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D S Fair

Publications and source records attributed to D S Fair.

At least 37 records · Page 2Linked to original sources
PubMed1988

Monoclonal antibody analysis of purified and cell-associated tissue factor.

Tissue factor (TF), one of the cell-surface initiators of blood coagulation, has been implicated as the major molecule of this type and as a critical controlling molecule in hemostasis, thrombosis and inflammation. Analysis of the expression of human TF by cells has been hampered by the lack of suitable molecular probes. We have prepared a library of twenty-four murine hybridomas which stably secrete monoclonal antibodies to human TF. Based on their characteristics, these monoclonals can be categorized into a minimum of five distinct groups. Twenty-three of the hybridoma antibodies strongly inhibited TF activity, which was attributable to blocking of formation of the bimolecular complex of TF and factor VII. We have used these antibodies to demonstrate directly that TF is the sole high affinity factor VII receptor on an intact cell. We have also demonstrated the immunologic relationship between constitutive and induced expression of the protein responsible for TF-like activity by several cells and tissues. Most of the antibodies were found to inhibit TF activity expressed by other primate species, and the potential in vivo therapeutic use of monoclonal antibodies of differing intramolecular specificity is discussed.

Animals↗
PubMed1988

Resolution of monomeric and heterodimeric forms of tissue factor, the high-affinity cellular receptor for factor VII.

Tissue factor (TF) is the high affinity cell surface receptor and obligatory cofactor for plasma coagulation factor VII. Purification of TF from human brain and placenta has recently been reported to yield both a monomeric 47 kDa protein as well as a 58 kDa heterodimeric form. In this study, the TF glycoprotein was isolated from human brain by immunoaffinity chromatography using a newly developed monoclonal antibody, TF8-5G9, and was compared to TF isolated by factor VII-affinity chromatography. Except for the greater efficiency of the immunoaffinity method, both methods yielded TF with similar specific activities, and both preparations contained the monomeric and heterodimeric forms of TF. The 58 kDa form was established to be a disulfide-linked heterodimer composed of TF and the alpha chain of hemoglobin. From these results and from studies of immunoprecipitation of TF from cultured fibroblast cells, we conclude that the 47 kDa monomeric form of TF is the naturally occurring cellular form of TF, and that heterodimer formation is a secondary event. The potential significance of the proclivity of TF to form a heterodimer with hemoglobin is discussed.

Antibodies, Monoclonal↗
PubMed1988

Regulation of the procoagulant activity within the bronchoalveolar compartment of normal human lung.

The nature of the procoagulant activity of normal bronchoalveolar fluid was examined both qualitatively and quantitatively. Unconcentrated, cell-free lavage freshly obtained from normal volunteers clotted normal plasma in a mean of 84 +/- 20 s. The procoagulant activity was initiated by Factor VII-tissue factor complexes as judged by differential activity in various plasmas genetically deficient in single clotting factors, by neutralization of the procoagulant activity with antibodies to either Factor VII or tissue factor, and by a Factor X activation assay. Preincubation of the lavage with calcium was required to demonstrate Factor VII activity in unconcentrated samples. The cell-free fluid contained about 8,500 thromboplastin units/mg protein, equivalent to a third of the thromboplastin standard and indicating high amounts of cofactor. Quantitation of Factor VII was estimated by functional analysis in coagulation and amidolytic assays with reference to dilutions of normal plasma of known Factor VII concentration. When lavage and diluted plasma were adjusted to yield equivalent amidolytic activities, the average ratio of the Factor VII-clotting activity of the alveolar fluid relative to plasma Factor VII was 19 +/- 7, suggesting the presence of Factor VIIa in lavage. In contrast to previous reports with serum or activated plasma, immunoblots of concentrated lavage revealed only single-chain Factor VII, and 125I-Factor VII added to the fluid was not converted to 125I-Factor VIIa, suggesting a unique control mechanism in the lung compartment which differs from plasma. When equivalent Factor VII amidolytic activities in diluted plasma and cell-free lavage were compared, the rates of Factor Xa formation were very similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation Factors↗
PubMed1988

Local abnormalities of coagulation and fibrinolysis and alveolar fibrin deposition in sheep with oleic acid-induced lung injury.

