An Overview of Hemostasis*

G. DANIEL BOON
Department of Pathology, College of Veterinary Medicine,
University of Missouri, Columbia, Missouri 65205

ABSTRACT
Hemostasis is a remarkable and a remarkably complex mechanism. It can maintain blood in a fluid state
intravascularly but very quickly changes blood to a jellylike mass upon disruption of the vasculature. This
review will give a synopsis of the 3 phases of hemostasis: platelet, vascular, and coagulation. Fibrinolysis
and control mechanisms of hemostasis will also be covered. In addition, brief descriptions of the clinical
and laboratory evaluation of patients and the diagnosis of bleeding disorders will be presented.
Keywords. Platelet function; coagulation; bleeding diatheses; coagulation testing; thrombocytopenia

INTRODUCTION
The hemostatic process is remarkable in its ability
to stop the flow of blood from a breach in any rea-
sonably sized vessel within minutes. Yet blood is
maintained in a fluid state within the vascular sys-
tem. This is accomplished by the coordinated efforts
of 3 distinct but intimately related mechanisms: the
vascular, the platelet, and the coagulation phases of
hemostasis (90). These same mechanisms, when
overactive or inappropriately activated, can result
in thrombosis, embolism, or disseminated intra-
vascular coagulation. In this article, I will provide
an overview of the hemostatic mechanism and an
approach to the diagnosis of hemorrhagic disorders.
Because of space limitations and the expertise of
other authors within this issue, less attention to
platelet function and fibrinolysis will be given here.
HEMOSTATIC MECHANISMS
Platelets
Platelets perform functions in all phases of he-
mostasis. Most apparently, they provide primary
hemostasis by producing the platelet plug that forms
almost immediately after a small vessel has been
disrupted (88, 90). As illustrated in Fig. 1, platelets
begin adhering to the subendothelium within sec-
onds after it has been exposed (84). After 30-60 sec
the first fibrin strands can be seen interspersed among
the platelets, and after several minutes the platelet
plug is completely formed and stabilized by fibrin
*
Address correspondence to: Dr. G. Daniel Boon, Department
of Pathology, College of Veterinary Medicine, University of Mis-
souri, Columbia, Missouri 65205.
170
(88). Within a few hours, the platelets lose their
integrity, and the plug appears as a mass of fibrin
strands (36, 84). This familiar sequence occurs many
times every day and involves all phases of hemo-
stasis, but platelets are central and all of their basic
functions are involved. The fibrin formation occurs
because the platelets, in addition to adhering to the
subendothelium (adhesion) (88) and each other (ag-
gregation) ( 11 ), provide a surface for the assembly
of coagulation factors that lead to the generation of
thrombin and ultimately fibrin (83, 100). Secretion
is of course involved in the recruitment of more
platelets into this activity, but it also supplies a large
number of other factors with a wide variety of ac-
tivities (11, 93). Thus, the 4 primary platelet func-
tions are adhesion, aggregation (cohesion), secre-
tion, and procoagulant activity.
Adhesion. When vessels are damaged, platelets
will adhere to different components of the suben-
dothelium, depending on the flow rate of the blood.
In low flow areas, platelets will adhere to and spread
upon collagen and fibronectin. In higher flow areas,
such as arterioles, platelet adhesion depends on the
presence ofvon Willebrand factor (vWF) in the sub-
endothelium (1, 2, 33, 73, 103). Von Willebrand
factor is a large multimeric protein (27) synthesized
by megakaryocytes (91) and endothelial cells (42).
Megakaryocytes, in humans, package vWF into the
alpha granules of platelets (14, 91 ), whereas endo-
thelial cells secrete it into both the plasma and the
subendothelium (94). In the dog, platelets contain
little vWF (72). In the subendothelium, vWF binds
to matrix and awaits vascular injury and the arrival
of platelets. In the plasma, vWF complexes with the
coagulation cofactor factor VIII, resulting in vWF

