10l
ally, the evaluation should address the key steps by
which platelets play an integral role in hemostasis after
vascular injury. Platelets attach to the exposed collagenous subendothelial matrix at the site of injury, un-
io2
dergo activation and shape change, and release potent
biochemical stimuli from the internal granules. Platelet
aggregation occurs through the binding of fibrinogen to
membrane glycoprotein
lib-Ifla
receptors. The biomol-
io
0
20
40
60
TIme (h)
80
100
Fig. 2. Leuclnetracer/traceeratiosfor apo B-containinglipoproteins
prepared at various times after an intravenousbolus injectionof
[H3JIeucine,6 mg/kg body wt.
variables with those derived from our previous VLDL double-turnover studies showedgoodagreement.
References
1. Demant T, Bedford D, Packard QJ, Shepherd J. The influence of
the apolipoprotein E polymorphism on apolipoprotein B-100 metabolism in normolipidemic subjects. J Clin Invest 1991;88:1490-501.
2. Demant T, Gaw A, Watts G, Bucidey B, Durrington P, Packard
CJ, Shepherd J. The metabolism of apolipoprotein B containing
lipoproteins in flmi1iR1 hyperchylomicronemia.
J Lipid Res 1993;
34:147-56.
3. Cryer DR., Matsushima T, Marsh JB, Yudkofl’ M, Coates PM,
Cortner PA. Direct measurement of apolipoprotein B synthesis in
human very lowdensity lipoprotein usingstableisotopesand mass
spectrometry. J Lipid Res 1986;27:508-15.
4. Parhofer KG, Barrett PHIl., Bier DM, Schonfeld G. Determination
of kinetic parameters of apolipoprotein B metabolism using amino
acids labeled with stable isotopes.J Lipid lIes 1991;32:1311-23.
5. Egusa G, Brady DW, Grundy SM, Howard By. Isopropanol
precipitation method for the determination of apolipoprotein B
specific activity and plasma concentrations
during metabolic studies of very low density lipoprotein apolipoprotein B. J Lipid Res
1983;24:1261-7.
6. Lindgren Fr, Jensen LC, Wills lID, Freeman NK Flotation
rates, molecular weights and hydrateddensitiesof the low density
lipoproteins. Lipids 1969;4:337-44.
7. CalderAG, Smith A. Stable isotope ratio analysis of leucineand
ketoisocaproic acid in bloodplasma by gas chromatography/mass
spectrometry. Use of tertiary butyldimethylsilyl derivatives.
Rapid Commun Mass Spectrom 1988;2:14-6.
& Cobelli C, ToffoloG, Bier DM, Nosadini R. Models to interpret
kinetic data in stable isotope tracer stuciies. Am J Physiol 1987;
253:E551-64.
9. Berman M, Belts WF, Greif PC, Chabay R Boston RC. CONSAM
user’s guide. Bethesda,MD: National Institutes of Health, Laboratory of Mathematical Biology, 1983:1.1-15.1.
Evaluation of Platelet Function by PFA-100,
Kundu,1
Reynaldo
(Baxter
33174)
Diagnostics,
Sourav
Mitu, and Roy Ostgaard
10555 W. Flagler St., Miami, FL
Sio, Angela
Evaluation of platelet function provides critical information to the clinician in charge of the hemostatic management of a patient with potential bleeding risk. Ide1
Corresponding author: fax 305-222-6485
ecules released help recruit more platelets from the systemic circulation to the site of injury. The platelet membrane surface also participates
in the activation of
several coagulation factors and modulates the complex
process of thrombin generation and fibrin formation.
Platelet aggregates, in combination with the fibrin
meshwork and other blood cells, form a clot that prevents blood loss through the site of injury and participates in healing of the wound. Impairment of any of the
functions in this intricately linked multistep biochemical process could result in clinically significant bleeding.
The goal of the present work was to develop a quantitative, simple, rapid, in vitro method for evaluation of
platelet function as an alternative to the current clinically accepted techniques
(e.g., in vivo bleeding-time
measurement, optical and impedance aggregometry, and
platelet count), which are either imprecise, impractical in
the clinical setting, or provide insufficient
information.
The PFA-100Th system2 is based on the principle originally described by Kratzer et al. (1, 2). In this system,
a motor-driven syringe aspirates anticoagulated
blood
samples under steady-flow conditions through a microscopic aperture
[150 jm (i.d.)1 cut into a membrane
placed in the test cartridge. The membrane is coated
with either fibrillar collagen type-I (2 g) and epinephrine (10 pg), or collagen and ADP (50 pg). Platelets
undergo adhesion, activation, release, and aggregation
reactions in response to the shear stresses and the agonists on the membrane. Platelets attach to the area
surrounding the aperture, eventually forming a platelet
plug that occludes the aperture. The movement of the
syringe is controlled by a microprocessor via a feedback
loop that maintains a constant pressure 4 kPa below
atmospheric pressure in the system. The volume (and
thus the flow rate) of blood passing through the aperture
is constantly monitored. The time for closure of the
aperture due to formation of the platelet plug is indicative of the platelet function in the blood sample.
We performed a series of in vitro studies to characterize the sensitivity of the PFA-100 system for various
types of impairment of primary
hemostasis (Table 1).
The normal reference ranges for the collagen-epinephrine and collagen-ADP test cartridges were determined
by evaluating several individuals who had been prescreened
for platelet abnormalities
by currently accepted techniques. To determine the effect of impairment of platelet adhesion in a platelet receptor GPIbvon Wifiebrand
factor (vWf) system, we obtained blood
samples from normal volunteers not taking any medications and incubated these with a monoclonal antibody
2Currently under development.
