FVIII Heavy Chain Enhances Tenase Activity Induced By FVIIIa Mimicking Bispesific Antibody, ACE910

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1481-1481
Author(s):  
Hiroaki Minami ◽  
Keiji Nogami ◽  
Takehisa Kitazawa ◽  
Kunihiro Hattori ◽  
Midori Shima
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Research Funding ◽  
Factor Xa ◽  
Factor X ◽  
Advisory Committees ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Physiological Structure

Abstract Background: ACE910, asymmetric bispecific monoclonal antibodies to activated factor IX (IXa) and factor X, mimics the cofactor function of activated factor VIII (VIIIa) by modulating an optimal position on the tenase assembly. The estimated therapeutic range of ACE910 shows ~30% of thrombin generation in native tenase assembly, supporting that the structure on ACE910-mimicking tenase assembly is different from that on native tenase. Being close to physiological structure consisting from factor IXa, factor X, and factor VIIIa is important for potentiating the clotting function. We examined the effects of factor VIII subunits (light chain, heavy chain, A1 and A2, C2) on ACE910-tenase. Materials/Methods: The factor VIII light chain and heavy chain were isolated from EDTA-treated recombinant factor VIII following chromatography on SP- and Q- Sepharose columns. The A2 and A1 subunits were purified from thrombin-cleaved factor VIII heavy chain by Heparin-, SP- Sepharose columns. Purified factor Xa generation assays was examined with (i) factor VIII subunit (0-40 nM), ACE910 (10 µg/ml), phospholipid (PL) (40 µM), factor IXa (1 nM) and factor X (200 nM), (ii, iii) the A2 or heavy chain (40 nM), ACE910 (10 µg/ml), PL (40 µM), factor IXa and factor X (1 or 0-80 nM, and 0-300 or 200 nM, respectively). These mixtures were reacted for five minutes (i, ii) or one minute (iii). These assays were conducted at 37 °C. Results: (i) The factor Xa generation in ACE910-tenase complex in the absence of factor VIIIa was 10.1±2.2 nM. With the intact heavy chain and A2, amounts of factor Xa were increased dose-dependently, resulting in 1.3- and 1.2-fold increases, respectively. While, the light chain and A1 subunit failed to increase at all. (ii) Vmax for factor X in ACE910-tenase was 173.0±7.0 nM and Km was 31.2±3.9 nM. Vmax obtained with the heavy chain or A2 was 175.9±6.1 or 159.0±6.1 nM, whilst Km was 17.0±2.2 or 31.9±3.5 nM, respectively, indicating that the heavy chain enhanced the binding affinity for factor X in ACE910-tenase. (iii) Vmax for factor IXa in ACE910-tenase was 43.8±2.7 nM and Km was 36.9±4.8 nM. With the heavy chain or A2, Vmax was 46.8±3.0 or 45.0±3.1 nM, and Km was 36.4±3.0 or 32.1±4.9 nM, respectively, indicating that either the heavy chain or A2 did not enhance the catalytic activity and the binding affinity for factor IXa in ACE910-tenase. Conclusion: ACE910-tenase assembly seems to be close to physiological structure by the presence of intact heavy chain interacting with factor X. In addition, ACE910 may substitute the position such as the factor VIII(a) light chain associated with FIXa and FX on ACE910-tenase assembly defecting factor VIII. Disclosures Minami: Chugai Pharmaceutical Co., Ltd.: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Cleavage of Factor VIII Heavy Chain Is Required for the Functional Interaction of A2 Subunit with Factor IXa

2001 ◽  
Vol 276 (15) ◽  
pp. 12434-12439 ◽  
Author(s):  
Philip J. Fay ◽  
Maria Mastri ◽  
Mary E. Koszelak ◽  
Hironao Wakabayashi
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Fluorescence Anisotropy ◽  
Factor Xa ◽  
Factor X ◽  
Functional Interaction ◽  
Factor Ixa ◽  
Factor Viiia ◽  
A2 Domain ◽  
Stimulation Of

Factor VIII circulates as a noncovalent heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B domains) and light chain (LC). Cleavage of HC at the A1-A2 and A2-B junctions generates the A1 and A2 subunits of factor VIIIa. Although the isolated A2 subunit stimulates factor IXa-catalyzed generation of factor Xa by ∼100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa and X resulted in an increase in factor IXa activity because of conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of ∼1% observed for thrombin. HC showed little inhibition of the A2 subunit-dependent stimulation of factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa in the presence of factor X, whereas thrombin-cleaved HC yielded a marked increase in this parameter. These results indicate that HC cleavage by either thrombin or factor Xa is essential to expose the factor IXa-interactive site(s) in the A2 subunit required to modulate protease activity.

