Human platelets antigens influence the viral load of platelets after the interaction of the platelets with HCV and HIV in vitro.

INTRODUCTION: In this study, we evaluated hepatitis C virus (HCV) and human immunodeficiency virus (HIV) - platelet interactions in vitro as well as human platelets antigen (HPA) polymorphisms. METHODS: Platelets were obtained from 100 healthy HPA-genotyped volunteer donors and incubated with HIV or HCV. The viral load after in vitro exposure was detected. RESULTS: The viral load in the platelets after exposure to the virus was higher in the HIV exposure than in the HCV exposure. CONCLUSIONS: HIV-platelet ligation could be more efficient than HCV-platelet interaction. Further, the HPA-1b allele seems to influence the interaction of platelets with HCV.

The αIIb p.Leu841Met (Cab3(a+) ) polymorphism results in a new human platelet alloantigen involved in neonatal alloimmune thrombocytopenia.

BACKGROUND: Fetal-neonatal alloimmune thrombocytopenia (FNAIT) diagnosis relies on maternofetal incompatibility and alloantibody identification. Genotyping for rare platelet (PLT) polymorphisms allowed the identification of three families with suspected or confirmed maternofetal incompatibility for the αIIb-c.2614C>A mutation (Halle et al., Transfusion 2008;48:14-15). STUDY DESIGN AND METHODS: A polymerase chain reaction-sequence-specific primers amplification assay was designed to genotype the αIIb-c.2614C>A mutation. HEK293 cells expressing αIIb-Leu841 or αIIb-Met841 αIIbß3 forms were used to probe the reactivity of maternal sera from these families and to study the effects of the substitution on αIIbß3 expression and functions. RESULTS: Tested by flow cytometry (FCM), one serum sample specifically reacted with αIIb-Met841 but not with αIIb-Leu841 αIIbß3. This specificity revealed the αIIb-Leu841 polymorphism as a new alloantigen named Cab3(a+) . Cross-match testing using FCM also showed the Cab3(a+) antigen to be expressed at the PLT surface. As for anti-human PLT alloantigen (HPA)-3a (or -3b) and anti-HPA-9bw, detection of anti-Cab3(a+) alloantibodies appeared difficult and required whole PLT assays when classical monoclonal antibody-specific immobilization of PLT antigen test failed. In our FNAIT set, the immune response to Cab3(a+) maternofetal incompatibility could induce severe thrombocytopenias and life-threatening hemorrhages. The p.Leu841Met substitution has limited effects, if any, on local αIIb structure, preserving both αIIbß3 expression and functions. CONCLUSION: The Cab3(a+) polymorphism is a new rare alloantigen (allelic frequency <1%) carried by αIIb that might result in severe life-threatening thrombocytopenias. In Sub-Saharan African populations, higher Cab3(a+) gene frequencies (up to 8.2%; Halle et al., Transfusion 2008;48:14-15) and homozygous people are observed.

Molecular insight into human platelet antigens: structural and evolutionary conservation analyses offer new perspective to immunogenic disorders.

BACKGROUND: Human platelet antigens (HPAs) are polymorphisms in platelet membrane glycoproteins (GPs) that can stimulate production of alloantibodies once exposed to foreign platelets (PLTs) with different HPAs. These antibodies can cause neonatal alloimmune thrombocytopenia, posttransfusion purpura, and PLT transfusion refractoriness. Most HPAs are localized on the main PLT receptors: 1) integrin αIIbß3, known as the fibrinogen receptor; 2) the GPIb-IX-V complex that functions as the receptor for von Willebrand factor; and 3) integrin α2ß1, which functions as the collagen receptor. STUDY DESIGN AND METHODS: We analyzed the structural location and the evolutionary conservation of the residues associated with the HPAs to characterize the features that induce immunologic responses but do not cause inherited diseases. RESULTS: We found that all HPAs reside in positions located on the protein surface, apart from the ligand-binding site, and are evolutionary variable. CONCLUSION: Disease-causing mutations often reside in highly conserved and buried positions. In contrast, the HPAs affect residues on the protein surface that were not conserved throughout evolution; this explains their naive effect on the protein function. Nonetheless, the HPAs involve substitutions of solvent-exposed positions that lead to altered interfaces on the surface of the protein and might present epitopes foreign to the immune system.

Regulation of Wnt/beta-catenin pathway by cPLA2alpha and PPARdelta.

