Ming Hou

Antibodies against platelet GPIb/IX, GPIIb/IIIa, and other platelet antigens in chronic idiopathic thrombocytopenic purpura

Abstract: Antibodies involved in the pathogenesis of chronic idiopathic thrombocytopenic purpura (ITP) react most frequently with platelet glycoprotein (GP) Ib/IX and GPIIb/IIIa. However, uncertainty as to the specificity, frequency, and clinical significance of such antibodies still remains. By using a modified antigen‐capture ELISA (MACE), an immunoprecipitation assay, and an immunoblot assay, sera from 60 patients with chronic ITP were analyzed. GP‐specific antibodies were found in 50% (30/60) of the patients, with 14 patients having antibodies directed solely to GPIIb/IIIa, 8 holding antibodies specific only for GPIb/IX, and 8 possessing antibodies against both antigens. Serum antibodies were more frequently (p<0.01) detected in either active and/or non‐splenectomized ITP patients. Moreover, in patients displaying antibodies against GPIb/IX, significantly (p<0.05) lower platelet counts were observed. Using the immunoblot assay, antibodies specific for a 30 kD platelet antigen were detected in 12 of 60 patients. This antigen could not be immunoprecipitated from surface labelled platelet membranes, indicating an intracellular location. We conclude that in chronic ITP, (1) the frequency of anti‐GPIIb/IIIa antibodies is close to that of anti‐GPIb/IX antibodies, (2) anti‐GP antibodies are more likely to be detected in patients with an active disease status and, (3) a 30 kD internal platelet protein is another frequent antigen.

Detection of platelet antibodies in chronic idiopathic thrombocytopenic purpura (ITP). A comparative study using flow cytometry, a whole platelet ELISA, and an antigen capture ELISA

Abstract: Chronic idiopathic thrombocytopenic purpura (ITP) is a consequence of rapid platelet destruction caused by circulating platelet antibodies. In this study we compared three methods for detecting serum platelet antibodies in a population of 65 patients with chronic ITP. In two of the techniques intact platelets were used as the antibody target, i.e. the whole platelet ELISA and the flow cytometric assay; in the third an antigen‐specific modified antigen capture ELISA (MACE) was employed. By using the whole platelet ELISA and the flow cytometric assay 35% and 45% of the patients, respectively, displayed an antiplatelet antibody. In most cases (26 of 29 patients) IgG was the predominant antiplatelet immunoglobulin. As analysed using the MACE‐technique glycoprotein (GP) Ib/IX‐specific antibodies occurred with the same frequency as antibodies specific for GPIIb/IIIa. Moreover, there was a poor correlation between the MACE results on the one hand and results from the intact platelet‐based techniques on the other, i.e. several patients were positive in one assay whereas they were negative in the other. We conclude that all three techniques have their merits and demerits; it appears reasonable that they should be used together in the evaluation of the autoimmune process of chronic ITP.

Immunoglobulins targeting both GPIIb/IIIa and GPIb/IX in chronic idiopathic thrombocytopenic purpura (ITP): evidence for at least two different IgG antibodies

Antiplatelet antibodies in chronic idiopathic thrombocytopenic purpura (ITP) mainly target glycoprotein (GP) IIb/IIIa and GPIb/IX. Previous studies, employing modern antigen‐specific assays, indicate that serum reactive with both GPIIb/IIIa and GPIb/IX is not an uncommon finding in chronic ITP. However, the mechanism behind this dual reactivity remains unclear. We studied sera from 72 patients with chronic ITP using modified GPIIb/IIIa‐ and GPIb/IX‐specific MAIPA assays. Among the 34 positive sera, seven showed strong reactivity against both GPIIb/IIIa and GPIb/IX. These seven dual reactive ITP sera were further analysed by absorption studies. It was found that sera absorbed with immobilized GPIb/IX lost nearly all serum IgG specific for GPIb/IX but fully retained the IgG specific for GPIIb/IIIa. Conversely, sera absorbed with immobilized GPIIb/IIIa retained their reactivity only with GPIb/IX. These findings demonstrate that ITP sera, reactive with both GPIIb/IIIa and GPIb/IX, contain at least two different IgG antibody populations, each reactive with only one of the GP complexes.

