hemostasis and thrombosis protocols

Hemostasis 3
3
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
1
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
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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 (see Subheading 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
Hemostasis 5
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
Intact endothelial cells are intrinsically nonthrombogenic, exerting a power-
ful inhibitory influence on hemostasis by a range of factors that they either
Fig. 1. A flow diagram representing the major events in the process of overall

hemostasis.
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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.
Hemostasis 7
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 × 10
9
/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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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.

Fig. 2. Stylized structural features of the platelet. See text for decription of indi-
vidual components.
Table 1
Important Platelet Membrane Glycoproteins
Glycoprotein 10
3
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
Hemostasis 9
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
2
) synthesized
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
2
, ADP release, and ultimately platelet aggregation. Thrombin also
activates the cofactors of coagulation factor V, factor VIII, and factor XIII.
Hemostasis 11
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
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.
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in coagulation, which in its activated form facilities the conversion of pro-

thrombin to thrombin. In Factor V, the rate of conversion of prothrombin to
thrombin is 200,000- to 300,000-fold.
Factor V circulates as a single-chain protein in a precursor inactive form. It is
converted into an active two-chain form by thrombin or factor Xa. Thrombin cleaves
factor V at three separate sites. Following cleavage, the two chains are linked via a
divalent metal ion bridge. Binding to phospholipid surfaces occurs via the light chain.
Factor V is inactivated by activated protein C and its cofactor protein S.
Although it is predominantly synthesized in the liver (plasma factor V),
megakaryocytes also synthesize factor V, which is stored in platelet α-gran-
ules (platelet factor V). Platelet factor V, which is released on platelet activa-
tion, accounts for approx 20% of total factor V. Factor V has a binding protein
in platelets (multimerin), which is analogous to vWF for factor VIII.
Plasma concentration of factor V is about 7–10 µg/mL with a half-life of
approx 12 h.
5.4. Factor VII
This is a vitamin K dependent plasma glycoprotein and serine protease of
mol wt 50 kDa (406 amino acids) coded for by a 13-kb gene on chromosome
13. It has 10 N-terminal glutamic acid residues that are terminal γ carboxylated
to form the Gla domain. Calcium binding properties of factor VII are crucial to
its normal function and biological activity.
Factor VII is involved in the initiation of blood coagulation, forming a com-
plex with tissue factor to generate an enzyme complex that activates factor X
and factor IX. Factor VII is activated by cleavage of the Arg
153
–Ile
153
peptide
bond. Activators include thrombin, activated factor X, and activated factor IX.
Activated factor VII has no catalytic activity until bound to tissue factor. It
circulates at a concentration of 0.5 µg/mL and half-life of 4–6 h.

5.5. Factor VIII
This is a plasma glycoprotein of approx mol wt 360 kDa (2351 amino acids)
coded for by a complex 26 exon 186-kb gene on the X chromosome. It has a
domain structure that is very similar to that of factor V and is related to the
copper protein ceruloplamsin. However, unlike factor V, the large B domain is
not required for coagulant activity. Factor VIII is one of the largest and least
stable coagulation factors with a complex polypeptide composition, circulat-
ing in plasma in a noncovalent complex with vWF. vWF functions to protect
factor VIII from premature proteolytic degradation and concentrate factor VIII
at sites of vascular injury. Factor VIII functions as a cofactor for factor IX,
facilitating the conversion of factor X to factor Xa. Factor VIII increases the
rate of conversion of factor X to Xa by factor IX by 200,000-fold.
Hemostasis 13
Liver synthesized single-chain molecule factor VIII is cleaved shortly after
synthesis, circulates as a heterodimer, and comprises an 80-kDa light chain
linked through a divalent metal cation bridge to a heavy chain (90–200 kDa).
Variable amounts of the B domain remain after this initial cleavage. On activa-
tion by thrombin (or factor Xa), factor VIII is cleaved at Arg
372
, Arg
740
, and
Arg
1689
, the Arg
740
cleavage removing residual B domain remnants. This cleav-
age yields a 90-kDa heavy chain. A rate-limiting Arg
372
cleavage yields two

smaller 50 and 40 kDa fragments, both of which are essential for factor VIII
clotting activity. At the same time, a small fragment is cleaved that removes
vWF from factor VIII. Activated factor VIII is very unstable and rapidly loses
cofactor function, owing to subunit disassociation. Inactivation of factor VIII
also occurs via activated protein C and its cofactor protein S, by cleavage at
Arg
336
and Arg
562
. Plasma concentration of factor VIII is about 100–200 ng/mL
and half-life of approx 12 h.
5.6. Factor X
This is a vitamin K-dependent plasma glycoprotein and serine protease of
mol wt 59,000 coded for by a 22-kb gene on chromosome 13. It has 11
N-terminal glutamic acid residues that are terminal γ carboxylated to form the
Gla domain. Calcium binding properties of factor X are crucial to its normal
function and biological activity. It is a central component in the common path-
way of blood coagulation. Factor X is synthesized as a single chain, but exists
in plasma as a heavy and light chain linked by a single disulfide bond. It is
activated by cleavage of the Arg51–Ile52 peptide bond. Activators include
activated factor VII/tissue factor complex and activated factor IX/factor VIII
complex in the presence of calcium ions.
Factor Xa, in conjunction forms a complex on phospholipid surfaces with
factor V to form the prothrombinase complex. This complex converts pro-
thrombin to thrombin. Factor X is inhibited by antithrombin and α
2
macro-
globulin.
Factor X circulates at a concentration of 8–10 µg/mL and has a half-life of
approx 36 h.

