Search PubMed⌕ Search

Biomedical subjects

Y Nemerson

Publications and source records attributed to Y Nemerson.

At least 109 records · Page 6Linked to original sources
PubMed1978

Activation of factor X by factors IXa and VIII; a specific assay for factor IXa in the presence of thrombin-activated factor VIII.

We studied the activation of factor X by the intrinsic pathway of blood coagulation using a new assay of factor X activation. When factor X tritiated in its sialic acid residues is activated, activation can be measured by the release of tritiated activation peptide, and the initial rate of activation can be determined under varying conditions. In the presence of phospholipid and calcium ions, factor IXa activated factor X slowly without factor VIII, and this activation was blocked by a specific factor IX inhibitor. These data provide strong evidence that factor IXa is the enzyme responsible for factor X activation by the intrinsic pathway. The role of factor VIII was also investigated. Factor VIII could be reproducibly thrombin activated and then stabilized by the addition of 2 mM benzamidine hydrochloride; this suggests that inactivation is due to proteolysis. Neither unactivated nor thrombin-activated factor VIII produced factor X activation without factor IXa. With a constant level of factor IXa, factor X activation was directly proportional to the level of activated factor VIII. With a constant level of activated factor VIII, factor X activation was proportional to the factor IXa concentration. This observation was exploited to develop a specific, sensitive assay for factor IXa.

Factor IX↗
PubMed1977

Activation of bovine factor VII by hageman factor fragments.

During the early events of coagulation of human blood by the intrinsic pathway, factor XII is activated to a form which can activate factor XI, and is proteolytically fragmented to smaller species (30,000 daltons and 70,000 daltons) which have lost most of the ability to activate factor XI but which can activate prekallikrein rapidly. The effect of these fragments on factor VII was studied. It was found that these Hageman factor fragments promoted rapid proteolysis of one-chain factor VII to a more active two-chain form. The amino-terminal sequences of the chains of activated factor VII were found to be Ala-Asx-Gly- and Ile-Val-Gly-, the same as were earlier observed after activation of factor VII by activated factor X. This finding indicates that initiation of coagulation by the intrinsic pathway also primes the extrinsic pathway.

Amino Acid Sequence↗
PubMed1976

Mechanism of activation of bovine factor VII. Products of cleavage by factor Xa.

Coagulation Factor VII from bovine plasma is a glycoprotein containing a single peptide chain. The NH2-terminal sequence of Ala-Asx-Gly-Phe-Leu- is homologous with the NH2 termini of prothrombin, Factor IX, and the light chain of Factor X. Factor Xa in the presence of calcium ions and phospholipid cleaves Factor VII at an Arg-Ile bond in the sequence Arg-Ile-Val-Gly-Gly-, producing a two-chain molecule with at least 85 times the coagulant activity of single-chain Factor VII and a new NH2-terminal sequence homologous with the corresponding chains of thrombin, Factor IXa and Factor Xa. A second slower cleavage at an Arg-Gly bond destroys Factor VII activity. Bovine Factor VII, unlike prothrombin, Factor IX, and Factor X, is rapidly inhibited by diisopropylphosphorofluoridate (iPr2PF). [3H]iPr2PF is readily incorporated into one-chain, two-chain, and three-chain forms of Factor VII up to ratios of approximately 0.9 moles of [3H]diisopropylphosphate per mole of protein. The radioactive peptides generated from each form of [32P]iPr2PF-inhibited Factor VII by tryptic and thermolytic digestion were found to migrate together on paper electrophoresis. This indicates that the iPr2PF is incorporated stoichiometrically into the same specific site in each form.

Amino Acid Sequence↗
PubMed1976

Biological control of factor VII.

The tissue factor pathway is initiated by factor VII in the presence of tissue factor. The first proteolytic reaction involves cleavage of factor X by factor VII. Activated factor X, the product of this reaction, can activate factor VII by cleavage of a specific bond. The apparent coagulant activity of factor VII then rises about 60-fold. Activated factor X can also inactivate factor VII by catalyzing the cleavage of a second bond which results in a three chain molecule. Fragments of Hageman factor and perhaps kallikrein can also activate factor VII. Hageman factor, however, does not catalyze the inactivating cleavage of factor VII at a significant rate. Recent data showing that the tissue factor pathway can activate the intrinsic system are discussed. We have shown that activated factor X, which can be generated by the tissue factor pathway, can feed back and activate factor IX in a calcium and phospholipid requiring reaction.

Binding Sites↗
PubMed1976

Tissue factor in cultured cells: metabolic control.

Tissue factor content of WISH amnion cells in spinner culture increases 3- to 10-fold within 12 hours after subculture, then declines to a basal level within 30 to 50 hours. Maximal development of activity requires fresh serum and fresh medium. When added at the time of subculture, actinomycin D and cycloheximide completely inhibit development of coagulant activity; when added several hours after transfer, these inhibitors suppress the development but do not affect the disappearance of activity. Of the oxidative phosphorylation inhibitors tested, dinitrophenol had no effect whereas carbonyl cyanide m-chlorophenylhydrazone inhibited the activity increase but did not alter the decline. The kinetics of development and decay are similar over a pH range of 6.7 to 7.6 and with fetal calf serum concentration between 5 and 30 per cent. At pH 6.7 or in 30 per cent fetal calf serum, cell division did not occur. 3H-leucine and 35SO4= incorporation into the cell surface coat did not change appreciably during the burst of coagulant activity nor did the levels of naphthylamidase or alkaline phosphatase; 3H-thymidine incorporation reached a peak within 2 hours of the tissue factor maximum.

Alkaline Phosphatase↗
PubMed1976

Tissue factor in cultured cells: pharmacologic effects.

Tissue factor activity in suspension cultures of WISH amnion cells is modulated by pharmacologic doses of agents which alter membrane structure and function. Lysosomal stabilizing steroids (hydrocortisone, dexamethasone, aldosterone, prednisolone, and estradiol) suppress the change in activity which follows subculture; lytic steroids (testosterone and progesterone) are ineffective. Chloroquine both increases the specific activity and extends the time before return to the basal level. Dimethyl sulfoxide and ouabain suppress the complete expression of activity but do not inhibit the subsequent decay. The effect of cytochalasin B is complex, the drug being either suppressive or slightly stimulatory depending on the time of addition. Cyclic nucleotides (AMP or GMP) or insulin do not regulate the expression of tissue factor in these cells. A dramatic increment and prolongation of activity occurs when colchicine or vinblastine is added to the cell suspension shortly after subculture; there is much less stimulation by griseofulvin. Lumicolchicine has no effect while deuterium oxide is inhibitory. From these experiments, we conclude that increased membrane fluidity or altered secretory processes resulting from microtubule disruption stabilize tissue factor in cultured cells. Since contradictory results were obtained with agents which stabilize lysosomes or inhibit transport, the role of these cellular functions in tissue factor production or decay is unclear.

Amnion↗
PubMed1975

The activation of coagulation factor X. Identity of cleavage sites in the alternative activation pathways and characterization of the COOH-terminal peptide.

Bovine Factor X can be activated by two alternative pathways. The first, favored at high concentrations of the complex of tissue factor and Factor VII, is initiated by the action of Factor VII on Factor X to cleave an activation peptide from the NH2 terminus of the heavy chain, to produce alpha-Xa. This is then converted autocatalytically to another form of Factor Xa, beta-Xa, by the loss of a 17-residue glycopeptide from the COOH terminus of the heavy chain, in a lipid-dependent reaction. The alternative pathway, favored at lower activator concentrations, is initiated by the action of Factor Xa on Factor X, in the presence of lipid, to release the same COOH-terminal peptide as is produced in the conversion of alpha-Xa to beta-Xa. The intermediate produced by the loss of this peptide from Factor X,I1, can be activated directly to beta-Xa by the tissue factor-Factor VII complex, with the loss of the same NH2-terminal peptide as is produced in the conversion of Factor X to alpha-Xa. The autocatalytic activation of Factor X by Factor Xa described previously occurs to a marked extent only at very low activator concentrations, and has been shown to proceed largely by the loss of the normal NH2-terminal peptide from the heavy chain of I1-Initial experiments show that neither peptide affects the rate of coagulation by either the extrinsic or intrinsic pathways. The amino acid sequences have been determined on both sides of the peptide cleavages, and it has been shown that the cleavage sites are the same, regardless of the pathway of activation. The amino acid sequence and carbohydrate composition of the COOH-terminal peptide have been determined. The carbohydrate moiety is attached via an O-glycosidic linkage at a threonine residue, and contains galactosamine but no glucosamine.

Amino Acid Sequence↗
PubMed1975

The control of prothrombin conversion. Kinetic control by mechanisms inherent in two activation pathways.

It is known that the activation of prothrombin to thrombin can proceed via two pathways: one initiated by the prothrombin-converting complex (factor Xa, factor V, phospholipid, and CA2+ ions) and the other initiated by the product, thrombin. A kinetic study has shown that the pathways do not proceed with equal ease under all conditions. At high levels of the converting complex, both go to completion: some prothrombin is always cleaved by thrombin, but the resulting intermediate is then activated to give quantitative conversion to thrombin. At slower rates of activation, the product-initiated pathway occurs to a relatively greater extent. Moreover, the intermediate then is not cleaved further but accumulates, so that the generation of thrombin is curtailed. The reason the intermediate is productive only at higher levels of activator may be partly that it is a poorer substrate for the converting complex than prothrombin. More importantly, the activity of the complex is also modulated by thrombin, which rapidly destroys the activity of factor V and factor Xa in a feedback reaction. These concerted controls ensure that prothrombin activation damps itself. Thus thrombin production occurs as a burst, the size of which is regulated by the amounts of factor Xa and factor V initially available.

Animals↗
PubMed1975

Activation and control of factor VII by activated factor X and thrombin. Isolation and characterization of a single chain form of factor VII.

Factor VII purified as previously described, was found to consist of two polypeptide chains joined by disulfide bridges. We now report the isolation and 200,000-fold purification of a single chain form of Factor VII. This was accomplished by protecting the molecule against proteolysis by including benzamidine during the entire purification. The purification was essentially as previously reported except that barium cirtate was substituted for barium sulfate as an absorbant for Factor VII as it resulted in a 4-fold increase in yield. Single chain Factor VII is rapidly hydrolyzed by Factor Xa in the presence of calcium ions and phospholipids, and by thrombin, to a two-chain form which possesses at least 85 times the Factor VII clotting activity of the single chain species. The two-chain form of the enzyme requires tissue factor in order to activate Factor X. From the observed rates of activation of Factor VII by Xa in the presence of calcium ions and phospholipids, it was calculated that at approximately physiological concentration, Factor VII activity would increase at an initial rate of 20-fold per min; this reaction is sufficiently rapid to constitute a feedback control mechanism. The action of thrombin is approximately 40-fold slower under these conditions. Diisopropylphosphorofluoridate inactivates the single chain and two-chain forms of Factor VII at approximately equal rates. After inhibition, the single chain species could be cleaved but not activated by proteolysis.

Animals↗
PubMed1975

Association of tissue factor activity with the surface of cultured cells.

Tissue factor occurs in a dormant state on the surface of cultured normal human fibroblasts and WISH 1 amnion cells. The activity of undisturbed monolayers or cells lifted with brief trypsin treatment (0.125 per cent trypsin for 1 min) increases up to 60-fold upon prolonged digestion with dilute trypsin (0.0025 per cent trypsin for 30 min); activity appears subsequent to cell detachment. Up to 70 per cent of the total cellular tissue factor becomes active under these conditions and is released from the cells. The ruthenium red staining coat of the cells is lost during detachment, but cell viability (more than 90 per cent exclude trypan blue) and cell morphology do not change during the subsequent development of tissue factor activity. Furthermore, less than 10 percent of four intracellular enzymes and less than 20 per cent of two plasma membrane enzymes are released during this period of time. We therefore conclude that cells in culture do have tissue factor activity, that it exists in a latent form, and that total cell disruption is not necessary for this activity to initiate blood coagulation.

Amnion↗