The isolation and characterisation of antiplatelet antibodies

The isolation and characterisation of antiplatelet antibodies in autoimmune thrombocytopenia purpura patients (ITP) is described. Autoimmune thrombocytopenia purpura is an autoimmune disease, clinically defined by low platelet counts, normal or increased megakaryocytopoiesis and antiplatelet antibodies in serum. This study used phage display to isolate Fab antiplatelet antibodies to study the structure-function relationships of pathogenic antibodies in ITP. Out of six randomly selected colonies, four colonies reacted strongly with whole platelets in enzyme-linked immunosorbent assay (ELISA). Sequence analysis showed that all four colonies had the same DNA sequence and were the same antibody. Results of Western blotting against non-reduced human platelet lysate showed that the Fab reacted with platelet proteins with apparent molecular weights of 116, 92 and 39 kD. Furthermore, Western blotting assay against purified membrane glycoprotein IIIa demonstrated reactivity against a band with a molecular weight of 92 kD. Results from Western blotting against platelet lysate and pure platelet glycoprotein confirmed the Fab fragment recognised the platelet glycoprotein IIIa. Three out of the four phage colonies produced soluble Fab, which demonstrated reactivity against platelet autoantigens in ELISA. Further sequence analysis showed that the Fab was somatically mutated suggesting antigen drive and therefore T-cell assistance was important in the development of this antibody. One of the somatic mutations introduced an RSD amino acid sequence in the complementary determining region 1 (CDR1) of the light chain, which may mimic the RGD motif of fibrinogen which binds integrin GPIIb/IIIa. This raises the possibility that somatic mutation and antigen drive have produced a pathogenic autoantibody.     

Immunofluorescent Staining of Platelet Suspensions and Detection of Antiplatelet Antibody in Patients with Idiopathic Thrombocytopenic Purpura

Direct immunofluorescent staining of 31 specimens of platelets obtained from 13 cases of idiopathic thrombocytopenic purpura (ITP) revealed positive staining on the surface of platelets for both immunoglobulins (Igs) and human B1C globulin in 9 specimens, for only Igs in 1 specimen and for human B1C alone in 5 specimens. The pattern of the positive immunofluorescent staining was granular. Indirect immunofluorescent staining of normal platelets in serum obtained from patients with ITP was positive for antiplatelet antibody in 9 out of 31 specimens. This suggests that platelets in patients with ITP may be damaged by an antiplatelet autoantibody acting directly on the platelet surface and/or by antigen antibody complexes binding via Fc IgG receptors on the surface of the platelets.     

Different Antiplatelet Antibody Specificities in Immune Thrombocytopenic Purpura

It is generally accepted that patients with immune thrombocytopenic purpura (ITP) produce antibody against platelet-associated antigens; however, it is not known if these antiplatelet antibodies are directed towards the same or different antigenic sites. In the present studies, quantities of antiplatelet antibody from different ITP patients, sufficient to saturate platelet antigenic sites, were simultaneously incubated with normal platelets and the quantity of platelet-binding IgG (PBIgG) was determined. In each of the five comparisons made, the amount of PBIgG bound after incubation of normal platelets with saturating quantities of two ITP antibodies approximated to the sum of the PBIgG bound after incubation with the antibodies separately. These data suggest that the antiplatelet antibody from these ITP patients differed in antigenic specificity.     

Identification of anti-platelet autoantibodies by western blot in 45 patients with idiopathic thrombocytopenic purpura (ITP).