scholarly journals Proteolytic interactions of factor IXa with human factor VIII and factor VIIIa

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3120-3126 ◽  
Author(s):  
BJ Lamphear ◽  
PJ Fay
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Prolonged Incubation ◽  
Heavy Chains ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Human Factor Viii ◽  
A1 Domain ◽  
Proteolytic Cleavages

Abstract Factor IXa was shown to inactivate both factor VIII and factor VIIIa in a phospholipid-dependent reaction that could be blocked by an antifactor IX antibody. Factor IXa-catalyzed inactivation correlated with proteolytic cleavages within the A1 subunit of factor VIIIa and within the heavy chain (contiguous A1-A2-B domains) of factor VIII. Furthermore, a relatively slow conversion of factor VIII light chain to a 68-Kd fragment was observed after prolonged incubation. Sites of cleavage were identified within the A1 domain at Arg336-Met337 and within the factor VIII light chain at Arg1719-Asn1720. Factor IXa failed to cleave isolated factor VIII heavy chains, yet cleaved isolated factor VIII light chain. In addition, the purified A1/A3-C1-C2 dimer derived from factor VIIIa was a substrate for factor IXa; however, cleavage of the A1 subunit occurred at less than 30% the rate of cleavage of A1 in trimeric factor VIIIa. These data suggest that factor VIII light chain contributes to the binding site for factor IXa and also support a role for a heavy chain determinant located within the A2 subunit in the association of factor VIIIa with factor IXa. Furthermore, the capacity of factor IXa to proteolytically inactivate its cofactor, factor VIIIa, suggests a mode of regulation within the intrinsic tenase complex.

Role of the Direct Interaction between Factor VIII C2 and Factor IXa Gla Domain in the Factor Xase Complex.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2687-2687
Author(s):  
Tetsuhiro Soeda ◽  
Keiji Nogami ◽  
Masahiro Takeyama ◽  
Kenichi Ogiwara ◽  
Kazuhiko Tomokiyo ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Dependent Manner ◽  
C2 Domain ◽  
Factor X ◽  
Calcium Dependent ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Gla Domain

Abstract Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 domains of factor VIII interact with the catalytic domain and EGF2 domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl2. This binding was competitively inhibited by the addition of excess of factor VIII or rC2 domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity (Kd) of rC2 domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 domain binding to EGR-factor IXa in a dose-dependent manner (IC50; 758 nM), suggesting the authentic of the C2 domain and factor IXa-GD interaction. The addition of rC2 domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC50; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 domain in the association between factor VIII and factor IXa in the factor Xase complex.

Regulation of Factor VIIIa in the Intrinsic Factor Xase

1999 ◽  
Vol 82 (08) ◽  
pp. 193-200 ◽  
Author(s):  
Philip Fay
Keyword(s):  
Factor Viii ◽  
Intrinsic Factor ◽  
Factor Xa ◽  
Clinical Importance ◽  
Factor X ◽  
Subunit Dissociation ◽  
Protein Factor ◽  
Catalytic Rate Constant ◽  
Factor Ixa ◽  
Factor Viiia

IntroductionHemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in the plasma protein factor VIII. The activated form of the protein serves as an essential cofactor for factor IXa in the conversion of factor X to factor Xa. This surface-bound complex of enzyme and cofactor is referred to as the intrinsic factor Xase. Factor VIIIa dramatically increases the catalytic rate constant for substrate conversion by an unclear mechanism. The activity and stability of the factor Xase appears to be regulated by the integrity of the cofactor. Factor VIIIa possesses a labile structure, and subunit dissociation results in the decay of Xase activity. Furthermore, factor VIIIa is a substrate for proteolytic inactivation by several enzymes, including factor IXa, the enzyme for which it serves as a cofactor. Although interest in the structure, function, and metabolism of factor VIII is commensurate with its biochemical and clinical importance, the molecular basis for its role in coagulation and the regulation of function through complex intramolecular and intermolecular interactions remain poorly understood.

scholarly journals Characterization of des-(741–1668)-factor VIII, a single-chain factor VIII variant with a fusion site susceptible to proteolysis by thrombin and factor Xa

