THROMBOSIS RESEARCH 62; 1651751991 0049-3848191 $3.00 + .OOPrinted in the USA. Copyright (c) 1991 Pergamon Press plc. All rights reserved.
PLATELET MEMBRANE GLYCOPROTEINS AND PLATELET FUNCTIONS DURING STORAGE IN THE PRESENCE OF A PROTEINASE INHIBITOR.
E. Mazoyer, B. Boizard-Boval, D. Pidard, J. Caen and J.L. Wautier. lnstitut des Vaisseaux et du Sang (IVS), INSERM UlSO-UA 334 CNRS, Hopital Lariboisiere, 8 rue Guy Patin, Paris 75010. France (Received 31.5.1987; accepted in revised form 7.1 .1991 by Editor M.C. Boffa) ABSTRACT The effect of the proteinase inhibitor aprotinin on membrane glycoproteins and functions of platelets stored for 5 days in platelet-rich plasma was tested. Platelet membrane glycoprotein content was determined by flow cytometry or immunoblot techniques using different monoclonal antibodies. ADP- and ristocetin-induced platelet aggregation and adhesion to collagen were tested in parallel. Using the flow-cytometry technique i) a progressive decrease in the percentage of platelets reacting with the different monoclonal antibodies was observed during storage ii) a 30% reduction of the GPlb mean fluorescence intensity (MFI) was observed after 5 days storage while the MFI of the GP Ilb-llla complex was not modified. Using the immunoblot technique, a decrease in the amount of both the GPlba and the component of Mr 100,000 was observed, while a 50,000 Mr fragment appeared progressively. Platelet adhesion and aggregation were reduced after 24 hours of storage. Aprotinin prevented neither the GPlba reduction nor the modifications of the functions of human platelets stored in their autologous plasma.
INTRODUCTION Platelet preservation has for a long time been the subject of an active debate. Different interfering factors and storage conditions have been widely studied (1,2). However, changes that occured on platelets have not yet been clearly explained. Platelets stored at room temperature rapidly lose their ability to respond in vitro to aggregating agents but survive normally when reinfused in vivo. Few reports stated the structural modifications of the platelet plasma membrane glycoproteins. Initially, after 3 days of storage at room temperature, a loss of the glycoprotein lb (GPlb), mesured by SDS-PAGE technique (3.4). a decrease in iodination of the GPlb as well as a decrease in thrombin binding to platelets (5) were reported. Such observations on a progressive toss of the GPlb were reported recently for platelets stored in autologous plasma (6). However, it has been also reported that no change of the GPlb following storage at room temperature in platelet concentrate could be observed when measured either by lectin (7) or by monoctonal antibodies bindings (8). In fact, the hypothesis of a cleavage of GPlb by a protease has been proposed. Platelets themselves contain a calcium-dependant protease; leukocytes may contaminate platelet-rich plasma and provide a source of proteolytic enzyme that could alter platelet surface; plasmin, trypsin and chymotrypsin have also been involved in the degradation of the GPb. KEY WORDS: Platelet storage, glycoprotein lb and Ilb-llla, 165
proteinase inhibitor (aprotinin).
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Aprotinin, a proteinase inhibitor, has been shown to inhibit plasmin in plasma (9,lO). The addition of aprotinin at a concentration of 400 KIU (Kallikrein Inactivator Units) per ml of blood (11) was shown to reduce markedly the formation of platelet microaggregates in stored whole blood. In banked blood, aprotinin was shown to inhibit the release of cellular mediators and enzymes as serotonin, histamine, LDH (12). It was suggested (12,13) that the proteinase inhibitor, aprotinin, exerts a membrane stabilizing effect on blood cells, especially on platelets. However, no direct evidence for such membrane stabilizing effect of aprotinin on blood cells under storage conditions has been presented so far, and the possible mechanism of action has remained a subject of speculation. To further investigate platelets modifications under storage conditions, their close relation and the role of aprotinin, two techniques were used: an indirect immunofluorescence technique with a study of the expression of monoclonal antibodies binding to the platelet membrane glycoproteins GPlb and GP Ilb-llla and an immunoblot technique with quantification of the GPlb by a monoclinal antibody against the GPlbu subunit; as a comparison, the response of stored platelets to ADP and ristocetin and platelet adhesion to collagen were investigated.
