Thrombocyte antigens of dogs]

Four platelet-specific antigens were identified in dogs by means of the platelet agglutination test; these antigens were not connected with the erythrocyte or leukocyte antigens, they were inherited by the mendelian type and controlled by several recessive genes.     

Fluorimetric coupled enzyme assay for lysoplasmalogenase activity in liver.

Two new proteins with apparent molecular masses of 53 kDa and 190 kDa have been identified in both sarcoplasmic reticulum and human blood platelets using a monoclonal antibody, FII1b5. The sarcoplasmic reticulum FII1b5 antigens were present in the terminal cisternae fraction, but were absent from light sarcoplasmic reticulum. The platelet and skeletal muscle proteins were not sensitive to digestion with endoglycosidase H under conditions that removed carbohydrate from the 53 kDa glycoprotein in sarcoplasmic reticulum or GPIIIa in platelet microsomes and did not bind 45Ca in a nitrocellulose overlay calcium-binding assay. These results distinguished the FII1b5 antigens from the 53 kDa glycoprotein and calsequestrin of sarcoplasmic reticulum. The 190 kDa platelet and sarcoplasmic reticulum proteins were extracted from membranes with high concentrations of NaCl, indicating that the high molecular mass FII1b5 antigens are peripherally associated with the bilayers. In contrast, the platelet and muscle 53 kDa proteins remained membrane-bound in the presence of high salt concentrations, suggesting that they are integral proteins.     

Management and cause analysis of platelet transfusion refractoriness

Platelet transfusion refractoriness (PTR) can be defined as the less increment of platelet count than expected after platelets transfusion, which is a challenging and expensive problem often observed in platelet-transfusion-dependent patients. Although PTR occurs most frequently due to non-immune causes, a significant minority is still caused by immune factors. The most important factor in immune dependent PTR is alloimmunization against Class I human leukocyte antigens (HLAs) or human platelet antigens (HPAs). The compatible platelets can be provided to immune-mediated patients using platelet crossmatching, HLA matching, and antibody specificity testing. These measures-aimed to eliminate donor-specific HLA antibodies will lead to the improved clinical management of PTR patients, caused by severe alloimmunization.     

Human platelets as a source of HL-A antigens : a study of various solubilization techniques.

The efficiency of various methods of solubilizing HL-A platelet antigens was investigated. The yield of soluble material was compared with that obtained from lymphocytes in culture in order to judge the quality of platelets as a source of HL-A antigens. The conclusion was reached that platelets, easily obtainable, can be considered as a good source of HL-A antigens.     

HLA compatibility and survival of 51 chromium-labeled allogeneic thrombocytes

In 37 patients with thrombocytopenia (mostly with ITP) the survival time of 51Cr-labeled allogeneic platelets was investigated. The HLA antigens were typed in donors and recipients and the presence of HLA cytotoxins and specific thrombocyte antibodies in sera of patients were examined. In 7 cases the identity of 2 HLA antigens, in 15 cases that of 1 HLA antigen and in 15 cases the HLA incompatibility between donor and patient were found. The survival of platelets did not depend on the degree of HLA compatibility, unless the HLA cytotoxins in sera of patients appeared. The HLA, as well the specific platelet antibodies brought about the shortened platelet survival to 1 day and less. The importance of these observations for platelet kinetics is discussed.     

Human monoclonal autoantibodies to characterize platelet antigens in immune-mediated thrombocytopenia

Idiopathic thrombocytopenic purpura is characterized by antiplatelet antibodies which mediate the rapid destruction of these cells by the reticuloendothelial cell system. Low serum titers of autoantibodies and the polyclonal nature of human serum make it difficult to identify platelet target antigens with plasma antibodies. To circumvent these problems, we have utilized the techniques of EBV transformation and somatic cell hybridization in order to isolate human monoclonal antibodies from patients with ITP. In this paper we describe the use of human monoclonal autoantibodies to characterize an activation specific antigen on GPIIIa and an autoantigen on the GPIb complex. Ultimately, we hope to determine whether these autoantibodies emerge from a pool of naturally occurring antibodies to activation or senescence antigens, or are triggered by environmental agents such as bacteria or virus, which are comprised of antigens similar to those found on the platelet membrane.     

Detection of phenylalanine hydroxylase protein in human platelets. Immunochemical detection

A protein reactive with anti-phenylalanine hydroxylase monoclonal antibody PH8 has been recovered from human platelet extracts. Two bands corresponding to molecular masses of about 60 kDa and 55 kDa were revealed by immunoblotting after electrophoresis according to Laemmli. Using the same antibody, a single band with a molecular mass of 60 kDa was demonstrated in extracts from human pineal gland; two similar antigens were found in human liver extracts and no antigen was found in adrenal gland extracts. Monoclonal antibodies, PH1 and PH3, did not react with these antigens during immunoblotting. Monoclonal antibodies, PH7 and PH9, reacted with the 55 kDa antigen in platelet extracts. The antigen content in platelet extracts was measured by ELISA with monoclonal antibodies relative to its content in the liver. The antigen content in platelet extracts was about 100 times as low as that in liver extracts and amounted to 100 ng/mg of protein. These findings suggest that the phenylalanine hydroxylase antigen is present in human platelets.     

