Antibodies to human neutrophil antigen HNA-4b implicated in a case of neonatal alloimmune neutropenia.

BACKGROUND: Maternal alloantibodies directed against human neutrophil antigens (HNAs) can cause moderate-severe neutropenia in the newborn in a condition known as neonatal alloimmune neutropenia (NAIN). Neonates with NAIN can present with sepsis or be asymptomatic. NAIN has previously been reported as caused by antibodies against HNA-1a, -1b, and -1c; CD16b, -2, -3a, -4a, and -5a; and HLA, but not by antibodies against HNA-4b. We report a case of NAIN due to anti- HNA-4b alloimmunization in a term neonate. CASE REPORT: An infant with persistent and marked neutropenia was suspected of having neonatal alloimmune neutropenia. Blood samples from both parents were investigated for HNA and HLA incompatibilities by molecular typing techniques and the mother for the presence of HNA and HLA antibodies by serologic techniques. RESULTS: Initial results indicated the presence of granulocyte antibodies in the maternal serum, the specificity of which were shown to be anti-HNA-4b. Subsequently, the mother was genotyped as HNA-4b negative and the father as heterozygous HNA-4ab. The child was shown to have inherited the incompatible HNA-4b allele. CONCLUSION: We have demonstrated the first case of NAIN due to maternal alloimmunization against HNA-4b, pending ratification by the International Granulocyte Immunobiology Workshop.

Drug-antibody-platelet interaction in quinine- and quinidine-induced thrombocytopenia.

Binding of quinine- and quinidine-dependent antibodies to platelets was studied using an electroimmunoassay to measure platelet-bound IgG. Antibodies from four patients with drug-induced thrombocytopenia differed significantly in their interaction with platelets: association constants for binding to platelets at high drug concentrations ranged from 0.29 to 2.6 x 10(7) M(-1), the maximum number of antibody molecules bound ranged from 36,000 to 161,000/platelet, the amount of drug necessary to achieve half-maximum binding of antibodies to platelets ranged from 2 to 60 muM, and only one of the antibodies cross-reacted with the stereoisomer of the drug to which the patient was sensitized. Binding of the antibodies to platelets was enhanced at the highest achievable molar ratio of drug:antibody, 10,000:1, rather than being inhibited, as would be expected in a conventional, hapten-dependent reaction. The drug-antibody-platelet reaction was unaffected by Factor VIII/von Willebrand protein, nonspecifically aggregated IgG, or heat-labile complement components. After pretreatment with tritiated quinine, platelets retained several hundred thousand molecules of drug each, but failed to bind detectable amounts of antibody. However, platelets treated simultaneously with quinine-dependent antibody and tritiated quinine retained significantly more drug after repeated washes than platelets treated with drug and normal serum. These findings support the proposition that in quinine- and quinidine-induced thrombocytopenia, drug and antibody combine first in the soluble phase to form a complex, which then binds with high affinity to a receptor on the platelet surface (innocent bystander reaction), and demonstrate that these antibodies are heterogeneous in respect to the amount of drug required to promote their binding to platelets, the number of platelet receptors they recognize, and their binding affinities.

Fab-mediated binding of drug-dependent antibodies to platelets in quinidine- and quinine-induced thrombocytopenia.

Platelets coated with quinine- or quinidine-induced antibodies form rosettes around protein A-Sepharose beads and normal platelets form rosettes about protein A-Sepharose beads coated with these antibodies. These reactions occurred only in the presence of sensitizing drug. Platelets also formed rosettes about protein A-Sepharose beads coated with an anti-PIA1 antibody, but drug was not required. Formation of rosettes between antibody-coated platelets and protein A-Sepharose was inhibited by F(ab')2 fragments of goat antibody specific for the Fc portion of human IgG, while rosette formation between antibody-coated protein A-Sepharose and platelets was inhibited by F(ab')2 fragments directed against the F(ab')2 portion of the IgG molecule. Since binding of IgG to protein A is known to occur via the Fc region, these findings suggest that binding of drug-induced antibodies to platelets occurs at the Fab domains of the IgG molecule.

Platelet GP IIIa Pl(A) polymorphisms display different sensitivities to agonists.