BACKGROUND. In sera and platelet eluates of ITP patients, antigen specificity was widely studied by means of sensitive methods including immunoprecipitation, monoclonal antibody immobilization, and immunoblot. These studies indicated that GPIIb-IIIa were the main epitopes of ITP autoantibodies. METHODS. We studied the specificity of antiplatelet autoantibodies in 45 patients with acute and chronic ITP. Patient sera were tested by Western blot on separated platelet proteins in non-reducing conditions; antibody binding was identified using biotinconjugated anti-human IgG and avidin-peroxidase. RESULTS. Two main nonspecific bands of 200 and 125 kD were visible using normal serum; the first referred to platelet IgG, and the second was due to a naturally occurring antibody towards an internal protein. Twenty-five sera (55%) stained one (n = 11), two (n = 7), three (n = 3) or four (n = 4) specific bands. In patients with chronic ITP there was a prevalence of multiple bands. The relative molecular weights of the recognized antigens were in the range of 140-160, 80-100, 50-70 and 40 kd. The 80-100 epitope was recognized as a membrane protein in only 40% of sera, and it was partially characterized as GPIIIa in 4 patients. The other stained epitopes were absorbed by platelet lysate and then identified as internal proteins. CONCLUSIONS. This finding might be related to sensitization to antigens exposed by platelets during immune damage, and may pose an important problem in the identification with the immunoblot technique of target antigens responsible for immune sequestration.     

Antiplatelet antibodies and their correlation with clinical findings in childhood immune thrombocytopenic purpura

The demonstration of antiplatelet antibodies (PAIgG, PAIgM) and decreased detection of platelet surface antigens (CD41, CD61, CD42b) in children with immune thrombocytopenic purpura (ITP) have a diagnostic role. This study was conducted to determine whether these parameters differed in acute and chronic ITP. Chronic ITP was defined as thrombocytopenia persisting for more than 6 months from the onset of illness. A total of 80 subjects were divided into three groups: group 1 included 39 patients with acute ITP; group 2 included 31 patients with chronic ITP, and group 3 included 10 healthy children. At diagnosis, blood samples were obtained for platelet count, mean platelet volume, plateletcrit and platelet distribution width along with platelet surface antigens and antiplatelet immunoglobulins. We found that platelet surface antigens were significantly decreased in both acute and chronic ITP when compared to the control group (p = 0.001). In contrast, PAIgG was increased in acute and chronic ITP patients compared to the control group. PAIgM was significantly higher in acute ITP. We conclude that decreased platelet surface antigens and increased antiplatelet antibodies are observed in both acute and chronic ITP. In patients with chronic progress, a relatively lower level of PAIgM can be identified.     

Influences of antiplatelet autoantibodies on platelet function in immune thrombocytopenic purpura

We investigated the characteristics of the antiplatelet autoantibodies in 60 patients with ITP. Using flow cytometry, the binding of monoclonal antibodies to the platelet glycoprotein (GP) IIb/IIIa complex and to GPIb was examined in these patients. The extent of binding was decreased in 15 patients (anti-GPIIb/IIIa in 12 patients and both anti-GPIIb/IIIa and anti-GPIb in 3 patients). Western blotting revealed that 10 of these 15 patients had either anti-GPIIb or anti-GPIIIa and 2 had anti-GPIb autoantibodies, ADP-induced aggregation of normal platelets was inhibited by autoantibodies in 12 of 60 patients, and 11 of these had anti-GPIIb/IIIa antibodies. Ristocetin-induced aggregation was inhibited in 4 of these patients, and 2 with prominent inhibition had anti-GPIb antibodies. There was a significant relationship between platelet-associated IgG value and ATP secretion. These results suggest that some antiplatelet autoantibodies can affect platelet function and thus have an influence on the pathophysiology of ITP.     

Specific antiplatelet autoantibodies in patients with antiphospholipid antibodies and thrombocytopenia

