Frequency of allotype "b" in human platelet antigen 1 to 29 systems among blood donors in Japan estimated using high-resolution melt analysis.

BACKGROUND: Antibodies against human platelet antigens (HPAs) cause thrombocytopenias. It is thus important to know the frequency of "b" allotypes in each HPA system for the diagnosis and treatment of anti-HPA antibody-mediated thrombocytopenia. STUDY DESIGN AND METHODS: Genomic DNA was extracted from peripheral blood cells obtained from 2170 blood donors in Japan and was subjected to high-resolution melt (HRM) analysis using polymerase chain reaction for each of the HPA genes, using 23 primer pairs. For genotyping, the resulting amplicons were classified based on their HRM curves. In some cases, direct sequence analysis was performed after HRM analysis to determine nucleotide substitutions. In cases where amino acid substitutions were predicted, protein expression levels were examined in a cell line using 293T cells. RESULTS: The frequencies of each of the HPA-b genotypes were as follows: HPA-1b, 0.4%; HPA-2b, 11.8%; HPA-3b, 41.3%; HPA-4b, 0.8%; HPA-5b, 4.3%; HPA-6b, 1.9%; HPA-15b, 48.8%; HPA-21b, 0.6%; and "b" allotype in the other HPA systems, 0.0%. Twenty-eight variants were found; nine of them were predicted to cause amino acid substitution. However, expression analysis revealed that they did not affect protein expression levels on the cell surface. CONCLUSION: Nine HPA systems are of primary importance in Japan in potentially triggering thrombocytopenia via the HPA antibodies. Similar studies in other countries or races, together with ours, could provide basic information for clinicians in multiethnic societies.

Cytofluorimetric platelet analysis.

Blood platelets are highly specialized cells that drive hemostatic events and tissue repair mechanisms at the site of vascular injury. Their peculiar morphology and certain functional characteristics can be analyzed by flow cytometry (FCM). Specifically, platelet activation, a hallmark of prothrombotic states and inflammatory conditions, is associated with changes in expression of both surface and intracellular antigens that are recognized by specific monoclonal antibodies. Assessment of platelet activation status as ex vivo or in vitro reactivity to specific agonists has become relevant in particular conditions (namely, cardiovascular diseases, hematological malignancies, monitoring of pharmacological antiaggregation). In addition, aberrant surface marker expression that characterizes inherited and acquired platelet function disorders is also detected by FCM. This review discusses the main applications of FCM in platelet analyses, which are relevant for both research and clinical settings.

Evaluation of platelet function during extended storage in additive solution, prepared in a new container that allows manual buffy-coat platelet pooling and leucoreduction in the same system.

BACKGROUND: A novel and practical storage container designed for manual buffy-coat pooling and leucodepletion was evaluated to assess its filtration performance and to analyse the quality of stored leucoreduced buffy-coat-derived platelet pools. MATERIALS AND METHODS: To analyse the grifols leucored transfer PL system, blood was collected from random donors into standard triple bag systems, and fractionated using standard procedures to obtain buffy-coats. Ten leucodepleted platelet pools were prepared each from five units of buffy-coats in additive solution. Concentrates were stored for 10 days at 22 °C on an end-over-end agitator. On days 0, 5, 7, and 10 of storage, samples were tested using standard in vitro platelet parameters. RESULTS: The use of this novel system for volume reduction and leucodepletion of buffy-coats resuspended in additive solution led to platelet pools that met the European requirements. pH was maintained well, declining from an initial value of 7.11±0.04 to 6.88±0.08 after 10 days. Parameters of cell lysis, response to a hypotonic stimulus and aggregation induced by agonists (arachidonic acid, ristocetin, collagen or thrombin receptor activating peptide) were also well-preserved. During storage, the quality profile of the platelet pools remained very similar to that previously reported in platelet concentrates in terms of metabolism, platelet activation (CD62, CD63, sCD62), expression of glycoproteins Ib and IIb/IIIa, capacity of glycoprotein IIb/IIIa to become activated upon ADP stimulation, and release of biological response modifiers (sCD40L and RANTES). DISCUSSION: This new system allows the preparation of leucodepleted buffy-coat platelet pools in additive solution with good preservation of platelet function. The logistics of the procedure are relatively simple and it results in good-quality components, which may reduce costs and ease the process of buffy-coat pooling and leucocyte reduction in transfusion services.

