Posttransfusion purpura due to an alloantibody reactive with glycoprotein Ia/IIa (anti-HPA-5b).

A 38-year-old woman (JT) was diagnosed with posttransfusion purpura and significant posthysterectomy vaginal bleeding 9 days after the transfusion of 2 U of packed red blood cells. Analysis of JT's serum by a monoclonal antibody-antigen capture enzyme-linked immunosorbent assay method showed the presence of anti-HPA-5b (anti-Bra) antibodies directed against an epitope on platelet glycoprotein (GP) la of the GPIa/IIa complex. The patient's serum immunoprecipitated two proteins from 125I-labeled HPA-5b positive platelets that migrated under both nonreducing and reducing conditions on sodium dodecyl sulfate polyacrylamide gels at molecular weights characteristic of GPIa (150 Kd and 165 Kd, respectively) and GPIIa (120 Kd and 145 Kd, respectively). These bands were not precipitated when 125I-labeled HPA-5b negative platelets were used. Platelet typings performed on JT and her three children showed that the patient was HPA-5b negative and one of her children was HPA-5b positive. Platelets obtained from one of the donors who provided blood for the inciting transfusion also typed as HPA-5b positive. These findings demonstrate that posttransfusion purpura may be induced by antibodies directed against an alloantigenic epitope, namely HPA-5b (Bra), located on GPIa/IIa. Moreover, clinically significant bleeding can be associated with antibody reactions directed against this GP complex.

Antigenic polymorphism of human very late activation protein-2 (platelet glycoprotein Ia-IIa). Platelet alloantigen Hca.

We have found evidence for a human alloantigenic system on the very late activation protein -2 (VLA-2) heterodimer (platelet GPIa/IIa). Sera from two patients with systemic lupus erythematosus (SLE) contained antibodies that immunoprecipitated surface molecules from platelets and fibroblasts that comigrated on SDS-PAGE and two-dimensional O'Farrell gels with platelet GPIa (VLA-alpha2 chain) and platelet GPIIa (VLA-beta chain). These SLE antibodies were alloreactive as they precipitated VLA molecules from only 5 of 22 normal donors' platelets and did not react with the lupus patients' own platelets, despite the expression of apparently normal amounts of VLA on the donors' cells. Two-dimensional O'Farrell analysis demonstrated no differences in the molecular weight or isoelectric point of GPIa and GPIIa obtained from platelets of alloantibody reactive or unreactive donors. Sequential immunoprecipitation experiments with VLA chain-specific monoclonal antibodies, and the pattern of immunoprecipitation of several different VLA heterodimers demonstrated that the alloantibody-reactive determinant was present on the VLA-2 heterodimer, and not other VLA molecules. Thus, these SLE sera demonstrate a previously unrecognized antigenic polymorphism of the VLA-2 (platelet GPIa/IIa) heterodimer, platelet alloantigen Hca.

Platelet glycoproteins Ia, Ic, and IIa are physicochemically indistinguishable from the very late activation antigens adhesion-related proteins of lymphocytes and other cell types.

The very late activation antigens (VLA) are a subset of the superfamily of cell surface glycoproteins that serve as receptors from extracellular matrix proteins. One or more of the VLA heterodimers are present on T lymphocytes and most other cell types, including platelets. We have used VLA-specific monoclonal antibodies to isolate the reactive platelet membrane molecules. We have identified them as previously characterized platelet surface glycoproteins and have compared them with VLA molecules isolated from lymphocytes and other cells. Utilizing one-dimensional SDS-PAGE, two-dimensional O'Farrell gel electrophoresis, and nonreduced-reduced two-dimensional gel electrophoresis, we show that reduced VLA molecules of platelets are composed of three chains of molecular weights 165,000, 145,000, and 140,000 that possess the physicochemical properties of platelet glycoproteins GPIa, GPIc alpha, and GPIIa. GPIa corresponds to the VLA 165,000 alpha 2-chain, GPIIa corresponds to a 145,000 Mr VLA beta-chain, and GPIc alpha corresponds to a 140,000 Mr VLA alpha-chain. The polypeptide structure of VLA molecules on platelets and lymphocytes are very similar or identical. Platelet proteins GPIa and GPIIa exist as a mixed heterodimer in detergent lysates and correspond with the VLA-2 heterodimer found on activated T lymphocytes and other cell types. The platelet glycoproteins GPIIa and GPIc form a second mixed heterodimer. The mAb A-1A5, which binds to the VLA beta chain, binds to platelet GPIIa and precipitates both the GPIIa-GPIa and GPIIa-GPIc heterodimers, and binds to 4,926 +/- 740 sites per platelet. A VLA-2-specific mAb, 12F1, which binds to the VLA alpha 2-chain reacts with GPIa and immunoprecipitates only the GPIIa-GPIa heterodimer, and binds to 1,842 +/- 449 sites per platelet. The similarity of VLA chains and platelet GPIIa, GPIa, and GPIc molecules suggests that these molecules may have similar functions on various cell types.

