Sensitive enzyme immunoassay for the measurement of platelet factor 4 in blood plasma

Clrnrccr Chuwrco AC/U. 138 (1984) 151-161 Elsevier

151

CCA 02810

Sensitive enzyme immunoassay for the measurement of platelet factor 4 in blood plasma Tetsuo Shimizu a,*, Iwazo Hasegawa ‘, Tsuneo Kanefusa Kato ’

Fukuda a and

” Lohoraroty for Blood

Reseurc~h. Aichr Prefecture Red Cross Blood Center. 3.2 2. San nomaru, Nukuku. Nag-oyu 460 und h Depcrriment of Buxhemistry, Insriture for Deuelopmenral Research. Archr Prefectural Colony. Kum~_vu, Krrsugai, A ichi 480 03 (Japan) (Received

July 21s~ revision November

Key words: Platelet Jucior 4; Enzyme immunoassay;

30th. 1983)

Plasma level; Blood cell; Platelet umcentrates

Summary A sandwich enzyme immunoassay method for the measurement of platelet factor 4 (PF4) was developed with the use of polystyrene balls with immobilized antibody F(ab’), fragments and the same antibody Fab’ fragments labeled with P-D-galactosidase from E. co/i. The measurable range was 30 pg to 3 ng of PF4 per tube. Within-run and between-run coefficients of variation were less than 10%. The results obtained with the enzyme immunoassay correlated well with those of a radioimmunoassay (r = 0.952, slope = 0.954, y-intercept = 2.43 ng/ml). Platelets contained large amounts of PF4 (7.21 f 1.97 ng/106 cells or 2.51 & 1.13 ng/mg protein), whereas the PF4 levels in red blood cells and lymphocytes were negligible, confirming the specific localization of PF4 in platelets. The applicability of the immunoassay method was tested to determine the in vitro release of PF4 during preparation and storage of platelet concentrates.

Introduction Platelet factor 4 (PF4) is a platelet-specific protein with a molecular weight of 27 100, consisting of four identical subunits [1,2]. The amino acid sequence of PF4 is very similar to that of j.%thromboglobulin (PTG), another platelet-specific protein [3-71. PF4 is synthesized in megakaryocytes [8] and packed in the platelet a-granules

* Address correspondence and reprint requests to Dr. Tetsuo Shimizu. Laboratory for Blood Research, Aichi Prefecture Red Cross Blood Center, 3-2-2, San-nomaru. Nakaku, Nagoya 460, Japan. 0009-8981/84/$03.00

0 1984 Elsevier Science Publishers

B.V.

152

[9,10]. PF4 is released from platelets as a complex molecule with proteoglycan carriers having a combined molecular weight of 358000 [2]. Although some biological functions of PF4 have been reported to include a strong heparin-neutralizing property and inhibitory effects on collagenase [1,11,12], the physiological significance of PF4 has not been fully elucidated. Specific radioimmunoassay systems for PF4 have been developed by a number of investigators [1,13-16] and these were applied not only to the analysis of in vitro platelet release reaction but also clinically as an index of in vivo platelet activation [15-201. Despite the advantages of high sensitivity and specificity of radioimmunoassay systems for PF4, there are a number of drawbacks to radioimmunoassays, including instability, the high cost of labeled compounds, and the short half-life of ‘2sI. In this report a sandwich enzyme immunoassay method is described for measurement of PF4 in plasma and blood cells, together with a discussion of its applicability to determine the degree of in vitro platelet damage during the preparation and storage of platelet concentrates. Materials and methods Preparation of platelet concentrates Whole blood from healthy donors was put into 200-ml plastic bags (Terumo Company, Tokyo, Japan) containing 28 ml of citrate-phosphate-dextrose (CPD) solution. After 35 min, the blood was centrifuged at 1350 x g for 6 min at 22°C. The resulting platelet-rich plasma was then centrifuged at 2055 x g for 6 min at 22°C and the platelet concentrates were obtained by the removal of about 80 ml of the supernatant. After 1 h storage at 22-23*C, the precipitated platelets were resuspended by shaking on a flat bed shaker (Taiyo recipro shaker, T-337, Osaka, Japan) at 100 osc!llations/min for 30 min and then at 60 oscillations/min for up to 72 h at 22-23°C. Blood samples (0.5 ml) were taken at each preparative step into pre-cooled blood sampling tubes containing a mixture of 0.1 ml of acid-citrate-dextrose (ACD) solution, 0.01 ml acetyl salicylic acid (final concentration 30 mmol/l), and 0.01 ml prostaglandin E, (final concentration, 1 pmol/l). The tubes were placed in crushed ice for at least 15 min and then centrifuged at 1500 x g for 30 min at 4°C. and the supernatant was kept frozen at -20°C for a few days until analysis. Purification of PF4 from human pi~telets Human platelet preparations used for purification of PF4 were 24-h old platelet concentrates obtained as described above from the Aichi Prefecture Red Cross Blood Center. PF4 and PTG were isolated from the released products of platelets according to the method of Rucinski et al [l]. The final preparation of PF4 showed a single band on SDS gel electrophoresis with 15% polyacrylamide (Fig. 1). About 2 mg of PF4 and 1 mg of PTG were obtained from 2000 ml of platelet concentrates. Its protein content was measured by the method of Lowry et al f21J using crystalline bovine serum albumin as a standard. The purified PF4 were stored at - 30°C in a 50% glycerol solution and used as immunogen to produce rabbit anti-PF4 serum, and as standard for immunoassay.