By means of immunoblotting and monoclonal antibody immobilization of platelet antigens (MAIPA) we have studied the specificity of antiplatelet antibodies in patients with antiphospholipid antibodies and thrombocytopenia defined as presence of anticardiolipin IgG and a platelet count below 100 × 10⁹/l. The study group consisted of 10 patients with systemic lupus erythematosus (SLE), 8 patients with primary anti-phospholipid syndrome (PAPS) and 16 patients with idiopathic thrombocytopenic purpura (ITP). The comparison group was formed by 17 patients with classical chronic ITP without anticardiolipin IgG. We identified the 80–100, 130–150 and 150–170 KD surface proteins that comigrate with GPIIIa, GPIIb and GPIb and a 50–70 KD cytoplasm band by immunoblot. In patients with classical chronic ITP, the prevalence of the antiplatelet antibodies against GPIIIa was 53% on immunoblot assay and 47% on MAIPA. In ITP patients who had also anti-phospholipid antibodies in serum, the percentage of reactivity to GPIIIa declined to 37% on immunoblot and 21 % on MAIPA but it was not statistically different from the percentage observed in patients with classical ITP. Autoantibodies to platelet surface glycoproteins were almost absent in SLE and PAPS patients, who showed a significant prevalence (78%) of IgG reactivity to the 50–70 KD internal platelet protein which was frequently encountered also in patients with ITP and aPL (56%). Our study provides additional evidence that platelet antigens in patients with phospholipid-associated secondary immune thrombocytopenia are different from those of primary ITP, and that surface glycoproteins were not involved.     

Prospective screening of 205 patients with ITP, including diagnosis, serological markers, and the relationship between platelet counts, endogenous thrombopoietin, and circulating antithrombopoietin antibodies

Immune thrombocytopenia purpura (ITP) is characterized by destruction of circulating platelets and the presence of antiplatelet antibodies. Many of the current immunomodulatory therapies act by reducing platelet destruction and usually do not have a lasting effect. This prospective, exploratory study characterized patients with ITP by identifying their demographic and comorbid clinical factors, use of treatments, serologic markers of autoimmunity, and possible relationships between platelet counts, concentrations of endogenous thrombopoietin (eTPO), and the presence of circulating anti-TPO antibodies. Data including medical history and laboratory evaluations were collected at a single patient visit on 205 patients (19 children, 186 adults). Reported histories revealed a 5% rate of thrombotic/ischemic events. Autoimmune markers including direct antiglobulin test and antinuclear antibodies were found more frequently than in the normal population; antiplatelet antibody testing was not done. eTPO concentrations were comparable to concentrations found in healthy volunteers. Our study confirmed that no significant inverse correlation occurred between circulating concentrations of eTPO and platelet counts in patients with ITP (Spearman r = -0.15). Two of the 205 patients tested (1%) had neutralizing activity of recombinant human TPO in a biological assay; however, this activity was confirmed to be anti-TPO antibody in only 1 patient.     

Cumulative experience with a simplified solid-phase radioimmunoassay for the detection of bound antiplatelet IgG, serum auto-, allo-, and drug-dependent antibodies

A simplified, sensitive, solid-phase radioimmunoassay employing 125I- staphylococcal protein A has been developed that is capable of detecting bound antiplatelet IgG as well as serum auto-, allo-, and drug-dependent antiplatelet antibodies. The simplified assay employs a ratio of test over control platelet counts per minute (cpm) for detection of positive results. All reagents are commercially available. The assay can be performed with as little as 10(6) washed platelets (10 microliters of whole blood) that have been stored for as long as 8 wk at 4 degrees C in microtiter plates. The assay time, employing stored platelets, is 4 hr. Bound platelet IgG is positive in 93% of 46 thrombocytopenic patients with autoimmune disease and correlates inversely with their platelet count, r = -0.65, p less than 0.001. The ability of this assay to detect serum antibody was studied with a rabbit anti-human platelet antibody capable of giving optimal immunoprecipitation with solubilized platelet membranes at a tier of 1:10. The present assay increases the sensitivity of antibody detection 256-fold to a titer of 1:2560. Human serum antiplatelet membrane antibody was positive in 2 of 2 patients with anti-PLA-1 antibody (titers of 1:256 and greater than 1:64); 7 of 12 multiply transfused patients who were refractory to platelet transfusion (2 had titers of greater than 1:256 and greater than 1:32); 5 of 19 patients with autoimmune thrombocytopenic purpura (2 had titers of 1:64 and 1:32); and 10 of 14 patients with clinical histories of drug-dependent antiplatelet antibody (2 had titers of 1:1280 for quinidine and 1:384 for phenazopyridine).     