Polymorphism of lymphocyte function-associated antigen-1 demonstrated by a lupus patient's alloantiserum.

We have found a human serum, E27, obtained from a multiply transfused patient with systemic lupus erythematosus, which immunoprecipitates the lymphocyte function associated antigen-1 (LFA-1). The immunoprecipitated molecules were identified as the LFA-1 alpha and beta chains by their comigration on SDS-PAGE, two-dimensional SDS-PAGE, and by sequential clearance experiments. Serum E27 did not immunoprecipitate LFA-1 from autologous cells, though LFA-1 molecules were present. In contrast, serum E27 immunoprecipitated LFA-1 from most but not all normal donor lymphocytes. Thus, serum E27 defines two serological phenotypes of LFA-1. 95% of normal individuals tested exhibited the LFA-1 phenotype precipitated by serum E27. Serum E27 appears to be directed at determinants of the LFA-1 alpha-chain and not the beta-chain since it immunoprecipitated LFA-1 molecules but not the Mac-1 molecules. Additional evidence for the alpha chain specificity was provided by immunoprecipitation of mouse-human heterohybridoma cells. LFA-1 was immunoprecipitated by serum E27 from mouse-human heterohybridoma cells expressing the human alpha-chain, not from a hybrid cell line expressing the human beta-chain. Together these findings demonstrate an antigenic polymorphism of the human LFA-1 alpha-chain molecule.

Use of the monoclonal antibody 12F1 to characterize the differentiation antigen VLA-2.

A monoclonal antibody, 12F1, has been produced that specifically immunoprecipitates the human cell surface structure VLA-2 from platelets and long-term activated T cells, as well as from fibroblast and neuroblastoma cell lines. Cross-linking studies indicate that the VLA-2 structure exists on the cell surface as a 165,000 Mr heavy chain (alpha 2) in noncovalent 1:1 association with a 130,000 Mr light chain (beta). The monoclonal antibody A-1A5, which reacts with the beta subunit common to all VLA structures, was able to completely preclear VLA-2, indicating that all of the alpha 2 subunit was associated with VLA beta-chain. The specificity of 12F1 for VLA-2 allowed independent immunoprecipitation and flow cytometry analysis of this alpha 2 beta structure separate from any other VLA structures that may have been present such as VLA-1 or free beta-subunit. Subunit dissociation studies were used to demonstrate that 12F1 recognizes an epitope on the alpha 2 chain on VLA-2, which is consistent with the 12F1 specificity for VLA-2 alone among the VLA proteins. Analysis of activated T cells indicated that VLA-2, like VLA-1, is another "very late" appearing T cell activation antigen that arises concurrently with VLA-1 starting at day 7 and increasing through 2 wk. VLA-2 was found on many of the same cells as VLA-1 (inactivated T cells, T cell leukemia cells, fibroblasts, SK-N-SH neuroblastoma cells), but VLA-1 and VLA-2 can be expressed independently, because VLA-2 was also present on VLA-1-negative cells such as HSB and platelets, and VLA-1 was present on VLA-2-negative C8215 cells.

Very late activation antigens (VLA) are human leukocyte-neuronal crossreactive cell surface antigens.

