Genetic variant of endothelial protein C receptor genes and its serum level in B thalassemic children
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Background Due to their chronic hypercoagulable status, thalassemic individuals are at an elevated risk of developing thromboembolic sequence consequences. The goal of the current study is to assesses the EPCR gene polymorphism and soluble EPCR in Egyptian thalassemic children and its role in hypercoagulable state.Research design and methods Eighty children diagnosed as thalassemia major and 80 healthy youngsters as a control group. The EPCR gene was identified using a restriction fragment length polymerase chain reaction (RFLP PCR). Additionally, we assessed the soluble EPCR levels using an enzyme-linked immunosorbent assay (ELISA).Results Frequency of 1651C-G EPCR, the GC genotype was strongly related with an increased risk of coagulation (OR = 1.83 (0.64–5.26), P = 0.0.016). In addition, soluble EPCR was considerably higher in patients with thalassemia than in controls, P value <0.001. Our study revealed significance difference between soluble EPCR and different genotypes.Conclusion Polymorphisms in the EPCR gene and an elevated soluble EPCR level in patients with β-thalassemia major may contribute to these patients’ hemostatic derangement in thalassemic Egyptian children.ABSTRACT A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C . parvum isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C . parvum .
Cryptosporidium parvum
Single-strand conformation polymorphism
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The Endothelial Protein C receptor (EPCR) is essential for the anticoagulant and cytoprotective functions of the Protein C (PC) system. Selected variants of the malaria parasite protein, Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) associated with severe malaria, including cerebral malaria, specifically target EPCR on vascular endothelial cells. Here, we examine the cellular response to PfEMP1 engagement to elucidate its role in malaria pathogenesis. Binding of the CIDRα1.1 domain of PfEMP1 to EPCR obstructed activated PC (APC) binding to EPCR and induced a loss of cellular EPCR functions. CIDRα1.1 severely impaired endothelial PC activation and effectively blocked APC-mediated activation of protease-activated receptor-1 (PAR1) and associated barrier protective effects of APC on endothelial cells. A soluble EPCR variant (E86A-sEPCR) bound CIDRα1.1 with high affinity and did not interfere with (A)PC binding to cellular EPCR. E86A-sEPCR used as a decoy to capture PfEMP1, permitted normal PC activation on endothelial cells, normal barrier protective effects of APC, and greatly reduced cytoadhesion of infected erythrocytes to brain endothelial cells. These data imply important contributions of PfEMP1-induced protein C pathway defects in the pathogenesis of severe malaria. Furthermore, the E86A-sEPCR decoy provides a proof-of-principle strategy for the development of novel adjunct therapies for severe malaria.
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Objective— To confirm the effect of the endothelial protein receptor gene ( PROCR ) haplotypes H1 and H3 on venous thromboembolism (VTE), to study their effect on endothelial protein C receptor (EPCR) expression in human umbilical vein endothelial cells, and to investigate the functionality of H1 tagging single-nucleotide polymorphisms in an in vitro model. Approach and Results— Protein C (PC), activated PC, and soluble EPCR (sEPCR) levels were measured in 702 patients with VTE and 518 healthy individuals. All subjects were genotyped for PROCR H1 and H3. Human umbilical vein endothelial cells isolated from 111 umbilical cords were used to study the relation between PROCR haplotypes, PROCR mRNA, cellular distribution of EPCR, and rate of PC activation. Finally, the functionality of the intragenic PROCR H1 single-nucleotide polymorphisms was analyzed using a luciferase-based method. We confirmed that individuals carrying H1 have reduced VTE risk, increased plasma activated PC levels, and reduced plasma sEPCR levels and that individuals with the H3H3 genotype have an increased VTE risk and increased plasma sEPCR levels. In cultured human umbilical vein endothelial cells, H1 is associated with increased membrane-bound EPCR, increased rate of PC activation, and reduced sEPCR in conditioned medium, but does not significantly influence PROCR mRNA levels. In contrast, H3 is associated with reduced membrane-bound EPCR and increased sEPCR in human umbilical vein endothelial cell–conditioned medium, higher levels of a truncated mRNA isoform, and a lower rate of PC activation. Finally, we identified the g.2132T>C single-nucleotide polymorphism in intron 1 as an intragenic H1-specific functional single-nucleotide polymorphism. Conclusions— These results support a protective role of PROCR H1 against VTE and an increased risk of VTE associated with the H3 haplotype.
