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D S Fair

Publications and source records attributed to D S Fair.

15 recordsLinked to original sources
PubMed1992

Molecular recognition sites on factor Xa which participate in the prothrombinase complex.

Coagulation factor X, when activated to factor Xa by proteolytic cleavage, itself becomes an active serine protease which participates as a component of the macromolecular prothrombinase complex along with factor Va, phospholipid, and calcium ions. To identify specific structural regions on factor Xa responsible for mediating its function in activating prothrombin, we used 21 synthetic peptides corresponding to 65% of the primary structure of factor X as potential inhibitors of prothrombin activation. Using purified components, thrombin formation was inhibited by seven peptides in a dose-dependent noncompetitive manner. Antibodies to selected inhibitory peptides affinity purified on a factor Xa-agarose column inhibited thrombin formation in a dose-dependent manner, indicating that the corresponding regions on factor Xa are surface-exposed. Kinetic analyses varying the order of reagent addition suggested that peptides 211-222, 254-269, and 263-274 were highly effective in preventing the factor Xa-factor Va interaction. Peptides 275-287 and 415-425 were considered to derive from a distal region involved in substrate binding, based upon mixed inhibition kinetic analyses and assuming that inhibitory peptides not inhibitory in factor Va binding are related to a specific region of substrate interaction. Cross-linking studies confirmed that peptides 263-274 and 263-276 could bind specifically to the light chain of factor V/Va. These findings provide the basis for further pursuing the precise definition of interactive sites on factor Xa using site-directed mutagenesis and molecular modeling.

Amino Acid Sequence↗
PubMed1991

Structurally homologous ligand binding of integrin Mac-1 and viral glycoprotein C receptors.

Three spatially distant surface loops were found to mediate the interaction of the coagulation protein factor X with the leukocyte integrin Mac-1. This interacting region, which by computational modeling defines a three-dimensional macromotif in the catalytic domain, was also recognized by glycoprotein C (gC), a factor X receptor expressed on herpes simplex virus (HSV)-infected endothelial cells. Peptidyl mimicry of each loop inhibited factor X binding to Mac-1 and gC, blocked monocyte generation of thrombin, and prevented monocyte adhesion to HSV-infected endothelium. These data link the ligand recognition of Mac-1 to established mechanisms of receptor-mediated vascular injury.

Amino Acid Sequence↗
PubMed1991

Molecular defect in coagulation factor XFriuli results from a substitution of serine for proline at position 343.

Our previous findings suggested that coagulation factor XFriuli could be functionally defective owing to a point mutation in the portion of the factor X gene coding for the fully activated heavy chain. To verify the existence of this postulated change, we analyzed all eight exons of the normal and Friuli factor X gene. Each exon was amplified from genomic DNA using the polymerase chain reaction and cloned into the plasmid pUC19. The amplified DNA inserts were subjected to direct sequencing by the dideoxy chain termination method with forward and reverse oligonucleotide sequencing primers. A point mutation (C to T transition at nucleotide position 19,297) that results in coding for serine (TCC) in place of proline (CCC) at amino acid position 343 was found. This substitution involves a highly conserved proline residue oriented spatially close to both the cleavage site of the zymogen and the active site of the enzyme and explains the previous observations of discrete biochemical and functional differences between factor XFriuli and normal factor X. The mutation abolished an HgiCI restriction site present in the normal factor X gene, and this change constitutes the basis for a convenient method for screening individuals carrying this molecular defect. Proline343 is in conserved region 5 of the serine protease superfamily to which factor X belongs and is part of a 14-residue L*****P******C motif that occurs in at least 16 other enzymes. Computer analysis suggests that the motif may be an essential aspect of conformational features important to functional properties of factor X as well as other serine proteases.

Amino Acid Sequence↗
PubMed1991

Identification of molecular sites on factor VII which mediate its assembly and function in the extrinsic pathway activation complex.

