Platelet Alloantigen Research Articles

Platelet reactive alloantibodies responsible for immune thrombocytopenia in Malay population

Background: Alloantibodies against human platelet alloantigens (HPAs) are responsible for the development of platelet transfusion refractoriness (PTR) in patients receiving random platelets and bleeding disorder in babies with fetal neonatal alloimmune thrombocytopenia (FNAIT). Recently, our results based on the analysis of the allelic distribution of HPAs indicated that immunization may occur among Malay. In this study, we sought to analyze the frequencies of platelet reactive alloantibodies responsible for FNAIT and PTR in Malaysia.Methods: Sera from suspected FNAIT (n = 295) and PTR (n = 74) were collected in five years period (2008-2013) and tested for the presence of platelet reactive antibodies by the use of antigen capture assay.Results: In 5/74 (5.41%) platelet specific antibodies against HPA-2b (n = 1), HPA-5a (n = 1), HPA-5b (n = 1), HPA-15b (n = 2) could be identified in our PTR cohort. In FNAIT cohort, platelet specific alloantibodies could be detected in 18 sera (6.10%) consisting anti-HPA-1a (n = 1), anti-HPA-3a (n = 3), anti-HPA-5a (n = 6), anti-HPA-5b (n = 6), anti-HPA-15a (n = 1), and anti-HPA-15b (n = 1).Conclusion: Our study indicates that anti-HPA-3, -HPA-5 and -HPA-15 antibodies seems to be the most platelet specific antibodies involved in FNAIT and PTR cases in Malaysian population. Since similar HPA allelic distribution among Malaysian and Indonesian populations have been observed, immunization against these three HPA systems are expected to be the most potential risk of alloimmune mediated platelet disorders in Indonesia.

Thrombophilic disorders in migraine.

Migraine is a widespread disorder thataffects about 18% of the people, in 2/3 ofcases women (1).The concept that migraine could berelated to some vascular diseases, such asmyocardial infarction or a stroke, havegrown over the years, mainly in migrainewith aura (2–6). In the last decades, severalstudies have been carried out to under-stand if and what the linkage could be.Among these studies, we have found manyresearches on thrombophilic disorders. Inthiscontext,thestudiesfocusonalterationsof coagulation factors, coagulation pro-teins,homocysteine with MTHFR variants,andauto-immunedisordersof coagulationsuch as anti-phospholipid antibodies. Theavailable data are sometimes difficult tointerpret. The first problem we meet is thatthrombophilic disorders usually have a lowprevalence, so we need larger sample stud-ies to reach significant results. The secondproblem could be how to correctly selectthe proper study population,because somedeficits could be inherited and some othersacquired,andresultscouldchangedepend-ing on contextual factors such as preg-nancyoranti-coagulantorestroprogestinictherapy. The third problem, mainly deal-ing with auto-immune coagulative disor-ders,couldbedifferentlaboratorymethodsand the different positivity criteria used.Another problem is that the prevalence ofthe coagulative deficit could change withage, so we find variable results in study-ing children or adults. Therefore, we needto calculate properly the sample size ofthe study population. Another chance tostudy the correlation between migraineand thrombophilic disorders could be byusing the data collected through spe-cific disease database. The main Interna-tional Disease Database is the EuropeanRegister on anti-phospholipid syndrome(APS). These data show that the first andmost frequent symptom of the syndromeis a headache, probably a migraine con-sidering the reported descriptions (7, 8).Headaches are so important in APS tolead expert hematologists to recommendcarrying out tests for anti-phospholipidantibodies (aPL) in all patients present-ing severe headaches (9). The Italian Reg-istry of Rare Diseases is another impor-tant source of information. It was activatedby the National Health Institute (NHI)almost 10years ago, through the creationof Regional Registries. In the last Data-base Report of Rare Diseases’ 2013 data-base of the Piedmont Region, we find howthe number of the diagnosis of a throm-bophilic disease performed in an headachecenter is equal or higher than the onerecorded by those centers taking care ofpatients who had experienced a throm-botic event (10). Articles published morerecently correlate migraine and throm-bophilic alterations, so we can presumethey could be co-morbid.Coagulative factors II, V, VII, VIII, IX,and von Willebrand have been studied inmigraineurs. The correlation between fac-tor II mutations and migraine has beenstudied throughout several smaller studies,always with negative results (11, 12). Onlyrecently,a population based study reporteda higher prevalence of factor II mutationin women suffering from migraine, but aselection bias could be suspected becausethis population had been selected amongwomen with a positive family history ofvenous and arterial thrombosis (13). Wecan find a few reports of deficit of fac-tor VII, which seems to be non-relevant(14, 15), and of factors von Willebrand,VIII e IX, and various platelet alloantigens(11, 16), topics that would be interestingto investigate further. Most authors studiedthe alterations of factor V in migraineurs,and also in these studies the sample sizeseem to be unrepresentative, and resultsare controversial (11, 15, 17–20). Further-more, some authors considered homozy-gous mutation alone, while others consid-ered both homozygous and heterozygousmutation. Among the coagulative factors,the V seems to be the most interestingone, but larger and better designed stud-ies are required, with more severe selectionof inclusion criteria.Still little is known on deficit of coagu-lation proteins C and S. The main prob-lem of studying protein C deficit is itslow prevalence, which is around 0.4%, thatrequires larger sample sizes to study anddetect any significant difference in preva-lence between migraineurs and healthycontrols. The few available studies are toosmall to clearly understand this topic (11,18, 19). The prevalence of protein S deficitis higher, but this type of deficit is morecomplex to understand and diagnose. Firstof all, laboratory methods that are usedshould be clearly defined, such as the sen-sitivity of the laboratory kit used andwhat type of protein S was dosed, and isit the total or only the free protein, allelements needed to make a proper diag-nosis of the type of defect (genetic oracquired). It would be better to distinguishinherited from acquired types of deficit,because of the different clinical relevanceand etiopathogenesis. Inherited deficits areeasiertodetectandthelevelofproteinScanbe stable over time, on the contrary of theacquired deficits. It is also very importantto consider and exclude iatrogenic deficits,such as those due to anti-coagulant orestroprogestinic therapy,or the physiologic