FIG. I. -Formation of the platelet plug in primary hemostasis.
serving as a carrier protein for factor VIII and pro-
longing its half-life (37, 67, 106).
Aggregation. After a layer of platelets has accu-
mulated on the injured subendothelium, additional
platelets participate in the formation of a complete
plug in a process known as platelet aggregation (90,
102). This occurs through active platelet metabo-
lism (11, 38) and stimulation by specific agonists
(11, 30, 102). Platelet aggregation can be studied in
vitro with instruments called aggregometers (30).
Aggregometers measure the transmission of light
through a suspension of stirred platelets. Aggrega-
tion is initiated by the addition of agonists and, as
clumps of platelets form, light passes more freely as
the turbidity of the suspension decreases (30, 102).
Aggregation can be divided into primary and sec-
ondary phases. Primary aggregation occurs as the
direct consequence of the agonist, is reversible, and
is not accompanied by secretion. Secondary aggre-
gation occurs with secretion and is manifested by
the formation of large irreversibly aggregated plate-
lets. At high agonist concentrations, primary and
secondary aggregation may occur simultaneously (30,
102).
Platelet agonists can be classified according to
whether or not they stimulate secretion indepen-
dently of secondary aggregation. Strong agonists can
and weak agonists cannot stimulate secretion in-
dependently of aggregation. That is, strong agonists
171
can stimulate platelets to secrete their granule con-
tents even in the presence of aspirin and other in-
hibitors of cyclooxygenase; weak agonists cannot.
Obviously there is another pathway to release be-
sides cyclooxygenase. Strong agonists include
thrombin and collagen. Weak agonists include ADP
and epinephrine (11). Miscellaneous agonists are
platelet-activating factor, serotonin, and vasopres-
sin.
The signal produced by the agonists and the re-
sponse of the platelets, especially aggregation and
secretion, are coupled by complex biochemical re-
actions that are not allcompletely clear.
Briefly, phospholipase C hydrolyzes membrane
phosphatidylinositol 4,5-biphosphate to diacylglyc-
erol (DAG) and inositol 1,4,5-triphosphate (IP3) (see
Fig. 2). Both of these are second messengers. DAG
activates protein kinase C, which is active in both
aggregation and secretion, although its mechanism
is not entirely clear. IP3 causes the release of calcium
from the dense tubular system, one of the intracel-
lular storage depots for calcium. The elevated cy-
toplasmic calcium, in turn, activates phospholipase
A2, which then hydrolyzes the membrane lipids and
releases arachidonic acid (50, 56).
Secretion. Secretion is mediated by many of the
same second messengers discussed under aggrega-
tion. The increased concentration of cytoplasmic
Ca++ is involved in the assembly of microtubules
 172

FIG. 2.-A schematic diagram of platelet metabolism. (See text for discussion.) lib and Illa membrane proteins that
=

form the fibrinogen receptor; Va activated coagulation factor V; VaR activated coagulation factor V receptor; A
= = =

agonist; Ca++ ionized calcium; DG diacylglyceride; G guanine nucleotide-binding protein; IP3 inositol 1,4,5-
= = = =

triphosphate ; PIP2 phosphatidylinositol 4,5-biphosphate; R receptor.

