Factor Xa then binds to
Va on the membrane surface to generate prothrombinase, which
converts prothrombin to thrombin. As thrombin is formed, two
small prothrombin fragments, termed molecules F1 and F2, are
released and can be used as markers of serum thrombin forma-
19
tion.
The intrinsic pathway is monitored through the activated
partial thromboplastin time (APTT), which relies on foreign substances such as glass or silicates to activate factor XII to initiate
the pathway. Deficiencies in the earliest states of the intrinsic
pathway, when prekallikrein, HMWK, and factor XII are involved,
are not associated with bleeding tendencies and therefore do
not lead to a bleeding diathesis, even though there is an elevation in partial thromboplastin time. Mutations in factor XII have
been reported in a group of patients with hereditary angioedema,
although there does not appear to be a bleeding diathesis with
this disorder. Some initial studies have suggested that factor XII
polymorphisms may be associated with an increased propensity
for thrombosis, but this has not been validated.
When factor Xa generates thrombin, the intrinsic and extrinsic pathways have merged into the common pathway. Thrombin
is essential for fibrinogen to generate fibrin, which is released
through proteolytic cleavage.
22
The fibrin molecules that are
generated have polymerization sites exposed, making it easier
for fibrin to cross-link noncovalently. This cross-linking enables
platelets to be entrapped in a meshwork of fibrin strands to form
the secondary clot through the action of factor XIII, activated by
thrombin.
23
In the process of cross-linking, there is also an inherent mechanism of autoregulation, with the binding sites necessary to initiate fibrinolysis being blocked so the clot does not
self-destruct.
This process of platelet activation and up-regulation of the coagulation cascade occurs in a swift and efficient manner to prevent excessive bleeding. It can, however, lead to thrombosis if left
unchecked, so there are other mechanisms in place whose main
role is to modulate coagulation activities to avoid such complications. These mechanisms involve mechanical means such as
dilution of coagulation factors in blood and removal of factors
after activation through the reticuloendothelial system, as well as
antithrombotic pathways that are separate from the coagulation
cascade. Patients with deficiencies in these natural antithrombotic mechanisms often present with thrombosis. These pathways
include antithrombin, protein C and S, and tissue factor pathway
inhibitor (TFPI).
Antithrombin is a serine protease inhibitor that binds specifically to factors IXa, Xa, and thrombin, thereby inactivating them.
Antithrombin has two main binding sites that maintain its functionality: the reactive center at Arg 393/Ser 394 and the heparin
binding site at the amino-terminal end of the molecule. Binding
of both endogenous and exogenous heparins at this site causes
18
Activated factor XII
20,21
a conformational change in antithrombin that enables it to inactivate its targets at an accelerated rate. The glycosaminoglycan
heparan sulfate (HS), present on the surface of ECs, mediates antithrombin's ability to increase its activity and functions as the physiological equivalent to heparin.
associated with a genetic propensity to form venous thrombosis,
discussed in Chapter 10.
25
24
Deficiency of antithrombin is
Activated protein C (APC) and protein S are also important
mechanisms for preventing excessive clotting. During the clotting
process, thrombin binds to thrombomodulin, which is also present on the EC surface. It then undergoes a conformational change
leading to activation of protein C.
26
Activated protein C complexes
with protein S and proteolytically cleaves factors Va and VIIIa,
resulting in their inactivation and a decrease in generation of factors Xa and thrombin. Cleavage of factor Va occurs at Arg 506, Arg
306, and Arg 679 by APC in a sequential manner such that the cleavage at Arg 506 exposes cleavage sites at the other sites through a
conformational change. Mutation of the arginine located at position 506 to glutamine leads to factor V Leiden, which is associated
with a hypercoagulable state.
Another important natural anticoagulant is TFPI, which acts as a
multivalent protease inhibitor to inactivate both factor Xa and IXa.
Tissue factor pathway inhibitor is also present within ECs, with the
majority remaining localized to the endothelial surface and very
little circulating in plasma. The concentration in plasma, however, is
increased in the presence of heparin, which modulates its release
from the endothelial surface.
Fibrinolysis
The importance of fibrinolysis lies in its removing blood clots
and maintaining hemostasis without excessive clotting. The
mechanism of serine protease activity is preserved in the fibrinolytic system and accounts for the mechanism of action of many
of its components (
nolysis is plasmin. The process begins when plasminogen in its
inactive form is converted to the active enzyme, plasmin, which
functions to covert fibrin to soluble fibrin degradation products.
Two molecules that mimic this function include tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). The motif responsible for its action is the kringle
domain, which resides in the amino-terminal end. Kringles are 80
amino acids in length and have a unique folded sheet structure
that results from disulfide linkages, which yields a homotypic
binding site specific for plasminogen, fibrinogen, and fibrin.
There is homology between the kringles contained in all three
of these molecules.
These kringle domains are essential for providing a mechanism for binding many components of the developing thrombus, including fibrinogen and fibrin. The kringle domains
shared by tPA and plasminogen allow fibrinogen and fibrin to
bind and therefore be incorporated into the developing clot.
Plasminogen is converted to plasmin through the proteolytic
cleavage achieved by tPA and uPA at the Arg 560 and Val 561
27
sites.
The plasmin generated can then bind to a number
of proteins involved in the process of fibrinolysis. Relevant
properties include its high affinity for fibrin, ability to cleave Gluplasminogen to Lys-plasminogen, ability to activate factor XII, and
ability to inactivate factors V and VIII in the coagulation cascade.
Plasmin cleaves the fibrin molecule into differentially sized degradation or split products (FDP), the smallest of which is D-dimer,
which is used as a marker of venous thromboembolism and disseminated intravascular coagulopathy (DIC).