Modeling and molecular dynamics simulations of the V33 variant of the integrin subunit β3: Structural comparison with the L33 (HPA-1a) and P33 (HPA-1b) variants

The human plateletalloantigen (HPA)-1 system, the first cause of alloimmune thrombocytopenia in Caucasians, results from leucine-to-proline substitution (alleles 1a and 1b) of residue 33 in β3 subunit of the integrin αIIbβ3. A third variant with a valine (V33) has been described. Although leucine and valine share similar physicochemical properties, sera containing alloantibodies to the HPA-1a antigen variably reacted with V33-β3, suggesting structural alterations of β3. To analyze the effect of the L33V transition, molecular dynamics simulations were performed on a 3D structural model of the V33 form of the whole β3 extracellular domain (690 residues). Dynamics of the PSI (carrying residue 33), I-EGF-1, and I-EGF-2 domains of β3 were compared to previously obtained dynamics of HPA-1a structure and HPA-1b structural model using classical and innovative developments (a structural alphabet). Clustering approach and local structure analysis showed that L33-β3 and V33-β3 mostly share common structures co-existing in different dynamic equilibria. The L33V substitution mainly displaces the equilibrium between common structures. These observations can explain the variable reactivity of anti-HPA-1a alloantibodies suggesting that molecular dynamic plays a key role in the binding of these alloantibodies. Unlike the L33P substitution, the L33V transition would not affect the structure flexibility of the β3 knee, and consequently the functions of αIIbβ3.

Polymorphisms in human leukocyte antigens, human platelet antigens, and cytokine genes in hantavirus cardiopulmonary syndrome patients from Ribeirão Preto, Brazil

Hantavirus cardiopulmonary syndrome is a severe human disease associated with hantavirus infection. The clinical course of illness varies greatly among individuals, possibly due to viral and immunological elements and the influence of host genetic factors on clinical outcome. As the magnitude of immune activation has been associated with disease severity, polymorphisms in genes involved in the immune response that may affect the development of this syndrome were investigated. Polymorphisms in the TGF-β, IL-10, IL-6, and IFN-γ genes, human leukocyte antigens (HLA), and human platelet alloantigens (HPA) genes were investigated in 122 patients with Araraquara virus infection from Ribeirão Preto, Brazil. Patients were divided into two groups: hantavirus cardiopulmonary syndrome (HCPS group; n = 26) and hantavirus seropositive only (n = 96). The frequencies of HLA alleles, cytokines and platelet antigen genotypes were evaluated in both groups and compared to a control group. The data demonstrated no significant influence of the HLA alleles, HPA, IL-6, and IL-10 genotypes on susceptibility to hantavirus infection. However, the hantavirus seropositive group presented a significantly higher frequency of a polymorphism associated with a high IFN-γ production than the HCPS group. In addition, a genotype associated with high TGF-β production was found more frequently in individuals infected with hantavirus than in the control group. This phenotype was associated with a less intense thrombocytopenia in the HCPS group and may be protective against the most severe form of hantavirus disease. More studies are required to quantify further the influence of the high TGF-β producer phenotype on the outcome of hantavirus infection.