5.7. Factor IX
This is a vitamin K-dependent plasma glycoprotein and serine protease of
mol wt 57,000 (415 amino acids) coded by a 34-kb gene on the long arm of the
X chromosome. It is the largest of the family of vitamin K dependent proteins.
Twelve N-terminal glutamic acid residues are terminal γ carboxylated to form
the Gla domain. Calcium binding properties of factor IX are crucial to its nor-
mal function and biological activity.
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Factor IX circulates as a single-chain polypeptide. Activation occurs via cleav-
age of two peptide bonds, Arg
145
–Ala
146
and Arg
180
–Val
181
, by either activated
factor XI or activated factor VII, complexed to tissue factor. Arg
180
–Val
181
cleav-
age is rate-limiting. Cleavage into factor IXa generates a heavy and light chain
bound together via a single disulfide bond. A 24 amino acid activation peptide is
removed during cleavage. Together with factor VIII, factor IXa can then proceed
to activate factor X. In addition, factor IXa may also activate factor VII.
The plasma factor IX concentration is about 5 µg/mL with a half-life of
approx 24 h.
5.8. Factor XI

This is plasma glycoprotein and serine protease of mol wt 160 kDa coded by
a 23-kb gene on chromosome 4. Factor XI is a homodimer, comprising two
identical subunits bound together by disulfide bond, that circulates bound to
high-mol-wt kininogen. The plasma factor XI concentration is about 5 µg/mL
with a half-life of approx 72 h.
Factor XI is cleaved to active factor XIa by activated factor XII in the pres-
ence of high-mol-wt kininogen. Activation cleavage occurs within each sub-
unit at Arg
369
–Ile
370
in a region bounded by a disulfide linkage, so yielding two
heavy chains and two light chains in the active molecule. Only the light chains
possess catalytic activity. Factor XIa activates factor IX in the presence of cal-
cium. No specific additional cofactors are required for this reaction. Both fac-
tor XI and factor XIa bind to platelets.
5.9. Factor XII
This is a plasma glycoprotein and serine protease of mol wt 80 kDa (596
amino acids) coded for by a 12-kb gene located on chromosome 5. Factor XII
has a half-life of approx 2 d and a plasma concentration of approx 30 µg/mL.
In the process of contact activation factor XII is absorbed on to negatively
charged surfaces and undergoes limited proteolysis at specific sites to yield
active factor XII'. This slowly converts prekallikrein to kallikrein, which spe-
cifically cleaves factor XII to yield fully active factor XIIa. In addition, factor
XIIa can autoactivate factor XII. Factor XIIa can activate factor XI to promote
downstream activation of the coagulation cascade.
5.10. Factor XIII
This is a tetramer of two a and b chains. The b chains function as the carrier
for the a chains. On activation by thrombin, factor XIII crosslinks fibrin and
other proteins involved in the clot via a transglutamase reaction. The factor

XIIIa subunit has a plasma concentration of 15 µg/mL and the b subunit a
concentration of 14 µg/mL.
Hemostasis 15
5.11. von Willebrand Factor (vWF)
This is a multimeric glycoprotein of basic subunit of 400 kDa mol wt (2813
amino acids) coded for by a large complex gene of 178-kb on the short arm of
chromosome 12. vWF is an important component in primary platelet hemosta-
sis. Following translation, it undergoes extensive intracellular processing and
exists as a series of multimers of the basic subunit, ranging from mol wt 800 to
20,000 kDa. It is produced in both endothelium and megakaryocytes. Endothe-
lial cells secrete a vWF into the plasma constitutively, but store the majority of
vWF synthesized (in Wieble Palade bodies) for regulated secretion. Platelet
vWF is released from α-granules locally when they aggregate. vWF functions:
as a carrier protein for coagulation factor VIII and as an adhesive protein
involved in endothelial-platelet interaction, via platelet surface membrane gly-
coprotein Ib and IIb–IIIa complex. Its function as an adhesive protein is par-
ticularly important in situations of high shear stress.
6. Coagulation Cascade
The classic “waterfall” hypothesis for coagulation proposes the intrinsic and
extrinsic pathways (see Fig. 4) (5,6). The intrinsic system assumes that expo-
sure of contact factors (factor XII, high-mol-wt kininogens, prekallikrein) to
an abnormal/injured vascular surface leads to activation of factor XI, which in
turn activates factor IX. Activated factor IX, in the presence of its cofactor
factor VIII, then activates factor X to factor Xa in the presence of phospho-
lipid. In turn, factor Xa, with its cofactor factor V, together form the
prothrombinase complex, which converts prothrombin to thrombin. Thrombin
then converts fibrinogen to fibrin. The extrinsic system assumes that factor VII
and tissue factor, released from damaged vessels, directly activate factor X,
and coagulation factor lying below factor X in the final common pathway.
The division into extrinsic and intrinsic systems and the ability to test these