The plasminogen pathway is complex and tightly regulated.28
The main proteins involved in its modulation are plasminogen
activator inhibitors (PAI)-1 and PAI-2. The activators and inhibitors
of plasminogen regulate fibrinolysis upon release from ECs. These
activators of the fibrinolytic process are under the control of PAIs,
which complex with tPA and uPA to inactivate them and therefore
block plasmin generation.
Fig. 5-2). The main factor responsible for fibri-
Fibrinolysis
Plasminogen activator
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inhibitor I & II
73
Fibrin
Urokinase
Plasminogen Plasmin
Factor XIa, XIIa, kallikrein
FIGURE 5-2 Pathway of fibrinolysis. Inhibition is signified by red arrows and stimulation is signified by green arrows.
Tissue plasminogen
activator (tPA)
Evidence for why tPA is more important than uPA for normal
hemostasis is how ECs up-regulate production of this protein when
injured. It is stimulated by a variety of substances, including thrombin, serotonin, bradykinin, cytokines, and epinephrine. This binding affords tPA some protection from degradation and enables it
to survive for longer than its expected half-life of only 4 minutes.
Its role in hemostasis is of such significance that recombinant tPA
(alteplase) and its derivatives that incorporate the kringle domains
(e.g., reteplase, tenecteplase) are used as thrombolytic agents
in patients with acute thrombotic events, including myocardial
infarction.
29
The other essential plasminogen activator in this process is
uPA, which exists in a high-molecular-weight and a low-molecularweight form, both of which have the ability to activate plasminogen
through cleavage at Arg 560/Val 561. Urokinase is present in high
concentration in urine. Whereas tPA is mainly important for intravascular fibrinolysis, urokinase has more of a role in the extravascular compartment. Unlike tPA, however, uPA does not bind to fibrin
and therefore is not involved in activation of plasminogen incorporated into clots through fibrin binding.
30
As its name implies, uPA
is derived from urokinase, which consists of a single-chain precursor molecule termed scuPA that is hydrolyzed by plasmin or kalli-
krein to the two-chain active uPA, which is biologically active.
plasma, scuPA does not activate plasminogen, but in the presence
of fibrin, it is actually scuPA that induces clot lysis. Interestingly, the
role of urokinase has been expanded to include support of invasion and metastasis in malignancy
32,33
; uPA has been shown to play
a role in extracellular matrix degradation, allowing for migration
and invasion of metastatic cells. There is now a growing interest in
developing targeted therapy that blocks this pathway as a means
of controlling metastasis.
Streptokinase does not participate in normal hemostasis but is
used as a therapeutic agent for acute thrombosis. It is isolated from
β-hemolytic streptococci, and since it is not an enzyme, must complex with plasminogen to form an active molecule which then has
the ability to cleave plasminogen to plasmin.
34
Its use as a thera-
peutic agent, however, is limited; as a foreign substance, it is often
-Antiplasmin 2-Macroglobulin
2
Fibrin Degradation Products
factors, lipids, insulin, angiotensin II (ANGII), and endotoxin.35
Recently the role of PAI-1 as an inhibitor of tissue factor has
been postulated to regulate hemostasis in inflammatory conditions such as sepsis or acute lung injury.
platelets release PAI-1 as a mechanism of preventing premature
clot dissolution. Patients who are deficient in PAI-1 have a bleeding diathesis when confronted with trauma or surgery.
Another important mechanism for regulation of fibrinolysis is
thrombin-activatable fibrinolysis inhibitor (TAFI), which is not a
member of the serpin family. It is known for its ability to cleave the
carboxy-terminal lysine in fibrin, impairing plasminogen binding.
Activation of TAFI is dependent upon the thrombin-thrombomodulin
complex, which can expedite the inhibitory process in a similar
manner to thrombin.
be inhibited by platelet factor 4, which is secreted by activated
platelets.
39
If the feedback mechanisms of thrombin generation
through factors V, VIII, and XI is impaired—leading to diminution
of the thrombin-thrombomodulin complex and therefore
decreased activation of TAFI—clinical consequences can occur. It
has been suggested that in chronic liver disease where coagulation factors are decreased, low amounts of TAFI may account for
31
the low-grade fibrinolysis typically observed.
also occur, as is seen in patients with the G20210A prothrombin
In
gene mutation in which thrombin generation is increased leading
to increased activation of TAFI and an increased thrombotic propensity through a inhibition of fibrinolysis.
Recently it has been shown that there is yet another important
mechanism by which to regulate the fibrinolytic process via matrix
metalloproteinases (MMPs). Matrix metalloproteinases (including MMP-3, -7, -9, and -12) are found in ECs and have the ability to
cleave uPA and plasminogen. The importance of MMPs in downregulating cellular fibrinolysis remains to be elucidated, but it is
clear they function by reducing availability of plasminogen. MMP-3
and -7 also have the ability to degrade fibrinogen and cross-linked
fibrin; MMP-11 can degrade fibrinogen but not fibrin. Matrix metalloproteinases also can modulate the activity of many inhibitors of
fibrinolysis, including α2-antiplasmin and PAI-1.
38
This process has recently been shown to
recognized by the immune system, and antistreptokinase antibodies are generated.