Identification of potential platelet alloantigens in the Equidae family by comparison of gene sequences encoding major platelet membrane glycoproteins

Platelet alloantigens in horses may play an important role in the development of neonatal alloimmune thrombocytopenia (NAIT). The objective of this study was to evaluate genes encoding major platelet glycoproteins within the Equidae family in an effort to identify potential alloantigens. DNA was isolated from blood samples obtained from Equidae family members, including a Holsteiner-Oldenburg cross, a Quarter horse, a donkey, and a Plains zebra (Equus burchelli). Gene sequences encoding equine platelet membrane glycoproteins IIb, IIIa (integrin subunits αIIb and β3), Ia (integrin subunit α2), and Ibα were determined using PCR. Gene sequences were compared to the equine genome available on GenBank. Polymorphisms that would be predicted to result in amino acid changes on platelet surfaces were documented and compared with known alloantigenic sites documented on human platelets. Amino acid differences were predicted based on nucleotide sequences for all 4 genes. Nine differences were documented for αIIb, 5 differences were documented for β3, 7 differences were documented for α2, and 16 differences were documented for Ibα outside the macroglycopeptide region. This study represents the first effort at identifying potential platelet alloantigens in members of the Equidae Family based on evaluation of gene sequences. The data obtained form the groundwork for identifying potential platelet alloantigens involved in transfusion reactions and neonatal alloimmune thrombocytopenia (NAIT). More work is required to determine whether the predicted amino acid differences documented in this study play a role in alloimmunity, and whether other polymorphisms not detected in this study are present that may result in alloimmunity.

Human platelet antigens – 2013

To date, 33 human platelet alloantigens (HPAs) have been identified on six functionally important platelet glycoprotein (GP) complexes and have been implicated in alloimmune platelet disorders including foetal and neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura (PTP) and multitransfusion platelet refractoriness (MPR). The greatest number of recognized HPA (20 of 33) resides on the GPIIb/IIIa complex, which serves as the receptor for ligands important in mediating haemostasis and inflammation. These include HPA-1a, the most commonly implicated HPA in FNAIT and PTP in Caucasian populations. Other platelet GP complexes, GPIb/V/IX, GPIa/IIa and CD109, express the remaining 13 HPAs. Of the recognized HPAs, 12 occur as six serologically and genetically defined biallelic 'systems' where the -a form designates the higher frequency allele and the -b form, the lower. Twenty-one other HPAs are low-frequency or rare antigens for which postulated higher frequency -a alleles have not yet been identified as antibody specificities. In addition to the HPA markers, platelets also express ABO and human leucocyte antigen (HLA) antigens; antibodies directed at the former are occasionally important in FNAIT, and to the latter, in MPR.

Health Outcomes in Vaso Occlusive Crisis of Sickle Cell Disease - Retrospective Indicators

AbstractAbstract 4699 BackgroundRetrospective data analysis is relatively inexpensive & allows evaluation of rare conditions such as Vaso Occlusive Crisis (VOC) of Sickle Cell Disease (SCD) with ease, as compared to conducting actual prospective studies. A particularly useful application of a retrospective data analysis is to act as a pilot study that can be completed in anticipation of a prospective study. The analysis can help to identify feasibility issues & factors which affect study outcomes that need to be adjusted for in prospective studies. ObjectiveTo identify health outcome measures employed in retrospective database analysis of individuals suffering from VOC of SCD, from reviewed literature. MethodPotentially relevant articles were searched electronically using terms related to VOC & pain crisis in SCD, across PubMed, COCHRANE & CINAHL databases. Dissertations, guidelines to management of VOC, abstracts, conference proceedings & case reports were excluded for lack of sufficient information. Reference lists of prior literature reviews, as well as reference lists of studies included in this review, served to identify additional articles. However, review articles were not included for further analyses. Studies analyzing retrospective databases such as chart reviews, hospital records, electronic medical records (EMR), claims data, health care cost & utilization project database (HCUP) were included for further analyses. Studies qualifying for full review were searched for relevant clinical, humanistic, economic outcomes & clinical endpoints, which have previously been defined elsewhere. Result1,917 potentially relevant study abstracts were reviewed. Of which, 13 studies qualified for further analyses. 8 clinical outcomes, 6 humanistic outcomes, 3 economic outcomes & 1 clinical endpoint were found. Codes used to identify patients with VOC of SCD in claims databases were (282.42), (282.60), (282.62), (282.64) & (282.69). Four studies used HCUP database, 3 studies used EMRs, & 2 studies each used Medicaid claims data, chart reviews & hospital records for retrospective assessment of a VOC event. Majority of the studies captured length of stay (LOS) (8 studies) & readmission rates (5 studies) as clinical outcomes measure while Wong-Baker Faces scale in children (2 studies) & Numeric Rating scale (2 studies) in adults, were most common measures recorded. Type of hospital admission, type of SCD admission, severity of pain, age, hemoglobin count & polymorphonuclear leukocyte count, associated significantly with length of stay in patients with VOC of SCD (Table 1). Disease severity, severity of pain, use of steroids, age, type of visits & having co-morbid history of asthma associated significantly with 14- & 30-day readmission rates. Number of pain episodes, intensity of pain, LOS, presence of Human Platelet Alloantigen polymorph (HPA-3), amount of analgesic administered, cost of healthcare & 14- & 30-day readmission rates were found to be significant predictors of severity of a VOC (Table 2). ConclusionResearch evidence available, while scarce, is applicable, in population of all ages with VOC of SCD. Findings suggest using LOS & readmission rates for assessment of a VOC event in a retrospective database as well as prospective studies aimed to assess treatment efficacies. Care should be taken during design of pilot & retrospective studies to ensure collection & adjustment for variables known to associate significantly with LOS & readmission rate before reaching a clinical conclusion. Further research is necessary to support the use of suggested retrospective indicators of a VOC event.Table 1Factors significantly associating with LOS & 14-, 30-day readmission rates in VOC of SCDLength of stay14- & 30-day readmission ratesType of hospital admissionDisease severityType of SCD admissionUse of steroidsSeverity of painSeverity of painAgeAgeHemoglobin countType of visitsPolymorphonuclear leukocyte countHistory of asthmaTable 2Significant predictors of severity of VOC eventPredictorsNumber of pain episodesIntensity of painLength of stayAmount of analgesic administeredPresence of HPA-3Total cost of healthcare14- & 30-day readmission rates Disclosures:Shafer:Pfizer: Employment. Sivamurthy:Pfizer: Employment. Kollmer:Pfizer: Employment, Equity Ownership. Vendetti:Pfizer: Employment.

