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

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.

THE PHOSPHOLIPID-BINDING SITE OF FACTOR VIII IS LOCATED ON THE 80 kD LIGHT CHAIN

1987 ◽  
Author(s):  
G Kemball-Cook ◽  
S J A Edwards ◽  
K Sewerin ◽  
L-O Andersson ◽  
T W Barrowcliffe
Keyword(s):  
Binding Site ◽  
Light Chain ◽  
Binding Sites ◽  
Immunoaffinity Chromatography ◽  
The Other ◽  
Immunological Methods ◽  
Electrophoretic Separation ◽  
Antigenic Sites ◽  
Reducing Conditions ◽  
Sds Page

The binding of Factoi. VIII (F.VIII) peptides to phospholipid (PL) vesicles has been studied by two different methods involving the use of fractionated anti-F.VIII:C I-Fab123’pre viously reported, i-Fab123’ was fractionated by immunoadsorptionwith F.VIII-PL complexes into two pools:one binding only to PL-binding sites on F.VIIIsAg (PL-site antibody), the other directed against other antigenic sites (non-PL-site antibody).The first technique used was a modification of the method of Weinstein et al. (Proc.Natl.Acad.Sci.USA, 78, 5137-5141, 1981), and involved incubation of the two anti-F.VIII pool swith F.VIII-containing samples, followed by electrophoretic separation of the complexes on the basis of size in non-denaturing SDS gels: this technique allows qualitative analysis of antibody reactive peptides in highly impure samples. Non-PL-site pool reacted with a range of peptides with MrMapparent Mr 90 kD up to 280 kD, a similar pattern to that of ’heavy chain’(HC) peptides of F.VIII seen on SDS-PAGE under reducing conditions; the PL-site antibody, however, reacted only with peptides at apparent Mrs of 80 kD and sometimes150 kD, but not with bands of higher Mr a pattern more consistent with binding to light chain (LC) peptides. Thesame patterns with the two labels were seen in both plasma and F.VIII concentrateThe second approach employed the two labels described above in direct immunoradiometric assays (IFMA’s) on purified human F.VIII peptides prepared by immunoaffinity chromatography and ion exchange on Mono Q gel. Both PL-site and non-PL-site labels measured similar amounts of F.VIII m a sample containing both HC and LC peptides; however, on assaying a sample containing purified HC peptides alone, PL-site antibody measured only 2% of F.VIII:Ag found by non-PL-site label, indicating that PL-binding sites present in samples containing both HC and LC are absent in HC alone.Results from both these immunological methods indicate that the 80 kD LC peptide of F.VIII carries the PL-binding site.

Evidence That the COOH-Terminal Region of Factor Va Heavy Chain Is Involved in the Regulation of Prothrombinase Activity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1025-1025
Author(s):  
Jamila Hirbawi ◽  
Melissa A. Blum ◽  
Michael A. Bukys ◽  
Tivadar Orban ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Thrombin Generation ◽  
Amino Acid Residues ◽  
Terminal Portion ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Sds Page ◽  
Carboxyl Terminal ◽  
Prothrombin Activation