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To the Editor, In addition to the anticoagulant property of activated protein C (APC), it has been shown that APC also has profibrinolytic, anti-inflammatory and anti-apoptotic roles. At the endothelial cell surface, endothelial protein C receptor (EPCR) augments APC formation by representing the thrombin-thrombomodulin (TM) complex to protein C. The influence of EPCR on APC generation has been shown by cell culture and animal model studies in vitro. It has been demonstrated that a recombinant human APC (rhAPC) upregulates the synthesis of cyclooxygenase (COX)-2 expression via EPCR binding and protease-activated receptor-1 (PAR-1) signaling mechanism [1]. The results of this study have shown in vitro effects of TNFalpha (TNF-α), EPCR and PAR-1 on APC response. Three different antibodies raised against the inhibition of EPCR binding with APC, an antibody for the detection of normal human EPCR and anti-human PAR-1 antibody were used in human umbilical vein endothelial cells (HUVEC) cell lines. When the stimulated endothelial cells were treated with anti-human EPCR blocker, the expression level of COX-2 decreased, and the same was true for the anti-human PAR-1 antibody that blocks the expression of COX-2 protein. Additionally, TNF-α ??levels also increased the expression level of this protein with a synergistic effect with rhAPC [1]. This study has shown the effect of EPCR-APC binding on protein expression, especially on the very important one, COX-2, that regulates prostaglandin synthesis and TNF-α-APC response relationship. The in vitro effects of TNF-α on EPCR have been reported in human endothelial cells [2]. The effect of TNF-α on TM and EPCR expression has been evaluated by measuring APC formation. It has been suggested that APC and TNF-α induce microparticle-associated EPCR formation in HUVEC and monocytes [3]. However, there is no study that elicits the possible effects of TNF-α on APC formation via EPCR. The three inflammatory cytokines --interleukin-1‚, TNF-α and endotoxin -have been claimed to reduce TM, EPCR and protein S levels [4]. A recent study [5] showed that there was no association between the interleukin-6 (IL-6) and TNF-α gene promotor polymorphisms in a Turkish pediatric stroke group when compared to healthy controls. A rare 23 bp insertion mutation leading to a truncated protein generation has been reported to decrease the membrane expression of EPCR. Most recently, two studies revealed that plasma soluble (s)EPCR levels are genetically controlled by a set of haplotypes, including 6936 A-G (Ser219Gly) or the A3 haplotype and several single nucleotide polymorphisms (SNPs)
Thrombomodulin
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Pathogenesis
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Restriction fragment
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<i>Background:</i> The endothelial cell (EC) protein C receptor (EPCR) negatively regulates coagulation and inflammation. Factors and mechanisms regulating the expression of cell-bound EPCR and the release of soluble (s) EPCR are still unclear. <i>Methods:</i> We investigated the reciprocal regulation of membrane-bound and sEPCR upon inflammation using primary cultures of vascular EC. The impact of 2 parameters, gender and <i>EPCR </i>gene A3 haplotype, on sEPCR plasma basal level and endothelial expression was examined by Elisa and flow cytometry. <i>Results:</i> Exposure of EC to tumor necrosis factor α causes a rapid downregulation of membrane-associated EPCR expression without affecting markedly the spontaneous release of sEPCR by EC. In a cohort of 100 healthy donors, we show that males express significantly higher basal sEPCR in plasma than females (194 ± 12 vs. 145 ± 9 ng/ml, respectively, p < 0.01). Both gender and <i>EPCR</i> A3 haplotype affect sEPCR plasma levels but have no apparent effect on EPCR expression by EC. No quantitative correlation between cellular expression and circulating blood sEPCR was observed, suggesting that endothelial expression may not reflect the plasma level. <i>Conclusion:</i> Male gender is another parameter with A3 haplotype associated with elevated sEPCR levels in blood, and both parameters may contribute to selective regulatory mechanisms of EPCR release upon inflammation.
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Abstract The interaction between the P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of parasitized red blood cells (pRBC) and the endothelial cells (EC) receptors during P. falciparum infection results in the sequestration of pRBC from blood circulation. The amount of sequestration is determined by specific interactions among PfEMP1 and several host adhesion receptors, including intercellular adhesion molecule 1 (ICAM-1), CD36, and endothelial protein C receptor (EPCR). PfEMP1 is composed of multiple domains such as the cysteine-rich inter domain region (CIDR) and Duffy binding –like (DBL) domains. CIDRα1 competitively binds to EPCR with activated protein C (APC) and impair cytoprotective and anticoagulant effects by APC, which plays an important role in severe malaria (SM) pathogenesis such as cerebral malaria (CM) and severe malaria anemia (SMA). The strategy to inhibit EPCR binding to pRBC while to concomitantly strengthen its binding to APC may be crucial in restoring impaired protein C (PC) system’s function. The purpose of this study is to evaluate the association between severity of malaria and the EPCR genotypes as well as the soluble EPCR (sEPCR), and the study also addresses the physiological relevance of EPCR genetic polymorphism. In this study, we conducted meta-analysis on the eligible studies by comparing the frequency of EPCR rs867186-GG versus rs867186- GA and -AA genotype in SM, mild malaria (MM) or uncomplicated malaria (UM) patients and healthy individuals from Thailand, Uganda, Benin, Tanzania, and Ghana. We also determined the relationship between rs867186 genotype and sEPCR levels. Our results showed that the gene type of rs867186-GG is higher in MM/UM than in SM patients. SM patients carrying the rs867186-GG genotype have higher plasma soluble EPCR (sEPCR) levels than in rs867186-AG and rs867186-AA carriers. A significant difference is seen with the higher plasma sEPCR expression among MM/UM patients carrying the rs867186-AG genotype compared to those carrying rs867186-AA. Similarly, the rs867186-GG is associated with sEPCR level in healthy individuals. In conclusion, this meta-analysis demonstrates that pRBCs and EPCR interactions are associated with malaria severity, and treatments that block pRBC binding to EPCR via PfEMP1 CIDRα1 could be a potential therapy for SM.
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