153

Fig. 1. SDS polyacrylamide gel electrophoresis (15%) of purified PF4. Anode at bottom, cathode at top. The sample contained about 6 pg of purified PF4.

Antibodies to PF4 Anti-PF4 serum was raised in rabbits by with complete Freund’s adjuvant at multiple were administered at 3-week intervals for.at injection, the rabbits were bled and the sera

injection 0.4 mg of purified PF4/rabbit intracutaneous sites. Booster injections least 3 months. After 7 days of the last were pooled.

Purijication of antibodies to PF4 The pooled antiserum (about 80 ml) was diluted 2-fold with 0.15 mol/l NaCl and the immunoglobulin G (IgG) fractions were precipitated at 0°C by adding solid (NH,),SO, (50% saturation). The precipitate was dissolved with the original serum volume of 0.01 mol/l sodium phosphate buffer (pH 7.0) containing 0.1 mol/l NaCl and 0.1% NaN,, and dialyzed overnight against the same buffer at room temperature. After removing the insoluble proteins, the IgG fractions were applied to the column (0.9 x 20 cm) of PF4-coupled Sepharose 4B, which had been equilibrated with the above buffer, at a flow rate of 5 ml/h at room temperature (about 6 mg of the purified PF4 was coupled to 5 g of CN-Br activated Sepharose 4B). The column was washed successively with the phosphate buffer described above (30 ml) and then the same buffer containing 1 mol/l NaCl (70 ml). The antibodies bound to the column were eluted with 0.1 mol/l glycine-HCl buffer (pH 2.5), containing 1 mol/l NaCl at a flow rate of 18 ml/h at room temperature. Each 3-ml fraction was collected in a tube containing 1 ml of 0.1 mol/l sodium phosphate buffer (pH 7.0). The purified antibody fractions were pooled, neutralized and concentrated with Amicon Centriflo (CF-25, Amicon Corp., Lexington, MA, USA).

Fig. 2. Double immunodiffusion antibody to PF4. Ten microliters the thrombin-aggregated, washed antibodies (about 4 gg/lO ~1) to

of the purified PF4 and a crude platelet extract against the purified containing about 3 gg each of purified PF4 (a, c). the supernatant from platelet (b). or /3TG (d) were layered in the side well. The purified PF4 were applied to the center well.

