bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Detection of SAP Deficiency in Cytotoxic Lymphocytes from Patients with X-Linked Lymphoproliferative Disease and Their Family Members.
Authors: Tabata Yasuhiro , Villanueva Joyce , Lee Susan M. , Zhang Kejian , Kanegane Hirokazu , Miyawaki Toshio , Sumegi Janos , Filipovich Alexandra H.
Published: October 2019

Background: X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency, which is characterized by an extreme susceptibility to Epstein-Barr virus (EBV). In half of the XLP patients, primary EBV infection can be fatal with explosive activation and proliferation of lymphocytes in many organs, which leads to fluminant hepatitis and bone marrow failure with hemophagocytosis. Mutations in the SH2D1A gene, which encodes the SAP protein, have been described in a proportion of patients with the clinical syndrome of XLP. The diagnosis of XLP is still difficult given its clinical heterogeneity, and the lack of a readily available rapid diagnostic laboratory test, particularly in cases without a family history of XLP. XLP should always be a consideration in males with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH).

Methods: Four-color flow cytometric analysis was used to establish normal patterns of SAP expression for control subjects, and patterns of SAP staining in cytotoxic lymphocytes (CD8+ T cells, CD56+ T cells and natural killer [NK] cells). This assay was used to study patients with clinical syndromes consistent with XLP, and in some cases; their family members.

Results: 6 patients with XLP confirmed by detection of mutations in the coding regions of SH2D1A showed lack of intracellular SAP in all cytotoxic cell types; one was tested presymptomatically. Carriers of SH2D1A mutations tested (mother and sister) had decreased SAP staining patterns below normal levels. Eleven males with clinical syndromes consistent with XLP, predominantly EBV-HLH who were demonstrated to have normal or increased perforin expression were also studied. All patients showed normal SAP expression including one case with a convincing X-linked family history; all were subsequently shown to have no mutations in SH2D1A. Additionally, one asymptomatic adult male with an intronic mutation previously published as causing XLP was found to have normal SAP expression.

Conclusions: Four-color flow cytometry provides diagnostic information that may speed the identification of this fatal disease, differentiating it from other EBV-HLH. We also suspect it will prove useful in substantiating or excluding disease-causing genetic variants in the SH2D1A gene.
Driving T Cell Development in the Thymus.
Authors: Joao Cristina M. , Ogle Brenda M. , Cascalho Marilia , Platt Jeffrey L.
Published: October 2019

Background: Classic reports on lymphocyte development hold that B and T cells develop independently. This concept derives in part from the observation that patients with pure B cell immunodeficiency and hypogammaglobulinemia have a normal thymus and T cell numbers. Our recent findings however challenge this concept. We found that T cell development depends not only on the interaction of T cell precursors with thymic epithelial cells but also on other cells. Here we report that those other cells are B cells.

Aims: The purpose of this study was to determine whether B cells drive T cell development and TCR diversification in the thymus.

Methods: We compared the number of sub-populations of thymocytes and TCR repertoire diversity in B-cell deficient and B-cell proficient mice and in B cell deficient mice following immunoglobulin (Ig) injections. Total leucocytes numbers were determined with a Coulter counter and numbers of thymocytes sub-populations were calculated by flow cytometry analysis. TCR repertoire diversity was measured by a novel method based on hybridization of TCR Vβ specific cRNA on a gene chip platform.

Results: In B-cell deficient mice the number of thymocytes was four times reduced and TCR Vβ chain diversity was up to one million times lower compared with wild type mice. Numbers and diversity were restored by treatment of the mice with gamma globulin (see table).

Conclusions: T cell development and diversification is driven by B cells.

Mice Number of total thymocytes (mean ± standard deviation) p Value β V TCR diversity of thymocytes (median; min.-max.) p Value C57BL/6 (wild mice) 1.3 x 108 ± 5.1 x 107N=7 4.7 x 106; 1.0 x 105 − 1.1 x 108N=5 JH−/− (B cell immunodeficient mice) 3.1 x 107 ± 1.7 x107N=7 0.002 5.9 x 102; 3.6 x 102 − 1.1 x 103N=5 0.0002 JH−/− treated with Ig 3.9 x 107 ± 1.4 x106N=2 0.20 1.1 x 105; 2.7 x 100 − 7.7 x 105N=4 0.08
Structural Analysis of Janus Kinases.
Authors: Boggon Titus J. , Li Yiqun , Eck Michael J.
Published: October 2019

Janus Kinase 3 (Jak3) plays an essential role in hematopoietic signaling. Primarily expressed in B-, T- and Natural killer cells, it is activated through the γc chain of interleukin-2 like cytokine receptors (IL-2, -4, -7, -9, -15, -21). Deficiency of catalytically active Jak3 or disruption of the Jak3:IL-2γc interaction results in severe combined immunodeficiency (SCID). Jak3-deficient humans demonstrate defects restricted to the immune system, suggesting that selective inhibition of Jak3 catalytic activity or interruption of the Jak3:IL-2γc interaction are potentially exploitable strategies to achieve immunosuppression. Jak kinases contain four defined regions; a catalytically active carboxy-terminal kinase, a pseudo-kinase, a SH2-like region and a N-terminal Ferm domain. To date, no direct structural information has been reported for portions of any of the Jak family kinases. Structural studies are underway to define crystallographically the kinase domain of Jak3 and the Jak3-Ferm:IL-2γc interaction. Structural insights into the mechanism of Jak activation and routes to specific inhibition will be discussed.
Cytotoxic Activity of Cytokine-Induced Killer Cells Correlates with Expression of SAP and SLAM.
Authors: Mehrle Stefan , Frank Susanne , Schmidt Jan , Buchler Markus W. , Schmidt-Wolf Ingo G.H. , Marten Angela
Published: October 2019

