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chapter 36
Biochemistry of Hemostasis
plasm a sam ples from individuals w ith no know n bleed -
ing or throm botic tendencies. C onsequently, both a de-
creased concentration o f a factor (protein) and the pres-
en ce o f the factor in a functionally inactive form result
in functional deficiency. D eficien cies in w hich the co n -
centration o f the norm al factor is less than present in the
pool are now categorized as type I. D eficien cies in w hich
the normal concentration is present but som e m olecules
are inactive or less than fully active are categorized as
type II. Subcategories exist but are beyond the scope
o f this description. Frequencies o f each o f the coagu -
lation factor d eficien cies are given in Table 36-1. D efi-
cien cies in procoagulant subsystem com ponents that lead
to hem orrhage are relatively rare. D eficien cies o f anti-
coagulant subsystem com ponents are found w ith m uch
higher frequency in individuals presenting with venous
throm bosis. Prospective, epid em iological studies indicate,
however, that heterozygous individuals deficient in sin-
g le com ponents do not have a substantial additional risk
o f throm bosis. It is now generally believed that
throm-
bophilia
(a substantial risk o f throm bosis) is a m ultigenic
disorder.
Laboratory Assessment of Coagulation
System Functions
Sam ple collection and sam ple handling are particularly
important in coagulation factor testing. The venipuncture,
or capillary puncture for som e d evices, is a blood vessel
injury that elicits hem ostatic response. W hen a tourniquet
is em ployed to facilitate venipuncture, w hich is alm ost
universally the case, the tourniquet should be in place for
the shortest tim e p ossible. Plasm inogen activator (t-PA) is
released from the vessel in response to the com pression
and activates plasm inogen. Badly collected sam ples from
extensive tourniquet com pression can be rendered unclot-
table because o f the fibrinogenolysis that occurs from plas-
m in action. N orm ally, all abnormal coagulation system
functional tests should be reconfirm ed.
Prothrombin Time
The prothrombin tim e (PT), as proposed by Q uick, is the
m ost com m only perform ed coagulation function test. It
is used to m onitor oral anticoagulant therapy and also as
a preoperative screening test to warn o f possible b leed -
ing risk in patients w ith a personal or fam ily history o f
bleeding (Table 36-2). M easured clotting tim es are ex -
trem ely dependent on the anim al and tissue source and
the quality o f the throm boplastin used. Variability can
be expected because o f the assay’s dependence on the
num ber o f tissue factor m olecules and the quantity o f
m em brane surface provided by the throm boplastin. To
im prove the com parability o f prothrombin tim e m easure-
m ents, throm boplastins are standardized by com parison
with an international reference throm boplastin. Standard-
ized throm boplastins are described by a number, the In-
ternational Standardization Index (ISI), w hich relates the
throm boplastin to the international standard. A lthough this
has im proved com parisons o f oral anticoagulant therapy
m onitoring, it is still difficult to accurately com pare the re-
sults o f different laboratories and different thromboplastin
manufacturers.
Further im provem ent in m easurem ent o f oral antico-
agulation therapy has been achieved by relating the ratio
o f the prothrombin tim e o f the patient to the m ean value
for “norm al” individuals w ho are not on oral anticoagu-
lant therapy. This ratio, called the International N orm al-
ized Ratio (IN R ), better relates the anticoagulation assays
from different laboratories. M onitoring oral anticoagulant
therapy is critical to m aintaining a balance betw een the
risk o f throm bosis and the risk o f bleeding.
Throm bin Time
Purified thrombin is added to plasm a sam ples and the time
for clotting is m easured. T his test, the thrombin time (TT),
primarily reflects the concentration o f fibrinogen. H ow -
ever, it also reflects the ability o f the fibrinopeptides to
be cleaved and the polym erization o f fibrinogen. Sepa-
ration o f these three contributions requires a quantitative
m easurem ent o f the concentration o f fibrinogen that is not
related to tim e for clot form ation. The thrombin tim e can
be prolonged if heparin is present in the patient’s plasm a
sam ple because it w ill prom ote preferential inactivation
o f the added thrombin by antithrombin and reduce the
am ount o f thrombin that can act on fibrinogen.
Activated Partial Thromboplastin Time
Intrinsic pathway com ponents are m easured in the acti-
vated partial throm boplastin tim e (A PT T). The designa-
tion “activated” refers to the perform ance o f the test in two
stages. In the first stage, the citrate-anticoagulated plasm a
is preincubated with the surface activator (see “The C on-
tact Phase o f the
In Vitro
Intrinsic Pathway o f C oagula-
tion”) to form activated factor XI. In the second stage,
Ca2+ is added to initiate the activation o f factor IX and
the rem aining reactions o f the “intrinsic” pathway. The
clotting tim e is determ ined from the tim e o f addition o f
Ca2+. T he m ost com m on uses o f the A PT T are screening
for p ossib le factor VIII or factor IX d eficiencies and for
m onitoring heparin therapy. B ecause the rate o f activation
o f factor IX and the clotting tim e are very dependent on the