Abstract The proteolytic conversion of prothrombin to thrombin is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. Incorporation of fVa into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. A first cleavage of prothrombin by prothrombinase at Arg320 produces the active intermediate meizothrombin, while the second cleavage at Arg271 produces thrombin. It has been demonstrated that elimination of the carboxyl terminal portion of the heavy chain of fVa by proteolytic enzymes results in a cofactor molecule with decreased clotting activity and slightly increased to normal chromogenic activity. In addition, we have previously shown that the carboxyl terminal portion of the heavy chain of fVa is involved in the interaction of the cofactor with prothrombin. To further ascertain the importance of this region of the molecule for cofactor activity we used PCR based methods to produce recombinant fVa molecules with several portions of the COOH-terminus deleted. Recombinant fV653 has amino acids 653–709 deleted, recombinant fV696 has amino acid residues 680–696 deleted, recombinant fV680 has amino acid residues 653–680 deleted, while recombinant fV709 has amino acid residues 680–709 missing. These recombinant molecules along with wild type fV (fVWT) were transiently expressed in COS7L cells and assessed for their capability to promote prothrombin activation following activation by Russell’s Viper Venom factor V activator (RVV-V activator). Thrombin generation was evaluated by SDS-PAGE and the kinetic parameters of the reactions were determined. While fVa653 and fVa680 were devoid of clotting activity, fVa696 and fVa709 had reduced clotting activities compared to fVaWT and plasma-derived fVa. This level of clotting activity was similar to the clotting activity of a fV molecule that was treated with thrombin and human neutrophil elastase (HNE) resulting in fVaHNE. fVaHNE is cleaved at Ala677/Thr678 resulting in a cofactor with a shorter heavy chain. Further analyses revealed that all mutant recombinant molecules as well as fVaHNE have similar KD values for fXa when compared to plasma fVa and fVaWT. SDS-PAGE analyses of prothrombin activation time courses revealed that the overall cleavage of prothrombin by prothrombinase assembled with fVa696, fVa709, or fVaHNE was slower resulting in accumulation of meizothrombin. This data confirm our previous findings and suggest that this region on the heavy chain of fVa contribute to cofactor function. A logical explanation for these findings is that the COOH-terminus of the heavy chain of fVa participates in the regulation of the rates of appearance/disappearance of meizothrombin. Increased persistence of meizothrombin in the reaction mixture can explain the slower clotting times since it is well known that meizothrombin has poor clotting activity. Thus at a given time point there will be more meizothrombin present in a sample where prothrombinase was assembled with fVa709, or fVa696, or fVaHNE than in a sample where prothrombinase was formed with fVaWT. Overall the data suggests that the COOH-terminal portion of the factor Va heavy chain contributes to the appropriate orientation of prothrombin with respect to the catalytic site of fXa resulting in efficient cleavages at Arg320 /Arg271 and competent thrombin formation.

Lysine-Binding Site-Dependent Interaction with Plasmin and Factor Va Responsible for Plasmin-Catalyzed Factor Va Inactivation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2016-2016
Author(s):  
Katsumi Nishiya ◽  
Keiji Nogami ◽  
Kiyotaka Okada ◽  
Osamu Matsuo ◽  
Kenichi Ogiwara ◽  
...  
Keyword(s):  
Binding Site ◽  
Heavy Chain ◽  
Light Chain ◽  
Active Form ◽  
Limited Proteolysis ◽  
Coagulation Factor ◽  
Dependent Manner ◽  
Competitive Binding Assay ◽  
Factor Va ◽  
Sds Page