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is a rate-limiting key enzyme that releases arachidonic acid (AA) from membrane phospholipid for the production of biologically active lipid mediators including prostaglandins, leukotrienes and platelet-activating factor. cPLA(2)alpha is translocated to nuclear envelope in response to intracellular calcium increase and the enzyme is also present inside the cell nucleus; however, the biological function of cPLA(2)alpha in the nucleus remains unknown. Here we show a novel role of cPLA(2)alpha for activation of peroxisome proliferator-activated receptor-delta (PPARdelta) and beta-catenin in the nuclei. Overexpression of cPLA(2)alpha in human cholangiocarcinoma cells induced the binding of PPARdelta to beta-catenin and increased their association with the TCF/LEF response element. These effects are inhibited by the cPLA(2)alpha siRNA and inhibitors as well as by siRNA knockdown of PPARdelta. Overexpression of PPARdelta or treatment with the selective PPARdelta ligand, GW501516, also increased beta-catenin binding to TCF/LEF response element and increased its reporter activity. Addition of AA and GW501516 to nuclear extracts induced a comparable degree of beta-catenin binding to TCF/LEF response element. Furthermore, cPLA(2)alpha protein is present in the PPARdelta and beta-catenin binding complex. Thus the close proximity between cPLA(2)alpha and PPARdelta provides a unique advantage for their efficient functional coupling in the nucleus, where AA produced by cPLA(2)alpha becomes immediately available for PPARdelta binding and subsequent beta-catenin activation. These results depict a novel interaction linking cPLA(2)alpha, PPARdelta and Wnt/beta-catenin signaling pathways and provide insight for further understanding the roles of these key molecules in human cells and diseases.

[3 polymorphisms of gene GP IIb and the function of GP IIb T13959 G in the platelet transfusion refractoriness].

OBJECTIVE: To investigate the relationship among 3 polymorphisms of GP IIb and the function of GP IIb T13959 G in the platelet transfusion refractoriness(PTR). METHODS: The 26th exon, the 30th exon and the 21st intron of gene GP IIb in 110 individuals were amplified by polymerase chain reaction (PCR), and the PCR products were analyzed with single-strand conformation polymorphism(SSCP) and sequenced to investigate whether there was linkage among the polymorphisms of the gene. Human platelet antigen-3 (HPA-3) gene frequency was detected by Fok I enzyme in 147 patients with hematologic diseases, and was compared with that in 110 normal individuals. Forty-four patients who received apheresis platelet transfusion repeatedly were randomly divided into the HPA-3 homotype group and the control group. The antibodies of the platelet were detected after 3 times of platelet transfusion. RESULTS: There were polymorphisms of gene GP IIb in the 26th, 30th exon and the 21st intron, and the mutations were: T changed into G in 13,959 th of the 26th exon; C changed into T in 16,997 th of the 30th exon; the 9 bps deletion occurred in 11,996-12,004 th of the 21st intron. The 3 polymorphisms had synchronization in the individuals. The results of Fok I enzyme indicated that the frequency of HPA-3a was 83.6% (92/110)and 81.9%(119/147), and that of HPA-3b was 16.4%(18/110) and 19.1%(28/147) in the normal individuals and the patients respectively. There was no significant difference between the patients and normal individuals (P>0.05). After the platelet transfusion, the antibodies of all the cases of the homotype platelet transfusion were negative, but the antibodies in 2 cases of the control group were positive, and there was antibody to HPA-3a in one of the antibodies positive cases. CONCLUSION: (1)There is close linkage among the polymorphisms of gene GP IIb, which is T->G in 13 959 th of the 26th exon, C->T in 16,997 th of the 30th exon, and the 9 bps deletion in 11,996-12,004 th in the 21st intron. (2)The gene frequency of HPA-3a/3b is similar in the normal individuals and patients with hematologic diseases. (3) HPA-3 system may be one of the reasons for PTR in Chinese.

Platelet receptors and their role in diseases.

The absence or deficiency of specific platelet glycoprotein receptors has a well-defined role in causing several rare bleeding disorders such as Bernard-Soulier syndrome or Glanzmann's thrombasthenia. Several new rare disorders caused by defects in other receptors or their signalling pathways have recently been described. Platelet receptors are also often targets for antibodies in pathological conditions. The roles of platelet receptors or their polymorphism variants in diseases such as cardiovascular disorders have started to be intensively investigated over the last 5 years. Many of these findings still remain controversial. Recent evidence points to a fundamental role for platelets and their receptors in the origins of atherosclerosis. Studies on the role of platelet receptors in diseases such as asthma, diabetes and HIV are still at an early stage.