There are multiple endogenous proteins that can rap-
idly inhibit the fibrinolytic response. These include PAI-1, α
antiplasmin, α
inhibitors act through serine protease inhibition (serpin) and
-antitrypsin, and C1 inhibitor. Most of these
2
therefore affect many aspects of coagulation. The most important of these inhibitors is PAI-1, which is expressed by ECs or
platelets after exposure to thrombin; inflammatory mediators such as tumor necrosis factor alpha (TNF-α); and growth
Summary
-
In this chapter, we have described the intricate pathways involved
2
in coagulation and fibrinolysis, with specific emphasis on regulation of hemostasis. Future endeavors focused on understanding the complex nature of these processes and how they relate
to human disease processes, including inflammation, malignancy,
and arterial and venous thrombotic events, will provide targeted
therapies to modulate hemostasis and thrombosis.
Thrombin
Thrombin-activatable
fibrinolysis inhibitor (TAFI)
36
It has been shown that
40
The opposite can
41
42,43
CH
5
NoRmAl mECHANisms of VAsCulAR HEmosTAsis
37
74
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32. Mekkawy AH, Morris DL, Pourgholami MH: Urokinase plasminogen activator system as a
potential target for cancer therapy, Future Oncol 5(9):1487–1499, 2009.
33. Hildenbrand R, Allgayer H, Marx A, et al: Modulators of the urokinase-type plasminogen
activation system for cancer, Expert Opin Investig Drugs 19(5):641–652, 2010.
34. Bell WR: Present-day thrombolytic therapy: therapeutic agents–pharmacokinetics and
pharmacodynamics, Rev Cardiovasc Med 3(Suppl 2):S34–S44, 2002.
35. Kohler HP, Grant PJ: Plasminogen-activator inhibitor type 1 and coronary artery disease,
N Engl J Med 342(24):1792–1801, 2000.
36. Sen P, Komissarov AA, Florova G, et al: Plasminogen activator inhibitor-1 inhibits factor VIIa
bound to tissue factor, J Thromb Haemost 9:531–539, 2010.
37. Zhao L, Buckman B, Seto M, et al: Mutations in the substrate binding site of thrombinactivatable fibrinolysis inhibitor (TAFI) alter its substrate specificity, J Biol Chem
278(34):32359–32366, 2003.
38. Mosnier LO, Meijers JC, Bouma BN: Regulation of fibrinolysis in plasma by TAFI and
protein C is dependent on the concentration of thrombomodulin, Thromb Haemost
85(1):5–11, 2001.
39. Mosnier LO: Platelet factor 4 inhibits thrombomodulin-dependent activation of thrombinactivatable fibrinolysis inhibitor (TAFI) by thrombin, J Biol Chem 286:502–510, 2010.
40. Van Thiel DH, George M, Fareed J: Low levels of thrombin activatable fibrinolysis inhibitor
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41. Colucci M, Binetti BM, Tripodi A, et al: Hyperprothrombinemia associated with prothrombin
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42. Lijnen HR, Van Hoef B, Collen D: Inactivation of the serpin alpha(2)-antiplasmin by
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2000.
CHAPTER
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6 Vascular Pharmacology
David G. Harrison, James M. Luther
Vascular pharmacology has traditionally focused on drugs that
modulate vasomotion, and such agents continue to be commonly
used for treatment of disorders such as hypertension, myocardial ischemia, vasospasm, cardiovascular shock, and orthostatic
hypotension. In the past 2 decades, new drug targets have been
recognized, including inflammation, angiogenesis, and thrombosis.
In some cases, drugs affect these targets by unexpected off-target
effects that are nevertheless pharmacologically important.
Vascular Smooth Muscle Activation
The actions of many drugs discussed in this chapter affect vascular smooth muscle cell (VSMC) contraction, and a basic understanding of contractile regulation is essential to understanding
their mechanism of action. Contraction of vascular smooth muscle involves a sliding filament mechanism similar to that observed
in other smooth muscle or in skeletal muscle. This topic has been
reviewed in depth previously,
and is therefore only briefly discussed here. The classical paradigm, depicted in
calcium lead to formation of a calcium-calmodulin complex.
Calcium--CaM then binds and activates myosin light chain kinase
(MLCK), which then phosphorylates myosin light chain (LC-20).
Phosphorylation of LC-20 increases myosin adenosine triphosphatase (ATPase) activity, which leads to cross-bridge cycling
and contraction. Myosin light chain phosphatase negatively regulates this process by dephosphorylating LC-20. Myosin light chain
phosphatase is in turn inhibited by the small G-protein Rho and
Rho kinase, which phosphorylates a subunit of myosin light chain
phosphatase known as the myosin-binding subunit (MBS), leading to inhibition of phosphatase activity and favoring contraction.
Myosin phosphatase is also inhibited by a 17-kDa protein known
as CPI-17 (protein kinase C [PKC]–potentiated inhibitory protein
of 17 kDa) that in turn is activated by PKC. Thus, activation of
PKC can indirectly reduce myosin phosphatase activity, increase
myosin phosphorylation, and promote vasoconstriction.
An important counterregulatory pathway in this scheme is the
nitric oxide (NO) pathway. Nitric oxide acts on soluble guanylyl
cyclase (sGC), which catalyzes the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). In turn,
cGMP acts as the only substrate for type 1 protein kinase G (PKG),
which phosphorylates MBS, increasing its phosphatase activity
and promoting vasodilation. Protein kinase G also phosphorylates
and inhibits Rho, further reducing the propensity for vasoconstriction and promoting vasodilation. These pathways are targets of myriad vasoactive drugs that will be considered in greater depth in this
chapter and are depicted in
Traditionally, precapillary arterioles with diameters of approximately 25 μm were thought to regulate blood flow. While this is true
in some organs such as the kidney, in many organs, vascular resistances are distributed over a wider range of vessel sizes. In the coronary circulation, fully half of this resistance lies in vessels between
100 and 300 μm in diameter, and the remainder exists in vessels
smaller than 100 μm and in venules. Interestingly, many vasoactive
agents variably affect these different-sized vessels. As an example,
organic nitrates act predominantly on the larger arteries and veins
and have minimal effect on arterioles. In contrast, adenosine
potently dilates resistance vessels and has less effect on larger
vessels. Vasopressin is a potent constrictor of resistance arterioles
and causes endothelium-dependent vasodilation of conductance
arteries. These differential effects cause various drugs and hormones to selectively affect factors such as venous capacitance,
large (conductance) vessel diameter, and blood flow in the intact
circulation.