= =

that areinvolved in the central movement of the
granules (50). This brings them into close proximity
to the open canalicular system. There granule and
canalicular membranes fuse and granular contents
are emptied. The open canalicular system com-
municates to the exterior, allowing granular con-
tents access to the plasma.
There are 4 types of granules in platelets: alpha
granules, dense or 6 granules, lysosomes, and mi-
croperoxisomes (93). The alpha granules contain a
large number of constituents. A few examples are
platelet factor 4 (26, 87), thromboglobulin (87),
platelet-derived growth factor (44), and thrombo-
spondin (107, 108). Dense granules also contain a
variety of components including ADP, ATP, cal-
cium, pyrophosphate, and serotonin (32, 93). The
ADP and ATP are present in a ratio of 3:2 and
exchange very slowly if at all with the metabolic
pool of nucleotides. ADP is the agent responsible
for recruitment of additional platelets into the plug
formed during primary hemostasis (32, 93).
Procoagulant Activity. Platelets contain coagu-
lation factors including fibrinogen, factor V, and
factor VIII (45, 63, 71, 87, 98). In fact, 20% of whole
blood factor V is contained in platelets (98), sug-
gesting some importance. However, their most im-
portant contribution to coagulation is surfaces and
specific receptors upon which complexes of coagu-
lation factors form (89, 99, 100). These complexes
will be discussed later.
Coagulation Cascade
The coagulation cascade has classically been con-
sidered to be a series of sequential zymogen acti-
vation steps (Fig. 3). Each factor is the substrate for
the previous enzyme and the activator of the sub-
sequent proenzyme (15, 5 5). Although this concept
is basically correct, it has been enlarged a great deal.
Surprisingly, the mechanism of initiation of the co-
agulation sequence is still uncertain.
The coagulation cascade can be intimidating. It
is complex, and the roman numeral system of no-
menclature can be confusing. The numerals seem-
ingly were assigned at random. However, if the cas-
cade is subdivided into some functional areas, each
can be addressed with much less trepidation.
Contact System. The intrinsic system (so named
because all factors are found in blood) has been
thought to start with the contact activation system.
Factor XII is activated (factor XIIa) by contact with
negatively charged surfaces. This probably occurs
through autoactivation after contact with negatively
charged surfaces, collagen, or other contact activa-
tors (80). Factor XIIa activates factor XI to factor
XIa and prekallikrein to kallikrein. Kallikrein sub-
sequently activates more factor XII. All of these
reactions require the presence of high molecular
weight kininogen (13, 31, 54, 60, 66, 80, 85, 96).
The factor XII-prekallikrein amplification system
results in a great deal of factor XIIa to activate factor
XI and initiate the intrinsic system. However, the
clinical relevance of this sequence is in serious doubt.
Deficiency of any or all of the factors involved in
the contact system is not accompanied by clinical
bleeding (13, 23, 31, 34, 54). This has always cast
a shadow on the importance of contact activation
and suggested that there must be another pathway
for the activation of factor XI. Indeed, as will be