SAP is a small protein, consisting of a single SH2 domain which is mutant in humans with X-linked lymphoproliferative disease. Patients with XLP are affected by fatal EBV infections and malignant B cell lymphomas. The increased risk for B cell lymphomas is suggested to result from impaired immunosurveillance of B cell proliferation by T cells. Here, we investigated the role of SLAM/SAP for activation of effector cells with cytotoxic activity (CIK cells), which are generated by unspecific stimulation of the T cell receptor and addition of exogenous IL-2 as described previously. The TCR activation on day +1 resulted not only in a short peak of activated cells, but activation continued and increased in combination with IL-2. We observed a striking peak of SLAM (Signaling Lymphocyte Activation Molecule) on day +6 in form of extracellular detectable CD150 as well as at the level of proteins and mRNA. Interestingly, the cytotoxic activity and the amount of SHP-2 protein showed a similar pattern as the parameters mentioned above but were shifted one day. Comparing these data for correlation, we observed a significant correlation between cytotoxic activity and CD150 expression pattern (P<0.001) and amount of SLAM protein (P<0.02) as well as between amount of SHP-2 protein and SLAM parameters (P<0.03). IL-10 secretion did not correlate with any of the parameters investigated. Secretion of Th1/Th2 cytokines was determined using a cytometric bead assay. There was no change in the amount of IL-6 and TNF-alpha. IL-4 was below the detection threshold and IL-2 could not be analyzed due to exogenous addition.

IFN-gamma levels increased during cultivation, peaked on day +3 and then remained at about 4ng/ml. IL-10 secretion started after stimulation of cells by anti-CD3, peaked on day +3 and then decreased continuously. Between day +6 and day +7, only 0.86 ± 0.02ng/ml/24hrs/3x106cells were secreted. In summary, activation of peripheral blood cells with agonistic anti-CD3 antibody and exogenous IL-2, as used for generation of CIK cells, results in significant SLAM and SAP activation five days after TCR stimulation. This peak correlates with cytotoxic activity against tumor cells. SLAM expression and binding by SAP seems to be important in the activation of cytotoxic effector cells.
The Effects of Chloride Channel Blockers on Platelet Cytoplasmic Free Calcium Concentration and Platelet Aggregation.
Authors: Yin Songmei , Chen Xiaolin , Nie Danian , Xie Shuangfeng , Ma Liping , Wang Xiujiu , Wu Yu-Dan , Li Yiqing , Feng Jianhong
Published: October 2019

Objective To explore the effects of chloride channels on the regulations of platelet cytoplasmic free calcium concentration ([Ca2+]i) and platelet aggregation (PAG).

Methods Platelet were separated freshly and then activated by thrombin; The chloride channel blockers 4,4′-diisothiocyano-2, 2′-disulfonic acid stilbene (DIDS) or niflumic acid (NFA), and calcium channel blockers 1-{β-[3-(4-methoxyphenyl)propoxy]- 4-methoxyphenethyl}- 1H - imidazole hydrochloride (SK&F96365) or Nifedipine were added to react with the activated platelets. The effects of each agent on platelet [Ca2+]i and PAG were detected. The combine effects and the interactions among chloride channel blockers (DIDS, NFA) and calcium channel blockers (SK&F96365, Nifedipine) were also investigated.

Results Both DIDS and NFA [the concentration were12.5, 25, 50, 100 and 200μmol•L−1 respectively] could inhibit the PAG induced by thrombin (1U/ml) and the effect was dose-dependent. Compared with the control, they had no significant effects on resting [Ca2+]i. Compare with the control group, DIDS (100μmol•L−1), SK&F96365 (100μmol•L−1) and Nifedipine (100μmol•L−1) could significantly reduce the PAG, Ca2+ release and Ca2+ influx activated by thrombin in platelet (P<0.05). DIDS (100μmol•L−1) and SK&F96365 (100μmol•L−1) could enhance each other’s effect on reducing the PAG, Ca2+ release and Ca2+ influx (P<0.05). DIDS (100μmol•L−1) and Nifedipine (100μmol•L−1) could enhance each other’s effect on reducing Ca2+ release (P<0.05). NFA (100μmol•L−1) and SK&F96365 (100μmol•L−1) could weaken each other’s effect on Ca2+ release (P<0.05). NFA (100μmol•L−1) and Nifedipine (100μmol•L−1) could weaken each other’s effect on PAG, Ca2+ release and Ca2+ influx activated by thrombin in platelet (P<0.05).

Conclusion The chloride channel blockers DIDS and NFA have no effect on the resting [Ca2+]i and the leak calcium influx of platelet. DIDS can inhibit the Ca2+ release, Ca2+ influx and PAG of platelet induced by thrombin, while NFA can only inhibit the Ca2+ release of platelet induced by thrombin. There are interactions between chloride channel blockers and calcium channel blockers in resting [Ca2+]i and PAG of platelet. The opening of chloride channel can influence the cellular calcium movement of platelet.
Variability of PECAM-1 Expression on Blood Cells.
Authors: Berken Melanie S. , Newman Debra K. , Newman Peter J.
Published: October 2019

