We studied changes in the expression of P-selectin on the blood platelet plasma membrane. The aim of the study was to determine the influence of renal carcinoma on P-selectin expression associated with changes in platelet morphology. Venous blood was collected from 30 patients with renal carcinoma and from 24 control subjects for cytometric analysis and to evaluate platelet morphology. P-selectin being the CD62P receptor on blood platelets was marked by anti-CD61/62P MoAb, and the results were presented as the percentage of CD62P-positive cells. Changes in the expression of the CD62P on the platelet plasma membrane during activation were investigated by flow cytometry in a comparative study of in vivo activation and in vitro platelet reactivity. Platelet activation reflected by P-selectin expression was higher in the group of patients (4.45 +/-1.96), compared to control (2.48 +/-1.66) (p < 0.05). However, adenosine diphosphate [ADP] -stimulated platelet reactivity in renal cancer patients increased only by 0.24% (p>> 0.05), while following activation by thrombin by 0.54% (p < 0.05). Moreover, a higher (4.72 +/-2.02), statistically significant percentage of platelets with P-selectin expression was found in patients with disseminated neoplastic changes in renal parenchyma, compared to patients with a single localized neoplastic lesion (4.17 +/-1.89) (p < 0.05). A statistically significant difference was noted in the platelet count and anisocytosis in renal cancer patients. Renal cancer enhances P-selectin expression. It is due to the presence of intensified thrombinogenesis and other platelet agonists in the blood.

We examined cytokine-stimulated proliferation and survival of human megakaryocyte progenitor cells. We used a reliable, immunoenzymatic method of labeling CD41a-, CD42b-stained megakaryocytes in intact agar cultures to specifically identify and enumerate all megakaryocyte-containing colonies. We examined a previously defined population of cells enriched for megakaryocyte progenitors that coexpress CD34 and the megakaryocyte/platelet marker CD61. These CD34+61+ cells displayed clonogenicity of approximately 30% and contained myeloid, erythroid and megakaryocyte progenitor cells. With single CD34+61+ cells, megakaryocyte growth and development factor ([MGDF], also known as mpl-ligand or thrombopoietin) stimulated 9% of cells to complete their first cell division by day 2 (versus 21% with stem cell factor [SCF], or 13% with interleukin 3 alone). MGDF showed an additive effect with SCF and interleukin 3 to increase this number at least twofold. In purified CD34+61+ cells, MGDF stimulated the survival of megakaryocyte-colony forming cells (CFC) when addition of other proliferative factors was delayed for 5, 10 and 15 days (all p < 0.0001 versus saline control). MGDF also promoted survival of BFU-E and granulocyte-macrophage-CFC for at least 10 days (p < or = 0.0013 and p < or = 0.0362, respectively). SCF alone prolonged survival of CD34+61+ progenitor cells, however, MGDF + SCF was significantly more active. Whereas the action of MGDF on megakaryocyte-CFC was evident both in stimulating proliferation and survival, its ability to promote survival was between two- and fivefold greater than its action to stimulate proliferation. Thus MGDF alone, and in combination with SCF, was active in promoting the survival and proliferation of human progenitor cells of multiple hemopoietic lineages.

BACKGROUND

Binding of integrins to the extracellular matrix elicits various responses. We have previously reported a megakaryocytic-erythroid cell line (JAS-R) that showed phenotypic changes after adhesion to plastic dishes. However, the matrix protein and the mechanism responsible for megakaryocytic differentiation still remain unknown.

METHODS

JAS-REN (erythroid) cells were cultured on dishes coated with various proteins. The cells were treated with RGDS, a tetrapeptide ligand to integrins, or phorbol ester (12-o-tetradecanoylphorbol-13-acetate, TPA) for 48 hours and then were harvested. Subsequently, the cell surface markers were analyzed using flow cytometry and gene expression was studied by RT-PCR.

