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PubMed1995

The impact of three-dimensional structure on the expression of PlA alloantigens on human integrin beta 3.

We have compared the binding of affinity-purified anti-PlA1 IgG from seven nonrelated donors with chimeric integrin subunit beta 3 molecules expressed in the baculovirus-Spodoptera frugiperda insect cell system. Beta 3 chimeras were engineered to include segments of antigenic human beta 3 sequences spliced to intervening segments of nonantigenic Xenopus beta 3 sequence. Our results clearly show that antibodies from all seven donors will bind to nondenatured molecules containing the antigenic human beta 3 Cys26-Cys38 loop only when it is presented in a correct orientation that must be maintained by noncontiguous human sequences. Key downstream sequences are located within the region beta 3(288-490), flanking either side of the putative long-range disulfide at Cys435. Although our results confirm unambiguously that the Leu/Pro polymorphism at position 33 in human beta 3 is necessary for the expression of PlA epitopes, they also indicate that this polymorphic sequence alone is not sufficient. The requirement for additional human beta 3 sequence transcends the need to maintain a correct orientation within the Cys26-Cys38 loop itself, because the murine monoclonal antibody SZ21, which recognizes the sequence beta 3(28-35) contained within the Cys26-Cys38 loop, binds to all chimeras containing this loop, even if the same chimeras are not recognized by anti-PlA1. Our results indicate that additional noncontiguous residues encompassed by the sequence 288-490 either directly contribute to the composition of the PlA1 epitope or, more likely, maintain the Cys26-Cys38 loop in a proper orientation with respect to the remainder of the beta 3 molecule and thereby maintain proper antigenic presentation of the sequences in that loop.

Amino Acid Sequence↗
PubMed1995

50-kD integrin-associated protein does not detectably influence several functions of glycoprotein IIb-IIIa complex in human platelets.

A 50-kD integrin-associated protein (IAP) has been reported to be associated with beta 3 integrins and to modulate their function, especially vitronectin receptor in human erythroleukemia (HEL) cells and leukocyte response integrin in neutrophils. We studied the involvement of IAP in the function of platelet beta 3 integrin, glycoprotein (GP) IIb-IIIa complex. IAP was a widely distributed protein and was also expressed in the cells that do not have beta 3 integrin. Platelets from a patient with thrombasthenia, which lack GPIIb and IIIa, expressed IAP as well as normal platelets. Neither platelet aggregation nor intracellular Ca2+ elevation after stimulation was influenced by the anti-IAP antibody, B6H12, which was reported to be inhibitory for other beta 3 integrins. The expression level of GPIIb-IIIa complex was not influenced by coexpression of human IAP in the transfected Chinese hamster ovary (CHO) cells. IAP did not facilitate the binding of soluble fibrinogen to the CHO cells expressing GPIIb-IIIa complex. Furthermore, cell adhesion onto the immobilized fibrinogen via GPIIb-IIIa complex was not inhibited by B6H12 in HEL cells and was not altered by coexpression of human IAP in CHO cells. We concluded that expression of IAP is regulated independently with that of GPIIb-IIIa complex and that IAP does not influence the function of GPIIb-IIIa complex.

Animals↗
PubMed1995

Involvement of the cysteine-rich domain of glycoprotein IIIa in the expression of the human platelet alloantigen, PlA1: evidence for heterogeneity in the humoral response.

To assess the potential contribution of the cysteine-rich domain of human platelet glycoprotein (GP) IIIa to the structure of the PlA1 epitope, we used site-directed mutagenesis to substitute alanines for cysteines at key positions potentially involved in PlA1 alloantigen formation. One of these GPIIIa isoforms in which alanine replaced Cys435 reacted normally with a series of well-characterized murine MoAbs directed against the 250 amino-terminal residues of GPIIIa, whereas binding of MoAbs specific for residues 348-692 was diminished or lost. Interestingly, of eight PlA1-specific antibodies tested, two recognized Ala435GPIIIa, two did not, and four bound to it less well than to wild-type (WT) GPIIIa. However, disruption of disulfide bonds located at or near the N-terminus of GPIIIa abolished the binding of all the anti-PlA1 alloantibodies tested. These findings provide evidence that the humoral response to the PlA1 antigen is (1) heterogenous, ie, the binding site on GPIIIa for human anti-PlA1 antibodies differs from one individual to another and (2) complex--although some anti-PlA1 alloantibodies bind to an epitope comprised solely of the amino-terminus of GPIIIa, others combine most efficiently with a more complex determinant that involves the cysteine-rich domain of GPIIIa. These findings may have implications for diagnostic and therapeutic use of synthetic or recombinant PlA1 mimetics.

