PAICA: a method for characterizing platelet-associated antibodies--its application to the study of idiopathic thrombocytopenic purpura and to the detection of platelet-bound c7E3

In idiopathic thrombocytopenic purpura (ITP), autoantibodies reacting with antigens on the platelet membrane bring about accelerated platelet destruction. We now report PAICA ("Platelet-Associated IgG Characterization Assay"), a method for detecting autoantibodies bound to specific membrane glycoproteins in total platelet lysates. This monoclonal antibody (MAb) capture assay takes into account the fact that antibodies on circulating platelets may be translocated to internal pools as well as being on the surface. A total of twenty ITP patients were examined by PAICA, and the results compared with those obtained by measuring (i) serum antibodies bound to paraformaldehyde-fixed control platelets by ELISA, (ii) IgG bound to the surface of the patient's own platelets by flow cytometry (PSIgG), (iii) total platelet-associated IgG (PAIgG) by ELISA and (iv) serum antibodies reacting with control platelets by MAIPA ("Monoclonal Antibody-specific Immobilization of Platelet Antigens"). Of twelve patients with elevated PAIgG, nine had increased PSIgG yet eleven reacted positively in PAICA. Of these, eight possessed antibodies directed against GP IIb-IIIa, two against GP Ib-IX and one patient possessed antibodies directed against GP IIb-IIIa and GP Ia-IIa respectively. Only seven of the patients possessed serum antibodies detectable by MAIPA. PAICA was also able to detect platelet-associated c7E3 (the chimeric form of Fab fragments of the MAb 7E3) following its infusion during antithrombotic therapy, when it proved more sensitive over a seven-day period than a MAIPA assay adapted for assessing surface-bound antibody. We propose that PAICA provides added sensitivity to the detection of platelet-associated antibodies in immune thrombocytopenias or following therapy with humanized MAbs.     

Autoimmune thrombocytopenic purpura (AITP) and acquired thrombasthenia due to autoantibodies to GP IIb-IIIa in a patient with an unusual platelet membrane glycoprotein composition

The subject (E.B.) is a 63-year-old woman with autoimmune thrombocytopenic purpura (AITP) who was first examined some 6 years ago with symptoms of epistaxis and gum bleeding, severe thrombocytopenia, and large platelets. Her serum tested positively with control platelets in the MAIPA assay performed using monoclonal antibodies (MoAb) to glycoprotein (GP) IIIa (XIIF9, Y2/51), yet was negative in the presence of MoAbs to GP IIb (SZ 22) or to the GP IIb-IIIa complex (AP2, P2). The patient's platelets failed to aggregate with all agonists tested except for ristocetin. IgG isolated from the patient's serum inhibited ADP-induced aggregation of control platelets. Unexpectedly, flow cytometry showed an altered expression of membrane glycoproteins on the patient's platelets. Levels of GP Ib-IX were much higher than previously located by us in platelets. In contrast, the expression of GP IIb-IIIa was about half that seen with control subjects. When Western blotting was performed, a striking finding was a strong band of 250 kDa recognized by a series of MoAbs to GP Ib alpha in addition to the band in the normal position of GP Ib alpha. Finally, ADP-stimulated (E.B.) platelets failed to express activation-dependent epitopes on GP IIb-IIIa as recognized by PAC-1, AP6, or F26 and additionally gave a reduced P-selectin expression after thrombin addition. In conclusion, we present a novel patient with a severely perturbed platelet function where an altered membrane GP profile is associated with the presence of an autoantibody recognizing a complex-dependent determinant on GP IIb-IIIa and inhibitory of platelet aggregation.     