MATERIALS
AND METHODS
Whole blood (400 ml) was withdrawn from volunteer donors of the Blood Bank and was collected in a quadruple bag containing 63 ml of CPD in the first bag only. All satellite bags designated PL 1240 were made of new material containing a tri (2-ethylhexyl) trimetillate plasticizer (TEHTM) (supplied by Travenol Laboratory, 36400 La Chatre, France). Plateletrich plasma (PRP) was prepared by centrifugation of whole blood at 2000g for 3.5 minutes at room temperature. The PRP was then transferred into 2 satellite bags in an equal final mean volume of 109f3.5 ml. A sterile “sampling site coupler” (Travenol Laboratory) was adapted to each bag for daily sampling. The bags were stored at 20°C for 5 days and continuously stirred with a rotator revolving at 6 rpm (PR-1, Fenwall Laboratory, Deerfield, IL). Each day a 14 ml aliquot was removed. Platelet count and pH determination of the PRP, platelet aggregation, platelet adhesion, indirect immunofluorescence and immunoblot tests were performed.
Aprotinin substance (Bayer AG, FRG) was dissolved under sterile conditions in saline. The filtered solution (lml) (0.22 pm Millipore, France) was then injected into one of the two bags. The final concentration was 7000 KIU of aprotinin per ml of PRP (150pM). No supplement was injected into the control bag.
Each day, platelets were counted under phase contrast microscopy after dilution in a Malassez cell and the pH was detemined using a standard pH meter (Metrohm AG, Switzerland).
Platelet aggregation was performed in a Chrono-Log aggregometer (Chrono-Log Corporation, Dual Aggro-meter). Two aggregating reagents were used: ADP (Sigma, final concentration from 1.2 pLM to 100 PM); ristocetin (Sigma, final concentration 1 mglml). y Platelet adhesion to collagen was performed using a technique previously described (14). ’ PRP (10 ml) was centrifuged for 15 Platelet preparation and labelling: minutes at 1500 g at room temperature after the addition of PGEl (100 nM, Sigma). Platelet concentrate was resuspended into 2 ml of platelet-poor plasma (PPP) and incubated for 40 minutes at room temperature with 80 PC/ of Sodium Chromgte 5lCr (CEA, Orsay, France). Labelled platelets were washed in a buffer (36 mM Citric Acid, ;5 mM Glucose, 5 mM KCI, 2mM CaCl2, 1mM MgCl2, 103mM NaCI, 0.35% BSA, 1OOnM PGEI, pH 8.5) and resuspended in Tyrode’s buffer(pH 7.4) with BSA (0.35%) at a final concentration of 400 000 plateletslpl.
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Platelet adhesion: The 51Cr-labelled platelet suspension (0,4ml) was incubated at 33°C for 150 seconds without stirring either in the absence (control) or the presence of 50 ug of fibrillar collagen (Stago, France). The mixture was transferred at room temperature onto a column packed with Sepharose 28 (Pharmacia Fine Chemical, Upsala , Sweden) and eluted with the same Tyrode’s buffer containing BSA. Ffve fractions of lml were collected and the radioactivity was measured in a gamma counter (Gamma 7000, Beckman, France). The percentage of adherent platelets to collagen was calculated as follows : 100(x-y)/x. (x = cpm in fractions 1 to 5 of control platelets, y P cpm in fractions 1 to 5 of platelets incubated with collagen). m Platelet immunofluorescence tests were performed according to a technique previously described (1516). Platelet-specific antibodies: Platelet glycoproteins were characterized using monoclonal antibodies directed against: the glycocalycin part of the glycoprotein lb: APl(supplied by T.Kunickt, the Blood Center of SE Wisconsin, Milwaukee, WI, USA) (17), AN51 (supplied by G.Tobelem, U150 Inserm, Paris France) (16) and 6Dl (supplied by B.Coller, SUNY at Stony Brook, Stony Brook, NY, USA) (19); the glycoprotein Ilb-llla complex, but unresponsive to individual glycoproteins: AP-2 (supplied by D.Pidard, Ul50 Inserm, Paris, France) (20); the glycoprotein Illa: AP-3 (supplied by P. Newman, The Blood Center of WE Wisconsin, Milwaukee, WIJSA) (21). All these monoclonal antibodies were tested under saturation conditions using dilutions which yielded the highest fluorescence intensity after binding to normal platelets. Antiglobulins: A goat anti-mouse immunoglobulin labelled with fluoresceinisothiocyanate (FITC) (Nordic-Tebu,France) was used to reveal the