scholarly journals Human Neutrophil Elastase Activates Human Factor V but Inactivates Thrombin-Activated Human Factor V

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1065-1074 ◽  
Author(s):  
John A. Samis ◽  
Marilyn Garrett ◽  
Reginald P. Manuel ◽  
Michael E. Nesheim ◽  
Alan R. Giles
Keyword(s):  
Sequence Analysis ◽  
Heavy Chain ◽  
Human Neutrophil ◽  
Human Factor ◽  
Time Dependent ◽  
Human Neutrophil Elastase ◽  
Factor V ◽  
Terminal Sequence ◽  
Sds Page ◽  
Cofactor Activity

Abstract The effect of human neutrophil elastase (HNE) on human factor V (F.V) or α-thrombin–activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and NH2 -terminal sequence analysis. Incubation of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in a time-dependent increase in its cofactor activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of F.V activation accomplished by incubation with HNE was approximately 65% to 70% of that observed with α-thrombin in presence of Ca2+. The extent of both the HNE-dependent enhancement in F.V cofactor activity and the HNE-dependent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 μmol/L). The HNE-derived cleavage products of F.V, which correlated with increased cofactor activity, as demonstrated by SDS-PAGE under reducing conditions, were different from those generated using α-thrombin. Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in the production of three closely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judged by densitometry. Treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted in the cleavage of both the 96 kD heavy chain and the 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 kD heavy chain was more extensive during the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2 -terminal sequence analysis of F.V treated with HNE indicated cleavage at Ile819 and Ile1484 under conditions during which the procofactor expressed enhanced cofactor activity in the prothrombinase complex. NH2 -terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala341, Ile508, and Thr1767 under conditions, which the cofactor became inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological activator and inactivator of F.V and F.Va, namely α-thrombin (Arg709 and Arg1545) and Activated Protein C (APC) (Arg306 and Arg506), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express significantly enhanced procoagulant cofactor activity similar to that observed following activation with α-thrombin. In contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed following inactivation by APC.

scholarly journals Human Neutrophil Elastase Activates Human Factor V but Inactivates Thrombin-Activated Human Factor V

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1065-1074 ◽  
Author(s):  
John A. Samis ◽  
Marilyn Garrett ◽  
Reginald P. Manuel ◽  
Michael E. Nesheim ◽  
Alan R. Giles
Keyword(s):  
Sequence Analysis ◽  
Heavy Chain ◽  
Human Neutrophil ◽  
Human Factor ◽  
Time Dependent ◽  
Human Neutrophil Elastase ◽  
Factor V ◽  
Terminal Sequence ◽  
Sds Page ◽  
Cofactor Activity

The effect of human neutrophil elastase (HNE) on human factor V (F.V) or α-thrombin–activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and NH2 -terminal sequence analysis. Incubation of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in a time-dependent increase in its cofactor activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of F.V activation accomplished by incubation with HNE was approximately 65% to 70% of that observed with α-thrombin in presence of Ca2+. The extent of both the HNE-dependent enhancement in F.V cofactor activity and the HNE-dependent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 μmol/L). The HNE-derived cleavage products of F.V, which correlated with increased cofactor activity, as demonstrated by SDS-PAGE under reducing conditions, were different from those generated using α-thrombin. Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in the production of three closely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judged by densitometry. Treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted in the cleavage of both the 96 kD heavy chain and the 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 kD heavy chain was more extensive during the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2 -terminal sequence analysis of F.V treated with HNE indicated cleavage at Ile819 and Ile1484 under conditions during which the procofactor expressed enhanced cofactor activity in the prothrombinase complex. NH2 -terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala341, Ile508, and Thr1767 under conditions, which the cofactor became inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological activator and inactivator of F.V and F.Va, namely α-thrombin (Arg709 and Arg1545) and Activated Protein C (APC) (Arg306 and Arg506), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express significantly enhanced procoagulant cofactor activity similar to that observed following activation with α-thrombin. In contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed following inactivation by APC.