Factor VII-VIIa, in association with tissue factor, participates in the complex which initiates blood coagulation through the extrinsic pathway. To identify functional domains on factor VII which mediate the activation of factor X, 16 synthetic peptides corresponding to 55% of the primary structure were assayed for their ability to inhibit factor VII function. Factor Xa formation was inhibited by eight of the peptides in a dose-dependent manner. Kinetic analyses indicated noncompetitive inhibition of factor X activation by seven of these peptides. Peptide-(347-361) inhibited factor Xa cleavage of a chromogenic substrate by a competitive mechanism and was excluded from further analysis in this study. Among the seven inhibitory peptides which have the ability to prevent the factor VIIa-tissue factor-mediated conversion of factor X to factor Xa, peptide-(285-305) was most inhibitory, with a Ki value of 2.4 microM. The Ki values were in the range of 42-65 microM for peptides-(44-50), -(194-214), -(208-229), and -(376-390). The least inhibitory peptides were at positions 170-178 and 330-340, with a Ki value greater than 200 microM. Polyclonal antibodies were raised against four of these peptides; and when antisera were assayed by a solid-phase radioimmunoassay, they bound not only to their respective immunizing peptides, but also to factor VII. The Fab fragments of specific IgG preparations, affinity-purified on a factor VII-agarose column, inhibited the rate of factor X activation in a dose-dependent manner. Six of the seven inhibitory peptides represent amino acid sequences within the heavy chain of factor VII, and the remaining one corresponds to a sequence within the light chain. The corresponding regions in the x-ray crystal structure of chymotrypsin represented by the six heavy chain inhibitory peptides are found to be located in three distinct regions, one region located spatially distal to the active site and the other two regions located relatively closer to the active site and the substrate-binding pocket. The results suggest that at least three specific regions in the heavy chain and one region in the light chain of factor VII mediate its interaction with the factor X activation complex.

Amino Acid Sequence↗
PubMed1991

Serial abnormalities of fibrin turnover in evolving adult respiratory distress syndrome.

We studied the changes of coagulation and fibrinolysis in bronchoalveolar lavage (BAL) and plasma obtained serially at intervals after the onset of adult respiratory distress syndrome (ARDS). BAL procoagulant activity was increased at 3 days and tended to decrease thereafter. Tissue factor associated with factor VII was the major BAL procoagulant. Fibrinopeptide A was increased, indicating increased thrombin-mediated conversion of fibrinogen to fibrin. Fibrinolytic activity was usually undetectable in BAL at 3 days post-ARDS and remained depressed for up to 14 days despite unchanged concentrations of urokinase and variably detectable tissue plasminogen activator. Depressed fibrinolytic activity was associated with increased antiplasmin activity and plasminogen activator inhibitor 1 (PAI-1) while PAI-2 concentrations approximated those of control samples and did not change during evolving ARDS. Evidence of systemic coagulopathy and increased systemic fibrin degradation were commonly found in serial ARDS plasma samples, consistent with accelerated vascular and/or extravascular fibrin deposition in these patients. The data indicate that intra-alveolar as well as systemic derangements of fibrin turnover are common features of evolving ARDS. Concurrent local abnormalities of both coagulation and fibrinolytic pathways favor persistence of alveolar fibrin for up to 14 days after clinical recognition of ARDS.

Adult↗
PubMed1979

Combined functional and immunochemical analysis of normal and abnormal human factor X.

Human Factor X was isolated from Cohn fraction III and characterized by polyacrylamide gel electrophoresis, amino acid composition, and isoelectric focusing. Two molecular forms with biological activity were observed at isoelectric points of 4.8 and 5.0. Antisera generated to Factor X was monospecific and used to establish an equilibrium competitive inhibition radioimmunoassay. This assay was specific for human Factor X and did not cross-react with human prothrombin or bovine Factor X within the sensitivity range of 6-300 ng Factor X antigen/ml. The mean concentration of Factor X based on the antigen was 11.9 mug/ml, whereas concentration values based on coagulant activity was 7.8 mug/ml. This 30% difference in measurement appears to result from the presence of a subpopulation of Factor X molecules devoid of coagulant activity. The radioimmunoassay was used to qualitatively and quantitatively compare purified Factor X to plasmic Factor X obtained from normal, warfarintreated, acquired Factor X-deficient, and congenitaldeficient patients. In all but one case, the Factor X present in these plasmas was immunochemically identical to the purified Factor X and permitted precise quantitation of these abnormal Factor X molecules. Factor X procoagulant activity was analyzed relative to Factor X antigen and the specific activities were used to characterize normal and abnormal Factor X molecules. Reduced Factor X activity in plasmas from warfarin-treated and acquired Factor X-deficient patients was attributed to both decreases in Factor X antigen and decreased function of the Factor X molecules. Congenitally deficient patients, in general, showed a reduction in Factor X antigen in parallel with Factor X procoagulant activities resulting from comparable decreases in specific biological activity of the molecules.

Amino Acids↗
PubMed1977

Circulating tumor cells in murine myeloma.