Extravascular, primarily intra-alveolar, fibrin deposition is a histologic hallmark of acute lung injury in humans and experimental animals, but the mechanisms leading to this finding are poorly understood. To determine whether local abnormalities in the fibrinolytic-procoagulant balance contribute to alveolar fibrin deposition in acute lung injury, we studied bronchoalveolar lavage (BAL) fluids of anesthetized sheep that received intravenous oleic acid. Prominent alveolar fibrin deposition was observed within 2 h after oleic acid-induced lung injury. Procoagulant and fibrinolytic activities were determined in BAL samples of anesthetized, mechanically ventilated sheep before and 2 h after intravenous oleic acid or saline. BAL procoagulant activity was found to be due mainly to tissue factor associated with Factor VII. In baseline BAL samples, we found relatively low levels of procoagulant activity and relatively high levels of fibrinolytic activity. After induction of oleic acid-induced lung injury, the procoagulant activity of BAL was markedly increased, whereas fibrinolytic activity was either depressed or undetectable. Antiplasmin activity was detectable in BAL of sheep after oleic acid-induced lung injury, which contributed at least in part to the depressed fibrinolytic activity observed. These perturbations occurred with the appearance of extensive alveolar fibrin deposition. In control sheep, BAL fibrinolytic activity was decreased, and antiplasmin activity increased modestly after 2 h of mechanical ventilation, but procoagulant activity was unchanged and alveolar fibrin was not observed. Procoagulant activity in lung lymph and plasma after lung injury did not differ from baseline values, and fibrinolytic activity was undetectable in lymph or plasma samples. These data indicate that increased procoagulant activity and concurrent disruption of the balance of coagulation and fibrinolysis establish local conditions that promote acute fibrin deposition in the alveoli of mechanically ventilated, oleic acid-injured sheep.

Animals↗
PubMed1987

Cooperative interaction between factor VII and cell surface-expressed tissue factor.

Assembly of the extrinsic pathway on cell surfaces was investigated by studying the binding and activity of factor VII on the bladder carcinoma cell line J82 which expressed 18,800 milliunits of tissue factor activity/10(6) cells. In binding studies, the association of factor VII to monolayers of cells was time-, temperature-, and calcium-dependent. The ligand binding was specific, reversible, and saturable. This interaction was inhibited by a monoclonal antibody to human brain tissue factor. Factor VII added to the cells was recovered as factor VII rather than factor VIIa when incubated in the presence of factor X neutralizing antibodies, suggesting that these cells produced factor X. Specific factor VII binding to the cell revealed a sigmoidal binding isotherm with half-maximal binding occurring at 314 +/- 145 pM to 38,300 +/- 14,300 sites/cell. Hill plots of the binding data indicated an average slope of 2.1. Binding parameters were also determined kinetically. At maximal factor VII-tissue factor complex formation the apparent Km for factor X was 274 nM, the Vmax was 4.15 nM/min, and the kcat was estimated to be 14 s-1. In the presence of excess tissue factor and factor X, increasing amounts of factor VII added to the J82 cells demonstrated a sigmoidal relationship with the rate of factor Xa formation. Hill plots indicated a slope of 2.0 at the lower factor VII concentrations which changed to 1.0 at the higher input amounts of factor VII. Hanes plots were used to determine the apparent dissociation constant of the interaction (222 +/- 85 pM). The Vmax was 5.54 +/- 1.04 nM/min for the cleavage of factor X. These data are consistent with factor VII binding to at least two sites on tissue factor (receptor) with positive cooperativity. Because at saturation the stoichiometry of the factor VII-tissue factor complex is 1:1, tissue factor must be expressed as a dimer on the surface of the J82 cells.

Antibodies, Monoclonal↗
PubMed1987

Initiation of the extrinsic pathway of coagulation. Association of factor VIIa with a cell line expressing tissue factor.

We have examined initial assembly of the extrinsic pathway of blood coagulation on cell surfaces with radiolabeled human factor VIIa and a human fetal lung cell line possessing abundant functional tissue factor activity. Binding of factor VIIa to these cells was observed and was time- and temperature-dependent. Binding of factor VIIa was quantitatively equivalent at 37 and 6 degrees C, although the kinetics of binding differed. The radiolabeled ligand bound by the cell was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel analysis from the factor VIIa offered. Factor VIIa binding was influenced by calcium ions. The binding appears to involve at least two classes of calcium-dependent binding sites. Optimal binding occurred at 2 mM calcium for both classes of sites, and there was inhibition of binding to the high affinity sites at higher calcium. Association of factor VIIa was specific, saturable, had a Kd of 123 +/- 37 pm, and factor VIIa interacted with about 100,000 binding sites per cell. Once established, specific binding was rapidly reversible. Direct cellular binding of human factor X also was observed and was calcium, time- and temperature-dependent. Factor X binding was specific and saturable with half-maximal binding at 87.6 +/- 27.4 nM to 6.03 +/- 1.03 X 10(6) sites per cell. Specific high affinity binding of factor VIIa correlated with generation of factor Xa. A direct linear relationship was observed at low factor VIIa binding; however, at higher bound factor VIIa, the relationship was nonstoichiometric, i.e. less factor Xa was formed per mole of factor VIIa. Expression of specific binding sites for factors VIIa and X provides further substantiation for the molecular assembly hypothesized to initiate the extrinsic coagulation protease cascade on cells.