Mechanism of the Potent Activated Protein C Resistance of Novel Factor V Mutation with W1920R (FV Nara) Relative to R506Q (FV Leiden)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 537-537
Author(s):  
Kenichi Ogiwara ◽  
Keiko Shinozawa ◽  
Keiji Nogami ◽  
Tomoko Matsumoto ◽  
Katsumi Nishiya ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Protein C ◽  
Activated Protein C ◽  
Vena Cava ◽  
Hek293 Cells ◽  
Factor V ◽  
Activated Protein C Resistance ◽  
Sds Page ◽  
Fv Leiden ◽  
Cofactor Activity

Abstract Abstract 537 Introduction: Factor V (FV) functions both as procoagulant and anticoagulant factors. FV R506Q (FV Leiden) showing activated protein C resistance (APCR) is popular among Caucasians, whilst it has not been reported in Asians. Recently, we have reported a Japanese boy with deep venous thrombosis (DVT) who has a novel FV W1920R mutation (FV Nara, ISTH 2009, 2011). Although he showed APCR in activated partial thromboplastin time (APTT)-based assay, low levels of FV activity (10%) and antigen (40%) made it difficult to explain his severe DVT (both lower extremities and inferior vena cava). Here, we show the detailed mechanisms of his thrombotic diathesis via APCR relative to FV Leiden. Methods: We performed the detection of PC pathway inhibition with Thrombopath® (Protac-induced coagulation inhibition %; PiCi%) or the detection of APCR with calibrated automated thrombogram (CAT) in patient's plasma. FV-deficient plasmas containing varying concentrations of FV wild-type (WT) or W1920R were evaluated by an APCR-assay that specifically can measure the APC cofactor activity of FV in activated FVIII (FVIIIa) inactivation and by the APTT-based assay that probes both the susceptibility and APC cofactor components (Castoldi E, 2004). Recombinant FV proteins (FV-WT, -R506Q, and -W1920R) under pMT2/FV vector were expressed in HEK293 cells. In purified assays, activated FV (FVa) was inactivated by APC in the presence of protein S (PS). FVIIIa were also inactivated by APC/PS in the presence of FV. Cleavages of FV heavy chain were observed in SDS-PAGE and Western blotting. Results: PiCi%, a decrease ratio of thrombin generation by the addition of PC activator (protac) was low in patient's plasma (protac absent/present 794/221 mOD/min; PiCi% 72.2%) (v.s. normal plasma absent/present 834/76.1 mOD/min; PiCi% 90.9%). In CAT using platelet-rich plasmas, peak thrombin level of patient was lower than control (169 and 191 nM), but was greater in the presence of 40 nM APC (103 and 71 nM). In FV-deficient plasma containing FV WT or W1920R, W1920R exhibited no cofactor activity in FVIIIa inactivation, likely supporting the contribution to W1920R-associated APCR more significantly rather than poor susceptibility to APC. In purified assays, FVa (8 nM) activity in FV-WT, -R506Q and -W1920R after 5-min reaction with APC (25 pM), PS (30 nM), PL (20 μM) was decreased to 2.6%, 16.7%, and 62.8%, respectively. SDS-PAGE analysis for FVa heavy chain revealed little cleavage of Gln506 in FV-R506Q and markedly delayed cleavage of Arg506 in FV-W1920R. Of surprise, cleavage of Arg306 was little observed in FV-W1920R. In addition, FVIIIa (10 nM) activity after 20-min reaction with APC (0.5 nM), PS (5 nM), PL (20 μM) and FV (0.5 nM) in FV-WT, -R506Q, and -W1920R was diminished to 18.0%, 52.8% and >95% of those without FV, respectively. Although Trp1920 in FV is close to PL-binding site of the C1 domain, the binding ability of FV-W1920R to immobilized PL retained ∼70-80% of that of FV-WT. Conclusion: The novel FV mutant, W1920R possessed the potent APCR relative to FV Leiden, by reducing the susceptibility of FVa to APC-mediated inactivation and/or by interfering with the FV cofactor activity in APC-catalyzed FVIIIa inactivation. This mechanism might be associated with the impaired cleavages of Arg506 and Arg306 due to the conformational change of this FV mutant. Disclosures: Nogami: Bayer Award 2009: Research Funding.