1995 ◽  
Vol 312 (1) ◽  
pp. 49-55 ◽  
Author(s):  
M J S H Donath ◽  
R T M de Laaf ◽  
P T M Biessels ◽  
P J Lenting ◽  
J W van de Loo ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Heavy Chain ◽  
Light Chain ◽  
Factor Xa ◽  
Single Chain ◽  
Factor Ixa ◽  
Fusion Site ◽  
Von Willebrand ◽  
Willebrand Factor

A factor VIII variant has been characterized in which the heavy chain is directly fused to the light chain. Des-(741-1668)-factor VIII lacks the processing site at Arg1648, as Arg740 of the heavy chain is fused to Ser1669 of the light chain. The sequence of the fusion site is similar to that of other cleavage sites in factor VIII. The fusion site of des-(741-1668)-factor VIII was readily cleaved by both thrombin and factor Xa, and the same result was obtained for heavy chain cleavage. In contrast, des-(741-1668)-factor VIII cleavage by thrombin at position Arg1689 proceeded at a lower rate than the analogous cleavage by factor Xa, which presumably takes place at position Arg1721. The rate of cleavage at position Arg1689 by thrombin was also lower than that at the other processing sites. When des-(741-1668)-factor VIII was activated by thrombin, initial rates of factor Xa formation were similar to the rates obtained when plasma-derived factor VIII was activated by thrombin or factor Xa. Remarkably, activation of des-(741-1668)-factor VIII proceeded at a higher rate by factor Xa than by thrombin. These results indicate that factor VIII activation is strongly associated with cleavage at position Arg1689 or Arg1721. For the interaction between des-(741-1668)-factor VIII and von Willebrand factor, a Kd value of (0.8 +/- 0.3) x 10(-10) M was determined, which is similar to that of heterodimeric factor VIII. The affinity of single-chain des-(741-1668)-factor VIII for factor IXa was found to be 27 +/- 6 nM. The in vivo recovery and half-life of des-(741-1668)-factor VIII were assessed in guinea pigs. Upon infusion of des-(741-1668)-factor VIII at a dosage of 50 units/kg body weight, a rise of 1.0 +/- 0.3 unit/ml in factor VIII activity was obtained. The same recovery was determined for wild-type factor VIII. The half-life of des-(741-1668)-factor VIII was found to be 3 +/- 1 h, compared with 4 +/- 2 h for heterodimeric recombinant factor VIII. In conclusion, des-(741-1668)-factor VIII displays normal activity, is readily cleaved by thrombin and factor Xa at its fusion site, binds with high affinity to von Willebrand factor and factor IXa, and behaves like heterodimeric recombinant factor VIII in guinea pigs. By virtue of these properties, des-(741-1668)-factor VIII may prove useful for the treatment of bleeding episodes in patients with haemophilia A.

scholarly journals Proteolytic interactions of factor IXa with human factor VIII and factor VIIIa

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3120-3126 ◽  
Author(s):  
BJ Lamphear ◽  
PJ Fay
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Prolonged Incubation ◽  
Heavy Chains ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Human Factor Viii ◽  
A1 Domain ◽  
Proteolytic Cleavages

Factor IXa was shown to inactivate both factor VIII and factor VIIIa in a phospholipid-dependent reaction that could be blocked by an antifactor IX antibody. Factor IXa-catalyzed inactivation correlated with proteolytic cleavages within the A1 subunit of factor VIIIa and within the heavy chain (contiguous A1-A2-B domains) of factor VIII. Furthermore, a relatively slow conversion of factor VIII light chain to a 68-Kd fragment was observed after prolonged incubation. Sites of cleavage were identified within the A1 domain at Arg336-Met337 and within the factor VIII light chain at Arg1719-Asn1720. Factor IXa failed to cleave isolated factor VIII heavy chains, yet cleaved isolated factor VIII light chain. In addition, the purified A1/A3-C1-C2 dimer derived from factor VIIIa was a substrate for factor IXa; however, cleavage of the A1 subunit occurred at less than 30% the rate of cleavage of A1 in trimeric factor VIIIa. These data suggest that factor VIII light chain contributes to the binding site for factor IXa and also support a role for a heavy chain determinant located within the A2 subunit in the association of factor VIIIa with factor IXa. Furthermore, the capacity of factor IXa to proteolytically inactivate its cofactor, factor VIIIa, suggests a mode of regulation within the intrinsic tenase complex.

