Hemostasis and Thrombosis Protocols

330 Pages·1999·2.532 MB·English

by  David J. Perry, K. John Pasi

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Hemostasis
Components and Processes
K. John Pasi
1. Introduction
Hemostasis is a host defense mechanism that protects the integrity of the
vascular system after tissue injury. It works in conjunction with other inflam-
matory, immune, and repair mechanisms to produce a coordinated response.
Hemostatic systems are generally quiescent, but following tissue injury or dam-
age these systems are rapidly activated.
Hemostasis has evolved to accommodate the conflicting needs of maintain-
ing vascular integrity and free flow of blood in the vascular tree. Given the
high pressures that exists in arterial circulation, it is clearly important that
procoagulant mechanisms exist that can minimize blood loss from a site of
vascular damage as rapidly as possible. However, this powerful procoagulant
response must be localized to prevent unwanted thrombosis and controlled to
prevent thrombosis in the slower low-pressure venous circulation. As a result
of these competing needs, hemostasis has evolved as a patchwork of inter-
related activating and inhibiting pathways that can either promote or suppress
other elements of the overall process. Hemostasis has therefore evolved to
integrate five major components: vascular endothelium, platelets, coagulant
proteins, anticoagulant proteins, and fibrinolytic proteins. The coordinated
hemostatic response ultimately produces a platelet plug, fibrin-based clot,
deposition of white cells at the point of injury and activation of inflammatory,
and repair processes, maintenance of blood flow, and vascular integrity.
2. Overview of Hemostasis
All components of the hemostatic mechanism exist under resting conditions
in an inactive form. A diagrammatic representation of the overall hemostatic
From:Methods in Molecular Medicine, Vol. 31: Hemostasis and Thrombosis Protocols
Edited by: D. J. Perry and K. J. Pasi © Humana Press Inc., Totowa, NJ
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response is shown in Fig. 1. Following injury, there is immediate vasoconstric-
tion and reflex constriction of adjacent small arteries. This slows blood flow
into the damaged area. The reduced blood flow allows contact activation of
platelets. On activation by tissue injury (or other agonists), platelets undergo a
series of physical, biochemical, and morphological changes. Platelets adhere
to exposed connective tissue, mediated in part by the von Willebrand factor
(vWF).Collagen exposure and local thrombin generation (seeSubheading 6.)
lead to the release of platelet granule contents. Release of platelet granule con-
tents, which include adenosine diphosphate (ADP), serotonin, and fibrinogen,
further enhances platelet activation, formation of platelet aggregates, and inter-
action with other platelets and leukocytes. This process leads to the formation
of the initial platelet plug.
The vascular endothelium also undergoes a series of changes moving from
its resting phase (with predominantly anticoagulant properties) to a more active
procoagulant and repair phase. In concert with these cellular changes, inactive
plasma coagulation factors are converted to their respective active species by
cleavage of one or two internal peptide bonds. In sequence, these active factors
generate thrombin, which leads to formation of fibrin from fibrinogen (to sta-
bilize the platelet plug), crosslinking of the fibrin formed (via activation of
factor XIII), further activation of platelets, and also activation of fibrinolytic
pathways (to enable plasmin to dissolve fibrin strands in the course of wound
healing). Additionally, thrombin interacts with other nonhemostatic systems to
promote cellular chemotaxis, fibroblast growth, and wound repair.
3. Components of the Hemostatic System
3.1. Vascular Endothelium
Vascular endothelium is the monolayer of cells that line the inner surface of
blood vessels. Since an uninterrupted vascular tree is necessary for survival,
the ability of the vasculature to maintain a nonleaking system is essential. If a
vessel is disrupted and leakage occurs, the coagulation system and platelets
close the defect temporarily until cellular repair of the defect takes place. If a
vessel is occluded by thrombus, blood flow may be re-established by lysing the
clot or recanalizing the occluded vessel. These properties are the main func-
tional characteristics of the vascular endothelial cell.
Endothelial cells are attached to and rest on the subendothelium, an
extracelluar matrix secreted by the endothelial cells. Subendothelium is com-
posed of collagen, elastin, mucopolysaccharides (including heparan sulfate,
dermatan sulfate, chrondroitin sulfate), laminin, fibronectin, vWF, vitronectin,
thrombospondin, and occasionally fibrin. All these components are synthesized
by the endothelial cells. Together, endothelium and subendothelium form a
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Fig. 1. A flow diagram representing the major events in the process of overall
hemostasis.
selectively impermeable layer, resistant to the passive transfer of fluid and cel-
lular elements of blood, but permeable to gases. Cells may pass through the
endothelium at sites of inflammation by a process of adherence and then
migration between endothelial cells. Subendothelium can act as a physical bar-
rier in the absence of endothelial cells. Endothelial cells have multiple func-
tions as outlined below (1).