RESULTS

The JAS-REN cells adhered to fibronectin-coated dishes, but showed poor adhesion to dishes coated with collagen, laminin or poly-D-lysine. The TPA-stimulated JAS-REN cells showed an increase in the expression of integrin alphaIIbbeta3 complex (CD41a) and integrin beta3 (CD61), while glycophorin A (CD235a) expression was decreased. JAS-REN cells that were adherent to fibronectin-coated dishes also showed a similar pattern of phenotype to TPA-treated cells, but the changes were not so prominent. RT-PCR revealed that TPA treatment altered the gene expression profile of JAS-REN cells, making it similar to that of JAS-RAD (megakaryocytic) cells. The RGDS-treated and fibronectin adherent JAS-REN cells also showed a mostly similar expression profile to JAS-RAD cells, but these two stimuli did not alter the gene expression profile as TPA stimulation did. Transcription factors, FLI1 and GFI1, were induced by all stimuli.

CONCLUSIONS

Signals triggered by adhesion to fibronectin result in the induction of FLI1 that may play a pivotal role in the lineage shift of JAS-REN cells from erythroid to megakaryocytic.

Circulating microparticles (cMPs) are small phospholipid-rich microvesicles shed by activated cells that play a pivotal role in cell signalling related to the pathogenesis of atherothrombosis. We aimed to investigate the prognostic value of cMPs released from different vascular cells for cardiovascular event (CVE) presentation in asymptomatic patients at high cardiovascular risk factors under nutritional and pharmacologic treatment. This is a nested case-control study of 50 patients from the five-year follow-up prospective PREDIMED trial enrolled in the nuts arm of the Mediterranean diet (MedDiet-nuts). We randomly selected 25 patients who had suffered a CVE during follow-up and pair-matched them for sex, age, and classical CV risk factors to 25 patients who remained asymptomatic (no-CVE). Total Annexin V-(AV)+ cMPs and cMPs from cells of the vascular compartment were quantified by flow cytometry at baseline and after one year follow-up. MedDiet-nuts and pharmacological treatment neither modified levels nor source of MP shedding in CVE patients. However, no-CVE patients showed 40-86 % decreased total AV+, PAC-1+/AV+, CD61+/AV+, CD142+/CD61+/AV+, CD62P+/AV+, CD146+/AV+, CD63+/AV+ and CD11a+/AV+ cMPs at one year follow-up (p≤0.046, all). CD142+/CD61+/AV+, CD146+/AV+ and CD45+/AV+ cMPs were decreased in no-CVE patients compared to CVE patients. A ROC-curve clustered model for CD142+/CD61+/AV+, CD45+/AV+ and CD146+/AV+ cMPs predicted a future CVE [p<0.0001, AUC=0.805 (0.672 to 0.938)]. In patients at high CV risk profile treated with a controlled MedDiet supplemented with nuts and receiving up-to-date CV drug treatment, reduced cMPs derived from activated platelets, leukocytes and endothelial cells are predictive of protection against CVE within the next four years.

Glanzmann's thrombasthenia is a rare congenital bleeding disorder characterized by lack of platelet aggregation induced by most agonists. The disease is caused by different mutations in either GPIIb or GPIIIa genes that lead to a lack or dysfunction of the αIIbβ3. Western blot analysis was performed on the platelet lysates of 95 patients with Glanzmann's thrombasthenia who were referred to the Iranian Blood Transfusion and Hemophilia Clinic. Glanzmann's thrombasthenia was diagnosed based on clinical findings and were classified according to Glanzmann's Thrombasthenia Italian Team (GLATIT) protocol. The platelet glycoprotein expression pattern in Iranian patients with Glanzmann's thrombasthenia was studied and the relationship between the platelet glycoprotein expression levels and clinical symptoms were investigated. Loss or severe reduction of platelet GpIIb and GpIIIa were observed in majority of patients (78%). The remaining patients (22%) showed a relatively sharp decline to the normal amounts of the glycoproteins. None of the patients showed expression of CD41 without CD61. Statistical analysis showed no significant relationship between clinical symptoms and expression of platelet glycoproteins. Different patterns of platelet glycoproteins expression suggest that there is variety of mutations in the patients. Unlike the universal Glanzmann's thrombasthenia database, the majority of Iranian patients may suffer from a GpIIIa gene mutation. This study also confirmed a lack of correlation between clinical manifestation and GPIIb/IIIa expression in the patients.