Binding, Competitive↗
PubMed1987

Molecular interactions of the intrinsic activation complex of coagulation: binding of native and activated human factors IX and X to defined phospholipid vesicles.

The assembly of proteins of the intrinsic activation complex has been partially elucidated. In the present study we examine the association of gamma-carboxylated serine proteinase zymogens factors IX and X, and their proteolytically activated counterparts factors IXa and Xa to unilamellar lipid vesicles of defined composition using three types of physical measurement. Utilizing relative light scatter to estimate the dissociation constants for binding in the presence of calcium ions, it appears that factor IXa (0.93 +/- 0.37 microM) may preferentially associate with phospholipids relative to factor IX (0.35 +/- 0.08 microM). In contrast, factor X (0.34 +/- 0.14 microM), the substrate for factor IXa, appears to bind to phospholipid with a higher affinity than factor Xa (0.58 +/- 0.13 microM). These observations are compatible with the hypothesized dynamics where the forward 'traffic' is facilitated by favoring the association of factor IXa with factor X. The dissociation constants were estimated by molecular exclusion chromatography (1.1 - 2.5 microM) and do not reflect these relative and ordered differences in association with lipid vesicles. Quasi-elastic light scatter analyses indicate that each protein appears to saturate the same vesicle surface, consistent with competition for similar surface lipids, although the molecular shell formed by factor Xa (36 A) is smaller, suggesting that it has a different packing on the phospholipid surface than the other proteins (64-79 A). The pattern of preferential affinities for phospholipid is consistent with a kinetically functional forward traffic through the reaction precursors to products, and suggests that these preferential affinities may assist in the ordering of the four proteins in the intrinsic activation complex.

Calcium↗
PubMed1987

Steady state levels of factor X mRNA in liver and Hep G2 cells.

In order to examine the control of human factor X biosynthesis we have molecularly cloned the cDNA and investigated the expression of the Factor X gene. A recombinant clone of approximately 1100 base pairs in length containing the sequence of factor X was identified in a lambda gt11 human liver cDNA library by screening with polyclonal antibodies. One plaque was selected and confirmed for specificity with a mixture of five factor X specific monoclonal antibodies (MoAbs). A partial nucleic acid sequence of the 5' end of the cDNA corresponded to the described amino acid sequence between residues 41 and 56 of the light chain of factor X. Northern blot analysis of RNA from human liver and the hepatoma cell line, Hep G2, identified the factor X mRNA as a single molecular species of approximately 1700 bases. Cell lines which do not secrete factor X did not contain factor X mRNA indicating restriction of transcription to hepatocytes. Slot-blot hybridization analysis of factor X and actin mRNA demonstrated no change in the levels of total or specific factor X mRNA in Hep G2 cells following treatment with warfarin or vitamin K. We conclude that modulation of factor X production by these drugs, known to influence gamma-carboxylation and total factor X secretion by these cells, is mediated by changes in posttranscriptional events rather than by effects on the steady state levels of factor X mRNA.

Actins↗
PubMed1986

Specificity of the thrombin-induced release of tissue plasminogen activator from cultured human endothelial cells.

The addition of thrombin (9 nM) to primary cultures of human endothelial cells induces a 6- to 7-fold increase in the rate of release of tissue plasminogen activator (tPA). Several other serine proteases which specifically interact with endothelial cells were also analyzed for their effect on tPA release. Gamma-thrombin, an autocatalytic product of alpha-thrombin, promoted tPA release but was less effective than alpha-thrombin. A maximum increase of 5.5-fold was observed, although a concentration of gamma-thrombin 20 times greater than alpha-thrombin was required. The response to Factor Xa was similar to alpha-thrombin, although the stimulation was significantly reduced by the addition of hirudin or DAPA suggesting that prothrombin activation was occurring. The simultaneous addition of prothrombin with Factor Xa resulted in enhanced tPA release equal to that observed with an equimolar concentration of active alpha-thrombin. Thus, under these conditions, Factor Xa-cell surface mediated activation of prothrombin can lead to a secondary effect resulting from cell-thrombin interaction. Activated protein C, which has been implicated as a profibrinolytic agent, was also tested. No change in tPA release occurred after the addition of up to 325 nM activated protein C in the presence or absence of proteins. Factor IXa and plasmin were also ineffective. The effect of thrombin on the endothelial cell derived plasminogen activator specific inhibitor was also studied. Thrombin produced a small but variable release of the inhibitor with an increase of less than twice that of non-thrombin treated controls.