Comparison of the direct platelet immunofluorescence test (direct PIFT) with a modified direct monoclonal antibody-specific immobilization of platelet antigens (direct MAIPA) in detection of platelet-associated IgG

Glycoprotein (GP)-specific platelet-associated IgG (PA-IgG) may be demonstrable in autoimmune-mediated thrombocytopenia. We studied 159 consecutive patients with histories of thrombocytopenia by a modified direct monoclonal antibody-specific immobilization of platelet antigens (direct MAIPA) assay, which immobilizes GP IIb/ IIIa, GP Ib/IX and GP Ia/IIa simultaneously. This modification requires smaller quantities of platelets than standard measurements performed separately. PA-IgG was present in 84/159 (53%) patients, as shown by the direct platelet immunofluorescence test (PIFT) with flow cytometry as a reference. PA-IgG against GP IIb/IIIa and/or GP Ib/IX and/or GP Ia/IIa was noted in 46 patients (29%), of whom 93% (43/46)-were also PA-IgG positive. The amount of PA-IgG detected by PIFT correlated directly with that detected by direct MAIPA (r = 0.71; P < 0.001). Only three patients 12548 with negative direct PIFT had GP-specific PA-IgG. GPV-specific PA-IgG was detected in 13 (10%) of the 125 patients, in whom further studies could be performed. In the subgroup of patients with GP-specific PA-IgG, the median fluorescence intensities of direct PIFT were higher than in patients with no GP-specific PA-IgG (P < 0.001). Direct PIFT and direct MAIPA divided the patients into asymmetric subgroups. However, the relative roles of these tests in the diagnosis of autoimmune-mediated thrombocytopenia await further studies.     

Anti-platelet antibodies in patients with systemic lupus erythematosus and the primary antiphospholipid antibody syndrome: their relationship with the observed thrombocytopenia

The role of antiphospholipid antibodies in the pathogenesis of the thrombocytopenia observed during primary antiphospholipid antibody syndrome (APAS) and systemic lupus erythematosus (SLE) remains controversial. We have used the MAIPA test to examine the frequency and specificity of anti-platelet antibodies directed against the major platelet membrane glycoproteins (GP IIb-IIIa, GP Ib-IX, GP Ia-IIa and GP IV) in patients where SLE and APAS were associated or not with thrombocytopenia. Results were compared with a series of 26 ITP patients, 46% of whom were shown to possess anti-platelet antibodies directed against one or more of the platelet surface glycoproteins. When APAS was associated with thrombocytopenia, 7/10 patients possessed antibodies against GP IIb-IIIa and/or GP Ib-IX. For SLE patients with thrombocytopenia, 6/10 patients were shown to have antiplatelet antibodies against GP IIb-IIIa, GP Ib-IX or GP IV. In contrast, for APAS (n=11) and SLE patients (n=11) without thrombocytopenia, only one patient had an antibody directed against GP IIb-IIIa and one patient had an antibody to GP IV. Our results suggest that antibodies directed against major platelet membrane glycoproteins may play a role in the thrombocytopenia that is seen during SLE and APAS.     

Identification and mapping of keratinocyte muscarinic acetylcholine receptor subtypes in human epidermis

We have previously shown that lethally irradiated normal strains of mice, radioprotected with severe combined immunodeficient (SCID) bone marrow, can be engrafted with human peripheral blood mononuclear cells (PBMC). The human/mouse radiation chimera can mount marked humoral and cellular responses to recall antigens, as well as primary responses. In the present study, we adoptively transferred splenocytes from patients with chronic immune thrombocytopenic purpura (ITP) into lethally irradiated BALB/c mice, radioprotected with SCID bone marrow. High titres of total human immunoglobulin appeared as early as 2 weeks post-transplant and declined after 6 weeks, while human anti-human platelet antibodies were detected 2-8 weeks after the transfer of splenocytes. The immunoglobulin G (IgG) fraction contained antibodies against glycoprotein (GP) IIb/IIIa (CD41) or GPIb/IX (CD42). The human platelet antibodies showed a low level of cross-reactivity with mouse platelets, and thrombocytopenia in the animals was not observed. Splenocytes from individual ITP patients differed in their capacity to produce either human platelet antibodies or total human immunoglobulin. Furthermore, antibodies produced in the murine system were not always identical to the original antibodies present in the serum of the patients. The study of the serological aspects of autoantibodies against human platelets in an animal model might be useful for the investigation of potential therapeutics in ITP.     