A journey to produce platelets in vitro.

Allogeneic platelet transfusions protect patients from bleeding episodes and also make aggressive medical procedures such as those involving marrow transplants requiring chemotherapy and/or radiotherapy possible. These patients are dependent upon an unfailing supply of platelets that can sometimes be in short supply due to high demands coupled with an extremely short expiration date for platelet products of only 5 days. One approach that is under investigation to overcome platelet shortages is to harness the extraordinary capabilities of stem cells to proliferate and differentiate into various cell types and to use this ability to specifically produce clinical scale quantities of functional platelets in bioreactors. To accomplish such an enormous and complex task requires an appreciation of the regulatory mechanisms that occur during the development of megakaryocytes (MKs) and the subsequent biogenesis of functional platelets from mature MKs. This means understanding the complex network of intracellular and extracellular regulatory mechanisms that act at each phase of a developmental process that ushers stem cells along the MK lineage to produce billions of platelets per day in a healthy individual.

Expression of the peroxisome proliferator-activated receptors-alpha, -beta, and -gamma in ovarian carcinoma effusions is associated with poor chemoresponse and shorter survival.

Peroxisome proliferator-activated receptors regulate lipid metabolism, affecting inflammation and cancer. The present study analyzed the anatomical site-related expression and prognostic role of peroxisome proliferator-activated receptors in ovarian carcinoma. Fresh-frozen effusions (n = 79), primary carcinomas (n = 44), and solid metastases (n = 16) were studied for peroxisome proliferator-activated receptor-alpha, -beta, and -gamma messenger RNA expression using reverse transcriptase polymerase chain reaction. Peroxisome proliferator-activated receptor-gamma messenger RNA expression was further assessed in 60 tumors (30 effusions, 20 primary carcinomas, 10 metastases) using in situ hybridization. Peroxisome proliferator-activated receptor-gamma protein expression was immunohistochemically analyzed in 160 effusions. All peroxisome proliferator-activated receptors were expressed in most tumors at all anatomical sites using reverse transcriptase polymerase chain reaction, but peroxisome proliferator-activated receptor-alpha (P = .004) and peroxisome proliferator-activated receptor-beta (P = .002) messenger RNA levels were higher in effusions compared with primary carcinomas and solid metastases. In situ hybridization localized peroxisome proliferator-activated receptor-gamma messenger RNA to carcinoma cells in both effusions and solid lesions. Peroxisome proliferator-activated receptor-gamma protein was detected in carcinoma cells in 102 of 160 (64%) effusions. Higher effusion messenger RNA levels of all peroxisome proliferator-activated receptors were associated with less favorable response to chemotherapy at diagnosis (P = .009). In univariate survival analysis, higher messenger RNA expression of all peroxisome proliferator-activated receptors was associated with poor progression-free (P = .045) and overall (P = .014) survival. Higher peroxisome proliferator-activated receptor-gamma protein expression was similarly associated with poor overall survival for the entire cohort (P = .046) and for patients with disease recurrence effusions (P = .009). Peroxisome proliferator-activated receptors were not independent predictors of survival in Cox multivariate analysis. Peroxisome proliferator-activated receptor members are frequently expressed in ovarian carcinoma, with upregulated expression in effusions. Peroxisome proliferator-activated receptor expression in effusions is associated with poor response to chemotherapy at disease recurrence and poor survival, suggesting a role in tumor biology at this unique microenvironment.

Induction of hematopoietic prostaglandin D synthase in hyalinated necrotic muscle fibers: its implication in grouped necrosis.