Alleles↗
PubMed1993

[Evaluation of the microplate enzyme-linked assay with isolation of glycoprotein IIb/IIIa from platelet membranes: comparison with immunofluorescent tests and monoclonal antibodies].

The enzyme test with GPIIb/IIIa has been used to detect antibodies in 500 sera from patients with thrombocytopenia and mothers of infants with thrombocytopenia. The results were compared with antibody detection in the platelet suspension immunofluorescence test (PIFT) and in the monoclonal antibody immobilization of platelet antigens (MAIPA). Platelet antibodies were detected in 125 sera (25%). In 46.2% the results were positive in all three tests. However, in 38.4% the ELISA was negative while the PIFT and MAIPA were positive. On the other hand, in 14.4% the ELISA was positive while the PIFT and/or the MAIPA were negative. The ELISA appears to be useful in the detection of anti-HPA-1a antibodies, which are the main cause of AIMN and PTP. This test, however, seems to be less useful in the detection of autoantibodies. The significance of the ELISA in the detection of platelet alloantibodies responsible for refractoriness to platelet transfusions is not clear yet and requires further investigations.

Antibodies, Monoclonal↗
PubMed1993

[Post-transfusion purpura: clinical and immunologic studies in 38 patients].

BACKGROUND: Posttransfusion purpura (PTP) is characterized by severe thrombocytopenia and hemorrhagic diathesis about 1 week following blood transfusion. Only few studies exceed the description of single cases. MATERIALS AND METHODS: Clinical data from 38 patients were analyzed. Platelet antibodies were studied by complement binding, immunofluorescence, MAIPA assay and acid elution techniques. RESULTS: All patients were female. The mean age at onset was 60.7 years (35-78 years). 4 patients received whole blood, 28 were transfused with packed RBC. 11 of 13 patients (85%) had febrile, non hemolytic adverse reactions during the transfusion. The interval between transfusion and onset of purpura extended from 2 to 14 days, with a peak at 7 and 8 days. Hemorrhagic symptoms lasted 10.1 days. The minimal platelet count was 7.0 x 10(3)/microliters. The platelet count increased to over 50 x 10(3)/microliters after 13.9 days (n = 26), and to over 100 x 10(3)/microliters after 17.0 days (n = 22). 2 patients died of hemorrhagic complications. 24 patients were treated with glucocorticoids, 20 with intravenous immunoglobulins (ivIG), 17 of these received both therapies. 14 of 19 patients (74%) responded well to ivIG. In contrast, platelet transfusions produced no adequate increment, in 6 cases they provoked febrile reactions. 35 sera (92.1%) contained anti-Zwa, either alone or together with anti-HLA or in 1 case with anti-Bra. Anti-Baka and anti-Bakb were found in 1 case each. In 5 patients, anti-Zwa could be eluted from autologous Zwa-negative platelets. CONCLUSIONS: The broad clinical spectrum of PTP could be shown. Since 5% of the patients succumb to bleeding complications, the application of ivIG as therapy of choice is recommended. The phenomenon of elution of alloantibodies from autologous platelets is interpreted as a consequence of 'pseudo-specificity' which may play a role in the pathogenesis of PTP.

Adult↗
PubMed1995

A human monoclonal antibody specific for the leucine-33 (P1A1, HPA-1a) form of platelet glycoprotein IIIa from a V gene phage display library.

IgG alloantibodies to polymorphic platelet glycoproteins (GPs) are known to be responsible for severe thrombocytopenia in the neonate and after transfusion. Platelet GPIIIa can have either a leucine or a proline at residue 33. The most immunogenic platelet alloantigen in thrombocytopenia is the leucine 33 form of GPIIIa. Here, we have generated human monoclonal antibody fragments that are specific for the leucine and not the proline form of GPIIIa and can inhibit the binding of polyclonal human IgG alloantibodies to GPIIIa leucine 33 on platelets. The antibody fragments were selected from a library of single chain Fv fragments displayed on the surface of filamentous phage. The VH gene repertoire was derived from the peripheral blood lymphocytes of an alloimmunized individual and recombined with a VL gene repertoire from a nonimmune source. Antibodies such as these, which are able to distinguish between two variant forms of a native antigen and which have been unobtainable by conventional hybridoma technology, have both diagnostic and potential therapeutic applications.

Amino Acid Sequence↗
PubMed1995

Function and regulation of the beta 3 integrins in hemostasis and vascular biology.

