gamma-Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function.

The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/gamma 40E is uncarboxylated at residue 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca (II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 125I-labeled plasma-derived factor IX at concentrations of 2-6 nM. Functionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These results demonstrate that the gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined.

Hymenolepis microstoma: mouse strain differences in resistance to a challenge infection.

Antibody responses and host resistance to the tapeworm, Hymenolepis microstoma, were investigated using AKR/J and C3HeB/FeJ strains of mice. AKR mice were significantly more resistant than controls to a secondary infection following exposure to a 3-, 21-, or 40-day primary infection. During a primary infection, intestinal anti-worm antibody responses measured by an enzyme-linked immunosorbent assay were elevated in the more resistant AKR strain, whereas serum antibody titers did not differ between the two strains. However, during a secondary infection, serum IgA titers were higher in AKR mice than C3H mice. Suppression of the serum IgA anti-worm response by oral administration of lipopolysaccharide also suppressed resistance to a secondary infection. Intraperitoneal immunization with worm antigen resulted in a minor degree of protection in AKR mice. This protection was associated with increased intestinal antibody titers compared to mice not demonstrating protection. These results suggest that the protective responses observed in AKR mice relative to C3H mice reflect differences in mucosal antibody responses to H.

Characterization of Factor V activation intermediates.

Bovine Factor V was partially activated with bovine beta-thrombin and activation intermediates, and end products were isolated either by column chromatography under nondenaturing conditions or electroelution from slab gels following electrophoresis in dodecyl sulfate or both. Electrophoresis of partially activated single-chain Factor V (Mr = 330,000) revealed intermediates designated B (Mr congruent to 205,000) and C (Mr congruent to 150,000), plus end products designated C1 (Mr congruent to 120,000), D (Mr congruent to 94,000), E (Mr congruent to 74,000), F (Mr congruent to 71,000), and G (Mr congruent to 31,000). All components except C1 were visualized readily by staining with Coomassie blue. C1, however, did not stain with this dye but was readily visualized with the Schiff-periodate or silver staining procedures. Chromatography of samples of partially activated Factor V on QAE (quaternary aminoethyl)-cellulose in Ca2+ yielded fractions consisting, respectively, of B plus C and B plus D. Both were biologically active, but the former pair required further exposure to thrombin to yield activity, whereas the latter did not. Either pair, when treated with EDTA, lost activity and could be resolved by further chromatography on QAE-cellulose into isolated components B, C, and D. Activity was recovered upon reconstitution of the pairs in Ca2+, suggesting that B plus C or B plus D comprise two subunit proteins, both subunits of which are required for biological activity. Gel electrophoretic analysis of isolated B and C, after further exposure to thrombin, indicated that B is the precursor of end products C1, E, and G, whereas C is the precursor of D and F. Conventional NH2-terminal sequence analysis and amino acid composition analysis indicated that C and D share the same NH2 terminus with Factor V and that the weighted composition of B plus C is the same as Factor V. Microsequence analysis of C1 and E indicated that C1 has the NH2-terminal sequence of intermediate B, which differs from that of end product E. These results indicate that: 1) a single cleavage of Factor V yields B and C from the COOH and NH2 termini, respectively, of the parent; 2) the pair B and C constitute a Ca2+-stabilized protein of two subunits, both of which are required for subsequent expression of biological activity; 3) the precursor-product relationship between B and C and the end products of activation are: B----C1 + E + G, and C----D + F; and 4) end product D is derived from the NH2 terminus of C, and end product C1 is derived from the NH2 terminus of B.

The factor Xa-catalyzed activation of factor V.

Factor V appears to be a procofactor with, at best, 1/400 the activity of fully activated Factor V (Factor Va). The proteolytic conversion of Factor V to Factor Va is catalyzed by thrombin. However, since Factor Va activity is required for thrombin generation, the initial participation of Factor V in the expression of prothrombinase activity is not well understood. In the present study, the activation of Factor V by Factor Xa has been investigated. Cofactor activation was assessed by monitoring the conversion of prethrombin-1 to thrombin in the presence of 5-dimethylamino-naphthalene-1-sulfonylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA). The DAPA not only provided a fluorescent signal for the formation of thrombin, but also attenuated the feedback activation of Factor V by thrombin. Trace quantities of Factor Va were removed from the Factor V preparations by immunoadsorption with immobilized murine monoclonal antibodies selective for Factor Va. The incubation of Factor V with Factor Xa in the presence of phosphatidylcholine/phosphatidylserine vesicles, CaCl2, and DAPA resulted in a time-dependent increase in cofactor activity. Phosphatidylcholine/phosphatidylserine vesicles were not absolutely required, but the rate of Factor V activation was significantly enhanced by inclusion of the vesicles. The activation was absolutely dependent upon Factor Xa and was eliminated by immunoadsorption of the Factor Xa preparation with a murine anti-Factor X (Xa) monoclonal antibody coupled to agarose. The activation was not affected by immunoadsorption of the Factor Xa and Factor V preparations with burro polyclonal anti-prothrombin IgG. Most of the products of the Factor Xa activation of Factor V differ from the products derived by the thrombin-catalyzed activation of the procofactor. The results demonstrate that Factor Xa catalyzes the activation of Factor V. Furthermore, these studies suggest that the Factor Xa activation of Factor V may be responsible for the advent of early prothrombinase activity.