BACKGROUND: Both inherited predisposition and platelet hyperreactivity have been associated with ischemic coronary events, but mechanisms that support genetic differences among platelets from different subjects are generally lacking. Associations between the platelet Pl(A2) polymorphism of GP IIIa and coronary syndromes raise the question as to whether this inherited variation may contribute to platelet hyperreactivity. METHODS AND RESULTS: In this study, we characterized functional parameters in platelets from healthy donors with the Pl(A) (HPA-1) polymorphism, a Leu (Pl(A1)) to Pro (Pl(A2)) substitution at position 33 of the GP IIIa subunit of the platelet GP IIb/IIIa receptor (integrin alpha(IIb)beta(3)). We studied 56 normal donors (20 Pl(A1,A1), 20 Pl(A1,A2), and 16 Pl(A2,A2)). Compared with Pl(A1,A1) platelets, Pl(A2)-positive platelets showed a gene dosage effect for significantly greater surface-expressed P-selectin, GP IIb/IIIa-bound fibrinogen, and activated GP IIb/IIIa in response to low-dose ADP. Surface expression of GP IIb/IIIa was similar in resting platelets of all 3 genotypes but was significantly greater on Pl(A2,A2) platelets after ADP stimulation (P=0.003 versus Pl(A1,A1); P=0.03 versus Pl(A1,A2)). Pl(A1,A2) platelets were more sensitive to inhibition of aggregation by pharmacologically relevant concentrations of aspirin and abciximab. CONCLUSIONS: Pl(A2)-positive platelets displayed a lower threshold for activation, and platelets heterozygous for Pl(A) alleles showed increased sensitivity to 2 antiplatelet drugs. These in vitro platelet studies may have relevance for in vivo thrombotic conditions.

Quinine-induced thrombocytopenia following intravenous use of heroin.

Profound thrombocytopenia developed in a 22-year-old man after intravenous use of heroin. A high-titer, quinine-dependent, platelet-specific antibody was detected in his serum using lysis of normal platelets labeled with chromium 51 and an electroimmunoassay for measurement of platelet-associated IgG. The antibody was specific for quinine and failed to react with platelets in the presence of quinidine hydrochloride or two structural analogues of heroin. Quinine, a common adulterant found in heroin, was detected in the patient's blood and urine. On the basis of these observations, the patient was judged to have quinine-induced immunologic thrombocytopenia. To our knowledge, this report is the first to confirm that quinine used as an adulterant can induce immunologic thrombocytopenia following an injection of heroin.

Specificity of drug-induced immune cytopenias.

What conclusions can be drawn concerning specificity of drug-induced immune reactions? We have seen that specificity of these reactions depends on several molecular features including the chemical nature of the drug, specific domains of particular membrane components, and as yet unidentified characteristics that determine selectivity for one or more cell types. This latter property does not seem to be related to shared membrane components because, for example, Rh antigens on RBCs, the peptide tail region of GPIb alpha on platelets, and the 85-kd GP on neutrophils are clearly not part of the same molecules. From multiple studies of quinine/quinidine-dependent and nomifensine-dependent antibody interactions with platelets and RBCs, respectively, we can conclude that these particular reactions are a function of specific features of the drug molecules and specific domains of various membrane glycoproteins. These characteristics strongly argue that the hypervariable regions of drug-dependent, platelet and RBC antibodies recognize simultaneously a specific domain of the membrane GP and a specific configuration of the drug molecule. If this is true, then it follows that together a specific domain of the cell membrane component plus the drug define an antigenic determinant or epitope for attachment of certain drug-dependent antibodies. We have also seen that some drug-dependent antibodies preferentially react with the drug alone when it is attached to cell membranes (eg, penicillin-dependent antibodies reacting with penicillin-coated RBCs or platelets). Some drugs elicit antibodies that react at specific sites on the cell membrane independently of drug (eg, nomifensine and the Rh antigens (E) or quinidine and platelet GPV). These three concepts of antibody specificity induced by drugs are presented in Fig 6, using RBCs as an example. Despite major advances in understanding drug-induced immune reactions during the past four decades, several important questions remain to be answered. For example, why are platelets involved more frequently than other cells of the circulation in these types of reactions? Why do some individuals develop drug-induced immune cytopenias that are specific for a single cell type, whereas others develop reactions involving multiple cell types with distinct antibodies? What mechanism directs the reaction toward platelets, RBCs, or neutrophils? How are drug-dependent antigens presented to the immune system? This latter question is particularly intriguing considering that most drugs known to induce immune cytopenias bind only weakly to target tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of antiplatelet drugs, heparin, and preanalytical variables on platelet function detected by the platelet function analyzer (PFA-100).

