The regulation of human factor V by a neutrophil protease

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 846-851
Author(s):  
AM Oates ◽  
HH Salem
Keyword(s):  
Light Chain ◽  
Neutrophil Elastase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Neutrophil Elastase ◽  
Ionophore A23187 ◽  
Factor V ◽  
Single Chain ◽  
Prolonged Incubation ◽  
Factor Va

Neutrophils activated with serum opsonized zymosan, soluble heat- aggregated IgG, and ionophore A23187 in the presence of calcium release a material capable of initially activating factor V. Subsequent inactivation of factor V was only observed with neutrophil releasate derived from IgG and ionophore. In this study we examine the nature of this neutrophil activity and investigate its role in the regulation of factor V/Va. From early in the fractionation it was apparent that the cells contained different enzymes capable of cleaving factor V. The most active of these was isolated and found to be an isomer of human neutrophil elastase. The purified protease caused a dose-dependent activation of isolated factor V to a maximum of threefold. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, single-chain factor V was cleaved to form intermediates of 100 and 91 kilodaltons (kD). Coagulant activity correlated with the formation of a 97-kD heavy and 77-kD light chain. On prolonged incubation the formed factor Va(e) was inactivated in association with proteolysis of the 97-kD band to smaller peptides and cleavage of the 77-kD light chain to a molecular weight of 75 kD, which is similar to thrombin-activated factor Va light chain. Neutrophil elastase also caused rapid inactivation of thrombin- activated factor V, factor Va(t). These observations suggest that elastase cleaves factor V at sites distinct from that by thrombin and therefore represents a novel factor V activation pattern. It is proposed that upon neutrophil activation elastase is secreted into the plasma milieu to initiate factor V activation. This serves to generate small amounts of thrombin that, in turn, by positive feedback fully activates factor V and thus amplifies the coagulation reaction.

scholarly journals The regulation of human factor V by a neutrophil protease

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 846-851 ◽  
Author(s):  
AM Oates ◽  
HH Salem
Keyword(s):  
Light Chain ◽  
Neutrophil Elastase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Neutrophil Elastase ◽  
Ionophore A23187 ◽  
Factor V ◽  
Single Chain ◽  
Prolonged Incubation ◽  
Factor Va

Abstract Neutrophils activated with serum opsonized zymosan, soluble heat- aggregated IgG, and ionophore A23187 in the presence of calcium release a material capable of initially activating factor V. Subsequent inactivation of factor V was only observed with neutrophil releasate derived from IgG and ionophore. In this study we examine the nature of this neutrophil activity and investigate its role in the regulation of factor V/Va. From early in the fractionation it was apparent that the cells contained different enzymes capable of cleaving factor V. The most active of these was isolated and found to be an isomer of human neutrophil elastase. The purified protease caused a dose-dependent activation of isolated factor V to a maximum of threefold. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, single-chain factor V was cleaved to form intermediates of 100 and 91 kilodaltons (kD). Coagulant activity correlated with the formation of a 97-kD heavy and 77-kD light chain. On prolonged incubation the formed factor Va(e) was inactivated in association with proteolysis of the 97-kD band to smaller peptides and cleavage of the 77-kD light chain to a molecular weight of 75 kD, which is similar to thrombin-activated factor Va light chain. Neutrophil elastase also caused rapid inactivation of thrombin- activated factor V, factor Va(t). These observations suggest that elastase cleaves factor V at sites distinct from that by thrombin and therefore represents a novel factor V activation pattern. It is proposed that upon neutrophil activation elastase is secreted into the plasma milieu to initiate factor V activation. This serves to generate small amounts of thrombin that, in turn, by positive feedback fully activates factor V and thus amplifies the coagulation reaction.

scholarly journals The isolation of human platelet factor V [published erratum appears in Blood 1987 Jul;70(1):339]

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1188-1195 ◽  
Author(s):  
RW Viskup ◽  
PB Tracy ◽  
KG Mann
Keyword(s):  
Human Plasma ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor V ◽  
Single Chain ◽  
Platelet Factor ◽  
Factor Va ◽  
Plasma Factor ◽  
End Products