Monoclonal antibodies to human coagulation factor V and factor Va.

BALB/c mice were immunized with human factor V. The immunogen was a mixture of procofactor (factor V) and thrombin-activated cofactor (factor Va). Spleen cells were obtained from an immunized animal and fused with NS-1 murine myeloma cells. Hybrid cell cultures were assayed for the production of antibodies to human factor V and factor Va by a solid-phase radioimmunoassay. Factor V and/or factor-Va-specific antibodies were detected in 38 of the 96 cultures assayed. The cells from 10 of these positive cultures were subcloned by limiting dilution and grown as ascites tumors in BALB/c mice. Ascitic fluids were obtained and characterized with respect to their binding interaction with human factor V and factor Va. Three hybridoma cell lines produce monoclonal antibodies that react equally well with factor V and factor Va. Another antibody reacts with both antigens, but the reactivity with factor V is better than with factor Va. An additional two antibodies react with factor Va better than factor V in the radioimmunoassay (RIA). The remaining four antibodies react exclusively with factor V. A previously described murine monoclonal antibody to human factor V (alpha HFV-1) has been used to study the peptides produced during the thrombin-catalyzed activation of human factor V. This antibody binds both factor V and factor Va, releases them at high ionic strength, and has an apparent dissociation constant for factor Va of 3 x 10(-9)M. When human factor V (mol wt 330,000) is activated by thrombin and passed over an alpha HFV-1-Sepharose affinity resin, factor Va binds and subsequently can be eluted. The eluate in 1.2 M NaCl contains two fragments of apparent mol wt 93,000 and 70,000. EDTA, which inactivates factor Va, promotes release of the mol wt 93,000 fragment from factor Va bound to the antibody. Subsequent elution with 1.2 M NaCl releases the mol wt 70,000 fragment. These observations indicate that human factor Va is a two subunit protein and that the epitope for alpha HFV-1 is on the mol wt 70,000 fragment.

Monoclonal antibodies selective for activated Factor V. Immunochemical probes for structural transitions occurring during the thrombin-catalyzed activation of the procofactor.

Monoclonal antibodies have been used to probe for structural transitions which potentially occur during the activation of bovine Factor V by thrombin. One of these antibodies (alpha 2D) is reactive with an epitope on the NH2-terminal segment of Factor V (i.e. the Mr = 94,000 component D), while a second antibody (alpha 2E) recognizes an epitope on the carboxyl-terminal segment of Factor V (the Mr = 74,000 component E). Neither antibody reacts with native unactivated Factor V. The alpha 2D-reactive epitope requires at least two proteolytic events for expression, and the alpha 2E-reactive epitope is expressed following the initial cleavage catalyzed by thrombin. In addition, the alpha 2E-reactive epitope is expressed upon the addition of chelators to Factor V. However, the alpha 2D epitope is not influenced by the removal of calcium ions. The interactions of these antibodies with Factor V and Factor V-derived peptides suggest that conformational changes occur in both the NH2-terminal and carboxyl-terminal regions of Factor V concomitant with activation by thrombin, which give rise to the antibody recognition sites.

A monoclonal antibody which inhibits the factor Va:factor Xa interaction.

An immunoprecipitation technique has been used to determine the subunit specificity of two of the monoclonal antibodies to bovine Factor V(Va) developed by this laboratory. One of the antibodies is specific for the 74,000-dalton subunit (the E chain) of Factor Va, and the other antibody is specific for the 94,000-dalton subunit (the D chain). The binding of Factor Va to phospholipid was studied by light scattering, and the interaction of Factor Xa with phospholipid-bound Factor Va was examined using 5-dimethylaminonaphthalene-1-sulfonyl-glutamyl-glycyl-arginyl-Xa (Dns-EGR-Xa). Neither the antibody specific for the E chain nor the antibody specific for the D chain inhibit the binding of Factor Va to phospholipid vesicles. The antibody specific for the E chain blocks the increase in fluorescence polarization seen when Factor Va is added to a solution of Dns-EGR-Xa, phospholipid vesicles and calcium. This antibody also inhibits the association of Dns-EGR-Xa with phospholipid-bound Factor Va as determined by gel-exclusion high pressure liquid chromatography. The antibody specific for the D chain of Factor Va does not block the increase in polarization seen when Factor Va is added to a solution of Dns-EGR-Xa, phospholipid, and calcium. It was concluded that the antibody specific for the E chain of Factor Va binds at or near the Factor Xa-binding site on the E chain and that the Factor Va E chain plays a significant role in binding Factor Xa.