Extreme leukoreduction of major histocompatibility complex class II positive B cells enhances allogeneic platelet immunity

In a murine model of platelet alloimmunization, we examined the definitive role that mononuclear cells (MC) have in modulating platelet immunity by using platelets from severe combined immunodeficient (SCID) mice. CB.17 (H-2(d)) SCID or BALB/c (H-2(d)) mouse platelets were transfused weekly into fully allogeneic CBA (H-2(k)) mice and antidonor antibodies measured by flow cytometry. MC levels in BALB/c platelets were 1.1 +/- 0.6/microL and SCID mouse platelets could be prepared to have significantly lower (<0. 05/microL) MC numbers. Transfusions with 10(8) BALB/c platelets (containing approximately 100 MC/transfusion) stimulated IgG antidonor antibodies in 100% of the recipients by the fifth transfusion, whereas 10(8) SCID mouse platelets (containing approximately 5 MC/transfusion) stimulated higher-titered IgG alloantibodies by the second transfusion. When titrations of BALB/c peripheral blood MC were added to the SCID mouse platelets, levels approaching 1 MC/microL reduced SCID platelet immunity to levels similar to BALB/c platelets. Characterization of the alloantibodies showed that the low levels of MC significantly influenced the isotype of the antidonor IgG; the presence of 1 MC/microL was associated with induction of noncomplement fixing IgG1 antidonor antibodies, whereas platelet transfusions, devoid of MC (<0. 05/microL), were responsible for complement-fixing IgG2a production. When magnetically sorted defined subpopulations of MC were added to the SCID platelets, major histocompatability complex (MHC) class II positive populations, particularly B cells, were found to be primarily responsible for the reduced SCID mouse platelet immunity. The presence of low numbers of MC within the platelets was also associated with an age-dependent reduction in platelet immunogenicity; this relationship however, was not observed with SCID mouse platelets devoid of MC. The results suggest that a residual number of MHC class II positive B cells within allogeneic platelets are required for maximally reducing alloimmunization.     

Confirmation that GP Ib-IX complexes have a reduced surface distribution on platelets activated by thrombin and TRAP-14-mer peptide

In 1990 we reported that GP Ib-IX complexes accumulated within the surface-connected canalicular system (SCCS) of thrombin-stimulated platelets. This conclusion was reached following investigations using monoclonal antibodies (MAbs) in flow cytometry and a polyclonal antibody to GP Ib alpha in electron microscopy with immunogold staining performed on ultrathin sections of resin-embedded platelets. Recent controversy concerning these results has prompted us to perform further studies using 14 anti-GP Ib-IX MAbs obtained from the 1993 Boston Workshop on Leukocyte Antigens. Features were the use of the MAbs in mixtures and the fact that immunogold staining was performed on frozen thin sections. Platelets were stimulated with either alpha-thrombin or TRAP-14-mer peptide. In all cases a decreased density of GP Ib-IX complexes on exposed areas of the activated platelet surface was accompanied by an increased expression within the SCCS. At the same time we noted that when platelets were stimulated with TRAP-14-mer they progressively exhibited a different internal morphology in comparison to that seen with thrombin. In particular, the dense central mass disappeared and large vacuoles were present throughout the cytoplasm. Overall, these studies confirm that changes in the distribution of GP Ib-IX complexes which follow thrombin-induced platelet activation (i) are indeed observed when antibody mixtures are used to detect them, and (ii) are mediated through the receptor recognized by the TRAP-14-mer peptide.     