two systems in the laboratory (the prothrombin time and activated partial
thromboplastin time, respectively) have been valuable in understanding clini-
cal bleeding problems, but fail to represent accurately what happens in in vivo
hemostasis. This may be shown by considering the following points. First,
patients who have an inherited deficiency of factor XII, prekallikrein or high-
mol-wt kininogen have no clinical bleeding problems, yet have extremely pro-
longed activated partial thromboplastin times. This clinical observation
demonstrates that these proteins are probably not important components of
blood coagulation in vivo and, therefore, should not be included in an in vivo
consideration of blood coagulation. Similarly, factor XI deficiency is not
always associated with bleeding and its role is therefore unclear, whereas
patients with factor VII deficiency bleed abnormally, although they have an
intact intrinsic system. Third, factor VII–tissue factor is known to activate not
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only factor X, but also factor IX. In the classic waterfall, this activation is not
required. Fourth, tissue factor is a natural constituent of many nonvascular cells.
Tissue factor on such cells is able to initiate blood coagulation. These points
suggest a more central role for the tissue factor–factor VII complex. Addition-
ally, the identification of an endogenous inhibitor of tissue factor-induced
coagulation (tissue factor pathway inhibitor; TFRI) and an increased understand-
ing of its properties have led to a questioning of traditional dogmas (7).
The revised cascade is outlined in Fig. 5. This revised cascade is believed to
represent more accurately the processes that occur in vivo (7–9). Coagulation
is initiated when tissue damage at the site of the wound exposes blood to tissue
factor, produced constitutively by cells beneath the endothelium. Factor VII
binds tissue factor forming the tissue factor–factor VII complex. This complex
directly activates factor X to factor Xa and some factor IX to factor IXa. It is
not clear what proteases initially activate factor VII in this complex, but once
coagulation is activated, other proteins are able in turn to activate factor VII,
including factor Xa and VIIa. This provides a mechanism for further amplifi-

cation and acceleration of coagulation.
Fig. 4. Classic “waterfall” hypothesis for coagulation with the intrinsic, extrinsic,
and final common pathways. Although useful in understanding coagulation pathways
in in vitro clotting assays this schema does not accurately represent in vivo coagula-
tion processes.
Hemostasis 17
Once formed, the complex of the factor VIIa, tissue factor, and factor Xa
binds TFPI forming a quaternary complex. This TFPI binding inhibits further
generation of factor Xa and factor IXa by tissue factor–factor VIIa complex.
Under these conditions, further factor Xa can only be generated by the factor
IXa, factor VIIIa pathway.
By this point in coagulation activation, enough thrombin usually exists to be
able to activate factor VIII to factor VIIIa (generated by direct activation of
factor Xa by factor VII–tissue factor). With activation of factor VIIIa and using
the initial generation of factor IXa (by tissue factor–factor VIIa), the factor
IXa, factor VIIIa route is able to move forward and allow further factor Xa
generation to proceed.
Further augmentation of factor IX activation is produced via thrombin acti-
vation of the factor XI pathway. This is proposed to be a process that occurs
later in coagulation.
The revised cascade assumes that tissue factor–factor VIIa is responsible for
the initial generation of factor Xa and thrombin, sufficient to activate factor V,
factor VIII, and platelet aggregation locally. Following inhibition by TFPI, the
Fig. 5. The revised coagulation pathway. See text for details. Note the central role
of TFPI and absence of contact factors.
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amount of factor Xa produced is insufficient to maintain coagulation, and there-
fore, factor Xa generation must be amplified using factor IXa and factor VIIIa
to allow hemostasis to progress to completion.
Unlike the waterfall hypothesis, the revised hypothesis does not assume that