About 40 mg of the purified antibody IgG was obtained from 80 ml of the antiserum. Upon double immunodiffusion the purified antibodies formed a single precipitin line with both the purified PF4 and the crude PF4 fraction, but not with purified j3TG (Fig. 2). Polystyrene balls with immobilized antiboc& F(ab’)_, frugments The purified antibody IgG was digested with pepsin to obtain the F(ab’)z fragments of the antibodies 1221. To increase the sensitivity of the assay 122,231 and to protect the non-specific interference with the assay by substances in biological samples [24], the antibody fragments were used to prepare the immunoassay reagents. The antibody F(ab’), fragments were coupled on polystyrene balls (3.18 mm in diameter, Precision Plastic Ball Co., Chicago, IL, USA) as described previously [25], and the balls were stored in sodium phosphate buffer, 0.01 mol/l, pH 7.0, containing 0.1 mol/l NaCl, 1 mmol/l MgCl,, 1 g/l bovine serum albumin and 1 g/l NaN, (buffer A) for at least 2 days to stabilize the assay. Antibody Fah’ fragments labeled with @-D-galactosidase The Fab’ fragments were coupled with B-D-galactosidase from E. co/i by use of N, ~‘-o-phenylenedimaleimide [22]. The amounts of labeled antibody were expressed in units of galactosidase activity (one unit = 1 (*mol pr~uct/min at 30°C) as described previously [25].

155

Immunoassay

procedures

The PF4 was assayed with the antibody F(ab’),-immobilized polystyrene balls and the same antibody Fab’ fragments labeled with fi-D-galactosidase. Ten microliters of samples or standards, otherwise mentioned, were mixed in duplicate in a glass test tube (1.2 X 7.5 cm) with 0.49 ml of sodium phosphate buffer, 0.01 mol/l, pH 7.0 containing 0.3 mol/l NaCl, 1 mmol/l MgCl,, 1 g/l bovine serum albumin, 5 g/l gelatin (Difco Laboratories, Detroit, MI, USA), and 1 g/l NaN, (buffer G). Then a piece of polystyrene solid-phase with immobilized antibodies was added to each tube, and the mixture was incubated at 30°C for 2 h with shaking. The reaction medium was discarded by aspiration, and the solid-phase was washed twice with 1 ml of chilled buffer A in each test tube. The solid-phase was transferred to a test tube (1 x 7.5 cm) containing 0.1 ml buffer A with 1 mU of the antibody Fab’ labeled with /?-D-galactosidase and left standing at 4°C overnight. The polystyrene ball was then washed with chilled buffer A as described above, and the galactosidase activity bound to the ball was assayed at 30°C with 0.1 mmol/l 4-methylumbelliferyl ,8-D-galactosidase (Sigma Chemical Co., St. Louis, MO, USA) as substrate [25]. Standard

PF4

The purified PF4 was used as a standard. Almost identical standard curves of the immunoassay were obtained with the purified PF4 and a standard PF4 of a radioimmunoassay kit described below. Radioimmunoassay

of PF4

A standard PF4 of a radioimmunoassay kit (Abbott Laboratories, North Chicago, IL, USA) was the kind gift of Mr. I. Mochizuki, Dinabbott RI Laboratories, Tokyo, Japan. Plasma samples which had been measured for PF4 content with a radioimmunoassay kit of Abbott Laboratories were the gift of Mr. C. Nashida, Special Reference Laboratory, Tokyo, Japan. Isa&ion

of blood cells

Platelets were isolated from platelet-rich plasma obtained by platelet apheresis by gel filtration on a Sepharose 2B column (1.9 X 25 cm) as described [26]. The red blood cells were obtained by centrifuging packed red cells at 1200 X g for 10 min at 4°C. and the supernatant and buffy coat phase were discarded by aspiration. The pellet of red blood cells was resuspended in 0.15 mol/l NaCl solution and centrifuged at 1200 x g for 10 min at 4’C. These procedures were repeated six times to obtain the final red blood cell fractions. Lymphocytes were separated by centrifugation of the blood with Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala. Sweden) at 400 X g for 20 min at 22’C. Remaining platelets were removed by adding thrombin, and fractions containing lymphocytes were washed four times with the balanced salt solution by centrifugation at 100 X g according to the instructions of Pharmacia. The cells were kept frozen at - 20°C for a few days before analysis. The thawed fractions were diluted lo-10000-fold with buffer G, and 0.01-0.1 ml aliquots of the diluted sample were subjected to immunoassay.