Figure 6-1, is that increases in intracellular
1
is covered in detail in Chapter 3,
Figure 6-1.
It is now apparent that many pharmacological agents not
only modulate vascular tone but also vascular growth, remodeling, inflammation, thrombosis, and vascular repair. As examples,
many components of the contractile pathway discussed earlier
exist in endothelial cells (ECs), including actin, myosin light chain,
MLCK, Rho, and Rho kinase. These regulate endothelial shape,
migration, cell-cell contact, and permeability. Myosin light chain
kinase activation controls EC calcium entry, NO production, and
release of endothelium-derived hyperpolarizing factor (EDHF).
The Rho/Rho kinase pathway works in concert with other GTPases
to modulate endothelial production of NO and reactive oxygen
species (ROS) and gene expression.
control have been the subject of substantial recent research, and
new drugs have been developed to affect these targets. In addition,
these pathways seem to be affected in an off-target fashion by several existing pharmacological agents.
3
These aspects of vascular
Pharmacokinetics and Pharmacodynamics
Before discussing specific pharmacological agents, the basic
concepts of pharmacology should be reviewed. Drug absorption, distribution, metabolism, and clearance are the principal
concepts of pharmacokinetics, and these concepts are detailed
elsewhere in pharmacology texts. Certain disease states, such as
renal insufficiency, liver insufficiency, or heart failure, may have
important effects on drug pharmacokinetics within individuals, and relevant situations will be discussed with specific drugs.
Pharmacodynamics is the study of drug mechanism of action
and physiological effects. Because some agents produce persistent effects beyond their clearance from the circulation, a drug
with a short half-life can have a longer dosing interval than
that predicted by its clearance. For example, aspirin irreversibly
inactivates the cyclooxygenase (COX) enzyme and achieves
long-lasting platelet inhibition despite rapid clearance from the
circulation. Diuretic agents may have a persistent antihypertensive effect after drug cessation, at least until a new level of sodium
balance is achieved. Other effects may become evident only after
drug withdrawal. For example, β-blockers increase receptor sensitivity as well as circulating catecholamines, and sudden drug
withdrawal of their β-blockade can result in rebound hypertension. Therefore, consideration of both pharmacokinetic properties and pharmacodynamic effects is essential for understanding
drug action.
The nature of a drug response helps classify the drug as a full
or partial agonist, antagonist, or an inverse agonist (
and may provide insight into the mechanism of drug action. For
receptor conformation–specific drugs, pure antagonists stabilize the active and inactive conformations equally and have no
net effect on basal activity. Inverse agonists preferentially stabilize the receptor's inactive form, and agonists stabilize the active
conformation.
The potency
essary to achieve a desired response (e.g., 50% maximal stimulation or inhibition;
maximal response relative to other agents (Fig. 6-2C). Clinical
differences in drug potency may be overcome by increasing the
dosage, whereas differences in drug efficacy cannot.
Receptor antagonists can be assessed by the response to a
known stimulus in the presence of increasing antagonist concentration (
tor can be overcome with increasing concentration of agonist
(
Fig. 6-3A). Antagonists that irreversibly bind their target impair
the maximal response with increasing concentration (
A number of drugs act in an allosteric manner by binding to a site
of a drug refers to the molar concentration nec-
Fig. 6-2B), whereas efficacy reflects the drug's
Fig. 6-3). Antagonists that reversibly bind to the recep-
Fig. 6-2A)
Fig. 6-3B).
2
75
76
Minoxidil
Agonist/Antagonist
Potency
Efficacy
2
2
Ca
Ca
cGMP-gated
ion channels
Ca
2
Ca
2
LC20
Rho kinase
CaM
2
Ca
CaM
ATP
MLCK
Myosin
Phosphatase
CPI-17
PKC
PKG-1
GMP
cGMP
PDE
K
CCBs
CH
6
2
Ca
Sarcoplasmic
reticulum
FIGURE 61 Vascular smooth muscle contractile regulation. ATP, adenosine triphosphatase; CaM, calmodulin; cAMP, cyclic adenosine monophosphate; CCB,
calcium channel blocker; cGMP, c yclic guanosine monophosphate; CPI-17, protein kinase C-potentiated inhibitory protein of 17 kDa; GMP, Guanosine monophosphate;
GTP, guanosine triphosphate; MLCK, myosin light chain kinase; NO, nitric oxide; PDE, phosphodiesterase; pGCase, particulate guanylyl cyclase; PGI2, prostacyclin; PKC,
protein kinase C; PKG-1, type 1 protein kinase G; sGC, soluble guanylyl cyclase.
Pinacidil
cAMP
Actin-myosin cross-bridge
cycling and contraction
–Pi
NO
sGC
pGCase
PGI
GTP
Natriuretic
peptides
2
200
150
100
50
% Basal Response
0
ABC
FIGURE 62 Comparison of receptor agonist activity. A, Conformation-specific receptor agonists (see text). B, Drug potency is compared by the concentration
necessary to achieve 50% maximal drug response (EC50). C, Drug efficacy is compared by the observed maximal response achieved.