Prostaglandin (PG) D(2) exerts pro-inflammatory and anti-inflammatory functions under various pathological conditions. We found that the immunoreactivity of hematopoietic PGD synthase (HPGDS) appeared in necrotic muscle fibers, mainly in foci of grouped necrosis, in Duchenne's muscular dystrophy (DMD) and polymyositis (PM), but not in Becker's muscular dystrophy or in Fukuyama-type congenital muscular dystrophy. The HPGDS expression in DMD and PM was observed to be transient in hyalinated fibers at the early necrotic stage but not detected in opaque fibers, in fibers infiltrated by monocytes/lymphocyte, or in regenerating fibers. The immunoreactivities of a cytosolic form of phospholipase A(2) and cyclooxygenase-2, the upstream enzymes of the arachnoid acid cascade, were similarly observed in the HPGDS-positive fibers, suggesting that PGD(2) was produced in situ in those necrotic muscle fibers.

CD34+ cells derived from fetal liver contained a high proportion of immature megakaryocytic progenitor cells.

Endoreplication and maturation of the megakaryocyte (MK) may be retarded or delayed during ontogenesis. In this study, CD34+ cells were isolated from both human fetal liver and adult bone marrow and incubated with thrombopoietin (TPO). The cell number, morphological characteristics, platelet-associated antigen phenotype, maturation stage and DNA ploidy of CD41+ cells were examined from day 0 to day 12 in culture. 1) TPO stimulated the proliferation of fetal liver (FL)-derived CD34+ cells with a mean 73.14-fold increase of CD41+ cells after 12 d in culture. Adult BM-derived CD34+ cells increased only slightly, with a mean 8.18-fold increase of CD41+ cells. 2) Although the membrane phenotype of both FL CD34+-derived MKs and BM CD34+ -derived MKs analyzed with CD41a, CD42a, CD61 and CD34 were similar, all FL CD34+-derived MKs were in maturation stage I and II and in low ploidy (<4N) class. By comparison, BM CD34+ MKs possessed 15% MKs in maturation stage III and IV and with 23% MKs in high ploidy class ( > 4N). 3) Most of cultured FL-derived CD34+ cells did not have a well developed demarcation system (DM) and numerous alpha-granules after 12 d incubation. von Willebrand factor (vWF) appeared earlier on the cultured BM-derived CD34+ cells than on FL-derived CD34+ cells. 4) The expression of both cyclin E and cyclin B1 progressively increased in FL CD34+ cells induced by TPO during 12 d in culture. 5) The expression of cyclin D1 gradually decreased in FL CD34+ cells induced by TPO over 12 d incubation. 6) Immunocytochemical analysis showed that cyclin D3 was detected only in cytoplasm of cultured FL-derived CD34+ cells, whereas in both cytoplasm and nuclei of cultured BM-derived CD34+ cells. These data suggest that FL-derived CD34+ cells contain a high proportion of immature megakaryocytic progenitor cells. It further suggests that TPO can push these progenitor cells into proliferation by upregulating the expression of cyclins B1 and E, and drive a high proportion of cells into megakaryocytic lineage.

Soluble fibrin augments platelet/tumor cell adherence in vitro and in vivo, and enhances experimental metastasis.