Glanzmann thrombasthenia (GT) is a rare monogenic hemorrhagic disorder involving aggregation defect of non-nuclear platelets. In this study we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of a GT patient with complex heterogeneous mutations of ITGA2B gene. GT-iPSCs could be successfully differentiated into platelets (GT-iPS-platelets). GT-iPS-platelets were CD41-/CD42b+/CD61- and were platelet activation marker (PAC-1) negative after adenosine diphosphate (ADP) activation. Furthermore, GT-iPS-platelets were defective in platelet aggregation tests in vitro. Moreover, exogenous expression of the wild-type ITGA2B gene in GT-iPS platelets restored CD41 expression and normal platelet aggregation. Our study suggested that patient-specific iPSCs could be a potential target of stem cell based gene therapy for platelet diseases.

Using monoclonal antibodies against proliferating cell nuclear antigen or PCNA (PC10) and the Ki-67 antigen (MIB1), an immunohistochemical and morphometric study was performed on routinely processed splenic tissue from ten patients with primary (idiopathic) osteomyelofibrosis (OMF). To determine the proliferation capacity of erythroid precursors and the endoreduplicative activity of megakaryocytes, corresponding antibodies (Ret40f and CD61) were applied in combination with the cell-cycle markers (sequential double-immunostaining). Morphometric analysis revealed no significant differences in PCNA or Ki-67 reactivity in either cell lineages. In comparison with previous studies on normal bone marrow, in splenic tissue showing myeloid metaplasia, the numbers of PCNA-labelled proerythroblasts, erythroblasts and megakaryocytes were conspicuously increased. Considering the ineffective erythropoiesis in OMF, there seemed to be a disproportional enhancement in PCNA and Ki-67 immunostaining of the red cell lineage. Similarly, the small size of megakaryocytes in advanced, OMF-associated myeloid metaplasia was in keeping with an impairment of endoreduplicative activity. In addition to various other contributory factors, anaemia in OMF may be partially caused by secondary folate (haematinic) deficiency. From experimental studies this defect is known to cause an abnormal arrest in the S-phase of the cell-cycle, comparable to that characterising pernicious anaemia. As a sequel of this pathomechanism, an undue overexpression of PCNA and Ki-67 has to be assumed, that is not necessarily associated with DNA synthesis or cell cycling.

Enumerating and phenotyping of platelets, resting and activated, from whole blood is important for both the identification and verification of many disease states. Microvolume laser scanning cytometry (MLSC) has been shown to be a simple method for enumerating and phenotyping peripheral blood cells. Here, the utility of MLSC, in conjunction with an anticoagulant containing platelet activation inhibitors, for simultaneously measuring platelet count, phenotype and responsiveness directly from non-fixed whole blood was examined. CTAD or EDTA anticoagulated blood was collected from five to 20 healthy volunteers, stained with fluorescence-labeled antibodies specific for platelet antigens, and run on an in-house modified MSLC device. MLSC was able to measure antigens CD9, CD29, CD36, CD41, CD42a, CD42b, and CD61 on platelets and determine an average of 2.3 x 10(5) +/- 7 x 10(4) platelets per microliter. Counts correlated well with those obtained from the Cell-Dyn 3500 (r(2)=0.84). Agreeing with previous data, less than 2% of platelets from peripheral blood of normal individuals expressed the activation markers CD62P or CD63. After in vitro thrombin activation, >93% of the platelets expressed activation markers. Data presented here shows the benefits of using MLSC in combination with platelet inhibitors to quantitate and phenotype platelets while maintaining a viable responsive state.

OBJECTIVE

Previous studies have shown that germ-like cells can be induced from human umbilical cord mesenchymal stem cell (hUC-MSCs) in vitro. However, induction efficiency was low and a stable system had not been built. CD61, also called integrin-β3, plays a significant role in cell differentiation, in that CD61-positive-cell-derived pluripotent stem cells easily differentiate into primordial germ-like cells (PGC). Here, we have explored whether overexpression of CD61 would promote hUC-MSC differentiation into PGC and male germ-like cells.

METHODS

hUC-MSCs were cultured and transduced using pCD61-CAGG-TRIP-pur (oCD61) and pTRIP-CAGG plasmid (Control), and hUC-MSCs overexpressed CD61 were induced by bone morphogenetic protein 4 (BMP4, 12.5 ng/ml), to differentiate into PGC and male germ cells. Quantitative real-time PCR (RT-qPCR), western blotting and immunofluorescence staining were used to examine PGC- and germ cell-specific markers.