Mechanisms of Human Neutrophil Elastase-Catalyzed Inactivation of the Factor VIII(a).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2125-2125
Author(s):  
Katsumi Nishiya ◽  
Keiji Nogami ◽  
Tomoko Matsumoto ◽  
Kenichi Ogiwara ◽  
Masahiro Takeyama ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Serine Proteases ◽  
Conflicts Of Interest ◽  
Protein S ◽  
Cathepsin G ◽  
Human Neutrophil Elastase ◽  
Sds Page

Abstract Abstract 2125 Poster Board II-103 Inflammation and coagulation are linked in a variety ways. Since both neutrophil elastase and cathepsin G bind to activated platelets, they can be localized on the platelet membrane providing negatively-charged phospholipid, that is essential for assembly of tenase complex. Although it has been reported that cathepsin G provides some procoagulant effect by activating factor (F)VIII, effect of elastase on FVIII is poorly understood. We now examine the effect of human neutrophil elastase (HNE) on FVIII(a). FVIII activity (used 100 nM) was rapidly decreased and was undetectable within 10 min after the addition of HNE (0.5 nM) in a one-stage clotting assay. This time-dependent inactivation was little or mildly affected (∼35% inhibition at 10 μg/ml) in the presence of phospholipid or von Willebrand factor, respectively. Rate constant of FVIII inactivation by HNE possessed ∼15-fold greater than that by APC/protein S (PS). SDS-PAGE analysis revealed that HNE proteolyzed the 90∼210-kDa heavy chain of FVIII to 47- and 37-kDa terminal products, via some proteolyses within the B domain. Western blot and N-terminal sequence analyses showed that these fragments derived from the heavy chain were identified as the A11-357 and A2375-708, respectively, by cleavages at Val357 and Val374 in A1 and Val708 in A2. The cleavages at Val374 and Val708 by HNE were faster than that at Val357. The 80-kDa light chain was proteolyzed to 75-kDa fragment by cleavage at Val1670, quite different with the pattern of FVIII cleavage by known serine proteases. Of note, HNE-catalyzed inactivation of FVIIIa was much slower (by ∼25-fold) than that of FVIII, supporting that HNE preferably attacked to FVIII. This discrepancy was attributed to much slower cleavage at Val708 in FVIIIa. Rate of inactivation of FVIIIa by HNE was ∼30-, ∼8-, and ∼3-fold lower than those by plasmin, APC/PS, and FXa, respectively. SDS-PAGE using FVIII(a) fragments revealed that each cleavage at Val357 and Val708 was regulated by the presence of the light chain and heavy chain (intact A1-A2), respectively. These results demonstrate that the importance of cleavage at Val708, constitutes a FIXa-interactive site in tenase complex, for the mechanism of HNE-catalyzed FVIII inactivation. Furthermore, this cleavage appears to be selectively modulated by the A1-A2 domain that might interact with HNE. Disclosures: No relevant conflicts of interest to declare.

The Neutrophil Proteases, Elastase and Cathepsin G, May Modulate the Activity of Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1716-1716
Author(s):  
Andrew J. Gale ◽  
Diana Rozenshteyn ◽  
Justin Riceberg
Keyword(s):  
Factor Viii ◽  
Human Neutrophil ◽  
Cleavage Site ◽  
Maximum Level ◽  
Cathepsin G ◽  
Human Neutrophil Elastase ◽  
Factor V ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
A1 Domain

Abstract Neutrophils and monocytes express cathepsin G and elastase and also can bind to activated platelets, thus they can be localized to the site of active coagulation. Early studies suggested that cathepsin G and elastase inactivated factor VIII (FVIII) and were thus anticoagulant. But other studies have suggested procoagulant functions for cathepsin G and elastase in activation of factor V or activation of platelets among other possible mechanisms. Therefore, we investigated the effects of human neutrophil elastase and human neutrophil cathepsin G on FVIII/VIIIa. Elastase does inactivate both FVIII and FVIIIa but cathepsin G activates FVIII while having very little effect on FVIIIa. Cathepsin G activation of FVIII is enhanced by phospholipid vesicles, apparently due to enhanced rate of cleavage and stabilization of the resulting molecule. The maximum level of activation is less than that of thrombin, but it is still four-fold as measured in an APTT assay. Cleavage sites for both proteases in FVIII were identified by Edman degradation and gel analysis. FVIII cleavages are limited to a few specific sites that are mostly located near known activating and inactivating cleavage sites. A notable exception is a cleavage site for elastase after valine 26 in the A1 domain. Cathepsin G cleavage sites near to thrombin cleavage sites likely contribute to the partial activation of FVIII. The unique elastase cleavage site at valine 26 likely contributes to the inactivation of FVIII and FVIIIa. Therefore, it is possible that neutrophils and monocytes may provide some pro-coagulant effect by activating FVIII and may also provide negative feedback by inactivating FVIIIa as well.

