British lourrial ofHaeniatology, 1982, 50, 351-359

Serological and biochemical analysis of the PIA1 alloantigen of human platelets

JUDY LANE, MELISSA BROWN, IRWIN BERNSTEIN, PAULA K. WILCOX, SHERRILL J. SLICHTER ANDROBERT C. NOWINSKI FredHutchinson Cancer Research Center and the Puget Sound Blood Center, Seattle, Washington

Received 29 April 7 981; accepted forpublication 1 OJulg 1981

SUMMARY. A solid phase platelet antibody assay has been developed which rapidly and sensitively detects PIA1 antibodies. The three-step assay is performed by: (1) adhering platelets to the wells of a microtitre plate, (2) incubating the platelets with test serum, and ( 3 ) adding radiolabelled Staphylococcal protein A which binds to the Fc domain of IgG antibodies. Immune reactions are detected by overnight autoradiography .

Characterization of the PIA1 antigen was performed by using PIA1 antisera in immune precipitation assays. A 90 000 dalton molecular weight species was precipitated from PIA’ positive human and dog platelets.

Human histocompatibility antigens (Colombani et al, 1967; Svejgaard, 1969), red cell antigens (Pfisterer et al, 1968; Aster, 1965; Duquesnoy et al, 1979) and platelet specific antigens DUZOa+ (Moulinier, 19581, PIA1 (van Loghem et al, 1959; Shulman, 1961), PIAL (van der Weerdt et al, 1963), Koa (van der Weerdt et al, 1962), Kob (van der Weerdt, 1965a, b), PIE1 and PlE2 (Shulman et al, 1964), and recently reported Bak” (von dem Borne et al, 1980) are represented on the platelet surface. The platelet specific antigen PIA1 carried by 98% of Caucasians is the most common antigen found in platelet specific antibody problems. PIA1 negative individuals are subject to immunization to PIA1 antigens through either transfusion or pregnancy. Occasionally these individuals develop post-transfusion purpura, a syndrome characterized by profound thrombocytopenia 5-1 0 d following a transfusion of incompatible platelets (Shulman, 1961). Also, pregnancy can be complicated by the production of an alloantibody to incompatible paternal antigens on fetal platelets resulting in neonatal isoimmune thrombocytopenia (Shulman et al, 1962). Over 90% of the reported cases of post-transfusion purpura (Zeigler et al, 1975) and neonatal isoimmune thrombocyto- penia (Shulman et a2,1964; von dem Borne et al, 1981) are caused by incompatibilities for the PIA1 antigen.

Correspondence: Ms Judy Lane, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle. Washington 98104, U.S.A.

0007-1048/82/0200-0351$02.00 @ 1982 Blackwell Scientific Publications

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3 52 Iudy Lane et a1

The antibodies have usually been complement-fixing, although blocking antibodies have been described (Shulman et al, 1964). PIA1 antibodies have often been only weakly positive in antiglobulin consumption, agglutination, and inhibition of clot retraction methods (Shul- man et al, 1964; Kissmeyer-Nielsen & Jensen, 1969; von dem Borne et al, 1981) but have been reliably measured by 51chromium lysis (Abramson et al, 19 74), immunofluorescence tests (von dem Borne et al, 1981), and platelet radioactive antiglobulin (Soulier et al, 1979).

We have used the blood from a recent case of post-transfusion purpura to develop a platelet antibody assay system in which radiolabelled Staphylococcal protein A is used to detect IgG molecules by binding to the Fc domain (Goding, 1978). In addition, we have used this antiserum to characterize further the PlA1 antigen which was identified earlier as a membrane glycoprotein of subclass IIla (Kunicki & Aster, 1978, 1979).

MATERIALS AND METHODS

Development of antibody assay

Platelet antibody samples. Plasma (or serum) from a patient (E.M.) with post-transfusion purpura was used as a source of PIA1 antibody. E.M., a 32-year-old female, was admitted to the hospital after sustaining multiple fractures in an automobile accident. Four units of blood were administered over the 2 d following surgery. On day 8 epistaxis developed and widespread petechiae and ecchymoses were observed on day 9. Over the next 2 d, 16 platelet concentrates prepared from random blood donors were transfused without a rise in platelet count. The patient had no history of pregnancy, but she had received two units of blood 7 years previously for an undiagnosed anaemia. A diagnosis of post-transfusion purpura was made on the basis of history, a bone marrow showing increased number and size of megakaryocytes, and positive platelet antibody test. E.M.’s platelets were typed as PIA1 negative through the courtesy of Dr Richard Aster of the Milwaukee Blood Center, Milwaukee, Wisconsin.