Cells, Cultured↗
PubMed1986

Specific association of thrombin-antithrombin complexes with a human hepatoma cell line.

The clearance of thrombin-antithrombin (TAT) complexes from blood by the liver is through a receptor mediated pathway. We have used the established human hepatoma cell line, Hep G2, to determine if these hepatocytes have the capacity to bind this enzyme-inhibitor complex. The TAT complex was bound to the cells in a time and temperature dependent manner, reaching an apparent steady state at 90 minutes at both 4 and 37 degrees C. Binding at 4 degrees C was 5-7-fold less extensive than at 37 degrees C. The bound TAT was structurally similar to the added ligand. This interaction was specific, as it was inhibited by nonlabeled TAT but not by 50-fold molar excesses of unrelated proteins or by the individual constituents, thrombin or antithrombin. Factor Xa- antithrombin complex inhibited the binding reactions slightly. Specific binding isotherms at 37 degrees C were subjected to Scatchard plots. The apparent dissociation constant was 247 +/- 74 nM, and the number of TAT molecules bound per cell was 5.19 +/- 0.89 X 10(5). Bound TAT complexes did not undergo degradation at 4 or 37 degrees C for up to 2.5 hr, as greater than 85% of the bound ligand was acid precipitable during the time course of binding. Internalization of the TAT complex was compared with transferrin, a molecule known to be internalized by Hep G2 cells, by resistance of the cell-bound ligands to degradation by trypsin or pronase. In contrast to transferrin, most of the TAT complexes remained cell-surface associated for at least 2 hr at both 4 degrees and 37 degrees C, indicating that TAT was not substantially internalized by the Hep G2 cells.

Antithrombins↗
PubMed1986

Immunologic relationship between platelet membrane glycoprotein GPIIb/IIIa and cell surface molecules expressed by a variety of cells.

A polyclonal antiserum to platelet membrane glycoprotein GPIIb/IIIa was used to detect antigenically related molecules on a diverse panel of human cells. Umbilical vein endothelial cells, erythroleukemic HEL cells, and diploid fetal lung GM1380 fibroblasts expressed GPIIb/IIIa-related molecules, as judged by immunofluorescence and immunoprecipitation of surface-labeled proteins. The GPIIb and GPIIIa subunits were both present and were of similar molecular weight in these cell types. These molecules were synthetic products of the cells, as shown by immunoprecipitation of intrinsically labeled proteins. Promyeloid U937 cells could be induced by 4 beta-phorbol 12-myristate 13-acetate to synthesize and express GPIIb/IIIa-related molecules on their cell surface. The GPIIb/IIIa-related molecules were not precisely identical in the various cell types, based on slight differences in electrophoretic mobility and their failure to react with monoclonal antibodies specific for each subunit of platelet GPIIb/IIIa. These results suggest the existence of a widely distributed family of GPIIb/IIIa-related molecules. This family of "cytoadhesins" may share a common function in cellular adhesive reactions.

Antibodies, Monoclonal↗
PubMed1986

Characterization and comparison of immune-depleted and hereditary factor-VII-deficient plasmas as substrate plasmas for factor VII assays.

The functional activity of coagulation factors usually is determined using bioassays dependent on substrate plasmas deficient in the factor being measured. These are obtained most often from patients with hereditary deficiency and are frequently poorly characterized. The authors characterized seven commercially available hereditary Factor-VII-deficient substrate plasmas in terms of Factor VII activity and antigen level, and then compared them with a Factor-VII-deficient plasma prepared by immunoadsorption. All of the hereditary Factor-VII-deficient plasmas contained significant quantities of Factor VII antigen (120-217 ng/mL; normal, 250-600 ng/mL). One of the commercial substrate plasmas showed significant factor VII coagulant activity with a human thromboplastin. The sensitivity of the Factor VII assay system was dependent on both the source of Factor-VII-deficient plasma and the origin of the thromboplastins; human thromboplastin was more sensitive than rabbit thromboplastin, and immune-depleted plasma gave the most sensitive assay with each thromboplastin. These results indicate that immunoadsorbed plasma may be a superior reagent for coagulation factor assays.