Platelet membrane glycoproteins: role in primary hemostasis and component antigens

The biochemical details of the platelet surface as they relate to normal platelet function have been elucidated through study of labeled membranes from both normal platelets and those with congenitially defective function. Several cytoadhesive glycoprotein complexes which are integral components of the platelet membrane have been demonstrated to act as important receptors for extracellular matrix macromolecules. Glycoproteins Ia/IIa (collagen receptor), Ic/IIa (fibronectin receptor), and IIb/IIIa (fibrinogen receptor) belong to a family of cytoadhesive complexes called the integrins, while glycoprotein Ib/IX, the major von Willebrand receptor, has different features. These same major glycoproteins comprise all of the alloantigens and most of the autoantigens that have been characterized. Glycoprotein IIb/IIIa contains the alloantigens, PlA (Zw), Bak (Lek), and Pen (Yuk), as well as the most frequent target antigenic sites for anti-platelet autoantibodies. Because a number of platelet alloantigens were discovered independently by more than one group, nomenclature is confusing at present, although a system analogous to that used for histocompatibility antigens has been proposed. Precise identification of the antigenic epitopes has not yet been accomplished for all of the platelet antigens. Current research efforts include characterization of antigenic epitopes, elucidation of mechanisms by which platelet immunization occurs, and determination of the clinical implications of the presence of various platelet antibodies.     

Recent trends in platelet antigen/antibody detection

The detection of platelet antigens and platelet antibodies has always been difficult. Recent technical achievements are due to the availability of more specific and sensitive reagents (i.e. F(ab)2 fragments; higher specific activity of labels; monoclonal antibodies directed against platelet membrane constituents etc.) and the application of advanced immunological assays to platelet immunology (i.e. blotting assays, "capture"-ELISA's and radioimmunoprecipitation in conjunction with SDS polyacrylamide gel electrophoresis). These techniques have permitted the definition and immuno-chemical characterization of new platelet allo- and autoantigens, have assisted in clinical diagnosis and promoted our understanding of pathogenic mechanisms. Illustrative examples are presented and discussed.     

Pathogenesis of the delayed phase of Rauscher virus-induced thrombocytopenia

BALB/c (H-2d) mice infected with Rauscher murine leukemia virus (RMuLV) developed two phases of thrombocytopenia: an acute phase, probably due to direct virus-platelet interactions, and a delayed phase, starting 2 to 3 wk after virus injection, which was associated with the infection of megakaryocytes by RMuLV and with the expression of RMuLV gp70 and p30 antigens on platelet membranes. This study was concerned with the pathogenesis of this second phase of thrombocytopenia. During this period, the number of marrow megakaryocytes was increased. A peripheral platelet destruction was further indicated by reduced platelet life span. It was shown that radiolabeled platelets, either normal or infected, were submitted to a more rapid clearance in infected recipients than in normal recipients. This might be due to the splenomegaly observed in infected recipients. However, the immediate clearance of gp70+ platelets was more accelerated in infected recipients with high titers of serum anti-gp70 antibodies than in infected recipients without detectable serum anti-gp70 antibodies. In addition, the passive transfer of anti-RMuLV serum to normal BALB/c mice induced a rapid and specific clearance of previously injected radiolabeled platelets expressing RMuLV antigens. In H-2d mice, viral gp70 antigen expression on platelets correlated with the development of delayed thrombocytopenia; but H-2k strains of mice, although susceptible to RMuLV and expressing RMuLV-related antigens on their platelets, did not develop any anti-RMuLV antibodies nor any delayed thrombocytopenia. These results suggest that specific clearance of gp70+ platelets in the presence of significant amounts of serum antiviral antibodies and nonspecific hypersplenism play a role in the development of delayed thrombocytopenia in RMuLV-infected mice.     