Review of neonatal alloimmune thrombocytopenia

Neonatal alloimmune thrombocytopenia (NAIT), with an incidence of one in 1000 live births, is the most common cause of severe thrombocytopenia and intra-cerebral haemorrhage in term neonates. NAIT results from trans-placental passage of maternal antibodies against a paternally derived fetal platelet alloantigen. Clinical presentation varies from unexpected thrombocytopenia on a blood film in a well newborn to intracranial haemorrhage (ICH). In contrast to haemolytic disease of the newborn, NAIT can present in a first pregnancy, and subsequent pregnancies are usually more severely affected. The role of antenatal screening for maternal alloantibodies instead of fetal blood sampling to identify at-risk fetuses remains uncertain, but there is a trend towards less invasive maternally directed treatment for at-risk pregnancies. Neonatal management is aimed at preventing or limiting thrombocytopenic bleeding with transfusion of antigen-matched platelets.

Prevalence of the HPA‐18w to ‐21w alleles in the Chinese Han population

Recently, four new platelet alloantigen (HPA) systems HPA-18w to-21w were identified. However, genotyping for HPA-18w to -21w alleles was rarely reported. Here, we established a polymerase chain reaction sequence-based typing (PCR-SBT) method and investigated the distribution of HPA-18w to -21w alleles in the Chinese Han population. The specific primers of HPA-18w, -19w, -20w and -21w were designed, and the PCR products were bidirectionally sequenced. 855 randomly selected platelet donors were genotyped for HPA-18w to -21w with the PCR-SBT method. The results showed that all individuals were monomorphic for HPA-18w to HPA-20w with a/a homozygous frequency of 1.0 and absence of HPA-18bw to -20bw alleles. The frequencies of the HPA-21a/21a and HPA-21a/21b genotypes were 0.981(839/855) and 0.019(16/855), respectively. Seven mutations were confirmed on sequenced region separate from HPA polymorphisms, including ITGA2 (IVS17+48G>A and IVS17+72G>A), ITGA2B (IVS19-26C>G) and ITGB3 (IVS4+234C>T, IVS11-19 T>C, IVS11-104T>C and GT repeats from IVS11-131 to IVS11-109). These data will provide useful information for diagnosis, prevention and treatment of alloimmune thrombocytopaenia.

The αIIb p.Leu841Met (Cab3a+) polymorphism results in a new human platelet alloantigen involved in neonatal alloimmune thrombocytopenia

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). 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 αIIbβ3 forms were used to probe the reactivity of maternal sera from these families and to study the effects of the substitution on αIIbβ3 expression and functions. Tested by flow cytometry (FCM), one serum sample specifically reacted with αIIb-Met841 but not with αIIb-Leu841 αIIbβ3. 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 αIIbβ3 expression and functions. 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.

A point mutation in the EGF-4 domain of β3 integrin is responsible for the formation of the Seca platelet alloantigen and affects receptor function

Neonatal alloimmune thrombocytopenia (NAIT) is caused by fetomaternal platelet incompatibility with maternal antibodies crossing the placenta and destroying fetal platelets. Antibodies against human platelet antigen-1a (HPA-1a) and HPA-5b are responsible for the majority of NAIT cases. We observed a suspected NAIT in a newborn with a platelet count of 25 G/l and petechial haemorrhages. Serological analysis of maternal serum revealed an immunisation against αIIbβ3 on paternal platelets only, indicating the presence of an antibody against a new rare alloantigen (Sec(a)) residing on αIIbβ3. The location of Sec(a) on αIIbβ3 was confirmed by immunoprecipitation. Nucleotide sequence analysis of paternal β3 revealed a single nucleotide exchange (G(1818)T) in exon 11 of the β3 gene (ITGB3), changing Lys(580) (wild-type) to Asn(580) (Sec(a)). Two additional members of the family Sec were typed Sec(a) positive, but none of 300 blood donors. Chinese hamster ovary cells expressing Asn(580), but not Lys(580) αIIbβ3, bound anti-Sec(a), which was corroborated by immunoprecipitation. Adhesion of transfected cells onto immobilised fibrinogen showed reduced binding of the Asn(580) variant compared to wild-type αIIbβ3. Analysis of transfected cells with anti-LIBS and PAC-1 antibody showed reduced binding when compared to the wild-type. No such effects were observed with Sec(a) positive platelets, which, however, are heterozygous for the Lys(580)Asn mutation. In this study, we describe a NAIT case caused by maternal alloimmunisation against a new antigen on αIIbβ3. Analysis with mutant transfected cells showed that the Lys(580)Asn mutation responsible for the formation of the Sec(a) antigenic determinant affects αIIbβ3 receptor function.