BALB/c mice bearing subcutaneous ADJ-PC5 myelomas had hematologic findings suggestive of a preleukemic syndrome: thrombocytopenia, anemia, leukocytosis, and megakaryocytic hyperplasia. Six BALB/c myelomas were successfully transplanted sc by fragments or cell suspensions of spleens from mice bearing a subcutaneous tumor, though typical myeloma cells were difficult to visualize by light microscopy in these spleens. The fidelity of transmission of the ADJ-PC5 myeloma by this procedure was shown by the retention of idiotypic specificity of the immunoglobulin produced by the tumor in a radioimmunoassay. The tumorigenic cell that homed to the spleen was apparent as early as 8 days after sc transplantation of the myeloma. The spleens of tumor-bearing mice, however, could destroy or suppress the expansion or growth of a limited number of cells that had migrated to the spleens. Tumorigenic cells present in the peripheral circulation constituted 2-3% of the leukocytes. These cells, however, had reduced levels of the murine myeloma viral and cell-associated antigens, were difficult to detect by an indirect immunofluorescence assay, and did not rapidly divide in this environment, as indicated by the very low number of cells detected by autoradiography.

Animals↗
PubMed1976

Antibody differentiation: apparent sequence identity between variable regions shared by IgA and IgG immunoglobulins.

We have analyzed a pair of human myeloma immunoglobulins (biclonal proteins) of the IgG and IgA classes from a single patient, GR. The light chains are identical in amino-acid sequence over 40 residues at their NH2-terminus, hwereas the heavy chains are identical throughout 45 residues of their NH2-terminus. Additional chemical and serological studies suggest the light chains and variable regions of the heavy chains (VH) are very similar, if not identical. The implications of these and of other published studies are discussed with regard to (i) the association of one VH region with multiple constant regions of the heavy chain (CH regions), (ii) two alternative types of V-C joining mechanisms, (iii) the differentiation of antibody-producing cells, and (iv) three categories of biclonal immunoglobulins.

Amino Acid Sequence↗
PubMed1976

Identification of multiple cytolytic components associated with the beta-LT class of lymphotoxins released by mitogen-activity human lymphocytes in vitro.

Cytotoxic activity (lymphotoxin (LT) associated with the 50,000 dalton beta-LT class of human lymphotoxins has been resolved into multiple components by chromatography on DEAE-cellulose or electrophoresis on polyacrylamide gels. The two components, termed beta-LT, and beta-LT2, are separable from the 90,000 m.w. alpha-LT, since neither component is neutralized by incubation with heterologous rabbit anti-alpha-LT antisera. However, the two components differ in their overall charge and relative heat stability with the betaLT1 component being more positively charged than the beat-LT2 molecule and extremely heat labile.

Cells, Cultured↗
PubMed1976

Development of monoclonal IgA and an apparent IgG in a patient with macroglobulinemia: sharing of individually specific antigenic determinants among IgM, IgA, and IgG.

Patient CM, who initially was diagnosed as having macroglobulinemia (IgM, kappa) was subsequently found to develop a monoclonal IgA(kappa) protein. Rabbit antisera directed against the patient's IgAm and IgM were rendered specific for individual antigenic (ind) determinants. The anti-IgAm and IgM ind sera reacted with both 131I labeled monoclonal proteins in a double antibody radioimmunoassay (RIA). In addition, both monoclonal immunoglobulins inhibited the reaction between labeled immunoglobulin and both ind antisera, and statistical analysis of the data suggested that the shared ind determinants were identical. The IgG fraction of patient CM's serum also contained a component which competed with both monoclonal IgA (CM) and IgM (CM) in the RIA specific for ind determinants. Analysis of serum samples taken over a 2-year period revealed that, in addition to IgM, both the IgA and IgG components possessing the shared ind determinant(s) were present in low concentrations in the earliest sample, although not detected by conventional techniques. The monoclonal IgA and the IgG component were found to increase in concentration over this time interval with a concomitant decrease in IgM. The regulation of immunoglobulin expression with respect to the proposed models of gene organization in antibody-producing cells was discussed.

Antibody-Producing Cells↗
PubMed1975

Studies on IgA and IgA monoclonal proteins derived from a single patient. Evidence for identical light chains and variable regions of the heavy chain.

Two immunoglobulins, IgA(K) and IgG(K), were isolated from the serum of a single patient with two monoclonal components (biclonal proteins). After chain separation, the light chains from each molecule were found to be identical by the following criteria: electrophoretic mobilities under various pH and dissociating conditions, amino acid compositon, fingerprint analysis of tryptic peptides and of 14C-succinylated chymotryptic peptides, and amino acid sequence of the N-terminal 40 residues. The heavy chains were indistinguishable for the N-terminal 45 amino acid residues. These data are consistent with the hypothesis that a single heavy chain variable (VH) region may be associated with two different heavy chain constant (CH) genes.

Amino Acid Sequence↗