Caspase inhibition causes hyperacute tumor necrosis factor-induced shock via oxidative stress and phospholipase A2.

Dysregulated apoptotic cell death contributes to many pathological conditions, including sepsis, prompting the suggestion that caspase inhibition to block apoptosis could have useful therapeutic applications. Because the cytokine tumor necrosis factor (TNF, also known as TNF-alpha) is both pro-apoptotic and pro-inflammatory and is involved in septic shock, we tested whether caspase inhibition would alleviate TNF-induced toxicity in vivo. General caspase inhibition by the protease inhibitor zVAD-fmk exacerbated TNF toxicity by enhancing oxidative stress and mitochondrial damage, resulting in hyperacute hemodynamic collapse, kidney failure and death. Thus, survival of TNF toxicity depends on caspase-dependent processes. Our results demonstrated the pathophysiological relevance of caspase-independent, ROS-mediated pathways in response to lethal TNF-induced shock in mice. In addition, survival of TNF toxicity seemed to require a caspase-dependent protective feedback on excessive reactive oxygen species (ROS) formation and phospholipase A2 activation.

The ESAT-6/WXG100 superfamily -- and a new Gram-positive secretion system?

ESAT-6 is a small secreted protein of unknown function from Mycobacterium tuberculosis that is of fundamental importance in virulence and protective immunity. A PSI-BLAST search has identified distant homologues of ESAT-6 in more tractable bacteria, including Bacillus subtilis, Bacillus anthracis, Staphylococcus aureus and Clostridium acetobutylicum. The genes for ESAT-6-like proteins often cluster with genes encoding homologues of B. subtilis YukA. I speculate that the ESAT-6-like and YukA-like proteins form a novel Gram-positive secretion system potentially driven by the FtsK/SpoIIIE ATPase domains in the YukA-like proteins. The way is now open to investigate this hypothesis in organisms that are easier to manipulate than pathogenic mycobacteria.

A functional platelet fibrinogen receptor with a deletion in the cysteine-rich repeat region of the beta(3) integrin: the Oe(a) alloantigen in neonatal alloimmune thrombocytopenia.

This report describes a new low-frequency alloantigen, Oe(a), responsible for a case of neonatal alloimmune thrombocytopenia (NAIT). In a population study none of 600 unrelated blood donors was an Oe(a) carrier. By immunochemical studies the Oe(a) antigen could be assigned to platelet glycoprotein (GP) IIIa. Sequencing of GPIIIa complementary DNA from an Oe(a) (+) individual showed deletion of a lysine residue at position 611 (DeltaLys(611)). Analysis of 20 Oe(a) (-) and 3 Oe(a) (+) individuals showed that the DeltaLys(611) form of GPIIIa was related to the phenotype. Anti-Oe(a) reacted with the DeltaLys(611), but not with the wild-type isoforms on stable transfectants expressing GPIIIa, indicating that DeltaLys(611) directly induces the expression of Oe(a) epitopes. Under nonreducing conditions the Pro(33)DeltaLys(611) variant migrated with a slightly decreased molecular weight compared to the Pro(33)Lys(611) isoform suggesting that DeltaLys(611) has an influence on the disulfide bonds of GPIIIa. The Pro(33)DeltaLys(611) GPIIIa could undergo conformational changes and bind to fibrinogen in a similar manner as the Pro(33)Lys(611) isoform. No difference was found in the tyrosine phosphorylation of pp125(FAK), suggesting that DeltaLys(611) has no effect on integrin function. In contrast to all other low-frequency antigens, the DeltaLys(611) isoform was associated with the HPA-1b, but not with the high frequency HPA-1a allele. Comparison with GPIIIa DNA from nonhuman primates indicated that the HPA-1a allele represents the ancestral form of GPIIIa. It can be assumed that the Oe(a) form did arise as a result of a mutational event from an already mutated GPIIIa allele.

Role of 4 platelet membrane glycoprotein polymorphisms on experimental arterial thrombus formation in men.

This study investigates whether the polymorphisms of 3 important platelet receptors affected experimental thrombus formation in men. Forty healthy male volunteers randomly recruited were genotyped for the variable number of tandem repeat (VNTR) of GPIbalpha, the -5T/C polymorphism in the Kozak sequence of GPIbalpha, the 807C/T polymorphism of GPIa, and the PI(A1)/PI(A2) polymorphism of GPIIb/IIIa. Platelet thrombus formation was induced ex vivo by exposing a collagen-coated coverslip in a parallel plate perfusion chamber to native blood for 4 minutes. The shear rates at the collagen surface were 650 and 2600 x s(-1). At 2600 x s(-1) platelet thrombus formation was significantly related only to the 807C/T polymorphism. In contrast, at 650 x s(-1) thrombus formation was significantly altered only by the Kozak sequence polymorphism. The VNTR and the PI(A1)/PI(A2) polymorphisms did not influence thrombus formation. Thus, platelet thrombus formation is significantly influenced by genetic variations of the GPIbalpha and GPIa receptors. The effect of these polymorphisms was dependent on the blood flow rate.