Antiplatelet glycoprotein autoantibodies in patients with autoimmune diseases with and without thrombocytopenia

The presence and specificity of antiplatelet autoantibodies in 32 patients with primary and 18 patients with secondary autoimmune thrombocytopenic purpura (AITP), as well as 11 non-thrombocytopenic patients with systemic autoimmune diseases, were studied. By means of the direct and indirect monoclonal antibody immobilization of platelet antigen (MAIPA) assay, antiplatelet autoantibodies were detected using monoclonal antibodies specific for platelet glycoproteins (GPs) Ib, IIb/IIIa, Ia/IIa, and IV. Serum antiplatelet autoantibodies were found in 18 of 32 primary AITP patients (56%), 6 of 18 secondary AITP patients (33%), and 5 of 11 nonthrombocytopenic patients (45%). Platelet-associated autoantibodies were detected in five of eight patients with primary (62%) and in four of eight patients with secondary AITP (50%) and in two of four patients without thrombocytopenia (50%). Multiple antibody reactivity, mainly against GPs IIb/IIIa and Ib and, in a few patients, against Ia/IIa, was found. Using MAIPA, platelet xylene eluates from 20 patients were also studied. Antiplatelet elutable autoantibodies were related to thrombocytopenia; autoantibodies against membrane GPs Ib and IIb/IIIa were demonstrable in 84 and 63% of eluates from patients with primary and secondary AITP, respectively, but not in eluates from nonthrombocytopenic patients. The presence of antiplatelet antibodies thus appears to be a common feature of many autoimmune diseases apart from the thrombocytopenia, but the (primary or secondary) etiology of the immune thrombocytopenia cannot be differentiated on the grounds of their specificity.     

Inhibition of bispecific monoclonal antibody (bsAb)-targeted cytolysis by human anti-mouse antibodies in ovarian carcinoma patients treated with bsAb-targeted activated T-lymphocytes

T lymphocytes of 8 patients with ovarian cancer were targeted to the tumor cells using F(ab')2 fragments of a bispecific monoclonal antibody (bsAb), specific for CD3 (a component of the T lymphocyte receptor for antigen) and for the folate receptor MOv18 (overexpressed by ovarian carcinoma cells) as part of a phase I/II study. Phase I (days 0 to 3) consisted of increasing intraperitoneal (i.p.) numbers (10(6)-10(9)) of bsAb-targeted T lymphocytes plus low-dose interleukin-2 (IL-2). Phase II (days 6 to 13, and 27 to 33) consisted of daily i.p. infusions of 10(9) targeted T lymphocytes, 2 mg soluble bsAb, and low-dose IL-2. Using enzyme-linked immunosorbent assays (ELISA), human anti-mouse antibodies (HAMA) were detected in all patients: in the serum from day 13 onwards and in the peritoneal fluid from day 20 onwards. A significant proportion of the HAMA appeared to be directed against the idiotypes of the bsAb specific for CD3 and MOv18, as suggested by (1) the clearly higher ELISA titers against OC/TR bsAb as compared to those against a monoclonal antibody (MAb) with unrelated specificity, and (2) failure to abrogate the capacity of peritoneal fluid containing HAMA to block the binding of OC/TR bsAb to MOv18+ or CD3+ cells by absorption of human anti-mouse IgG-framework antibodies in peritoneal fluid to immobilized mouse IgG. The OC/TR-targeted cytolysis of the MOv18+ ovarian carcinoma cell line Igrov-I by autologous T lymphocytes was inhibited by peritoneal fluid samples containing relatively high HAMA titers. Such inhibitory activity was never detected at the start of phase II, but coincided with the last series of i.p. infusions of targeted T lymphocytes in 2 patients.     

Immunochemical study of incomplete platelet autoantibodies by the anti-globulin consumption test (AGCT) with specific anti-immunoglobulin sera

The indirect anti-globulin consumption test (AGCT) with specific immunoglobulin antisera (anti-IgG and anti-IgM) has been applied to the immunochemical characterization of incomplete platelet auto-antibodies in 33 patients with idiophatic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE) and autoimmune hemolytic anemia (AHA). In these cases indirect AGCT on platelets was positive with polyvalent gamma antiserum. The test with anti-IgG was positive in all cases except two, while always negative with anti-IgM, with no relation to the presence of complete platelet antibodies, the type of disease and the immunochemical type of erythrocyte autoantibodies in AHA patients. These results indicate that the incomplete platelets auto-antibodies were of the IgG class.     