platelet antibody binding. The optimal dilution was determined as the dilution that gave maximal fluorescence with the antibody but no fluorescence with normal sera. Technique: Each day 10 ml of PRP was sampled from the bag for immunofluorescence tests. Platelets were prepared by centrifugation, washed three times in EDTA phosphate buffer(9mM EDTA-Na2, 26.4mM NapHPOg-2H20, 140mM NaCI, pH 7) and fixed with 1% paraformaldehyde (PFA l%, pH 7). Platelets, after three washes, were resuspended in PBSEDTA buffer at a final concentration of 200 000 /PI. Fixed platelets were incubated with each antibody for 30 minutes at 37°C. Two negative controls were used at the same time: phosphate’s buffer saline (PBS) and normal mouse Ig (Coulter Immunology). After incubation, platelets were washed and then incubated 30 minutes in the dark at room temperature, with a goat antimouse antibody conjugated with fluorescein. After two washes, the final platelet suspension was examined with a Zeiss vertical fluorescence microscope (Zeiss, Wetzlau, FRG) and the antibody binding quantified using a cytofluorograph (Ortho 50H, Boulogne, France). Flow cytometry analysis: Platelet-associated fluorescence was quantified with a cytofluorograph (Ortho 50) linked to a computerized system (computer 2150, soft Ortho, “three pitdif”). The cytofluorograph was equipped with an Argon ion Laser 16405 Spectrophysic (500mW, 466nm). Calibration was performed using ATC-labelled 2.Oum microspheres. Each sample was introduced in a fluid capillary; 10,000 platelets were analyzed. A threshold was deliberately defined excluding the first channels representing the background and the two last channels corresponding to the platelet aggregates. For each sample were calculated the percentage of fluorescent platelets and the mean fluorescence intensity (MFI) expressed as arbitrary units of fluorescence (AUF) (obtained by dividing the total fluorescence recorded by the number of fluorescent platelets analyzed) SDS-PAGE SDS-PAGE: Each day 10ml of PRP was sampled from each bag. Platelets were washed in a modified Tyrode’s buffer pH 6.5 containing apyrase (25ug/ml), PGEl (lOOnM), BSA (3.5mg/ml), leupeptine (50pg/ml) and finally resuspended in Tris 10mM NaCl 150mM EDTA 3mM pH 6.6 at the concentration of 2xl09/ml. Platelets were solubilized in the presence of SDS 2% NEM 5mM for 5 min. at 100°C. Protein concentration was determined by the method of Lowry et al (22). Single dimension SDS-PAGE was performed using a 7% acrylamide gel. All samples were diluted to the same protein concentration. Samples of 25pg of proteins were
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reduced by incubation 5 min. at 100°C with 5% Bmercaptoethanol (v/v). Electrophoris was performed overnight at 30 Volts (23). lmmunoblotting The .reduced.~ proteins were electrophoretically _ . . technique: . . .. . . . transferred from unstained polyacrylamide gels to nitroCelluIOSe membrane as reported by Kieffer et al (24). After saturation with BSA (1.5%) for 2 hours, the proteins transferred on the nitrocellulose membrane were incubated for 2 hours at room temperature firstly with a monoclonal antibody against the GPlba subunit (WM23) (25), secondly with a rabbit immunoglobulin against the mouse IgG (RAM, Nordic-Tebu, France) and finally with the 1251Protein A (CEA, France). In between the successive incubations, the nitrocellulose membrane was washed with a buffer pH 7.4 (Tris lOmM, NaCl 150mM, CaC12 2mM, MgCI2 lmM, Tween O,l%, Azide de Na 0.05%, Ficoll 400 0.04% ). Autoradiography of the dried nitrocellulose membrane was perfomed against Kodak X-OMat MA films and autoradiograph scanned using an Ultroscan laser densitometer (LKB-Production AB, Sueden). w
. .
Statistical analysis was done using Student’s unpaired or paired t test.
RESULTS PRP m
(Table 1). Storage in platelet-rich plasma (PRP) induced a 56% decrease in the platelet count from 4.5 f 0.15~108 platelets per ml the first day to 2.06 f 0.18~108 /ml the fifth day. When platelets were stored in plasma containing aprotinin, the average platelet count similarly decreased. Platelets aggregates were observed after 3 days of storage in both bags. A slight PRP alkalinisation occured (from 7.22 to 7.55) in the absence or the presence of aprotinin.