3.1.1. Maintenance of Blood Flow
Endothelial cells influence vascular tone, blood pressure, and blood flow by
induction of vasoconstriction and vasodilatation. This is achieved by secretion
of renin, endothelin, endothelial-derived relaxing factor (EDRF) or nitrous
oxide, adenosine, prostacyclin, and surface enzymes that convert or inactivate
other vasoactive peptides, such as angiotensin and bradykinin.
3.1.2. Antiplatelet and Anticoagulant Properties
Intactendothelial cells are intrinsically nonthrombogenic, exerting a power-
ful inhibitory influence on hemostasis by a range of factors that they either
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synthesize or express on their surface. For example, platelets adhere to
subendothelium rather than endothelial cells. This is due to endothelial pro-
duction of components that inhibit platelet aggregation, such as prostacylin,
EDRF, and adenosine.
Cell-surface heparan sulfate enhances the effect of antithrombin in forming
thrombin–antithrombin complexes. Perhaps the major anticoagulant proper-
ties of endothelium are via the endothelial expression of thrombomodulin and
tissue factor pathway inhibitor (TFPI). Thrombomodulin enhances the ability
of thrombin to activate protein C. Enhancement of protein C activation leads to
increased inactivation of factor Va and factor VIIIa. Endothelium also secretes
protease nexin 1. This inactivates thrombin by covalent binding to the throm-
bin active site. This complex formation is enhanced by heparan sulfate.
3.1.3. Coagulant Properties
In contrast to the above, in the setting of damage to blood vessels, the endothelium
functions as an important component to coagulation pathways. Central to this role is
endothelial cells production of tissue factor in response to injury. In addition, they
bind factors IX, X, V, high-mol-wt kininogen (HMWK), contain factor XIII activity,
and produce endothelin to induce vasoconstriction. Importantly, endothelial cells also
produce the natural inhibitor of tissue factor mediated coagulation, TFPI.
3.1.4. Fibrinolytic Properties
Endothelial cells secrete several components active in fibrinolysis. These include
plasminogen activators and plasminogen activator inhibitor. These components are
bound to the endothelial cell surface to enable assembly of active complexes.
3.1.5. Repair Properties
Endothelial cells are capable of significant repair of blood vessels. Simple
minor injuries are repaired by migration of adjacent cells and subsequent
endothelial cell proliferation. More severe vessel wall injuries require migra-
tion and proliferation of smooth muscle cells and fibroblasts. Endothelium
secretes components that are active in the repair process by enhancing smooth
muscle migration and fibroblast function. These include a protein resembling
platelet-derived growth factor, vascular permeability factor, and fibroblast
growth factor. Endothelial cells are also responsive to platelet-derived endot-
helial growth factor and transforming growth factor β.
3.1.6. Interactive Properties
The endothelium interacts with leukocytes. This is critical in the migration
of leukocytes into area of inflammation. Adhesion molecules present on both
endothelial cells and leukocytes mediate this interaction.
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4. Platelets
Platelets are nonnucleated fragments of cytoplasm that have a crucial role in
primary hemostasis. They are derived from bone marrow megakaryocytes and
are smooth biconvex disks of approx 1–4 mm diameter. Normal circulating
numbers are approx 140–400 × 109/L.
4.1. Production
In the production of platelets, megakaryocytes undergo specialized cellular
division.The megakaryocyte nucleus divides, but the cell itself does not divide
(endomitosis)(2), although there is formation of new membrane and cytoplas-
mic maturation. This cytoplasmic maturation includes development of plate-
let-specific granules, membrane glycoproteins, and lysosomes. Mature
megakaryocytes are therefore variably polyploid, with up to 64 N. They are
large at approx 60 µm diameter. As a part of the endomitosis process, there is
increased membrane. This excess membrane is accommodated by invagina-
tion. The invagination process continues, thereby clipping off individual plate-
lets (cytoplasmic fragmentation) from the main megakaryocyte body. It is
suggested that circulating megakaryocytes undergo cytoplasmic fragmentation
in the pulmonary capillary bed.