PECAM-1 is a 130 kDa member of the Immunoglobulin gene superfamily that is expressed on circulating platelets, neutrophils, monocytes, and certain lymphocyte subpopulations. Deficiency of PECAM-1 in mice results in hyper-reactive platelets, B cells, and mast cells, compromised vascular integrity, and endothelial cells that are more susceptible to radiation-induced apoptosis in vivo. Whether or not PECAM-1 expression levels vary in the human population, and whether variable PECAM-1 expression renders one more or less susceptible to cardiovascular disease, autoimmune disorders, or sepsis has not been investigated to date. As a first step in examining these questions, we have developed a quantitative flow cytometry-based assay to determine the number of PECAM-1 molecules expressed on the surface of blood and vascular cells. Whole blood samples obtained from anonymous normal volunteer blood donors were stained with saturating amounts of fluorochrome-conjugated anti-PECAM-1 antibody, and relative fluorescence intensities were determined for platelets, neutrophils, monocytes, and lymphocytes by flow cytometry. Polystyrene microbeads coated with calibrated numbers of antibody binding sites were saturated with the same fluorochrome-conjugated anti-PECAM-1 antibody and used to create a standard curve that allowed us to relate relative fluorescence intensities to known numbers of antibody binding sites. By plotting the fluorescence intensity of anti-PECAM-1 antibody binding to each cell population against this standard curve, we were able to quantify the number of PECAM-1 molecules on the surface of each cell population. We found that PECAM-1 expression levels most often ranged between 5,000–12,000 molecules/platelet, 6,000–10,000/lymphocyte, 30,000–40,000/neutrophil, and 50,000–80,000/monocyte. Some individuals, however, expressed 2–5 times more PECAM-1 on a given blood cell subtype. Interestingly, individuals who expressed high levels of platelet PECAM-1 did not necessarily also express high levels of leukocyte PECAM-1, and vice versa. Since a number of naturally-occurring alleles of the PECAM-1 gene have recently been associated with the progression of atherosclerosis, myocardial infarction, and/or coronary artery disease, correlation of genetic variation within the PECAM-1 locus with its variable expression on the surface of blood and vascular cells may permit addition of PECAM-1 to the growing list of cell adhesion and signaling receptors whose expression is linked to bleeding and clotting disorders in humans.
Effects of Estrogen Replacement Therapies on Mouse Platelet Funcion.
Authors: Leng Xing-Hong , Zhang Wei , Nieswandt Bernhard , Lopez Jose A. , Bray Paul F.
Published: October 2019

Clinical trials of hormone replacement therapy (HRT) have demonstrated an increase in stroke and myocardial infarction despite the beneficial changes in LDL and HDL cholesterols, and it has been suggested that HRT may adversely affect thrombosis and/or inflammation. Platelets participate in both of these processes and play a central role in thrombosis formation in high shear arterial vasculature. The effects of HRT on platelet function have been controversially reported, probably due to different hormone regimes used (e.g. different estrogenic compounds, different dosages and routes of administration). We used a murine model of menopause to test the hypothesis that HRT alters platelet reactivity. Bilaterally ovariectomized female littermates were treated with various estrogen regimes or matching placebo for 21 days. Three estrogen replacement therapy (ERT) regimes were studied: 1) oral conjugated equine estrogen (CEE) vs. placebo (n=5 pairs), 2) oral 17-beta estradiol (E2) vs. placebo (n=4 pairs), and 3) subcutaneously implanted E2 (SQ E2) vs. placebo (n=8 pairs). Washed platelets were used to focus on intrinsic platelet reactivity in the absence of plasma coagulation factors. Platelet reactivity was assessed by flow cytometric quantification of agonist-induced fibrinogen binding. Oral CEE, but not oral E2 enhanced the platelet response to collagen-related-peptide (CRP) (P<0.001 and P=0.87 for oral CEE and oral E2, respectively). This enhancement of platelet function was agonist specific since neither oral CEE (P=0.862) nor oral E2 (P=0.917) affected the platelet response to thrombin. We also tested whether the route of hormone administration affected platelet function. SQ E2 induced a condition different from that of oral E2: the platelet response to thrombin was enhanced (P=0.028), but only to low (<10 mU/ml), not high concentrations of thrombin. In contrast to oral E2, SQ E2 caused a reduction in CRP-induced platelet fibrinogen binding (P=0.030). Platelet surface expression of GPVI was reduced in response to SQ E2 (P<0.001), and the level of GPVI correlated with CRP-induced platelet fibrinogen binding (P<0.0001). In conclusion: 1) the effects of different estrogen replacement therapies on platelet function differ significantly, perhaps explaining some of the conflicts in the literature, 2) the effects of ERT on platelet function are dependent upon the ERT preparations and routes, 3) the effects of ERT on platelet function are agonist and agonist dose-dependent, and 4) SQ E2 induces lower platelet GPVI expression, most likely explaining the reduced response to CRP. A potential practical consequence of our findings is that oral E2 may be less prothrombotic than either oral CEE or transdermal E2.
Leptin-Mediated Activation of Human Platelets: Involvement of a Leptin Receptor and Phosphodiesterase 3A Cellular Signaling Complex.
Authors: Elbatarny Hisham S. , Maurice Donald H.
Published: October 2019

Leptin, a 16-KDa peptide-hormone produced primarily by adipocytes, was reported previously to promote platelet aggregation by a yet undefined mechanism. In this study, we investigated the effect of leptin on human platelet activation and delineate a molecular basis for this effect. Thus, we report that leptin potentiates ADP-induced platelet aggregation and directly stimulates adhesion of platelets to fibronectin. Using a combination of pharmacological and biochemical approaches we demonstrate that leptin mediates these effects by activating a signaling cascade that includes the leptin receptor (OB-R) and the kinases Jak2, phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB/Akt). Consistent with an important role for PKB-mediated phosphorylation and activation of the dominant cAMP hydrolyzing phosphodiesterase, phosphodiesterase 3A (PDE3A), in this system, incubation of platelets with leptin activated PDE3A and stimulated a PKB-mediated phosphorylation of PDE3A. A selective PDE3 inhibitor, cilostamide, attenuated leptin effects in these cells. While leptin did not alter platelet cAMP levels, our identification of an OB-R/PDE3A complex is consistent with leptin acting locally to regulate a “pool” of cAMP that controls platelet activity. This is the first report of leptin-induced platelet adhesion which provides a mechanistic basis for this potentially important effect of this peptide hormone.
Murine Platelets Expressing a Polymorphism of Integrin β3 Associated with Acute Coronary Thrombosis in Humans.
Authors: Wilcox David A. , Fang Juan , Johnson Bryon D.
Published: October 2019