The antigenic relationship between human neuronal and lymphocyte cell surface antigens has been analyzed using heteroantisera raised against human peripheral blood mononuclear cells (PBMC). The specificities of the crossreactive antigens were examined by immunoprecipitation of 125I-labeled SK-N-SH cultured neuronal cells using rabbit anti-PBMC (RAPBMC) sera and compared to known specificities using mAb. The predominant reactivity of each rabbit antiserum tested against SK-N-SH cells was with three molecules of 130,000, 160,000, and 180,000 Mr. These three chains comigrated with three molecules precipitated with the very late activation antigen (VLA)-specific mAb A-1A5. Sequential precipitations with mAb A-1A5 established that the three RAPBMC-precipitated bands were members of the VLA complex. This was confirmed by two-dimensional PAGE of the RAPBMC and A-1A5 immunoprecipitates, which were indistinguishable from one another. The two-dimensional pattern was more complex than was anticipated from the heterodimeric model of VLA chain association, and suggests an additional 130,000 Mr component of VLA. The three chains of the VLA complex precipitated by RAPBMC or mAb A-1A5 from SK-N-SH neurons closely resembled the VLA pattern present on activated T cells, including the 180,000 Mr activation-specific alpha 1 chain recognized by mAb TS2/7. Normal brain cell membranes also contain VLA molecules that are precipitated by RAPBMC and mAb A-1A5. Thus the VLA complex provides potentially important shared immunogens on human neurons and T cells.

Unique features of group B streptococcal arthritis in adults.

Group B streptococci have rarely been reported to cause serious infection in adults and even less frequently to result in septic arthritis. We reviewed our clinical and radiologic experience with septic arthritis and uncovered five cases of infectious arthritis caused by group B streptococci. Unlike previously described patients with monoarthritis and complete recovery, our patients displayed polyarticular involvement. The infection in some cases was aggressive, resulting in destruction of multiple joints. Therefore, the series supports the view that this uncommon pathogen may produce an aggressive polyarthritis with the potential for serious functional damage and permanent morbidity. Culture and identification of a group B streptococcal pathogen and prompt institution of therapy can help avoid these complications.

Simultaneous occurrence of polyarteritis nodosa and leukocytoclastic vasculitis.

We report a patient with cutaneous vasculitis and mononeuritis multiplex. He was found to have both medium size arterial disease, characteristic of polyarteritis nodosa, and small vessel involvement, characteristic of leukocytoclastic vasculitis. This represents a rarely reported combination and serves to emphasize their usual nonoverlapping patterns. The apparent limitation upon the distribution of involved vessels may represent underreporting or signify that different pathophysiologic processes damage vessels of different sizes.

Limited trypsin cleavage distinguishes MHC and thymus-leukemia antigens.

A simple technique is presented for the identification of particular cell membrane antigens. The method employs labeled membrane antigens that are isolated immunospecifically and subjected to limited trypsin digestion followed by polyacrylamide gel electrophoresis in detergent. A large "core" peptide is produced by proteolysis of murine thymus-leukemia antigens (TLA) and from antigens of the major histocompatibility complex (MHC). The tryptic cores from H-2K and H-2D are regularly distinguishable from the thymus-leukemia antigens (TLA) by gel electrophoresis in one dimension. This chemical distinction is particularly important in the analysis of antigen mixtures isolated with antisera specific for beta 2 microglobulin. These techniques have been used to identify thymus-restricted beta 2 microglobulin-associated antigens on cell membranes from mouse, man, guinea pig, and monkey. In appropriate inbred mouse strains, these are the TLA and it is proposed that in the three other species examined they may be analogues, although not necessarily homologues, of TLA. The broad species distribution of these thymus-restricted cell membrane antigens suggests that they are involved in the differentiation of thymus-dependent lymphocytes (T cells).

Shared chemical properties of different murine thymus-leukemia antigens.

Immunochemical studies of murine thymus-leukemia antigens (TLA) have confirmed that the subunit structure consists of a 45,000-dalton heavy chain and a beta 2 microglobulin (beta 2m) light chain. Similar structural features are exhibited by the TLA from thymocytes of Tlaa, Tlac, Tlad, and a leukemia cell derived from C57BL/6, a Tlab strain. In addition to the similar subunit structure from the four haplotypes, each TLA shows a similar pattern of trypsin proteolysis. This procedure yields a major heavy chain cleavage product of approximately 37,000 daltons that remains associated with beta 2m and retains most or all of the antigenic determinants of the intact TLA. Evidence is presented that TLA do not exhibit Fc receptor properties, nor do they adsorb to murine leukemia virus antigens under the conditions of isolation for analysis on polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). Taken together these findings strongly support the hypothesis that TLA comprise a family of chemically similar antigens belonging to a structurally and genetically related group that includes H-2D, H-2K, and Qa-2,3.