Cleavage at Arg740 Appears Essential for Thrombin-Catalyzed Cleavage at Arg372 during the Activation of Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1692-1692
Author(s):  
Jennifer Newell ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Time Course ◽  
Specific Activity ◽  
Factor Xa ◽  
Factor X ◽  
Wild Type ◽  
Thrombin Cleavage ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Initial Cleavage

Abstract Factor VIIIa serves as an essential cofactor for the factor IXa-catalyzed activation of factor X during the propagation phase of coagulation. The factor VIII procofactor is converted to factor VIIIa by thrombin-catalyzed proteolysis of three P1 positions at Arg372 (A1–A2 junction), Arg740 (A2–B junction), and Arg1689 (a3–A3 junction). Cleavage at Arg372 exposes a cryptic functional factor IXa-interactive site, while cleavage at Arg1689 liberates factor VIII from von Willebrand factor and contributes to factor VIIIa specific activity, thus making both sites essential for procofactor activation. However, cleavage at Arg740, separating the A2–B domainal junction, has not been rigorously studied. To evaluate thrombin cleavage at Arg740, we prepared and stably expressed two recombinant factor VIII mutants, Arg740His and Arg740Gln. Results from a previous study examining proteolysis at Arg372 revealed substantially reduced cleavage rates following substitution of that P1 Arg with His, whereas replacing Arg with Gln at residue 372 yielded an uncleavable bond at that site (Nogami et al., Blood, 2005). Specific activity values for the factor VIII Arg740His and Arg740Gln variants as measured using a one-stage clotting assay were approximately 50% and 18%, respectively, that of the wild type protein. SDS-PAGE and western blotting following a reaction of factor VIII Arg740His with thrombin showed reduced rates of cleavage at His740 as well as at Arg372 relative to the wild type. Alternatively, factor VIII Arg740Gln was resistant to thrombin cleavage at Gln740 and showed little, if any, cleavage at Arg372 over an extended time course. The mutant proteins assayed in a purified system by factor Xa generation showed a slight increase in activity for the Arg740His variant compared with the Arg740Gln variant in both the absence and presence of thrombin, and the activities for both variants were reduced compared with wild type factor VIII. These results suggest that cleavage at residue 740 affects subsequent cleavage at Arg372 and generation of the active cofactor factor VIIIa. Preliminary results obtained evaluating proteolysis of these mutants by factor Xa, which cleaves the same sites in factor VIII as thrombin, also revealed slow proteolysis at the P1 His and no cleavage at the P1 Gln. However, subsequent cleavage at Arg372 exhibited less dependence on initial cleavage at residue 740. These observations may explain the higher than predicted specific activity values obtained for the two variants and suggest a different mechanism of action for the two activating proteinases. Overall, these results support a model whereby cleavage of factor VIII heavy chain by thrombin is an ordered pathway with initial cleavage at Arg740 required to facilitate cleavage at the critical Arg372 site to yield the active cofactor.

Protein S Down-Regulates Factor VIIIa by Competing with Factor IXa.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1714-1714
Author(s):  
Masahiro Takeyama ◽  
Keiji Nogami ◽  
Kohei Tatsumi ◽  
Yuri Fujita ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Protein S ◽  
Factor X ◽  
Cleavage Rate ◽  
Physiological Concentration ◽  
C2 Domains ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Km Value

Abstract Factor VIII functions as a cofactor in the factor Xase complex responsible for phospholipid surface-dependent conversion of factor X to factor Xa by factor IXa. Factor VIIIa, activated form by thrombin and factor Xa, is down regulated by activated protein C (APC), and the reaction is enhanced by the presence of protein S, a cofactor for APC. It was previously reported that protein S inactivated directly factor Xa or factor Va, however, the direct regulation of factor VIII by protein S remains to be investigated. In the present study, surface plasmon resonance (SPR)-based assay showed that factor VIII bound directly to immobilized protein S (Kd; 70 nM). The isolated A2 and A3 domains also bound to protein S with similar modest affinity (Kd; 15 and 17 nM, respectively), whilst the isolated A1 and C2 domains failed to bind, suggesting the presence of protein S-binding sites within the A2 and A3 domain. Since it is known that factor IXa also interacts with the A2 and A3 domains in factor VIII, we examined the inhibitory effect of factor IXa on the factor VIII and protein S interaction in a SPR-based assay. Active-site modified (EGR−) factor IXa competitively inhibited the binding of protein S to both the A2 and A3-C1-C2 domains dose-dependently. Furthermore, Western blotting analysis using an anti-A1 monoclonal antibody revealed that Arg336 cleavage in factor VIII by factor IXa in the presence of protein S was slower with an ~1.8-fold lower cleavage rate than that in its absence, supporting that protein S competed the factor IXa interaction with factor VIII. Of interest, the reaction with protein S to factor VIII inhibited the generation of factor Xa dose-dependently in a factor Xa generation assay (IC50; 150 nM). The Km value for factor X obtained with factor Xase complex in the presence of physiological concentration of protein S was 19 nM, which was ~2-fold lower than that in its absence (45 nM). Whilst, the Km value for factor IXa in the presence of protein S was greater than 100 nM, which was ~5000-fold higher than that in its absence (21 pM). We demonstrate that protein S not only contributes to down-regulate factor VIIIa activity as a cofactor for APC, but also impairs the factor Xase complex by competing the binding of factor IXa to factor VIII.