Abstract Human platelet factor V has been isolated using either a monoclonal or polyclonal antibody directed against human plasma factor V. The largest peptide observed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified human platelet factor V comigrates with purified human plasma factor V. However, a significant portion of the isolated protein is represented by peptides of lower apparent molecular weight (Mr). These lower Mr species that copurify with platelet factor V have been shown to be platelet factor V components by their immunological cross-reactivity with monoclonal and polyclonal antibodies to purified human plasma factor V. Platelets isolated from whole blood drawn directly into inhibitors to prevent proteolysis and platelet activation demonstrate the pattern of fragmented platelet factor V. The components of purified platelet factor V demonstrate apparent Mr ranging between 115 K and 330 K and are detectably different from the intermediates and end products observed during the thrombin cleavage of single-chain plasma factor V. Upon treatment with thrombin the platelet factor V components are cleaved and the end products are indistinguishable from those obtained upon thrombin activation of plasma factor V to plasma factor Va. Examination of the components by immunoblotting demonstrates that some of the cleavages which have occurred in the platelet factor V molecule are within the 150-K activation peptide. Bioassay indicates that platelet factor V exists as a procofactor and cleavage by thrombin yields the active cofactor, platelet factor Va. These data suggest that human platelet factor V is stored in the platelet as a partially fragmented procofactor that can be activated by thrombin to yield human platelet factor Va, the active cofactor in the human prothrombinase complex.

scholarly journals The isolation of human platelet factor V [published erratum appears in Blood 1987 Jul;70(1):339]

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1188-1195 ◽  
Author(s):  
RW Viskup ◽  
PB Tracy ◽  
KG Mann
Keyword(s):  
Human Plasma ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor V ◽  
Single Chain ◽  
Platelet Factor ◽  
Factor Va ◽  
Plasma Factor ◽  
End Products

Human platelet factor V has been isolated using either a monoclonal or polyclonal antibody directed against human plasma factor V. The largest peptide observed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified human platelet factor V comigrates with purified human plasma factor V. However, a significant portion of the isolated protein is represented by peptides of lower apparent molecular weight (Mr). These lower Mr species that copurify with platelet factor V have been shown to be platelet factor V components by their immunological cross-reactivity with monoclonal and polyclonal antibodies to purified human plasma factor V. Platelets isolated from whole blood drawn directly into inhibitors to prevent proteolysis and platelet activation demonstrate the pattern of fragmented platelet factor V. The components of purified platelet factor V demonstrate apparent Mr ranging between 115 K and 330 K and are detectably different from the intermediates and end products observed during the thrombin cleavage of single-chain plasma factor V. Upon treatment with thrombin the platelet factor V components are cleaved and the end products are indistinguishable from those obtained upon thrombin activation of plasma factor V to plasma factor Va. Examination of the components by immunoblotting demonstrates that some of the cleavages which have occurred in the platelet factor V molecule are within the 150-K activation peptide. Bioassay indicates that platelet factor V exists as a procofactor and cleavage by thrombin yields the active cofactor, platelet factor Va. These data suggest that human platelet factor V is stored in the platelet as a partially fragmented procofactor that can be activated by thrombin to yield human platelet factor Va, the active cofactor in the human prothrombinase complex.

scholarly journals Activation/inactivation of human factor V by plasmin

Blood ◽  
1989 ◽  
Vol 73 (1) ◽  
pp. 185-190 ◽  
Author(s):  
CD Lee ◽  
KG Mann
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Coagulation Factor ◽  
Temporal Correlation ◽  
Procoagulant Activity ◽  
Factor V ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Active Fragments ◽  
Transient Intermediates