Inheritance of a naturally occurring polymorphism of the human integrin β3-subunit at amino acid 33 causes a change from Leu to Pro that has been implicated as a risk factor for thrombotic complication in humans. It is unclear; however, if Pro (also known as the platelet alloantigen PlA2 form of β3) can alter integrin activation to significantly affect platelet function in vivo, since some clinical studies do not corroborate the initial findings. Since PlA2 is a frequent variant of β3 expressed on 28% of platelets from Americans the impact of this genetic variation on platelet function could have widespread clinical relevance; therefore, we developed a murine model to determine if expression of this common polymorphism of human integrin β3 can increase platelet function and lead to an increased propensity for thrombosis within a homogeneous population of mice. This model eliminates issues arising from variation in the genetic make-up, physical environment, and/or lifestyles of humans and allows for in vivo analysis not possible with human subjects. To accomplish this, cDNA encoding each form of the human β3-subunit was subcloned into an HIV type-1 lentivirus-derived vector under the transcriptional control of the human αIIb gene promoter to direct synthesis of β3 specifically to the megakaryocyte lineage. Bone marrow was isolated from β3-deficient mice and transduced with β3 virions encoding either the PlA1 or PlA2 form of human β3, and then transplanted into lethally irradiated β3-deficient littermates. Flow cytometric analysis demonstrated stable expression of the hybrid murine/human αIIbβ3 integrin complex on the surface of circulating platelets. Immunoanalysis using monoclonal antibodies and human serum that react specifically with the PlA1 or PlA2 confirmed the identity of each alloantigen of human β3. The lentivirus contained a second transgene encoding a drug-selectable marker (P140K MGMT) that was used for in vivo enrichment of transduced cells in mice treated with two regimens of cytotoxic reagents, O6-BG/BCNU. Flow cytometric analysis showed that use of drug-selection resulted in increased expression of the integrin αIIbβ3 complex to equal levels on nearly 100% of platelets using either form of human β3. Similar to platelets from normal mice, platelets expressing each PlA form of β3 could be induced to form aggregates ex vivo upon treatment with a cocktail of physiological agonists of platelet activation (adenosine diphosphate, epinephrine and the thrombin receptor activating peptide).

These results demonstrate the feasibility for targeting expression of altered forms of the human integrin β3-subunit to murine platelets and pave the way for future studies to examine and compare the effect of integrin αIIbβ3 structure on platelet function and thrombosis in vivo.
Platelet Derived Microparticles (PMP) Interfere with Rolling, Adhesion and Transmigration of Hematopoietic Progenitor Cells (HPC).
Authors: Bistrian Roxana , Ruster Brigitte , Gottig Stephan , Seifried Erhard , Henschler Reinhard
Published: October 2019

We wished to investigate if small particles derived from platelet membranes (PMP) which are present in blood may influence the migration behavior of HPC. PMP were isolated from pooled platelet concentrates derived from human whole blood donations via the buffy coat or PRP methods. After activation of platelets with thrombin or calcium ionophore, PMP were quantitatively enriched by sequential centrifugation at 1.250 and 50.000g. Flow cytometric analysis showed separated forward and side scatter profiles of the larger platelets and smaller PMP, with both platelets and PMP staining highly positive for CD41 and CD61. Purity of PMP was 89–92%. Binding of PMP to HPC was performed at 37°C in cell culture medium at ratios between 3 and 100 PMP/HPC. Binding was assessed flow cytometrically using PMP which were fluorescence-marked with PKH26, or by appearance of CD41/CD61 complex on the surface of HPC. We found that PMP dose-dependently bound human KG-1 or primary CD34+ HPC, with half-saturation occurring within seconds to a few minutes, and 10–20 PMPs binding per HPC.

Compared to controls, the binding of PMP-preloaded HPC to immobilized fibronectin showed a mean 10.4-fold reduction with KG-1 cells and a mean 4.2-fold reduction with CD34+ primary HPC. The interaction of PMP-pretreated HPC with endothelial cells (EC) was assessed under laminar shear flow in a parallel plate flow chamber at defined wall shear stresses using TNF-alpha prestimulated human umbilical vein endothelial cells (HUVEC). We found a 1.6-fold (KG-1) and 2.2 fold (CD34+ HPC) reduction in numbers of rolling and subsequently binding cells at shear stresses of 0.1 dyn/cm2, and a 1.5-fold (KG-1) or 1.7-fold (CD34+ HPC) reduction at 2 dyn/cm2 compared to untreated controls. Both binding and rolling of HPC were dependent on P-selectin in this model, as shown by blocking of the interaction of HPC with EC with MoAB AK-4, and the abrogation of this block with PMP-HPC. Finally, SDF-1alpha-induced transmigration of HPC in transwell chemotaxis chambers was reduced > 3-fold after preincubation of HPC with PMP when more than 10–20 PMP were bound per HPC. Taken together, highly purified PMP are able to bind HPC and to modulate their interaction with endothelium. This process is mediated by (i) suppression of P-selectin mediated rolling and adhesion of HPC to endothelium, (ii) suppression of integrin-mediated adhesion of HPC and (iii) suppression of SDF-1alpha mediated transmigration of HPC. PMPs may therefore be part of a physiological mechanism which can modulate the adhesion behaviour of circulating HPC and protect them from recruitment into sites of inflammation or activated coagulation under conditions of postcapillary or venous shear stress.
The Platelet Specific Chemokine Platelet Factor 4 Induces Expression of E-Selectin in an NF-κ B Dependent Manner.
Authors: Sachais Bruce S. , Ma Peihong , Rux Ann H. , Yu Guangyao
Published: October 2019

