Specific Interaction of HLA Antibodies (Eluates) with Washed Platelets
(Rcceivcd I 3 April 1976; n c c c p d f ; ) r prih!icatiori I 5 Septenrber 1976) SUMMARY. The mechanism of complement-independent action of HLA-A2 antibodies (eluates) on washed platelets was investigated. HLA-specific alteration was confirmed by serological (platelet riiicro-complement fixation), morphological (platelet spreading) and functional parameters (platelet aggregation, inhibition of collagen-induced platelet aggregation, [ 1 4 C ] ~ e r ~ t o nrelease). in In the presence of fibrinogen and calcium ions, HLA antibodies induced instantaneous platelet aggregation and release. Although no morphological (spreading) and functional changes (collagen-induced aggregation) were seen, these platelets did not aggregate or release when fibrinogen was subsequently added. When platelets-in the presence of fibrinogen-were incubated with antibody concentrations too low to induce platelet aggregation or release, specific reduction of platelet reactivity was observed by subsequent collagen aggregation. HLA-specific action of antibodies on washed platelets was inhibited by apyrase and acetyl-salicylic acid, indicating an active participation of platelets in HLA antibody-induced platelet alteration. Sera of polytransfused patients have been shown to inhibit [14C]serotoninuptake by platelets (Bridges ct al, 1963), inhibit clot retraction (Shulman et a], 1964), induce platelet aggregation (Dc Gaetano ct nl, 1970), release ADP (Hirschman & Shulman, 1972),['4C]serotonin (Hirschman e f nl, 1973 ; Gockernian Pt nl, 1975), platelet factor 3 (De Gaetano et a\, 1970) and factor 4 (Praga c't al, 1974) as well as 51Cr from platelets (MacDonald et all 1975). I n the majority of tliese studies the specificity of platelet alloantibody has not been determined. However, from studies on antibody differentiation in tliese sera it can be assumed that isoimmunizatioii of polytransfused patients most probably is directed against HLA antigens (Heinrich et al, 1973). Concerning the effect of HLA antibodies on platelets two different mechanisms have been suggested: ( I ) a complement-independent action of HLA antibodies 011 platelets resulting in specific platelet aggregation and ['4C]serotonin release (Hirschman ct all 1973 ; De Gaetano c't nl, 1970); ( 3 ) a cell-dependent alteration of platelets resulting in platelet lysis (MacDonald a/, 1975). A complement-dependent action of HLA antibodies on platelets has not been established so far. Although it has been suggested that tliere exist similarities between inimunologic and non-immunologic aggregation and 'release' reaction (Shulman et 01, 1973) only scanty inCorrmpondence: Dr Dieter Heinrich, Department of Internal Medicine, Justus Liebig-University, Klinikstr. 36, D-6300Giessen, FKG. 4-41
442
D. Heinrich, U. Stephinger and C . Mueller-Eckhnrdt
formation is available as to the preconditions of complement-independent platelet alterations specifically induced by HLA-antibodies. In the present study pertinent data will be presented with special emphasis on the kinetics of antibody-induced immune reactions of human platelets and the essential role of cofactors in these reactions. MATERIALS AND METHODS Seventeen ml of fresh human blood were anticoagulated with 3 ml of ACD (Formula A). Platelet-rich plasma (PRP) was obtained by differential centrifugation for 20 min at I I O g. Platelets were washed according to Mustard et a/ (1972) with minor modifications as depicted in Table I. TABLE I. Washing and suspending fluids
Susperision
First wash Second wash Suspending fluid
Tyrodelalbumin Tyrode/albumin Tyrodelalbumin
Heparin (Ulml)
Apyrase
25
20
-
I0
-
4
(mg%)
Temp. PH
("C)
6.5 6.S 7.3
37 37 37