initial generation of factor Xa and thrombin is the end of the hemostatic pro-
cess. Rather it assumes that following initial generation the hemostatic response
must be reinforced and/or consolidated by a further progressive generation of
factor Xa and thrombin. This allows the hypothesis to encompass the compet-
ing influences of inhibitors of coagulation, blood flow washing away activated
coagulation factors, and also thrombin-activated fibrinolysis. Additionally, it
does not all factor known to be involved in blood coagulation.
The revised hypothesis also allows a better explanation of bleeding seen in
hemophilia A and hemophilia B. In these two conditions, bleeding occurs both
spontaneously (intrinsic system) and after trauma (extrinsic system), which
cannot easily be reconciled on the classical waterfall hypothesis. Using the
revised schema, it is clear that without factor VIII or factor IX, bleeding will
ensue because the amplification and consolidating generation of factor Xa is
insufficient to sustain hemostasis.
7. Anticoagulant Pathways
Natural, physiological anticoagulants fall into two broad categories, serine
protease inhibitors (SERPINS) and those that neutralize specific activated
coagulation factors (protein C system). These systems are of major physiologi-
cal significance. They are active from the very outset of the coagulation pro-
cess and often brought fully into play before fibrin deposition has occurred.
7.1. Serine Protease Inhibitors (SERPINS)
Serpins include many of the key inhibitors of coagulation, such as antithrom-
bin, heparin cofactor II, protein C inhibitor, plasminogen inactivators, and α
2
-
antiplasmin. Of these, antithrombin is perhaps the most important (10). AT is a
single-chain glycoprotein of mol wt 58,000 (432 amino acids) coded for on
chromosome 1. It will inhibit all the coagulation serine proteases (II, VII, IX,
X, XI, XII), but it is its antithrombin and anti-Xa activity that are physiologi-
cally important. AT activity/inhibition is increased 5- to 10,000-fold in the

presence of heparin and other sulfated glycosaminoglycans. Heparin is not
normally found in the circulation, and physiologically, antithrombin probably
binds to heparan sulfate on the vascular endothelial cells.
7.2. Protein C System
Factors Va and VIIIa are powerful cofactors in coagulation-enhancing
activity of serine proteases. Both Va and VIIIa are specifically inactivated by
Hemostasis 19
components of the protein C pathway. Protein C is the key inactivating enzyme
(11). It is a single-chain vitamin K-dependent protein synthesized by the liver.
Together with its cofactor, protein S it inactivates factors Va and VIIIa.
Thrombin generated during coagulation binds to thrombomodulin (Tm) on
the surface of vascular endothelial cells. Thrombin/Tm complex is a potent
activator of Protein C, Tm accelerating Protein C activation approx 20,000-
fold. Protein C is activated by cleavage at Arg
169
–Leu
170
. Activated protein C
(APC) is inhibited by the specific inhibitor, protein C Inhibitor (PCI) and since
it is a serine protease, it is inhibited by antithrombin.
Protein S, the cofactor for protein C, is vitamin K-dependent. It circulates in
plasma as a single-chain glycoprotein of mol wt 60,000 and is synthesized by
the liver, endothelial cells, and megakaryocytes. Approximately 60% of pro-
tein S is complexed to C4b binding protein, Only the unbound or “free” protein
S is physiologically active.
8. Fibrinolysis
Fibrinolysis principally exists to ensure that fibrin deposition in excess of
that which is required to prevent blood loss from damaged vessels is either
prevented or degraded and removed (12).
Plasminogen, the inactive form of the enzyme plasmin, has a mol wt of 92

kDa (790 amino acids), and is synthesized in the liver and coded on chromo-
some 6. Plasminogen contains five homologous looped structures called
“kringles,” four of which contain lysine binding sites through which the mol-
ecule interacts with its substrates and its inhibitors. Internal autocatalytic cleav-
age occurs during activation of plasminogen with the release of an activation
peptide. This changes the N-terminus from containing a glutamic acid residue
(Glu-plasminogen) to a form that contains a lysine residue (Lys-plasminogen).
Conversion of plasminogen to plasmin can occur via two routes. Most acti-
vators cleave plasminogen at Arg
560
to generate a two-chain protein Glu-plas-
min, the two chains linked by a single disulfide bridge. The light chain is
derived from the C-terminus of the protein and contains the active serine cata-
lytic site, whereas the heavy chain is derived from the N-terminus and contains
the kringle domains. Glu-plasmin is functionally inactive, since its lysine bind-
ing sites are masked. It is activated when it is converted to Lys-plasmin by
autocatalytic cleavage between Lys
76
–Lys
77
. This cleavage exposes the lysine
binding sites on the kringle domains, dramatically increasing the affinity of the
protease for fibrin. Both Glu-plasmin and Lys-plasmin attack the Lys
76
-Lys
77
bond to form Lys-plasminogen. This is capable of binding to the fibrin clot
before it develops protease activity, and it is, therefore brought into close prox-
imity with the physiological activators.
Plasminogen is activated by a number of endogenous proteins. Of these, tis-