Involvement of platelet glycoprotein Ib in platelet microparticle mediated neutrophil activation

Summary Platelet microparticles (MPs) are membrane vesicles shed by platelets after activation, and carry antigens characteristic of intact platelets, such as glycoprotein (GP) IIb/IIIa, GPIb and P-selectin. Elevated platelet MPs have been observed in many disorders in which platelet activation is documented. Recently, platelet GPIb has been implicated in the mediation of platelet–leukocyte interaction via binding to its ligand Mac-1 on leukocyte. The role of GPIb for mediating adhesion-activation interactions between platelet MPs and leukocytes has not been clarified. In this study we investigate the role of GPIb in the interplay between platelet MPs and neutrophils. Platelet MPs were obtained from collagen-stimulated platelet-rich plasma (PRP). In a study model of neutrophil aggregation, platelet MPs can serve a bridge to support neutrophil aggregation under venous level shear stress, suggesting that platelet MPs may enhance leukocyte aggregation, which would bear clinical relevance in diseases where the platelet MPs are elevated. The level of aggregation can be reduced by GPIb blocking antibodies, AP1 and SZ2, but not by anti-CD18 mAb. The GPIb blocking antibodies also decreased platelet MP-mediated neutrophil activation, including β2 integrin expression, adherence-dependent superoxide release and platelet MP-mediated neutrophil adherence to immobilized fibrinogen. Our data provide the evidence for the involvement of GPIb–Mac-1 interaction in the cross-talk between platelet MPs and neutrophils.     

Leukocyte- and platelet-derived microvesicle interactions following in vitro and in vivo activation of toll-like receptor 4 by lipopolysaccharide

Background

Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS).

Methodology/Principal Findings

Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens.

Conclusions/Significance

Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood.     

N-terminal sequence of human leukocyte glycoprotein Mo1: conservation across species and homology to platelet IIb/IIIa

Mo1 and gp160-gp93 are two surface membrane glycoprotein heterodimers present on granulocytes and monocytes derived from humans and guinea pigs, respectively. We purified both antigens and found that their alpha subunits had identical N-termini which were significantly homologous to the alpha subunit of the human adhesion platelet glycoprotein IIb/IIIa.     

"Anti-platelet antibodies" in HIV infected haemophiliacs.

We have studied anti platelet antibodies and circulating immunocomplexes in 16 haemophiliacs with mild thrombocytopenia eight of which were infected by human immunodeficiency virus (HIV). No difference in platelet count was observed between HIV+ (143 +/- 31 x 10(9)/l) and HIV- patients (148 +/- 30 x 10(9)/l). On the contrary, HIV+ haemophiliacs had serum platelet bindable IgG (SPBIgG), normal platelet associated IgG (PAIgG), high serum IgG and circulating immunocomplexes (CIC). Considering all 16 patients serum IgG correlated with CIC (r = 0.7 p less than 0.01) and SPBIgG (r = 0.6 p less than 0.01) respectively. We obtained also a positive correlation between serum CIC and SPBIgG (r = 0.51 p less than 0.05). Immunoblotting of patients' sera showed no specific binding to target platelet antigens. In conclusion there is no evidence of HIV related immune thrombocytopenia in our haemophiliacs but the study confirms the appearance of immunocomplexes in the HIV+ subjects.     

Recombinant rabbit Fab with binding activity to type-1 plasminogen activator inhibitor derived from a phage-display library against human α-granules

The display of panels of antibody (Ab) fragments on the surface of filamentous bacteriophage offers a way of making Ab with defined binding specificities. Because rabbit Ab are routinely utilized as immunologic probes in a variety of biological techniques, the aim of this study was to design and utilize primers for the amplification of mRNAs encoding rabbit κ light and λ heavy chains for the construction of an Ab library from this species. Using the polymerase chain reaction, a diverse Ab library with a repertoire of 2 × 10⁷ clones was derived from the spleen and bone marrow of a rabbit that had been immunized with purified human platelet α-granules. From this library, specific clones were isolated after three rounds of affinity selection with binding activity to type-1 plasminogen activator inhibitor, a trace protein contained in platelet α-granules. These data indicate that recombinant phage-displayed Ab libraries obtained after immunization with complex biological antigens can be employed for the isolation of rabbit monoclonal Fab against specific antigens contained in the biological sample.     

Expression of platelet membrane glycoproteins and alpha-granule proteins by a human erythroleukemia cell line (HEL)

We demonstrate that HEL, a human erythroleukemic cell line, has numerous megakaryocytic markers which were markedly enhanced following the addition of the inducers dimethyl sulfoxide or 12-O-tetradecanoylphorbol-13-acetate to the culture medium. Ultrastructural and cytochemical studies showed: (i) the presence of organelles morphologically resembling the platelet alpha-granules; and (ii) a peroxidase activity with the same characteristics as that specifically found in platelets. The platelet alpha-granule proteins (von Willebrand factor, platelet factor-4 and beta-thromboglobulin) were immunologically detected in the HEL cell cytoplasm and their amounts increased after induction. Of particular interest was the presence of platelet membrane proteins. A monoclonal antibody specific for glycoprotein Ib bound to HEL cells. Platelet membrane glycoproteins IIb and IIIa were identified on intact cells using specific antibodies in a binding assay or in cell lysates using either crossed immunoelectrophoresis or an immunoblotting procedure following SDS-polyacrylamide gel electrophoresis. Most HEL cells also expressed the platelet alloantigen PIA1. All of the platelet membrane proteins were present in higher amounts after induction. Glycophorin A, specific for the erythroid lineage, was also detected on HEL cells. Thus, while confirming the presence of erythroid markers, our studies provide evidence that the HEL cell line also expresses platelet antigens. As such, HEL cells represent a unique system with which to study the biosynthesis of platelet-specific proteins and glycoproteins.     