The platelet function analyzer (PFA)-100 is a newly developed instrument that provides a rapid, in vitro, quantitative measurement of platelet adhesion and aggregation in whole blood flowing through a small aperture under high shear conditions. Thirty patients undergoing percutaneous transluminal coronary angioplasty (PTCA) and ten normal individuals were included in this study. In vitro and in vivo studies were conducted to discern the effect of combinations of antiplatelet drugs (aspirin, ticlopidine, abciximab) and heparin on the performance of the device as well as the effects of preanalytical variables, such as method of sample collection and ex vivo anticoagulants. Studies were also conducted examining the effect of aperture size (standard 150 microns vs. smaller 120 microns) on the ability of the device to detect the effect of antiplatelet drugs. There was no difference in mean PFA-100 closure time with citrate versus PPACK anticoagulants or with venipuncture vs. sheath sampling. Closure times did not vary with heparin administration. Closure times were slightly longer for patients taking aspirin plus ticlopidine compared to aspirin alone (p = NS). In contrast adenosine disphosphate (ADP) induced platelet aggregation was significantly less in patients that took aspirin plus ticlopidine vs. aspirin alone (p = .0005). In vitro, there was a dose-dependent increase in closure time for both aperture sizes with increasing abciximab concentration. Although both cartridges showed infinite closure times at an abciximab concentration of 2.25 micrograms/mL, there was a slight benefit to using the 120 microns aperture cartridges at abciximab concentrations of 1.75 to 2.0 micrograms/mL. In ten patients who were followed during abciximab therapy to assess the effect of aperture size, the PFA-100 was able to detect in vivo platelet inhibition by abciximab, but detection of recovery from abciximab-induced platelet dysfunction was slightly better for the PFA-100 with the 120 microns aperture compared to the standard 150 microns aperture collagen/ADP cartridge.

Detection of antiplatelet antibody with a platelet immunofluorescence assay.

An indirect platelet immunofluorescence assay (PIFA) was developed for detection of circulating antiplatelet antibody in dogs with suspected immune-mediated thrombocytopenia (ITP). The PIFA was performed on 10 healthy dogs with normal platelet counts; 76 thrombocytopenic dogs, 20 of which were suspected of having ITP; and 18 dogs with other diseases and normal platelet counts. All normal dogs and negative test results. Fourteen (70%) of 20 dogs suspected of having ITP had positive test results. Fifteen of the remaining 56 thrombocytopenic dogs had positive test results, 9 had cancer and 6 had other immune-mediated diseases including systemic lupus erythematosus (SLE). In this study, the PIFA assay seemed to be more sensitive (70%) than the megakaryocyte immunofluorescence assay (41%) in the diagnosis of ITP. Of the 9 PIFA-positive dogs with neoplasia, 6 had lymphoproliferative disorders. The PIFA was positive in 5 of 18 diseased dogs with normal platelet counts. There was an inverse relationship between the platelet count and the intensity of fluorescence in the PIFA-positive dogs. We conclude that the PIFA is a sensitive screening method for detecting circulating antiplatelet antibody.

Comparison of microscopic and flow cytometric detection of platelet antibody in dogs suspected of having immune-mediated thrombocytopenia.

A flow cytometric platelet immunofluorescence assay (FC-PIFA) was compared with a previously developed microscopic platelet immunofluorescence assay (MI-PIFA) for detection of circulating platelet antibody. Both assays were performed on serum from 10 healthy dogs with normal platelet count, and on serum from 27 thrombocytopenic dogs--18 had primary immune-mediated thrombocytopenia (IMT), and 9 had IMT in addition to other immune-mediated disease (secondary IMT). Both assays yielded negative results for all control dogs. The MI-PIFA and FC-PIFA results were in agreement in 23 dogs with IMT (14 positive and 9 negative). There was linear correlation between MI-PIFA scores and FC-PIFA results (r = 0.873). Positive results were obtained for 55.5% of the dogs with suspected IMT, using the MI-PIFA, compared with 67%, using the FC-PIFA; however, the difference was not statistically significant. Use of fresh or frozen fixed donor platelets as the antigen source yielded similar results in the FC-PIFA.

Detection of drug-dependent platelet antibodies using immobilized Staphylococcal protein A.

Serum samples from eight patients suspected of having drug-induced immunologic thrombocytopenia provoked by a variety of medications were tested for drug-dependent platelet antibodies with a newly developed assay that forms rosettes of antibody-coated platelets around Staphylococcal protein A-Sepharose beads. The same samples were then tested with immunofluorescence and 51Cr-release assays. Seven of eight patients (87.5%) tested positive for drug-dependent antibodies by the rosette assay. In contrast, two of eight (25.0%) and one of eight (12.5%) patients tested positive for drug-dependent antibodies by immunofluorescence and 51Cr release, respectively. These results demonstrate that the new rosette method is significantly more sensitive and specific for the detection of drug-dependent platelet antibodies than either immunofluorescence or 51Cr release.

Human platelet activating antibodies.