FIG. 3.-A schematic diagram of the coagulation cascade. (See text for a more complete discussion.) The dotted arrow
from unactivated factor VII to the conversion of X to Xa indicates a small degree of enzyme activity. This may represent
the initial activation of the coagulation cascade in vivo.
discussed, another pathway for the activation of fac-
tor XI has been recently described (24).
Intrinsic System. Factor Xla activates factor IX
to factor IXa in the presence of ionized calcium (21 ).
However, the most prominent reaction of the in-
trinsic system is the activation of factor X by factor
IXa (23, 37, 39-41, 61, 69, 82, 104) and its cofac-
tors. This reaction occurs on asurface that is prob-
ably provided by platelets (23, 41). The enzyme
(factor IXa) and the substrate (factor X) are brought
into close proximity on the surface by binding to
Ca++. Factor V also enters the reaction and accel-
erates it 500-fold (61). This is 1 of 3 complexes
formed by coagulation factors, and it illustrates sev-
eral important points. First, a cofactor is very im-
portant in allowing the reaction to take place at a
maximum rate. Loss or inactivation of the cofactor
is a very effective way of controlling the reaction
(see discussion on protein in the later section Con-
trol of Coagulation). Second, each of the complexes
formed involves at least 1 of the factors of the &dquo;pro-
thrombin complex.&dquo; The prothrombin complex is
a group of coagulation factors (factors II, VII, IX,
and X) that have been modified by a reaction in-
volving vitamin K so that they will bind calcium
(22, 68, 95). The binding of calcium is important
173
in the formation of the complexes (64). This par-
ticular complex of factors VIII, IX, and X, as well
as Ca++ and membrane (probably platelet mem-
brane), is sometimes called ten-ase because it cleaves
factor X (ten). These complexes are very important
to the understanding and control of coagulation.
Common Pathway. The common pathway con-
tains the second of the 3 complexes. Factor Xa forms
a complex with its substrate prothrombin (factor II),
its cofactor factor V, a membrane surface (platelet),
and Ca++ (99, 100) to form a complex sometimes
referred to as prothrombinase. The prothrombin is
cleaved and thrombin is released when the reaction
is complete. The primary action of thrombin is, of
course, to convert fibrinogen to fibrin. However,
thrombin has a wide range of other actions (40, 49,
52, 75), one of which is the activation of factor XI
(24). This casts a whole new light on the importance
of the extrinsic system for in vivo coagulation (see
later).
Extrinsic System. The extrinsic system consists
almost entirely of the third enzyme-coenzyme-sub-
strate complex. The extrinsic system is also where
one of the more important additions to knowledge
of the coagulation cascade has been made. Factor
VII forms a complex with tissue factor and Ca-+
174
that enzymatically activates factor X to factor Xa
by limited proteolysis (7, 9, 23, 25, 65, 69, 109).
Factor VII is the only coagulation enzyme that pos-
sesses activity without undergoing activation (7, 75,
76, 113). It does indeed do that. It also activates
factor IX (70, 112), providing further amplification
of factor Xa production and perhaps explaining why
hemophiliacs have clinical bleeding problems. If
factor IX activation were not initiated by the ex-
trinsic system and the extrinsic system was a pri-
mary initiator of in vivo coagulation, there would
be no reason for a hemophiliac to have bleeding
problems.
Coagulation Cascade Rearrangements. The 2
relatively recent discoveries of thrombin activation
of factor XI and factor Vlla activation of factor IX
have resulted in some new ways to think about the
coagulation cascade. In addition, some old enigmas
may have cleared up. It has long been a mystery
why individuals deficient in contact activation fac-
tors do not have a clinical bleeding problem. It may
be that the contact activation system is not impor-
tant in coagulation in vivo. The extrinsic system may
be the primary initiator of coagulation. This makes