Platelet Aggregation Test with Measles Antigen in Multiple Sclerosis

With the measles platelet aggregation test, a new technique recently developed for measuring virus antibody, 153 serum specimens from patients with multiple sclerosis and 164 controls were tested. With one of the three measles antigens used in the test a significantly higher positive rate (P<0·001) was obtained in the specimens from the patients with multiple sclerosis (40%) than in those from the controls (11%). The other two measles antigens also yielded slightly but not significantly higher positive rates in the patients with multiple sclerosis.     

Stimulation of lymphocytes by platelet-antibody-complexes and their role in the pathogenesis of idiopathic thrombocytopenia

The effect of washed human platelets, platelet lysates, and platelet antibody complexes on 14C-thymidine incorporation by human lymphocytes was studied. For sensitization of platelets, HLA-specific alloantibodies as well as platelet autoantibodies were used. Lymphocytes for in vitro cultures were collected from unsensitized individuals, healthy women with proven fetomaternal immunization against HLA antigens and patients with idiopathic thrombocytopenic purpura (ITP). Unsensitized platelets have a dose-dependent inhibitory effect on the in vitro proliferation of normal lymphocytes induced by mitogens (PHA, ConA, PWM). Platelet antibody complexes (allo- and autoantibodies; allogenic and autologous lymphocyte-platelet combinations) did not stimulate 14C-thymidine incorporation. Lymphocytes from ITP patients showed a significantly reduced stimulatory response toward PHA compared to normal persons. These findings are discussed in the light of our present knowledge regarding the role of cellular immune reactions in the pathogenesis of ITP.     

The problem of preparing avid cytotoxic anti-B-lymphocyte serums]

There were anti-B-lymphocyte toxins in 40 samples (= 7.58%) from 527 examined sera of pregnant women and polytransfused persons without HLA cytotoxins and 84 samples (= 33.2%) from 253 anti-HLA sera. Of 49 specific anti-B sera 27 were suitable for typing B-lymphocyte antigens; with the help of 13 of these sera 5 specific antigens of B-lymphocytes called 1-5 could be determined. These 27 sera produced a positive cytotoxic reaction, mainly with the strength of +++ or ++. Attempts of absorbing HLA antibodies from anti-B lymphocyte sera led to unsatisfactory results. Sometimes the content of anti-B-lymphocyte toxins could also be diminished by a platelet absorption, in other cases the absorption was insufficient and had to be repeated therefore.     

Co-localization of CD9 and GPIIb-IIIa (·IIb ·3 integrin) on activated platelet pseudopods and ·-granule membranes

Summary CD9 is a 24-kDa membrane glycoprotein expressed on the surface of human platelets and potentially involved in cellular activation and adhesion functions. This protein belongs to a recently delineated family of cell-surface antigens that span the membrane four times, called tetraspans, and found mainly in leucocytes and tumour cells. As a first approach to clarify the function of CD9, we used immunoelectron microscopy to determine the localization of this antigen in human platelets, and compared its distribution with that of the GPIIb-IIIa integrin, the platelet receptor for fibrinogen. Monoclonal antibodies against CD9 (MAb7) and GPIIb-IIIa (HP1-1D) coupled to colloidal gold of different sizes (5 and 15 nm) were incubated with intact platelets in suspension or on ultrathin sections of platelets embedded in LR white. CD9 was found in association with GPIIb-IIIa on the inner face of ·-granule membranes. These two antigens also co-localized on pseudopods of activated platelets and in contact regions between adjacent platelets. CD63, another member of the tetraspan family, was absent from ·-granules but was associated with lysosomal structures. Flow cytometric analysis of platelet CD9 with a series of monoclonal antibodies revealed an increased expression upon thrombin stimulation, confirming the presence of an intracellular granular pool. The observation that CD9 and GPIIb-IIIa are stored in the same intracellular structures and migrate to the same activation zones after platelet stimulation lends support to previous suggestions of a close association between CD9 and GPIIb-IIIa in human platelets and of a possible involvement of CD9 in adhesive functions of platelets. This revised version was published online in November 2006 with corrections to the Cover Date.