Platelets ( 1997) 8, 159- 162

Soluble P-selectin assay: importance of correct anticoagulant choice

G. Kirk, M. McLaren, J. F. Belch

P-selectin is stored preformed in the a-granules of platelets. Previous studies show P-thromboglobulin, also stored in a-granules can be readily released from platelets during the processing of whole blood. This artefactual release was rectified by using combination of various anti-platelet and anticoagulant compounds placed in the collecting tube. We investigated the levels of sP-selectin from 40 volunteers, comparing two anticoagulants, tri-sodium citrate, CTAD (a mixture of sodium citrate and citric acid, theophylline, adenosine and dipyridamole) plus iloprost and serum. Iloprost, a stable prostacyclin analogue, is a potent anti-platelet agent. We found significantly lower levels of sP-selectin (P < 0.0001, paired t-test) measured from blood collected into CTAD and iloprost compared to levels measured from either citrated plasma or serum. We suggest that plasma levels obtained from the blood collected into a CTAD tube containing iloprost are likely to more accurately reflect the true levels of circulating sP-selectin than those obtained when test-tube activation of platelets is allowed to continue in vitro.

Introduction

At sites of vascular injury and thrombosis, platelet, endothelial cell and leucocyte interactions occur. These interactions depend on the participation of the cell adhesion molecules (CAMS) which are present on all three cell types.' Platelets and leucocytes must adhere to the endothelium to initiate their roles in haemostasis and in the inflammatory and immune response.

P-selectin is a member of the selectin family of cell adhesion molecules. This selectin is a transmembrane glycoprotein which is expressed on platelets, mega- karyocytes and endothelial cells. P-selectin is stored preformed in a-granules (in platelets and megakar- yocytes) and in Weibel Palade bodies (in endothelial cells).* The CAM can be rapidly mobilised to the cell surface following stimulation by various mediators, for example, thrombin, histamine and hydrogen p e r ~ x i d e . ~ P-selectin has been found to exist in a soluble form in normal individuals4 and in individuals with thrombotic diseases.' This soluble form of P-selectin (sP-selectin)

Correspondence to: Gwen Kirk, Department of Medicine, Ninewells Hospital & Medical School, Dundee DDI 9SY. UK, e-mail: g.kirk@dundee.ac.uk

can be measured using an enzyme linked immunosorbent assay (ELISA).

Previous studies have shown that another platelet a-granule product, P-thromboglobulin, can be readily released from platelets during collection and centrifuga- tion of whole blood leading to artefactually high levels; this problem being mainly rectified by the use of combinations of various anti-platelet and anticoagulant compounds placed in the collecting tube.6 If P-selectin was also released during the above procedures this would be of considerable importance and impair accuracy of results and subsequent conclusions drawn from these results regarding the levels of sP-selectin in health and disease. The aim of this study was to find the anticoagu- lantlanti-platelet chemical combination which when placed in the collecting tube allowed minimal in vitro activation of the platelets, and thus, reflect the true level of circulating P-selectin.

Materials and methods

After ethical permission had been obtained, 40 volun- teers gave their informed consent for this study. Twenty healthy normal individuals and 20 patients with insulin dependent diabetes mellitus (IDDM) each had 15 ml of

0953-7 I04/97/020l59-04 0 I997 Journals Oxford Ltd

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I60 SOLUBLE P-SELECTIN ASSAY

c E . p 200- I

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blood removed. Ten of the patients with IDDM were on a daily dose 75 mg of aspirin, the other 30 volunteers had not taken aspirin or aspirin containing compounds for at least 14 days prior to blood sampling. The blood samples were drawn from the antecubital fossa using a 19 gauge needle. Light tourniquet pressure was applied if required to assist venepuncture. The pressure was released for at least 10s prior to the blood being drawn.

Aliquots of 4.5ml of blood were added to a tube containing 0.5 ml 0. I % tri-sodium citrate dihydrate and another 4.5 ml of blood were added to a tube containing CTAD (a mixture of sodium citrate and citric acid, theophylline, adenosine and dipyridamole), this tube is a commercially available tube called Diatube H (Diagno- stica Stago, France). The CTAD tube had a further 20 pl of iloprost ( I mg/ml) (Schering AG, Germany) added to it. lloprost, a synthetic prostacyclin analogue, is a potent antiplatelet agent.' The remaining 5 ml of blood were added to a tube containing clotting beads for serum preparation. Within 10 min of sampling (samples kept on ice until this time) the CTAD and citrate tubes were centrifuged at 3500 rpm for I5 min at 4°C. The plasma was then removed and aliquoted. The blood in the serum tube was left to clot for 1 h in a water bath at 37"C, after this period the serum samples were also centrifuged as above. The serum was then removed and aliquoted. All samples were stored at -70°C until sP-selectin levels were measured using an ELlSA (Takara Biomedicals, Japan).