RESULTS

High expression levels of PGC-specific markers were detected in oCD61 hUC-MSCs compared to controls. After BMP4 induction, expression levels of male germ cell markers such as Acrosin (ACR), Prm1 and meiotic markers including Stra8, Scp3 in oCD61 were significantly higher than those of the Control group.

CONCLUSIONS

Under induction of BMP4, CD61-overexpressing hUC-MSCs, which had turned into PGC-like cells, could be further differentiated into male germ-like cells. Thus, a simple and efficient approach to study male germ cell development by using hUC-MSCs has been established.

BACKGROUND

Mature human mast cells express several types of adhesion molecules on their surface. Interactions between extracellular matrix (ECM) and adhesion molecules may be important for the migration and localization of mast cells and their precursors in tissues. Little is known about the regulation of adhesion molecules on mast cells during their differentiation.

OBJECTIVE

To clarify the evolution of adhesion phenotype and function, we examined the expression of adhesion molecules during cultured human mast cell (CHMC) differentiation and tested adhesion of mature CHMCs to various ECM proteins.

METHODS

CHMCs were obtained by culturing human cord blood-derived CD34(+) cells in the presence of stem cell factor and IL-6. Indirect immunofluorescence and flow cytometry was used to study cell surface expression of adhesion molecules and other markers. Mature CHMCs were tested for adhesion molecule function with immobilized matrix proteins.

RESULTS

At 1 week of culture, cells expressed CD11a, CD18, CD29, CD49d, and CD49e. At 14 weeks of culture, more mature CHMCs expressed CD11b, CD11c, CD29, CD49b, CD49c, CD49d, CD49e, CD51, CD61, and CD54 and weakly expressed CD18 and CD11a. CD11c, CD51, and CD61 appeared de novo by 4 weeks of culture, whereas CD49b and CD49c appeared by 8 weeks. CD29 decreased at 4 weeks but returned to the identical levels of 1-week-old cells by 8 weeks. Compared with levels at week 1, the levels of CD11a, CD18, CD49d, and CD49e at 4 weeks and beyond decreased during culture. Expression of CD49a, CD49f, and alphad integrin was never detectable during CHMC differentiation. Fourteen-week-old CHMCs significantly adhered to the leucine-aspartic acid-valine-containing connecting segment 1 fragment of fibronectin, the 120-kd argine-glycine-aspartic acid-containing fragment of fibronectin, vitronectin, and laminin through specific integrins.

CONCLUSIONS

Expression of integrins and CD54 is differentially regulated during CHMC differentiation, and mature CHMCs can adhere to many ECM proteins. These changes may facilitate emigration from the bone marrow into the circulation and ultimately contribute to the tissue homing and localization pattern seen with mature mast cells.

To find the best immunohistochemical marker for megakaryocytes in normal marrow, myelodysplastic syndrome (MDS), and chronic myeloproliferative disorders (CMPD), 57 marrow biopsy specimens were studied semiquantitatively with immunohistochemical methods using a panel of 7 antibodies. The staining intensity was graded 0 to 3 for scoring 100 consecutive megakaryocytes in each stained section. The final score for each stain was the sum of these 100 megakaryocytes individually multiplied by their corresponding grade. In normal marrow (11 cases), the average scores for antivon Willebrand factor (vWF) and Ulex europaeus agglutinin-1 (UEA-1) were 177.1 and 195.1, respectively. The scores for the other 5 markers, including anti-platelet-derived growth factor-BB, 2 anti-transforming growth factor-beta 3, anti-CD61, and anti-CD79a ranged from 96.1 to 124.1. In MDS (27 cases), the scores were 200.8 (vWF), 152.6 (UEA-1), and 28.7 to 98.5 (others). In CMPD (19 cases), the scores were 220.5 (vWF), 179.2 (UAE-1), and 64.8 to 101.2 (others). These results show that vWF and UEA-1 are good immunohistochemical markers for megakaryocytes in normal marrow, and vWF is the best marker in MDS and CMPD. For routine practice, vWF is the most reliable marker for identifying atypical megakaryocytes, especially in the cases of 5q-syndrome and agnogenic myeloid metaplasia.

OBJECTIVE

Platelet reactivity may be important in the management of patients with stroke. However, degree of platelet reactivity has not been correlated with Thrombelastography (TEG(®)) parameters in stroke. We sought to detect a correlation between TEG(®) values and clot platelet reactivity in ex vivo clots of stroke patients.