Abstract The effect of human plasmin on human coagulation factor V was studied using isolated proteins. Incubation of factor V with plasmin resulted in a rapid increase in procoagulant activity, followed by a subsequent decline in the ability of factor V to serve as a cofactor in the prothrombinase complex. Identical results were obtained when these reactions were conducted in the presence of dansylarginine-N-(3-ethyl- 1,5-pentanediyl) amide (DAPA), indicating that the changes observed could not have occurred as a consequence of cleavage by alpha-thrombin. Analysis of the products of the reaction by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) revealed a temporal correlation between the rise and fall in factor V activity and the presence of several transient intermediates. These fragments are distinct from the subunits of alpha-thrombin-activated factor V (factor Va). The activation phase of the reaction was not significantly affected by the presence of phospholipid. In contrast, the rate of degradation of active fragments of factor V and the accompanying loss of activity were markedly enhanced in the presence of phospholipid vesicles. These data suggest that the action of plasmin upon factor V results in the transient formation of proteolytic fragments which express significant procoagulant activity.

scholarly journals Sulfation of tyrosine residues in coagulation factor V

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 946-952 ◽  
Author(s):  
GL Hortin
Keyword(s):  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Coagulation Factor ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor V ◽  
Factor Va ◽  
Alkaline Hydrolysate ◽  
Coagulation Factor V

Abstract Sulfation of human coagulation factor V was investigated by biosynthetically labeling the products of HepG2 cells with [35S]sulfate. There was abundant incorporation of the sulfate label into a product identified as factor V by immunoprecipitation, lability to proteases, affinity for the lectin jacalin, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two or more sites in factor V incorporated sulfate as indicated by labeling of different peptide chains of factor Va. The 150-Kd activation fragment of factor Va incorporated the greatest amounts of sulfate. This fragment of factor Va was bound selectively by jacalin-agarose, reflecting its content of O-linked oligosaccharides. Analysis of an alkaline hydrolysate of sulfate-labeled factor Va by anion-exchange chromatography showed that the sulfate occurred partly in tyrosine sulfate residues and partly in alkaline-labile linkages. Sulfate groups are potentially important structural and functional elements in factor V, and labeling with [35S]sulfate provides a useful approach for examining the biosynthesis and processing of this protein. The hypothesis is advanced that sites of sulfation in factor V and several other plasma proteins contribute to the affinity and specificity of thrombin for these molecules, just as it does for the interaction of thrombin with the potent inhibitor hirudin from leeches.

scholarly journals Activation/inactivation of human factor V by plasmin

Blood ◽  
1989 ◽  
Vol 73 (1) ◽  
pp. 185-190 ◽  
Author(s):  
CD Lee ◽  
KG Mann
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Coagulation Factor ◽  
Temporal Correlation ◽  
Procoagulant Activity ◽  
Factor V ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Active Fragments ◽  
Transient Intermediates

The effect of human plasmin on human coagulation factor V was studied using isolated proteins. Incubation of factor V with plasmin resulted in a rapid increase in procoagulant activity, followed by a subsequent decline in the ability of factor V to serve as a cofactor in the prothrombinase complex. Identical results were obtained when these reactions were conducted in the presence of dansylarginine-N-(3-ethyl- 1,5-pentanediyl) amide (DAPA), indicating that the changes observed could not have occurred as a consequence of cleavage by alpha-thrombin. Analysis of the products of the reaction by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) revealed a temporal correlation between the rise and fall in factor V activity and the presence of several transient intermediates. These fragments are distinct from the subunits of alpha-thrombin-activated factor V (factor Va). The activation phase of the reaction was not significantly affected by the presence of phospholipid. In contrast, the rate of degradation of active fragments of factor V and the accompanying loss of activity were markedly enhanced in the presence of phospholipid vesicles. These data suggest that the action of plasmin upon factor V results in the transient formation of proteolytic fragments which express significant procoagulant activity.

scholarly journals Sulfation of tyrosine residues in coagulation factor V

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 946-952 ◽  
Author(s):  
GL Hortin
Keyword(s):  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Coagulation Factor ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor V ◽  
Factor Va ◽  
Alkaline Hydrolysate ◽  
Coagulation Factor V