scholarly journals Functional assembly of intrinsic coagulation proteases on monocytes and platelets. Comparison between cofactor activities induced by thrombin and factor Xa.

1992 ◽  
Vol 176 (1) ◽  
pp. 27-35 ◽  
Author(s):  
M P McGee ◽  
L C Li ◽  
M Hensler
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Cascade ◽  
Factor X ◽  
Regulatory Pathways ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Human Factor Viii

Generation of coagulation factor Xa by the intrinsic pathway protease complex is essential for normal activation of the coagulation cascade in vivo. Monocytes and platelets provide membrane sites for assembly of components of this protease complex, factors IXa and VIII. Under biologically relevant conditions, expression of functional activity by this complex is associated with activation of factor VIII to VIIIa. In the present studies, autocatalytic regulatory pathways operating on monocyte and platelet membranes were investigated by comparing the cofactor function of thrombin-activated factor VIII to that of factor Xa-activated factor VIII. Reciprocal functional titrations with purified human factor VIII and factor IXa were performed at fixed concentrations of human monocytes, CaCl2, factor X, and either factor IXa or factor VIII. Factor VIII was preactivated with either thrombin or factor Xa, and reactions were initiated by addition of factor X. Rates of factor X activation were measured using chromogenic substrate specific for factor Xa. The K1/2 values, i.e., concentration of factor VIIIa at which rates were half maximal, were 0.96 nM with thrombin-activated factor VIII and 1.1 nM with factor Xa-activated factor VIII. These values are close to factor VIII concentration in plasma. The Vsat, i.e., rates at saturating concentrations of factor VIII, were 33.3 and 13.6 nM factor Xa/min, respectively. The K1/2 and Vsat values obtained in titrations with factor IXa were not significantly different from those obtained with factor VIII. In titrations with factor X, the values of Michaelis-Menten coefficients (Km) were 31.7 nM with thrombin-activated factor VIII, and 14.2 nM with factor Xa-activated factor VIII. Maximal rates were 23.4 and 4.9 nM factor Xa/min, respectively. The apparent catalytic efficiency was similar with either form of factor VIIIa. Kinetic profiles obtained with platelets as a source of membrane were comparable to those obtained with monocytes. These kinetic profiles are consistent with a 1:1 stoichiometry for the functional interaction between cofactor and enzyme on the surface of monocytes and platelets. Taken together, these results indicate that autocatalytic pathways connecting the extrinsic, intrinsic, and common coagulation pathways can operate efficiently on the monocyte membrane.

scholarly journals The Role of Factor IXa and Factor VIII in the Activation of Factor X.

1977 ◽  
Author(s):  
Earl W. Davie ◽  
Gordon Vehar ◽  
Kazuo Fujikawa ◽  
Richard Di Scipio
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Serine Proteases ◽  
Factor Xa ◽  
Basic Mechanism ◽  
Factor X ◽  
Amino Terminal ◽  
Factor Ixa ◽  
A Charge

Factor IXa and factor VIII participate in the middle phase of blood coagulation. These two proteins convert factor X to factor Xa in the presence of calcium ions and phospholipid. The coagulant activity of factor VIII is increased 50-100 fold by the addition of thrombin, and this activity is stabilized in the presence of CaCl2. The activated product (tentatively identified as activated factor VIII) was readily inhibited by diisopropyl phosphorofluoridate or antithrombin III, suggesting that it is a serine enzyme. The exact role of this enzyme in the conversion of factor X to factor Xa, however, is not known. When factor X (bovine or human) is converted to factor Xa, an activation peptide is cleaved from the amino-terminal end of the heavy chain. This gives rise to a new amino-terminal sequence of Ile-Val-Gly-Gly-in the heavy chain. No change occurs in the light chain during the activation reaction. These data indicate that the basic mechanism involved in the conversion of human and bovine factor X to factor Xa appears to be essentially identical and probably involves the formation of a charge relay system characteristic of the pancreatic serine proteases.

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