Plasma Glycocalicin in Platelet Concentrates

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 transfusion. 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 Willebrand factor (vWf). The reduction in GPIb integrity 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 concentration in PCs has been associated with evidence of platelet activation and with leakage of platelet cytosol [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], although protease inhibitors added to the concentrates 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 dehydrogenase; vWf, von Willebrand factor; GPIb, glycoprotein Ib. Corresponding author: Harvey R. Gralnick, Room 2C/390, Building 10, NIH, Bethesda, MD, 20892. Tel: 11 (301) 496 6891; Fax: 11 (301) 402 2046.

Another question regarding GC is how consistently it rises in PCs, as compared with other commonly 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 Healthcare Corporation, Deerfield, IL). They were stored on a horizontal rotator (Model PR70; Hoefer Scientific 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-

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0.04 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 20.73 0.85 0.54 0.62 0.56 20.79

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73621 0.8660.23 250061160 6.4263.49 61.09645.4 6.1160.11 77623 0.6860.13 23906760 5.5362.40 44.0639.4 6.1160.11 75620 0.5560.18 20206440 4.9461.3 29.57630. 6.1860 84631 0.4660.14 16006660 4.1861.27 17.9612.7 6.3860.27 75620 0.3060.15 12206590 2.526.6 14.467.0 6.7360.32 84627 0.1360.04 8906230 0.7060.13 12.1465.7 7.1360.14

Coefficient for the correlation of the parameter with time. b p value calculated by Fisher’s r to z transformation.

Correlation coefficients (r) were calculated using StatView (Abaacus Concepts, Inc., Berkley, CA).

a

1.4. Statistical Analyses

1

PF4 was measured by ELISA using Asserachrom PF4 Kits (Diagnostica Stago, Asnieres-Sur-Seine, France). vWf activity (ristocetin co-factor activity) and multimeric distribution were analyzed by previously published methods [13]. Lactate levels were measured with a Dupont ACA Star (Dupont, Newark, DE), and LDH with a Hitachi 917 System (Boehringer Mannheim, Indianapolis, IN). The pH of each sample was estimated using pHydrion paper (Micro Essential Laboratory, Brooklyn, NY).

0

1.3. Additional Assays

Table 1. Change in parameters over time (mean61 SD)

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 a glycocalicin index (GCI, mg/L), which is a unit adjusted for platelet count [12]: GCI5GC concentration3250,000/mL4platelet count of the PC.

Day

1.2. Glycocalicin ELISA

3

4

trifugation at 2000g for 30 minutes at room temperature and passage through 0.22 mM filters, the plasma from each collection was frozen in aliquots at 2708C until analyzed.

vWf Rcof (%) GCI (mg/ml) PF4 (IU/ml) Lactate (mmol/L) LDH (U/L) pH

5

Fig. 1. The changes in GCI over 6 days of storage for nine platelet storage bags.

69618 0.9260.24 22806800 6.2363.10 67.7645.4 6.1160.11

pb ra

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

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

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Table 2. Correlation of GCI with PF4, lactate, LDH and pH

3. Discussion

GCI vs.

r

p

PF4 Lactate LDH pH

0.43 0.53 0.74 20.63

0.0025 0.0001 ,0.0001 ,0.0001

The appearance of plasma glycocalicin in the platelet packs was more consistent than changes in the other parameters we measured. Furthermore, the GCI on day 1 of storage predicted the GCI on day 5 (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 relatively early in their storage period. This possibility, however, will require careful monitoring of the clinical response to transfusions of platelet concentrates with known GCIs. While all of the parameters we measured correlated with the rise of GCI in the PCs (Table 2), the correlation between GCI and LDH was the greatest (r50.74, p,0.0001). Therefore removal of glycocalicin from the platelets is associated with leakage of cytosol. Whether this leakage is the source of proteases that cleave glycocalicin is unknown. Sloand and Klein demonstrated that the neutrophil content of platelet packs lowers platelet responsiveness to ristocetin, which requires intact GPIb [8]. Although this influence might be exerted directly via neutrophil-derived proteases, others have reported that a variety of protease inhibitors 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 multimers 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 storage [14].

To determine whether r was significantly different from zero, Fisher’s r to z transformations were carried out on the correlations also using StatView.

2. Results Figure 1 shows the changes in GCI over the 6 days of storage for each of the nine PCs studied. Although all of the parameters changed significantly with time the rise in GCI was the most consistant (r50.85; Table 1). Changes in LDH and pH most strongly correlated with the rise in GCI (r50.74 and 20.63 respectively; Table 2). Analysis of plasma vWf in the PCs revealed decreasing ristocetin cofactor activity over time (Table 1), while the vWf multimeric pattern fluctuated minimally (Figure 2).

References

Fig. 2. vWf multimeric pattern in the plasma from a single storage bag, day 0–day 6. The normal plasma vWf pattern is shown in the lanes labeled P.

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