Thrombosis Research 92 (1998) 195–198
BRIEF COMMUNICATION
Plasma Glycocalicin in Platelet Concentrates:
Relationship to Other Parameters of the Storage Lesion
Masayuki Sano, Sybil Williams, Nikisha Smith, McDonald Horne and Harvey R. Gralnick
The Hematology Service, Clinical Pathology Department, Warren G.
Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland.
(Received 22 April 1998 by Editor D.A. Triplett; revised/accepted 13 July 1998)
Key Words: Glycocalicin; Platelets; Platelet Concentrates
P
latelets collected for transfusion develop a
“storage lesion” during their shelf life of 5
days [1]. This is evident in their decreased
in vitro aggregability and may affect the function
and/or the survival of the platelets after transfu-
sion. A feature of the storage lesion is the loss of
intact platelet membrane glycoprotein Ib (GPIb),
which is a critical protein for binding of von Wille-
brand factor (vWf). The reduction in GPIb integ-
rity results from proteolytic cleavage of glycocalicin
(GC), the carbohydrate-rich amino terminus of
GPIb. GC has been shown to increase in the plasma
of stored platelet concentrates (PCs) [2–4].
The processes underlying the proteolysis of
GPIb have remained unclear. The GC concentra-
tion in PCs has been associated with evidence of
platelet activation and with leakage of platelet cy-
tosol [3–5]. It has also been correlated with the rise
in lactate levels in the concentrates, but adding
exogenous lactate does not increase GC production
[6,7]. There is also evidence that GC is released
by the proteases derived from neutrophils [8], al-
though protease inhibitors added to the concen-
trates have been reported not to prevent the rise
in GC [4,9].
Abbreviations: GC, glycocalicin; GCI, glycocalicin index; PC,
platelet concentrates; PF4, platelet factor 4; LDH, lactate dehy-
drogenase; vWf, von Willebrand factor; GPIb, glycoprotein Ib.
Corresponding author: Harvey R. Gralnick, Room 2C/390, Build-
ing 10, NIH, Bethesda, MD, 20892. Tel: 11 (301) 496 6891; Fax:
11 (301) 402 2046.
0049-3848/98 $–see front matter Published by Elsevier Science Ltd. Printed in the USA.
PII S0049-3848(98)00128-5
Another question regarding GC is how consis-
tently it rises in PCs, as compared with other com-
monly measured parameters, such as lactate and
LDH. In an attempt to answer this question and
to clarify the events associated with the increases
of GC, we have measured GC as well as markers of
platelet activation (plasma platelet factor-4 [PF4]),
anaerobic metabolism (lactate, pH), and platelet
lysis (lactate dehydrogenase [LDH]) in the plasma
of platelet concentrates over a period of 6 days and
have analyzed the correlation of these parameters
with the changes in GC.
1. Materials and Methods
1.1. Preparation of Platelet Concentrates
PCs were collected from normal volunteer donors
by apheresis (Fenwal CS-3000 Plus; Baxter Health-
care Corporation, Deerfield, IL). They were stored
on a horizontal rotator (Model PR70; Hoefer Sci-
entific Instruments, San Francisco, CA) at 228C
in polyvinylch loride bags (Fenwal Transfer Pack
Container PL1813 or Platelet Storage Pack PL 732,
Baxter) with acid-citrate-dextrose, formula A (A
CD-A; Fenwal) following standard blood banking
procedures. Initial white cell and platelet counts
were performed with a Partical Data cell counter
(Elmhurst, IL) and varied from 15,000–40,000/mL
and 575,000–1,865,000/mL, respectively.
Each day for 6 days of storage a 3-mL sample
was removed aseptically from each PC. After cen-
196 M. Sano et al./Thrombosis Research 92 (1998) 195–198

,0.0001
,0.0001
,0.0001
,0.0001
,0.0001
0.04
pb

20.79
20.73
0.85
0.54
0.62
0.56
ra

0.9260.24

6.2363.10
67.7645.4
6.1160.11
22806800
69618
6

250061160
0.8660.23

6.4263.49

6.1160.11
61.09645.4
73621
Fig. 1. The changes in GCI over 6 days of storage for nine

5
platelet storage bags.