Monoclonal antibodies selective for the functional states of bovine factor V and factor Va.

Hybridoma technology has been used for the production of murine monoclonal antibodies to bovine coagulation Factor V and its thrombin-activated product, Factor Va. Hybrid cell cultures were assayed for the production of anti-Factor V and anti-Factor Va antibodies by a solid-phase radioimmunoassay. Antibody-producing cell lines were selected, cloned and grown as ascites tumors. Gel filtration chromatography (Ultrogel AcA34) and affinity chromatography (protein A-Sepharose) were used to isolate the monoclonal immunoglobulins from the ascites fluids. Thirteen monoclonal antibodies have been characterized with respect to their binding to Factor V and Factor Va and their effect on cofactor bioactivity. Six of these thirteen antibodies react with both Factor V and Factor Va. One of these antibodies is strongly inhibitory, while a second antibody is only moderately inhibitory. The antibody produced by another cell line binds Factor V but not Factor Va and is not inhibitory. The remaining six cell lines each produce an antibody that reacts preferentially with Factor Va, and each of these antibodies is inhibitory to some extent. Both a radioimmunoassay and light scattering have been used to study the interaction of the immunoglobulins with Factor V and Factor Va. The light scattering technique has proven useful to study the interaction of isolated antibodies and antigens and permits the determination of interaction stoichiometries. Each of the interactions studied was characterized by a stoichiometry of two antigens per antibody. These monospecific immunochemical reagents will be useful in the study of structure and function relationships of Factor V, Factor Va and activation fragments.

Calcium oxalate deposition in the periodontium secondary to chronic renal failure.

Deposition of calcium oxalate in the periodontium with accompanying bone loss and tooth resorption, occurred in a 55-year-old woman on long-term renal dialysis for end-stage renal disease. These deposits were birefringent upon examination with polarized light. Scanning electron microscopy of the deposits revealed clusters of sharply angular oblong and needlelike crystals. Microincineration followed by staining with alizarin red S was positive for calcium oxalate. X-ray diffraction studies of the crystals in the tissue specimen revealed a pattern identical to that of commercially prepared calcium oxalate monohydrate. It is postulated that pre-existing inflammatory periodontal disease may have provided the appropriate milieu for the deposition and precipitation of crystals. These deposits, acting as a foreign body, further intensified the inflammatory response, resulting in extensive alveolar bone loss and external tooth resorption.

Affinity labeling of the active site of Escherichia coli glutamine synthetase by 5'-p-fluorosulfonylbenzoyladenosine.

The interaction of Escherichia coli glutamine synthetase with the adenosine 5'-triphosphate analogue, 5'-p-fluorosulfonylbenzoyladenosine (5'-FSO2BzAdo), has been studied. This interaction results in the covalent attachment of the 5'-FSO2BzAdo to the enzyme with concomitant loss of catalytic activity. Although adenine nucleotides interact with glutamine synthetase at three distinct sites--a noncovalent AMP effector site, a regulatory site of covalent adenylylation, and the catalytic ATP/ADP binding site--our studies suggest that reaction with 5'-FSO2BzAdo occurs only at the active center. When glutamine synthetase was incubated with 5'-FSO2BzAdo, the decrease in catalytic activity obeyed pseudo-first order kinetics. The plot of the observed rate constant of inactivation versus the concentration of 5'-FSO2BzAdo was hyperbolic, consistent with reversible binding of the analogue to the enzyme prior to covalent attachment. Protection against inactivation was afforded by ATP and ADP; L-glutamate did not protect the enzyme against inactivation, but rather enhanced the rate of inactivation, consistent with the observations of others (Timmons, R. B., Rhee, S. G., Luterman, D. L., and Chock, P. B. (1974) Biochemistry 13, 4479-4485) that there is synergism in the binding of the two substrates to the enzyme. The incorporation of approximately 1.09 mol of the 5'-FSO2BzAdo/mol of glutamine synthetase subunit resulted in the total loss of enzymatic activity. The results suggest that 5'-FSO2BzAdo occupies the ATP binding site at the active center of glutamine synthetase and binds covalently to an amino acid residue nearby.