156

Plasma samples Normal plasma was collected from healthy individuals of both sexes, 22-33 years old, who had received a regular health examination 1 week before venipuncture. Venous blood (2.5 ml) was drawn into pre-cooled blood sampling tubes (Amersham International Limited, Amersham, UK). The tubes were placed in crushed ice for 30 min and then centrifuged at 1500 X g for 30 min at 4°C. The top-most 0.5 ml of the supernatant was transferred to a storage tube and kept frozen at -20°C until analysis. Other methods The levels of lactic dehydrogenase (LDH) in blood were measured by a kit (Wako Pure Chemical Industries, Co., Ltd, Osaka, Japan). The isolated red blood cells and lymphocytes were counted using a microcell counter (Sysmex CC-108, Tokyo, Japan), and platelets were counted by means of a platelet counter (Sysmex PL-100, Tokyo, Japan). Protein concentrations of the cell extracts were determined with the fluorometric assay [27]. SDS polyacrylamide gel electrophoresis was performed according to the method described by Weber and Osborn [28]. Results Sensitivity and precision of the immunoassay for PF4 The standard curve for the assay of PF4 and the cross-reactivity of this assay with /?TG are shown in Fig. 3. Dose-response of the enzyme activity bound was seen between 30-3000 pg of the standard PF4. The PTG, another protein specific to platelet, showed little cross-reactivity (less than 1% that of PF4) on the assay system. When human plasma samples were subjected to the immunoassay with various sample volumes (5-40 PI), the slopes of the curve were almost parallel with the standard curve in the assay system (data not shown). The results indicate that the human PF4 protein in plasma can be determined by the present method with a sample volume of 10 ~1.

PF4

(nglassay

tube)

Fig. 3. The standard curves of the immunoassay for PF4. Indicated amounts of PF4 (0) or /3TG (0) incubated in duplicate with the solid-phase coated with immobilized antibodies to PF4.

were

757 TABLE

I

Recovery

of PF4 added

No. of plasma

samples

to plasma *

PF4 recovery

10 10 * Plasma levels of PF4 ranged ** The purified PF4 was added

TABLE

samples (ng/ml)

added **

recovered

mean+

30 100

29.7*1.5 100 *4

99&5 lOOk4

from 5.1 to 6.7 ng/ml. to the test tube together

with 10 ~1 of plasma

1 SD

samples

II

Within-assay

and between-assay

variation

of the enzyme immunoassay

No. of assays

for PF4

Mean * 1 SD

cv

(ng/mI)

(a)

20 20 20

3.4kO.2 34.2 i 2.1 110.3 +5.7

6.8 6.1 5.1

10 10 10

8.1 * 0.8 19.4* 1.9 40.2 + 3.5

9.9 9.8 8.7

Wirhin assay sample 1 sample 2 sample 3 Berween - assuy sample 4 sample 5 sample 6

I 100 t

0

ci,

0

20

LO

PF4

assayed

60

by RIA

80

1

100

(nglml)

Fig. 4. Correlation between the enzyme immunoassay and radioimmunoassay for PF4. Plasma samples were assayed by both enzyme immunoassay (EIA) and radioimmunoassay (RIA). Regression equation was y = 0.954x + 2.43 ng/ml and the correlation coefficient was 0.952.