Megakaryocyte maturation is controlled in a simple negative feedback loop,
under the influence of the growth factor thrombopoietin and cytokines, such as
interleukin-3 (IL-3) and interleukin-11 (IL-11). When platelet production is
increased, megakaryocytes undergo a more rapid cytoplasmic maturation than
nuclear maturation. Under such circumstances, platelets may be produced from
octaploid or even tetraploid cell megakaryocytes. Such platelets are often larger
than normal and more metabolically active.
Once released from the bone marrow, platelets are sequestered in the spleen
for 24–48 h. The spleen may contain upto 30% of the normal circulating mass
of platelets. Significant platelet pools may also exist in the lungs.
The normal life-span of platelets is approx 8–14 d. Platelets are removed
from the circulation by the reticuloendothelial system on the basis of senes-
cence rather than by random utilization. However, there is a small fixed com-
ponent that exists owing to random utilization of platelets that maintain
vascular integrity.
4.2. Structure
Stylized structural features are shown diagrammatically in Fig. 2. A range of
glycoproteins molecules partially or completely penetrate cell-membrane lipid
bilayer. These glycoprotein molecules function as receptors for different ago-
nists, adhesive proteins, coagulation factors, and for other platelets. Important
membrane glycoproteins are listed in Table 1 with their associated functions.
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Fig. 2. Stylized structural features of the platelet. See text for decription of indi-
vidual components.
Table 1
Important Platelet Membrane Glycoproteins
Glycoprotein 103 copies/platelet Receptors
Ia 2–4 Collagen
IIa 5–10 Fibronectin, laminin
Ic 3–6 Fibronectin, laminin
Ib/IX 25–30 vWF, thrombin
IIb/IIIa 40–50 Fibrinogen, vWF, Fibronectin, vitronectin
IV Collagen, thrombospondin
V Thrombin
The most abundant glycoproteins on the platelet surface are glycoproteins
IIb and IIIa. These two glycoproteins form a heterodimer and carry receptors
for adhesive proteins (fibrinogen, vWF, fibronectin). The IIb-IIIa complex is a
member of the integrin family of adhesion receptors. Glycoprotein Ib contains
a receptor for vWF and thrombin. This receptor is essential in the platelet ves-
sel wall interaction. The cell membrane also has importance as a source of
phospholipid (prostaglandin synthesis), site of calcium mobilization, and
localization of coagulant activity to the platelet surface.
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Platelet structure is complex (3). Below the plasma membrane lies a periph-
eral band of microtubules, which function as the cellular cytoskeleton. The
microtubules maintain the discoid shape in the resting platelet, but disappear
temporally (disassemble?) on platelet aggregation.
The surface-connected canalicular system is an extensive system of plasma
membrane invaginations. This system vastly increases the surface area across
which membrane transport occurs and through which platelet granules dis-
charge their contents during the secretory phase of platelet aggregation.
The dense tubular system probably represents the smooth endoplasmic
reticulum. It is thought to be the site of prostaglandin synthesis and sequestra-
tion/release of calcium ions.
Platelets contain many organelles (mitochondria, glycogen granules, lysos-
omes, peroximsomes) and two types of platelet-specific storage granules: dense
bodies (d-granules) or a-granules. The contents of the platelet-specific gran-
ules are released when platelets aggregate.
Dense bodies contain 60% of the platelet storage pool of adenine nucle-
otides (such as adenosine diphosphate) and serotonin. Dense body adenine
nucleotides do not readily exchange with other adenine nucleotides in the plate-
let metabolic pool. α-Granules contain multiple different proteins. These pro-
teins may be platelet specific or proteins that are found in the plasma or other
cell types (such as coagulation factors). The major contents of α-granules are
vWF, platelet factor 4, β-thromboglobulin, thrombospondin, factor V, fibrino-
gen, fibronectin, platelet derived growth factor, high-mol-wt kininogen, and
tissue plasminogen activator inhibitor-1.
4.3. Function
Platelets are crucial components of the hemostatic system. When a vessel
wall is damaged, platelets escaping from the circulation immediately come into
contact with and adhere to collagen and subendothelial bound vWF (through
glycoprotein Ib). Glycoprotein IIb-IIIa is then exposed, via the binding of vWF.