METHODS

We collected venous blood from 40 patients with stroke. TEG(®) measurements were carried out and residual clots were fixed in 10% formalin immediately following completion of TEG(®). The formalin specimens were embedded in paraffin blocks, cut at 4 micrometers, and stained with CD 61 (immunohistochemical stain used to detect platelets) with appropriate controls. Under light microscopy, three pathologists blinded to TEG(®) results independently graded CD61 intensity (how aggregated/intense the CD61 stained) into a low and high group, as a proposed measurement representing the platelet reactivity of the clot. We compared pre-tPA-TEG(®) values among groups with different CD 61 intensities.

RESULTS

After adjusting for confounding factors, we found statistically significant correlation between CD61 staining and several TEG(®) parameters (Delta and CD61 staining intensity (p=0.047); Angle and CD61 staining intensity grade (p=0.04); and G and CD61 staining intensity grade (p=0.04)).

CONCLUSIONS

Clot strength on TEG(®) as measured by Delta, Angle, and G correlates with a clot with greater platelet reactivity.

Here, we aim at exploring the effect of CST5 on bone resorption and activation of osteoclasts in osteoporosis (OP) rats through the NF-κB pathway. Microarray analysis was used to screen the OP-related differentially expressed genes. Osteoporosis was induced in rats by intragastric retinoic acid administration. The serum levels of tartrate-resistant acid phosphatase (TRAP), bone alkaline phosphatase (BALP) and osteocalcin (OC) and the expression of CD61 on the surface of osteoclasts were examined. The number of osteoclasts and the number and area of resorption pits were detected. Besides, the pathological changes and bone mineral density in bone tissues of rats were assessed. Also, the relationship between CST5 and the NF-κB pathway was identified through determining the expression of CST5, RANKL, RANK, OPG, p65 and IKB. Poorly expressed CST5 was indicated to affect the OP. CST5 elevation and inhibition of the NF-κB pathway decreased serum levels of TRAP, BALP and OC and expression of CD61 in vivo and in vitro. In OP rats, CST5 overexpression increased trabecular bones and bone mineral density of bone tissues, but decreased trabecular separation, fat within the bone marrow cavities and the number of osteoclasts through inhibiting the NF-κB pathway. In vivo experiments showed that CST5 elevation inhibited growth in number and area of osteoclastic resorption pits and restrained osteoclastic bone absorption by inhibiting the NF-κB pathway. In summary, overexpression of CST5 suppresses the activation and bone resorption of osteoclasts by inhibiting the activation of the NF-κB pathway.

A perfect microenvironment facilitates the activated circulating tumor cells (CTCs) to spark the adhesion-invasion-extravasation metastatic cascade in their premetastatic niche. Platelet-CTC interaction contributes to the progression of tumor malignancy by protecting CTCs from shear stress and immunological assault, aiding CTCs entrapment in the capillary bed, enabling CTCs to successfully exit the bloodstream and enter the tissue, inducing epithelial-mesenchymal-like transition (EMT), and assisting in the establishment of metastatic foci. To prevent the cascade from sparking, we show that, the multifunctional S-nitrosocaptopril (CapNO) acts on both CTCs and platelets to interrupt platelet/CTCs interplay and adhesion to endothelium, thus inhibiting CTC-based pulmonary metastasis in vivo. The activated platelets cloak cancer HT29 cells, resulting in HT29-exhibiting platelet biomarkers CD61 and P-selectin positive. CapNO inhibits both sialyl Lewisx (Slex) expression on HT29 and ADP-induced activation of platelets through P-selectin- and GPIIb/IIIa-dependent mechanisms, confirmed by the corresponding antibody assay. CapNO inhibits platelet- or interleukin (IL)-1β-mediated adhesion between HT29 and endothelial cells, and micrometastatic formation in the lungs of immunocompetent syngeneic mouse models. CapNO have also shown the effects of vasodilation, anticoagulation, inhibition of matrix metalloproteinase-2 (MMP2) expression on cancer cells, and inhibition of cell adhesion molecules (CAMs) expression on vascular endothelium. Due to a series of the beneficial effects of CapNO, CTCs remain exposed to the hostile bloodstream environment and are vulnerable to death induced by shear stress and immune elimination. This new discovery provides a basis for CapNO used for cancer metastatic chemoprevention, and might suggest regulation of the CTCs bloodstream microenvironment as a new avenue for cancer metastatic prevention.