sue plasminogen activator (t-PA) is perhaps the most important. t-PA is synthe-
20 Pasi
sized primarily by vascular endothelial cells although many other cells, are
capable of its synthesis. t-PA is synthesized as a single-chain glycoprotein (sct-
PA) and contains two kringle domains through which it binds fibrin. Although
sct-PA has significant proteolytic activity, its biological activity is low until
bound to fibrin. When bound, its affinity for plasminogen is increased approx
400-fold. Plasmin generated is capable of cleaving sct-PA into a two-chain tPA.
Two-chain t-PA has more exposed binding sites and a significantly increased
activity through increased binding of fibrin and plasminogen. t-PA has a short
half-life (5 min) and is rapidly cleared from the circulation.
sct-PA and tct-PA are inhibited by the SERPIN plasminogen activator
inhibitor type 1 (PAI-1). A second inhibitor of t-PA, plasminogen activator
inhibitor type 2 (PAI-2) is found in plasma in significant amounts during preg-
nancy. Normally free t-PA is rapidly inactivated because of an excess of PAI-
1 and any free plasmin generated is rapidly inactivated by α
2
-antiplasmin.
A second endogenous activator of fibrinolysis is urokinase. Urokinase is
synthesized as an essentially inactive single-chain protein (scu-PA/pro-uroki-
nase). It must be converted to the two-chain form (tcu-PA or U-PA) before it is
functionally active. scu-PA is converted to tcu-PA (U-PA) by plasmin and kal-
likrein. tcu-PA activates plasminogen to plasmin by a cleaving at Arg
560
–
Val
561
. Inhibition of the active enzyme occurs via PAI-1, PAI-2, and also by
protease nexin 1. Although urokinase can activate plasminogen in plasma it is
thought that its major role is an extravascular activator of plasminogen, espe-

cially where tissue destruction or cell migration occurs.
9. Summary
Hemostasis is clearly a complex interactive system involving numerous compo-
nents. The revised hypothesis of coagulation has helped to unify the whole pro-
cess. The recent improved understanding has in part been brought about by
improved knowledge of the individual components of the different elements of the
overall process of hemostasis and cellular repair. Although increasing by appreci-
ated to be complex, attempts have been made to model and reproduce this system
in vitro to validate research findings and increase understanding of the interactions.
For all its complexity, many of these models of hemostasis, both laboratory and
mathematical, have proven to be useful, and show that for all the interactions and
complexity of different systems combined with flow and cellular interaction, we
do have a considerable understanding of the processes of hemostasis.
Suggested Reading
Bloom, A. L., Forbes, C. D., Thomas, D. P., and Tuddenham, E. G. D. (1994)
Haemostasis and Thrombosis, 3rd ed. Churchill Livingstone, Edinburgh, UK.
Tuddenham, E. G. D. and Cooper, D. N. The Molecular Genetics of Haemostasis and
its Inherited Disorders. Oxford Medical Publications, Oxford, UK.
Hemostasis 21
References
1. Cines, D. B., Pollak, E. S., Buck, C. A., Loscalzo, J., Zimmerman, G. A., McEver,
R. P., Pober, J. S., Wick, T. M., Konkle, B. A., Schwartz, B. S., Barnathan, E. S.,
McCrae, E. R., Hug, B. A., Schmidt, A. S., and Stern, D. M. (1998) Endothelial
cells in physiology and in the pathophysiology of vascular disorders. Blood 91,
3527–3561.
2. Gerwitz, A. (1995) Megakaryocytopoiesis: the state of the art. Thomb.
Haemostasis 74, 204–209.
3. White, J. G. (1994) Anatomy and structural organisation of the platelet, in Hemo-
stasis and Thrombosis: Basic Principles and Clinical Practice (Coleman, R. W.,
et al., eds.), 3rd ed., J. B. Lippencott Co., Philadelphia, PA.

4. Levy-Toledano, S., Gallet, C., Nadel, F., Bryckaert, M., Macloug, J., and Rosa,
J P. (1997) Phosphorylation and dephosporylation mechanisms in platelet func-
tion: a tightly regulated balance. Thromb. Haemostasis 78, 226–233.
5. Macfarlane, R. G. (1964) An enzyme cascade in the blood clotting mechanism
and its function as a biochemical amplifier. Nature 202, 498,499.
6. Davie, E. W. and Ratnoff, O. D. (1964) Waterfall sequence for intrinsic blood
clotting. Science 145, 1310–1312.
7. Broze, G. J. Jr., Warren, L. A., Novotny, W. F., Higuchi, D. A., Girrad, J. J., and
Miletich, J. P. (1988) The lipoprotein associated coagulation inhibitor that inhib-
its the factor VII-tissue factor complex also inhibits factor Xa: insight into its
possible mechanism of action. Blood 71, 335–343.
8. Furie, B. and Furie, B. C. (1992) Molecular and cellular biology of blood
coagualtion. N. Engl. J. Med. 326, 800–806.
9. Rapaport, S. I. and Rao, L. V. (1995) The tissue factor pathway: how it has bevome
a “prima ballerina.” Thromb. Haemostasis 74, 7–17.
10. Perry, D. J. (1994) Antithrombin and its inherited deficiencies. Blood Rev. 8, 35–37.
11. Dadhlback, B. (1995) New molecular insights into the genetivcs of thrombophilia:
resistance to activated Protein C caused by the Arg
506
to Gln mutation in factor V as
a pathogenic risk factor for venous thrombosis. Thromb. Haemostasis 74, 139–148.
12. Collen, D. and Lijen, H. R. (1991) Basic and clinical aspects of fibrinolysis and
thrombolysis. Blood 78, 3114–3124.
Isolation of DNA and RNA 25
25
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
2
Isolation of DNA and RNA

