Amino Acids Glu323, Tyr324, Glu330, and Val331 of Factor VA Heavy Chain Are Essential for Expression of Cofactor Activity

We have recently demonstrated that amino acid region 323-331 of factor Va heavy chain (9 amino acids, AP4\u27) contains a binding site for factor Xa (Kalafatis, M., and Beck, D. O. (2002) Biochemistry 41, 12715-12728). To ascertain which amino acids within this region are important for the effector and receptor properties of the cofactor with respect to factor Xa, we have synthesized three overlapping peptides (5 amino acids each) spanning the amino acid region 323-331 and tested them for their effect on prothrombinase complex assembly and function. Peptide containing amino acids 323EYFIA327 alone was found to increase the catalytic efficiency of factor Xa but had no effect on the fluorescent anisotropy of active site-labeled factor Xa (human factor Xa labeled in the active site with Oregon Green 488; [OG488]-EGR-hXa). In contrast, peptide containing the sequence 327AAEEV331 was found to interact with [OG488]-EGR-hXa with half-maximal saturation reached at approximately 150 microm, but it was unable to produce a cofactor effect on factor Xa. Peptide 325FIAAE329 inhibited prothrombinase activity and was able to partially decrease the fluorescent anisotropy of [OG488]-EGR-hXa but could not increase the catalytic efficiency of factor Xa with respect to prothrombin. A control peptide with the sequence FFFIA did not increase the catalytic efficiency of factor Xa, whereas a peptide with the sequence AAEMI was impaired in its capability to interact with [OG488]-EGR-hXa. Two mutant recombinant factor Va molecules (Glu323 --\u3e Phe/Tyr324 --\u3e Phe, factor VaFF; Glu330 --\u3e Met/Val331 --\u3e Ile, factor VaMI) showed impaired cofactor activity when used at limiting cofactor concentration, whereas the quadruple mutant (Glu323 --\u3e Phe/Tyr324 --\u3e Phe and Glu330 --\u3e Met/Val331 --\u3e Ile, factor VaFF/MI) had no cofactor activity under similar experimental conditions. Our data demonstrate that amino acid residues Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are critical for expression of factor Va cofactor activity

The Contribution of Amino Acid Region ASP695-TYR698 of Factor V to Procofactor Activation and Factor VA Function

There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690-699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC(50) values of approximately 12 and approximately 10 microm, respectively. The two peptides were unable to interfere with the binding of factor Va to active site fluorescently labeled Glu-Gly-Arg human factor Xa, and kinetic analyses showed that HC3 and HC4 are competitive inhibitors of prothrombinase with respect to prothrombin with K(i) values of approximately 6.3 and approximately 5.3 microm, respectively. These data suggest that the peptides inhibit prothrombinase because they interfere with the incorporation of prothrombin into prothrombinase. The shared amino acid motif between HC3 and HC4 is composed of Asp(695)-Tyr-Asp-Tyr-Gln(699) (DYDYQ). A pentapeptide with this sequence inhibited both prothrombinase function with an IC(50) of 1.6 microm (with a K(D) for prothrombin of 850 nm), and activation of factor V by thrombin. Peptides HC3, HC4, and DYDYQ were also found to interact with immobilized thrombin. A recombinant factor V molecule with the mutations Asp(695) --\u3e Lys, Tyr(696) --\u3e Phe, Asp(697) --\u3e Lys, and Tyr(698) --\u3e Phe (factor V(2K2F)) was partially resistant to activation by thrombin but could be readily activated by RVV-V activator (factor Va(RVV)(2K2F)) and factor Xa (factor Va(Xa)(2K2F)). Factor Va(RVV)(2K2F) and factor Va(Xa)(2K2F) had impaired cofactor activity within prothrombinase in a system using purified reagents. Our data demonstrate for the first time that amino acid sequence 695-698 of factor Va heavy chain is important for procofactor activation and is required for optimum prothrombinase function. These data provide functional evidence for an essential and productive contribution of factor Va to the activity of prothrombinase

Leprosy perceptions and knowledge in endemic districts in india and indonesia: Differences and commonalities