To gather further information about the effects on blood platelet activation of in vivo exposure to nitric oxide (NO), platelet reactivity was studied in blood from healthy, non-smoking male volunteers before and after 30 min inhalation of 40 ppm NO. Whole blood was stimulated in vitro with adenosine diphosphate or thrombin receptor activation peptide (TRAP-6). In an ex vivo perfusion model, non-anticoagulated blood was exposed to immobilised collagen at arterial blood flow conditions (2600 s(-1)). Blood samples from both the in vitro and ex vivo experiments were stained with fluorochrome-labelled Annexin-V and antibodies against CD42a, CD45, CD49b, CD61, CD62P and fibrinogen, and analysed with a three-colour flow cytometry technique. NO inhalation reduced the platelet activation response to adenosine diphosphate (ADP) stimulation by decreasing platelet-platelet aggregation, alpha-granule release and platelet-leukocyte conjugate formation. TRAP-stimulated platelet activation, collagen-induced platelet activation and thrombus growth was unaffected by NO inhalation. We therefore suggest an ADP receptor inhibitor mode of action of inhaled NO, selective on the newly suggested G protein- and phospholipase C-coupled P2Y1 receptor. Our results demonstrate that blood platelet activation in healthy subjects is modulated by inhalation of NO in therapeutically relevant doses, although the clinical impact of our findings remains unclear.

The induction of leukemic cell differentiation is a hopeful therapeutic modality. We studied the effects of monochloramine (NH2Cl) on erythroleukemic K562 cell differentiation, and compared the effects observed with those of U0126 and staurosporine, which are known inducers of erythroid and megakaryocytic differentiation, respectively. CD235 (glycophorin) expression, a marker of erythroid differentiation, was significantly increased by NH2Cl and U0126, along with an increase in cd235 mRNA levels. Other erythroid markers such as γ-globin and CD71 (transferrin receptor) were also increased by NH2Cl and U0126. In contrast, CD61 (integrin β3) and CD42b (GP1bα) expression, markers of megakaryocytic differentiation, was increased by staurosporine, but did not change significantly by NH2Cl and U0126. NH2Cl retarded cell proliferation without a marked loss of viability. When ERK phosphorylation (T202/Y204) and CD235 expression were compared using various chemicals, a strong negative correlation was observed (r = -0.76). Paradoxically, NH2Cl and staurosporine, but not U0126, induced large cells with multiple or lobulated nuclei, which was characteristic to megakaryocytes. NH2Cl increased the mRNA levels of gata1 and scl, decreased that of gata2, and did not change those of pu.1 and klf1. The changes observed in mRNA expression were different from those of U0126 or staurosporine. These results suggest that NH2Cl induces the bidirectional differentiation of K562. Oxidative stress may be effective in inducing leukemic cell differentiation.

Thrombosis is the prognostic factor with the greatest effect on survival in patients with paroxysmal nocturnal hemoglobinuria (PNH), who lack dozens of membrane surface proteins. We recently described a primary homozygous Cys89Tyr congenital nonfunctioning CD59 in humans with clinical manifestation in infancy, associated with chronic hemolysis, recurrent strokes, and relapsing peripheral demyelinating neuropathy. Here we investigated hypercoagulability mechanisms characterizing the syndrome.

Membrane attack complex (MAC) deposition (anti-SC5b-9) and free hemoglobin (colorimetric assay) were assessed. Platelet activation was identified (anti-CD61, anti-CD62P), and microparticles (MPs) of 0.5-0.9 μm, were characterized (Annexin V, anti-human GlyA, anti-CD15, anti-CD14, anti-CD61). Platelet-monocyte aggregation was assessed with FlowSight.

2/7 patients (29%) with homozygosity for Cys89Tyr and 6/12 (50%) with any of four described CD59 mutations had recurrent strokes. In plasma samples from four patients carrying identical mutations, MAC deposition was increased on RBCs (p < 0.0003), neutrophils (p < 0.009), and platelets (p < 0.0003). Free-plasma hemoglobin levels were abnormally high, up to 100 mg/dl. Patients with CD59 mutation had RBC-derived MP levels 9-fold higher than those in healthy controls (p < 0.01), and 2-2.5 fold higher than PNH patients (p < 0.09). Leukocyte-activated platelet aggregation was increased (p < 0.0062). Loss of CD59 was shown in the endothelium of these patients.

