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Protein C (PC) is a vitamin K-dependent plasma glycoprotein, which upon activation by thrombin in complex with thrombomodulin (TM), regulates the coagulation cascade through a feedback loop inhibition mechanism. PC deficiency is associated with an increased risk of venous thromboembolism (VTE). A recent cohort study aimed at establishing a normal PC range identified a healthy PC-deficient subject whose PC antigen level of 65% and activity levels of 50% (chromogenic assay) and 36% (clotting assay) were markedly low. The proband has a negative family history of VTE. Genetic analysis revealed the proband has a heterozygous missense mutation in which Thr-315 of the PC heavy chain has been substituted with Ala. We expressed this mutant in HEK-293 cells and purified it to homogeneity. A similar decrease in both anticoagulant and anti-inflammatory activities of the activated protein C mutant was observed in plasma- and cell-based assays. Interestingly, we discovered if functional assays were coupled to PC activation by the thrombin-TM complex, the variant exhibits improved activities in all assays. Sequence analysis revealed Thr-315 is a consensus N-linked glycosylation site for Asn-313 and that its elimination significantly (∼four- to fivefold) improves the maximum velocity of PC activation by the thrombin-TM complex, explaining the basis for the proband's negative VTE pedigree. [ABSTRACT FROM AUTHOR]

High-molecular weight heparins promote the protein Z-dependent protease inhibitor (ZPI) inhibition of factors Xa (FXa) and XIa (FXIa) by a template mechanism. To map the heparin-binding site of ZPI, the role of basic residues of the D-helix (residues Lys-113, Lys-116, and Lys-125) in the interaction with heparin was evaluated by either substituting these residues with Ala (ZPI-3A) or replacing the D-helix with the corresponding loop of the non-heparin-binding serpin a1-proteinase inhibitor (ZPI-D-helixa1-PI). Furthermore, both the C-helix (contains two basic residues, Lys-104 and Arg-105) and the D-helix of ZPI were substituted with the corresponding loops of a1-proteinase inhibitor (ZPI-CD-helixa1-PI). All mutants exhibited near normal reactivity with FXa and FXIa in the absence of cofactors and in the presence of protein Z and membrane cofactors. By contrast, the mutants interacted with heparin with a lower affinity and the ∼48-fold heparin-mediated enhancement in the rate of FXa inhibition by ZPI was reduced to 30-fold for ZPI-3A, 15-fold for ZPI-D-helixa1-PI, and ∼8-fold for ZPI-CD-helixa1-PI. Consistent with a template mechanism for heparin cofactor action, ZPI-CD-helixa1-PI inhibition of a FXa mutant containing a mutation in the heparin-binding site (FXa-R240A) was minimally affected by heparin. A significant decrease (∼2-5-fold) in the heparin template effect was also observed for the inhibition of FXIa by ZPI mutants. Interestingly, ZPI derivatives exhibited a markedly elevated stoichiometry of inhibition with FXIa in the absence of heparin. These results suggest that basic residues of both helices C and D of ZPI interact with heparin to modulate the inhibitory function of the serpin. [ABSTRACT FROM AUTHOR]

Introduction The antiphospholipid syndrome (APS) is an acquired autoimmune disease characterized by recurrent vascular thrombosis and obstetric complications. However, the precise mechanisms by which the autoantibodies mediate disease remain to be elucidated. Moesin is an intracellular protein that links the cell membrane and cytoskeleton, mediating the formation of microtubules and cell adhesion sites as well as ruffling of the cell membrane, which is crucial for platelet activation. Materials and methods We screened the serums from patients with APS for the presence of anti-moesin antibodies (anti-moesin Abs) recognizing antigens derived from prokaryotic expression system, and investigated the effect of murine monoclonal anti-moesin Abs (anti-moesin mAbs) on platelet activation and aggregation by flow cytometry and platelet aggregation assay in vitro to study their potential pathogenic role in APS. Results The presence of anti-amino (N)-terminal portion of moesin antibodies (anti-moesin-N Abs) was observed in 73% (73/100) patients with APS, which was significantly higher than anti-cardiolipin antibodies (aCL, 49%) and anti-β 2 glycoprotein I antibodies (anti-β 2 GPI, 43%). Moreover, the elevated anti-moesin-N Abs levels significantly correlated with plasma levels of anti-β 2 GPI (rs = 0.474, P < 0.001) rather than aCL (P = 0.203). The murine anti-moesin mAbs promote platelet activation and aggregation in vitro, which could be effectively neutralized by moesin-N. Conclusions In combination of the detection of aCL and anti-β 2 GPI, screening for the presence of anti-moesin-N Abs might has its value in facilitating the laboratory diagnosis of APS. The pathogenic role of anti-moesin-N Abs in the serums of APS patients needs to be further studied. [ABSTRACT FROM AUTHOR]