Recovery of purified PF4 added to plasma was tested using 10 normal plasma samples. As shown in Table I the recovery was nearly 100%. The precision of the assay system was tested by assaying three plasma samples 20 times in one assay (within-run) or in duplicate in 10 consecutive assays (between-run). The coefficients of variation in each assay were less than 10% (Table II). To evaluate the accuracy of the present method, PF4 concentrations in plasma from 36 subjects were determined by both the enzyme immunoassay and a radioimmunoassay. As shown in Fig. 4. there was a good correlation between the two methods over the entire range studied. The regression equation was .v = 0.954-x + 2.43 ng/mI and the correlation coefficient was 0.952. PF4 levels in plasma from normal subjects and in isolated blood cells Normal plasma levels of PF4 were determined in six healthy adults. The plasma contained about 6 ng/ml (Table III). Isolated blood cells from four to six donors were examined. The cells were destroyed by the repeated freezing and thawing procedures. The extract obtained by centrifugation was subjected to the assay for PF4. Red blood cells and lymphocytes had very little PF4 when expressed on the basis of either per mg protein or per 10h cells (Table III). Platelets, however. contained a great amount of PF4 (2.51 pg/mg protein and 7.21 ng/106 cells). PF4 release during preparu~ion and storage 0~pta~elet ~otl~e~?r~~e.~ In order to determine the applicability of the present method, PF4 release during preparation and storage of platelet concentrates was serially assayed. As shown in Table IV, little PF4 release was observed in whole blood in the bag immediately after blood collection. The storage for 35 min at room temperature and the following centrifugation of the whole blood at 1350 x g for 6 min at 22°C to prepare the platelet-rich plasma resulted in a slight release of PF4. However, a large amount of released PF4 was detected in the platelet concentrates, which were obtained by the high-speed centrifugation of platelet-rich plasma at 2055 x g for 6 min at 22’C, and

TABLE

III

Levels of PF4 in normal Blood components

plasma

and isolated

blood cells determined

No. of samples

with the enzyme PF4

_.

( W/~~~) Normal

plasma

irnrnun~~~ssay

6

6.4

Red blood cells Lymphocytes Platelets

6 4 5

(“‘q/rq 0.19 0.49 2 509

Red blood cells Lymphocytes Platelets

6 4 5

( q/10’ cell.7 ) 0.003 * 0.001 0.062 * 0.003 7.21 +1.97

+ 1.4 pr&vn) & 0.06 i 0.23 k1128

159 TABLE IV Release of PF4 and leakage of LDH during preparation and storage of platelet room temperature

concentrates

for

72 h at

Phase

Total volume (ml)

PF4 (ng/ml)

LDH rl

Immediately after phlebotomy 35 min after phlebotomy Platelet-rich plasma Platelet concentrates After 1 h 30 min after agitation ’ 24 h after storage’ 48 h after storage 72 h after storage

232.8 + 9.3

1.4h 16.0 + 7.0 27.3 + 4.2 230.5 i 133.2 119.2 + 445.8 1308.9 + 570.2 3093.7 rt 926.5 5 105.9i 1748.5 6 306.1 i 1234.6

221.3 + 40.3 h 233.6k44.1 243.3 k 31.7 254.1 + 24.9 260.4 + 34.8 262.4+ 32.9 323.6 + 55.1 361.4i57.9 398.9 I 71.9

It ’ ’ ’

112.9 k 7.6 27.6 * 2.0

6.4 *

Lactic dehydrogenase activity; Wroblewski units/ml [31]. Mean of 10 preparations k 1 SD. Pelleted platelets were shaken on a flat bed shaker at 100 oscillations/min for 30 min. Resuspended platelets were continuously agitated on a flat bed shaker at 60 oscillations/min

the further PF4 release was noted when left standing at room temperature for 1 h. The mean volume of platelet concentrates in this step was 27.6 k 2.0 ml (n = lo), and the mean number of platelets was 1.317 rt 0.391 x 106/yl. The pH in products was 7.42 &-0.06. The total amount of PF4 in the platelet concentrates, which was determined after destruction of platelets by repeated freezing and thawing, was 19.33 F 10.07 pg/ml. After agitation with 100 oscillations/min for 30 min to resuspend the precipitated platelets, the concentration of released PF4 was about 1.7 times higher than before agitation. Storage for 24, 48, and 72 h with agitation of 60 oscillations/min produced a further release of PF4, which was calculated to be 20.4 _t 11.2, 33.3 rf 19.5, and 41.1 _t 19.4%. respectively, of the total PF4 amount in platelet concentrates. The initial level of LDH in whole blood in the collection bag was 227.7 + 40.3 Wroblewski units/ml. Total amounts of LDH were 6183.7 + 2539.7 units/ml in Triton X-100 lyzed platelets in platelet concentrates (n = 10). However, LDH leakage remained relatively stable during preparation of platelet concentrates. and the percent leakage of LDH was maintained within 3% of the total LDH amount during storage for 3 days. Discussion The sandwich enzyme immunoassay system for the measurement of PF4 described here had a sensitivity of 30 pg/assay tube, which is higher than that of radioimmunoassay systems reported by a number of investigators [13-161. The high sensitivity could have resulted from the use of purified antibodies. The assay system showed a small but significant cross-reactivity with PTG, which is another plateletspecific protein. Similar cross-reactivities have been found in radioimmunoassay