Platelet adhesion to decorin but not collagen I correlates with the integrin 2 dimorphism E534K, the basis of the human platelet alloantigen (HPA)-5 system

A single nucleotide polymorphism in the integrin α2 gene ITGA2 (rs1801106; G1600A) creates the non-conservative amino acid substitution E534K, the basis of the human platelet alloantigen system HPA-5. Yet HPA-5 alleles do not influence binding of α2β1 to its primary ligand collagen I, and the effect of HPA-5 on platelet function has not been determined. We used a direct platelet adhesion assay to evaluate whether differential inheritance of HPA-5 alleles influences platelet adhesion to collagen I or an alternative ligand, decorin. Platelets from donors bearing one or more minor allele HPA-5b showed attenuated adhesion to purified decorin but not collagen I. Adhesion to decorin was significantly inhibited by human alloantibodies specific for HPA-5a but not by the collagen I sequence GFOGER or α2-specific inhibitory monoclonal antibodies. The minor allele 534K attenuates platelet adhesion to decorin but not collagen I, providing the first evidence of a functional effect of HPA-5 alleles.

The Integrin α2 Dimorphism E534K Modulates Platelet Binding to Decorin but Not Collagen I,

Abstract 3256 Background.Integrin a2b1-mediated adhesion to collagens supports cellular attachment, while decorin binding by a2b1 often attenuates adhesion. Collagen I binds to the a2 I-domain via the triple-helical sequence GFOGER, but the decorin binding site is not within the a2 I-domain and has not yet been identified. A single nucleotide polymorphism in the a2 gene ITGA2 (rs1801106) (G1600A) resulting in the amino acid substitution glutamate-534 to lysine-534 (E534K) is the basis for one of the most important human platelet alloantigen (HPA) systems, HPA-5, yet HPA-5 alleles do not influence the binding of a2b1 to collagen I, and the effect of HPA-5 on platelet function, if any, has not been determined. We sought to determine whether the minor allele HPA-5b (534K) might influence the a2b1-mediated adhesion of platelets to a physiologically relevant ligand other than collagen I. One such alternative ligand is decorin, an extracellular matrix small leucine (Leu)-rich proteoglycan (SLRP). Methods/Results. Two groups of normal subjects were compared. The first group (GG) consisted of ten donors homozygous for the major allele rs1801106G. The second group (GA+AA) included one donor homozygous for the minor allele rs1801106A and nine donors heterozygous for these alleles. Aside from this, there were no significant differences between the two groups with respect to platelet count, mean platelet volume, surface expression of four selected platelet receptors, GPIba, GPVI, aIIbb3 or a2b1, or the allelic distribution of five receptor genes, ITGA2B rs5911, ITGB3 rs5918, GP1BA rs6065, GP6 rs1613662 and P2RY1 rs1065776. In direct platelet adhesion assays, we determined that platelets from GG donors bound more strongly to decorin than those from GA+AA donors (p < 0.01), while platelets from either group bound equally well to collagen I (p = 0.73). Using platelets from donors homozygous for the major allele rs1801106G, adhesion to decorin was significantly inhibited by human alloantibodies specific for HPA-5a but not by GFOGER or an a2-specific monoclonal antibody 6F1 known to inhibit cell adhesion to collagen I. Conversely, GFOGER and 6F1 inhibited adhesion to collagen I but not decorin. Conclusions. The simplest explanation of our findings is that 534E, located in the a2 b-propeller domain, is necessary for maximal binding of a2b1 to decorin but not collagen I. These results suggest that 534E contributes to the decorin binding site and that this amino acid represents a potential target for interventions that can modify cell adhesion to decorin and perhaps other proteoglycans. This also represents the first instance of a functional difference attributable to the HPA-5 alleles. These results expand our understanding of the decorin and collagen I interactions described in murine and human studies of cell adhesion and cancer biology, where decorin and collagen I often have opposite effects that are both mediated by a2b1. Decorin knockout (Dcn(−/−)) murine embryonic fibroblasts exhibit greater adhesion to collagen and greater migration on collagen substrates, while decorin attenuates the aggressiveness and metastasis of tumor cells with diverse histologic backgrounds. Further studies of the relationship between the collagen I and decorin binding sites on integrin a2 are warranted, particularly regarding its influence on thrombosis and hemostasis. Disclosures:No relevant conflicts of interest to declare.