Abstract Plasmin (Plm), an active form of plasminogen (Plg), functions as a key enzyme in the fibrinolytic system. Furthermore, this enzyme directly inactivates various coagulation factors such as factor V (FV) and factor VIII (FVIII) by limited proteolysis, suggesting another role of Plm in the regulation of the coagulation system. We recently reported that Plm/Plg interacts with FVIII and its active form (FVIIIa), both dependently and independently of lysine-binding site (LBS) (Blood2007; 110, 522a). In this study, we attempted to localize a factor Va (FVa)-interactive region on Plm (and Plg) using Plm/Plg kringle fragments. Surface plasmon resonance-based assays showed that FVa directly bound to active-site modified Plm (anhydro-Plm) with an ~2-fold higher affinity, compared to Plg (Kd; 97 and 198 nM, respectively). In particular, FVa bound to the immobilized-Plg fragment consisting of kringle 1-2-3 domains (K1-3) (Kd: 706 nM), whilst FVa failed to bind both the kringle 4 domain (K4) and Plg fragment consisting of kringle 5 and catalytic domains (K5-CD). A similar experiment using immobilized FVa also revealed that the K1-3 solely bound to FVa. These results were quite different from those obtained by FVIII and Plm/Plg binding experiment that the K5-CD bound to FVIII(a) more preferably. Competitive binding assay using 6-aminohexanoic acid (6-AHA), a competitor of LBS of Plm/Plg, showed that 6-AHA markedly inhibited (by >90%) the K1-3 binding to FVa (IC50; ~25 μM), suggesting that interaction of FVa with Plm is mostly dependent upon LBS. According to the one stage-clotting assay, 6-AHA inhibited (>90%) Plm-catalyzed inactivation of FVa in a dose-dependent manner (IC50; ~10 μM). Furthermore, Plm-catalyzed inactivation of FVa was blocked by an anti-K1-3 monoclonal antibody (mAb), not by either anti-K4 or anti-K5-CD mAb, although Plm-catalyzed inactivation of FVIII was blocked by anti-K5-CD mAb. In order to confirm that the inhibitory effect of 6-AHA on the Plm-catalyzed inactivation, we performed SDS-PAGE experiment. Plm cleaves FVa at Lys309 and Arg348 in the heavy chain, and at Arg1752 in the light chain. SDS-PAGE analysis revealed that 6-AHA blocked Plm-catalyzed cleavages of the light chain more prominently than that of the heavy chain (IC50; ~10 and ~>100 μM, respectively). These findings suggest that the K1-3 of Plm (and Plg) interacts with the FVa through the LBS-dependent mechanisms, and these interactions likely contribute to the FVa-catalyzed inactivation by proteolytic cleavages at Arg1752 in the light chain. Present study indicated that plasmin-catalyzed protelytic inactivation of coagulation factor is complicatedly regulated by the LBS dependency in the protein and protein interaction.

scholarly journals Detection of in vitro and in vivo cleavage of high molecular weight kininogen in human plasma by immunoblotting with monoclonal antibodies

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 455-462 ◽  
Author(s):  
M Berrettini ◽  
B Lammle ◽  
T White ◽  
MJ Heeb ◽  
HP Schwarz ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Heavy Chain ◽  
Light Chain ◽  
Plasma Samples ◽  
Single Chain ◽  
Sds Page ◽  
Immunoblotting Technique

Abstract Purified human high-mol-wt kininogen (HMWK), the cofactor of the contact phase of blood coagulation, migrated as a single band (approximately 110,000 mol wt) in a continuous buffer sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), but appeared as two separated bands (approximately 120,000 and 105,000 mol wt) when analyzed in a discontinuous buffer SDS-PAGE system. After elution from SDS polyacrylamide gels, each of the two bands showed coagulant activity. Six murine monoclonal antibodies (Mabs) against HMWK were produced and purified. In immunoblotting studies, three Mabs bound to the isolated alkylated heavy chain and one to the alkylated light chain of HMWK, whereas the remaining two bound only to the single-chain or unreduced two-chain molecule. None of the Mabs inhibited the clotting activity of HMWK or its binding to kaolin. Two of the Mabs, one directed against the light chain and one against the heavy chain, were used as specific probes to study HMWK in plasma samples using an immunoblotting technique. The anti-light chain Mab identified two distinct bands (approximately 120,000 and approximately 105,000 mol wt) in normal human plasma, but not in plasma from patients with hereditary HMWK deficiency. The anti-heavy chain Mab detected two additional bands (approximately 60,000 and approximately 54,000 mol wt) corresponding to low-mol-wt kininogen (LMWK) in normal plasma. A sensitive and specific quantitative immunoblotting assay of HMWK antigen in plasma was developed. Moreover, the immunoblotting technique with the anti-light chain Mab was used to detect the cleavage of HMWK in plasma samples after in vitro or in vivo activation of the contact system. The anti- light chain Mab demonstrated in vivo activation and cleavage of HMWK during an angioedema attack in a patient with hereditary angioedema and C1-inhibitor deficiency.