CLINICALCHEMISTRY, Vol. 40, No. 9, 1994
1827
to platelet receptor GPIb (10 mgIL blood) for 8 mm with
periodic mixing; we then evaluated the platelet function
in the resulting mixtures with both collagen-ADP and
collagen-epinephrine test cartridges. Severe impair-
ment of platelet plug formation was noted for all subjects. Additionally, to determine the effect of vWf impairment, we incubated blood samples with various
amounts of a monoclonal antibody to vWf. A dose-dependent prolongation in closure time was observed by
the PFA-100 (Table 1). Similarly, when blood samples
were incubated with monoclonal antibodies (10 mg/L of
blood) to platelet receptor GPIIb-llIa,
complete absence
of platelet plug formation was noted. These experiments
indicate that the PFA-100 test system can be used to
detect abnormalities of both adhesion and aggregation
mechanisms.
The establishment of clinical correlation between PFA100 results and platelet dysfunction is currently underway. Table 1 provides the results of some of the preliininary investigations conducted on patients with clinically
diagnosed disorders of prmiaiy hemostasis. In all pathological cases,the PFA-100 closure times remained outside
the normal reference range (mean ± 2SD). For the aspirin
tolerance study, seven normal individuals were given 325
mg of acetylsalicylic
acid orally. PFA-100 tests were performed with collagen-epinephrine
test cartridges before
intake of aspirin (baseline), and then at 4 h, and 1, 3, and
7 days after aspirin ingestion. Immediately after aspirin
intake all individuals demonstrated no platelet plug for-
Table 1. Summary of studies performed with PFA-100
prototype test system.
Mean closure time, $
______________
Collagen.plnephrlne
test cartridge
Normal referencerangea
In vitro Incubation of bloodsamples
with monodortal antibody to
PlateletreceptorGPIb
PlateletreceptorGPIIb-llIa
vWf, mg/L blood
0.9
0.45
0.23
97-188
(28)b
CollagenADP test
cartridge
63-123 (35)
(5)
(5)
>300(2)
>300 (1)
262
day
+3 days
+7 days
+1
von Willebrand disease
Mild
Severe
Storage pool disease
Bemard-Souliersyndrome
>
>
monoclonal antibodies as a generous gift.
References
1. Kratzer MAA, Born GVR. Simulation of primary haemostasis
in vitro. Haemostasis 1985;15:357-62.
2. Kratzer MAA, Bellucci S, Caen ,JP. Detection of abnormal
platelet functions with an in vitro model of primary haemostasis.
Haemostasis 1985;15:363-70.
Enhancement of Peroxidase-Catalyzed
Chemlluminescent Oxidation of Cyclic Diacyl
Hydrazides by Arylboronic AcIds, Larry J. Kricka1”
and Xiaoying Jj1 ( Dept. of Pathol. and Lab. Med.,
Univ. of Pennsylvania. 3400 Spruce St., Philadelphia,
PA
19104, and 2 Hosp. of the Univ. of Pennsylvania,
Philadelphia, PA 19104; author for correspondence, fax
215-662-7529)
The chemiluminescent
horseradish
peroxidase-catalyzed oxidation of luminol is enhanced by certain phenols
(e.g.,4-iodophenol),naphtho]s
(e.g., 6-bromo-2-naphthol),
benzothiazoles (e.g., firefly luciferin), and amines (e.g.,
4-anisidine) (1-5). A range of boromc acid derivatives
are
also effective as enhancers,
and we have determined the
properties and enhancement characteristics of several
members of this new class of enhancer (6).
We screened a series of mono-, di-, and polysubstituted
boronic acids and found several compounds that are effective as enhancers, including 4-chloro-, 4-bromo-, 4-iodo-,
174(7)
mmol/L) (7) and hydrogen peroxide (2.73 zmol/L) was
prepared and protected from light. To test the effect of the
boronate on the background light emission, we mixed the
237 (7)
182 (7)
luminol-hydrogen
(7)
(7)
200 (2)
>300 (7)
252 (1)
>300 (1)
CUNICAL CHEMISTRY, Vol. 40, No. 9, 1994
peroxide reagent (100 L) and either
of boronate, or as a control, 10 iL of Tris buffer (0.1
mol/L, pH 8.6), in a cuvette. The light emission was recorded for 60 mm with a Berthold (Nashua, NH) Biolumat,
LB9500C luminometer or an Amerlite microplate reader
(Amersham Diagnostics, Amersham, UK). The effect of
boronate on the signal in the presence of horseradish peroxidase (50 fmol) was determined by mixing the luminolperoxide reagent (100 pL), peroxidase [10 L of a 5000fold dilution of Sigma (St. Louis, MO) Type VIA, 1 mg/mL
10 L
158 (1)
Mean ± 2SD, establishedwith volunteers with normai platelet count and
plateletfuricdon.
b No. of subjects is listed in parentheses.
1828
We thank Rasheed Aishameeri and Eberhard F. Mainmen of
Wayne State University School of Medicine, Detroit, MI, for conducting some of the patient studies. We are also indebted to Barry
S.Coller of Mt. Sinai Hospital, New York, NY, for providing the
4-phenyl-, 4-chloro-3-nitrophenyl-,
and 3-amino-2,4,6,trichlorophenylboromc acid (Fig. 1). The different boronates were screened as follows. A Tris-buffered
reagent
(0.1 mol/L, pH 8.6) containing
sodium luminol (1.25
>
Patients’studies
Volunteerstakingaspirin
Baseline
+4 h
mation. Afterwards, however, a time-dependent response
was observed. The interindividual
differences observed
suggest substantial variability
in the rate of platelet synthesis.
The studies conducted so far indicate potential utility
of PFA-100 system as a screening tool for platelet dysfunction. Its simplicity, precision, and cost-effectiveness
in comparison with the current technology should make
it attractive
to the clinical community.