The data were analysed by two parametric tests, the data being normally distributed. A paired t-test was performed to compare the anticoagulants. For each individual anticoagulant an unpaired t-test was per- formed to compare the diabetic patients with the normal control subjects. This test was also used to compared sP- selectin levels from patients on aspirin with levels of sP- selectin from those not on aspirin.

Results

Demographic details of the study populations are shown in Table I . From Figure I , i t can be seen that the CTAD plus iloprost samples had a significantly lower sP- selectin level than the samples taken into tri-sodium citrate for both the control and diabetic patient groups. The increase in the citrate sample levels was seen for all paired results. Using paired t-test this was highly statistically significant P < 0.0001, for both patients and

Table 1. Demographic details

Normal controls Diabetic patients

Number of subjects 20 20 Sex, male:female 14:6 16:4 Age 46 years 54 years (median, range) (26-67 years) (29-74 years) Aspirin, yes:no 0:20 1O:lO

Control Diabetic patients 300r *Lf < 0.0001~ y P < 0.00011

** ** ** 2 5 4

CTAD Citrate CTAD Citrate + iloprost + iloprost

Figure 1. Paired sP-selectin results from 40 volunteers ( n = 20 healthy volunteers and n = 20 patients with diabetics) showing differences in two anticoagulants * anti-platelet agents.

controls. CTAD plus iloprost gave a mean value of 98.9 ng/ml (range 32.3-180.8 ng/ml) for the control subjects and a mean of 139.8ng/ml (range 62.9-201.9 ng/ml) for the patient group. The citrate samples gave mean levels for control and patient groups of I 19.2 ng/ml range (49.8-206.2 ng/ml) and 162.6 ng/ ml (range 69.3-266.3 ng/ml) respectively (Table 2).

Serum levels were significantly higher (paired t-test, P < 0.0002) in both the normal controls (n = 10, mean level 616.2 ng/ml, range 427.8-93 I .8 ng/ml) and in the diabetics ( n = 20, mean level of sP-selectin 438.9 ng/ml,

Controls Diabetic patients

*** T

" CTAD Citrate Serum CTAD Citrate Serum

+ iloprost + iloprost

Unpaired t-test * P = 0.006; ** P = 0.008; ** P = 0.0075

Figure 2. Comparing sP-selectin levels from 40 volunteers (n = 20 healthy controls and n = 20 patients with diabetes) showing differences in three anticoagulants.

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PLATELETS 16 1

Table 2. sP-selectin levels

CTAD + iloprost Citrate mean (range) mean (range)

Serum mean (range)

Total volunteers (n = 40) Healthy normals ( n = 20) Diabetic patients (n = 20) 139.8 (62.9-201.9) ng/ml 162.6 (69.3-266.3) nglml 438.9 (186.1-684.6) nglml No aspirin (n = 10) 141.8 (62.9-201.9) nglml 168.1 (69.3-250.5) nglml 452.4 (186.1-684.6) nglml Aspirin (n = 10) 137.9 (82.5-201.9) nglml 160.3 (91.2-266.3) nglml 422.0 (200.9-661.7) ng/ml

119.4 (32.3-201.9) nglml 98.9 (32.3-180.8) nglml

140.9 (49.8-266.3) nglml 119.2 (49.8-206.2) nglml

498.0 (186.1-931.8) nglml ( n = 30) 616.2 (427.8-931.8) nglml (n = 10)

range 186.1-684.6 ng/ml) compared to either CTAD plus iloprost or citrate.

An unpaired ?-test was used to compare patients and healthy controls. This gave P values of P = 0.006 when comparing CTAD plus iloprost sP-selectin levels, P = 0.008 for citrate and P = 0.0075 when comparing serum sP-selectin levels between the two subject groups (Figure 2). The sP-selectin levels from healthy controls were significantly lower in CTAD plus iloprost and citrate, and were significantly higher in serum, when compared with the diabetic patient group.

When sP-selectin levels from 10 patients with diabetes, who took a daily low dosage of aspirin, were compared to 10 patients with diabetes who had not taken aspirin for at least 14 days, sP-selectin levels tended to be lower in the group of patients who were on aspirin on a daily basis for both anticoagulants and serum, although the results did not reach statistical significance (Figure 3).