The Role of Amino Acids 700–701 of the Factor Va Heavy Chain During Prothrombin Activation by Factor Xa

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2203-2203
Author(s):  
Jamila Hirbawi ◽  
Paul Y Kim ◽  
Michael E. Nesheim ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Heavy Chain ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Human Origin ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Sds Page ◽  
Carboxyl Terminal ◽  
Prothrombin Activation

Abstract Abstract 2203 Blood coagulation is initiated after vascular injury, promoting formation of the fibrin plug. The prothrombinase complex plays a crucial role during activation of prothrombin (Pro) to thrombin. The complex is composed of the enzyme, factor Xa (fXa), along with its non-enzymatic cofactor, factor Va (fVa), in the presence of calcium on a phospholipid surface. The incorporation of fVa into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. Prothrombinase activates prothrombin through initial cleavage at Arg320 followed by cleavage at Arg271 to yield human alpha-thrombin. This pathway is responsible for the generation of a transient catalytically active intermediate, meizothrombin. Recent data has suggested a differential effect of bovine and human factor Va on prothrombin-1 (Pre-1) activation by prothrombinase. This difference was localized within the last ten amino acids from the carboxyl-terminal region of fVa heavy chain. The only amino acid difference between the two cofactor molecules is localized at position 700–701 where the Asn-Arg dipeptide in the fVa of human origin is replaced by the Asp-Glu sequence in the carboxyl-terminal region of the cofactor of bovine origin. We have therefore constructed a recombinant human mutant fVa molecule with these amino acids mutated to their bovine counterpart. We have created a recombinant fVa molecule with the mutation700NR701 →DE. This recombinant cofactor molecule (fVDE) along with wild type factor V (fVWT) were transiently expressed in COS7 cells, purified to homogeneity, and assessed for their capability to by assembled in prothrombinase and promote Pro activation. Thrombin generation was evaluated by SDS-PAGE in a system using all proteins of human origin and the kinetic parameters of the reactions were determined using a chromogenic substrate to assess for thrombin activity. Kinetic analyses revealed that the Kd of fVaDE for human fXa, as well as the kcat and Km values of prothrombinase assembled with fVaDE for human Pro activation were similar to the values obtained following Pro activation by prothrombinase assembled with fVaWT. Surprisingly, SDS-PAGE analyses of prothrombin activation time courses revealed that the overall rate of cleavage of Pro by prothrombinase assembled with fVaDE was significantly delayed with significant accumulation of the intermediate meizothrombin, and delayed thrombin generation when compared to the rate of activation of Pro by prothrombinase assembled with fVaWT. Two-stage clotting assays (PT times) also revealed that fVaDE had reduced clotting activity when compared to fVaWT. Comparison of the rate of cleavage of two recombinant Pro mutant molecules, rMZ-II a recombinant Pro molecule that cannot be cleaved at Arg271 and rP2-II a recombinant Pro molecule that cannot be cleaved at Arg320, by prothrombinase assembled with fVaDE demonstrated impaired rate of cleavage of both substrates when compared to the rate of cleavage of the mutant recombinant Pro molecules by prothrombinase assembled with fVaWT. These findings were verified by experiments using active-site blocked purified human meizothrombin (FPR-meizo). Prothrombinase assembled with fVaDE was considerably impaired in its ability to cleave FPR-meizo at Arg271 as compared to the ability of prothrombinase assembled with fVaWT for the same cleavage. In fact, gel electrophoresis analyses demonstrated that prothrombinase assembled with fVaDE cleaves FPR-meizo with a rate similar to the cleavage of FPR-meizo by fXa alone. All these data together strongly suggest that the 700NR701 portion of the COOH-terminus of the fVa heavy chain plays a significant role in enzyme-substrate recognition/interaction during Pro activation by prothrombinase and thus regulates the rates of thrombin formation locally at the place of vascular injury. Disclosures: No relevant conflicts of interest to declare.

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.