Blood Coagulation Tests↗
PubMed1986

Human endothelial cells synthesize protein S.

Human umbilical vein endothelial cells were analyzed for the presence of prothrombin, factor VII, protein C, and protein S in culture supernatants and cell extracts using specific radioimmunoassays. Only protein S was detected. Conditioned medium from 24-hour cultures and cell lysates contained 21.7 ng/mL and 88.8 ng/10(7) cells of protein S, respectively. Intrinsic labeling and immunoprecipitation indicated that protein S was synthesized and secreted as a 75,000 molecular weight protein. Vitamin K, phorbol myristate acetate, and thrombin increased the production or specific activity (determined from activity/antigen ratios of 0.99 to 1.07, 0.93 to 1.04, and 0.90 to 1.04, respectively) of protein S. While untreated cells secreted a partially active protein S (activity/antigen = 0.40), warfarin greatly decreased the specific activity (less than 0.10) of this molecule, suggesting that endothelial cells contain the enzymes required for the carboxylation of selected glutamic acid residues. The production of protein S by these cells supports the hypothesis that cofactor production and expression by the endothelial cells may play a significant regulatory role in the initiation, propagation, and suppression of hemostasis and thrombosis.

Cells, Cultured↗
PubMed1986

Biosynthesis and secretion of factor VII, protein C, protein S, and the Protein C inhibitor from a human hepatoma cell line.

Using specific radioimmunoassays, 8 day cultures of Hep G2 cells were shown to contain in their supernatants 16, 74, and 828 ng/mL and in their cell lysates, 8, 55, and 48 ng/2 X 10(8) cells of factor VII, protein C, and protein S, respectively. These proteins and the protein C inhibitor were functionally active, and each of these activities was neutralized by their respective polyclonal antibodies. Although vitamin K had a modest effect, warfarin decreased the activity of secreted factor VII, protein C, and protein S by 50% to 90%. Protein C and protein S antigens were reduced three- to fourfold by warfarin. The protein C inhibitor antigen and activity were unaffected by vitamin K or warfarin treatment. Intrinsic labeling and immunoprecipitation indicated that factor VII, protein S, and the protein C inhibitor were secreted as 52,000, 77,000, and 58,000 molecular weight (mol wt) proteins, respectively. Protein C was secreted as a single-chain protein of about 65,000 mol wt, indicating that all of the vitamin K-dependent proteins are translated and secreted as single-chain molecules. Each of the four proteins studied represented their plasma protein counterparts structurally, functionally, and immunochemically. Thus, all of the known soluble components of the protein C pathway are produced by liver parenchymal cells.

Animals↗
PubMed1985

Factor B, the complement alternative pathway serine proteinase, is a major constitutive protein synthesized and secreted by resident and elicited mouse macrophages.

Factor B, the complement alternative pathway serine proteinase, a class III gene product of the major histocompatibility complex, is a major constitutive secretion product of mouse mononuclear phagocytes. This glycoprotein was synthesized and secreted by macrophages as a doublet of Mr 90,000 and 93,000 polypeptides that were immunoprecipitable with antibodies raised to human serum factor B, and that were indistinguishable from plasma factor B by immunoreactivity, peptide mapping, and molecular weight. Macrophage factor B was cleaved and activated to factor Bb- and Ba-like fragments by factor D and cobra venom factor. Some conversion of macrophage factor B to Bb-sized fragments occurred spontaneously in the conditioned culture medium after several hours. Factor B represented approximately 0.5% of newly synthesized protein and 4-6% of the secreted protein of resident peritoneal macrophages and macrophages elicited with thioglycollate broth, pyran copolymer, NaIO4, bacillus Calmette-Guerin, or Corynebacterium parvum. We detected synthesis of factor B immediately upon explanting these macrophages in culture; synthesis continued for several days in culture. The rate of secretion of factor B, as a proportion of total protein secretion in culture, remained constant with time. By radioimmunoassay, factor B antigens accumulated in the 24-h macrophage-conditioned culture medium at 2-10 nM, and was present in cell lysates at 4-8 nmol per 10(6) cells. We detected synthesis of factor B in bone marrow-derived macrophages as early as 5 d of culture. The P388D1 macrophage line synthesized factor B, but mouse L cells did not. In contrast, apolipoprotein E, another secreted protein of macrophages, was secreted by resident and thioglycollate-elicited macrophages but not by freshly harvested pyran copolymer-activated macrophages. Its synthesis was initiated at day 9 in culture of bone marrow-derived macrophages. These data support the classification of factor B as a constitutive biosynthetic and secreted protein of immature and mature macrophages in various states of activation. Production of factor B was modulated by treatment of macrophages in vivo or in culture with bacterial lipopolysaccharide endotoxin, which increased the synthesis, secretion, and accumulation of factor B up to 11-fold.