Nonfunctioning CD59 is a major risk factor for stroke and hypercoagulability. Uncontrolled hemolysis causes massive MP release and endothelial heme damage. MAC attack on unprotected endothelium and platelet activation and aggregation with leukocytes mediate additional mechanisms leading to vascular occlusion. It is suggested that CD59 loss represents a major arterial prothrombotic factor in PNH and additional diseases.

Integrins are heterodimeric cell adhesion molecules with major roles in a variety of biological processes ranging from cell migration to tissue organization, immune and non-immune defense mechanisms and oncogenic transformation. Members of the beta(3) integrin subfamily are composed of a beta(3) subunit (CD61) non-covalently associated with two alpha subunits, alpha(IIb) (CD41) and alpha(v) (CD51), to constitute a group of transmembrane glycoproteins that participate in many physiologically important events. This investigation has focused on the molecular characterization of the cDNA encoding the porcine beta(3) integrin subunit. The deduced 762-amino acid sequence was 93, 92, 91, 89, 79 and 73% homologous to human, dog, rabbit, mouse, chicken and Xenopus laevis CD61 protein, respectively. Porcine CD61 molecule shares many structural features with human CD61, including a region containing a metal ion-dependent adhesion site (MIDAS) folding into an I domain-like structure. Through PCR-SSCP analysis and sequencing, six polymorphic positions were detected in the cDNA sequence of porcine CD61, and their frequencies were observed from a collection of 47 pigs. Expression analysis was done at two different levels: expression of the CD61 mRNA by RT-PCR and localization of the protein by immunohistochemistry. Our results show that CD61 transcripts were detected mainly in platelets and hematopoietic tissues. The immunohistochemical tissue localization of CD61 protein by a specific monoclonal antibody against CD61 recombinant protein showed that CD61 was expressed on vascular and non-vascular smooth muscle, epithelium and myeloid cells, being undetectable in cells of the lymphoid lineage. Furthermore, pulmonary intravascular macrophages (PIM), a subpopulation of macrophages which seem to play an important role in blood clearance, expressed much more CD61 when compared to pulmonary alveolar macrophages (PAM). The knowledge of the structure and distribution of the CD61 provides insight into the physiological function of the porcine beta(3) integrins and should be of importance in understanding the role of this integrin family in biological processes.

Microparticles (MP) are proposed to play a role in the pathogenesis of rheumatoid arthritis (RA). This study aimed to profile cell lineage-specific MP in patients with RA, osteoarthritis (OA), and healthy controls (HC) in synovial fluid and circulation. Patients with RA (n = 40), OA (n = 30) and HC (n = 33) were included. Cell-free synovial fluid (SF) and platelet-poor plasma samples were stained with annexin V APC and antibodies against CD45, CD20, CD14, CD4, CD8, CD66b, and CD61 for multicolor flow cytometry. Mann-Whitney U test/unpaired T test was used to assess intergroup differences among RA and OA SF and clinical, serological phenotypes of RA based on normality distribution; Kruskal-Wallis test with Dunn's multiple comparisons for comparing plasma MPs among RA, OA, and HC. Correlation between MP proportions and disease parameters was assessed by Spearman's correlation. The proportion of annexin V⁺ MP in SF of patients with RA [5 (6.35)] [median (IQR)] was higher compared to OA [1.8 (1.35), p < 0.001] and plasma of patients with RA [3.45 (5.63)] compared to OA [1.85 (1.4)] and HC [0.9 (1.1), p < 0.001]. Leukocyte-derived [0.85 (1.17)], granulocyte-derived [0.4 (2.05)], monocyte-derived [0.4 (0.4)], and T cell-derived MP [CD4⁺ - 0.1 (0.1); CD8⁺ - 0.1(0.1)] were higher in RA SF (p < 0.001). Platelet-derived MP (PMP) were the major fraction [1.5 (4.23), p < 0.001] in RA plasma. Leukocyte-derived MP were higher in RA plasma [0.1 (0.2); p < 0.001) than OA and HC. Annexin V⁺ MP and PMP were higher in the SF of RA with extra-articular manifestations (n = 15), as compared to those without (n = 25) (p = 0.02; p < 0.01, respectively). High SF granulocyte-derived MP were observed in patients with established RA (n = 24), ACPA-positive RA (n = 32) compared to their negative counterparts (p = 0.03; p = 0.02, respectively). Our observations of higher proportions of cell-derived MP in the plasma and synovial fluid of DMARD-naïve RA patients, their clinical and serological phenotypes suggest their role in dynamic cross talk between the joint and systemic circulation, disease pathology, and progression.