The renal effects of Bothrops jararacussu venom and the role of PLA(2) and PAF blockers.

The most common complication in the lethal cases of ophidian bites in Brazil is acute renal failure, but its pathogenesis is obscure. The effects of Bothrops jararacussu venom (3, 10 and 30 microg/ml) were examined using the isolated perfused kidney from Wistar rats. Dexamethasone, and WEB 2086, a triazolobenzodiazepine substance, which is a platelet activating factor receptor antagonist, were tested for a possible blockade of the renal effects in the presence of 10 microg/ml of venom. The most intense effects of the venom were noticed at 120 min after using 30 microg/ml. We observed a decrease in the perfusion pressure and in the renal vascular resistance. However, the glomerular filtration rate (GFR) and the urinary flow (UF) increased significantly. The percent of sodium (%Na(tot)(+)) and potassium (%K(tot)(+)) tubular transport were also decreased. Dexamethasone was unable to block the effects of B. jararacussu in the kidney, while WEB 2086 blocked its effect in glomerular filtration rate, urinary flow and in the percentage of total tubular potassium reabsorption. We suggest that this venom promotes diuresis independently of perfusion pressure drop. The alterations in GFR, UF and %K(tot)(+) are probably mediated by platelet activating factor. Dexamethasone did not block the renal effects maybe because of the concentration used in this work or maybe the renal effects are promoted by the myotoxin, which does not have PLA(2) activity.

Platelet glycoproteins and their role in diseases.

The role of platelet glycoprotein receptors in disorders caused by their absence or defects such as in Bernard-Soulier syndrome or Glanzmann's thrombasthenia has been known for many decades now. Their function as targets for pathological antibodies is also well established. The possible roles of platelet receptors or their polymorphism variants in the origins of diseases such as cardiovascular disorders are less well studied. Investigation of this area began about five years ago and many findings still remain controversial. The involvement of platelet receptors in other diseases like asthma, diabetes and HIV are only starting to be studied.

Superimposed levels of regulation of the 4-hydroxyphenylacetate catabolic pathway in Escherichia coli.

The regulation of the Pg promoter, which controls the expression of the meta operon of the 4-hydroxyphenylacetic acid (4-HPA) catabolic pathway of Escherichia coli W, has been examined through in vivo and in vitro experiments. By using Pg-lacZ fusions we have demonstrated that Pg is a promoter only inducible in the stationary phase when cells are grown on glucose as the sole carbon and energy source. This strict catabolite repression control is mediated by the cAMP receptor protein (CRP). This event does not require the presence of the specific HpaR repressor or the 4-HPA permease (HpaX), excluding the involvement of a typical inducer exclusion mechanism. However, the acetic acid excreted in the stationary phase by the cells growing in glucose acts as an overflow metabolite, which can provide the energy to produce cAMP and to adapt the cells rapidly to the utilization of a new less preferred carbon source such as the aromatic compounds. Although Pg is not a final sigma(38)-dependent promoter, it is activated by the global regulator integration host factor (IHF) in the stationary phase of growth. Gel retardation assays have demonstrated that both CRP and IHF simultaneously bind to the Pg upstream region. DNase I footprint experiments showed that cAMP-CRP and IHF binding sites are centered at -61.5 and -103, respectively, with respect to the transcription start site +1 of the Pg promoter.

Effect of the Pl(A2) alloantigen on the function of beta(3)-integrins in platelets.