Animals↗
PubMed1985

Leukocyte complement: interleukin-like properties of factor Bb.

It has been previously shown that the activated form of Factor B (Factor Bb) of the alternative pathway of complement activation stimulates monocyte spreading and killing of xenogenic erythrocytes and staphylococci. Factor Bb also stimulates lymphocyte blastogenesis in vitro, and native (uncleaved) Factor B is a major constitutive product of murine macrophages. To evaluate the possible "monokine" or "lymphokine"-like properties of Factor Bb, a radioimmunoassay was developed to measure the quantities of Factor B in phytohemagglutinin (PHA)-mitogen-stimulated cultures of human peripheral blood mononuclear cells. Nonstimulated mononuclear cell cultures from human peripheral blood (containing 10-14% monocytes and greater than 85% lymphocytes) at a density of 3 X 10(6) cells/ml (in serum-free medium) released less than 7 X 10(-10) M/liter (60 ng/ml) of Factor B antigen in 24 hr at 37 degrees C, and when mononuclear cells were stimulated with PHA mitogen in serum-free medium, the levels of Factor B antigen in media at 24 hr were significantly higher 1-3 X 10(-8) M/liter (0.9-2.8 micrograms/ml). The molecular size of Factor B in these media was 50-65 kDa by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size appropriate for Factor Bb (60 kDa). Since pathological effects of macrophages in autoimmune disease may result from the release of lysosomal hydrolases, the effects of purified Factor Bb on mononuclear phagocytes were investigated in an in vitro system of murine peritoneal exudate macrophages. Factor Bb induced secretion of marker lysosomal hydrolases N-acetyl-beta-D-glucosaminidase (hexosaminidase) and beta-glucuronidase from thioglycollate-elicited murine peritoneal exudate macrophages in a dose-response and kinetic manner. Hydrolase release was induced in serum-free medium without a known particulate activator at a concentration of 80-200 nM (5-13 micrograms/ml) Factor Bb. Maximal release occurred in 3-5 hr at 37 degrees C and extracellular enzyme activity of hexosaminidase and glucuronidase increased as intracellular enzyme levels decreased, suggesting that Factor Bb triggers release of these enzymes from intracellular lysosomal pools. These results provide an example of a complement protein which is synthesized, released, and activated during mononuclear cell culture and which induces release of lysosomal enzymes from macrophages. In conventional terminology, Factor B or Factor Bb might be termed a "lymphokine," "monokine," or "interleukin".

Complement C3↗
PubMed1985

Heterogeneity of hereditary and acquired factor X deficiencies by combined immunochemical and functional analyses.

Limited information is available regarding molecular abnormalities associated with derived factor X deficiencies. In order to assess the types of molecular aberrations that may occur in this group of haemostatic diseases we have analysed plasmas of 33 individuals from 28 kindred exhibiting factor X deficiency. These included those of hereditary type, transiently acquired deficiency as well as factor X deficiency associated with amyloidosis. Plasmas were analysed by one-stage assays for factor X activation by the extrinsic coagulation pathway, intrinsic coagulation pathway and Russell's viper venom. Selected plasmas were analysed in two-stage assays. Normal factor X concentration by specific radioimmunoassay was 6.38 +/- 1.29 micrograms/ml. Factor X associated with factor X deficiency were grouped by their specific activities measured for the three activation pathways and antigen concentration. The results suggest that a broad spectrum of molecular aberrations exist in the factor X deficiency states. The most common group of factor X deficiency was associated with abnormal activation of factor X by all three pathways. Variants of factor X associated with primary amyloidosis and transient acquired deficiency appeared to be abnormal molecules and not just reduced factor X concentration. Hereditary abnormal factor X molecules include the spectrum of potential defective molecules. The relationship of factor X structure to function and the heterogeneity of these defective molecules is discussed.

Antigens↗