Target antigens of anti-platelet antibodies in patients with steroid-resistant ITP of recent onset. Canadian Apheresis Study Group.

Recent application of Western blotting procedures in the detection of anti-platelet antibodies has permitted more refined definition of reactivity. We report on the results of anti-platelet antibody assays in a series of 19 patients with recently diagnosed (less than 6 months duration) steroid-resistant, idiopathic thrombocytopenia purpura (ITP). At presentation, six of the 19 patients had a positive test for platelet-associated IgG (PAIgG) as measured by the direct radial-immunodiffusion (RID) assay, whereas three of the 19 were positive with the direct-platelet-suspension-immunofluorescence test (DPSIFT). The indirect-platelet-suspension-immunofluorescence test (IPSIFT) demonstrated antibodies in seven of the sera. Following Western blot (WB) analysis, the serum of 7/19 patients (only three of which were positive in the IPSIFT) could be shown to react with platelet antigens. Two patterns were seen: in four cases there were bands of apparent molecular weights of 60,000, 55,000 and 50,000; in the other three samples, a single band near 90,000 was demonstrated. Unlike the situation reported for chronic ITP, no reactivity was seen against higher molecular weight antigens other than the 'non-specific' binding at apparent molecular weight 200,000 which is also seen with normal sera. The data suggest that antibodies reacting against specific platelet antigens are present in the serum of some patients with recent onset ITP.     

Qualitative Platelet Abnormalities in a Patient with Chronic Idiopathic Thrombocytopenic Purpura (ITP)††

A patient is described with active, chronic idiopathic thrombocytopenic purpura (ITP) and qualitative platelet abnormalities. An antiplatelet factor was demonstrated by the reduced survival of both autologous and isolo-gous platelets, and its identification as antibody suggested by positive tests for antiplatelet antibody. The major abnormalities of platelet function were reduced platelet adhesion in vivo and to glass, and reduced platelet aggregation with ADP, Ristocetin and collagen. ADP-induced aggregation of normal platelets was reduced by prior incubation of the normal platelets with the patient's platelet-poor plasma (PPP). It is suggested that abnormal platelet function in ITP may be an index of antibody activity, a determinant of premature platelet removal by the reticulo-endothelial system (RES) and a contributing factor to impaired haemostasis.     

Morphological differences between GPIb antibody-induced and shear stress-induced platelet aggregates

We determined the morphological differences between NNKY5-5-induced and shear stress-induced platelet aggregates using confocal laser scanning microscopy, and investigated the effects of cytokines on these aggregates. As shear stress was increased from low to high, many aggregates were generated. Some platelets and aggregates already exhibited PAC-1 binding at low shear stress. And finally, aggregates were clearly stained by PAC-1 at high shear stress. Low shear stress-induced aggregates included fibrinogen, but not von Willebrand factor (vWF). In contrast, high shear stress-induced aggregates included both fibrinogen and vWF. NNKY5-5-induced platelet aggregates exhibited both PAC-1 binding and fibrinogen, but vWF was not found in them. The aggregate formation in NNKY5-5-treated platelet-rich plasma (PRP) at low shear stress was clearly more pronounced than that in untreated PRP. In addition, aggregates in NNKY5-5-treated PRP formed after 6 min under low shear stress were clearly stained by PAC-1 and included fibrinogen, but vWF was not found in them. In order to investigate the effects of cytokines on platelet activation under NNKY5-5 stimulation, changes in light transmission and light scatter were assessed with an AG-10. G-CSF, IL-6 and thrombopoietin (TPO) each enhanced light scatter compared to control PRP, although IL-3, GM-CSF, erythropoietin and stem cell factor did not. In particular, TPO significantly enhanced the '%-T' and 'S-Max' of NNKY5-5-treated PRP. TPO clearly enhanced the aggregate formation with high shear stress or NNKY5-5 stimulation. These results suggest that the glycoprotein Ib (GPIb)-unmediated aggregates can be formed with only fibrinogen, but the GPIb-mediated aggregates by vWF and fibrinogen synergistically. In particular, the latter is formed more firmly, and both aggregates are enhanced by TPO.     