THROMBIN-ACTIVATED FACTOR VIIIa IS COMPOSED OF A NONCOVALENT 73/51 kD DIMER

1987 ◽  
Author(s):  
P J Fay
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Similar Extent ◽  
Factor Viiia ◽  
Polypeptide Patterns ◽  
Human Factor Viii ◽  
Sds Page

Human factor VIII purified from plasma concentrates consists of a series of heterodimers composed of a light chain of 83 kD noncovalently bound to a heavy chain which varies in size from 93 to 170 kD. Previously, we showed that each of the purified heterodimers wasactivated by thrombin to a similar extent. Activation to factor VIIIa was correlated with proteolysis of the light chain generating a73 kD polypeptide and cleavage of the heavy chain(s) generating polypeptides of 51 and 43 kD, whereas subsequent inactivation of factor VIIIa occurred in the absence of further proteolysis (Biochim Biophys Acta 871:268-278, 1986). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of reduced or nonreduced samples showed similar polypeptide patterns indicating that there were no covalent linkages between the 51 and 43 kD chains. However, prior data does not distinguish between a factor VIIIa complex of the 73, 51 and 43 kD polypeptides and a subset of these chains. To identify factor VIIIa, thrombin- treated factor VIII at peak activity was subjected to rapid gel filtration on Superose 12. Factor VIII activity eluted as a single peak representing about 30% of the applied activity after correction for spontaneous inactivation. SDS-PAGE followedby silver staining showed that activity was correlated to fractionscontaining the 73 and 51 kD polypeptides, which co-eluted and which were separated from both the 43 kD fragment and thrombin. Densitometric scans of the stained gel indicated the stoichiometry of the 73:51 kD polypeptides in eachactive fraction to be 1:1. Addition of EDTA(50 mM) to a similar thrombin-factor VIII mixture resulted in rapid inactivation of factor VIIIa. Gel filtration followed by SDS-PAGE analysis of this sample showed that the 73 and 51 kD polypeptides eluted separately and were more included, while the elution position of the 43 kD polypeptide was unchanged. These results suggest that factor VIIIa is represented by a noncovalent dimer consisting of a 73 kD polypeptide derivedfrom the light chain plus a 51 kD polypeptide derived from the heavy chain.

scholarly journals Detection of in vitro and in vivo cleavage of high molecular weight kininogen in human plasma by immunoblotting with monoclonal antibodies

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 455-462 ◽  
Author(s):  
M Berrettini ◽  
B Lammle ◽  
T White ◽  
MJ Heeb ◽  
HP Schwarz ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Heavy Chain ◽  
Light Chain ◽  
Plasma Samples ◽  
Single Chain ◽  
Sds Page ◽  
Immunoblotting Technique

Purified human high-mol-wt kininogen (HMWK), the cofactor of the contact phase of blood coagulation, migrated as a single band (approximately 110,000 mol wt) in a continuous buffer sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), but appeared as two separated bands (approximately 120,000 and 105,000 mol wt) when analyzed in a discontinuous buffer SDS-PAGE system. After elution from SDS polyacrylamide gels, each of the two bands showed coagulant activity. Six murine monoclonal antibodies (Mabs) against HMWK were produced and purified. In immunoblotting studies, three Mabs bound to the isolated alkylated heavy chain and one to the alkylated light chain of HMWK, whereas the remaining two bound only to the single-chain or unreduced two-chain molecule. None of the Mabs inhibited the clotting activity of HMWK or its binding to kaolin. Two of the Mabs, one directed against the light chain and one against the heavy chain, were used as specific probes to study HMWK in plasma samples using an immunoblotting technique. The anti-light chain Mab identified two distinct bands (approximately 120,000 and approximately 105,000 mol wt) in normal human plasma, but not in plasma from patients with hereditary HMWK deficiency. The anti-heavy chain Mab detected two additional bands (approximately 60,000 and approximately 54,000 mol wt) corresponding to low-mol-wt kininogen (LMWK) in normal plasma. A sensitive and specific quantitative immunoblotting assay of HMWK antigen in plasma was developed. Moreover, the immunoblotting technique with the anti-light chain Mab was used to detect the cleavage of HMWK in plasma samples after in vitro or in vivo activation of the contact system. The anti- light chain Mab demonstrated in vivo activation and cleavage of HMWK during an angioedema attack in a patient with hereditary angioedema and C1-inhibitor deficiency.