sense in that injury would expose tissue factor, which
is an absolute requirement for the activation of fac-
tor X and factor IX by factor VII (66). It may also
be that the thrombin activation of factor XI is an-
other amplification system for the production of
thrombin. Certainly, the importance this places on
factors IX, VIII, X, and Ca++-phospholipid com-
plexes makes sense of the clinical picture presented
by hemophiliacs. The contact activation system
should not be dismissed too quickly, however. There
is still more to be learned. Passovoy factor is some-
how involved in the contact system, and patients
deficient in it do have a bleeding diathesis (34,
35, 58).
Control of Coagulation. With all the amplifica-
tion that takes place in the coagulation cascade, it
would seem that once coagulation was initiated it
would continue out of control. There are several
mechanisms to prevent this. The complexes that
occur in the coagulation cascade not only allow the
enzymes involved to act more efficiently, but they
also result in the localization of the coagulant ac-
tivity (23, 57). Some of this complexing takes place
on endothelial cell membranes but most probably
occurs on platelet membranes (23, 83, 99). Regard-
less, the receptors involved are exposed by injury
or activation and are localized to the area where
hemostasis is needed. All of these complexes in-
volve the vitamin K-dependent coagulation factors.
These are prothrombin and factors VII, IX, and X.
Vitamin K mediates a posttranslational modifica-
tion of these factors that involves the addition of
gamma-carboxyl groups to a series of glutamyl
groups. This gamma-glutamyl carboxylation allows
the factors involved to bind calcium and therefore
participate in complex formation (64, 68, 95).
Protein C and its cofactor protein S are also im-
portant controls of coagulation. Both are also vi-
tamin K-dependent. Protein C is activated by
thrombin in a reaction that requires the binding of
thrombin by a membrane protein, thrombomodu-
lin, of endothelial cells. Activated protein C enzy-
matically cleaves factor Va and factor Villa into
forms that will no longer support coagulation. This
is a very effective inhibition of the coagulation cas-
cade. Interestingly, while thrombin bound to throm-
bomodulin will activate protein C, it will no longer
activate platelets or factor V or convert fibrinogen
to fibrin. Thrombomodulin transforms it from a
coagulant to an anticoagulant intermediate. Protein
C also promotes fibrinolysis by protecting plasmin-
ogen activator from inhibition (12, 18, 19, 23, 47,
59, 105).
A variety of other control mechanisms also affect
the coagulation cascade. These include antithrom-
bin III (10, 101), heparin cofactor II (5, 6, 29, 97),
and (X2-macroglobulin (51).
Vascular Phase
The blood vessel has traditionally been thought
to be an inert conduit through which blood flows
but which did not participate in hemostasis. Grad-
ually, discussions of hemostasis have come to in-
clude comments on endothelial contributions. Many
of the interactions are detailed in the discussions of
the various phases of hemostasis. As can be seen,
endothelial cells participate extensively. Some he-
mostatic contributions are vasoconstriction (43, 53),
production of vWF (42, 103), and tissue factor pro-
duction (23). Antithrombotic contributions include
prostacyclin production (62), thrombomodulin pre-
sentation (23, 59), and tissue plasminogen activator
production (48).
Fibrinolysis
Once hemostasis is complete and healing has oc-
curred, clots must be disposed of and vascular lu-
mina maintained. This is accomplished by the fi-
brinolytic system. The operative enzyme of the
fibrinolytic system is plasmin. Plasmin is a rather
nonspecific protease and is generated by the action
of plasminogen activators on plasminogen. Plas-
minogen activators present in normal tissues are
tissue-type plasminogen activator and urokinase-
type plasminogen activator. Plasminogen is incor-
porated into fibrin clots. Plasminogen activator is
released from endothelium in response to, among
other things, high concentrations of thrombin. This

