The tissue factor region that interacts with factor Xa in the activation of factor VII

Tissue factor is the cell membrane-anchored cofactor for factor VIIa and triggers the coagulation reactions. The initial step is the conversion of factor VII to factor VIIa which, in vitro, is efficiently catalyzed by low concentrations of factor Xa. To identify the tissue factor region that interacts with the activator factor Xa during this process, we evaluated a panel of soluble tissue factor (1-219) mutants for their ability to support factor Xa-mediated activation of factor VII. The tissue factor residues identified as most important for this interaction (Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, and Tyr185) were identical to those found to be important for the interaction of substrate factor X with the tissue factor.factor VIIa complex. The residues form a continuous surface-exposed patch with an area of about 500 A(2), which appears to be located outside the tissue factor-factor VII contact zone. In agreement, the two monoclonal antibodies 5G6 and D3H44-F(ab')(2), whose epitopes overlap with this identified region, inhibited the rates of factor VII activation by 86% and 95%, respectively. These antibodies also strongly inhibited the conversion of (125)I-labeled factor VII when cell membrane-expressed, full-length tissue factor (1-263) was employed. Together the results suggest the usage of a common surface region of tissue factor in its dual role-as a cofactor for factor Xa-mediated factor VII activation and as a cofactor for factor VIIa-mediated factor X activation. The finding that factor Xa and factor X may engage in similar, if not identical, molecular interactions with tissue factor further indicates that factor Xa and factor X are similarly oriented toward their respective interaction partners in the ternary catalytic complexes.     

The restricted expression of granzyme M in human lymphocytes

We have analyzed the expression of human granzyme M (Gzm M) in various human leukocyte subsets using the specific mAb 4H10. Using FACS and Western blotting analysis we compared the expression of Gzm M with that of other granzymes (Gzm A and Gzm B) and the lytic protein perforin. Human Gzm M was constitutively highly expressed in NK cells as was perforin and Gzm A. Surprisingly, freshly isolated NK cells had very low (sometimes undetectable) levels of Gzm B. In contrast to Gzm B and perforin, Gzm M was not detected in highly purified CD4(+) and CD8(+) T cells either constitutively or after short term activation in vitro. However, low levels of Gzm M were observed in some T cell clones on prolonged passage in vitro. Gzm M was not detected in highly purified neutrophils, monocytes, or tumor cells of the myelomonocytic lineage. Examination of minor T cell subsets from human peripheral blood showed detectable Gzm M in CD3(+), CD56(+) T cells and gammadelta T cells. A histological staining procedure was developed that demonstrated a granular staining pattern for Gzm M and a cellular distribution similar to that observed by Western blotting. These data indicate that the expression of Gzm M does not always correlate with the lytic activity of cytotoxic cells. However, expression of Gzm M in NK cells, CD3(+), CD56(+) T cells, and gammadelta T cells suggests that this enzyme may play some role in innate immune responses.     

Sterols in blood of normal and Smith-Lemli-Opitz subjects

Smith-Lemli-Opitz syndrome (SLOS) is a hereditary disorder in which a defective gene encoding 7-dehydrocholesterol reductase causes the accumulation of noncholesterol sterols, such as 7- and 8-dehydrocholesterol. Using rigorous analytical methods in conjunction with a large collection of authentic standards, we unequivocally identified numerous noncholesterol sterols in 6 normal and 17 SLOS blood samples. Plasma or erythrocytes were saponified under oxygen-free conditions, followed by multiple chromatographic separations. Individual sterols were identified and quantitated by high performance liquid chromatography (HPLC), Ag(+)-HPLC, gas chromatography (GC), GC-mass spectrometry, and nuclear magnetic resonance. As a percentage of total sterol content, the major C(27) sterols observed in the SLOS blood samples were cholesterol (12;-98%), 7-dehydrocholesterol (0.4;-44%), 8-dehydrocholesterol (0.5;-22%), and cholesta-5,7,9(11)-trien-3beta-ol (0.02;-5%), whereas the normal blood samples contained <0.03% each of the three noncholesterol sterols. SLOS and normal blood contained similar amounts of lathosterol (0.05;-0.6%) and cholestanol (0.1;-0.4%) and approximately 0.003;-0.1% each of the Delta(8), Delta(8(14)), Delta(5,8(14)), Delta(5,24), Delta(6,8), Delta(6,8(14)), and Delta(7,24) sterols.The results are consistent with the hypothesis that the Delta(8(14)) sterol is an intermediate of cholesterol synthesis and indicate the existence of undescribed aberrant pathways that may explain the formation of the Delta(5,7,9(11)) sterol. 19-Norcholesta-5,7,9-trien-3beta-ol was absent in both SLOS and normal blood, although it was routinely observed as a GC artifact in fractions containing 8-dehydrocholesterol. The overall findings advance the understanding of SLOS and provide a methodological model for studying other metabolic disorders of cholesterol synthesis.     