([Drug]) log
Full Agonist
Partial Agonist
Antagonist
Inverse Agonist
on the receptor that is distinct from the native ligand, inducing a
conformational change. Allosteric modulators can either increase
or decrease agonist response by binding to a site distinct from the
agonist binding site. An allosteric antagonist dose-response curve
appears similar to that of a noncompetitive antagonist. Allosteric
potentiators shift the agonist curve to the left (see
competitive antagonists shift the curve to the right.
100
80
60
40
% Max Response
20
0
Fig. 6-3A), while
([Agonist]) log
This pathway is illustrated in the right portion of Figure 6-1, and
involves the guanylyl cyclase enzymes, cGMP, and the binding
targets of cGMP, which include the cGMP-dependent PKGs, ion
channels regulated by cGMP, and phosphodiesterases (PDEs).
The guanylyl cyclase/cGMP pathway is affected by a variety
of agents, including NO and NO donors (the nitrovasodilators);
other agents that activate guanylyl cyclase; agents that modulate
100
80
60
40
20
0
([Agonist]) log
degradation of cGMP; and agents that directly activate PKG.
Drugs That Affect Nitric Oxide/Guanylyl
Cyclase/cGMP–Dependent Protein
Kinase Pathway
The NO pathway plays a major role in modulating vascular
reactivity; however, NO represents only one step in a complex
pathway that can be affected by a variety of signaling molecules.
Endogenously, NO is produced by the nitric oxide synthase
(NOS) enzymes, and serves myriad signaling roles depending
on the cell and tissue in which it is produced.
4
Experimental
studies have shown that NO produced by the endothelium not
only mediates vasodilation, but also inhibits expression of adhesion molecules, reduces platelet adhesion, inhibits vascular
smooth muscle growth and hypertrophy, and prevents vascular
remodeling.
77
Competitive Antagonist
Non-Competitive Antagonist
Maximal % Response
AB
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FIGURE 63 Receptor antagonism.
A, With increasing concentration of a
competitive antagonist, the agonist dose
response curve is shifted to the right.
Allosteric potentiators produce a left
shift of this curve. B, Noncompetitive
antagonists and allosteric antagonists
shift the agonist response curve to the
right and impair maximal response in a
nonlinear manner.
Guanylyl cyclases convert GTP to cGMP. When first discovered,
this enzymatic activity was found in both the particulate or membrane fractions and in the soluble or cytoplasmic fractions of cell
homogenates. Shortly after this first discovery, it was recognized
that the soluble enzyme was activated by sodium azide, sodium
nitroprusside, and nitroglycerin in a heme-dependent fashion.
It has subsequently been confirmed that NO allosterically binds
a prosthetic heme group in sGC, which in turn alters enzyme
conformation and activates the enzyme. Removal of the heme
group eliminates the ability of NO to stimulate enzyme activity. Ten
years later, the particulate form was found to be activated not by
NO-like compounds but by atrial natriuretic peptide (ANP), and
that the particulate forms are in fact receptors for the natriuretic
peptides. Thus, NO donors and the natriuretic peptides share common downstream signaling pathways, albeit via activation of different upstream enzymes.
Nitrovasodilators
The nitrovasodilators produce their biological effects either by
releasing NO or closely related molecules that are converted to NO
in cells. The most commonly employed nitrovasodilators are the
organic nitrates and sodium nitroprusside. It is useful to begin a
discussion of these agents by comparing sodium nitroprusside and
nitroglycerin, which are illustrated in
oxidation state of the nitrogen that is ultimately released as NO differs in these molecules, and this basic structural property provides
insight into their pharmacological profiles. Sodium nitroprusside
requires a one-electron reduction to release NO, and this is readily accomplished nonenzymatically by a variety of reductants
in the circulation, the interstitial space, and the cell. Thus, when
infused intravenously, nitroprusside begins to release NO throughout the circulation and potently dilates all vessels. Moreover, given
O
N
O
N
Nitroglycerin MolsidomineNitroprusside
FIGURE 64 Nitrovasodilator agents. Structure of nitric oxide (NO) donor
agents. The nitroprusside cyanide ligands (C≡N) are highlighted in red.
100
80
60
40
20
10x 100x
0
([Agonist]) log
100
80
60
40
20
CH
6
VAsCulAR PHARmACology
0
([Agonist]) log
the short half-life of NO, the vasodilation caused by nitroprusside is
short-lived once its infusion is discontinued.
As is apparent from its structure, nitroprusside possesses five
cyanide groups in each molecule (highlighted in red in
Fig. 6-4),
and prior studies have shown that each of these is reduced prior
to the release of NO. The cyanide radicals react with hemoglobin
(Hb) to form methemoglobin and are converted to thiocyanate
in the liver. When these metabolic pathways are depleted, cyanide
toxicity occurs, characterized by central nervous system (CNS)
dysfunction, metabolic acidosis with a base deficit, and elevated
plasma lactic acid concentrations.
5
Fortunately, cyanide toxicity is
infrequent during brief administration of sodium nitroprusside but
occurs more commonly when infusion rates exceed 2 μg/kg/min
and when the drug is infused for prolonged periods. In addition,
the risk of cyanide toxicity is increased in patients with renal or
hepatic failure, so sodium nitroprusside should be avoided in
patients with these conditions. Owing to its capacity to rapidly
release NO, sodium nitroprusside produces potent systemic vasodilation and is effective as an antihypertensive. It is still used for
treatment of severe hypertension and, in some cases, for afterload
reduction in patients with severe heart failure; however, newer
agents with less potential toxicity are now more commonly used.