David J. Perry
1. Introduction
Blood samples for most coagulation tests are collected into 3.8% trisodium
citrate in a ratio of 1 part anticoagulant to 9 parts blood. Whole-blood samples
for DNA isolation can be stored at –50°C and the DNA prepared at a later
stage. A more convenient method requiring less freezer space is to store buffy
coats—the interface between the red cells and the plasma that is seen following
centrifugation of whole blood. This latter method also allows isolation of the
plasma fraction.
There are numerous methods for isolating DNA. The methods described in
this Chapter are routinely used to prepare high molecular weight DNA for
Southern blot analysis or for amplification by the polymerase chain reaction
(PCR) technique. The first method employs a phenol/chloroform step to dena-
ture proteins, whereas the second employs a salt precipitation step to precipi-
tate proteins. Both methods can be readily adapted to processing small-volume
samples (e.g., 100 µL). The first method has been successfully used to isolate
DNA from a wide variety of cells, including whole blood, buffy coats, plate-
lets, various cell lines, spleen, lymph nodes, and bone marrow.
As with the isolation of DNA, there are many techniques for isolating RNA
from a wide variety of cells, some of which can be adapted to allow the simul-
taneous isolation of both RNA and DNA. Many of the methods in current use
employ strong chaotropic agents (e.g., guanidinium thiocyanate) to disrupt cell-
ular membranes and inactivate intracellular RNases. The method described has
been in routine use for several years and generates high-quality RNA suitable
for a wide variety of uses. A number of commercial kits are now available for
rapid RNA isolation, e.g, RNeasy™ kit (Qiagen Ltd, UK). Although often more
expensive than “in-house” methods, these kits are capable of isolating high-
26 Perry
quality RNA from a wide variety of cells, including whole-blood, leukocyte
buffy coats, platelets, and tissue-culture cells.

Methods are also included in this chapter for the isolation of lymphocytes
and platelets from whole blood.
2. Materials
Molecular-grade reagents should be used whenever possible. Sterile dispos-
able polypropylene is used for most steps, but if glassware is used, it should be
baked at 280°C for at least 3 h to inactivate any RNases.
2.1. Isolation of Mononuclear Cells from Whole Blood
1. Phosphate-buffered saline: 120 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer,
pH 7.4. Autoclave before use and store at 4°C.
2. Density gradient medium, e.g., Histopaque 1077 (Sigma).
2.2. Isolation of Platelets from Whole Blood
1. Phosphate-buffered saline: 120 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer,
pH 7.4. Autoclave before use and store at 4°C.
2. 38–40% Bovine serum albumin (BSA).
2.3. DNA Isolation Using the Phenol/Chloroform Method (1)
1. Sucrose lysis buffer: sucrose 0.32 M, 1% (v/v) Triton X-100, 5 mM MgCl
2
, 10 mM
Tris-HCl, pH 7.5. Store at 4°C.
2. TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0. Autoclave before use.
3. Nuclear lysis buffer: 0.32 M lithium acetate, 2% (w/v) lithium dodecyl sulfate
(LiDS), 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0. Store at room temperature.
4. Phenol: Equilibrated phenol for both DNA and RNA isolation can be purchased
commercially (e.g., CamLabs, Cambridge, UK) and avoids many of the potential
risks associated with its use. If crystalline phenol is used, 0.1% hydroxyquinoline
should be added as an antioxidant and it should be extracted initially with 1 M
Tris-HCl, pH 8.0, and then repeatedly with 0.1 M Tris-HCl, pH 8.0, until the pH
of the aqueous phase is 8.0 (2). Phenol should be stored at 4°C.
5. Phenol:chloroform: A 1:1 mixture phenol and chloroform is made by mixing
equal volumes of chloroform and equilibrated phenol. This may be purchased