Expression of markers of platelet activation and the interpatient variation in response to abciximab

Our study concerns the biological effects of abciximab (c7E3 Fab, ReoPro), a powerful new antiplatelet drug that blocks glycoprotein (GP) IIb-IIIa complexes. Samples were examined from 6 patients with coronary artery disease who received a bolus of abciximab followed by a 10- microg/min infusion for at least 18 hours before percutaneous transluminal coronary angioplasty. Inhibition of ADP-induced PA was maximal for 4 patients but partial (79% and 53%) for 2 others during the infusion. Flow cytometry performed with monoclonal antibodies (PAC-1, AP-6, and F26) specific for the "activated" GP IIb-IIIa complex revealed large decreases in the expression of activation markers on platelets during therapy, but these decreases were less marked when inhibition of ADP-induced PA was incomplete. Residual aggregation was seen for all patients during the infusion when TRAP 14-mer peptide or thrombin was the stimulus. Unblocked GP IIb-IIIa complexes were detected on thrombin-stimulated platelets from the patients by immunoelectron microscopy performed using the monoclonal antibody AP-2. Unblocked GP IIb-IIIa complexes were also detected by flow cytometry when platelets preincubated for 1 hour in vitro with abciximab under saturating conditions were (1) incubated with TRAP 14-mer or (2) permeabilized with Triton X-100. In confirming interpatient variation in the platelet response to a standard dose of abciximab, our results also show that an uninhibited internal pool of GP IIb-IIIa complexes may mediate a residual response to strong agonists.     

Ultrastructural analysis of the distribution of the vitronectin receptor (alpha v beta 3) in human platelets and megakaryocytes reveals an intracellular pool and labelling of the alpha-granule membrane

The vitronectin receptor (VnR or alpha v beta 3) belongs to the cytoadhesin subclass of the integrin family. This subclass consists of two receptors which have the beta 3 subunit in common: GP IIb-IIIa complexes (or alpha IIb beta 3) and VnR. We report the subcellular distribution of VnR within human platelets as determined by immunogold staining of ultrathin frozen sections and transmission electron microscopy. Monoclonal antibodies directed against: (i) the alpha v subunit (LM142, AMF7, CLB-706), or (ii) an epitope specific to the complex (LM609) were used. Although VnR is present on platelets, it is a minor component. We therefore first compared several different staining procedures to detect this integrin. Optimal localization of VnR was obtained using a multistep procedure in which biotinylated anti-mouse IgG and a monoclonal anti-biotin antibody provided staining enhancement. Results showed that although present on the surface, alpha v beta 3 was mostly detected in internal membrane systems including those of alpha-granules. Occasionally, platelet sections showed special vesicular structures covered by gold particles. These were often localized at the edge or immediately under the plasma membrane and their origin remains unclear. An internal pool of alpha v beta 3 was confirmed by flow cytometry and by using platelets from a patient with type I Glanzmann's thrombasthenia arising from a GP IIb gene defect. We also investigated the presence of VnR in megakaryocytes (MK) obtained from normal human bone marrow. A fluorescence study showed VnR in small MK with unilobulated nuclei, suggesting that synthesis of this integrin occurs early during megakaryocytopoiesis. In mature cells, VnR expression had decreased relative to GP IIb-IIIa, although intracellular staining was present in EM and alpha-granules were again labelled.     

Steam-sterilizable, fluorescence lifetime-based sensing film for dissolved carbon dioxide