In contrast to sodium nitroprusside, nitroglycerin and other
organic nitrates require a 3-electron reduction to yield NO. In
Figure 6-4. As apparent, the
the last several years, it has become clear that this is in large part
accomplished by the action of the mitochondrial enzyme aldehyde reductase-2 (ADH2).
6
Mice lacking this enzyme are resistant
to the actions of nitroglycerin. Notably, about 40% of East Asians
have a dominant negative mutation of ADH2 that causes intolerance to ethanol and markedly impaired vasodilator responses to
nitroglycerin.
7
As mentioned earlier, organic nitrates preferentially dilate larger
arteries and veins while having less effect on arterioles, particularly at lower doses.
8
This response profile is likely beneficial in
alleviating angina because potent arteriolar dilators are prone to
cause coronary steal and paradoxically worsen myocardial isch-
O
O
O
O
N
O
O
O
NN
CC
NNCCFe
N
2
ONC
O
N
O
O
N
N
N
O
emia. Moreover, venous dilatation reduces left ventricular (LV)
filling, alleviates pulmonary congestion, and can improve subendocardial perfusion in ischemic regions of the myocardium.
Traditionally, organic nitrates have been employed to either
alleviate or prevent the chest pain associated with myocardial
ischemia. For acute angina, nitroglycerin is administered either
as a sublingual tablet or an oral spray. For prevention of angina,
long-acting nitroglycerin preparations or related organic nitrates
(e.g., isosorbide mononitrate, isosorbide dinitrate, pentaerythritol tetranitrate, transdermal nitrates) are commonly employed.
Nitroglycerin is often administered intravenously for treatment of
acute coronary syndromes (ACS).
Experimental studies have shown that NO inhibits platelet adhesion, expression of adhesion molecules, and vascular smooth
muscle proliferation and migration. Thus, one might expect that
NO donors such as nitroglycerin would reduce atherosclerosis
78
progression and potentially reduce major cardiovascular events
in patients with coronary artery disease (CAD). Despite extensive
use for alleviation of myocardial ischemia for almost a century and
a half, no clinical trials have shown that these drugs reduce ischemic cardiovascular events. The GISSI-3 and ISIS-4 trials examined
the effect of nitrates following myocardial infarction (MI) but failed
CH
to show a significant improvement in outcome.
6
observed patients for 5 weeks to 6 months following MI, and therefore did not determine whether long-term nitrates might have a
beneficial effect on outcome in patients with ischemic heart disease. Given the many putative beneficial effects of NO on vascular function, longer-term treatment might impart a beneficial effect
on atherosclerosis, inflammation, vascular remodeling, or plaque
stability. Indeed, a recent analysis of the GRACE registry, which
includes patients admitted for ACS, showed that chronic nitrate
users were much more likely to present with non–ST-segment elevation MI NSTEMI) than non-nitrate users.
11
preted with caution because the use of nitrates was not randomized,
and conclusions were derived from a retrospective analysis.
In addition to their use as antianginal agents, the long-acting
nitrates are now often employed for treatment of congestive
heart failure (CHF), commonly in combination with hydralazine.
Unlike the case for treatment of CAD, prospective randomized trials have shown that long-acting nitrates improve survival, reduce
hospitalizations, and enhance quality of life in patients with CHF,
particularly among African Americans.
underlying the beneficial effects are unclear; however, longacting nitrates appear to synergize with hydralazine as afterloadand preload-reducing agents. These agents might also improve
renal hemodynamics and promote diuresis and, via release of
NO, have beneficial effects on vascular and cardiac remodeling.
A major limitation to prolonged use of organic nitrates is development of tolerance. Within about 12 hours of administration,
the hemodynamic effects of organic nitrates begin to abate, in
part due to extravascular adaptations such as volume redistribution and neurohormonal activation. After several days of continuous nitrate therapy, the direct vascular actions of nitrates are lost,
even when vessels are removed from the animal or human. The
mechanisms of nitrate tolerance, and in particular this latter form
of true vascular tolerance, remain uncertain but have been attributed to formation of ROS, nitrosation and oxidation of guanylyl
cyclase, and changes in activity of ADH2.
gies have been proposed to prevent nitrate tolerance, but the only
approach accepted clinically is to allow a drug “holiday”; that is,
to withdraw the nitrate for about 12 hours daily. The commonly
employed isosorbide mononitrate preparations accomplish this
by increasing blood levels of the drug for about 12 hours during
waking hours, after which blood levels fall to near-undetectable
levels. Experimental studies have shown that hydralazine prevents nitrate tolerance by reducing oxidative stress,
explain the benefit of hydralazine when added to long-acting
nitrates in the treatment of heart failure. The long-acting nitrate
pentaerythritol tetranitrate seems not to cause tolerance in experimental animals, but this has not been proven in clinical studies.
Intravenous nitroglycerin has occasionally been used for treatment of hypertensive emergencies. This condition is often associated with a contracted blood volume. Owing to nitroglycerin's
propensity to produce venular dilation rather than arteriolar dilation, it has the potential to reduce cardiac output in this setting
and may produce untoward effects in patients with compromised
coronary, renal, or cerebral perfusion.
might be useful in combination with other agents in treating a
hypertensive emergency, particularly in patients with acute pulmonary edema, but other agents are available and likely more
effective.
In addition to its reaction with sGC, NO can react with other
heme proteins and radicals. Higher oxides of NO can also react
with thiols, leading to formation of nitrosothiols.
example of these reactions is the reversible NO reaction with
cytochrome C, which modulates mitochondrial respiration and
9,10
These trials only
These data must be inter-
12
Precise mechanisms
13
A number of strate-
13
which might
14
Low-dose nitroglycerin
15
An important
superoxide production.
16
It is uncertain as to how important these
reactions are in the overall response to nitrovasodilators.