The involvement of platelets in the pathogenesis of atherosclerosis has recently gained much attention. Platelet factor 4 (PF4) is a platelet specific chemokine released upon platelet activation. PF4 has been localized to atherosclerotic lesions, including macrophages and endothelium. In this report, we demonstrate that E-selectin, an adhesion molecule involved in atherogenesis, is up-regulated in human umbilical vein endothelial cells exposed to PF4. Induction of E-selectin mRNA is time and dose dependent, and requires the presence of cell surface glycosaminoglycans. Surface expression of E-selectin, as measured by flow cytometry, is also increased by PF4. Activation of NF-κB is critical for PF4 induced E-selectin expression, as demonstrated by promoter activation studies and electrophoretic mobility shift assays. In summary, our data demonstrate that PF4 can increase expression of E-selectin by endothelial cells by activation of NF-κB. PF4 induction of endothelial E-selectin expression represents another mechanism by which platelets may participate in atherosclerotic lesion progression. These data also suggest that PF4 may participate in the proinflammatory functions of activated platelets.
Influence of Multimer Size and a Natural Dimorphism on the Activity of Convulxin.
Authors: Kato Kazunobu , Cheli Yann , Furihata Kenichi , Radis-Baptista Gandhi , Kunicki Thomas J.
Published: October 2019

Convulxin (CVX), a C-type lectin derived from the venom of Crotalus durissus terrificus, is a potent activator of human platelets, binding predominantly to platelet glycoprotein (GP) VI, but also to GP Ibα. Native CVX is an octamer composed of four αβ-heterodimers. It has been speculated that multimer size contributes greatly to CVX function, but proof of this hypothesis has not yet been reported. Additionally, two different native sequences have been reported (GenBank protein accession numbers CAA76182 and AAQ11362), one bearing lysine (K), the other glutamic acid (E), at β chain residue 89, but the physiological relevance of this difference is unknown.

In an earlier report, we have described the production of monomeric recombinant CVX (rCVX) heterodimers using the Drosophila S2 system. We now employ this recombinant protein and site-directed mutagenesis to evaluate the influence of multimer size and the substitution βK89E on CVX function.

By flow cytometry, native CVX (αβ)4 and both recombinant forms of CVX (αβ89K and αβ89E) bind to CHO cells expressing human recombinant GPVI (hrGPVI) and to human platelets in whole blood. Surface plasmon resonance (BIAcore) was employed to assess the kinetics of the interaction between hrGPVI and native or recombinant CVX. The calculated equilibrium dissociation constants (KD) were: for rCVX αβ89K, 11.3 x 10−8 M; for rCVX αβ89E, 9 x 10−8 M; and for native CVX (αβ)4, 2.8 x 10−8 M. These results indicate that the affinities of the two rCVX forms for hrGPVI are essentially the same and that the relative affinity of native CVX is about three-fold higher. Next, aggregation of human PRP was used to assess the relative function of the CVX forms. The minimum concentration of native CVX that induces platelet aggregation (70 picomolar) is roughly 400-fold lower than that of either rCVX αβ89K or rCVX αβ89E (each, 29 nanomolar).

Based on these findings, it is apparent that: 1) There is little, if any difference, in the affinity or activity of the two recombinant forms of CVX; 2) the octameric form of native CVX results in roughly a three-fold increase in affinity for GPVI; and 3) this increase in affinity of the octameric form of native CVX cannot account for the substantial increase in its ability to induce platelet aggregation relative to the recombinant forms. These results are consistent with the hypothesis that the ability of native CVX octamer to cluster mobile GPVI molecules within the platelet membrane may be the single most important factor that contributes to the efficiency with which CVX is able to induce signal transduction and platelet activation. Moreover, smaller forms of CVX, such as the rCVX heterodimers, may prove to be efficient inhibitors of platelet activation by native CVX and other GPVI agonists.
Platelet Counts Vary by Ethnicity, Sex, and Age: Analysis of NHANES III Data.
Authors: Segal Jodi B. , Molterno Alison R.
Published: October 2019

Background: The possibility of variation in platelet count by age and genetic background has not been examined. Identification of subpopulations with elevated platelet counts, within what is traditionally considered a normal range, may identify a group with excessive morbidity or mortality. Furthermore, platelet count differences may suggest populations in which genetic polymorphisms in regulatory proteins such as the thrombopoietin receptor influence platelet production. We hypothesized that there were differences in platelet count by ethnicity, sex and age not explained by environmental factors. Objective: To demonstrate differences in mean platelet counts by ethnicity, sex, and age while controlling for variables known to influence platelet count.

Methods and Design: We used data from the National Health, Nutrition and Examination Survey III (NHANES III), which is a multistage probability sample of the United States population with data collected between 1988 and 1994. Using appropriate weighting for the complex sampling design, the geometric mean platelet count was calculated for the total population and the population stratified by ethnicity, sex, and age, while controlling for C-reactive protein, white blood cell count, iron-deficiency, serum folate, markers of alcohol intake, presence of hepatitis B or C antibodies, and diabetes mellitus. Other potential influences, such as medications, were found not to affect the predicted counts and not included in the models.

Results: The lowest mean platelet counts were among whites (259 K/ml [95% C.I. 255–264 K/ml]) and the highest were in non-Hispanic blacks (275 K/ml [95% C.I. 270–280 K/ml]) with Mexican-Americans having intermediate values (266 K/ml [95% C.I. 261–272 K/ml]), when controlled for age and sex. Older men and women of each ethnicity consistently had lower mean platelet counts, with 60–69 years olds having mean counts approximately 7 K/ml lower than young adults (p=0.015) and 70–90 year olds having mean counts 19 K/ml lower than young adults (p<0.001). Even with controlling for iron deficiency, women had significantly higher platelet counts than men (273 K/ml [95% C.I. 269 – 278]) versus 251 K/ml [95% C.I. 245 – 256])