An additional reference alloantiserum from a multiply transfused patient (A. W.), with known anti-PIA1 antibody, was also provided by Dr Aster.

Platelets from two patients (K.B. and P.M.) who were delivered of babies with neonatal isoimmune thrombocytopenia were utilized to further assess the specificity of antibodies in the plasma of patients E.M. and A.W.

Plasma samples were obtained from acid citrate dextrose (ACD) solution (USP Formula B, Travenol Lab. Inc., Deerfield, Ill.) anticoagulated blood (1 ml ACD/9 ml blood) while serum was obtained from clotted blood. Pooled plasma or serum samples from random, non-transfused male group A or AB donors were used as controls.

Platelet collection. Platelets were collected from patients and normal donors into either ACD solution or 3.8% (w/v) sodium citrate solution (1 ml of anticoagulant per 9 ml of blood). An additional 0.1 ml of citrate was added to reduce the pH to 6.5 to reduce clumping during washing and resuspension. The blood was centrifuged at 300 g for 10 min and the upper two-thirds of the platelet-rich plasma (PRP) was removed. Examination of 20 PRPs from normal individuals showed no contaminating red or white cells. The platelets were

Analpis of the PIA1 Alloantigen 3 5 3

sedimented at 1800 g for 10 min and then washed twice in acidified Ringer’s citrate dextrose (RCD, pH 6.5) (Abramson, 1964) prior to resuspension in RCD at a concentration of 2 x lo7 platelets per ml. Platelets stored in RCD for up to 3 d at 4°C performed appropriately in the test system.

12SI-ZabeZZed protein A. Purified Staphylococcal protein A (Pharmacia Ltd, Uppsala, Sweden), was radioiodinated with Na1251 (New England Nuclear, Boston, Massachusetts) by the chloramine-T method (Greenwood et aZ, 1963). This iodinated protein A (IPA) was stored at - 80°C and used within 2 weeks of preparation.

Antibody-binding (AB) assay. Platelet antibodies in plasma (or serum) were detected by a binding assay with 1251-labelled protein A (IPA). For the AB assay, loh platelets in 50 pl of RCD were dispensed into the individual wells of a Linbro tissue culture plate (Flow, Hamden, Connecticut). Platelets adhered to the bottom of the wells after centrifugation at 1800 g for 10 min at room temperature. Non-adherent platelets were then removed by washing the plates with RCD. The wells of the plate were blocked from further non-specific protein adsorption by 30 min incubation with 5% human serum albumin (HSA, w/v) in RCD pH 6.5. The AB assay was performed in three steps: (1) 50 p1 of plasma or serum diluted in 2% bovine serum albumin (BSA, w/v) in RCD pH 7.2 was incubated in each of the platelet-coated wells for 45 min at 37°C. Non-bound immunoglobulins and other serum proteins were then removed from the wells by washing five times with RCD; ( 2 ) lo5 cpm of IPA in 50 pl of RCD containing 2% BSA was added to each of the platelet-coated wells for 45 min at 37°C. The non-bound IPA was then removed from the w e b by washing five times with RCD; ( 3 ) the immune reactions were detected by autoradiography of the plate for 16 ha t - 80°C on Kodak NS-2T film with enhancement by X-ray intensifying screens. If desired, soft laser densitometry could be employed for quantification of the autoradiogram.

Immune precipitation of cell extracts

Radiolabelling of cell membranes by the lZ5I lactoperoxidase method was performed by the method of Vitetta et aZ (1971). Briefly, 108 platelets were suspended in 1 ml of phosphate buffered saline (PBS, pH 7-2) containing 100 mg of lactoperoxidase and 3 mCi of 1251. This mixture was activated by adding pulses of 0.06% H202 at 5 min intervals. The entire labelling procedure was performed on ice with continuous shaking. The labelled platelets were then washed to remove free lzSI and disrupted in PBS with gentle vortex mixing in 0.5% Nonidet P-40 (NP40) for 30 min at 0°C. Non-solubilized cellular structures were removed by centrifugation at 100 000 g for 45 min.