The polymorphism responsible for the Pl(A2) alloantigen on the beta(3)-component of beta(3)-containing integrins is reported to be a risk factor for coronary thrombosis. This study examined the effect of Pl(A2) on the function of beta(3)-integrins using platelets from subjects homozygous and heterozygous for Pl(A1) and Pl(A2). There was overlap in the distribution of the dissociation constant (K(d)) and maximum fibrinogen binding (B(max)) values for fibrinogen binding to alpha(IIb)beta(3) on platelets from Pl(A1) and Pl(A2) homozygotes and Pl(A1)/Pl(A2) heterozygotes. However, whereas there was no statistical difference in these values for the Pl(A1) homozygotes and Pl(A2) heterozygotes, the K(d) for the Pl(A2) homozygotes was significantly lower than that for the Pl(A1)/Pl(A2) heterozygotes, but was not statistically different from that for the Pl(A1) homozygotes. No differences were detected in ADP sensitivity between platelets from Pl(A1) homozygotes and Pl(A1)/Pl(A2) heterozygotes, in the IC(50) for RGDS inhibition of fibrinogen binding to alpha(IIb)beta(3), in the alpha(v)beta(3)-mediated adhesion of platelets to osteopontin and vitronectin, and in the phorbol ester-stimulated adhesion to fibrinogen of B lymphocytes expressing alpha(IIb)beta(3) containing either the Pl(A1) or the Pl(A2) polymorphism. Finally, no differential effects of Pl(A2) on turbidometric platelet aggregation, platelet secretion, or platelet thrombus formation were found as measured in the PFA-100. Because no differences were detected in the ability of beta(3)-integrins to interact with ligands based on the presence or absence of the Pl(A2) polymorphism, the results suggest that factors unrelated to beta(3)-integrin function may account for the reported association of the Pl(A2) allele with coronary thrombosis.

Adhesive and signaling properties of a naturally occurring allele of glycoprotein IIIa with an amino acid substitution within the ligand binding domain-the Pena/Penb platelet alloantigenic epitopes.

Platelet membrane glycoprotein IIIa (GPIIIa) is the most polymorphic integrin subunit in man, with at least seven recognized allelic isoforms present in the human gene pool. Whether these allelic variants of the GPIIb-IIIa complex differ in the ability to interact with the adhesive ligand fibrinogen (Fg) is still unknown. Since the Pena and Penb allelic forms of GPIIIa are distinguished by a single Arg143Gln amino acid substitution within the RGD binding domain of GPIIIa and anti-Pena human alloantibodies have been shown to bind GPIIb-IIIa on the platelet surface and inhibit ADP-induced platelet aggregation, we expressed both forms of this integrin in Chinese hamster ovary (CHO) cells and examined the relative adhesive properties. Both allelic forms of GPIIb-IIIa were expressed on the cell surface and were recognized by a well-characterized panel of murine and human monoclonal and polyclonal antibodies. Like Pena, the Penb form of GPIIb-IIIa could undergo conformational changes in response to RGD peptide binding, and could be induced by activating antibodies to bind Fg and the Fg mimetic antibody P1-55. The binding affinity for Fg of the Pena form of the GPIIb-IIIa complex was not significantly different from that of the Penb form, nor was its ability to signal to focal adhesion kinase, suggesting that Arg143Gln polymorphism has little or no effect on integrin function. Examination of the functional consequences of other integrin polymorphisms may be necessary to determine whether they constitute a risk factor for thrombosis or hemorrhage.

The human platelet membrane glycoprotein IIb/IIIa complex: a multi functional adhesion receptor.

The glycoprotein GPIIb/IIIa complex is a major constituent of the platelet membrane; it plays an important role in platelet adhesion and aggregation. The complex is a member of the integrin superfamily. Integrins are related membrane receptors which mediate the adhesive interactions of a variety of cells; they specifically recognize the arginine-glycine-aspartic acid (RGD) sequence present in several adhesive proteins. The GPIIb/IIIa complex of activated platelets can bind fibrinogen, von Willebrand factor, fibronectin, vitronectin and thrombospondin. Platelets are activated by a variety of signals including extracellular matrix molecules and soluble factors; upon platelet activation the complex undergoes a conformational change, thus permitting the macromolecular ligands access to their binding sites. In turn, fibrinogen binding results in a receptor modification and neoantigens exposure; such events may participate in signal transduction. The adhesive proteins compete reciprocally for binding to GPIIb/IIIa, and the complex binds to different domains of them, thus creating multiple interactions with the ligands.

Molecular genetic aspects of human platelet antigen systems.

Recent advances in molecular and cellular biology have made it possible to build upon previous serological and biochemical studies of human platelet alloantigen systems in important and exciting ways. In addition to providing a detailed basic understanding of the polymorphisms that are responsible for eliciting an alloimmune response, the molecular characterization of platelet membrane glycoprotein polymorphisms is expected to have an increasingly large clinical impact. As the molecular basis of the remaining platelet antigen systems becomes known, our ability to design novel diagnostic and therapeutic approaches for the care and management of patients with PTP and NATP should improve.