A combined method for the preparation of washed human platelets using prostacyclin and gel filtration

THROMBOSIS RESEARCH 50; 733-738, 1988 0049-3848/88 $3.00 t .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.

BRIEF

COMMUNICATION

A COMBINED METHOD FOR THE PREPARATION OF WASHED HUMAN PLATELETS USING PROSTACYCLIN AND GEL FILTRATION K. Yamamoto, A. Hirai, Y. Tamura, and S. Yoshida The 2nd Department of Internal Medicine, School of Medicine, Chiba University, Chiba 280, Japan (Received 28.9.1987; Accepted in revised form 4.2.1988 by Editor S. Okamoto) (Received 15.3.1988 by Executive Editorial Office)

INTRODUCTION Several methods for the separation of platelets from plasma proteins have been developed (l-7). Recently Vargas et al. developed a new technique to separate platelets from plasma proteins by the addition of prostacyclin in order to prevent platelet activation during the procedure of platelet separation and washing from whole blood (6). Although this method has several advantages such as good preservation of platelet function, the procedure was rather complicated because repeated centrifugation and addition of prostacyclin were required. Timmons et al. established a method of preparing washed platelets by use of albumin cushion during centrifugation process followed by gel filtration though prostacyclin was not used (5). This method can provide concentrated washed platelets from a relatively small volume of blood sample in a short time, though the washed platelets had low sensitivity to various aggregants such as adenosine diphosphate (ADP) and epinephrine. Accordingly we have developed a combined method for the preparation of washed human platelets by use of prostacyclin and albumin cushion-gel filtration technique and-investigated the morphology and the function of washed platelets prepared by this method. MATERIALS AND METHODS Blood samples were obtained from healthy volunteers who did not take any medications at least 2 weeks before blood sampling. Nine ml of venous blood was collected in a 10 ml vacuum glass tube containing 1 ml of 3.8% (w/v> sodium citrate and 20 pg of PG12-Na (Ono Pharmaceutical Co., Ltd., Osaka, Japan). The sample was centrifuged at 15Oxg for 10 min at 20°C to obtain platelet rich plasma (PRP). Four ml of PRP was gently layered on 750 ~1 of 40% (w/v) bovine serum albumin solution in a glass tube and then centrifuged at 1500xg for 10 min at 2O'C. The clear supernatant and albumin cushion were carefully removed with a glass capillary pipette and the remaining platelets were gently resuspended with 300 pl of HEPES-Tyrode's buffer (NaCl 129 mM, NaHC03 8.9 mM, KH7_PO40.8 mM, MgC12 0.8 mM, glucose 5.6 mM, HEPES 10 mM, bovine serum albumin 0.035%, pH 7.35) and stored at 20°C till use. Key words:

Human

platelets,

prostacyclin 733

and

gel

filtration

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Sepharose 2B (Pharmacia Fine Chemicals, Uppsala, Sweden) was washed with 3 volumes of acetone and 6 volumes of saline (0.154 M NaCl) and was suspended in 2 volumes of saline. A 10 ml disporsable plastic syringe with a 40 vm The gel nylon net at the bottom was used as a column for gel filtration. suspension was poured into the column and allowed to pack by gravity flow to a height of 9 cm. The column was then equilibrated with 100 ml of HEPESPlatelets suspended in HEPES-Tyrode's buffer were carefully Tyrode's buffer. applied to the column and eluted with 4-5 ml of HEPES-Tyrode's buffer. Elution fluid was collected by every 500 ~1. The two fractions (fraction 5 and 6) were collected and used for aggregation study. Platelet counts of gel filtered platelet suspension were measured using a particle counter Model PC-6034 (Erma Optical Works Co., Ltd., Tokyo, Japan) and adjusted to 3x10' platelets/ml with HEPES-Tyrode's buffer. Then gel filtered platelets were stored at 20°C till use. Aggregation studies of gel filtered platelets were carried out between 120 and 180 min after blood sampling unless otherwise stated. Aggregation studies of PRP which was prepared without PGI2 were carried out between 60 and 90 min as previously reported (8). Platelet aggregation study was performed according to the method of Born (9) with minor modifications using a 4 channel aggregometer HEMA TRACER 1 (Niko Bioscience, Tokyo, Japan). For scanning electron microscopic analysis, platelets were fixed in 0.1 M sodium phosphate buffer (pH 7.3) containing 1% glutaraldehyde. Samples were then filtered through a glass fiber filter paper and washed with 0.1 M sodium phosphate buffer (pH 7.3). Filters were dehydrated in a graded series of ethanol, dried in a JEOL critical point drier (JEOL, Ltd., Tokyo, Japan) with liquid CO2 as the transition fluid, and coated with Au in a JEOL ion sputter coater. Platelets were examined in a JEOL JSM-35CF scanning electron microscope at an accelerating voltage of lo-15 KV. RESULTS Scanning electron microscopy of gel filtered platelets prepared without PG12 showed that most of the platelets were spherical shape with numerous pseudopods (Fig. l-a). The number of pseudopods were decreased and the shape of platelets became discoid by increasing the doses of PGI2 added to titrated blood from 0.05 to 2.0 ug/ml blood (Fig. 1-b,c,d). Most of gel filtered platelets prepared with 2.0 ng PGIp/ml blood were discoid shape with no pseudopods or one or two pseudopods (Fig. l-d). There was no further improvement in the shape of gel filtered platelets by increasing the doses of PGI2 up to 5 ug/ml of blood (data not shown). Total platelet yield was 92.4+ 11.8% (meantS.D., n=l7). The protein concentration in the suspension of gel filtered platelets (3x10* platelets/ml) with the platelets removed by centrifugation was 41+- 31 ug/ml (mean* S.D., n=5) compared to 70000 ug/ml in normal plasma. Collagen (0.125-1.0 ng/ml) (Hormon Chemie, Munchen, West Germany) and epinephrine (0.5-5.0 nM) (Sigma Chemical Co., St. Louis, MO, USA) induced a dose-dependent aggregation at concentrations similar to those used in PRP (Fig. 2-a,b, 3-a,b). ADP (2-20 nM) (Sigma Chemical Co.) induced platelet aggregation in a dose-related manner (data not shown). The addition of human fibrinogen (600 ug/ml) (Kabi Vitrum AB, Stockholm, Sweden) to the platelet suspension potentiated aggregatory response to ADP (0.2-2.0 PM) (data not shown). Thrombin (0.025-0.1 U/ml) (Mochida Pharmaceutical Co., Ltd., Tokyo, Japan) induced platelet aggregation in a dose-dependent manner (data not shown).

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GEL FILTERED PLATELETS WITH PG12

(b)

(a>

Cd) FIG. 1.

Scanning electron microscopic analysis of gel filtered platelets. Morphological changes of gel filtered platelets were examined by scanning electron microscopy to estimate platelet activation during washing procedure. Gel filtered platelets were prepared with (a) 0, (b) 0.05, (c) 1, (d) 2 ug PGIz/ml blood as described in "Materials and Methods." Magnification 4,000.

10

s

9

W 1.0

0.5

-_L 6 ._ z ._ E e e c)

z .z a

0.33

0.25 0.125

0

9

1-min

COllagt3fl

FIG. 2.

a) Aggregation of gel filtered platelets to collagen. b) Aggregation of PRP to collagen. Numbers beside traces show concentrations of collagen in ug/ml.

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FIG. 3.

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a) Aggregation of gel filtered platelets to epinephrine. b) Aggregation of PRP to epinephrine. Numbers beside traces show concentrations of epinephrine in pM.

The time course of platelet aggregability to collagen after blood sampling was studied. Gel filtered platelets were stimulated by threshold dose of collagen at the concentration ranging from 0.33 to 0.75 ng/ml. It was noted that there was an initial progressive increase in percent aggregation during the first 120 min followed by stable response for additional 60 min. Similar changes were observed when gel filtered platelets were stimulated with the threshold doses of epinephrine alone or ADP in the presence of fibrinogen (600 vg/ml> (data not shown). DISCUSSION Several methods have been developed to prepare washed platelets which were separated from plasma proteins (l-5). Recently Radomski and coworkers reported that addition of PGI2 at the initial step of blood sampling and also subsequent each step of centrifugation could prevent platelet activation and resulted in good preservation of platelet function (6,7,10). However the washing procedure reported by them was rather complicated because repeated centrifugation and addition of PGIz at each step of centrifugation and washing were required. Timmons et al. described a method for the preparation of washed platelets using albumin cushion-gel filtration technique which could provide concentrated washed platelet suspension from a relatively small amount of blood (5). They employed albumin cushion technique for the protection of the platelets from large forces of centrifugation, though PGI, was not used. The