160

systems for PF4 [1,16,18]. However, this may be accounted for by contamination of the purified @TG with PF4. The plasma levels of PF4 in normal subjects determined with the present method are consistent with those reported by various investigators [14,16]. The concentrations of PF4 in isolated platelets are in good accordance with those reported by Kaplan et al [lo] and Rucinski et al [l]. The specific localization of PF4 in platelet a-granules has been shown by studies on density gradient fractionations [9,10] and immunofluorescent study [17]. Using a radioimmunoassay system, Kaplan et al [15] found that this protein is specific to platelets. The present study confirmed that red blood cells and lymphocytes contained but very small amounts of PF4 (Table III). PF4 levels in platelets were over SOOO-fold more than those in other blood cells when compared on the basis of mg protein. The present assay system was used for monitoring the PF4 release during the preparation of platelet concentrates. The PF4 level in whole blood in the bag immediately after blood collection was low and values were similar to those measured in healthy subjects, suggesting that no significant in vitro release occurred in blood collection. High-speed centrifugation caused an increase in release of PF4 with little LDH leakage. These findings are in good accord with those of Snyder et al [29], who measured the release of PTG during preparation of platelet concentrates. Storage of platelet concentrates with agitation for 3 days at room temperature led to the release of large amounts of PF4 from platelets (about 40% of the total PF4 on the final day), whereas the LDH leakage was less than 3% that of the total. Similar findings were reported in PTG release from platelets stored as platelet concentrates at room temperature [29,30]. The sandwich enzyme immunoassay system for the measurement of PF4 reported here may also be useful for monito~ng changes in the plasma level of this protein with platelet activation in vivo. References Rucinski B, Niewiarowski S, James P, Walz DA, Budzynski AZ. Antiheparin proteins secreted by human platelets. Purification, characterization, and radioimmunoassay. Blood 1979; 53: 47-62. Moore S, Pepper DS, Cash JD. Platelet antiheparin activity. The isolation and characterization of platelet factor 4 released from thrombin-aggregated washed human platelets and its dissociation into subunits and the isolation of membrane-bound antiheparin activity. Biochim Biophys Acta 1975; 379: 370-384. Levine SP, Wohl H. Human platelet factor 4: purification and characterization by affinity chromatography. Purification of human platelet factor 4. J Biol Chem 1976; 251: 324-328. Handin RI, Cohen HJ. Purification and binding properties of human platelet factor four. J Biol Chem 1976; 251: 4273-4282. Hermodson M, Schmer G, Kurachi K. Isolation. crystallization, and primary amino acid sequence of human platelet factor 4. J Biol Chem 1977; 252: 6276-6278. Deuel TF, Keim PS, Farmer M. Heinrikson RL. Amino acid sequence of human platelet factor 4. Proc Ntl Acad Sci USA 1977; 74: 2256-2258. Begg CS, Pepper DS, Chesterman CN, Morgan FJ. Complete covalent structure of human ,0-thromboglobulin. Biochemistry 1978; 17: 173991744. Ryo R, Nakeff A, Huang SS, Ginsberg M, Deuel TF. New synthesis of a platelet-specific protein: platelet factor 4 synthesis in a megakaryocyte-enriched rabbit bone marrow culture system. J Cell Biol 1983; 96: 515-520. Da Prada M, Jakabova M. Liischer EF, Pletscher A. Richards JG. Subcellular localization of the