Related to the chemistry mentioned earlier are reactions of
inorganic nitrate (
are oxidation products of endogenously produced NO, they are
−
) and nitrite (
NO
3
−
). Although these
NO
2
also derived from dietary sources such as green leafy vegetables.
Nitrate is rapidly converted to nitrite by bacteria in the oral cavity
and gastrointestinal tract. Nitrite, in turn, can be reduced by various heme proteins, including deoxyhemoglobin, to NO. Studies
have shown that the reaction of nitrite with deoxyhemoglobin
promotes NO formation and vasodilation in regions of the circulation where oxygen tension is low, thereby improving oxygenation
of hypoxic tissues.17 Thus, once considered an inactive metabolite
of NO, nitrite likely has physiological significance and might have
therapeutic utility.
18
Molsidomine (see Fig. 6-4) has also been used as an NO donor
for treatment of angina, but it is not commonly employed clinically.
The liver metabolizes molsidomine to release SIN-1, which in turn
decomposes to NO and superoxide in equimolar amounts. These
species can react rapidly with one another to yield the strong oxidant peroxynitrite. Because of this chemistry, SIN-1 oxidizes lipoproteins, damages DNA, and depletes antioxidants. This capacity to
generate peroxynitrite has dampened enthusiasm for clinical use
of molsidomine and related drugs, but SIN-1 is commonly used to
produce peroxynitrite in experimental settings.
There are other agents used experimentally as NO donors. Two
classes that deserve mention are the S-nitrosothiols (SNOs) and
the NONOates. S-nitrosothiols can be formed either by reactions
of thiols with higher oxides of NO or by the reaction of NO with
thiyl radicals. There is substantial evidence that SNOs are formed
in vivo, where they serve as reservoirs for NO, and that the attachment of NO to thiols in proteins affects protein function. As
an example, S-nitrosylation of Hb has been implicated in oxygen affinity and delivery. S-nitrosothiols are simple to synthesize
and, depending on the thiol backbone, have different stabilities
such that they can release NO in times ranging from seconds to
minutes. S-nitrosothiols can also undergo heterolytic scission,
yielding the nitrosonium cation (NO
+
), which acts as a nitrosating
agent to form various nitroso compounds. The NONOates are
commercially available nucleophilic/NO complexes often used
experimentally as NO donors. These release only NO and are
very useful because their varying structures permit controlled NO
delivery over widely varying times, ranging from seconds to hours.
Neither SNOs nor NONOates are clinically used at present.
Unique Modulators of Soluble Guanylyl Cyclase
Soluble guanylyl cyclase contains a heme group that is responsible for binding and activation by NO. Agents such as 1 H-[1,2,4]
oxadiazolo[4,3-a]quinoxalin-1 (ODQ), ferricyanide, or methylene
blue can inactivate sGC by oxidizing the heme group. Owing to
this enzymology, ODQ and methylene blue have been used as
pharmacological probes to prove that a biological response is
dependent on guanylyl cyclase. In a similar vein, oxidation of
the heme group by superoxide or hydrogen peroxide might alter
NO-dependent enzyme activity and therefore impair endothelium-dependent vasodilation under conditions where endogenous production of ROS is increased. Thus, in addition to
oxidative inactivation of NO, superoxide and related oxidants
can impair NO function by inactivating sGC.
Compounds have been developed that activate sGC in an
NO-independent fashion.
lopyridine BAY 41-2272 and YC-1, interact with the heme group
independent of NO, or can markedly enhance NO-stimulated
enzyme activity. Others, such as BAY 58-2667 and HMR-1766,
activate sGC in a heme-independent fashion and can stimulate
cGMP formation even when the heme group is oxidized. Because
these agents do not depend on endogenous production of NO,
they have potential advantages over PDE inhibitors (see later
discussion) in diseases where NO production is impaired. They
19
Some of these, such as the pyrazo-
also potentially bypass the problem of tolerance observed with
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various NO donors. These agents produce vasodilation, lower
blood pressure, inhibit platelet aggregation, and have been shown
to have therapeutic benefit in experimental models of systemic
hypertension, pulmonary hypertension (PH), and heart failure.
19
Like NO, they inhibit neointima formation following balloon injury
in rats and therefore might be effective in treatment or prevention
of restenosis and atherosclerosis. They also hold promise for treatment of erectile dysfunction (ED), liver fibrosis, and renal disease.
Currently, clinical trials are underway to examine the efficacy of
some of these agents in the treatment of heart failure and PH.
Natriuretic Peptides
Natriuretic peptides, including atrial (ANP), brain (BNP), and
C-type (CNP) natriuretic peptides, are 17-amino-acid ring structures with an internal disulfide bond and are secreted as prohormones. Atrial natriuretic peptide and BNP are predominantly
produced by atrial and ventricular myocytes; CNP is produced by
vascular endothelial cells, the brain, and other peripheral tissues.
Urodilatin, a related peptide processed from the ANP prohormone,
is released from distal tubular cells of the kidney.
21
The A and B
natriuretic peptide receptors are homodimers that are widely distributed, particularly in the cardiovascular system and kidney.
The cytoplasmic tails of these contain a guanylyl cyclase domain
that is activated by binding with natriuretic peptides.
20
There also
exists a C-type natriuretic receptor that has a short cytoplasmic
tail without a guanylyl cyclase domain and seems predominantly
involved in clearing natriuretic peptides from the circulation.
As mentioned, ANP and BNP are produced predominantly in
atrial myocytes. In the setting of a variety of conditions (e.g., heart
failure, cardiac inflammation, fibrosis, hypoxia), BNP is expressed
in large amounts by ventricular myocytes, leading to an elevation
of circulating BNP. Thus, BNP and pro-BNP are commonly used as
biomarkers for detection of various cardiac pathologies, and in particular for diagnosis and management of volume overload states.