Conclusion: Mean platelet counts differ by ethnicity, sex, and age and these differences are not explained by covariates known to influence platelet count. This suggests that genetic influences on the platelet count are operative. These data also suggest that there may be a hormonal regulation of platelet count. The decline with age may reflect decreased stem cell function with age; alternatively, there may be a survival advantage to having a lower platelet count. Longitudinal studies of platelet count with aging are indicated.
Production of Monoclonal Antibodies Against ADAMTS13 Determining Its Expression in Different Human Tissues.
Authors: Gao Weiqiang , Chow Xiaojun , Bai Xia , Su Jiang , Ruan Changgeng
Published: October 2019

The deficiency of ADAMTS13/von Willebrand factor-cleaving protease in cancer patients, which can lead to the accumulation of ultra-large von Willebrand factor (UL-vWF) in plasma, has been confirmed by several previous reports. Nevertheless, the results of them did not avoid the heterogeneity between different tumors and the reasons accounted for its deficiency still remain to be elucidated. Since ADAMTS13 is mainly synthesized and released from liver and tumors derived from liver often accompanies with metastasis, we have studied the expression levels of ADAMTS13 in patients with primary hepatocarcinoma and in different hepatic cancer cell lines. Firstly, we detected the ADAMTS13 mRNA and protein levels in paired primary cancer tissues and adjacent normal tissues using semi-quantitative reverse transcription PCR (RT-PCR) and immunohistochemistry. It showed that most of the cancer tissues expressed lower ADAMTS13 levels compared with their paired adjacent normal tissues. Significant low expression levels of ADAMTS13 had a close relationship with distal metastasis of hepatocarcinoma, but the abnormal of hepatic function did not have an association with the deficiency of ADAMTS13 expression. Interestingly, most of the patients suffering with hepatitis B and cirrhosis did not show significantly decreased ADAMTS13 levels, and most of the cancer patients with distal metastasis while without hepatitis B and cirrhosis accompanied markedly deficiency of the protease. The hepatocarcinoma cell lines, HepG2 and BEL7702, also expressed lower ADAMTS13 mRNA levels compared with SMMC7721 and a normal hepatic cell line L02. However, when the hepatocarcinoma cell lines were treated with a demethylation drug 5-aza-2′-deoxycytidine, its ADAMTS13 mRNA expression levels elevated with the increase of treatment dosage, especially in HepG 2 (P<0.05). It seems that the deficiency of ADAMTS13 in hepatocarcinoma might be associated with DNA methylation. The data above revealed that the ADAMTS13 expression levels were lower in hepatocarcinoma tissues. Furthermore, the activity of ADAMTS13 was significantly decreased in aged people older than 65 years, and the extent of methylation of genes are closely related with age. Therefore, based on our results, the deficiency of ADAMTS13 in hepatocarcinoma patients might not be caused by the abnormal of hepatic function, but be related to the DNA methylation of the protease.
Treatment of Relapsed Acute Promyelocytic Leukemia by Arsenic-Based Strategies without Hematopoietic Stem Cell Transplantation in Hong Kong: A Seven-Year Experience.
Authors: Au Wing Y. , Chim James C. , Lie Albert K. , Kumana Cyrus R. , Leung Anskar Y. , Tse Eric W. , Ma Shing Y. , Liang Raymond H. , Kwong Yok L.
Published: October 2019

Background: The optimal therapy for relapsed acute promyelocytic leukemia (APL) after arsenic trioxide (As2O3)-induced remission is unclear. Hematopoietic stem cell transplantation (HSCT) is associated with high morbidity and mortality. Moreover, lasting remission is observed in many patients who are not candidates for HSCT, owing to advanced age or lack of donors, implying that HSCT is not mandatory for durable remission. We evaluated our results of an As2O3-based, non-HSCT regimen for patients with relapsed APL.

Materials and methods: Forty-two consecutive patients (18 men, 24 women, median age: 35 years, 12–72) with relapsed (relapse 1, R1=39, R2=3) APL were treated with an-As2O3 based, non-HSCT regimen. The time from last complete remission (CR) was 22 (6–243) months (mo). Initial treatment was As2O3 (10 mg/day) either intravenously (n=16) or orally (n=28) until CR, followed by idarubicin consolidation (6 mg/m2/day x 9). Twenty-five patients received oral-As2O3 maintenance. Post-As2O3 relapses were treated with oral As2O3 + all-trans retinoic acid (ATRA, 45 mg/m2/day) until CR, followed by maintenance (two weeks of ATRA+As2O3 every 2 mo. for 2 years). Post-As2O3/ATRA relapses were treated with oral As2O3+ATRA+ascorbic acid (1g/day) until CR, followed by consolidation/maintenance with the same regimen (2 weeks every 2 mo. for 2 years). Part of the induction and all of the maintenance therapies were given in the outpatient clinic.

Results: Forty-one patients (98%) achieved CR after initial As2O3 treatment. One 72-year old man with XYY syndrome, diabetes and mental retardation died of pneumonia. Thirteen relapses occurred at a median of 15 (6–22) mo. As2O3-maintenance significantly decreased further relapses (3/24 with versus 10/17 without As2O3-maintenance, p=0.003). Two relapses died of cerebral APL before further treatment could be administered. Of eleven patients treated with As2O3+ATRA, 10 achieved CR, 8 of whom have remained in remission (median follow-up: 33 mo.). Two post-As2O3/ATRA relapses achieved CR again with As2O3+ATRA+ascorbic acid, and have remained in remission after maintenance treatment with As2O3+ATRA+ascorbic acid. All patients in continuous remission (n=38) were PML/RARa negative on polymerase chain reaction (sensitivity 10−3 to 10−4).