0.6860.13

5.5362.40
44.0639.4
6.1160.11
trifugation at 2000g for 30 minutes at room temper-

23906760
77623
ature and passage through 0.22 mM filters, the

4
plasma from each collection was frozen in aliquots
at 2708C until analyzed.

1.2. Glycocalicin ELISA

0.5560.18
20206440
4.9461.3
29.57630.
75620
Day

6.1860
3

GC was purified from human platelets following

the methods of Loscalzo and Handin [10]. An
ELISA for GC was then developed as previously
described [11]. GC concentration was expressed as
0.4660.14

4.1861.27
17.9612.7
6.3860.27
16006660

a glycocalicin index (GCI, mg/L), which is a unit

84631
2

adjusted for platelet count [12]: GCI5GC concen-

tration3250,000/mL4platelet count of the PC.
Table 1. Change in parameters over time (mean61 SD)

1.3. Additional Assays

6.7360.32
0.3060.15
12206590

14.467.0
75620

2.526.6
1

PF4 was measured by ELISA using Asserachrom

Coefficient for the correlation of the parameter with time.

PF4 Kits (Diagnostica Stago, Asnieres-Sur-Seine,

p value calculated by Fisher’s r to z transformation.

France). vWf activity (ristocetin co-factor activity)

and multimeric distribution were analyzed by pre-
0.1360.04

0.7060.13

7.1360.14
8906230

12.1465.7

viously published methods [13]. Lactate levels were

84627
0

measured with a Dupont ACA Star (Dupont, New-

ark, DE), and LDH with a Hitachi 917 System
(Boehringer Mannheim, Indianapolis, IN). The pH
of each sample was estimated using pHydrion paper
Lactate (mmol/L)

(Micro Essential Laboratory, Brooklyn, NY).

vWf Rcof (%)
GCI (mg/ml)
PF4 (IU/ml)

LDH (U/L)

1.4. Statistical Analyses

Correlation coefficients (r) were calculated using

pH

StatView (Abaacus Concepts, Inc., Berkley, CA).

a
 M. Sano et al./Thrombosis Research 92 (1998) 195–198 197

Table 2. Correlation of GCI with PF4, lac- 3. Discussion

tate, LDH and pH
GCI vs. r p The appearance of plasma glycocalicin in the plate-
PF4 0.43 0.0025 let packs was more consistent than changes in the
Lactate 0.53 0.0001 other parameters we measured. Furthermore, the
LDH 0.74 ,0.0001 GCI on day 1 of storage predicted the GCI on day 5
pH 20.63 ,0.0001 (r50.75, p50.035). Therefore, early measurements
of GCI might aid in estimating the severity of the
storage lesion in individual PCs and offer guidance
in identifying platelets that should be transfused
To determine whether r was significantly different relatively early in their storage period. This possi-
from zero, Fisher’s r to z transformations were bility, however, will require careful monitoring of
carried out on the correlations also using StatView. the clinical response to transfusions of platelet con-
centrates with known GCIs.
While all of the parameters we measured corre-
2. Results lated with the rise of GCI in the PCs (Table 2),
the correlation between GCI and LDH was the
Figure 1 shows the changes in GCI over the 6 greatest (r50.74, p,0.0001). Therefore removal of
days of storage for each of the nine PCs studied. glycocalicin from the platelets is associated with
Although all of the parameters changed signifi- leakage of cytosol. Whether this leakage is the
cantly with time the rise in GCI was the most con- source of proteases that cleave glycocalicin is un-
sistant (r50.85; Table 1). Changes in LDH and known. Sloand and Klein demonstrated that the
pH most strongly correlated with the rise in GCI neutrophil content of platelet packs lowers platelet
(r50.74 and 20.63 respectively; Table 2). Analysis responsiveness to ristocetin, which requires intact
of plasma vWf in the PCs revealed decreasing risto- GPIb [8]. Although this influence might be exerted
cetin cofactor activity over time (Table 1), while directly via neutrophil-derived proteases, others
the vWf multimeric pattern fluctuated minimally have reported that a variety of protease inhibitors
(Figure 2). do not block the loss of GPIb during storage [9].
Therefore, there is the possibility that neutrophils
promote platelet lysis, which in turn leads to GPIb
degradation by enzymes protected from inhibitors
in the milieu.
Of interest was the observation that vWf mul-
timers were preserved in the plasma of the PCs. In
fact in five of nine PCs there was a suggestion that
higher molecular weight multimers appeared late
in storage (Figure 2). Ristocetin cofactor activity
only fell slightly from dDay 0 to day 6 (z84% to
z69%; Table 1). In contrast, others have reported
evidence of vWf degradation in PCs during stor-
age [14].