Rlstocetln-Induced platelet aggregation (Fig.1). Platelet aggregation induced by ristocetin (lmg/ml) was tested each day. The results were expressed as a percentage of the first day value(D0). After 24 hours storage, the maximal intensity of aggregation was slightly reduced compared to fresh platelets and was 5% of the initial value on the fourth day (D3). The addition of aprotinin did not modify this reduction. ADP-induced platelet aggregation. Minimal final concentration of ADP required to obtain an aggregation was recorded. Increasing concentrations of ADP were necessary ranging from 1.2uM the first day to 100uM the fifth day. The presence of aprotinin did not modify the results.
TABLE I Platelet-rich plasma (PRP) volume and platelet count during 5 days of storage. __________----------~-----~--~-~~-~~~~~~~-~~-~--Platelet count (x log/ml) Volume of PRP (ml) Storage PRP+Aprotinin PRP __________________-_-----------~-~------~-----4.10 f 0.40 4.50 f 0.15 96 f 3.5 DO 3.82 + 0.45 82f4 3.90 f 0.14 Dl 2.30 It 0.15 2.90 f 0.23 69 f 4 2.40 f 0.30 2.30 f 0.25 :3 55k.5 1.45 f 0.55 2.06 f 0.18 42 f 5.8 D4 ___-l----_-_-_l_____-_-___------~--~-Each value represent the mean f SEM. Platelet storage in PRP (n=9), platelet storage in PRP + aprotinin (n=3).
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Platelet adhesion to collagen (Fig.1). Platelet adhesion was studied during the first three days of storage. The results were expressed as a percentage of the first day vaiue(D0). A rapid alteration of the adhesion to collagen was observed after two days of storage ( 76% decrease). Aprotinin did not modify the results.
Giycoprotein lb (GPib): &&l Given the conditions of the cytofiuorograph channel selection, a decrease in the percentage of fluorescent platelets reacting with AP-1 was observed to be significantly different on the third day (02) (Fig.2). This diminution started (70.2&6%) compared with the initial value on the first day (DO) (p
PLATELET MEMBRANE GLYCOPROTEINS AND PLATELET FUNCTIONS DURING STORAGE IN THE PRESENCE OF A PROTEINASE INHIBITOR.
E. Mazoyer, B. Boizard-Boval, D. Pidard, J. Caen and J.L. Wautier. lnstitut des Vaisseaux et du Sang (IVS), INSERM UlSO-UA 334 CNRS, Hopital Lariboisiere, 8 rue Guy Patin, Paris 75010. France (Received 31.5.1987; accepted in revised form 7.1 .1991 by Editor M.C. Boffa) ABSTRACT The effect of the proteinase inhibitor aprotinin on membrane glycoproteins and functions of platelets stored for 5 days in platelet-rich plasma was tested. Platelet membrane glycoprotein content was determined by flow cytometry or immunoblot techniques using different monoclonal antibodies. ADP- and ristocetin-induced platelet aggregation and adhesion to collagen were tested in parallel. Using the flow-cytometry technique i) a progressive decrease in the percentage of platelets reacting with the different monoclonal antibodies was observed during storage ii) a 30% reduction of the GPlb mean fluorescence intensity (MFI) was observed after 5 days storage while the MFI of the GP Ilb-llla complex was not modified. Using the immunoblot technique, a decrease in the amount of both the GPlba and the component of Mr 100,000 was observed, while a 50,000 Mr fragment appeared progressively. Platelet adhesion and aggregation were reduced after 24 hours of storage. Aprotinin prevented neither the GPlba reduction nor the modifications of the functions of human platelets stored in their autologous plasma.