Platelet antibodies identified in the plasma of three multiply transfused patients and a woman who had delivered a baby with neonatal alloimmune thrombocytopenia were investigated for their platelet activating properties. Three patients possessed multispecific HLA antibodies reactive with 90 to 100% of the cells on a lymphocytotoxic panel. These antibodies were also detected using the MAIPA assay and MAb w6/32, which recognizes an epitope common to all HLA class I molecules. In addition to HLA antibodies, three of the patients possessed platelet-specific antibodies that were identified by the MAIPA assay as anti-HPA-1a and anti-HPA-3a (one patient) and anti-HPA-1b (two patients). Each of the HLA antibodies when reacted with platelets expressing the corresponding HLA antigens, potently induced aggregation and release of ATP from dense granules. In contrast, the HPA-1b antibodies induced platelet agglutination, but failed to trigger ATP release. However, platelets coated with these latter antibodies were now refractory to subsequent stimulation by ADP. Similarly, when HLA antibodies were reacted with platelets to produce suboptimal activation, the platelets could now be stimulated only poorly or not at all by either epinephrine or thrombin. This was also true for anti-HPA-1b, which, although not inducing aggregation or ATP release by itself, was capable of almost completely blocking thrombin-induced platelet activation. The thrombin-inhibiting activity of these antibodies could partially be reversed by pretreating antibody-coated platelets with epinephrine immediately followed by stimulation with thrombin. These findings suggest that transfused platelets may either be activated or inhibited by reaction with various platelet antibodies. Therefore it is conceivable that the presence of platelet reactive antibodies in multiply transfused recipients may contribute to the increased thrombotic and hemorrhagic symptoms often observed among these patients.

Protein A immunoadsorption treatment in hematology: an overview.

Staphylococcal protein A efficiently binds immunoglobulins and circulating immune complexes (CIC) and provides an effective medium to remove immunoglobulins and CICs from plasma while sparing albumin and most coagulation proteins. Although it activates the complement system its clinical use abrogates the need for plasma expanders necessitated by plasma exchange. Despite anecdotal reports of utility in several hematologic syndromes, publications of clinical trials are available only for autoimmune thrombocytopenic purpura (AITP) and refractoriness to platelet transfusions (RFT) associated with alloimmunization. In the former situation Snyder et al. (Blood 79:2237-2245, 1992) reported on 72 patients with AITP all of whom had failed at least two previous therapies including splenectomy in 68%. Forty-six percent achieved improved platelet counts following treatment. The response was durable (8-26 mo) in all but 10%. Spleen-intact patients could not be differentiated from those who had been splenectomized. Both responders and nonresponders showed significant decreases in CIC and platelet-directed immunoglobulin (PDIG), but responders achieved near-normal levels. The beneficial response of these factors, particularly in spleen-intact patients, warrants a prospective study. In our studies at the University of Minnesota twelve patients with thrombocytopenia secondary to bone marrow failure who were refractory to platelet transfusion were treated with protein A immunoadsorption. Ten had demonstrable antiplatelet Abs (Anti-HLA, HPA, ABO). Seven of 12 demonstrated improved platelet counts and post-transfusion corrected count increments after treatment. This was associated with decreased platelet utilization and clinical bleeding. A prospective controlled clinical trial is justified.

Antibody-mediated platelet destruction by quinine, quinidine, and their metabolites.

Metabolites of quinine and quinidine, along with the parent compounds, were investigated for their ability to promote complement-mediated platelet destruction when combined with drug-dependent platelet antibodies from five patients with quinine- and quinidine-induced thrombocytopenia. In all, eight metabolites and four closely related structural analogues were studied. These included the desmethyl, 2'-oxo, 10,11-dihydroxy, N-oxide, N'-oxide, and diN,N'-oxide derivatives. When we used the cytotoxic chromium 51 release assay, the parent compounds were typically from 10 to greater than 300 times more effective than the corresponding metabolites and structural analogues in promoting antibody-mediated platelet lysis. Reaction patterns varied significantly among all antibodies and compounds studied, strengthening previous evidence that drug-dependent platelet antibodies are extremely heterogeneous in their reactions with platelets. Although most of the metabolites were much less potent than the parent compounds in promoting antibody-mediated platelet lysis, one quinidine-induced antibody was significantly inhibited in its quinidine-mediated lytic activity by the addition of desmethylquinidine, an essentially nonreactive metabolite with this particular antibody. These findings support the hypothesis that the native structures of quinine and quinidine are sufficient to provoke drug-dependent antibody formation and subsequent platelet destruction independently of their metabolites. They also suggest a possible protective role for some of these metabolites in certain individuals who are susceptible to this allergic drug reaction.