Association of autoantibodies against platelet glycoproteins Ib/IX and IIb/IIIa, and platelet-reactive anti-HIV antibodies in thrombocytopenic narcotic addicts

The levels of platelet-associated Igs (PAIgs) and plasma circulating antiplatelet antibodies were evaluated by a flow cytometric immunofluorescence assay (FCIFA), an enzyme-linked immunoassay (ELISA), and a platelet radioactive antiglobulin test (PRAT), in a group of 45 human immunodeficiency virus (HIV)-infected intravenous drug users (IVDUs), with or without thrombocytopenia (TCP). Direct tests demonstrated an increased amount of PAIgs in 40% of the patients, irrespective of their platelet count. These PAIgs were mainly of IgG class and could not be eluted with ether. Plasma IgG with antiplatelet activity was found in 70% of the thrombocytopenic individuals, whereas it was detected in only one patient without TCP. The relative frequencies of antibodies against the platelet glycoproteins (GPs) Ib/IX and IIb/ IIIa were assessed in plasma from all patients by means of the monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). Plasmas from all non-thrombocytopenic patients were negative when tested by indirect MAIPA. In contrast, 10/23 plasmas from thrombocytopenic patients reacted with either GP IIb/IIIa, GP Ib/IX, or both GPs. Finally, aiming to investigate whether HIV antibodies from these patients are reactive with normal platelets, we performed absorption-elution experiments, followed by evaluation of HIV antibodies in the indirect eluates by ELISA and Western blot. Interestingly, we detected anti-HIV antibodies that bind to normal platelet antigens in 50% of the ether eluates prepared from control platelets sensitized with plasma from patients with TCP, but in only 5% of eluates obtained from platelets sensitized with plasma from non-thrombocytopenic patients. The present study provides direct evidence that specific autoantibodies against platelet membrane GPs Ib/IX and IIb/IIIa are common in HIV positive thrombocytopenic individuals. The finding in these patients of HIV antibodies cross-reactive with normal platelets, suggests that mimicry of human antigens by HIV could play a key role in the pathophysiology of the HIV-related TCP.     

Pathophysiology of platelet destruction in immune (idiopathic) thrombocytopenic purpura

The mechanism of platelet destruction in immune (idiopathic) thrombocytopenic purpura (ITP) is thought to involve production of autoantibody to platelet surface antigens. Once coated with antibody, circulating platelets undergo sequestration via interaction with Fc receptors of macrophages in the reticuloendothelial system. A number of questions remain about the mechanism of platelet destruction in this disease: 1) What is the nature of the stimulus to the immune system that generates antiplatelet antibodies? 2) What is the role of interactions between T-helper lymphocytes and antigen-presenting cells in ITP? 3) What role, if any, is played by the targeting of single or multiple platelet surface glycoproteins by the autoimmune response? 4) Is the site of platelet destruction, intravascular or extravascular, or the state of activation of platelets important in the destruction of platelets?     

Immunochemical analysis of platelet autoantibodies in HIV-related thrombocytopenic purpura: a study of 68 patients

Serum antiplatelet IgG and platelet-associated IgG (PAIgG) were studied in 68 AIDS-free human immunodeficiency virus (HIV)-infected patients with severe immunologic thrombocytopenic purpura (ITP), for the presence of platelet autoantibodies. Serum IgG with antiplatelet activity was found in 72% of the sera. However, the presence of autoantibodies against platelet surface glycoproteins was not found in these sera by means of Western blot and immunoprecipitation procedures. Nevertheless, an immunoblot immunoassay and an indirect immunofluorescence test against semi-permeabilized platelets demonstrated the presence of antibodies in the patient sera, that reacted with intracytoplasmic platelet components, and which might participate in the elimination of platelet fragments. Direct immunofluorescence tests demonstrated an increased amount of PAIgG in 75% of the patients; the bound antibodies could be eluted with ether in 44% of the cases. These eluates were found to bind to normal platelets but not to Glanzmann type I platelets. Finally, immunoprecipitation procedures demonstrated the presence of platelet autoantibodies in six of the 35 eluates studied. These antibodies recognized GPIIb in two cases, GPIIIa in one case, and an unidentified platelet protein of 150 kDa in the three other cases. The discrepancy between sera and platelet eluates was interpreted as being due to the low titre of the antibodies and to their dilution in polyclonal hypergammaglobulinaemia. The present study provides direct evidence that isolated ITP in some HIV-positive patients is due to the presence of platelet autoantibodies. These results, however, do not exclude either direct or indirect involvement of HIV in the platelet destruction.     