Sulfation of human coagulation factor V was investigated by biosynthetically labeling the products of HepG2 cells with [35S]sulfate. There was abundant incorporation of the sulfate label into a product identified as factor V by immunoprecipitation, lability to proteases, affinity for the lectin jacalin, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two or more sites in factor V incorporated sulfate as indicated by labeling of different peptide chains of factor Va. The 150-Kd activation fragment of factor Va incorporated the greatest amounts of sulfate. This fragment of factor Va was bound selectively by jacalin-agarose, reflecting its content of O-linked oligosaccharides. Analysis of an alkaline hydrolysate of sulfate-labeled factor Va by anion-exchange chromatography showed that the sulfate occurred partly in tyrosine sulfate residues and partly in alkaline-labile linkages. Sulfate groups are potentially important structural and functional elements in factor V, and labeling with [35S]sulfate provides a useful approach for examining the biosynthesis and processing of this protein. The hypothesis is advanced that sites of sulfation in factor V and several other plasma proteins contribute to the affinity and specificity of thrombin for these molecules, just as it does for the interaction of thrombin with the potent inhibitor hirudin from leeches.

Screening of Single-Chain Variable Fragments against TSP50 from a Phage Display Antibody Library and Their Expression as Soluble Proteins

2006 ◽  
Vol 11 (5) ◽  
pp. 546-552 ◽  
Author(s):  
Jingyan Wei ◽  
Yang Liu ◽  
Songchuan Yang ◽  
Junjie Xu ◽  
Hangtian Kong ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Phage Display ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sandwich Elisa ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Library ◽  
Single Chain ◽  
Single Chain Variable Fragments ◽  
Phage Display Antibody

A novel gene, testes-specific protease 50 ( TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein in Escherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.

Human platelets contain forms of factor V in disulfide-linkage with multimerin

2004 ◽  
Vol 92 (12) ◽  
pp. 1349-1357 ◽  
Author(s):  
Nola Fuller ◽  
Shilun Zheng ◽  
Frédéric Adam ◽  
Samira Jeimy ◽  
Ian Horsewood ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Human Platelets ◽  
Factor V ◽  
Single Chain ◽  
Platelet Factor ◽  
Disulfide Linkage ◽  
Factor Va ◽  
Plasma Factor ◽  
Large Platelet ◽  
Large Factor

SummaryFactor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factorV is stored complexed with the polymeric α-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains, were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Since unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that unusually large factor V was associated with multimerin and it remained associated in 0.5 M salt. Moreover, platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage.The disulfide-linked complexes of multimerin and factor V in platelets, which are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Factor Va generation and function from unusually large platelet factor V is only speculative at this time.

Exosite-dependent regulation of factor VIIIa by activated protein C

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4802-4807 ◽  
Author(s):  
Chandrashekhara Manithody ◽  
Philip J. Fay ◽  
Alireza R. Rezaie
Keyword(s):  
Protein C ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Coagulation Cascade ◽  
Factor Va ◽  
Factor Viiia

AbstractActivated protein C (APC) is a natural anticoagulant serine protease in plasma that down-regulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. Recent results have indicated that basic residues of 2 surface loops known as the 39-loop (Lys37-Lys39) and the Ca2+-binding 70-80–loop (Arg74 and Arg75) are critical for the anticoagulant function of APC. Kinetics of factor Va degradation by APC mutants in purified systems have demonstrated that basic residues of these loops are involved in determination of the cleavage specificity of the Arg506 scissile bond on the A2 domain of factor Va. In this study, we characterized the properties of the same exosite mutants of APC with respect to their ability to interact with factor VIIIa. Time course of the factor VIIIa degradation by APC mutants suggested that the same basic residues of APC are also critical for recognition and degradation of factor VIIIa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the factor VIIIa cleavage reactions revealed that these residues are involved in determination of the specificity of both A1 and A2 subunits in factor VIIIa, thus facilitating the cleavages of both Arg336 and Arg562 scissile bonds in the cofactor.

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