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D N Fass

Publications and source records attributed to D N Fass.

At least 55 records · Page 3Linked to original sources
PubMed1984

Coagulation factors V and VIII and ceruloplasmin constitute a family of structurally related proteins.

Computer searches of the National Biomedical Research Foundation protein and nucleic acid sequence data bases using the NH2 terminus of the bovine factor Va 94-kilodalton heavy chain, the NH2 terminus of the 74-kilodalton factor Va light chain, and an internal 98-residue segment of porcine factor VIII revealed that both bovine factor V and porcine factor VIII are statistically homologous to human ceruloplasmin. The NH2-terminal segment of bovine factor Va heavy chain is homologous to three segments of ceruloplasmin sequence starting at residues 1, 351, and 713; the NH2-terminal sequence of bovine factor Va light chain is homologous to the same human ceruloplasmin sequence segments beginning at residues 1, 349, and 711. The longer porcine factor VIII sequence is homologous to three segments of human ceruloplasmin, residues 1-77, 400-433, and 683-791. These data indicate that factor V, factor VIII, and ceruloplasmin comprise a group of evolutionarily linked protein structures that possibly resulted from multiplication of ancestral precursor genes.

Amino Acid Sequence↗
PubMed1984

Synthesis of coagulation factor V by cultured aortic endothelium.

Bovine aortic endothelium has been examined with respect to the synthesis of coagulation factor V. After cultured cells reached confluency, samples of supernatant culture media and solubilized cells were analyzed for factor V in a two-stage bioassay and in a double-antibody radioimmunoassay. In addition, preconfluent cells were pulsed for 4 days with 35S-methionine in methionine-free media. After the 4-day pulse, supernatant media were chromatographed on a factor V monoclonal antibody-Sepharose resin to isolate 35S-labeled factor V. The isolated material and 125I-factor V standards were analyzed by electrophoresis and autoradiography. The bioassay indicated an increase, with time, of unactivated factor V in the culture supernatant, whereas solubilized cells were negative for factor V. The radioimmunoassay indicated an increase, with time, of factor V antigen in the culture supernatants, and the solubilized cells yielded a constant level of antigen per cell. Autoradiograms of electrophoretograms of immunoadsorbed 35S-culture supernatant with 125I-factor V/Va standards revealed labeled proteins with electrophoretic mobilities compatible with 125I-factor V/Va standards. The data obtained from three different sources-bioassay, radioimmunoassay, and 35S-methionine incorporation-all indicate that factor V is synthesized by cultured bovine aortic endothelium.

Animals↗
PubMed1984

Stabilization of thrombin-activated porcine factor VIII:C by factor IXa phospholipid.

The activation of porcine factor X by an enzymatic complex consisting of activated factor IX (factor IXa), thrombin-activated factor VIII:C (factor VIII:Ca), phospholipid vesicles, and calcium was studied in the presence of an irreversible inhibitor of factor Xa, 5-dimethylamino-naphthalene-1-sulfonyl-glutamyl-glycyl-arginyl- chloro met hyl ketone ( DEGR -CK). The formation of factor Xa was measured continuously by monitoring the increase in solution fluorescence intensity that occurs upon formation of DEGR -factor Xa. Omission of any component from the enzymatic complex reduced the reaction rate to a negligible level. In the presence of fixed excess factor IXa, the velocity of factor X activation was linearly dependent on the concentration of factor VIII:C, and thus, provided a plasma-free assay of factor VIII:C. Activation of factor VIII:C by 0.1 NIH U/ml thrombin in the presence of factor IXa, phospholipid vesicles, and calcium, followed at variable time intervals by the addition of factor X and DEGR -CK, was complete within 5 min, as judged by the fluorometric assay, and resulted in little or no loss of factor VIII:C activity over a period of 20 min; whereas, activation in the absence of either IXa or phospholipid vesicles decreased the half-life of factor VIII:C to approximately 5 min. Analysis of 125I-factor VIII:C-derived activation peptides by sodium dodecyl sulfate polyacrylamide gel radioelectrophoresis revealed identical results, regardless of whether factor IXa and/or phospholipid vesicles were included in the activation, suggesting that the lability of factor VIII:Ca is not due to a major alteration of its primary structure. We conclude that the activated porcine factor VIII:C molecule is stabilized markedly because of its interaction with factor IXa and phospholipid.

Amino Acid Chloromethyl Ketones↗
PubMed1983

Porcine cardiac valvular endothelial cells in culture. A relative deficiency of fibronectin synthesis in vitro.