Plasma Procofactor Activation by Factor Xla

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1024-1024
Author(s):  
Matthew F Whelihan ◽  
Kenneth G. Mann
Keyword(s):  
Sequence Analysis ◽  
Heavy Chain ◽  
Light Chain ◽  
Thrombin Generation ◽  
Coagulation Cascade ◽  
Sequencing Analysis ◽  
Reducing Conditions ◽  
Sds Page ◽  
Initial Cleavage ◽  
Contact Pathway

Abstract The procofactors FV and FVIII are activated by thrombin, FXa and plasmin. During contact pathway-initiated thrombin generation, FXIa activates FIX thus feeding into the coagulation cascade; however, the procofactors FVIII and FV must be activated to achieve a robust level of thrombin generation. We tested the hypothesis that FXIa can activate FV and FVIII. FV (1uM) was subjected to FXIa (100nM) proteolysis. During the reaction the relative activity and integrity were measured at selected time points using a one stage PT clotting assay and SDS-PAGE. Over the 60 minute time course, FV showed a transient 50% activation followed by a reduction in activity to 20%. SDS-PAGE analyses showed that proteolysis of FV by FXIa initially generated fragments with mobilities similar to those produced by α-thrombin. This activation and inactivation pattern suggested that FXIa makes the required activation cleavages at R709, R1018 and R1545 coincidently with inactivating proteolyses. FV was activated with α-thrombin and the reaction quenched by the addition of hirudin prior to FXIa proteolysis. FVa’s cofactor activity was reduced by 50% after 30 min and 80% after 60 min. Analysis of the FXIa cleavage process by SDS-PAGE under reducing conditions showed no intact heavy chain and significant proteolysis of the light chain after 30 minutes. In addition to the Mr = 105000 (HC) and Mr = 75000 (LC), six new products were identified by SDS-PAGE under reducing conditions: Mr = 54000, 50000, 48000, 30000, 22000 and 20000. NH2-terminal sequence analysis indicated a single cleavage in the light chain at R1765 yielding the products Mr = 50000, 48000: (1766à2196) and the Mr = 30000 (1546à1764), also seen with plasmin and FXa. A cleavage in the heavy chain represented by the Mr = 22000, 20000 (R511à709) fragments is also observed. Sequence analysis determined that the Mr = 54000 fragment represented the NH2-terminus of the heavy chain. Western analysis using a heavy chain antibody showed a transient band Mr = 75000 lying under the light chain that is consistent with an initial cleavage R510. A subsequent cleavage, which is coincident with a decrease in the cofactor’s activity, results in a Mr = 30000 product which is consistent with a cleavage somewhere COOH-terminal to R306(R316?). Activity analyses suggest that the initial cleavage in the heavy chain at R510 leads to a FVaXla 1 molecule with similar activity (50%) to that seen in FVa cleaved by APC in the absence of phospholipid. The FVaXla 1 was treated with APC resulting in complete inactivation of the cofactor. We have also observed an analogous FXIa cleavage pattern in FVIII; however sequencing analysis has not yet been attempted. These data suggest that factor XIa may play a role in procofactor activation and inactivation of FV and FVIII in the context of contact pathway-initiated blood coagulation. Figure Figure

Effect of Heterologous Factor V Heavy Chain Sequences on the Secretion of Recombinant Human Factor VIII

1996 ◽  
Vol 75 (01) ◽  
pp. 036-044 ◽  
Author(s):  
Thomas L Ortel ◽  
Karen D Moore ◽  
Mirella Ezban ◽  
William H Kane
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Human Factor ◽  
Secreted Protein ◽  
Factor V ◽  
Minimal Effect ◽  
Repetitive Domain ◽  
Human Factor Viii ◽  
A1 Domain ◽  
A2 Domain

SummaryFactor VIII and factor V share a repetitive domain structure of A1-A2-B-A3-C1-C2. To define the region(s) within the factor VIII heavy chain that result in inefficient expression of the recombinant protein, we expressed a series of factor VIH/factor V chimeras that contained heterologous sequences from the A1 and/or A2 domains. Substitution of the factor VIIIA1 domain dramatically reduced secretion of factor V ~ 500-fold, whereas substitution of the factor VIII A2 domain had minimal effect on secretion. Conversely, substitution of the factor V A1 domain increased secretion of factor VIII ~3-fold, whereas substitution of the factor V A2 domain actually reduced secretion ~4-fold. Pulse chase experiments confirmed that reduced expression levels were due to decreased secretion rather than instability of secreted protein. Smaller substitutions did not further localize within the A1 domain the regions responsible for inefficient secretion.