INTRODUCTION Platelet preservation has for a long time been the subject of an active debate. Different interfering factors and storage conditions have been widely studied (1,2). However, changes that occured on platelets have not yet been clearly explained. Platelets stored at room temperature rapidly lose their ability to respond in vitro to aggregating agents but survive normally when reinfused in vivo. Few reports stated the structural modifications of the platelet plasma membrane glycoproteins. Initially, after 3 days of storage at room temperature, a loss of the glycoprotein lb (GPlb), mesured by SDS-PAGE technique (3.4). a decrease in iodination of the GPlb as well as a decrease in thrombin binding to platelets (5) were reported. Such observations on a progressive toss of the GPlb were reported recently for platelets stored in autologous plasma (6). However, it has been also reported that no change of the GPlb following storage at room temperature in platelet concentrate could be observed when measured either by lectin (7) or by monoctonal antibodies bindings (8). In fact, the hypothesis of a cleavage of GPlb by a protease has been proposed. Platelets themselves contain a calcium-dependant protease; leukocytes may contaminate platelet-rich plasma and provide a source of proteolytic enzyme that could alter platelet surface; plasmin, trypsin and chymotrypsin have also been involved in the degradation of the GPb. KEY WORDS: Platelet storage, glycoprotein lb and Ilb-llla, 165
proteinase inhibitor (aprotinin).
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Vol. 62, No. 3
Aprotinin, a proteinase inhibitor, has been shown to inhibit plasmin in plasma (9,lO). The addition of aprotinin at a concentration of 400 KIU (Kallikrein Inactivator Units) per ml of blood (11) was shown to reduce markedly the formation of platelet microaggregates in stored whole blood. In banked blood, aprotinin was shown to inhibit the release of cellular mediators and enzymes as serotonin, histamine, LDH (12). It was suggested (12,13) that the proteinase inhibitor, aprotinin, exerts a membrane stabilizing effect on blood cells, especially on platelets. However, no direct evidence for such membrane stabilizing effect of aprotinin on blood cells under storage conditions has been presented so far, and the possible mechanism of action has remained a subject of speculation. To further investigate platelets modifications under storage conditions, their close relation and the role of aprotinin, two techniques were used: an indirect immunofluorescence technique with a study of the expression of monoclonal antibodies binding to the platelet membrane glycoproteins GPlb and GP Ilb-llla and an immunoblot technique with quantification of the GPlb by a monoclinal antibody against the GPlbu subunit; as a comparison, the response of stored platelets to ADP and ristocetin and platelet adhesion to collagen were investigated.
MATERIALS
AND METHODS
Whole blood (400 ml) was withdrawn from volunteer donors of the Blood Bank and was collected in a quadruple bag containing 63 ml of CPD in the first bag only. All satellite bags designated PL 1240 were made of new material containing a tri (2-ethylhexyl) trimetillate plasticizer (TEHTM) (supplied by Travenol Laboratory, 36400 La Chatre, France). Plateletrich plasma (PRP) was prepared by centrifugation of whole blood at 2000g for 3.5 minutes at room temperature. The PRP was then transferred into 2 satellite bags in an equal final mean volume of 109f3.5 ml. A sterile “sampling site coupler” (Travenol Laboratory) was adapted to each bag for daily sampling. The bags were stored at 20°C for 5 days and continuously stirred with a rotator revolving at 6 rpm (PR-1, Fenwall Laboratory, Deerfield, IL). Each day a 14 ml aliquot was removed. Platelet count and pH determination of the PRP, platelet aggregation, platelet adhesion, indirect immunofluorescence and immunoblot tests were performed.
Aprotinin substance (Bayer AG, FRG) was dissolved under sterile conditions in saline. The filtered solution (lml) (0.22 pm Millipore, France) was then injected into one of the two bags. The final concentration was 7000 KIU of aprotinin per ml of PRP (150pM). No supplement was injected into the control bag.
Each day, platelets were counted under phase contrast microscopy after dilution in a Malassez cell and the pH was detemined using a standard pH meter (Metrohm AG, Switzerland).
Platelet aggregation was performed in a Chrono-Log aggregometer (Chrono-Log Corporation, Dual Aggro-meter). Two aggregating reagents were used: ADP (Sigma, final concentration from 1.2 pLM to 100 PM); ristocetin (Sigma, final concentration 1 mglml). y Platelet adhesion to collagen was performed using a technique previously described (14). ’ PRP (10 ml) was centrifuged for 15 Platelet preparation and labelling: minutes at 1500 g at room temperature after the addition of PGEl (100 nM, Sigma). Platelet concentrate was resuspended into 2 ml of platelet-poor plasma (PPP) and incubated for 40 minutes at room temperature with 80 PC/ of Sodium Chromgte 5lCr (CEA, Orsay, France). Labelled platelets were washed in a buffer (36 mM Citric Acid, ;5 mM Glucose, 5 mM KCI, 2mM CaCl2, 1mM MgCl2, 103mM NaCI, 0.35% BSA, 1OOnM PGEI, pH 8.5) and resuspended in Tyrode’s buffer(pH 7.4) with BSA (0.35%) at a final concentration of 400 000 plateletslpl.