Human Platelet Alloantigens (HPA)1, HPA2 and HPA3 SNPs in Tunisian Sickle Cell Disease Patients

Abstract 4852Sickle Cell Disease (SCD) is a complex disease with various complications such as stroke, vaso-occlusive crisis (VOC), acute chest syndrome and leg ulcers. Sickle cell anemia (SCA; homozygous hemoglobin SS) is the most common form of SCD. Genetic variations and/or environmental modifiers may modulate clinical presentation of SCD. Few studies have examined hemoglobinopathies in Tunisia, North Africa. However, recently, frequencies of beta-thalassemia and sickle cell trait were estimated at 2.21% and 1.89%, respectively (Fattoum, 2006). In order to identify genetic factors that may predispose patients to SCD complications in this population, a pilot case control study was designed to assess polymorphisms in Human Platelet Antigen (HPA) Genes. HPA polymorphisms were recently associated with severe coronary artery disease in the general population in Tunisia (Abboud et al, 2010) and VOC presentation in SCA patients from Bahrain (Al-Subaie et al, 2009). We present here a study conducted in collaboration with the Department of Clinical Hematology at the Aziza Othmana Hospital in Tunis (Tunisia). The National Medical Ethics committee of Tunisia as well as the Institutional Review Board (IRB) of Morehouse School of Medicine (MSM) approved the study. Blood samples, clinical history and DNA samples were collected from SCD adult patients and healthy controls after informed consent. Previously validated questionnaires for genetic risks in patients with SCD (courtesy of Dr Telen, Duke University) were adapted to French. The Helena test kit was used to generate hemoglobin variant data in conjunction with cellulose acetate electrophoresis. Blood samples were collected in EDTA vacutainer tubes and genomic DNA was isolated,stored at −80°C and then shipped to MSM. Single nucleotide polymorphisms, SNPs (Table 1) were genotyped using PCR-RFLPs and compared with different clinical sub-phenotypes such as, onset age, strokes, cardiac problems, splenectomies, etc. as defined in the questionnaire. Pearson Chi-Square was used for comparison and a P<0.05 value was considered significant. A total of 98 DNA samples were collected and 54 questionnaires were filled (Table 2). Age of patients at time of sample collection ranged from 19 to 61 (n = 49) with a mean ± sd (standard deviation) of 29 ± 7. The reported age at onset ranged from 1 to 30 (n = 44) with a mean ± sd of 12 ± 9. No significant differences were found in the HPA alleles and genotype frequencies in SCD versus healthy controls. There was significant association between HPA1 polymorphism with patient defined cardiac problems in all SCD patients (P = 0.002) as well as in the SS sub-group separately (P = 0.01). There was significant association between HPA1 and reported age of onset in SCD patients (P = 0.05) as well as in the non-SS sub-group alone (P = 0.04). The HPA1 variant was linked to self-reported age of disease onset and heart complications in adult SCD patients in Tunisia. This present study is one of the first genetic studies in a seldom-studied group of Tunisian adult SCD patients. These results show that identification of biomarkers of SCD disease severity may be possible using a validated self-reporting instrument. This kind of approach could help to improve early diagnosis of at risk patients and enable development of early interventions.Table 1:SNPs screened.SymbolRs#Ch.Gene (GeneID)MutationPeptide changeRestriction Enz.HPA1rs591817ITGB3 (3690)196 T>CLeu33ProMspIHPA2rs606517GP1BA (2811)524 C>TMet145ThrSfaNIHPA3rs591117ITGA2B (3674)2622 T>GIle843SerFokITable 2:Tunisian cohort collection took place from January to December 2009. This table summarizes the number and genotypes of patients enrolled and questionnaires collectedSexMaleFemaleTotal (%)TOTAL455196 (100)SCD Patients403575 (78)Healthy Controls51621 (22)Hemoglobin Genotype of SCD patientsSS141529 (38.6)Sβ010919 (25.3)Sβ+112 (2.6)SC112 (2.6)SO101 (1.3)unknown13922 (29.3)Total (%)403575 (100)Questionnaires filled282654No questionnaires111021 Disclosures:No relevant conflicts of interest to declare.

Clinical Applications of Platelet Antibody and Antigen Testing

Immune thrombocytopenia occurs when antibodies targeting specific glycoproteins (GPs) on the platelet surface lead to their destruction. Tests of the patient’s platelets and serum for antibodies and typing of DNA for human platelet alloantigens (HPA) can be helpful in confirming a clinical diagnosis of immune thrombocytopenia. A thorough workup includes 1) serum tests using intact platelets; 2) antigen-capture assays to identify the specific HPA targeted by platelet antibodies; and 3) HPA genotyping of the patient’s DNA. For patients suspected of having autoimmune thrombocytopenia, a direct test of an eluate of the patient’s platelets for autoantibodies targeting the most common GPs can be performed. Properly interpreted platelet serologic test results, taken together with a good clinical history and other laboratory data, will ensure a more accurate diagnosis and appropriate treatment.

