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chapter 36
Biochemistry of Hemostasis
Specific Factor Assays
S pecific factor assays are variations on the A PTT or PT
tests. In the A PTT and PT, dilutions o f the patient’s plasm a
are m ade into a deficient, or depleted, “substrate” plasm a.
The assays are then perform ed in the usual way. The clot-
ting tim es are com pared w ith those obtained from dilutions
o f pooled normal plasm a, com m only 1:10, 1:20, 1:50,
and 1:100. A graph o f the logarithm o f the clotting tim e
(y axis) versus the logarithm o f the concentration as per-
centage o f normal
(x
axis) is used to determ ine the am ount
o f the factor activity in the patient’s plasm a. The normal
pooled plasm a is conventionally assigned a value o f
1 0 0
%
activity. M any variations exist for specific factor assays,
e.g., the venom o f
V ipera r u s s e llii
and phospholipids may
be substituted for throm boplastin in a PT-like assay. An
en zym e in R u ssell’s viper venom rapidly and relatively
specifically activates factor X . In conjunction with a fac-
tor X -deficient “substrate” plasm a, this provides a specific
factor X assay.
Im m unoglobulin inhibitors to factor VIII and factor IX
are found in patients with hem ophilia A and B, respec-
tively as a consequence o f their treatment with factor VIII
and factor IX preparations to arrest bleeding. E vidence
for inhibitors can be seen from A PTT assays using spe-
cific factor-deficient substrate plasm as. This evidence is
obtained by m aking dilutions o f the patient’s plasm a and
observing that higher dilutions give higher levels o f ac-
tivity for the factor in the patient’s plasm a. This reflects
the sim ultaneous dilution o f the antibody inhibitor and the
decreased inhibition at the higher dilution.
Assay of Heparin
Heparin therapy m ay be m onitored by its increases in clot-
ting tim e in the APTT, although this m ethod for measuring
heparin is difficult to standardize. Heparin is m ore sp ecif-
ically assayed by its effect on factor X a inactivation by
antithrombin. Such factor X a-based heparin assays usu-
ally em ploy purified factor X a as a reagent and factor X -
deficient substrate plasm a as the source o f anti thrombin.
The prolongation o f the clotting tim e that results from the
heparin in the patient’s plasm a is com pared with pooled
norm al plasm a that is know n to be free o f heparin. M any
variations o f this heparin assay are available. Heparin as-
says can use thrombin rather than factor X a, however, the
low -m olecular-w eight heparins are not reliably m easured
in throm bin-based assays.
factor XIa, the APTT is sensitive to deficiencies in factor
XII, prekallikrein, high-molecular-weight kininogen, and
factor XI.
36.12 Case Studies
Fibrinogen
Testing on a plasm a sam ple from a patient gave a PT
o f 12.9 s (norm al 1 0 .5 -1 2 .2 s) and an A PTT o f 38 s (nor-
m al 2 0 -3 2 s). A TT w as determ ined and the clotting tim e
w as 15 s (normal 1 0 -1 2 s). The patient’s plasm a sam ple
w as incubated w ith an en zym e that digests heparin. A f-
ter this treatment, the TT w as 14.7 s. The concentration
o f fibrinogen w as determ ined by a m ethod that w eighs
the fibrin after clotting. The fibrinogen w as 340 m g/dL, a
value higher than the normal range. W hat m ight explain
these results?
•
F ib rin o g e n p r o te o ly s is o r fib r in m o n o m e r
p o ly m e r iz a tio n m a y b e im p a ir e d a s th e re su lt o f a
m u ta tio n th a t a lte r s th e fib r in o g e n m o le c u le .
Factor XIII
A 60-year-old w om en is referred to you because o f
bleeding that has been a problem since infancy. Childbirth
(m ultiple) and surgery have required treatment with cryo-
precipitate and other plasm a protein fractions that contain
fibrinogen, vonW illebrand factor, factor VIII, factor XIII,
and many other plasm a proteins. A ll ordinary coagulation
functions tests w ere determ ined by you and found to be
normal. Her platelet count w as m easured and found to
be at the high end o f the normal range. The addition o f
plasm in to the plasm a clot resulted in alm ost im m ediate
dissolution (lysis) in contrast to ~
1 0
m inutes for a control
clot form ed from pooled normal plasm a. Plasm in w as not
detectable in a freshly collected plasm a sam ple from the
patient. A fter addition o f Ca2+ and incubation for 1 h, the
patient’s plasm a clot w as m ixed w ith an equal volum e o f
8
M urea and the clot dissolved. A plasm a clot prepared
in the sam e m anner from normal plasm a did not d issolve
upon addition o f
8
M urea. W hat is the likely cause o f the
patient’s bleeding disorder and her laboratory findings?
•
F a c to r X III d efic ie n c y . T h e re is n o e x p e c te d e ffe c t o n
a n y o f th e c lo ttin g tim e m e a su re m e n ts. T he
s u s c e p tib ility to p la s m in s u g g e s ts a n u n s ta b iliz e d clot.
T h e s o lu b ility in u re a is d ia g n o s tic f o r u n -c r o ss-lin k e d
fib rin .
Activated Protein C Resistance
A
30-year-old man with recurrent throm bosis is referred
to you for diagnosis. The PT and A PT T results determined
on his freshly drawn plasm a sam ple are
1 0
.
1
s (normal
1 0
-
12 s) and 18 s (norm al 2 0 -3 2 s). W hich com ponents o f
the hem ostatic system w ould you initially suspect as being