Presence of cross-reactive antibody between human immunodeficiency virus (HIV) and platelet glycoproteins in HIV-related immune thrombocytopenic purpura.

We previously reported the presence in platelet eluates of autoantibodies directed against epitopes of the platelet glycoprotein (GP)IIb/IIIa complex in acquired immunodeficiency syndrome (AIDS)-free human immunodeficiency virus (HIV)-infected patients with immunologic thrombocytopenic purpura (ITP). We investigated whether HIV antibodies recognized platelet membrane antigens to determine whether the virus might be directly or indirectly responsible for the thrombocytopenia in this context. Direct eluates of platelets from 25 patients with HIV-related ITP contained IgG reacting with HIV-GP160/120 and also, in 45% of patients, detectable antiplatelet antibodies, immunochemically characterized as anti-GPIIb and/or anti-GPIIIa in 5 patients. Furthermore, serum HIV-GP160/120 antibodies could be absorbed on and eluted from platelets from normal non-HIV-infected healthy blood donors (indirect eluates). In contrast, GP160/120 antibodies present in the serum of nonthrombocytopenic HIV-infected patients were not absorbable on normal platelets in most patients, suggesting a pathogenic role in HIV-related ITP. We performed detailed studies of a patient with the highest titer of both HIV-GP160/120 and GPIIb/IIIa antibodies in direct and indirect platelet eluates. No antibody binding to GPIIb/IIIa-deficient Glanzmann thrombasthenic platelets was detected. Furthermore, binding/elution experiments conducted with insoluble recombinant GP160 (expressed in baculovirus) and purified platelet GPIIb/IIIa demonstrated that the patient's IgG bound specifically, through the F(ab')2 portion, to a common epitope of HIV-GP160/120 and platelet GPIIb/IIIa. This common epitope was present on a recombinant GP160 expressed in baculovirus but absent from another recombinant GP160 expressed in vaccinia virus, suggesting that the cross-reactivity is dependent on the glycosylation or conformational structure of the GP. We conclude that molecular mimicry between HIV-GP160/120 and platelet GPIIb/IIIa may explain at least some cases of ITP in AIDS-free HIV-infected patients.     

Autoantibodies and autoantigens in chronic immune thrombocytopenic purpura

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder in which antiplatelet autoantibodies bind to antigens on the surface of platelets, resulting in their destruction. The newer antigen-specific (phase III) assays can detect platelet-associated and plasma autoantibodies in approximately 75% and 50% of patients, respectively. Antiplatelet autoantibodies bind to both platelets and megakaryocytes and preliminary evidence suggests that they not only cause platelet destruction but can also decrease platelet production either by interfering with megakaryocyte proliferation/maturation or by causing intramedullary platelet destruction. Autoantibodies are capable of activating complement and causing platelet phagocytosis both in vitro and in vivo. Many platelet-associated and plasma autoantibodies from ITP patients are light chain-restricted, which suggests a clonal origin. Approximately 75% of platelet autoantigens are localized to either the platelet glycoprotein (GP) IIb/IIIa or Ib/IX complex. Inhibition of the binding of autoantibodies from several ITP patients by either another ITP autoantibody or by a monoclonal anti-GPIIb/IIIa antibody suggests that the antigenic repertoire in chronic ITP may be limited. Most autoantigens on GPIIb/IIIa appear to be conformational since they are dependent on the presence of divalent cations. A variety of new investigative techniques have localized a few autoantigens to specific regions of the cytoplasmic or extracellular regions of both GPIIb/IIIa and GPIb/IX.     