Heparin-induced thrombocytopenia (HIT) is a common adverse effect of heparin therapy that carries a risk of serious thrombotic events. This condition is caused by platelet aggregation, which is mediated by anti-heparin/platelet factor 4 antibodies. Sera from patients with HIT in the presence of platelets, induced the expression of E-selectin, VCAM, ICAM-1 and tissue factor and the release of IL1beta, IL6, TNFalpha and PAI-1 by human umbilical vein endothelial cells (HUVECs) in vitro and initiated platelet adhesion to activated HUVECs. These effects which occurred in a time-dependent manner were significant in the first 1-2 h of incubation and reached a maximum after 6 to 9 h. The GP IIb-1IIa receptor antagonist SR121566A which has been shown to block platelet aggregation induced by a wide variety of agonists including HIT serum/heparin, reduced in a dose-dependent manner the HIT serum/heparin-induced, platelet mediated expression and release of the above mentioned proteins. The IC50 for inhibition of HIT serum/ heparin-induced platelet dependent HUVEC activation by SR121566A was approximately 10-20 nM. ADP, but not serotonin release, also appeared to be involved as apyrase and ATPgammaS blocked platelet-dependent, HIT serum/heparin-induced cell surface protein expression and cytokine release by HUVECs. Increased platelet adherence to HIT serum/heparin-activated HUVECs was inhibited by SR121566A and, to a lesser extent, by apyrase and ATPgammaS, showing that platelet activation and release was at the origin of the HIT serum/heparin-induced expression of these proteins by HUVECs. Thus, sera from patients with HIT induced the expression of adhesive and coagulation proteins and the release of cytokines by HUVECs through the activation of platelets which occurred in a GP IIb-IIIa-dependent manner, a process that could be selectively blocked by SR121566A.     

Effects of administration of monoclonal antibodies (anti-CD4 or anti-CD8) on the development of autoimmune diseases in (NZW x BXSB)F1 mice

(NZW x BXSB)F1 (W/BF1) mice spontaneously develop autoimmune diseases, characterized by lymphadenopathy, lupus nephritis, and immune thrombocytopenia associated with various autoantibodies such as anti-DNA, anti-platelet and anti-cardiolipin antibodies (Abs). In the present study, we investigate the effects of administration of monoclonal Abs (anti-CD4 or anti-CD8 mAb) on the development of autoimmune diseases in W/BF1 mice. MAb was administered from the age of 7 weeks. Prolongation of survival rate and reduction of severity of autoimmune diseases were observed after treatment with anti-CD4 mAb. However, anti-CD8 mAb treatment accelerated the diseases. Serum levels of IFN-gamma and IL-10 in old W/BF1 mice were significantly high, whereas IL-4 levels were low in comparison with those of young W/BF1 mice; the expression of mRNA of IFN-gamma, IL-4 or IL-10 in CD4+ T cells of old W/BF1 mice was parallel to the serum levels of each cytokine. These observations suggest that CD4+ cells are involved in the development of autoimmune diseases in W/BF1 mice, and that CD8+ cells have a suppressive effect on the development of autoimmune diseases in W/BF1 mice.     

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.     

Comparative study of three methods to detect free plasma antiplatelet antibodies

We have compared three techniques for the detection of plasma circulating antiplatelet antibodies, i.e., the platelet suspension immunofluorescence test (PSIFT), the platelet radioactive antiglobulin test (PRAT), and the monoclonal antibody immobilization of platelet antigens (MAIPA). Frozen plasma samples from patients with idiopathic thrombocytopenic purpura or HIV-associated thrombocytopenia were used in the study. The PSIFT and PRAT showed the appropriate ease of performance necessary for screening purposes. The PSIFT is free of radioactivity hazards, but seemed to be less sensitive than the PRAT. The MAIPA is a useful tool to detect antibodies against glycoproteins (GPs) Ib/IX and IIb/IIIa. However, in comparison to PSIFT and PRAT, MAIPA is more time consuming, requires considerable technical expertise, and the identification of antiplatelet activity is highly dependent on the selection of an appropriate primary anti-GP monoclonal antibody. This could explain the lower prevalence of antiplatelet activity detected by MAIPA, in comparison to the frequency provided by the PSIFT and PRAT.     