There is considerable evidence for a relationship between hemostasis and malignancy. Since platelet adhesion to tumor cells has been implicated in the metastatic process and plasma levels of fibrinogen (Fg) and soluble fibrin (sFn) monomer are increased in cancer, we hypothesized that these molecules might enhance tumor-platelet interaction. We therefore studied binding of sFn monomer to tumor cells in a static microplate adhesion assay and determined the effect of pre-treating tumor cells with sFn on tumor cell-induced thrombocytopenia and experimental metastasis. Soluble fibrin (produced by adding thrombin to FXIII- and plasminogen-free Fg in the presence of Gly-Pro-Arg-Pro-amide (GPRP-NH2) significantly increased platelet adherence to tumor cells. This effect was primarily mediated by the integrins alphaIIb beta3 on the platelet and CD 54 (ICAM-1) on the tumor cells. Platelets adhered to untreated A375 cells (28 +/- 8 platelets/tumor cell) and this was not significantly affected by pre-treatment of the tumor cells with fibrinogen or GPRP-NH2. Although thrombin treatment increased adherence, pre-incubation of the tumor cells with sFn resulted in a further increase in platelet binding to tumor cells. In contrast to untreated tumor cells, intravenous injection of sFn-treated A 375 cells reduced the platelet count in anticoagulated mice, supporting the in vitro finding that sFn enhanced tumor cell-platelet adherence. In a more aggressive model of experimental metastasis, treating tumor cells with sFn enhanced lung seeding by 65% compared to untreated cells. Extrapolation of our data to the clinical situation suggests that coagulation activation, and subsequent increase in circulating Fn monomer, may enhance platelet adhesion to circulating tumor cells and thereby facilitate metastatic spread.

Heterogeneity of antigen expression among human umbilical cord vascular endothelial cells: identification of cell subsets by co-expression of haemopoietic antigens.

First described as an interface between blood and tissues, the endothelial cells are now known to be also involved in haemostasis, inflammatory and immune responses. Recent studies have reported that the endothelial cells represent a heterogeneous population of cells with organ-specific properties. This report describes the phenotype analyzed by flow cytometry of human umbilical vascular endothelial cells (HUVEC). For this purpose, we use monoclonal antibodies (Mabs) recognizing B cell antigens (CD10, 19, 20, 21, 22, 23, 24, 38, 40), T cell antigens (CD1, 2, 3, 4, 5, 6, 7, 8), myeloid antigens (CD13, 14, 15, 34, 35, 64), platelet antigens (CD9, 31, 51, 62P), NK and non-lineage cell antigens (CD16, 45, 56, 57), activation antigens (CD25, 30, 69, 71) and adhesion molecules (CD11a, CD11b, 29, 44, 54, 62E, 102, 106). Mabs recognizing MHC ClI or ClII molecules are also tested. Firstly, we show that HUVEC co-express some haemopoietic antigens with different levels of expression. Secondly, this study reveals that the HUVEC population does not represent a homogeneous cell population. Different endothelial cell subsets are identified. These phenotypical differences could reflect specialization of HUVEC performing different functions. The significant of haemopoietic antigen expression on the HUVEC surface will be discussed.

Involvement of the cysteine-rich domain of glycoprotein IIIa in the expression of the human platelet alloantigen, PlA1: evidence for heterogeneity in the humoral response.

To assess the potential contribution of the cysteine-rich domain of human platelet glycoprotein (GP) IIIa to the structure of the PlA1 epitope, we used site-directed mutagenesis to substitute alanines for cysteines at key positions potentially involved in PlA1 alloantigen formation. One of these GPIIIa isoforms in which alanine replaced Cys435 reacted normally with a series of well-characterized murine MoAbs directed against the 250 amino-terminal residues of GPIIIa, whereas binding of MoAbs specific for residues 348-692 was diminished or lost. Interestingly, of eight PlA1-specific antibodies tested, two recognized Ala435GPIIIa, two did not, and four bound to it less well than to wild-type (WT) GPIIIa. However, disruption of disulfide bonds located at or near the N-terminus of GPIIIa abolished the binding of all the anti-PlA1 alloantibodies tested. These findings provide evidence that the humoral response to the PlA1 antigen is (1) heterogenous, ie, the binding site on GPIIIa for human anti-PlA1 antibodies differs from one individual to another and (2) complex--although some anti-PlA1 alloantibodies bind to an epitope comprised solely of the amino-terminus of GPIIIa, others combine most efficiently with a more complex determinant that involves the cysteine-rich domain of GPIIIa. These findings may have implications for diagnostic and therapeutic use of synthetic or recombinant PlA1 mimetics.