Venom-Derived Factor V from the Common Brown Snake P. Textilis Is Expressed as a Constitutively Active Cofactor.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1765-1765
Author(s):  
Mettine H.A. Bos ◽  
Michael Boltz ◽  
Liam St. Pierre ◽  
Martin F. Lavin ◽  
Rodney M. Camire
Keyword(s):  
Disulfide Bond ◽  
Factor Xa ◽  
Proteolytic Processing ◽  
Light Chains ◽  
Factor V ◽  
Single Chain ◽  
Processing Step ◽  
Sds Page ◽  
Cofactor Activity ◽  
Constitutively Active

Abstract Factor V (FV) circulates as procofactor with little or no procoagulant activity and is activated upon proteolytic removal of a central B-domain. Recently, we have shown that discrete B-domain sequences stabilize the inactive procofactor state and that their deletion drives the expression of procoagulant function without the need for proteolytic processing (JBC2007;282:15033). While the B-domain length is highly conserved in most mammals (∼800 aa), recent genomic data indicates that in some vertebrates the FV B-domain is dramatically shortened. The most striking example is found in an Australian snake family (P. textilis and O. scutellatus). These snakes, which are among the most venomous in Australia, have a powerful prothrombin activating complex in their venoms consisting of FXa-like and FVa-like components. Remarkably, both plasma FV and venom FV sequences of these snakes predict a B-domain of only 45 and 46 residues, suggesting that snake FV may have lost B-domain sequences that maintain the protein as procofactor. Alternatively, snake FV could use a different strategy to preserve the procofactor state. To gain insight into this, we expressed and purified venom-derived P. textilis FV (pt-FV) in BHK cells. Pt-FV expressed very well (4mg protein/L media), and SDS-PAGE analysis revealed that it migrated as a single-chain protein with a molecular mass of ∼180 kDa. Upon incubation with thrombin (IIa), pt-FV was completely processed to pt-FVa, yielding fragments similar to the human FVa heavy and light chains. Surprisingly, pt-FVa migrated as a single band on a non-reducing gel, indicating that the heavy and light chains are held together by a disulfide bond. Sequence analysis suggests that this disulfide bond is located between the A2 and A3 domains. Functional analysis using a PT-based clotting assay with human FV-deficient plasma demonstrated that pt-FV has low activity compared to human FVa. This is consistent with previous studies which showed that venom FV is a relatively ineffective cofactor when using bovine factor Xa (FXa), suggesting that it needs snake-derived FXa to optimally function. Despite the low reactivity towards human plasma, we were able to demonstrate that processing of pt-FV to pt-FVa did not significantly increase cofactor activity (activation quotient of 2 compared to 10–15 for human FV). This suggests that unlike all nonhuman FV variants characterized so far, pt-FV is expressed as a constitutively active cofactor. Consistent with this, assessment of cofactor activity using a purified prothrombinase assay with either human FXa or transiently expressed O. scutellatus FXa revealed that the initial rates of prothrombin activation were equivalent for pt-FV or pt-FVa. To further confirm these results we expressed and purified pt-FV in which IIa cleavage sites Arg709 and Arg1545 were eliminated (pt-FV-QQ). SDS-PAGE analysis showed that pt-FV-QQ was not cleaved by IIa, yet functional measurements revealed that its activity was similar to pt-FV or pt-FVa. These data indicate that pt-FV has constitutive cofactor activity and thus bypasses the normal proteolytic processing step to function within prothrombinase. This is in agreement with our previous findings that discrete sequences within the FV B-domain are necessary to maintain FV as procofactor. Thus, venom-derived P. textilis FV represents the first example of a naturally occurring FV variant that does not require proteolytic processing to be active.