Radioimmune precipitation (RIP) assay system consisted of 50 p1 of lZ5I labelled platelet extract ( lo7 cpm) and 2 pl of antiserum in a total volume of 200 pl of PBS containing 0.5% NP40. Reactions were terminated after a 1-h incubation at 0°C by the addition of 0.5 mg of formalin-fixed S. aureus. After a 30 min incubation at O O C , the bacteria and their bound immune complexes were removed by centrifugation through a cushion of 5% sucrose (in PBS) containing 3% NP40. The bacteria were then washed three additional times in PBS with 0.5% NP40 and the immune complexes eluted into sodium dodecyl sulphate (SDS)-contain-

3 54 Judy Lane et a1

ing sample buffer (3% SDS, 5% 2-mercaptoethanol, and 10% glycerol in 0.06 M Tris-HC1 (pH 6.8)) by incubation at 100°C for 2 min.

Polyacrylamide gel electrophoresis (PAGE) was performed by the method of Laemmli (1 9 70) in 15% polyacrylamide slab gels. Molecular weight markers for these gels included 1751 labelled proteins of murine leukaemia viruses:gp70 (70 000 daltons), p30 (30 000 daltons), and p15 (15 000 daltons).

RESULTS

Detection of platelet antibodies by the A B assay

The AB assay proved to be a rapid and sensitive method for the analysis of platelet antibodies in human plasma and serum. The AB assay was sufficiently sensitive to detect 1000 cpm of specifically bound IPA: control wells treated with IPA alone routinely bound less than 300 cpm of IPA. Fig 1 shows a representative autoradiogram derived from an AB assay performed with the platelets of a random donor and the plasma of patient E.M. For comparison, this assay also includes a parallel titration with a control specimen of ACD plasma. Dramatic differences were observed in the reactions of these two plasmas: the E.M. plasma had a titre of 1/1280, while the control plasma was non-reactive at a 1/10 dilution. Analysis of this autoradiogram by densitometry demonstrated a signal-to-noise ratio of > 100: 1 for the reactions with the plasma of E.M.

Serological identification of anti-PIA1 antibodies in human plasma

Since the plasma sample from E.M. was obtained during a period of post-transfusion purpura, it was presumed that the platelet antibodies had anti-PIA1 specificity (Zeigler et al, 1975). Accordingly, plasma E.M. was compared in the AB assay with a reference plasma (patient A.W.) known to contain anti-PIA1 antibodies against a panel of platelets from 127 random donors. Striking concordance was observed, with 124/127 (98%) of the platelet panel reacting with both E.M. and A.W. The remaining three platelet specimens (3/127; 2%) did

Fig 1. Antibody-binding (AB) assays with human platelets. Platelets from pooled random donors were made adherent to the wells of microtest plates by centrifugation. The adherent platelets served as solid phase antigens for detecting platelet antibodies. Serial dilutions of plasma samples from patient E.M. and a randomly selected ACD control were incubated in the antigen adsorbed wells. Immune reactions were detected by subsequent incubation with '251-labelled protein A, followed by overnight autoradiography of the test plate.

Analysis of the PIA1 Alloantigen 3 5 5

not react with either plasma sample. The ACD control plasma was consistently negative with all 127 platelet samples. Of the 124 samples reacting positively with E.M. and A.W., five showed concordantly weak reactions. Platelets from only 4/124 samples showed quantita- tive differences in their reactions with the two test plasmas.

Serological specifcity of an t i -PP antibodies

The specificity of antibodies in the plasma of patients E.M. and A.W. were investigated further by quantitative AB assays with (1) pooled platelets from random human donors (PIA1 positive controls), (2) platelets from patient E.M. (PIA1 negative control), ( 3 ) platelets from the two mothers (K.B. and P.M.) who were delivered of babies with neonatal isoimmune thrombo- cytopenia, (4) dog platelets, and (5) rat platelets.

As shown in Fig 2, antibodies in both E.M. and A.W. plasmas reacted with the platelets of random human and dog donors but not with the platelets of E.M. Furthermore, E.M. and A.W.'s plasma did not react with platelets from K.B. and P.M. or with rat platelets (data not shown). The ACD plasma control, also shown in Fig 2, did not react with any of the five platelet samples.

linmune precipitation and biochemical identification of PIA1 antigen

PIA1 antigen was initially isolated by immune precipitation using PIA1 reference antiserum

A B C

20 40 80 160 320 640 20 40 80 160 320 640 20 40 80 160 320 640

RECIPROCAL OF SERUM DILUTION

Fig 2. Serological identification of anti-PIA' antibodies by AB assays. Platelets from (A) a randomly selected donor (PIA1 positive), (B) patient E.M., and (C) a dog were tested in AB assays with plasma samples from patient E.M. ( O ) , patient A.W. ( A ) and ACD controls (0).