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gel filtered platelets prepared by their method had low sensitivity to several agonists such as ADP and epinephrine. The poor preservation of washed platelet function suggests that the platelets might be activated during washing procedure. So we have investigated whether PGI2 would be useful in preventing platelet activation during the separation from plasma using albumin cushion-gel filtration technique. The morphological change of platelet shape after washing procedure was analyzed by scanning electron microscopy to estimate platelet activation during the process of platelet separation. When gel filtered platelets were prepared without PGI2, most of the platelets possessed numerous pseudopods and were spherical shape. These morphological changes suggest that the gel filtered platelets were activated during washing procedure. Then the effect of addition of PGI2 (0.05-5.0 ng/ml blood) at blood sampling on the shape of gel filtered platelets was studied. The number of pseudopods of gel filtered platelets decreased and the shape of platelets became discoid with increasing the dose of PGI2. When gel filtered platelets were prepared with 2.0 pg PGIz/ml blood, most of the platelets were discoid shape with no pseudopods or one or two pseudopods. In addition, no further improvement in the shape of gel filtered platelets was observed by increasing the doses of PGI2 up to 5 pg/ml blood. Therefore, the present electron microscopic analysis indicates that the dose of 2 ng PGIz/ml blood might be sufficient to inhibit platelet activation during washing process and to preserve platelet shape. Total platelet yield of this method was 92.4 +11.8% and this value is similar to that reported by Radomski et al. (7). The protein concentration in the suspension of gel filtered platelets (3x10' platelets) was 41231 ug/ml similar to the value of 27*47 ng/ml reported by Robertson et al. (11). As one of the indices of platelet functions, the aggregatory responses of gel filtered platelets to several pro-aggregatory agents such as collagen, epinephrine, thrombin and ADP were studied (6). These gel filtered platelets demonstrated a dose-dependent aggregation to collagen and epinephrine at similar concentrations to those used in PRP. ADP induced platelet aggregation in a dose-related manner and the aggregatory response to ADP was potentiated by the prior addition of fibrinogen as reported by Radomski et al. (7). These results indicate that the aggregability of gel filtered platelets may be well preserved after the separation from plasma. An interesting finding is that aggregatory response to threshold dose of collagen was somewhat lower over the first 60-90 min and afterwards reached to the plateau level and maintained its level up to 180.min and tended to decline gradually. Similar results were obtained when platelets were stimulated with epinephrine or BP. The present results resemble with that reported by Radomski et al. (7), and this initial insensitivity seems to be most likely due to cytoprotective effect of PGI2. Therefore, in the present investigation, aggregation study of gel filtered platelets was performed between 120 and 180 min after blood sampling. In conclusion, the present combined method for the separation of platelets from plasma proteins can provide the washed human platelets which are morphologically intact and sensitive to various agonists and are suitable for physiological, biochemical and pharmacological studies.

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ACKNOWLEDGEMENT We are greatly indebted to Dr. H. Arita and Mr. N. Sunagawa of Shionogi Research Laboratories for their helpful discussion and technical assistance of scanning electron microscopy. REFERENCES 1.

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WALSH, P.M. Albumin density gradient separation and washing of platelets and the study of platelet coagulant activities. Brit. J. 22 205-217, 1972. Haematol., __,

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MUSTARD, J.F., PERRY, D.W., ARDLIE, N.H. and PACKHAM, M.A. Preparation of suspensions of washed platelets from humans. Brit. J. Haematol., 22, 193, 1972.

4.

LAGES, B., SCRUTTON, M.C. and HOLMSEN, H. Studies on gel-filtered human platelets: isolation and characterization in a medium containing no added Ca2+, Mg2+, or F?. J. Lab. Clin. Med., 85, 811, 1975.

5.

TIMMONS, S. and HAWIGER, J. Separation of human platelets from plasma proteins including factor VIIIVWF by combined albumin gradient-gel filtration method using Hepes buffer. Thromb. Es., l2, 297-306, 1978.

6.

VARGAS, J.R., RADOMSKI, M. and MONCADA, S. The use of prostacyclin in the separation from plasma and washing of human platelets. Prostaglandins, 22, 929-945, 1982.

7.

RADOMSKI, S. and MONCADA, S. An improved method for washing of human platelets with prostacyclin. Thromb. Res., 30, 383-389, 1983.

8.

HIRAI, A., TERANO, T., HAMAZAKI, T., SAJIKI, J., KONDO, S., OZAWA, A., FUJITA, T., MIYAMOTO, T., TAMDRA, Y. and KUMAGAI, A. The effect of the oral administration of fish oil concentrate on the release and the metabolism of [ 14C]arachidonic acid and [14C]eicosapentaenoic acid by Res > -3 28 285-298, 1982. human platelets. Thromb. __.__!

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BORN, G.V.R. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature, 13, 927, 1962.

10.

READ, N.G., RADOMSKI, M.W., GOODWIN, D.A. and MONCADA, S. An ultrastructural study of stored human platelets after washing using prostacyclin. Brit. J. Haematol., 60, 305-314, 1985.

11.

EGGERMAN, T.L., ANDERSEN, H. and ROPERTSON, R.P. Separate receptor for prostacyclin and prostaglandin E2 on human gel-filtered platelets. J.Pharmacol.__ Exp. Ther., 236, 568-573, 1986.