Loss of regional bone mineral density in the first 12 months following renal transplantation

We performed 2 studies aimed at developing a frozen platelet panel suitable for platelet cross-matching. The stability of the most important platelet membrane glycoproteins and the reactivity of antigens of the human platelet antigen (HPA) and of the human leukocyte antigen (HLA) systems were evaluated with the platelet suspension immunofluorescence test (PSIFT) in a panel of platelets frozen in microplates with 6% dimethylsulfoxide. In study No. 1 we evaluated platelet reaction with a broad-spectrum weak anti-HLA and a potent anti-HPA-1a antiserum and the expression of glycoproteins Ib and IIb/IIIa complex on platelet membrane before freezing and after 0.5, 1, 2, 3, 4, 5, 6 and 12 months of storage at -80 degrees C. In study No. 2 we examined platelet reactivity with anti-HPA-1b, -HPA-2a, -HPA-3a, -HLA-A2, -HLA-A3 of platelets stored frozen for 12 months in parallel with fresh platelets from the same donors. Study No. 1 showed that glycoprotein expression was stable and that the weak anti-HLA and the potent anti-HPA-1a antibodies were clearly detected during 12 months at -80 degrees C. Of the 35 paired PSIFT performed in study No. 2 with fresh and frozen/thawed platelets incubated with anti-HPA-1b, -HPA-2a, -HPA-3a, -HLA-A2, -HLA-A3 antisera and AB serum, concordant reactions were obtained in all cases with the exception of 1 case of HLA-A3-positive platelets incubated with anti-HLA-A3 antiserum, that was reactive with frozen/thawed platelets but nonreactive with fresh platelets from the same donor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific interaction of HLA antibodies (eluates) with washed platelets

The mechanism of complement-independent action of HLA-A2 antibodies (eluates) on washed platelets was investigated. HLA-specific alteration was confirmed by serological (platelet micro-complement fixation), morphological (platelet spreading) and functional parameters (platelet aggregation, inhibition of collagen-induced platelet aggregation, [14C]serotonin release). In the presence of fibrinogen and calcium ions, HLA antibodies induced instantaneous platelet aggregation and release. Although no morphological (spreading) and functional changes (collagen-induded 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.     

A case of acquired immune deficiency syndrome presenting acute lumbosacral polyradiculopathy due to opportunistic infection of cytomegalovirus

The binding of antiphospholipid antibodies to circulating platelets and the potential association with thrombocytopenia and platelet activation was investigated in 25 patients with primary antiphospholipid syndrome (APS). Fourteen patients had a platelet count above 150 x 10(9)/l, and 11 patients had mild to moderate thrombocytopenia of 50-150 x 10(9)/l. The presence of platelet autoantibodies was investigated by immunofluorescent binding. No correlation between the presence of autoantibodies on platelets and thrombocytopenia was found. The binding of antibodies in patients' serum and platelet eluates was investigated by performing enzyme-linked immunosorbent assays with phospholipids as antigens. In seven patients antibodies to negatively charged phospholipids were present in platelet eluates. Platelet activation was measured by flow cytometry using a fluorescein isothiocyanate (FITC) labeled monoclonal antibody to P-selectin (CD62). The binding of anti-P-selectin to patients' platelet surface P-selectin was not increased, compared with the binding to platelets obtained from normal donors. Platelet serotonin concentration in APS patients was significantly lower than that found in the platelets of normal controls. More studies are necessary to determine the exact role of antiphospholipid antibodies in the pathogenesis of thrombocytopenia, and to elucidate the cause of low serotonin levels in platelets of APS patients.     

Combination "sandwich" therapy for extensive renal calculi in 100 consecutive patients: immediate, long-term and stratified results from a 10-year experience

A relationship between the presence of platelet autoantibodies and major histocompatibilty complex class II alleles was determined in 27 patients with lupus anticoagulants. Twenty-two patients had a primary antiphospholipid syndrome' and five patients had lupus erythematosus (SLE). Platelet antibodies against the platelet glycoproteins (GP) IIb/IIIa were detectable in 20 patients. Anti-GPIb/IX or -GPIV antibodies were detectable only in patients with anti-GPIIb/IIIa antibodies. An increased frequency of HLA-DQB1*06 was demonstrable in the total patient population. The association between the lupus anticoagulants and HLA-DQB1*06 was even stronger if patients also had detectable platelet antibodies. This association was also seen if patients with a history of thromboembolic disease were considered separately. However, within the patient population there was no difference between frequencies of HLA alleles detectable platelet antibodies.