Conclusion: Our regimen resulted in a leukemia-free-survival of 89.3%. The results suggest that an oral and mainly outpatient As2O3-based, non-HSCT strategy is efficacious for relapsed APL. In terms of survival, costs, treatment side effects and patient tolerance, the results appear to be comparable to, if not more favorable than, other treatment options based on high dose chemotherapy, graft-versus-leukemia effect, or anti-myeloid antibody therapy.
Association Analysis of Interleukin-1 Gene Cluster Polymorphisms with Ischemic Stroke in a Chinese Population.
Authors: Lai Jiangtao , Pan Jiaqi , Hua Baolai , Zhu Yicheng , Cui Liying
Published: October 2019

Chronic inflammatory process acted an important role in atherothrombosis. Interleukin-1 (IL-1) is one of the key modulators of the inflammatory response, and its activity is critically regulated by its receptor antagonist (IL-1ra). Recently, several interleukin-1 cluster gene polymorphisms may associated with acute coronary syndrome have been reported, although with contrasting results. The associations of a variable number tandem repeat (86bp) polymorphism in intron 2 of IL-1ra and of the -511C/T polymorphism of IL-1β with the risk of the ischemic stroke were studied. 112 ischemic stroke patients of Chinese ancestry studied within ten days after presentation were compared with 95 ethnically matched healthy volunteers. 56 among these 112 stroke patients were followed for six months to have prognosis evaluated through measuring Modified Rankin Scale (RS) and Barthel Index (BI). 60 stroke patients had measured the plasma IL-1ra and C-reative protein (CRP) values. The frequency of the IL-1RN 1/1 genotype in the 112 stroke patients was significantly higher than those in healthy volunteers (93.7% vs 82.1%, p<0.05). The frequency of the IL-1RN allele 1 in the 112 stroke patients was significantly higher than those in healthy volunteers (96.4% vs 90.5%, p<0.01, OR=3.23, 95% CI 1.32–7.91). No significant association was seen for the IL-1β-511 C/T polymorphism. In the six months followed group, IL-1RN1/2 genotype carriers had higher BI scores compared with IL-1RN1/1 genotype carriers (98.00 vs 84.80, p<0.01), and patients with lower plasma IL-1ra value (below the median value 186.55pg/ml) had higher BI scores (95.00 vs 77.19, p<0.05), but no significant influence of CRP values and IL-1ra on prognosis or showed no significantly association on plasma IL-1ra and CRP values with IL-1β-511 C/T or IL-1RN genotpye studied in stroke patients. Our results implicate the IL-1ra gene in the susceptibility to ischemic stroke, which may be a useful clue for pharmacological intervention in IL-1 production.
Detection and Clinical Significance of Thrombomodulin in Both Plasma and Tissue Extracts of Cancer Patients.
Authors: Xu Xiaohua , Huang Liansheng , Lu Xingguo , Zhang Xiaohong , Zhao Xiaoying
Published: October 2019

Objective: To study the changes of thrombomodulin(TM) in both plasma and tissue extracts of cancer patients for evaluating its clinical significance.

Methods: Plasma TM levels were measured by enzyme-linked immunosorbent assay(ELISA) in both plasma of 188 cancer patients and 24 cancer tissue extracts including their adjacent non-cancer tissues.

Results: The plasma TM levels both in cancer patients and in metastasis patients were significantly higher than that in controls [(33.47±14.25)ug/L/(41.68±16.96)ug/L, vs (20.40±7.22)ug/L, P<0.01]. The plasma TM levels in cancer patients after operation decreased obviously than that before operation [(18.45±9.96)ug/L, vs (28.29±11.74)ug/L, P<0.01], whereas, the plasma TM levels in patients with recurrence and metastasis after operation increased obviously [(34.50±12.57)ug/L]. Among the type of cancer, the plasma TM levels in metastasis including lung cancers and gastric cancers and pancreatic cancers were significantly higher than that in non-metastasis respectively, but no significant differences were foundPOST http://www.call4abstracts.com/hem/body.pincluding gastric cancers and pancreatic cancers and nasopharyngeal cancers and large intestine cancers and laryngeal cancers (P>0.05). The TM levels in cancer tissue extracts were significantly lower than that in their adjacent non-cancer tissue extracts [(647.71±317.51)ug/L vs (1455.63±772.22)ug/L, P<0.01]. On the contrary, the plasma TM levels in these cancers were significantly higher than that in controls.

Conclusion: The rise of plasma TM levels in cancer patients was associated with metastasis and diffusion of cancers. The TM levels can be served as a sensitive index for judging progression and metastasis of cancers.
In-Vitro Stability of B-Domain Deleted Recombinant Factor VIII in Syringe-Based Continuous Infusion Devices.
Authors: Woloschuk Donna M.M. , Dyck Jennifer M. , Israels Sara J. , Toogood Peter
Published: October 2019

Continuous infusion (CI) factor VIII (FVIII) replacement is commonly used for serious bleeds in patients with FVIII deficiency. Clinicians lack stability information for Refacto®, a B-domain deleted recombinant FVIII (BDDrFVIII), when it is diluted or stored in CI devices for extended time periods. We studied BDDrFVIII to determine its concentration- and time-dependent stability and sterility when stored (reconstituted; reconstituted then diluted with normal saline) at ambient temperature in syringe-based CI devices (CADD-MicroTM; Baxter ColleagueTM). Three replicates of BDDrFVIII 125unit/mL and 25unit/mL admixtures were admixed aseptically then placed in CI devices that operated continuously for 48 hours. Samples (0.5ml) were obtained from control solutions (stored in the manufacturer’s glass vial) or expressed from the distal end of tubing at Hour −0.25 (priming volume), 0, 1, 2, 4, 8, 12, 24 and 48. Samples were stored at −70C until assayed for Factor VIII activity using a 1-stage automated assay and Refacto® [chromogenic equivalent] Standard. Results of FVIII assays for each trio of replicates were averaged. BDDrFVIII admixtures were considered stable if they retained ≥80% baseline (control) FVIII values. Recoveries for BDDrFVIII 125unit/mL admixtures remained stable and sterile for 48 hours when stored in the manufacturer’s glass vial, Baxter ColleagueTM or CADD MicroTM devices, sufficient for clinical use (Table). BDDrFVIII, when diluted to 25unit/mL, remained sterile but not stable when stored in Baxter ColleagueTM or CADD MicroTM devices, and is not recommended for CI in clinical settings. Loss of FVIII activity in BDDrFVIII 25unit/mL admixtures is thought to be due to binding of BDDrFVIII to device components.