The beta 3 integrins, alpha IIb beta 3 and alpha V beta 3, are prominently expressed on the surfaces of platelets and endothelial cells, respectively. This vascular localization places them in a strategic position to perform adhesive functions necessary for hemostasis, wound healing and angiogenesis. In addition, these integrins are now known to serve a signaling function, whereby environmental cues on either side of the plasma membrane can be transmitted across the membrane to the other side. Indeed, preliminary studies indicate that these dual function receptors are wired to other signaling pathways within platelets and endothelial cells, thus helping to explain how integrin-mediated cell anchorage can affect cytoskeletal organization and cell motility as well as endothelial cell growth, differentiation and apoptosis.

Animals↗
PubMed1996

[Post-transfusion purpura. Description of 2 cases diagnosed at the same hospital in a 6-month period].

Two new cases of postransfusional purpura diagnosed at the same hospital within the space of six months are described. This finding supports the idea that despite being an infrequent disorder, a substantial rise in PTP cases inside and outside our country, has been recorded. This increase has coincided with a greater interest in platelet immunology and, particularly, in complications associated with blood transfusion. Both cases constitute an example of the clinical epidemiological profile which characterizes the patients suffering from this disorder. The patients are two women aged 74 and 60 years who after 8 and 9 days, respectively, of being transfused with red cells developed a severe thrombocytopenia accompanied by generalized haemorrhagic diathesis. The serological studies performed revealed the presence, in both patients, of an HPA-1a platelet specific antibody. The platelet genotyping enabled us to confirm this specificity after detecting an HPA-1 (a-b+) platelet genotype. The treatment with immunoglobulins at high doses proved to be effective in both cases. The adsorption-elution experiments of the antibody versus HPA-1 (a + b) platelets were positive in the patient with the highest antibody titre (1024). This finding support the most recent hypothesis concerning the pathogenic mechanism of PTP. According to this theory, the antibody, which is detected in the acute phase of the PTP, would not yet have acquired the restricted specificity corresponding to it. This could enable it to react with a structure shared by the HPA-1a positive and HPA-1a negative platelets.

Aged↗
PubMed1996

Inhibition of binding of anti-PLA1 antibodies to platelets with monoclonal antibody LK-4. Evidence for multiple PLA1 receptor sites on platelet GPIIIa.

The PLA1 epitope on platelet GPIIIa has a sulfhydryl-dependent conformation and is dependent on a leucine 33/proline33 polymorphism. Monoclonal antibody LK-4 differentiates PLA1/PLA1 from PLA2/PLA2 platelet lysates on solid phase enzyme-linked immunosorbent assay (ELISA), as well as immunoblot. To determine whether LK-4 reacts at or near the binding site(s) for human anti-PLA1, nine such antibodies (Abs) (six neonatal; three posttransfusion) were examined in the presence and absence of LK-4 for binding to platelets, as well as rGPIIIa 1-66, a recombinant glutathione S-transferase fusion peptide. All nine human Abs bound to rGPIIIa 1-66, as well as platelets, in a saturation-dependent manner, employing both solid phase ELISA, as well as flow cytometry. Binding of all nine Abs to rGPIIIa 1-66 or platelets was inhibited by LK-4. IC50's for inhibition of binding of anti-PLA1 to rGPIIIa 1-66 varied from 8 to 160 micrograms/mL (5 x 10(-8)- 1 x 10(-6) mol/L). However, IC50's for LK-4 inhibition of binding to platelets was strikingly different. Six of the nine Abs had IC50's of 1 to 10 micrograms/mL (8-fold to 16-fold greater inhibition than with rGPIIIa 1-66), whereas three neonatal Abs had IC50's of 380 to 1,013 micrograms/mL (6-fold to 48-fold less inhibition than with rGPIIIa 1-66). Similar results were noted with intact GPIIIa, rGPIIIa 1-66 blocked the binding of anti-PLA1 Abs to platelets and served to segregate the nine patients into two groups: a sensitive group of anti-PLA1 Abs from six patients in which binding to platelets was progressively inhibited by increasing concentrations of rGPIIIa 1-66 with inhibition at 1 micrograms/mL of 18% and inhibition at 256 micrograms/mL of 78%; a second resistant group of three anti-PLA1 Abs from three patients in which inhibition was first noted at 16 micrograms/mL of 4% with 35% inhibition at 256 micrograms/mL. Thus, LK-4 binds to GPIIIa at the 1-66 N-terminal region, inhibits binding of anti-PLA1 Ab to platelets, and segregates, anti-PLA1 Abs into two groups. These data are compatible with two or more receptor sites for anti-PLA1 Ab: one that is present on rGPIIIa 1-66 and sensitive to LK-4 inhibition, another that is present on rGPIIIa 1-66, as well as other site(s) on platelet GPIIIa and insensitive to inhibition.