161 heparin-neutralizing factor in blood platelets. J Physiol 1976; 257: 495-502. 10 Kaplan KL. Broekmam MJ. Chernoff A, Lesznik CR. Drillings M. Platelet a-granule proteins: studies on release and subcellular localization. Blood 1979; 53: 604-618. 11 Nath N. Lowery CT. Niewiarowski S. Antigenic and antiheparin properties of human platelet factor 4 (PF4). Blood 1975; 45: 537-550. 12 Hiti-Harper J. Wohl H, Harper E. Platelet factor 4: an inhibitor of collagenase. Science 1978: 199: 991-992. 13 Bolton AE, Ludlam CA. Pepper DS, Moore S. Cash JD. A radioimmunoassay for platelet factor 4. Thromb Res 1976; 8: 51-58. 14 Levine SP. Krentz LS. Development of a radioimmunoassay for human platelet factor 4. Thromb Res 1977; 11: 673-686. 15 Kaplan KL, Nossel HL, Drillings M, Lesznik G. Radioimmunoassay of platelet factor 4 and P-thromboglobulin: development and application to studies of platelet release in relation to fibrinopeptide A generation. Br J Haematol 1978; 39: 129-146. 16 Chesterman CN, McGready JR, Doyle DJ, Morgan FJ. Plasma levels of platelet factor 4 measured by radioimmunoassay. Br J Haematol 1978; 40: 489-500. 17 Ginsberg MH, Taylor L, Painter RG. The mechanism of thrombin-induced platelet factor 4 secretion. Blood 1980; 55: 661-668. 18 Kaplan KL. Owen J. Plasma levels of /?-thromboglobulin and platelet factor 4 as indices of platelet activation in viva. Blood 1981; 57: 199-202. 19 Weiss HJ, Witte LD, Kaplan KL et al. Heterogeneity in storage pool deficiency: studies on granule-bound substances in 18 patients including variants deficient in a-granules, platelet factor 4. P-thromboglobulin, and platelet-derived growth factor. Blood 1979; 54: 1296-1319. 20 Niewiarowski S, Thomas DP. Platelet factor 4 and adenosine diphosphate release during human platelet aggregation. Nature 1969; 222: 1269-1270. 21 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-270. 22 Kato K, Fukui H, Hamaguchi Y. Ishikawa E. Enzyme-linked immunoassay: conjugation of the Fab’ fragment of rabbit IgG with P-D-galactosidase from E. co/i and its use for immunoassay. J Immunol 1976; 116: 1554-1560. 23 Kato K, Suzuki F, Semba R. Determination of brain enolase isozymes with an enzyme immunoassay at the level of single neurons. J Neurochem 1981; 37: 998-1005. 24 Kato K, Umeda Y, Suzuki F, Kosaka A. Interference in a solid-phase enzyme immunoassay system by serum factors. J Appl Biochem 1979; 1: 479-488. 25 Kato K, Hamaguchi Y. Okawa S, Ishikawa E, Kobayashi K, Katunuma N. Use of rabbit antibody IgG-loaded silicone piece for the sandwich enzymoimmunoassay of macromolecular antigens. J Biochem 1977; 81: 1557-1566. 26 Lages B, Scrutton MC, Holmsen H. Studies on gel-filtered human platelets: isolation and characterization in a medium containing no added Ca*‘, Mg*+, or K+. J Lab Clin Med 1975; 85: 811-825. 27 Bohlen P, Stein S, Dairman W. Udenfriend S. Fluorometric assay of proteins in the nanogram range. Arch Biochem Biophysiol 1973; 155: 213-220. 28 Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J Biol Chem 1969; 244: 440664412. 29 Snyder EL, Hezzey A, Katz AJ, Bock J. Occurrence of the release reaction during preparation and storage of platelet concentrates. VOX Sang 1981; 41: 172-177. 30 Scott NJ, Harris JR, Bolton AE. Effect of storage on platelet release and aggregation responses. VOX Sang 1983; 45: 359-366. 31 Wrhblewski F, LaDue JS. Lactic dehydrogenase activity in blood. Proc Sot Exp Biol Med 1955; 90: 210-213.