Pegylated Versus Glycopegylated G-CSFs and Their Biochemical and Physiological Properties

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4851-4851 ◽  
Author(s):  
Afsaneh Abdolzade-Bavil ◽  
Bridget A. Cooksey ◽  
Christian Scheckermann ◽  
Andreas Lammerich ◽  
Laurie Pukac ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Receptor Binding ◽  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Half Life ◽  
Binding Assay ◽  
Relative Activity ◽  
Human Neutrophil Elastase ◽  
Receptor Binding Assay ◽  
Sds Page

Abstract Background Lipegfilgrastim and pegfilgrastim are long-acting, once-per-cycle recombinant granulocyte colony-stimulating factors (G-CSFs) developed to reduce the duration of neutropenia and the incidence of febrile neutropenia in cancer patients receiving chemotherapy (CTx). Lipegfilgrastim is recombinant methionyl human G-CSF that is modified at its natural O-glycosylation site (threonine134) using a novel glycoPEGylation technology. Pegfilgrastim is a recombinant methionyl human G-CSF with a methoxy-polyethylene glycol-propionaldehyde covalently conjugated to its N-terminus. GlycoPEGylation generates functional PEGylated proteins with increased bioavailability and prolonged duration of action. Previous studies show that lipegfilgrastim has a 60% higher bioavailability and a 30% greater neutrophil response versus pegfilgrastim in healthy volunteers. When exposed to neutrophil elastase, G-CSF is rapidly cleaved and rendered inactive, suggesting that resistance to this degradation may result in an extended half-life. Objective The objectives of these studies were to characterize lipegfilgrastim versus pegfilgrastim with respect to bioavailability and affinity for the G-CSF receptor, to evaluate the effect of human neutrophil elastase on the degradation of lipegfilgrastim versus pegfilgrastim, and to evaluate the effect of degradation on the activity of these study drugs. Methods Lipegfilgrastim and pegfilgrastim binding to the G-CSF receptor was evaluated using: 1) a NFS-60-cell–based [125I]-G-CSF competitive G-CSF receptor binding assay; and 2) a label-free surface plasmon resonance (SPR) technology by Biacore™ platform using a recombinant human G-CSF receptor. For the competitive binding assay, cocktails of NFS-60 cells, free [125I]-G-CSF, and multiple concentrations of either (unlabeled) lipegfilgrastim or pegfilgrastim were incubated, and the cells were analyzed for bound [125I]-G-CSF. Dose-dependent inhibition of [125I]-G-CSF binding is indicative of the study drug binding to the G-CSF receptor. The SPR study evaluated binding kinetics and overall binding affinity between G-CSF receptor and lipegfilgrastim or pegfilgrastim. Degradation of lipegfilgrastim and pegfilgrastim by purified human neutrophil elastase was evaluated by incubation ± elastase and visualization using Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To evaluate the effect of elastase degradation on lipegfilgrastim or pegfilgrastim activity, samples treated with or without elastase for 15 or 120 minutes were incubated with NFS-60 cells for 20 hours, then pulsed with [3H]-thymidine for 4 hours. The level of active G-CSF–stimulated NFS-60 cell proliferation was measured by [3H]-thymidine incorporation. Results In the cell-based [125I]-G-CSF competitive G-CSF receptor binding assay, G-CSF receptor binding was equivalent between lipegfilgrastim and pegfilgrastim as indicated by inhibition of [125I]-G-CSF binding (0.70±0.09 nM IC50 versus 0.72±0.18 nM IC50 (mean±SD). In the SPR study, the affinity for lipegfilgrastim and pegfilgrastim was 481±84 nM and 516±153 nM (mean±SD), respectively. Lipegfilgrastim treated with purified neutrophil elastase for 15 minutes appeared minimally degraded in SDS-PAGE gels and demonstrated a 67% relative activity in [3H]-thymidine proliferation assays compared with undigested lipegfilgrastim. In contrast, pegfilgrastim treated under the same conditions appeared markedly degraded and demonstrated an ∼9% relative activity compared with undigested pegfilgrastim. After treatment with purified neutrophil elastase for 120 minutes, lipegfilgrastim was noticeably degraded and the protein retained an ∼18% relative proliferative activity compared with undigested lipegfilgrastim, versus pegfilgrastim, which was almost entirely degraded and retained no activity compared with undigested pegfilgrastim. Conclusions Based on the present studies, there is no apparent difference in G-CSF receptor-binding affinity between lipegfilgrastim and pegfilgrastim. In addition, lipegfilgrastim showed a greater time-dependent resistance to neutrophil elastase degradation and a greater retention of functional activity, which may provide an explanation for the longer in vivo half-life of lipegfilgrastim versus pegfilgrastim. Disclosures: Abdolzade-Bavil: Merckle GmbH (a Teva Pharmaceutical Inc., company): Employment. Cooksey:Teva Pharmaceuticals, Inc: Employment. Scheckermann:Merckle Biotec GmbH (a Teva Pharmaceuticals, Inc. company): Employment. Lammerich:Merckle/ratiopharm/Teva Pharm Industries: Employment. Pukac:Teva Pharmaceuticals, Inc: Employment. Krasney:Teva Pharmaceuticals, Inc: Employment. Allgaier:Merckle GmbH (a Teva Pharmaceuticals, Inc. company): Employment. Shen:Teva Pharmaceuticals, Inc: Employment. Müller:Teva Pharmaceuticals, Inc: Employment. Liu:Teva Pharmaceuticals, Inc: Employment. von Kerczek:2Teva Pharmaceuticals, Inc.: Employment.