%FVIII Activity of BDDrFVIII Stored in Infusion Devices

Hr 0 Hr 1 Hr 2 Hr 4 Hr 12 Hr 24 Hr 48 Mfr Glass Vial (Control) 92.5 93 90.7 90.8 93.2 76 80.7 CADD-Micro 125unit/ml 80 84 86.3 78.3 86.3 80 81.7 Baxter Colleague 125unit/ml 86.3 79 74 85 77.7 81 82.3 CADD-Micro 25unit/ml 65.7 61.3 56.3 59 57.5 59.7 56.3 Baxter Colleague 25unit/ml 18.7 19.3 33 24.3 27 27 27.3
TAFI and Diabetics.
Authors: Kubisz Peter
Published: October 2019

Background: A novel risk factor for thrombotic disorders is thrombin activatable fibrinolysis inhibitor (TAFI) which is also known as plasma procarboxypeptidase B. TAFI is a potent inhibitor of tissue plasminogen activator - induced fibrinolysis, impairing the process of clot lysis and causing a tendency to thrombosis development. The increased circulating plasma levels of TAFI were reported in type 2 diabetes mellitus (DM), obesity, insulin resistance and in hypercholesterolemic subjects. TAFI plasma level was significantly higher in diabetic patients with microalbuminuria than those in diabetic patients with normoalbuminuria and normal subjects. These authors suggest that increased plasma level of TAFI may be involved not only in hypofibrinolysis but also in the mechanism of vascular endothelial damage in patients with type 2 DM.

Aim of study: We evaluated the activity of activated TAFI (TAFIa) and its relation to another hemostatic and metabolic parameters in patients with diabetic nephropathy (DN).

Patients and methods: Eighteen patients (pts) with diabetic nephropathy (DN) − 14 pts type 2 DM / 4 pts type 1 DM, age 54,1±13,3 years (12 men, 6 women), fourteen pts with non-diabetic nephropathy (NDN), age 49,7±15,6 years (7 men, 7 women) and twenty healthy subjects were enrolled in the study. TAFIa (Actichrome Activity TAFI kit, American Diagnostica), soluble thrombomodulin TM (sTM), von Willebrand factor (vWF), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), homocysteine (Hcy), D-dimers plasma levels and metabolic parameters including glycosylated hemoglobin (HbA1c) were measured.

Results: TAFIa plasma levels in DN pts were significantly decreased compared to NDN pts (7,22 ± 3,54 vs. 12,67 ± 6,99 ug/ml, p<0,01, respectively). After analysis the significant TAFIa decrease was only in those DN pts with hypercreatinemia and proteinuria. The sTM levels showed significant increase in DN pts with hypercreatinemia (p<0,0001). There was a significant positive correlation between TAFIa and HbA1c (r=0,50931, p<0,05).

Conclusion: TAFIa plasma levels didn′t differ significantly in patients with diabetic nephropathy compared to those in control healthy group. Decreased TAFIa plasma levels in our DN pts with proteinuria and hypercreatinemia compared to NDN group may be due to the secondary (compensatory) increased fibrinolysis as a consequence of hypercoagulability in pts with DM. TAFIa was in a close relation to the metabolic compensation of DM (HbA1c) that can be of clinical importance for DN pts.
The Murine Genome Contains Two Functional Kininogen Genes Leading to Tissue Specific Expression of High Molecular Weight Kininogen (HK) and Expression of a Novel HK Splice Variant.
Authors: Merkoulov Sergei , Komar Anton A. , McCrae Keith R.
Published: October 2019

High molecular weight kininogen (HK) plays an important role in the assembly and activation of the kallikrein/kinin system. While the human genome contains only a single copy of the kininogen gene, three copies are present in the rat (one K-kininogen and two T-kininogen). Here, we report that the mouse genome contains two homologous kininogen genes (overall homology 91%), denoted mHK1 and mHK2. Both genes are located on chromosome 16 in a head-to-head orientation, and contain open reading frames. The size of intronic sequences between the 11 kininogen gene exons is similar (Figure). HK mRNA transcripts derived from the mHK1 and mHK2 genes differ slightly in size due to gaps of 33 and 18 nucleotides in exon 10 of mHK2. RT-PCR analysis of HK gene expression in adult and embryonic murine tissues revealed that HK mRNA was derived from mHK1 in liver, adrenal and embryo, but from mHK2 in kidney and lung. HK mRNA derived from both genes was present in testis, brain and muscle, though expression levels were low relative to those in other tissues. HK mRNA was not detected in ovary, bone marrow, heart or bladder. mHK1-derived HK mRNA was alternatively spliced, as demonstrated by the presence of an HK mRNA transcript encoding a novel HK1 isoform, ΔmD5, that lacked the portion of exon 10 encoding Thr400 - Asp582 of HK domains 5 and 6. Examination of the putative promoter regions of the two genes using the MatInspector Professional program (Genomatix) demonstrated distinct differences, perhaps explaining in part their tissue-specific expression patterns. Like domain 5 of human HK (hD5), domain 5 of murine HK (mD5), in which the histidine and lysine-rich C-terminal region of this domain previously shown to mediate the antiangiogenic activity of domain 5 is highly conserved, inhibited endothelial cell proliferation. While the function of each of the kininogen genes in the intact animal has yet to be defined, characterization of the two genes may provide new information concerning the role of high molecular weight kininogen in development, normal physiology, and pathological processes.

Figure Figure