We established culture lines of porcine endothelial cells derived from both the aortic valve and the ascending aorta. These cells had the typical morphology of vascular endothelial cells in culture and contained angiotensin-converting enzyme activity. The cells derived from the aorta grew well in standard tissue culture dishes; the valvular endothelial cells, however, could not be passed serially in vitro unless cultivated in dishes precoated with fibronectin. Moreover, under the conditions used, the valvular cells showed substantial changes in their morphology when deprived of fibronectin coating for several passages but reestablished typical vascular endothelial morphology when subcultured to fibronectin-coated dishes. The aortic cells did not show this behavior. Endogenous labeling of proteins synthesized in vitro by the two cell types with 35S-methionine revealed that a high molecular weight protein produced in substantial quantities by the aortic cells was synthesized only in trace amounts by the valvular cells. This protein could be isolated by gelatin-agarose chromatography and showed an apparent molecular weight under reduced conditions of 235,000 to 245,000 on sodium dodecyl sulfate gel electrophoresis. Synthesis of gelatin-binding proteins, an indirect estimation of fibronectin production, as well as direct measurement of porcine fibronectin present in the culture supernatants using an immunoradiometric assay, showed that aortic endothelial cells synthesized and released 10-fold more fibronectin in vitro than did valvular endothelial cells. We suggest that this relative deficiency of in vitro fibronectin synthesis by valvular cells, if paralleled in vivo, may have important implications for the pathogenesis of lesions unique to the cardiac valve.

Animals↗
PubMed1983

Functional studies of Willebrand factor using monoclonal antibodies.

Previously described monoclonal antibodies to porcine Willebrand factor were used to study various functional assays of Willebrand factor. These antibodies comprise at least five separate specificities as determined by differential reactivity in three distinct binding assays. The antibodies were tested for their effect on the in vivo bleeding time, in vitro bleeding time, and ristocetin cofactor activity. The titers of the antibodies in a binding assay did not correlate with any inhibitory activities, and the ability to inhibit a given functional test was independent of inhibitory activity in the other assays. These data suggest that the antibodies are able to detect specific structures on Willebrand factor involved in specific functional assays and that the structure(s) involved in ristocetin cofactor activity is not identical to those mediating the in vivo or in vitro bleeding time.

Animals↗
PubMed1982

Arteriosclerosis in normal and von Willebrand pigs: long-term prospective study and aortic transplantation study.

In a long-term prospective study, five normal control pigs and five pigs with homozygous von Willebrand's disease received a nonfatty diet from age 3 months to 4 years; then the aortas were analyzed. The fibrous arteriosclerotic plaques in the distal abdominal aortic region involved an average of 28% of the surface area in control pigs and only 7% of the surface area in pigs with von Willebrand's disease (P less than 0.01). In a subsequent study of 3-month-old pigs, the distal abdominal aortic segments from nine normal pigs were cross-transplanted with segments from nine other normal pigs (control study), and aortic segments from four normal pigs were transplanted into four host pigs with von Willebrand's disease (exchange study). All pigs received a 2% cholesterol diet for up to 6 months; then the transplanted aortic segments were analyzed. The donor normal aortic segments in the host normal pigs developed arteriosclerosis that involved an average of 20% of the surface; the endothelial fluorescent pattern of von Willebrand factor was identified. In contrast, the donor normal aortic segments in the host pigs with von Willebrand's disease had arteriosclerosis that involved an average of only 4% of the surface (P less than 0.01); the endothelial cell von Willebrand factor was not identified. The long-term prospective study indicates that pigs with von Willebrand's disease are resistant to the development of spontaneous arteriosclerosis. The aortic transplantation data are compatible with the hypothesis that the absence of von Willebrand factor in pigs with von Willebrand's disease may cause impairment of platelet-arterial wall interaction and resistance to arteriosclerosis.

Animals↗
PubMed1982

Monoclonal antibodies to porcine factor VIII coagulant and their use in the isolation of active coagulant protein.

Partially purified preparations of porcine factor VIII:C were used to immunize mice and spleen cells from the immunized animals were fused to NS-1 mouse myeloma cells. The ability of hybrid culture fluids to bind factor VIII:C was detected with a radiolabelled, affinity-purified, human antihuman VIII:C inhibitor. Three cloned hybrid lines have been obtained that preferentially bind to VIII:C when compared to von Willebrand factor binding. Two of these monoclonal antibodies partially inhibit VIII:C coagulant activity. The third antibody does not inhibit VIII:C, but it can be used as an affinity reagent to absorb dissociated VIII:C out of solution. Active coagulant can be recovered by elution in 50% ethylene glycol. The VIII:C obtained has a specific activity of 6 units/micrograms based on absorbance measurements. When analyzed on SDS gels, the unactivated VIII:C contains 3 bands of apparent molecular weight 166,000, 130,000 and 76,000. Thrombin treatment results in a 40 fold increase in activity and cleavage to products of 76,000, 67,000 an 50,000 and small amounts of lower molecular weight peptides. EDTA inactivation of the factor VIII:C results in the separation of the 166,000 and 130,000 chains from the 76,000 chain, suggesting a Ca++ dependent noncovalent interaction among the chains.