ready prepared from a number of manufacturers (e.g., CamLabs). Store at 4°C.
2.4. DNA Isolation Using the Salt-Precipitation Method (3)
1. Sucrose lysis buffer–see Subheading 2.3.1.
2. TKM 1: low-salt buffer–10 mM Tris-HCl, pH 7.6, 10 mM KCl, 10 mM MgCl
2
,
2 mM EDTA.
3. TKM 2: high-salt buffer–10 mM Tris-HCl, pH 7.6, 10 mM KCl, 10 mM MgCl
2
,
0.4 M NaCl, 2 mM EDTA.
4. 6 M NaCl: This is a supersaturated solution.
Isolation of DNA and RNA 27
5. 20% SDS: Dissolve 20 g SDS in 100 mL distilled water at 65°C. Store at room
temperature.
2.5. Isolation of Total Cellular RNA (4)
1. Solution D: 50 g guanidinium thiocyanate (GTC), 58.6 mL of distilled water,
3.52 mL 0.75 M trisodium citrate, 5.28 mL 10% sarkosyl NL30. Incubate at 65°C
to dissolve the GTC. Store at room temperature for up to 3 mo. Immediately
before use, add 3 5 µL of β-mercaptoethanol to 5 mL of solution D.
2. 2 M NaOAC, pH 4.0. Store at 4°C.
3. Phenol:chloroform (4:1): water-saturated phenol, pH 4.0 chloroform (4:1). This
can be purchased commercially (e.g., CamLabs). Store at 4° C.
3. Methods
3.1. Isolation of Mononuclear Cells from Whole Blood
1. Dilute 10–20 mL of whole blood 1:1 with ice-cold PBS.
2. Carefully layer onto an equal volume of the appropriate density gradient medium,
e.g., Histopaque 1077, in a 30-mL sterile tube.
3. Centrifuge at 600g for 30 min at 22°C.
4. Carefully collect the cellular interface using a sterile Pasteur pipet and re-sus-

pend the cells in 50 mL of ice-cold PBS.
5. Centrifuge at 800g for 30 min at 22°C to pellet the cells.
6. Re-suspend the cells in 1 mL of ice-cold PBS and store on ice until use.
3.2. Isolation of Platelets from Whole Blood (
see
Note 8)
1. Platelet rich plasma (PRP) is prepared from whole blood by centrifuging 10 mL
of whole blood at 600g for 10 min at 22°C. (The leukocyte count of PRP is gen-
erally <0.2 × 10
9
/L if carefully prepared.)
2. Carefully aspirate the PRP and layer onto 1 mL of 38% or 40% albumin.
3. Centrifuge at 1800g for 20 min at 22°C. The albumin acts as a cushion onto
which the platelets will form a lawn.
4. Collect the platelet lawn and carefully resuspend in 10 mL of PBS.
5. Centrifuge at 1800g for 20 min at 22°C to pellet the platelets.
6. Carefully decant the supernatant and resuspend the platelet pellet in 1 mL of PBS.
3.3. DNA Isolation Using the Phenol/Chloroform Method (1)
1. Add 10 mL of anticoagulated whole blood to 90 mL of ice-cold sucrose lysis
buffer, invert several times to mix and store on ice for 15 min. See Note 1 for use
of smaller volumes.
2. Centrifuge the samples at 1000g for 10 min at 4°C. Decant the supernatant into a
beaker and vigorously resuspend the pellet in 4.5 mL of TE, pH 8.0. The superna-
tant is potentially infectious and should be disposed of accordingly.
3. Lyse the cell pellet by adding 10 mL of nuclear lysis buffer and gently rotating
the sample on a mechanical rotator at approx 250 rpm until a clear viscous solu-
tion is obtained. Samples can be frozen at this stage for processing at a later date.
28 Perry
4. Add 5 mL of buffer-saturated phenol/chloroform and mix on a mechanical rota-
tor for 10 min (see Notes 2 and 3).

5. Centrifuge the samples at 1000g for 5 min at 20°C to separate the phases. Care-
fully remove the upper aqueous phase to a clean tube using a wide-bore pipet.
Avoid aspirating any of the lower phase which contains phenol/chloroform and
denatured proteins.
6. Add 5 mL of chloroform to the aqueous phase and mix on a mechanical rotator
for 5 min.
7. Centrifuge the samples at 1000g for 5 min at 20°C to separate the phases. Care-
fully remove the upper aqueous phase to a clean tube using a wide-bore pipet.
8. Precipitate the DNA by adding 21/2 vol of ethanol and gently inverting the tube.
Collect the DNA onto a sealed sterile glass Pasteur pipet, briefly rinse in 70%
ethanol and re-suspend in 50 µL of sterile, distilled water or TE, pH 8.0. The
DNA samples are stored at 4°C for 2–3 d to allow the DNA to go into solution
and then stored at –20°C.
3.4. DNA Isolation Using the Salt-Precipitation Method (3)
1. Add 5 mL of whole blood to 20 mL of sucrose lysis buffer and mix by inverting
several times.
2. Centrifuge the samples at 1000g for 10 min at 4°C. Decant the supernatant into a
beaker and add 5 mL of TKM 1 to the pellet. Centrifuge the samples at 1000g for
10 min at 4°C.
3. Decant the supernatant and resuspend the pellet in 0.8 mL of TKM 2. Transfer
the content into 2-mL labeled Eppendorf. Add 25 µL of 20% SDS to the tube and
gently mix. Incubate at 57°C in a waterbath/heating block for 30 min.
4. Transfer the Eppendorf(s) into a benchtop rack and add 300 µL of 6 M NaCl, mix thor-
oughly. Centrifuge in a benchtop microfuge at maximum speed (13,000 rpm) for 10 min.
5. Transfer the supernatant into a clean 2-mL Eppendorf and add an equal volume
of chloroform and mix by inversion. Centrifuge at 7,000 rpm for 5 min.
6. Transfer the supernatant into a sterile 20-mL tube and place on ice. Add an equal
volume of ice-cold absolute ethanol and gently invert to precipitate the DNA.
Collect the DNA onto a sealed sterile glass Pasteur pipette, rinse briefly in 70%
ethanol and re-suspend in 500 µL of sterile, distilled water or TE, pH 8.0. The