BACKGROUND

Diagnosing the cause of thrombocytopenia often requires a bone marrow aspiration or biopsy, an invasive procedure. Reticulated platelets (RP) are immature RNA containing platelets, accurate RP enumeration has yet to be achieved, partially due to the lack of a robust reference method.

OBJECTIVE

To refine previous work and gating strategies distinguishing RP from mature platelets while incorporating accurate platelet enumeration into the analysis. After reviewing previously published studies on Thiazole Orange (TO) staining of RP, we systematically evaluated CD41/CD61 in combination with a commercial source of TO (BDBiosciences). Previous RP methods have not taken advantage of platelet enumeration therefore our goal was to incorporate the ICSH platelet enumeration protocol into our method.

METHODS

TO concentration, incubation, and fixation method were determined to be 10% of stock concentration, 30 min, and 1% formaldehyde respectively. Gating strategy to determine RP fraction used an unstained control tube to set the limit of TO staining.

RESULTS

Normal range (n = 51) was 9.9 ± 3.1%. Analysis of 40 patients with immune-thrombocytopenia-purpura (ITP) showed a RP range from 4.3% to 81.2%. Platelet enumeration was consistent with our previous studies in this area.

CONCLUSIONS

Combining CD41/CD61 platelet enumeration with TO RP percentage is possible. Accurate RP percentage requires an effective gating strategy, as background fluorescence cursor placement is important. This method for enumeration of RP percentage combined with accurate platelet enumeration, particularly in the low range, should prove useful in differentiating production from consumption issues in thrombocytopenia and monitoring response to therapy.

Hormones drive mammary development and function and play critical roles in breast cancer. Epidemiologic studies link prolactin (PRL) to increased risk for aggressive cancers that express estrogen receptor α (ERα). However, in contrast to ovarian steroids, PRL actions on the mammary gland outside of pregnancy are poorly understood. We employed the transgenic NRL-PRL model to examine the effects of PRL alone and with defined estrogen/progesterone exposure on stem/progenitor activity and regulatory networks that drive epithelial differentiation. PRL increased progenitors and modulated transcriptional programs, even without ovarian steroids, and with steroids further raised stem cell activity associated with elevated canonical Wnt signaling. However, despite facilitating some steroid actions, PRL opposed steroid-driven luminal maturation and increased CD61+ luminal cells. Our findings demonstrate that PRL can powerfully influence the epithelial hierarchy alone and temper the actions of ovarian steroids, which may underlie its role in the development of breast cancer.

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

The aim of this study was to explore application value of detecting platelet associated antibody and platelet membrane glycoprotein in the diagnosis and prognosis for immune thrombocytopenia. The platelet associated immunoglobulin (PAIg) and platelet membrane glycoprotein (CD41, CD61, GPIIb/IIIa) in 76 cases of immune thrombocytopenia and 30 healthy subjects were determined by FCM. The results showed that PAIg level in ITP patients included PAIgG (31.25 +/- 18.06)%, PAIgM (32.41 +/- 15.51)%, PAIgA (23.39 +/- 16.67)% which were remarkedly higher than in health control (10.48 +/- 5.05)%, (9.40 +/- 4.42)% and (7.23 +/- 3.61)% (P < 0.001). In patients with secondary immune thrombocytopenia (chronic aplastic anemia, SLE, Evans syndrome, liver cirrhosis hypersplenism, etc), PAIg level was higher than that in control group, while the platelet membrane glycoprotein in the blood of these patients was lower than that in control group. The level of PAIg decreased (P < 0.05) after treatment, but platelet membrane glycoprotein increased (P < 0.01). The result suggested that measurements for platelet membrane glycoprotein and platelet associated antibody by FCM were practical with high sensitivity, rapidity and simplicity used as a routine method in diagnosis and evaluation of the therapeutic effects in immune thrombocytopenia patients.