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Platelet adhesion: The 51Cr-labelled platelet suspension (0,4ml) was incubated at 33°C for 150 seconds without stirring either in the absence (control) or the presence of 50 ug of fibrillar collagen (Stago, France). The mixture was transferred at room temperature onto a column packed with Sepharose 28 (Pharmacia Fine Chemical, Upsala , Sweden) and eluted with the same Tyrode’s buffer containing BSA. Ffve fractions of lml were collected and the radioactivity was measured in a gamma counter (Gamma 7000, Beckman, France). The percentage of adherent platelets to collagen was calculated as follows : 100(x-y)/x. (x = cpm in fractions 1 to 5 of control platelets, y P cpm in fractions 1 to 5 of platelets incubated with collagen). m Platelet immunofluorescence tests were performed according to a technique previously described (1516). Platelet-specific antibodies: Platelet glycoproteins were characterized using monoclonal antibodies directed against: the glycocalycin part of the glycoprotein lb: APl(supplied by T.Kunickt, the Blood Center of SE Wisconsin, Milwaukee, WI, USA) (17), AN51 (supplied by G.Tobelem, U150 Inserm, Paris France) (16) and 6Dl (supplied by B.Coller, SUNY at Stony Brook, Stony Brook, NY, USA) (19); the glycoprotein Ilb-llla complex, but unresponsive to individual glycoproteins: AP-2 (supplied by D.Pidard, Ul50 Inserm, Paris, France) (20); the glycoprotein Illa: AP-3 (supplied by P. Newman, The Blood Center of WE Wisconsin, Milwaukee, WIJSA) (21). All these monoclonal antibodies were tested under saturation conditions using dilutions which yielded the highest fluorescence intensity after binding to normal platelets. Antiglobulins: A goat anti-mouse immunoglobulin labelled with fluoresceinisothiocyanate (FITC) (Nordic-Tebu,France) was used to reveal the platelet antibody binding. The optimal dilution was determined as the dilution that gave maximal fluorescence with the antibody but no fluorescence with normal sera. Technique: Each day 10 ml of PRP was sampled from the bag for immunofluorescence tests. Platelets were prepared by centrifugation, washed three times in EDTA phosphate buffer(9mM EDTA-Na2, 26.4mM NapHPOg-2H20, 140mM NaCI, pH 7) and fixed with 1% paraformaldehyde (PFA l%, pH 7). Platelets, after three washes, were resuspended in PBSEDTA buffer at a final concentration of 200 000 /PI. Fixed platelets were incubated with each antibody for 30 minutes at 37°C. Two negative controls were used at the same time: phosphate’s buffer saline (PBS) and normal mouse Ig (Coulter Immunology). After incubation, platelets were washed and then incubated 30 minutes in the dark at room temperature, with a goat antimouse antibody conjugated with fluorescein. After two washes, the final platelet suspension was examined with a Zeiss vertical fluorescence microscope (Zeiss, Wetzlau, FRG) and the antibody binding quantified using a cytofluorograph (Ortho 50H, Boulogne, France). Flow cytometry analysis: Platelet-associated fluorescence was quantified with a cytofluorograph (Ortho 50) linked to a computerized system (computer 2150, soft Ortho, “three pitdif”). The cytofluorograph was equipped with an Argon ion Laser 16405 Spectrophysic (500mW, 466nm). Calibration was performed using ATC-labelled 2.Oum microspheres. Each sample was introduced in a fluid capillary; 10,000 platelets were analyzed. A threshold was deliberately defined excluding the first channels representing the background and the two last channels corresponding to the platelet aggregates. For each sample were calculated the percentage of fluorescent platelets and the mean fluorescence intensity (MFI) expressed as arbitrary units of fluorescence (AUF) (obtained by dividing the total fluorescence recorded by the number of fluorescent platelets analyzed) SDS-PAGE SDS-PAGE: Each day 10ml of PRP was sampled from each bag. Platelets were washed in a modified Tyrode’s buffer pH 6.5 containing apyrase (25ug/ml), PGEl (lOOnM), BSA (3.5mg/ml), leupeptine (50pg/ml) and finally resuspended in Tris 10mM NaCl 150mM EDTA 3mM pH 6.6 at the concentration of 2xl09/ml. Platelets were solubilized in the presence of SDS 2% NEM 5mM for 5 min. at 100°C. Protein concentration was determined by the method of Lowry et al (22). Single dimension SDS-PAGE was performed using a 7% acrylamide gel. All samples were diluted to the same protein concentration. Samples of 25pg of proteins were