Impact of endogenous thrombopoietin levels on the differential diagnosis of essential thrombocythaemia and reactive thrombocytosis

Abstract: By using the newly commercialized QuantikineTM human TPO immunoassay, plasma thrombopoietin (TPO) concentrations were measured in 12 patients with essential thrombocythaemia (ET), 13 patients with reactive thrombocytosis (RT) and 11 healthy volunteers. For the healthy volunteers the mean plasma TPO concentration was 21.1±11.0 pg/ml. The mean plasma TPO concentration in the group of RT was slightly lower (16.4±8.6 pg/ml) but did not differ significantly from the control group. The mean plasma TPO concentration in ET patients (44.1 ±45.2 pg/ml) was significantly (p<0.05) higher than the mean for RT patients, but did not differ statistically from the mean of healthy volunteers. These data suggest a defective clearance of plasma TPO in patients with ET.

Glycoprotein IIb/IIIa autoantigenic repertoire in chronic idiopathic thrombocytopenic purpura

Summary. The objective of the present study was to further disclose the autoantigenic repertoire carried by the platelet glycoprotein (GP) IIb/IIIa complex. IgG‐F(ab′)2 fragments were prepared from two prototype ITP patients, and their ability to block the binding of GPIIb/IIIa reactive antibodies derived from other patients with ITP was evaluated using a modified MAIPA assay; a P1A1 alloantiserum and 20 normal sera were included as controls. It was found that the two prototype IgG‐F(ab′)2 fragments were each able to significantly block the binding of serum IgG to GPIIb/IIIa in six (55%) and seven (64%) out of 11 patients with chronic ITP, respectively. No significant blocking effect was observed for IgG‐F(ab′)2 fragments prepared from normal subjects. Also, the binding of the P1A1 alloantiserum to its epitope on GPIIIa was not affected by any of the blocking IgG‐F(ab′)2 fragments exploited in the study. These data substantiate that in chronic ITP at least half of the GPIIb/IIIa reactive sera bind to homogenous autoepitopes.

Evidence for an expression of blood group A antigen on platelet glycoproteins IV and V.

Summary. Blood group ABO antigens are known to be carried by several platelet glycoproteins (GP), e. g. GPIb, GPIIa, GPIIb, GPIIIa and PECAM. Beside these proteins, we recently observed that blood group A antigen was also expressed on some other uncharacterized platelet proteins (70–90 kDa) having electrophoretic mobilities closely resembling those of GPIV and GPV. These findings prompted us further to characterize these latter ABO‐expressing platelet proteins. By antigen capture ELISA, wherein the monoclonal antibodies (mAbs) CLB‐IVC7 and CLB‐SW16 were used to hold the corresponding antigens GPIV and GPV, human anti‐A specifically bound to these proteins derived from A1‐platelets; neither GPIV nor GPV derived from A2‐, B‐ or O‐platelets bound anti‐A. In a Western blot assay using immunoprecipitated GPIV and GPV as antigens, mAb anti‐A immunostained GPIV and GPV precipitated from A1, but not from A2 and O platelets. These results conclusively demonstrate that blood group A antigen is expressed on platelet GPIV and GPV.

Platelet proteins as autoantibody targets in idiopathic thrombocytopenic purpura

Idiopathic thrombocytopenic purpura (ITP), caused by autoantibodies directed against certain platelet antigens, is the most common entity of the immune thrombocytopenias. ITP is an acquired disorder and can affect both children and adults. However, the clinical syndromes of ITP are distinct between children and adults. Childhood (acute) ITP characteristically is acute in onset, occurs within 1‐2 weeks of an infection, usually of viral origin, resolves spontaneously within 6 months. Adult (chronic) ITP has an insidious onset and rarely resolves spontaneously. Over the last decade considerable new information has accumulated as to the pathophysiological mechanisms of immune thrombocytopenias. In addition, most of the knowledge on this disorder has been obtained from studies of adult patients with chronic ITP. The present work gives an updated overview of the platelet autoantigens and the molecular immunological reactions in ITP.