Quinine- and quinidine platelet antibodies can react with GPIIb/IIIa.

Quinine- and quinidine-dependent antiplatelet antibodies are generally believed to bind to the membrane glycoprotein complex, GPIb/IX. However, we and others have found that some drug-dependent antibodies bind to platelets from patients with the Bernard-Soulier syndrome which lack these glycoproteins. We therefore studied the reactions of a group of these antibodies with normal and Bernard-Soulier platelets and their membrane proteins. As indicated by rosette formation of the sensitized platelets around protein A-Sepharose beads, two quinine- and two quinidine-dependent antibodies reacted with both normal and Bernard-Soulier syndrome platelets at a high (300 microM) concentration of drug. At a pharmacologic drug concentration (10 microM), all four antibodies reacted with normal platelets but only the two quinine-induced antibodies reacted with Bernard-Soulier platelets. Immunoprecipitation studies with solubilized, tritium-labelled normal platelets, at both high and low drug concentrations, showed that each of the four antibodies precipitated proteins corresponding to GPIb and GPIX. Fainter bands corresponding to glycoproteins IIb and IIIa, which do not label well with tritium, were also detected. With radioiodinated normal platelets, it was found that each of the four antibodies was capable of precipitating GPIIb/IIIa, but only in the presence of drug. The four antibodies also promoted drug-dependent precipitation of GPIIb and GPIIIa from lysates of radioiodinated Bernard-Soulier platelets. The two quinine-dependent antibodies precipitated these glycoproteins at both high and low drug concentrations, while the quinidine-dependent antibodies reacted much more strongly at the higher drug concentration. Precipitation of GPIb/IX was not observed with BSS platelets. Absorption of a quinine-induced antibody with Bernard-Soulier platelets in the presence of drug eliminated its ability to precipitate GPIIb and GPIIIa. However, the absorbed antibody retained the ability to precipitate GPIb from solubilized normal platelets. Thus, at least two drug-dependent antibodies were present, one specific for GPIb/IX and the other for GPIIb/IIIa. These findings indicate that glycoproteins IIb and/or IIIa, in addition to the GPIb/IX complex, can serve as targets for drug-dependent antibodies in both intact and detergent-solubilized platelet preparations.

Saccharide binding to transition metal ion free concanavalin A.

Saccharide binding has been observed with demetallized concanavalin A in the presence of Ca(2+) only, using the fluorescent sugar 4-methylumbelliferyl alpha-D-mannopyranoside. At pH 7.2 both the nicked and intact forms of concanavalin A bound 4-methylumbelliferyl alpha-D-mannopyranoside with similar affinities. Competitive binding with methyl alpha-D-mannopyranoside was demonstrated. The association constants at 5 degrees C were 9.6 +/- 0.6 X 10(4) M(-1) for 4-methylumbelliferyl alpha-D-mannopyranoside and 1.1 +/- 0.3 X 10(4) M(-1) for methyl alpha-D-mannopyranoside. 4-Methylumbelliferyl alpha-D-mannopyranoside binding was also observed if demetallized concanavalin A was remetallized with less than stoichiometric amounts of Ca(2+). The association constants with low Ca(2+) concentrations were similar to those determined with saturating Ca(2+). With less than stoichiometric levels of Ca(2+), the number of sugar molecules bound per protein subunit was a reflection of the fraction of activated lectin subunits. These results show that saccharide binding activity of concanavalin A does not require a transition metal ion at pH 7.2; only Ca(2+) is required. At pH values near 5, where most previous studies have been carried out, both a transition metal ion and Ca(2+) are necessary.

Synthesis of 10,11-dihydroxydihydroquinidine N-oxide, a new metabolite of quinidine. Preparation and 1H-nmr spectroscopy of the metabolites of quinine and quinidine and conformational analysis via 2D COSY nmr spectroscopy.

The first synthesis of 10,11-dihydroxydihydroquinidine N-oxide [7b], a recently isolated metabolite of quinidine, was accomplished in three steps from 1b. The related congener 7a in the quinine series was also prepared, as well as two other analogues 3a and 4a. In addition, the previously reported human metabolites 2a, 5a, and 6a of quinine [1a] and those 2b, 3b, 4b, 5b, and 6b of quinidine [1b] were synthesized. The chemical shift and coupling constants for all of the metabolites of quinine and quinidine were assigned via 2D COSY 1H-nmr spectroscopy. Moreover, the conformations of these metabolites in solution were found to parallel those of the parent alkaloids, quinine [1a] and quinidine [1b], respectively.