Antenatal interventions for fetomaternal alloimmune thrombocytopenia

Fetomaternal alloimmune thrombocytopenia results from the formation of antibodies by the mother which are directed against a fetal platelet alloantigen inherited from the father. The resulting fetal thrombocytopenia (reduced platelet numbers) may cause bleeding, particularly into the brain, before or shortly after birth. Antenatal treatment of fetomaternal alloimmune thrombocytopenia includes the administration of intravenous immunoglobulin (IVIG) and/or corticosteroids to the mother to prevent severe fetal thrombocytopenia. IVIG and corticosteroids both have short-term and possibly long-term side effects. IVIG is also costly and optimal regimens need to be identified. To determine the optimal antenatal treatment of fetomaternal alloimmune thrombocytopenia to prevent fetal and neonatal haemorrhage and death. We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (28 February 2011) and bibliographies of relevant publications and review articles. Randomised controlled studies comparing any intervention with no treatment, or comparing any two interventions. Two review authors independently assessed eligibility, trial quality and extracted data. We included four trials involving 206 people. One trial involving 39 people compared a corticosteroid (prednisone) versus IVIG alone. In this trial, where analysable data were available, there was no statistically significant differences between the treatment arms for predefined outcomes. Three trials involving 167 people compared IVIG plus a corticosteroid (prednisone in two trials and dexamethasone in one trial) versus IVIG alone. In these trials there was no statistically significant difference in the findings between the treatment arms for predefined outcomes (intracranial haemorrhage; platelet count at birth and preterm birth). Lack of complete data sets and important differences in interventions precluded the pooling of data from these trials. The optimal management of fetomaternal alloimmune thrombocytopenia remains unclear. Lack of complete data sets for two trials and differences in interventions precluded the pooling of data from these trials which may have enabled a more developed analysis of the trial findings. Further trials would be required to determine optimal treatment (the specific medication and its dose and schedule). Such studies should include long-term follow up of all children and mothers.

New K103 β3 allele identified in a context of severe neonatal thrombocytopenia

A new β3 allele was identified in a severe case of neonatal alloimmune thrombocytopenia (<7 × 10(9) /L). Diagnosis was done by use of monoclonal antibody-specific immobilization of platelet (PLT) antigen for serologic analyses and polymerase chain reaction (PCR)-sequence-specific primers (SSP) and PCR-restriction fragment length polymorphism (RFLP) for genotyping. Direct sequencing of PCR product was done and mutant αIIbβ3 expressed in HEK-293 cells. Serologic analysis revealed in the maternal serum an anti-human PLT alloantigen (HPA)-1a alloantibody associated to an anti-α2β1. Anti-HPA-1a alloimmunization diagnosis was confirmed by genotyping showing maternofetal incompatibility. However, investigation of rare HPA polymorphisms revealed discrepant HPA-16b assignation between PCR-RFLP and PCR-SSP. Sequencing revealed a new c.385C>A mutation in the β3 coding sequence resulting in a false assignation of the HPA-16b allele by PCR-RFLP. This mutation leads to a Q103K substitution in mature β3. The K103-β3 form of the complex was expressed in HEK-293 cells but did not react with the maternal serum. We have characterized a new rare allele (frequency < 1%) of β3 that yields false HPA-16b genotyping in PCR-RFLP. This new case of false typing assignation emphasizes the necessity to use two genotyping techniques in diagnosis. This particularly applies for rare HPA polymorphisms when PLT phenotyping cannot be used.

A new platelet alloantigen, Swia, located on glycoprotein Ia identified in a family with fetal and neonatal alloimmune thrombocytopenia

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disorder caused by transplacental passage of maternal antibodies to fetuses whose platelets (PLTs) express the corresponding human PLT antigen (HPA). We observed a fetus with FNAIT who died from a severe intracranial hemorrhage. Analysis of maternal serum in antigen capture assay with paternal PLTs showed reactivity with PLT glycoprotein (GP)IIb/IIIa (α(IIb) β(3) ) and GPIa/IIa (α(2) β(1) integrin), indicating the presence of anti-HPA-1a and an additional alloantibody against GPIa (termed anti-Swi(a) ). By immunochemical studies, the localization of the Swi(a) antigen on GPIa/IIa could be confirmed. Analysis of paternal GPIa full-length cDNA showed a single-nucleotide substitution C(3347) T in Exon 28 resulting in a Thr(1087) Met amino acid substitution. Testing of family members by polymerase chain reaction-restriction fragment length polymorphism using MslI endonuclease showed perfect correlation with phenotyping. Extended family and population studies showed that 4 of 10 members of the paternal family but none of 500 unrelated blood donors were Swi(a) carriers. Expression studies on allele-specific transfected Chinese hamster ovary (CHO) cells confirmed that the single-amino-acid substitution Thr(1087) Met was responsible for the formation of the Swi(a) epitope. Adhesion of CHO cells expressing the Swi(a) alloantigen to immobilized collagens was not impaired compared to the wild-type control and was not inhibited by anti-Swi(a) alloantibodies. In this study we defined a new PLT alloantigen Swi(a) that was involved in a case of additional immunization against HPA-1a. Our observations demonstrate that combinations of PLT-specific alloantibodies may comprise low-frequency alloantigens.