Discussion

As a member of the selectin family of adhesion molecules, P-selectin is involved in the initial capture and rolling of the leukocyte along the vessel wall. P-selectin is also involved in the adhesion of platelets to the endothelium. The platelets can bind either directly, by the P-selectin ligands on the platelets binding to the

Citrate Serum

T

loo[ n - Aspirin No Aspirin No Aspirin No

Figure 3. Comparing the effects of aspirin on sP-selectin levels in patients with IDDM.

aspirin aspirin aspirin

P-selectin expressed on the endothelium, or indirectly by binding to leukocytes that have already adhered to the endothelium.' The rapid mobilisation of P-selectin to the cell surface, following cell stimulation by inflammatory mediators, allows neutrophils and monocytes to bind to platelets and endothelial cells.' P-selectin has been found to exist in a soluble and functionally active form.'"." It has a diurnal variationI2 and normal volunteer studies have shown that sP-selectin is released from activated platelets. l o

The anticoagulant mixture of CTAD has been designed to prevent platelet aggregation in v i m , thus avoiding any release of platelet products that may occur between the time of blood sample collection and that of testing. This type of anticoagulant tube is used to assay platelet release molecules such as P-thromboglobulin and platelet factor 4.13 Both of these markers of platelet activation, like P-selectin, are stored and released from a - g r a n ~ l e s . ' ~ Platelet factor 4 and P-thromboglobulin levels may provide an index of endogenous activation and consumption of platelets."

Iloprost is a synthetic analogue of prostacyclin (PG12). In vitro studies have shown that iloprost like PG12 is able to suppress platelet activation, shape change and secretion of a - g r a n ~ l e s . ' ~ ~ ~ ~ Iloprost has been shown to inhibit the secretion of another a-granule product platelet derived growth factor (PDGF)," and it has been shown to attenuate thrombin stimulated P selectin expression on platelets.'

Since sP-selectin is released from platelets, statistically significant higher levels are found in serum samples when compared to plasma samples. Furthermore, it can be clearly seen from the results that the combination of CTAD and iloprost leads to less in vitro activation of the platelets when compared it to the anticoagulant tri-sodium citrate which is recommended by the ELISA manu- facturers. This degree of in vitro activation is the same for both the control and patient samples since the sP-selectin levels are attenuated by the same proportion when comparing citrate to CTAD plus iloprost.

These findings differ from Chong et al. who compared P-selectin levels from blood collected into tubes with or without ETP ( 1 10 mmol/l disodium EDTA, 12 mmol/l theophylline and 2.82 pmol/l prostaglandin El ) . They found differences between the two tubes for P-thrombo- globulin levels but no difference for sP-selectin levels.s

Dunlop et a1.I' also found that P-selectin was insensitive to different collection methods. They com-

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I62 SOLUBLE P-SELECTIN ASSAY

pared P-selectin levels in plasma which had been processed immediately after blood collection with levels from plasma that was processed 2 h after blood collec- tion.'" Our method of collection, in addition to using a combination of sodium citrate and citric acid, which is supplemented with the platelet aggregation inhibitors (theophylline, adenosine and dipyridamole), also has another potent platelet inhibitor present, iloprost. The method for sP-selectin sample collection is important so as to ensure that the levels measured at sP-selectin that has originated from either endothelial cells and/or circulating platelets, and not a consequence of in vitro platelet activation.

In the CTAD plus iloprost and the citrate group, the 20 diabetic patients had a higher mean value for sP-selectin compared with the healthy controls. Suggesting that patients with diabetes have activated circulating plate- lets. This agrees with others who have showed an increase in platelet activation in patients with diabetes." However when serum is used as anticoagulant there is a higher mean level of sP-selectin in the control group when compared to the patient group. This may reflect that platelets from a diabetic population are activated, allowing adhesion to neutrophils and endothelial cells and aggregation to other platelets. The consequence of this platelet hyperactivation,20 seen in the patients with diabetes, may be that when the blood is allowed to clot this could result in fewer P-selectin glycoproteins being available for release from the a-granules in the platelet which in turn results in less circulating P-selectin. Whereas in the platelets of the control subjects the optimum amount of P-selectin molecule can be released from its storage site, and therefore a larger amount of sP- selectin is measured.

When the effects of low dose daily aspirin (75mg) was investigated on sP-selectin levels in the patients with IDDM the group of patients on aspirin had lower sP- selectin levels in both anticoagulants and in serum. However, although the amounts of sP-selectin were lower there was no statistical difference between the two groups. This agrees with studies that used flow cyto- metric analysis to investigate the effects of aspirin in whole blood on the release products of a-granules from human platelets.2t,22 These studies showed that degranu- lation of the a-granules in response to thrombin, results in P-selectin expression on the surface of the platelet, which was paralleled by the release of P-thromboglobu- lin. P-selectin expression, after thrombin or adenosine diphosphate,21.22 was not affected by aspirin treatment (300 mg/day), whereas, the platelet aggregatory response to arachidonic acid was abolished by aspirin treatment.,'

We have shown that lower circulating levels of sP- selectin are obtained after collection of the blood into a CTAD tube that contains the PGI, analogue, iloprost, and suggests the levels obtained are likely to be a truer reflection of actual circulating sP-selectin levels than those obtained when test-tube activation of platelets is allowed to continue in vitro.

Acknowledgements GK has been supported by a PhD studentship and subsequently a post- doctoral fellowship kindly awarded by Ptizer Ltd. UK.

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