Animals↗
PubMed1981

The role of Willebrand factor in platelet - blood vessel interaction, including a discussion of resistance to atherosclerosis in pigs with von Willebrand's disease.

Von Willebrand pigs have all the manifestations of the severe human disease. The role of Willebrand antigen (VIII R:AG) and ristocetin cofactor (VIII: RWF) was assessed in these pigs by (1) transfusion and (2) "in vitro" bleeding time assay. The skin bleeding time became normal when the level of transfused Willebrand factor (VIII R:AG/RWF) was raised in the plasma above 30 U/dl. After single or repeated transfusions, skin capillary endothelium and platelets were still distinguished from normal by VIII R:AG deficiency. When incisions in excised porcine skin ("in vitro" bleeding time) were perfused with blood and plasma fractions, haemostasis occurred when plasmatic Willebrand factor exceeded 30 U/dl whether the skin or platelets came from normal or from von Willebrand pigs. The platelet plug occluding the skin incision contained VIII R:AG by immunofluorescence. Willebrand factor appears to coat surfaces and to serve as a platelet attachment protein. These bleeder pigs are resistant to atherosclerosis. If platelets are involved in early atherosclerotic lesions, the role of Willebrand factor in platelet - blood vessel interaction may be important.

Animals↗
PubMed1981

Monoclonal antibodies to von Willebrand's factor: reactivity with porcine and human antigens.

Monoclonal antibodies to porcine von Willebrand's factor (vWf) were obtained from hybridomas derived from the fusion of NS-1 plasmacytoma cells and spleen cells from immunized BALB/c mice. Twenty hybrids were positive in a radioimmunoassay based on the ability of immobilized hybridoma antibodies to bind porcine vWf as detected by the subsequent binding of 125I-polyclonal rabbit anti-vWf. Growth of the hybridomas as ascitic tumors yielded fluids having titers in this assay ranging from 8 x 10(7) to 8 x 10(11). All the antibodies were positive when normal, but not von Willebrand's, plasma was used as the source fo antigen in this RIA, and when normal, but not von Willebrand's, acetone-fixed platelets were used in an indirect immunofluorescence assay. Two of these antibodies inhibited both ristocetin-induced platelet aggregation and platelet aggregating factor. In a second radioimmunoassay, only 13 were of the 20 antibodies were reactive with human vWf, and two of these would not react with human antigen in the presence of 2M Nacl. Bases on these assays, six reactivity groups of antibodies could be identified.

Animals↗
PubMed1980

Hemostatic effect of transfused Willebrand factor in porcine von Willebrand's disease. Similarities to the human disease.

Cryoprecipitate was infused into pigs with severe von Willebrand's disease, and the bleeding time was corrected when the plasmatic level of VIII R:AG (WF) was above 30 U/dl. The VIII R:AG (WF) was not completely recovered but none was found in the plasma or endothelial cells. A new method for measuring the bleeding time is described--the ratio bleeding time. These results in pigs are compared with the reported results in humans.

Animals↗
PubMed1979

Factor VIII-related antigen (VIII R:AG) in haemophilic patients and in carriers.

All of fourteen patients with severe classic haemophilia and twelve of fifteen obligate carriers had a pre-peak ("rocket") above the sample well when factor VIII-related antigen was examined by crossed immunoelectrophoresis. The pre-peak may be found in other conditions--including von Willebrand's disease--but if these conditions can be excluded, the pre-peak may be helpful in identification of carriers or haemophilia.

Adolescent↗
PubMed1979

Increased von Willebrand factor in diabetes mellitus.

Abnormal platelet function may play a role in the genesis of vascular complications in diabetes mellitus. We measured plasma levels of ristocetin-Willebrand factor and factor VIII antigen in 75 subjects and also measured aggregation-enhancing factor in subsets. We found increased levels of ristocetin-Willebrand factor in all groups of diabetics studied, even in mild diabetics free of vascular disease. Factor VIII antigen was increased only in diabetics with vascular disease. We could not find an aggregation-enhancing factor in any group.

Adult↗
PubMed1979

Generation of factor VIII coagulant activity by isolated, perfused neonatal pig livers and adult rat livers.

Isolated, normal, neonatal pig livers released VIII:C into perfusates of porcine von Willebrand blood lacking VIIIR:AG and VIII:RWF. Neonatal von Willebrand livers released VIII:C only when partially purified VIIIR:AG was added to the von Willbrand blood. Adult rat livers, perfused with frozen-thawed erythrocytes suspended in Tyrode's solution containing 6% bovine serum albumin and hence no VIIIR:AG released VIII:C only when serum, adsorbed serum or cryoprecipitate from rat plasma was added to the perfusate. Cycloheximide blocked the release of VIII:C from rat liver.

Animals↗