Iron deficiency anemia is an extra-stomach disease experienced in H. pylori carriers. Individuals with type A blood are more prone to suffering from H. pylori infection than other individuals. To clarify the molecular mechanisms underlying H. pylori-associated anemia, we collected erythrocytes from A, B, O, and AB blood donors and analyzed morphology, the number of erythrocytes with H. pylori colonies attached to them, and iron contents in erythrocytes and H. pylori (NCTC11637 and SS1 strains) by means of optical microscopy, scanning electron microscopy, and synchrotron radiation soft X-ray imaging. The number of type A erythrocytes with H. pylori attached to them was significantly higher than that of other erythrocytes (P<0.05). Far more iron distribution was observed in H. pylori bacteria using dual energy analysis near the iron L2, 3 edges by soft X-ray imaging. Iron content was significantly reduced in host erythrocytes after 4 hours of exposure to H. pylori. H. pylori are able to adhere more strongly to type A erythrocytes, and this is related to iron shift from the host to the bacteria. This may explain the reasons for refractory iron deficiency anemia and elevated susceptibility to H. pylori infection in individuals with type A blood. [ABSTRACT FROM AUTHOR]

A severe coagulopathy is a life-threatening complication of acute promyelocytic leukemia (APL) and is ascribable mainly to the excessive levels of tissue factor (TF) in APL cells regulated in response to the promyelocytic leukemia/retinoic acid receptor α (PML/RARα) fusion protein. The underlying molecular mechanisms for this regulation remain ill-defined. With U937-PR9 cell lines stably expressing luciferase reporter gene under the control of different mutants of the TF promoter, both luciferase and ChIP data allowed the localization of the PML/RARα-responsive sequence in a previously undefined region of the TF promoter at position -230 to -242 devoid of known mammalian transcription factor binding sites. Within this sequence a GAGC motif (-235 to -238) was shown to be crucial because deletion or mutation of these nucleotides impaired both PML/RARα interaction and promoter transactivation. However, EMSA results showed that PML/RARα did not bind to DNA probes encompassing the -230 to -242 sequences, precluding a direct DNA association. Mutational experiments further suggest that the activator protein 1 (AP-1) sites of the TF promoter are dispensable for PML/RARα regulation. This study shows that PML/RARα transactivates the TF promoter through an indirect interaction with an element composed of a GAGC motif and the flanking nucleotides, independent of AP-1 binding. [ABSTRACT FROM AUTHOR]

Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FVIII vector under the control of a β-actin promoter with a cytomegalovirus enhancer (CB) and a bovine growth hormone (bGH) poly(A) sequence. The CB promoter plus bGH signal was shown to be 3- to 5-fold more potent than the mini-transthyretin (TTR) promoter with a synthetic poly(A) sequence for directing FVIII expression in the liver. Despite the 5.75-kb genome size of pAAV-CB-FVIII, sufficient AAV vectors were produced for in vivotesting. Approximately 3- to 5-fold more FVIII secretion was observed in animals receiving AAV-CB-FVIII vectors than in those receiving standard-sized AAV-TTR-FVIII vectors. Both the activated partial thromboplastin time assay and the whole blood thromboelastographic analysis confirmed that AAV-FVIII vectors fully corrected the bleeding phenotype of hemophilia mice. These results suggest that AAV vectors with an oversized genome should be useful for not only hemophilia A gene therapy but also other diseases with large cDNA such as muscular dystrophy and cystic fibrosis. [ABSTRACT FROM AUTHOR]

Coagulation factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy chain (HC) and a light chain (LC), which can be expressed independently and reassociate with recovery of biological activity. Because of the size limitation of adeno-associated virus (AAV) vectors, a strategy for delivering the HC and LC separately has been developed. However, the FVIII HC is secreted 10–100-fold less efficiently than the LC. In this study, we demonstrated that the F309S mutation and enhanced B-domain glycosylations alone are not sufficient to improve FVIII HC secretion, which suggested a role of the FVIII LC in regulating HC secretion. To characterize this role of the FVIII LC, we compared FVIII HC secretion with and without the LC via post-translational protein trans-splicing. As demonstrated in vitro, ligation of the LC to the HC significantly increased HC secretion. Such HC secretion increases were also confirmed in vivo by hydrodynamic injection of FVIII intein plasmids into hemophilia A mice. Moreover, similar enhancement of HC secretion can also be observed when the LC is supplied in trans, which is probably due to the spontaneous association of the HC and the LC in the secretion pathway. In sum, enhancing the secretion of the FVIII HC polypeptide may require the proper association of the FVIII LC polypeptide in cis or in trans. These results may be helpful in designing new strategies to improve FVIII gene delivery.Molecular Therapy (2007) 15 10, 1856–1862. doi:10.1038/sj.mt.6300268 [ABSTRACT FROM AUTHOR]