Cleavage of Factor Va Heavy Chain at Arg643 by Thrombin Partially Inactivates the Cofactor.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1703-1703 ◽  
Author(s):  
Evrim Erdogan ◽  
Michael A. Bukys ◽  
Thomas Orfeo ◽  
Kenneth G. Mann ◽  
Michael Kalafatis
Keyword(s):  
Endothelial Cells ◽  
Heavy Chain ◽  
Consensus Sequence ◽  
Factor V ◽  
Single Chain ◽  
Chain Fragment ◽  
Thrombin Cleavage ◽  
Factor Va ◽  
Plasma Factor ◽  
Cofactor Activity

Abstract Prothrombinase, the enzyme complex required to activate prothrombin, is composed of the serine protease factor Xa and the cofactor factor Va, associated in 1:1 stoichiometry on a phospholipid surface in the presence of Ca2+. Incorporation of factor Va in prothrombinase is required for any meaningful rate of thrombin generation and the arrest of hemorrhage. Factor Va inactivation down-regulates thrombin production resulting in the termination of the hemostatic response. The principal enzyme involved in this down-regulation is activated protein C (APC). Factor Va is formed following enzymatic cleavage of the single chain procofactor, factor V (Mr 330,000) by thrombin. Thrombin cleaves and activates the procofactor sequentially at Arg709, Arg1018, and Arg1545. The active cofactor, factor Va, is composed of heavy (HC105, Mr 105,000) and light (Mr 74,000) chains non-covalently associated in the presence of divalent ions. Previous studies of factor Va inactivation on human umbilical vein endothelial cells (HUVEC) have shown that thrombin cleaves the heavy chain at the COOH-terminus to produce a Mr 97,000 fragment containing the NH2-terminal portion of the heavy chain and a Mr 8,000 peptide representing the COOH-terminus of the molecule which remains attached to the heavy chain by a disulfide bond. The thrombin cleavage appeared to occur between residues 586 and 654. This region contains a consensus sequence for cleavage by thrombin located between residues 640–643 (S-P-R). To evaluate the functional importance of thrombin cleavage at Arg643 for factor Va inactivation, site-directed mutagenesis was used to create recombinant factor V molecules with mutations R643→Q (factor VR643Q) and R643→A (factor VR643A). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with thrombin. Under similar experimental conditions, cleavage of HC105 and appearance of the Mr 97,000 heavy chain fragment in the wild type molecule correlated with partial loss of cofactor activity, while following incubation of factor VR643Q and factor VR643A with thrombin no cleavage of HC105 at Arg643 was observed and no presence of the Mr 97,000 heavy chain fragment was noticed. Further, no loss in cofactor activity was observed using these mutant recombinant factor Va molecules following extended incubation with thrombin. The endothelial cell surface has been presumed to be the site of PC activation and factor Va inactivation in vivo. The relative phospholipid composition of endothelial membranes has been suggested to be consistent with their ability to support factor Va inactivation in a manner analogous to the commonly used phospholipid system composed of 25% phosphatidylserine and 75% phosphatidylcholine. In the experiments conducted on the HUVEC surface incubation of 20 nM plasma factor V with 0.1 nM thrombin resulted in almost complete cleavage of HC105 over a 60 minute thrombin treatment. In the experiments presented herein much higher concentrations of thrombin were necessary to obtain a similar effect. The combined data suggest the presence of a cofactor for thrombin on the surface of endothelial cells that would facilitate cleavage of factor Va heavy chain at Arg643. Collectively, the data demonstrate that cleavage of HC105 at Arg643 by thrombin results in a partially inactive cofactor molecule and provide for an APC-independent anticoagulant effect of thrombin.

Sign in / Sign up

Export Citation Format

Share Document