Activation of the A- and B-type natriuretic receptors leads to
vasodilation and a variable diuretic and natriuretic response,
depending on volume status. For this reason, a synthetic form of
BNP known as nesiritide has been marketed and employed for
treatment of decompensated heart failure. Like the nitrovasodilators, nesiritide infusion lowers pulmonary capillary wedge
pressure (PCWP), right atrial pressure, and systemic vascular
resistance, and improves symptoms of dyspnea.
23
This agent also
lowers circulating catecholamines, aldosterone, and angiotensin(Ang) II levels, and aids diuresis. One study suggested that nesiritide was more effective that intravenous nitroglycerin treatment
of patients with severe heart failure.
23
An early meta-analyses suggested that nesiritide therapy was associated with an increase
in mortality within 30 days of treatment, for uncertain reasons24;
however, more recent meta-analysis of six randomized clinical trials showed no change in outcome at 10, 30, or 180 days following
administration of this agent.25 A randomized trial of more than
7000 subjects has shown that treatment with nesiritide acutely
improves patients with class IV heart failure, without worsening
long-term outcome.
26
This positive study is tempered by a very
recent large study of 7143 patients with acute heart failure that
showed no benefit of nesiritide in reducing symptoms or improving outcome at 30 days.
27
20
21
22
Phosphodiesterase Inhibitors
As reflected in Figure 6-1, cGMP is rapidly inactivated to GMP by
cellular PDEs. There are 11 PDE isoenzymes with varying specificities for the different cyclic nucleotides. Phosphodiesterases
5, 6, and 9 are highly selective for cGMP, while PDEs 3 and 10
are preferentially activated by cyclic adenosine monophosphate (cAMP). Phosphodiesterases 1, 2, and 11 have dual substrate specificity.
PDEs are PDE1, 2, and 5. The PDEs are subject to substantial posttranslational regulation. As examples, PDE1 is calcium/CaMdependent, cGMP stimulates PDE2 inactivation of cAMP, and
binding of cAMP to PDE3 is inhibited by cGMP.
Several naturally occurring PDE inhibitors, such as caffeine, theophylline, and theobromines, are present in coffee, chocolates,
and tea, and have been used since antiquity as stimulants.
are among the most widely distributed drugs in the world. Like
cAMP and cGMP, the PDE inhibitors commonly contain a purine
structure with linked pyrimidine and imidazole rings. These
agents occupy the cAMP or cGMP PDE binding sites and inhibit
respective PDE isoenzymes with varying degrees of selectivity.
The immediate cardiovascular effects of nonselective PDE inhibition include vasodilation due to accumulation of cGMP and cAMP,
increases in cardiac contractility due to accumulation of cAMP,
and improvement in diastolic relaxation (lusitrophy) mediated by
cAMP and cGMP.
In the past 30 years, a variety of PDE5 inhibitors, including sildenafil, tadalafil, and vardenafil, have been developed and are now
used clinically (
the vasodilator effect of PDE5 inhibitors is almost exclusively
dependent on endogenous NO release, and is prevented by inhibition of NOS and in conditions in which endogenous NO production is impaired.
PDE5 inhibitors acutely reduce cardiac contractility and precondition cardiac myocytes to reduce necrosis and apoptosis caused by
experimental ischemia.
prevents experimental cardiac hypertrophy caused by transaortic
constriction.
The PDE5 inhibitors were developed as antihypertensive agents,
but because of their potent effect on the corpus cavernosa, they
were initially approved and have become widely employed for
treatment of erectile dysfunction. These agents are also potent
dilators of the pulmonary circulation. Sildenafil and tadalafil
been approved by the U.S. Food and Drug Administration (FDA)
for treatment of pulmonary arterial hypertension (PAH). This disorder, defined by the hemodynamic parameters of a mean pulmonary artery pressure (PAP) above 25 mmHg and a PCWP
15 mmHg or lower, occurs as a primary condition and in the setting of a variety of diseases that affect the pulmonary circula-
33
tion.
(Also see Chapters 56 and 57.) A single dose of sildenafil
was found to reduce PAP in patients with both primary and secondary PH and to augment the effect of inhaled NO in these sub-
34
jects.
Clinical studies have shown that chronic administration
of PDE5 inhibitors reduces PAP and right ventricular (RV) mass,
and improves exercise tolerance and functional status in patients
with PAH.
nafil improved 3-year survival in patients with PAH compared to
historical controls.
considered a mainstay of therapy for PAH. They have also been
28
In the cardiovascular system, the predominant
Table 6-1). Experimental studies have shown that
28
These agents also affect cardiac function. The
30,31
Chronic PDE5 inhibition with sildenafil
32
35
The recent SUPER-2 clinical trial showed that silde-
36
For these reasons, PDE5 inhibitors are now
29
These
79
CH
6
VAsCulAR PHARmACology
29
TABLE 6-1 Phosphodiesterase-5 Inhibitors
CLASS
PDE5 inhibitor Sildenafil (Viagra)
L, liver failure; PDE5, phosphodiesterase type 5; R, renal failure; T
DRUG
TRADE NAME
Tadalafil (Cialis) 17.5 L, R 2.5-20 24
Vardenafil (Levitra) 4-5 L 10-20 24
T
1/2
HOURS
4 L, R 25-100 24
, half-life.
1/2
DOSE
ADJUSTMENT
DOSE RANGE
TOTAL mg/ DAY
DOSING INTERVAL
HOURS
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