HLA-Az-specific eluates were used throughout the investigation. The eluateswere obtained by absorption of an HLA-Az-specific antiserum (Raub 11) to appropriate platelets and subsequent acid elution as reported previously (Heinrich et a / , 1974). Antibody titre of serum Raub I1 in platelet micro-complernent fixation (MCF) was 1:50. A weak crossreacting activity with the antigen HLA-A was present when tested in a final dilution of 1:s. For absorption, 2.5 x 10'' platelets were incubated with I ml of antiserum yielding 0.5 in1 of eluate with an anti-HLA-A2 titre of I :20 in platelet MCF. For antibody absorption and elution and for antibody screening platelets from healthy blood donors were used. Platelets were stored at 4°C up to 3 months. Platelet donors had been HLA typed applying two techniques : micro-lymphocytotoxicity (Terasaki & McClelland, 1964) and platelet microcomplement fixation (Colombani et a/, 1971). HLA antigens identified in our laboratory, the source of lytnphocytotoxic and complement-fixing antibodies employed and the evaluation of our typing results have been reported (Mueller-Eckhardt et al, 1974). To label platelets, 2 x 10' washed platelets were incubated with 3.5 x 10-' mol ['4C]serotonin (5-hydroxytryptamine creatinine sulphate, Amersham, England). [ 14C]Serotonin was dissolved in 70% ethanol to a final concentration of 0.145 nimol corresponding to 8 LiCi/nil. Specific ['4C]serotonin release from washed platelets by HLA-Aa-specific eluates was performed in microtitration plates (Greiner, System Cooke No. M 220-24A). The reacting mixture contained 150pl of washed platelets (200 x 109plateletsll.), 10p1 of human fibrinogen (I g%), and sop1 of HLA-Az-specific eluate or phosphate-buffered saline (PBS). Each sample was incubated for 15 min under constant stirring by two steel balls (diameter z mm) which were rotated by a magnetic stirrer below the plate at 1 8 0 rpm. Incubation was stopped by addition of 50 pl of EDTA ( 5 % ) and cooling (ice water). After centrifugation for I min at
HLA Atitibodics arid Platclots
443
8000 g [ I 4C]seroto~~in release was calculatcd froni the radioactivity of the supernatant of cach sample applying the following formula :
(%) release
=
Bx - B o x T - B,
I00
where B, = radioactivity of the supernatant of a sample treated with HLA-Az-specific eluate; B, = radioactivity of the supernatant o f a control sample treated with PBS; and T = total radioactivity of the sample. Aggregation of washed platelets by HLA-Aa-specific eluates was evaluated macroscopically (microtitration plate) and nephelometrically (Born, 1962). The reaction mixture for the Born aggregometer comprised 600 pl of washed platelets (200x 109 platelets/l.), 40 p1 of human fibrinogen ( I g%), and a00 p1 of HLA-Aa-specific eluate or PBS. Parallel to HLA antibody-induced [I 4C]serotonin release and platelet aggregation, platelet sprcading was investigated according to Breddin & Burck (1963). The reactivity of washed platelets was investigated regularly by collagen- and ADP-induced platelet aggregation. Comparing the maximal velocity of collagen-induced aggregation of washed platelets with 1’RP similar results were obtained (Table 11). Evaluation of washed platelets by light (platelet spreading) and electron microscopy cxhibited a good preservation of platelet morphology and platelet function (Fig I ) .
TABLE 11. Coniparisoii of collagen-induced platelet aggregation in platelet-rich plasma (PH1’) and washed platelet suspensions
No. nf sainples 1’KP Washed plateletst ~
Maxiriial velocity oj‘collagen-induced platelet a‘qgregation (%)* Mean
SE
I7
100.0
-
I7
100.2
21.6
~~
* 3.0
1111
washed platelets (containing 50 mg% fibrinogen) or 3.0
nil of PRP were incubated with 0 . 1 5 nil of acid soluble collagen ( 5 x I O - ~ M) in an aggregonieter. Maximal velocity of platelet
aggregation with PHP was set 100% and compared with corresponding results obtained with washed platelets. t I’latelet concentrations of washed platelet suspensions were adjustrd to the concentration in the corresponding PRI’.
RESULTS
Optirnn I Platc4ct Coiln t To determine the optimal platelet number for HLA antibody-induced [‘4C]serotonin release from washed platelets, platelet suspensions of 50-500 x 109 plateletsll. were incubated with HLA-Aa-specific eluates. Optimal platelet counts were between a00 and 400 x 109 platelets/l. exhibiting 7 7 1 21% release. With 50 x 109 platelets/l. [ ‘ 4 C ] ~ e r ~ t o nrelease in was 591t26% (N = 3), with 500x 109 platelets/l. 4 9 1 2 8 % (N= 3).