References

1. Seghatchian J, Krailadsiri P. The platelet stor-

age lesion. Transfus Med Rev 1997;11:130–44.
Fig. 2. vWf multimeric pattern in the plasma from a single 2. Michelson AD, Adelman B, Barnard MR, Car-
storage bag, day 0–day 6. The normal plasma vWf pattern roll E, Handin RI. Platelet storage results in
is shown in the lanes labeled P. a redistribution of glycoprotein Ib molecules.
198 M. Sano et al./Thrombosis Research 92 (1998) 195–198
Evidence for the large intraplatelet pool of gly-
coprotein Ib. J Clin Invest 1988;81:1734–40.
3. Järemo P, Rubach-Dahlberg E, Solum NO.
Correlation of light transmission changes to
changes of platelet glycoprotein 1b during stor-
age of platelet concentrates. Thromb Res
1993;69:467–77.
4. Bode AP. Platelet activation may explain the
storage lesion in platelet concentrates. Blood
Cells 1990;16:109–26.
5. Rinder HM, Snyder EL. Activation of platelet
concentrate during preparation and storage.
Blood Cells 1992;18:445–56.
6. Bessos H, Murphy WG, Robertson A, Vickers
M, Seghatchian MJ, Tandy NP, Cutts M, Pam-
philon DH. Quality of platelet concentrates
irradiated with UVB light: Effect of UV dose
and dose rate on glycocalicin release and corre-
lation with other ma rkers of the platelet stor-
age lesion. Transfus Med 1993;3:115–21.
7. Bertonlini F, Porretti L, Lauri E, Rebulla P,
Sirchia G. Role of lactate in platelet storage
lesion. Vox Sang 1993;65:194–8.
8. Sloand EM, Klein HG. Effect of white cells
on platelets during storage. Transfusion 1990;
30:333–8.
9. Bode AP, Miller DT. The loss of GPIb from
stored platelets is caused by platelet activa-
tion, not plasma enzyme activity. Transfusion
1988;28(Suppl):39S.
10. Loscalzo J, Handin RI. Platelet glycocalicin.
In: Hawiger J, editor. Methods in Enzymology.
Vol. 215. San Diego: Academic Press; 1992.
p. 289–94.
11. Williams SB, Sano M, Smith N, Horne M, Yar-
choan R, Wyvill K, Zeichner S, Taylor P,
Knudson T, Gralnick HR. Glycocalicin levels
in the plasma of HIV1 patients: An indicator
of platelet turnover. J Lab Clin Med. In
Press, 1998.
12. Beer JH, Buchi L, Steiner B. Glycocalicin: A
new assay—the normal plasma levels and its
potential usefulness in selected diseases. Blood
1994;83:691–702.
13. Gralnick HR, Williams SB, McKeown LP, Kri-
sek DM, Shafer BC, Rick ME. Platelet von
Willebrand factor: Comparison with plasma
von Willebrand factor. Thromb Res 1985;
38:623–33.
14. Cesar JM, Garcia-Avello A, Monteagudo J,
Espinosa JI, Lodos JC, Castillo R, Navarro JL.
Von Willebrand factor availability in platelet
concentrates stored for 5 days. Am J Hematol
1994;45:109–11.