Antibodies, Monoclonal↗
PubMedDate not supplied

Multiple use of a signal transduction pathway in tumor cell invasion.

Recent reports have indicated that a poorly characterized signaling pathway involving 12-lipoxygenase and PKC is involved in several steps of the metastatic cascade such as tumor cell adhesion to matrix, lysosomal enzyme release and migration. We investigated here the upstream elements of this signalling pathway looking for receptors which it might be coupled to. We described tumor cell alpha IIb beta 3-like integrin and the AMF receptor, gp78, as two transmembrane receptors which utilize the 12-lipoxygenase/PKC signaling. Furthermore, we compared the in vivo expression of the signal components of the "autocrine motility cycle" (gp78, 12-LOX and PKCa-Tímár et al 1993) in human prostate carcinoma cell lines (PC3 and DU145) with different invasive potential. We report here that all three components of this cycle are overexpressed three fold in vivo in the more aggressive tumor, suggesting that this signalling pathway might regulate the invasive phenotype-at least in the studied tumor cells.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1996

Evolution of bone marrow fibrosis and stromal antigenic expression in chronic myeloid leukemia on alpha interferon and Ara-C therapy.

We conducted a retrospective study to assess the changes in bone marrow (BM) stromal antigenic profile and fibrosis in chronic myeloid leukemia (CML) under combined interferon-alpha (IFN) and Ara-c therapy. Bone marrow biopsies were taken before therapy and twice (at 4 and 15 months) during therapy in 10 CML patients and compared with non-CML samples. Collagen and reticulin fibrosis was assessed by histochemical methods and phenotypic changes were studied by immunohistochemistry (APAAP) with antibodies directed against endothelial cell antigens, cell adhesion molecules, and HLA-DR. It was found that: (1) BM endothelial cells in patient and in control specimens showed a specific pattern of antigen expression: high expression of FVIII and CD34 (except on sinusoids for the latter), variable expression of UEA I, and no expression of HLA-DR and E-selectin. (2) Compared to non-CML controls, CML specimens at diagnosis showed an increased reticulin fibrosis and a decreased expression of CD61 on megakaryocytes and of CD31 on vessels and hemopoietic cells. (3) Treatment did not influence BM fibrosis, the vascular content of the BM, or the expression of the antigens tested except an increase in the number of CD34+ sinusoids (5/10 patients), an increase in the number of HLA-DR+, and a decrease in the number of CD34+ hemopoietic cells (6/10). (4) On therapy, difficulty in aspiration and/or reduced BM fragment numbers were noted in 8 of 10 patients whose bone marrow was still normocellular or slightly hypercellular. In conclusion, CML samples at diagnosis showed increased fibrosis and decreased CD31 and CD61 expression compared to controls. During the period of observation, combined therapy did not modify BM fibrosis; however, an increase in CD34+ sinusoids and a decrease in CD34+ hemopoietic cells were noted.

Adult↗
PubMed1997

Frequency of immune thrombocytopenia in newborns: a prospective study. Immune Thrombocytopenia Working Group.

Thrombocytopenia is a common condition in distressed newborns, but little is known about thrombocytopenia in an unselected cohort of neonates. In an attempt to address this issue, a multicenter prospective study was conducted in three obstetrical wards of AP-HP in Paris. We found the frequency of neonatal thrombocytopenia (<150 x 10(9)/L) to approximate 0.9% (48 of 5,632 appropriate samples). An immune mechanism was likely to be the cause of thrombocytopenia in 10 of the 33 cases studied, implying an incidence of 0.3% of immune neonatal thrombocytopenia in the general population. The frequency of alloimmune thrombocytopenia was 1.5/1,000 liveborn neonates, and 1/1,000 when considering anti-HPA-1a allo-immunization. Because thrombocytopenia, whatever its cause, was often silent and delayed, it appears that the only way to detect neonatal thrombocytopenia in time to prevent its potential disastrous complications would be to perform a systematic neonatal blood sampling for platelet count. All cases of ascertained thrombocytopenia should then be screened for an immune mechanism to enable early detection of autoimmune diseases in mothers and careful monitoring of subsequent pregnancies and deliveries, leading to appropriate prevention of potential severe deleterious effects in the offspring.

Adult↗