Bernard-Soulier syndrome due to the homozygous Asn-45Ser mutation in GPIX: an unexpected, frequent finding in Germany

Bernard-Soulier syndrome (BSS) is a rare inherited disorder with giant platelets, thrombocytopenia and a prolonged bleeding time. These abnormalities are caused by genetic defects of the glycoprotein (GP) Ib-IX complex that constitutes the von Willebrand factor receptor on the platelet surface. Here, we describe four unrelated German patients with low platelet counts and mild bleeding tendency. Three patients had been diagnosed with immune thrombocytopenia (ITP) and were treated with steroids without response. Another patient presented with easy bruising. Peripheral blood smears showed giant platelets. Ristocetin-induced platelet aggregation was almost absent, and quantitative flow cytometry and Western blotting disclosed a greatly reduced surface expression of GPIb-IX. Unexpectedly, sequencing the entire coding regions of GPIbalpha, GPIbbeta and GPIX revealed that all four unrelated patients were homozygous for an A to G mutation in position 1826 of the GPIX gene, constituting a Asn-45Ser change. This mutation has been described before and now represents by far the most often identified molecular defect causing BSS in Caucasians. Because BSS patients are likely to be misdiagnosed with ITP, treatment-resistant ITP patients should be re-evaluated thoroughly. Asn-45Ser genotyping may be a helpful tool for differential diagnosis.     

Flow cytometric analysis of changes in cytoskeletal proteins during platelet destruction and activation using a monoclonal antibody against platelet myosin

We developed a new monoclonal antibody directed against platelet myosin (NNKY6-19). Using this antibody, we analyzed platelet cytoskeletal changes related to stimulation with thrombin and to long-term storage. Immunoelectron microscopy showed increased binding of NNKY6-19 to pseudopods and the open canalicular system during treatment with thrombin (0.1 U/ml) and during storage for 7 days. Flow cytometry also showed increased binding to platelets by NNKY6-19 and an antiactin monoclonal antibody during storage. The binding of NNKY6-19 showed an increase greater than that with the antiactin antibody after storage of platelets for 7 days and after thrombin treatment. These findings indicated that the increased binding of NNKY6-19 had some relationship to changes in intracellular myosin and platelet morphology. Thus use of NNKY6-19 allowed analysis of subtle changes related to platelet activation, which differed from those detected by antibodies against platelet glycoproteins or by the antiactin antibody. This antibody appears to provide a simple method for studying changes in platelet cytoskeletal and surface proteins. © 1993 Wiley-Liss, Inc.     

Clinical significance of positive platelet immunofluorescence test in thrombocytopenia

The sensitivity and specificity of the platelet immunofluorescence test for the diagnosis of idiopathic thrombocytopenia (ITP) was studied in a series of 255 patients. Patients' platelets were tested directly. Diethyl-ether eluates of these platelets and patients' sera were tested indirectly with normal donor platelets. When all three tests were considered, positive results were obtained for 92.0% of the ITP patients with a platelet count of less than 150 X 10(9)/l and for 98.4% of the patients with a count of less than 100 X 10(9)/l. However, for many patients rather weak test results were obtained, with a score of 1/2-1 in 59.8% of the patients. Most patients (94.1%) with a positive direct test had a positive indirect test on the eluate. Thus, platelet-bound antibodies but not platelet-bound immune complexes were present in most, if not all, patients. Positive immunofluorescence tests were obtained for many patients with a diagnosis other than ITP. This resulted in a low specificity of the test for the diagnosis of ITP, evidently because autoimmune thrombocytopenia occurred together with many other diseases and also because antibodies against platelet cryptantigens (expressed by the action of EDTA or by platelet fixation) were present in many patients.