The characteristic decreased recovery and survival of transfused platelets in nonalloimmunized patients with idiopathic thrombocytopenic purpura (ITP) suggest that plasma antiplatelet autoantibodies (autoAbs) are present in almost all cases. Studies emphasizing reactions of IgG autoAbs with platelet glycoprotein (GP) IIb/IIIa indicate that less than 50% of ITP patients have detectable serum Abs, and that many of these Abs may not be pathogenic because they are directed against epitopes in the cytoplasmic domain of GPIIIa (Fujisawa et al, Blood 77:2207, 1991 and 79:1441, 1992). We evaluated the contribution of Ig classes other than IgG to the overall incidence of serum Abs in 47 patients with chronic ITP and the frequency of reactions with GPs IIb/IIIa, Ib/IX, IV, and Ia/IIa. Abs were further characterized by their reactions with cytosolic or exosolic GP epitopes and their titers and apparent affinities. Using immunobead techniques we found (1) anti- GPs in 85% of sera; (2) IgA and IgG Abs each in 68%, together in 51%; (3) IgM agglutinins in 15%, always with another Ab class; (4) GP Ib/IX, IIb/IIIa, IV, and Ia/IIa targets in 83%, 81%, 38%, and 28% of cases, respectively; (5) 93% of positive sera reactive with more than one GP; but GP IV or Ia/IIa never the sole target; (6) Abs against cytosolic epitopes on one or more of GPs IIIa, Ib alpha, and IIb beta in 66% of sera, always accompanied by Abs against exosolic epitopes of the same or a different GP; (7) autoAbs against cytosolic GP epitopes in 38% of 16 patients recovered from posttransfusion purpura and drug purpura; and (8) evidence that serum ITP Abs, often high-titered, saturate platelets less than alloAbs against the same GPs. Whereas Abs against external GP epitopes are a distinctive marker for ITP in 80% of patients, Abs against internal GP epitopes are likely a secondary phenomenon of platelet destruction and not pathogenic. Anti-GPs against exosolic epitopes were also found in eluates of patients platelets', suggesting that they have pathogenic significance.

Glycoproteins (GPs) IIb/IIIa and Ib/IX are principal targets of autoantibodies (autoAbs) in idiopathic thrombocytopenic purpura (ITP). Platelet-associated Abs against GPIIb/IIIa primarily recognize discontinuous or nonlinear epitopes (Fujisawa et al, Blood 81:1284, 1993). This study focused on whether Abs against the extracellular domain of GPIb/IX might react with short linear amino acid (aa) sequences of GPIb alpha. Complementary DNAs (cDNAs) coding for two overlapping fragments of GPIb alpha were amplified, cloned into pFLAG.2 plasmids, and expressed in Escherichia coli DH5 alpha competent cells as FLAG fusion proteins, which were purified by anti-FLAG immunoaffinity chromatography. Of 16 selected ITP sera containing anti- GPIb/IX, 6 reacted in microtiter radioimmunoassays (RIAs) with recombinant protein fragment 2 (aas 240 to 485); 1 also with fragment 1 (aas 1 to 247). When synthetic peptides corresponding to 4 segments of fragment 2 with high antigenic indices (P1 to P4) were used as targets in RIAs, all 6 sera reacted with P2 (aas 326 to 346); 1 also reacted with P4 (aas 389 to 412). P2 was shown to be present on the surface of intact platelets by adsorption studies, and anti-P2 was detected in direct eluates of platelets from ITP patients. Glycocalicin in solution effectively competed with immobilized P2 for anti-P2; P2 in solution was a less effective competitor. Epitope scanning with a panel of synthetic 15-mer peptides localized the P2 epitope to the sequence, TKEQTTFPP. Epitope definition may offer insight into the pathophysiology of and more specific treatments for ITP.

SummaryThe relative importance of different proteinases, and their inhibition, in the breakdown of human endothelial basement membrane (BM) by MDA-MB-231 and MCF7ADR human breast cancer cell lines has been studied using 35S-labelled (Fidler, 1990). Basement membrane degradation (BMD) is brought about by extracellular proteinases, and there is currently interest in inhibitors of these enzymes as potential therapeutic agents. Possible candidates for the proteinases involved in breast cancer invasion are found in each of the four major classes of proteinases, i.e. aspartic, cysteine, serine and matrix metalloproteinases (MMPs).The aspartic proteinase cathepsin D, a lysosomal enzyme, is oversecreted by breast cancer cells in vitro and may be a major proteinase involved in BMD (Briozzo et al, 1988). This view is supported by in vivo experiments in which transfection of cathepsin D cDNA resulted in a higher frequency of metastases in mice (Garcia et al, 1990). Nevertheless, some workers have questioned whether this lysosomal enzyme, which is only active at acid pH, has a role in invasion under physiological conditions (Johnson et al, 1993).The cysteine proteinases, cathepsins B, L and H, are also lysosomal enzymes that are overexpressed in breast cancer (Vasishta et al, 1988;Gabrijelcic et al, 1992). Cathepsin B from normal human liver and from human breast carcinomas has been shown to degrade components of BM at neutral pH as well as at acid pH (Buck et al,