DNA samples are stored at 4°C for 2–3 d to allow the DNA to go into solution
and then stored at –20°C.
3.5. Isolation of Total Cellular RNA
(4)
(
see
Notes 5 and 6)
1. Reusupend the mononuclear pellet from 10 mL of whole blood in 1.6 mL of
solution D and vortex vigorously for 30 s. Split into 2 × 800 µL aliquots in 2-mL
Eppendorfs (see Note 7).
2. To each tube add 80 µL of 2 M NaOAc, pH 4.0 and vortex briefly to mix.
3. Add 800 µL water-saturated phenol, pH 4.0 to each tube and vortex briefly to mix.
4. Add 160 µL chloroform:isoamyl alcohol (24:1) to each tube and vortex briefly to
mix. Leave one ice for 15 min.
Isolation of DNA and RNA 29
5. Centrifuge in a benchtop microfuge at 13,000 rpm for 10 min. Collect the upper
aqueous layer and transfer to a clean 2-mL Eppendorfs.
6. Add 800 µL of propan-2-ol to each tube and place at –20°C for 1–2 h (or over-
night).
7. Centrifuge in a benchtop microfuge at 13,000 rpm for 10 min. Remove the super-
natant and resuspend each pellet in 300 µL of solution D. Pool pairs of tubes.
8. Add 600 µL of propan-2-ol and place at –20°C for 1–2 h.
9. Centrifuge in a benchtop microfuge at 13,000 rpm for 10 min. Remove the super-
natant and resuspend the pellet in 400 µL of TE, pH 8.0.
10. Check the yield and quality of the RNA by electrophoresing 5–10 µL in a 1.5%
agarose gel in 1X Trios-Borate-EDTA (TBE). The 28S and 18S (and sometimes
the 5S) ribosomal bands should be clearly visible.
For long-term storage, the RNA should be precipitated by adding 2
1
⁄

2
vol of
ethanol and
1
/
10
vol of 3 M NaOAc and then placed at –70°C.
4. Notes
1. For packed cells, i.e., samples in which the plasma has been removed, 5 mL of
packed cells is added to 45 mL of cell lysis buffer. Whole blood samples which
have been frozen are thawed at 37°C and the tubes rinsed thoroughly with cell
lysis buffer. For small volumes (100–200 µL) of blood, packed cells, leukocyte
buffy coats, and so on, the cells should be lysed in 1.5 mL of cell lysis buffer,
centrifuged in a benchtop microfuge at 12,000 rpm for 30 s, the supernatant care-
fully aspirated, and the lysis step repeated. The pellet should be resuspended is
100–200 µL of TE, pH 8.0 and the nuclei lysed by adding twice the volume of
nuclear lysis buffer and vortexing the sample for 10–20 s. The lysed cells are
then extracted once with 200 µL of phenol-chloroform and once with 200 µL of
chloroform and the DNA precipitated by adding either 21/2 volume of ice-cold
ethanol or an equal volume of prop-2-ol. The DNA is collected by centrifugation at
12,00 rpm in a microfuge for 30 s, washed carefully in 1 mL of 70% ethanol,
allowed to dry at room temperature and then re-suspended in 50 µL of TE, pH 8.0.
2. Isoamyl alcohol (IAA) is commonly included in preparations of chloroform as a
24:1 (v/v) mixture to prevent foaming. However, this is not a problem using the
techniques described and is not routinely included.
3. Phenol is extremely toxic and should be handled with care in a fume hood, wear-
ing suitable protective clothing including goggles.
4. All solutions for RNA isolation must be treated with DEPC (diethylpyro-
carbonate). Tris-containing solutions cannot be treated in the manner and should
be made using DEPC-treated water and then autoclaved.

5. To make DEPC-treated water add 1 mL of DEPC to 1 L of distilled water. Incu-
bate at 37°C overnight and then autoclave. Store at room temperature.
6. DEPC is a potential carcinogen and must be handled with care.
7. Cells may be lysed in Solution D and then stored at –70°C until convenient. We have
combined with subsequent processing by the RNeasy™ kit, with great success.