Study on polymorphism of membrane glycoprotein genes related to human platelet alloantigens

To investigate the polymorphisms of platelet membrane glycoprotein genes related to human platelet alloantigen (HPA)-1 to 17w. The DNA segments of platelet membrane glycoprotein genes related to HPA-1 to 17w were amplified using author's designed primers. The amplification products were purified and directly sequenced to identify the HPA genotype and glycoprotein gene polymorphisms. Thirteen new single nucleotide polymorphisms (SNPs) and a micro-satellite sequence were found in the glycoprotein genes from the 112 random samples, in which two SNPs (1333G/A and 1960G/A) in ITGB3 gene result in two amino acid change (V419M and E628K) on glycoprotein GPIIIa. New variants in platelet membrane glycoprotein genes were identified, which may lead to structure change of platelet membrane glycoprotein and affect the accuracy of partial HPA genotyping method.

A third allele of the HPA‐5 (Br) platelet alloantigen system identified in investigating a case of neonatal thrombocytopenia

HPA-5a/b (Brb/a) was identified in the late 1980s as an antigen system capable of causing maternal immunization leading to neonatal alloimmune thrombocytopenia (NAIT).1 Subsequent studies showed that the HPA-5a/b antigens result from a single-nucleotide polymorphism (G>A1600; “A” of the ATG start codon is here designated Nucleotide 1) in the gene encoding platelet (PLT) glycoprotein (GP) Ia (α2 integrin), leading to a Glu (HPA-5a)>Lys (HPA-5b) amino acid substitution at Residue 505 in GPIa.2 Further studies showed that maternal-fetal incompatibility for HPA-5 antigens is second only to incompatibility for HPA-1a as a cause of NAIT.3 HPA-5a and -5b is thought to be a diallelic system with gene frequencies approximately 0.89 and 0.11, respectively. Here, we describe a third allele of HPA-5 identified in a Caucasian individual while studying paternal DNA in a suspected case of NAIT. A full-term male infant born to a primiparous woman had a normal blood count except for a PLT level of 38 × 109/L. PLTs were 39 × 109/L on Day 4, but increased to normal on Day 8. The infant had physical features of Down’s syndrome and Trisomy 21 was confirmed in genetic studies. No maternal antibody reactive with paternal PLTs was detected using flow cytometry and immunoprecipitation assays. Using polymerase chain reaction (PCR) with sequence-specific priming method, no incompatibility between father and mother was identified for HPA-1, –3, and –5 alloantigen systems. When this study was done, melting point analysis4 was being evaluated to type DNA for HPA antigens. In this technique, fluorescence is produced when PCR products containing the nucleotide sequence being typed anneal to sequence-specific “reporter probes.” As the reaction mixture is heated, the DNA complexes melt (with resulting loss of fluorescence) at temperatures that are allele-specific (Fig. 1). The mother’s DNA produced a single peak at 50°C, indicating that her genotype was HPA-5a/a. The father’s DNA produced two peaks, one corresponding to HPA-5a and the other intermediate between the HPA-5a peak and the one expected for HPA-5b (Fig. 1). Because of this atypical finding, the father’s DNA encoding the HPA-5 mutation was sequenced using standard methods. It was found that father’s DNA contained both the expected G at Position 1600 and an unexpected T substitution at Position 1601 (Table 1), providing an explanation for the atypical melting curves obtained when his DNA was typed for HPA-5 (Fig. 1). The subsitution of A at Nucleotide 1601 to T leads to amino acid substitution at Position 505 from glutamic acid into valine creating a third allele of HPA-5. “T” at position 1601 was identified in one individual in the course of screening 20 DNA samples from NAIT cases when the melting point technique was being evaluated. However, no similar examples of T1601 were identified in typing 128 normal Caucasian subjects for HPA-5 by direct sequencing. Additional normal subjects are being typed to obtain a better estimate of the frequency of this allele. Fig. 1 Melting curve analysis of HPA-5 alleles with LightCycler (Roche Diagnostic, Indianapolis, IN). The first negative derivative of the fluorescence versus temperature (−dF/dT) graph shows the melting temperature (Tm) of HPA-5a probe/DNA complex (50°C) ... TABLE 1 Alleles of HPA-5 It was not possible to obtain DNA from the infant to determine whether he inherited T1601. However, since no maternal antibody reactive with PLTs or GPIa/IIa from the father was identified and transient thrombocytopenia is common in newborns with Down’s syndrome,5 it seems unlikely that the low PLT counts were the result of maternal immunization against GPIa containing Val at Position 505. Knowledge of the GPIa mutation identified in this case will be helpful if other examples are identified in future evaluations of NAIT cases. It is possible, however, that T1601 will not be detected in assays that utilize sequence-specific primers to type for HPA-5a/b antigens.