mechanism serves to localize the fibrinolytic action
of plasmin and results in the production of the fa-
miliar fibrin split products (8, 20, 48, 52, 77, 78,
86, 92, 110).
CLINICAL APPROACH TO THE PATIENT
Perhaps the most important rule to keep in mind
when evaluating the bleeding patient is that blood
can be lost from vessels for reasons other than hem-
orrhagic tendencies. Because the most common cause
of bleeding is trauma, every bleeding animal does
not require a hemostatic workup. When a bleeding
diathesis is suspected clinically, a great deal can be
learned by performing a thorough physical exami-
nation and obtaining a good history.
History
History can, in clinical situations, be an effective
way of diagnosing hemostatic disorders. It is less
important in the controlled conditions of drug safety
assessment, but it should be mentioned that con-
genital disorders are occasionally seen in laboratory
animals. In those situations, age of onset, pedigree,
and other pertinent clinical historical information
may be of benefit. As noted, because trauma is the
most likely cause of any bleeding episode, the ap-
propriateness of the degree of hemorrhage can be
very helpful in determining whether or not a bleed-
ing tendency is present.
Physical Examination
The type of bleeding can be a clue as to the un-
derlying disorder. Petechiae are usually associated
with thrombocytopenia but can also be seen with
vascular defects. Ecchymoses are larger than pete-
chiae and are often thought to be confluent pete-
chiae. This is usually true, but they can also be seen
in coagulation disorders. Prolonged bleeding from
minor trauma (venepuncture, scratches, etc.) is as-
sociated with defective primary hemostasis-
thrombocytopenia. Rebleeding after a phase of ap-
parently normal hemostasis is characteristic of co-
agulation factor deficiencies (4, 79).
Although information about types of bleeding is
the primary goal of physical examination, additional
important information should not be overlooked.
Underlying diseases may be detected that can con-
tribute to or result in hemorrhage. Such abnormal-
ities including splenomegaly, lymphadenopathy, and
jaundice may be associated with diseases that cause
bleeding diatheses.
LABORATORY APPROACH TO THE PATIENT
General Concepts of Abnormal Bleeding
Levels of Platelets. Thrombocytopenia is the most
common cause of abnormal bleeding in both hu-
175
mans and animals. The degree of bleeding is quite
variable and often does not correlate with the degree
of thrombocytopenia. The reasons are probably re-
lated to the underlying cause of the disorder. Con-
ditions involving peripheral destruction cause a
compensatory increase in marrow production of
platelets. This results in a population of relatively
young platelets that may function more efficiently
than older platelets and prevent bleeding at lower
concentrations. Conversely, in situations where
marrow production is impaired, the platelets re-
maining in the circulation are relatively old and may
not prevent bleeding at higher concentrations. A
good rule of thumb for the platelet concentration at
which to expect bleeding is the familiar 40,000-
50,000/ ~l, although this will vary depending on the
hemostatic challenges the animal encounters. Per-
haps better rules are (a) spontaneous bleeding is
probably below platelet concentrations of 20,000/
wl, (b) spontaneous bleeding is unlikely more than
platelet concentrations of 50,000/jnl, and (c) bleed-
ing after challenge (e.g., surgery) is probably at plate-
let concentrations of 20,000-100,000/~1 (4, 79).
Levels of Coagulation Factors. The association
between levels of coagulation factors and the clinical
tendency to bleed is better than that between plate-
lets and bleeding. There is, however, considerable
variation from factor to factor. In general, the con-
centration needed to prevent bleeding is no more
than 25% of the normal, and some factors can be
even lower in concentration and not result in a prob-
lem. This is in contrast to the 50% concentrations
needed to maintain most coagulation screening tests
within normal limits. This means that, in general,
if the concentrations of factors are high enough for
the screening tests of coagulation to be normal, they
are high enough that clinical bleeding should not be
a problem (17, 46, 74, 81, 111).
Principles of Laboratory Examination
Platelets. Because thrombocytopenia is the most
common cause of abnormal bleeding, they should
be routinely evaluated in any abnormally bleeding
animal. In clinical practice, this evaluation is quick-
ly and easily done by examining a blood smear. In
most safety assessment laboratories, platelet counts
are routinely performed. This makes examination
of smears unnecessary for diagnosis, but it is in-
valuable in distinguishing true thrombocytopenia
from artificial thrombocytopenia due to aggrega-
tion. A thorough discussion of thrombocytopenia
and thrombopathy cannot be accomplished in this
short article.
Factors. A large variety of tests of
Coagulation
the coagulation cascade are available. Because of
the complexity of the cascade and the number of
176
factorsinvolved, a thorough test of coagulation in-
volving each factor would necessarily be cumber-
some and impractical. In spite of the mentioned
discoveries that have changed our view of the ap-
parent importance of the intrinsic and extrinsic sys-
tems for in vivo hemostasis, the old familiar scheme
is still useful for sorting out abnormalities. For
screening purposes, 2 simple tests still serve quite
well. These tests are the activated partial throm-
boplastin time (APTT) and the prothrombin time
(PT), sometimes called the 1-stage PT. Using these
tests, coagulation abnormalities (if severe enough)
can be detected as well as localized to a relatively
small area of cascade. The APTT tests the intrinsic
system and the common pathway by providing con-
tact activation, Ca++, and phospholipids for the in-
trinsic system reactions. The PT tests the extrinsic
system and common pathway by providing tissue
factor, Ca++, and phospholipid. Note that the phos-
pholipid portion of the APTT and PT reagents re-
places the platelet contribution. Thrombocytopenia
will therefore have no effect on these tests. Three
abnormal patterns are possible. Normal APTT and
prolonged PT localize the problem to factor VII.
Normal PT and prolonged APTT localize the prob-
lem to the intrinsic system. If the patient has no
clinical bleeding problem with this pattern, the ab-
normality is most likely in the contact activation
system. If the patient exhibits bleeding problems,
the abnormality most likely involves factor VIII,
IX, or XI. Prolongations of both the APTT and the
PT indicate either an abnormality in the common
pathway or multiple abnormalities involving the in-
trinsic and extrinsic systems. Abnormalities of the
common pathway are exceedingly rare, and this sce-
nario is almost invariably due to multiple abnor-
malities. In clinical medicine warfarin intoxication
is a common culprit (3, 4, 16, 28, 79).
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