356 Judy Lane et a1 and detergent lysates of 1251-surface-labelled platelets. The molecular weight of the precipitated antigen was identified by polyacrylamide gel electrophoresis of the immune precipitates in SDS-containing buffer with subsequent autoradiography of the gel.

Fig 3 presents the results of two immune precipitation assays performed with a detergent lysate of 1251-labelled platelets from a random donor and plasma samples from E.M., A.W., and randomly selected plasma controls. Antibodies in both the E.M. and A.W. plasmas precipitated a single 90 000 dalton protein from the 1251-labelled platelet lysate; the ACD control plasma, on the other hand, did not precipitate radiolabelled proteins from the same platelet lysates. In other experiments (data not shown) the E.M. plasma also showed a variable precipitation of a minor 100 000 dalton protein.

Fig 4 shows the results of additional tests performed with E.M. and control plasmas. In this instance the plasmas were reacted with detergent lysates of 12Wabelled platelets from a random human donor (PIA1 positive), patient E.M. (PIA1 negative), and a dog (PIA1 positive). The results of this test showed that antibodies in the E.M. plasma precipitated a 90 000 dalton protein from the 1251-labelled platelets of the randomly selected human donor and the dog, but not from patient E.M. In addition, the E.M. plasma also precipitated a 100 000 dalton protein from the dog platelets. Also shown is that the control ACD plasma did not precipitate radiolabelled proteins from any of the 1251-labelled platelet lysates.

Fig 3. Immune precipitation of PIA1 antigen. A detergent lysate of 1251-labelled platelets from a randomly selected donor was tested in two separate immune precipitation assays with plasmas from patient E.M. (lanes 1 and 3 ) , patient A.W. (lane 4) and the randomly selected ACD control (lanes 2 and 5 ) . The immune precipitates were analysed by PAGE in the presence of SDS; autoradiography was performed on the resultant slab gel.

Analysis of the PIA' Alloantigen 357

Fig 4. Immune precipitation of PIA1 antigen. Plasma samples from patient E.M. and the randomly selected ACD control were tested in immune precipitation assays with detergent lysates of 1251-labelled platelets from (A) a randomly selected donor, (B) patient E.M.. and (C) a dog. The immune precipitates were analysed by PAGE in the presence of SDS; autoradiography was performed on the resultant slab gel.

DISCUSSION

The AB assay with 1251-labelled protein A sensitively detected the PIA1 antibody in the plasma of a PIA1 antigen negative patient with post-transfusion purpura. The use of radiolabelled protein A as a marker for serological reactions with platelets has also been used by other investigators (Dorvall et al, 1975; Marier et al, 1979; Bergh & Solheim, 1978; Kekomaki, 1977). Modifications presented here for this assay now set forth an approach that can be utilized for the large-scale screening of human sera for anti-platelet antibodies.

The specificity of the anti-PIA1 antibody was confirmed with this assay by screening a random platelet panel in conjunction with another known PIA1 positive antiserum. These two antisera also reacted with dog platelets and not rat platelets, a characteristic found only with PIA1 antibodies (Shulman, 1961). The minor discordant results of 3% in quantitative reactivity between the two PIA1 antibody samples with the random normal platelet panel most likely represents either the presence of secondary antibodies in these plasmas of independent specificity, or perhaps minor variations in the PIA1 antigenic determinants detected by the two plasmas. Indeed, serum E.M. was also found to have anti-RBC and anti-lymphocyte antibodies (data not shown).

In immune precipitation studies two independent anti-PIA1 sera precipitated a predomi- nant 90 000 dalton molecular weight species from platelets of PIA1 positive human and dog platelets. In addition, the antisera also precipitated a minor 100 000 dalton protein. The variable precipitation of this minor protein could be clearly contrasted to the consistent

3 5 8 Ji& Lane et a1

reaction with the 90 000 dalton species. It was further noted that neither the 90 000 nor 100 000 dalton proteins were precipitated from PIA1 negative and rat platelets by these sera. These studies can be compared to those of Kunicki & Aster (1978, 1979) where with similar methods the PIA1 antigen was identified as a single protein of 100 000: 120 000 daltons molecular weight. The reasons for the discrepancies in the molecular weight of the PIA1 antigen are not immediately apparent, especially since one of our PIA' reference sera was initially classified by Aster as having PIA1 specificity. A possible reason for these differences could include differential migration of surface proteins (presumably glycoproteins) in different gel systems. Alternatively, there may be minor proteolytic degradation of certain antigenic forms in the cellular lysates. These questions can only be resolved by a direct exchange of reagents.

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