444
D. Heinrich, U. Stephitiger and C. Mtdler-Eckhardt
Optiriral Antibody Concentration When 150 p1 of washed platelets were incubated with increasing volumes of an HLA-Azspecific eluate (titre 1:20) optimal ['4C]serotonin release was obtained with 50 p1 eluate (90f4%; N = 3). Addition of IOO pl eluate exhibited considerably less release ( 6 6 ) 7:
(Rcceivcd I 3 April 1976; n c c c p d f ; ) r prih!icatiori I 5 Septenrber 1976) SUMMARY. The mechanism of complement-independent action of HLA-A2 antibodies (eluates) on washed platelets was investigated. HLA-specific alteration was confirmed by serological (platelet riiicro-complement fixation), morphological (platelet spreading) and functional parameters (platelet aggregation, inhibition of collagen-induced platelet aggregation, [ 1 4 C ] ~ e r ~ t o nrelease). in In the presence of fibrinogen and calcium ions, HLA antibodies induced instantaneous platelet aggregation and release. Although no morphological (spreading) and functional changes (collagen-induced aggregation) were seen, these platelets did not aggregate or release when fibrinogen was subsequently added. When platelets-in the presence of fibrinogen-were incubated with antibody concentrations too low to induce platelet aggregation or release, specific reduction of platelet reactivity was observed by subsequent collagen aggregation. HLA-specific action of antibodies on washed platelets was inhibited by apyrase and acetyl-salicylic acid, indicating an active participation of platelets in HLA antibody-induced platelet alteration. Sera of polytransfused patients have been shown to inhibit [14C]serotoninuptake by platelets (Bridges ct al, 1963), inhibit clot retraction (Shulman et a], 1964), induce platelet aggregation (Dc Gaetano ct nl, 1970), release ADP (Hirschman & Shulman, 1972),['4C]serotonin (Hirschman e f nl, 1973 ; Gockernian Pt nl, 1975), platelet factor 3 (De Gaetano et a\, 1970) and factor 4 (Praga c't al, 1974) as well as 51Cr from platelets (MacDonald et all 1975). I n the majority of tliese studies the specificity of platelet alloantibody has not been determined. However, from studies on antibody differentiation in tliese sera it can be assumed that isoimmunizatioii of polytransfused patients most probably is directed against HLA antigens (Heinrich et al, 1973). Concerning the effect of HLA antibodies on platelets two different mechanisms have been suggested: ( I ) a complement-independent action of HLA antibodies 011 platelets resulting in specific platelet aggregation and ['4C]serotonin release (Hirschman ct all 1973 ; De Gaetano c't nl, 1970); ( 3 ) a cell-dependent alteration of platelets resulting in platelet lysis (MacDonald a/, 1975). A complement-dependent action of HLA antibodies on platelets has not been established so far. Although it has been suggested that tliere exist similarities between inimunologic and non-immunologic aggregation and 'release' reaction (Shulman et 01, 1973) only scanty inCorrmpondence: Dr Dieter Heinrich, Department of Internal Medicine, Justus Liebig-University, Klinikstr. 36, D-6300Giessen, FKG. 4-41
442
D. Heinrich, U. Stephinger and C . Mueller-Eckhnrdt
formation is available as to the preconditions of complement-independent platelet alterations specifically induced by HLA-antibodies. In the present study pertinent data will be presented with special emphasis on the kinetics of antibody-induced immune reactions of human platelets and the essential role of cofactors in these reactions. MATERIALS AND METHODS Seventeen ml of fresh human blood were anticoagulated with 3 ml of ACD (Formula A). Platelet-rich plasma (PRP) was obtained by differential centrifugation for 20 min at I I O g. Platelets were washed according to Mustard et a/ (1972) with minor modifications as depicted in Table I. TABLE I. Washing and suspending fluids
Susperision
First wash Second wash Suspending fluid
Tyrodelalbumin Tyrode/albumin Tyrodelalbumin
Heparin (Ulml)
Apyrase
25
20
-
I0
-
4
(mg%)
Temp. PH
("C)
6.5 6.S 7.3
37 37 37