Background Understanding how knowledge, attitudes and practices regarding leprosy differ in endemic countries can help us develop targeted educational and behavioural change interventions. This study aimed to examine the differences and commonalities in and determinants of knowledge, attitudes, practices and fears regarding leprosy in endemic districts in India and Indonesia. Principle findings A cross-sectional mixed-methods design was used. Persons affected by leprosy, their close contacts, community members and health workers were included. Through interview-administered questionnaires we assessed knowledge, attitudes, practices and fears with the KAP measure, EMIC-CSS and SDS. In addition, semi-structured interviews and focus group discussions were conducted. The quantitative data were analysed using stepwise multivariate regression. Determinants of knowledge and stigma that were examined included age, gender, participant type, education, occupation, knowing someone affected by leprosy and district. The qualitative data were analysed using open, inductive coding and content analysis. We administered questionnaires to 2344 participants (46% from India, 54% from Indone-sia) as an interview. In addition, 110 participants were interviewed in-depth and 60 participants were included in focus group discussions. Knowledge levels were low in both countries: 88% of the participants in India and 90% of the participants in Indonesia had inad-equate knowledge of leprosy. In both countries, cause, mode of transmission, early

Abnormalities in Osteoclastogenesis and Decreased Tumorigenesis in Mice Deficient for Ovarian Cancer G Protein-Coupled Receptor 1

Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown to be a proton sensing receptor in vitro. We have shown that OGR1 functions as a tumor metastasis suppressor gene when it is over-expressed in human prostate cancer cells in vivo. To examine the physiological functions of OGR1, we generated conditional OGR1 deficient mice by homologous recombination. OGR1 deficient mice were viable and upon gross-inspection appeared normal. Consistent with in vitro studies showing that OGR1 is involved in osteoclastogenesis, reduced osteoclasts were detected in OGR1 deficient mice. A pH-dependent osteoclasts survival effect was also observed. However, overall abnormality in the bones of these animals was not observed. In addition, melanoma cell tumorigenesis was significantly inhibited in OGR1 deficient mice. OGR1 deficient mice in the mixed background produced significantly less peritoneal macrophages when stimulated with thioglycolate. These macrophages also showed altered extracellular signal-regulated kinases (ERK) activation and nitric oxide (NO) production in response to lipopolysaccharide. OGR1-dependent pH responses assessed by cAMP production and cell survival in macrophages or brown fat cells were not observed, presumably due to the presence of other proton sensing receptors in these cells. Our results indicate that OGR1's role in osteoclastogenesis is not strong enough to affect overall bone development and its role in tumorigenesis warrants further investigation. The mice generated can be potentially used for several disease models, including cancers or osteoclast-related diseases

Sequencing of Full-Length cDNA Encoding the Alpha and Beta Subunits of Human Casein Kinase II From Human Platelets and Megakaryocytic Cells. Expression of the Casein Kinase IIalpha Intronless Gene in a Megakaryocytic Cell Line

Casein kinase II (CKII) is a ubiquitous protein kinase composed of two subunits, alpha and beta, that can use both ATP and GTP as phosphoryl donors. Two genes located on two separate chromosomes were identified for CKIIalpha: one on chromosome 20 band 13 with an approximate size of 20 kb and a second on chromosome 11 band 15.5-p15.4 that is the same size as the cDNA of locus 20 kb (1.2 kb) and does not contain any introns. The two genes differ in four amino acids. Recently, it has been demonstrated that a membrane-associated platelet-derived CKII phosphorylates coagulation factor Va. The mRNA encoding the platelet CKII was isolated from fresh human platelets, and the corresponding cDNAs encoding the alpha and beta subunits of human platelet CKII were produced and sequenced. The cDNA for platelet CKIIalpha was found to be 99.7% homologous to the CKIIalpha intronless gene, having the same characteristic amino acid residues at positions 128, 256, 287, and 351. However, the cDNA of platelet CKIIalpha has a different amino acid at position 236 (Arg --\u3e His), which is not found in the intronless gene. The cDNA of the CKIIbeta subunit was completely identical with the sequence of the CKIIbeta subunit isolated from other tissues. Since platelets arise from megakaryocytes, mRNA was isolated from the megakaryocytic cell line MEG-01 and the cDNA for CKIIalpha was cloned and sequenced. The cDNA was found to be identical to the intronless gene found in platelets. We have also investigated the expression of the intronless gene in several other cell lines. Expression of the intronless gene was only found in cell line MEG-01. Our data demonstrate expression of the CKIIalpha intronless gene in megakaryocytes and platelets