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reduced by incubation 5 min. at 100°C with 5% Bmercaptoethanol (v/v). Electrophoris was performed overnight at 30 Volts (23). lmmunoblotting The .reduced.~ proteins were electrophoretically _ . . technique: . . .. . . . transferred from unstained polyacrylamide gels to nitroCelluIOSe membrane as reported by Kieffer et al (24). After saturation with BSA (1.5%) for 2 hours, the proteins transferred on the nitrocellulose membrane were incubated for 2 hours at room temperature firstly with a monoclonal antibody against the GPlba subunit (WM23) (25), secondly with a rabbit immunoglobulin against the mouse IgG (RAM, Nordic-Tebu, France) and finally with the 1251Protein A (CEA, France). In between the successive incubations, the nitrocellulose membrane was washed with a buffer pH 7.4 (Tris lOmM, NaCl 150mM, CaC12 2mM, MgCI2 lmM, Tween O,l%, Azide de Na 0.05%, Ficoll 400 0.04% ). Autoradiography of the dried nitrocellulose membrane was perfomed against Kodak X-OMat MA films and autoradiograph scanned using an Ultroscan laser densitometer (LKB-Production AB, Sueden). w
. .
Statistical analysis was done using Student’s unpaired or paired t test.
RESULTS PRP m
(Table 1). Storage in platelet-rich plasma (PRP) induced a 56% decrease in the platelet count from 4.5 f 0.15~108 platelets per ml the first day to 2.06 f 0.18~108 /ml the fifth day. When platelets were stored in plasma containing aprotinin, the average platelet count similarly decreased. Platelets aggregates were observed after 3 days of storage in both bags. A slight PRP alkalinisation occured (from 7.22 to 7.55) in the absence or the presence of aprotinin.
Rlstocetln-Induced platelet aggregation (Fig.1). Platelet aggregation induced by ristocetin (lmg/ml) was tested each day. The results were expressed as a percentage of the first day value(D0). After 24 hours storage, the maximal intensity of aggregation was slightly reduced compared to fresh platelets and was 5% of the initial value on the fourth day (D3). The addition of aprotinin did not modify this reduction. ADP-induced platelet aggregation. Minimal final concentration of ADP required to obtain an aggregation was recorded. Increasing concentrations of ADP were necessary ranging from 1.2uM the first day to 100uM the fifth day. The presence of aprotinin did not modify the results.
TABLE I Platelet-rich plasma (PRP) volume and platelet count during 5 days of storage. __________----------~-----~--~-~~-~~~~~~~-~~-~--Platelet count (x log/ml) Volume of PRP (ml) Storage PRP+Aprotinin PRP __________________-_-----------~-~------~-----4.10 f 0.40 4.50 f 0.15 96 f 3.5 DO 3.82 + 0.45 82f4 3.90 f 0.14 Dl 2.30 It 0.15 2.90 f 0.23 69 f 4 2.40 f 0.30 2.30 f 0.25 :3 55k.5 1.45 f 0.55 2.06 f 0.18 42 f 5.8 D4 ___-l----_-_-_l_____-_-___------~--~-Each value represent the mean f SEM. Platelet storage in PRP (n=9), platelet storage in PRP + aprotinin (n=3).
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Platelet adhesion to collagen (Fig.1). Platelet adhesion was studied during the first three days of storage. The results were expressed as a percentage of the first day vaiue(D0). A rapid alteration of the adhesion to collagen was observed after two days of storage ( 76% decrease). Aprotinin did not modify the results.
Giycoprotein lb (GPib): &&l Given the conditions of the cytofiuorograph channel selection, a decrease in the percentage of fluorescent platelets reacting with AP-1 was observed to be significantly different on the third day (02) (Fig.2). This diminution started (70.2&6%) compared with the initial value on the first day (DO) (p