HLA-Az-specific eluates were used throughout the investigation. The eluateswere obtained by absorption of an HLA-Az-specific antiserum (Raub 11) to appropriate platelets and subsequent acid elution as reported previously (Heinrich et a / , 1974). Antibody titre of serum Raub I1 in platelet micro-complernent fixation (MCF) was 1:50. A weak crossreacting activity with the antigen HLA-A was present when tested in a final dilution of 1:s. For absorption, 2.5 x 10'' platelets were incubated with I ml of antiserum yielding 0.5 in1 of eluate with an anti-HLA-A2 titre of I :20 in platelet MCF. For antibody absorption and elution and for antibody screening platelets from healthy blood donors were used. Platelets were stored at 4°C up to 3 months. Platelet donors had been HLA typed applying two techniques : micro-lymphocytotoxicity (Terasaki & McClelland, 1964) and platelet microcomplement fixation (Colombani et a/, 1971). HLA antigens identified in our laboratory, the source of lytnphocytotoxic and complement-fixing antibodies employed and the evaluation of our typing results have been reported (Mueller-Eckhardt et al, 1974). To label platelets, 2 x 10' washed platelets were incubated with 3.5 x 10-' mol ['4C]serotonin (5-hydroxytryptamine creatinine sulphate, Amersham, England). [ 14C]Serotonin was dissolved in 70% ethanol to a final concentration of 0.145 nimol corresponding to 8 LiCi/nil. Specific ['4C]serotonin release from washed platelets by HLA-Aa-specific eluates was performed in microtitration plates (Greiner, System Cooke No. M 220-24A). The reacting mixture contained 150pl of washed platelets (200 x 109plateletsll.), 10p1 of human fibrinogen (I g%), and sop1 of HLA-Az-specific eluate or phosphate-buffered saline (PBS). Each sample was incubated for 15 min under constant stirring by two steel balls (diameter z mm) which were rotated by a magnetic stirrer below the plate at 1 8 0 rpm. Incubation was stopped by addition of 50 pl of EDTA ( 5 % ) and cooling (ice water). After centrifugation for I min at
HLA Atitibodics arid Platclots
443
8000 g [ I 4C]seroto~~in release was calculatcd froni the radioactivity of the supernatant of cach sample applying the following formula :
(%) release
=
Bx - B o x T - B,
I00
where B, = radioactivity of the supernatant of a sample treated with HLA-Az-specific eluate; B, = radioactivity of the supernatant o f a control sample treated with PBS; and T = total radioactivity of the sample. Aggregation of washed platelets by HLA-Aa-specific eluates was evaluated macroscopically (microtitration plate) and nephelometrically (Born, 1962). The reaction mixture for the Born aggregometer comprised 600 pl of washed platelets (200x 109 platelets/l.), 40 p1 of human fibrinogen ( I g%), and a00 p1 of HLA-Aa-specific eluate or PBS. Parallel to HLA antibody-induced [I 4C]serotonin release and platelet aggregation, platelet sprcading was investigated according to Breddin & Burck (1963). The reactivity of washed platelets was investigated regularly by collagen- and ADP-induced platelet aggregation. Comparing the maximal velocity of collagen-induced aggregation of washed platelets with 1’RP similar results were obtained (Table 11). Evaluation of washed platelets by light (platelet spreading) and electron microscopy cxhibited a good preservation of platelet morphology and platelet function (Fig I ) .
TABLE 11. Coniparisoii of collagen-induced platelet aggregation in platelet-rich plasma (PH1’) and washed platelet suspensions
No. nf sainples 1’KP Washed plateletst ~
Maxiriial velocity oj‘collagen-induced platelet a‘qgregation (%)* Mean
SE
I7
100.0
-
I7
100.2
21.6
~~
* 3.0
1111
washed platelets (containing 50 mg% fibrinogen) or 3.0
nil of PRP were incubated with 0 . 1 5 nil of acid soluble collagen ( 5 x I O - ~ M) in an aggregonieter. Maximal velocity of platelet
aggregation with PHP was set 100% and compared with corresponding results obtained with washed platelets. t I’latelet concentrations of washed platelet suspensions were adjustrd to the concentration in the corresponding PRI’.
RESULTS
Optirnn I Platc4ct Coiln t To determine the optimal platelet number for HLA antibody-induced [‘4C]serotonin release from washed platelets, platelet suspensions of 50-500 x 109 plateletsll. were incubated with HLA-Aa-specific eluates. Optimal platelet counts were between a00 and 400 x 109 platelets/l. exhibiting 7 7 1 21% release. With 50 x 109 platelets/l. [ ‘ 4 C ] ~ e r ~ t o nrelease in was 591t26% (N = 3), with 500x 109 platelets/l. 4 9 1 2 8 % (N= 3).
444
D. Heinrich, U. Stephitiger and C. Mtdler-Eckhardt
Optiriral Antibody Concentration When 150 p1 of washed platelets were incubated with increasing volumes of an HLA-Azspecific eluate (titre 1:20) optimal ['4C]serotonin release was obtained with 50 p1 eluate (90f4%; N = 3). Addition of IOO pl eluate exhibited considerably less release ( 6 6 ) 7:
