Molecular Events in the Activation of Bovine Factor V

1979 ◽  
Author(s):  
C.T. Esmon ◽  
S.F. Gray
Keyword(s):  
The Other ◽  
Antigenic Determinants ◽  
Factor V ◽  
Factor Va ◽  
Molecular Events ◽  
Activation Products ◽  
Activation Peptide ◽  
Peptide Region ◽  
Bovine Factor

The conversion of bovine Factor V (FV) to Factor Va (FVa) by thrombin appears to proceed in two steps as shown below. FV(300,000) → thrombin Factor V intermediate (FVi) 2 sub-units; 220,000 & 100,000)→FVa (2 subunits; FVa-H.C., 100,000 & FVa-L.C., 73,000)+activation peptide/s. The 220,000 Mol. wt. subunit of FVi gives rise to the FVa-L.C. EDTA dissociates both FVi and FVa into subunits, Mn2+ facilitates reassociation. Even following dissociation with EDTA, 1 mole of Ca2+ remains bound to each of the FVa subunits.Support of the above model of FV activation comes from an immunological characterization of the FV. Antibodies to FV cross react with FVa, FVi and both subunits of Fva. Antibodies to FVa cross react with FVi both subunits of FVi and both subunits of FVa. Antibodies to the FVa-L.C. cross react with FV, FVj and FVa, but do not with the FVa-H.L. Antibodies to the FVa-H.C. cross react with FV, the FVi, FVa but not with the FVa-L. C.A protease from Russell’s viper venom activated FV by making a single proteolytic cleavage. This results in the formation of two subunits of Mol. wt., 250,000 & 73,000. The 73,000 Mol. wt. subunit is functionally and immunologically identical to the FVa-L.C. the 250,000 Mol. wt. subunit contains the antigenic determinants to the FVa-H.C. and can be converted into this subunit by incubation with thrombin. The appearance of two disdinct high Mol. wt. activation products from FV; one containing the FVa-L.C.; the other containing the FVa-H.C., is compatible with placing the activation peptide region between the two subunits of Fva.

Isolation And Properties Of Intermediates Involved In Thrombin-Catalyzed Conversion Of Bovine Factor V To Factor Va

1981 ◽  
Author(s):  
M E Nesheim ◽  
K G Mann
Keyword(s):  
Electrophoretic Analysis ◽  
Complete Loss ◽  
The Other ◽  
Factor V ◽  
The Third ◽  
Factor Va ◽  
Specific Activities ◽  
Cofactor Activity ◽  
The Individual ◽  
Bovine Factor

Bovine Factor V was incubated briefly with bovine β- thrombin, causing partial conversion to Factor Va. QAE- cellulose chromatography in Ca++ of the partially activated material yielded 3 fractions. Electrophoresis in DodSO4 indicated primarily starting material (Mr=330,000) in the first fraction; two peptides of Mr=150,000 and 200,000 in the second; and two peptides of Mr=200,000 and 90,000 in the third. The first of these latter fractions was active only after further incubation with thrombin, while the other was active without further thrombin catalyzed activation. After thrombin treatment both fractions had specific activities equal to that of fully activated, unfractionated Factor V. Incubation of either of these fractions with EDTA caused complete loss of activity and allowed isolation of the constituent polypeptides by further chromatography on QAE- cellulose in EDTA. The NH2-termini of the 150,000 and 90.000 apparent Mr peptides had sequences identical to that of Factor V. The 200,000 apparent Mr peptides of the two fractions obtained by chromatography in Ca++ had NH2- terminal sequences identical to each other but different from that of Factor V. The summed amino acid compositions of the Mr=200,000 and Mr=150,000 were equivalent to that of Factor V. Electrophoretic analysis indicated that thrombin converts the Mr=150,000 peptide to endproducts of Mr=90,000 and 71,000, and the Mr=200,000 peptide to endproducts of 83.000 and 31,000 apparent Mr. Recalcification of the individual isolated intermediates, followed by incubation with thrombin did not yield cofactor activity. Recombina- ion of the recalcified intermediates followed by incubation with thrombin, however, generated samples with activities approaching that of unfractionated, activated Factor V. These data indicate that the intermediates of Mr=150,000 and Mr=200,000 arise by a single cleavage of Factor V, and that Factor Va comprises peptides derived from both of these intermediates.

Activation of Bovine Factor V by Thrombin and A Protease From Russell's Viper Venom (RVV)

1979 ◽  
Author(s):  
M.J. Lindhout ◽  
C. M. Jackson
Keyword(s):  
Bond Cleavage ◽  
Peptide Bond ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor V ◽  
Peptide Bond Cleavage ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Activation Products ◽  
Ca Oxalate

In order to understand the function of activated factor V in the prothrombinase complex, we isolated the activation products obtained by action of thrombin and RVV-V on factor V and studied their functional properties. Factor V isolated from plasma by means of ion-exchange chromatography, a Ca-oxalate adsorption step and gelfiltration was homogenous in SDS-gelelectrophoresis (apparent MW 360,000, with and without reduction). Increase in factor V activity upon action by RVV-V is correlated with a single peptide bond cleavage, resulting in a 270,000 dalton and a 80,000 dalton component. Additional proteolysis of factor Va(RVV/V)’ by thrombin results in a further cleavage of the high MW component into peptides with MW's of 72,000, 94,000 and about 150,000 without a furth~r increase in factor V activity. Whereas none of the isolated peptides reveal factor Va activity, activity would be generated by a recombination in the presence of Ca2+ of the 94,000 MW or 270,000 MW component with the 80,000 component. Action of thrombin alone on factor V results in peptides of MW 72,000, 80,000, 94,000 and a peptide very rich in carbohydrate with an apparent MW of 150,000.

Thrombin-stimulated Human Platelets Express Molecular Forms of α-Granule Factor V (FV), which Differ from FVa

1994 ◽  
Vol 72 (06) ◽  
pp. 947-956 ◽  
Author(s):  
Edward G Wyshock ◽  
Gwendolyn J Stewart ◽  
Robert W Colman
Keyword(s):  
Human Platelets ◽  
Molecular Forms ◽  
Light Chains ◽  
Factor V ◽  
Platelet Surface ◽  
Specific Antibodies ◽  
Factor Va ◽  
Activation Peptide ◽  
Platelet Pellet ◽  
Incomplete Cleavage

SummaryBinding of 125I-Fab fragments of chain-specific antibodies indicate that both heavy and light chains of a-granule factor Va (FVa) were externalized on the platelet membrane after stimulation with thrombin. Using a Mab against the activation peptide of factor V (FV), the epitope appears on the stimulated platelet surface. Half as much light chain and heavy chain (FVa) was expressed compared to the activation peptide, suggesting that expression of α-granule FV occurs after thrombin stimulation. Using an ELISA, we find that 32% of a-granule FV was released and 68% is retained in the platelet pellet. Immunoblots of platelets indicate that FV exists in 200 kDa und 150 kDa forms, representing incomplete cleavage, while the releasate demonstrates a more complete cleavage by proteases. We conclude that expression of α-granule FV is quantitatively greater than that released and exists in molecular forms which cannot be completely explained by the binding of FVa.

scholarly journals Activation of Bovine Blood Coagulation Factor V, A Prerequisite for it of Bind Both Prothrombin and Factor Xa.

1977 ◽  
Author(s):  
M.C. Guillin ◽  
A. Bezeaud ◽  
J.P. Freeman ◽  
C.M. Jackson
Keyword(s):  
Factor Xa ◽  
Coagulation Factor ◽  
Factor V ◽  
Factor Va ◽  
Bovine Plasma ◽  
Coagulation Factor V ◽  
Russell’S Viper ◽  
Blood Coagulation Factor V ◽  
Bovine Factor ◽  
Plasma Heparin

It is known that prior to bind bovine prothrombin and to become fully functional, bovine Factor V must itself be “activated” by either thrombin or an enzyme isolable from Russell’s viper venom. The purpose of this work was to determine if Factor V activation is also required in order for it to bind bovine Factor Xa.This has been investigated by measuring the binding of both “native” (unactivated) Factor V and Factor V activated by the Russell’s viper venom activating enzyme, to a column of agarose-bound Factor Xa. The experiments were also performed using diisopropylfluorophosphate (DFP) inhibited Factor Xa covalently bound to agarose. Both purified bovine Factor V (Va) and bovine plasma were used and gave the same results. In order to prevent initiation of clotting in bovine plasma, heparin wad added to the plasma to promote inactivation of Factor Xa by antithrombin III.The results indicate that Factor V activation is a prerequisite for it to bind Factor Xa ; Factor Va binds both Factor Xa and DFP inhibited Factor Xa, unmodified Factor V does not.These experiments suggest that Factor V may not participate in prothrombin activation at all, until after some thrombin has been formed. If this is so, an alternate pathway by which the first thrombin is generated must be considered and may be proposed to be simply that involving Factor Xa, phospholipid and Ca2+ alone.

Production and Characterization of Monoclonal Antibodies Directed against the Laminin Receptor Precursor

1997 ◽  
Vol 12 (1) ◽  
pp. 1-5 ◽  
Author(s):  
S. Butò ◽  
C. Ghirelli ◽  
P. Aiello ◽  
E. Tagliabue ◽  
E. Ardini ◽  
...  
Keyword(s):  
Molecular Structure ◽  
Monoclonal Antibodies ◽  
The Other ◽  
Laminin Receptor ◽  
Antigenic Determinants ◽  
Native Protein ◽  
Partially Unfolded ◽  
Cross Competition ◽  
Membrane Level

The 67-kDa laminin receptor (67LR) is an important tumor marker whose molecular structure has not yet been fully elucidated. To shed new light on this molecule, we raised a series of eight new monoclonal antibodies, designated MPLR1 to 8, directed against the 37-kDa recombinant laminin receptor precursor (37LRP). Cross-competition experiments demonstrated that the epitopes recognized by MPLR2, 4 and 5 partially overlap, since MPLR4 and 5 compete with labelled MPLR2 for the binding to recombinant 37LRP. These three antibodies belong to the IgG1 class, whereas the other ones are all IgM. Presumably due to the fact that they are directed against partially unfolded antigenic determinants expressed on the recombinant protein, MPLRs did not recognize the native protein. Indeed, they showed no reactivity at the membrane level in cytofluorimetric analysis and they did not work in immunoprecipitation experiments. In contrast, these reagents are valuable tools in immunoblotting, since they clearly identify a 67-kDa protein (the mature laminin receptor) in addition to the 37-kDa precursor form. MPLRs are thus a new powerful tool which could help in the characterization of the still enigmatic 67LR molecule.

Electrophoretic Characterization of Porcine Pancreatic (Pro)elastases A and B

1975 ◽  
Vol 53 (4) ◽  
pp. 421-432 ◽  
Author(s):  
Michel Ledoux ◽  
François Lamy
Keyword(s):  
Gel Electrophoresis ◽  
Sucrose Gradient ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Species ◽  
The Other ◽  
Synergistic Activity ◽  
Fibrous Protein ◽  
Activation Products ◽  
Esterolytic Activity

Two porcine pancreatic zymogens can be separated by free electrophoresis on a sucrose gradient. After activation by trypsin, both enzymes can hydrolyze completely the fibrous protein elastin. One of the two proteins, proelastase B, has, in addition, an esterolytic activity towards N-acetyl-L-tyrosine ethyl ester. The other, proelastase A, does not possess it. The activation products of the zymogens have been tagged with radioactive diisopropylfluorophosphonate and separated by polyacrylamide-gel electrophoresis. Proelastase A gives only one active species, pancreatopeptidase E, but three distinct proteins can be obtained from proelastase B. Elastases A and B exhibit an important synergism when acting together upon a purified elastin lacking microfibrils. Trypsin has considerably less synergistic activity, and chymotrypsin has practically none.

scholarly journals The Factor V Activation Paradox

2004 ◽  
Vol 279 (19) ◽  
pp. 19580-19591 ◽  
Author(s):  
Thomas Orfeo ◽  
Nicole Brufatto ◽  
Michael E. Nesheim ◽  
Hung Xu ◽  
Saulius Butenas ◽  
...  
Keyword(s):  
Rate Constants ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Reaction Pathways ◽  
Factor V ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Activation Products ◽  
Initial Activation ◽  
Prothrombin Activation

The prothrombinase complex consists of the protease factor Xa, Ca2+, and factor Va assembled on an anionic membrane. Factor Va functions both as a receptor for factor Xa and a positive effector of factor Xa catalytic efficiency and thus is key to efficient conversion of prothrombin to thrombin. The activation of the procofactor, factor V, to factor Va is an essential reaction that occurs early in the process of tissue factor-initiated blood coagulation; however, the catalytic sequence leading to formation of factor Va is a subject of disagreement. We have used biophysical and biochemical approaches to establish the second order rate constants and reaction pathways for the activation of phospholipid-bound human factor V by native and recombinant thrombin and meizothrombin, by mixtures of prothrombin activation products, and by factor Xa. We have also reassessed the activation of phospholipid-bound human prothrombin by factor Xa. Numerical simulations were performed incorporating the various pathways of factor V activation including the presence or absence of the pathway of factor V-independent prothrombin activation by factor Xa. Reaction pathways for factor V activation are similar for all thrombin forms. Empirical rate constants and the simulations are consistent with the following mechanism for factor Va formation. α-Thrombin, derived from factor Xa cleavage of phospholipid-bound prothrombin via the prethrombin 2 pathway, catalyzes the initial activation of factor V; generation of factor Va in a milieu already containing factor Xa enables prothrombinase formation with consequent meizothrombin formation; and meizothrombin functions as an amplifier of the process of factor V activation and thus has an important procoagulant role. Direct activation of factor V by factor Xa at physiologically relevant concentrations does not appear to be a significant contributor to factor Va formation.

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

1982 ◽  
Vol 28 (5) ◽  
pp. 649-661 ◽  
Author(s):  
W.Barry Foster ◽  
Jerry A. Katzmann ◽  
Randall S. Miller ◽  
Michael E. Nesheim ◽  
Kenneth G. Mann
Keyword(s):  
Monoclonal Antibodies ◽  
Factor V ◽  
Factor Va ◽  
Functional States ◽  
Bovine Factor
Sign in / Sign up

Export Citation Format

Share Document