Blood group A antigen expression in platelets is prominently associated with glycoprotein Ib and IIb. Evidence for an A1/A2 difference

Blood group ABH antigens are associated with platelets as intrinsic determinants and extrinsically adsorbed antigens, and exist both on glycosphingolipids and on glycoproteins (GPs). We now provide direct evidence that the blood group ABH antigens are prominently associated with platelet GPIb and GPIIb. By immunoprecipitation, a murine monoclonal anti‐A antibody precipitated surface‐biotin‐labelled blood group A1 platelet membrane proteins with electrophoretic characteristics identical to those of GPIb/IX and GPIIb/IIIa. By immunoblotting of SDS‐PAGE separated blood group A1 platelet proteins the monoclonal anti‐A antibody bound to proteins with electrophoretic characteristics identical to those of GPIb and GPIIb. When immunoaffinity purified GPIb/IX and GPIIb/IIIa, derived from blood group O, A1 and A2 platelets, were employed for immunoblotting, GPIb and GPIIb only from A1 platelets bound the monoclonal anti‐A antibody. By ELISA, wherein monoclonal antibodies specific for GPIb (AP1) and the GPIIb/IIIa complex (AP2) were used to capture and hold antigens from platelet lysate, human anti‐A antibodies reacted with these proteins derived from blood group A1 platelets; proteins from blood group A2, O and B platelets showed no reactivity. These results indicate that blood group A antigen is associated with GPIb and GPIIb derived from blood group A1 but not A2 platelets.

Biotinylated Platelets Have an Impaired Response to Agonists as Evidenced by In Vitro Platelet Aggregation Tests

Biotinylation of platelets, using a water soluble biotin analogue which reacts with primary amines, has been proposed to be a reliable technique for study of in vivo survival of platelets and their subpopulations. The information about the influence of this technique on platelet function has been limited. In the present work we studied the effect of in vitro biotinylation on platelet function and activation. Washed human platelets, at a concentration of 1 x 10(9)/L, were biotinylated with five different concentrations of sulfo-NHS-biotin or NHS-LC-biotin, ranging from 0 to 5 mM. The degree of platelet activation during and after biotinylation was monitored by measuring the externalization of P-selectin, and the platelet function was evaluated by aggregometry. It was observed that biotinylated platelets, in a dose dependent manner, displayed an impaired aggregation response. A slight increase in platelet membrane P-selectin occurred during the labelling procedure.

Genotyping of the platelet‐specific alloantigen HPA‐5 (Bra/Brb) using polymerase chain reaction with sequence‐specific primers (PCR‐SSP)

Abstract:u2002 A DNA‐based one‐stage technique, polymerase chain reaction with sequence‐specific primers (PCR‐SSP) was developed for genotyping of the platelet specific alloantigen HPA‐5 (Bra/Brb). Sequence‐specific primers, matching the wild type and the point mutation responsible for the HPA‐5 (Bra/Brb) phenotype, were constructed. Conjointly a fragment of the gene coding for glycoprotein (GP) IIIa was amplified as an internal control of the enzyme reaction. Using these HPA‐5 (Bra/Brb) sequence‐specific primers the correct fragment of the GPIa gene was amplified, as evidenced by the PCR product size, the restriction map and by the nucleotide sequence. This assay was applied on 187 Swedish blood donors; 157 individuals were found to have a homozygous HPA‐5a (Brb/Brb) genotype and 30 individuals a heterozygous HPA‐5a,b (Bra/Brb) genotype. None of the donors was found to display a homozygous HPA‐5b (Bra/Bra) genotype. Thus, the (HPA‐5b) Bra antigen frequency in this population will be approximately 16.0% with a gene frequency of 8.0%. It is concluded that this assay is an attractive technique for genotyping of the HPA‐5 (Bra/Brb) alloantigens on genomic DNA. The technique can replace serological alloantigen typing, especially in cases where platelets and rare human alloantisera are not available.