Dietary n-3 polyunsaturated fat increases the fractional catabolic rate of medium-sized HDL particles in African green monkeys

We have previously described a novel pathway for the metabolism of HDL subfractions in which small [2 apolipoprotein (apoA-I) molecules per particle] HDL particles are converted in a unidirectional manner outside the plasma compartment to medium (3 apoA-I molecules per particle) or large (4 apoA-I molecules per particle) HDL particles, which are subsequently removed from the circulation by the liver (Colvin et al. 1999. J. Lipid Res. 40: 1782;-1792; Huggins et al. 2000. J. Lipid Res. 41: 384;-394). The purpose of the present study was to determine whether the reduction in concentration of medium HDL in African green monkeys consuming n-3 polyunsaturated versus saturated fat diets resulted from decreased in vivo production or increased catabolism. Tracer small LpA-I (HDL containing only apoA-I) were isolated, without ultracentrifugation, by gel filtration and immunoaffinity chromatography and radiolabeled. After injection, the specific activity of apoA-I in small, medium, and large HDL was determined, and the kinetic data were analyzed using our previously published multicompartmental model for HDL subfraction metabolism. We found a significant reduction of apoA-I concentration in medium HDL in the animals fed n-3 polyunsaturated fat (31.2 +/- 0.7 mg/dl) compared with animals fed saturated fat (85.4 +/- 11.9 mg/dl; P = 0.002). The production rates of apoA-I in small, medium, and large HDL were similar in both diet groups; however, there was a significant increase in the fractional catabolic rate of apoA-I in medium HDL in the animals fed n-3 polyunsaturated fat (2.188 +/- 0.501 pools/day) compared with animals fed saturated fat (0.714 +/- 0.191 pools/day; P = 0.02).We conclude that n-3 polyunsaturated fat reduces HDL cholesterol concentration by increasing the fractional catabolic rate of medium-sized HDL particles in African green monkeys.     

The Drosophila S3 multifunctional DNA repair/ribosomal protein protects Fanconi anemia cells against oxidative DNA damaging agents

Cells harvested from Fanconi anemia (FA) patients show an increased hypersensitivity to the multifunctional DNA damaging agent mitomycin C (MMC), which causes cross-links in DNA as well as 7,8-dihydro-8-oxoguanine (8-oxoG) adducts indicative of escalated oxidative DNA damage. We show here that the Drosophila multifunctional S3 cDNA, which encodes an N-glycosylase/apurinic/apyrimidinic (AP) lyase activity was found to correct the FA Group A (FA(A)) and FA Group C (FA(C)) sensitivity to MMC and hydrogen peroxide (H2O2). Furthermore, the Drosophila S3 cDNA was shown to protect AP endonuclease deficient E. coli cells against H(2)O(2) and MMC, and also protect 8-oxoG repair deficient mutM E. coli strains against MMC and H2O2 cell toxicity. Conversely, the human S3 protein failed to complement the AP endonuclease deficient E. coli strain, most likely because it lacks N-glycosylase activity for the repair of oxidatively-damaged DNA bases. Although the human S3 gene is clearly not the genetic alteration in FA cells, our results suggest that oxidative DNA damage is intimately involved in the overall FA phenotype, and the cytotoxic effect of selective DNA damaging agents in FA cells can be overcome by trans-complementation with specific DNA repair cDNAs. Based on these findings, we would predict other oxidative repair proteins, or oxidative scavengers, could serve as protective agents against the oxidative DNA damage that occurs in FA.