This forms a second binding site for vWF. In addition with glycoprotein IIb–
IIIa exposure, fibrinogen may be bound promoting platelet aggregation. Within
seconds of adhesion to the vessel wall, platelets undergo a shape change, ow-
ing to ADP released from the damaged cells or other platelets exposed to the
subendothelium. Platelets become more spherical and put out pseudopods,
which enable platelet–platelet interaction. The peripheral microtubules become
centrally apposed forcing the granules toward the surface and the surface-con-
nected canalicular system. Platelets then undergo a specific release reaction of
their granules, the intensity of the release reaction being dependent on the in-
tensity of the stimulus. With the shape change, there is also further exposure of
the glycoprotein IIb–IIIa complex and further fibrinogen binding. Since fi-
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brinogen is a dimer, it can form a direct bridge between platelets or act as a
substrate for the lectin-like protein thrombospondin. With the enhancement of
platelet–platelet interaction, platelet aggregation ensues. Platelet aggregation
causes activation, secretion, and release from other platelets, so leading to a
self-sustaining cycle that results in the formation of a platelet plug.
The binding of agonists to also leads to a series of signal transduction events
that mediate the platelet release reaction (see Fig. 3) (4). Agonist receptor
interaction activates guanine nucleotide binding proteins (G-protein) and
hydrolysis of plasma membrane phospholipids (phosphotidyl inositides) by
phospholipase C (PLC). Inositol triphosphates that are formed act as iono-
phores, and mobilize calcium ions into the cytosol from the dense tubular sys-
tem, and lead to an influx of calcium from outside. Diacylglycerol, also formed
within the G-protein/PLC pathway, activates protein kinase C, which in turn
phosphorylates a 47-kDa contractile protein. Together with the calcium-
dependent phosphorylation of myosin light chain, these reactions induce con-
traction and secretion of granule contents. Cyclic AMP/adenyl cyclase exert
regulatory control over these reactions (high levels of cAMP reduce cytosol
calcium concentration) and are in turn regulated by G-protein activity. In addi-
tion, prostaglandin (cyclic endoperoxides and thromboxane A ) synthesized
2
from membrane phospholipids may bind to specific receptors and further
stimulate these processes.
Platelet α-granules contain several coagulation factors (such as factor V,
fibrinogen, and high-mol-wt kininogen). On secretion from the α-granule, these
factors reach high local concentrations. Platelets provide a local phospholipid
surface for these factors to work on, particularly factor V. This procoagulant
activity of platelets is not seen in resting platelets.
4.4. Antigens
Platelets have a number of antigens on their surface specific to platelets.
Many of the platelet-specific antigens are associated with platelet membrane
glycoproteins (HPA IA—glycoprotein IIIa). Platelets also express HLA class I
antigens and ABO blood group antigens.
5. Coagulation Factors
5.1. Thrombin
Thrombin is the cornerstone of hemostasis. Prothrombin, its precursor, is a
vitamin K dependent plasma of mol wt 71 kDa (579 amino acids). Thrombin is
crucial to the conversion of fibrinogen to fibrin. It is the most potent physi-
ological activator of platelets causing shape change, the generation of throm-
boxane A , ADP release, and ultimately platelet aggregation. Thrombin also
2
activates the cofactors of coagulation factor V, factor VIII, and factor XIII.
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Fig. 3. Signal transduction events that mediate the platelet-release reaction. The
intermediate processes lead to the phosphorylation of 47kD protein and myosin light
chain, which together contract and lead to secretion of platelet granule contents.
Thrombin bound to thrombomodulin is a potent activator of protein C. In addition
to its procoagulant and anticoagulant activities, thrombin also has important roles
in cellular growth, cellular activation, and the regulation of cellular migration.
5.2. Tissue Factor
This is an integral transmembrane protein of mol wt 45 kDa (263 amino
acids) coded for by a short gene of 12.4 kb on chromosome 1. It is found on the
surface of vascular cells, but is also constitutively expressed by many
nonvascular tissues. It can be upregulated on monocytes and vascular endothe-
lium by inflammatory cytokines or endotoxin. Tissue factor (thromboplastin)
binds and promotes activation of factor VII, and is required for the initiation of
blood coagulation. It acts as a cofactor enhancing the proteolytic activity of
factor VIIa toward factor IX and factor X. It binds factor VII via calcium ions.
5.3. Factor V
This is a plasma glycoprotein of mol wt 330 kDa (2224 amino acids) coded
for by a complex 25 exon 80-kb gene on chromosome 1. It is a critical cofactor