The Acidic COOH-Terminal Region of Factor Va Heavy Chain Controls the Rate of Prothrombin Activation by Prothrombinase.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1745-1745
Author(s):  
Jamila Hirbawi ◽  
Michael A. Bukys ◽  
Melissa A. Barhoover ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Heavy Chain ◽  
Alternative Pathway ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Factor V ◽  
Divalent Metal Ions ◽  
Factor Va ◽  
Initial Cleavage ◽  
Acidic Region

Abstract The blood coagulation cascade is initiated at the site of vascular injury and results in the activation of prothrombin (Pro) to thrombin by the prothrombinase (IIase) complex. IIase is composed of the enzyme, factor Xa (fXa) bound to its cofactor, factor Va (fVa) on a phospholipid surface in the presence of Ca2+ ions. Two pathways for Pro activation are possible: membrane-bound fXa alone activates Pro following initial cleavage at Arg271 followed by cleavage at Arg320, while the fully assembled IIase activates Pro following the opposite pathway, initial cleavage at Arg320 followed by cleavage at Arg271. Activation of Pro via this latter pathway is characterized by the generation of a transient intermediate, meizothrombin (MzT) that has proteolytic activity. Initial cleavage of prothrombin at Arg320 resulting in MzT generation is absolutely fVa-dependent. Human factor V (fV) is activated by thrombin to produce a heterodimer consisting of a heavy chain and a light chain associated through divalent metal ions. The heavy chain of fVa contains an acidic hirudin-like region at the COOH-terminus (amino acids 680–709). We have shown using overlapping peptides from this region that a pentapeptide with the sequence DYDYQ inhibits Pro activation by IIase by inhibiting MzT generation. In has been reported that various proteases can cleave the acidic region of fVa heavy chain to produce a cofactor with a truncated heavy chain. All the studies revealed that removal of the acidic COOH-terminal portion of fVa heavy chain results in a cofactor molecule that is deficient in its clotting activity in a clotting assay using fV-deficient plasma, however IIase assembled with cofactor molecules missing the acidic COOH-terminus have significant higher kcat for Pro activation as assessed in an assay using purified reagents and a chromogenic substrate specific for thrombin. A molecular explanation for these paradoxical observations has not yet been provided. We have created a mutant recombinant fV molecule that is missing the last 30 amino acids from the heavy chain (fVΔ680-709). The clotting activity of the mutant molecule was impaired as compared to wild type fVa. IIase assembled with fVaΔ680-709 demonstrated a 30–40% increase in the kcat for the activation of Pro. Interstingly, gel electrophoresis revealed a delay in Pro activation with persistence of MzT during activation. Further experiments demonstrated that peptide DYDYQ inhibited MzT formation by IIase assembled with fVaΔ680-709. It has been well established that while MzT has poor clotting activity, its amidolytic activity is considerable increased towards small chromogenic substrates compared to thrombin. A logical explanation that will reconcile all the findings described above is that, the acidic COOH-terminus of fVa heavy chain regulates MzT concentration within IIase during the factor Xa catalyzed Pro activation. Thus, activation of Pro by IIase assembled with a cofactor that is missing the acidic region will result in increased MzT production. This result will be translated by a fVa molecule that is deficient in its clotting activity and produces an increase in kcat when introduced into IIase. In contrast, in the presence of an excess of DYDYQ no MzT is made by IIase resulting in the slow generation of thrombin through the alternative pathway. Our data are consistent with the interpretation that the acidic COOH-terminus of fVa heavy chain contributes a major productive interactive site for Pro within IIase.

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