Bryan J. Clarke

Expression and purification of recombinant rabbit factor VII.

To facilitate studies of the in vivo role of the extrinsic pathway of coagulation in experimental hemostasis and thrombosis, a full-length cDNA-encoding rabbit factor VII was isolated using polymerase chain reaction-mediated DNA amplification from plaque-purified lambda gt11 phage. Repeated DNA sequencing of both full-length rabbit factor VII cDNA and shorter cDNA fragments verified four changes in the previously reported amino acid sequence of mature rabbit factor VII, now predicted to be 405 amino acids in length. Rabbit factor VII cDNA was transfected into human embryonic kidney 293 cells and a cell line that permanently expressed rabbit recombinant factor VII was established. Rabbit recombinant factor VII was purified from tissue culture media using a combination of barium citrate precipitation, DEAE-sepharose FF chromatography, benzamidine agarose, and affinity chromatography using a sheep antirabbit factor VII polyclonal antibody. The purity and authenticity of rabbit recombinant factor VII was confirmed by polyacrylamide gel electrophoresis and Western blot analysis. Homogeneous rabbit recombinant factor VII was fully active biologically as determined by prothrombin time assay in factor FVII-depleted plasmas, of both human and rabbit origin, using either human or rabbit thromboplastin. Rabbit recombinant factor VII should prove useful for future in vivo investigations of experimental coagulopathies.

Activation and Active Site Occupation Alter Conformation in the Region of the First Epidermal Growth Factor-like Domain of Human Factor VII

The first epidermal growth factor-like domain (EGF-1) of factor VII (FVII) provides the region of greatest contact during the interaction of FVIIa with tissue factor. To understand this interaction better, the conformation-sensitive FVII EGF-1-specific monoclonal antibody (mAb) 231-7 was used to investigate the conformational effects occurring in this region upon both FVII activation and active site occupation. The binding affinity of mAb 231-7 was approximately 3-fold greater for the zymogen state than for the active state; a result affected by the presence of both calcium and the adjacent Gla domain. Once activated, active site inhibition of FVIIa with a variety of chloromethyl ketone inhibitors resulted in a 10-fold range of affinities of FVIIai molecules to mAb 231-7. Gla domain removal eliminated this variation in affinity, suggesting the involvement of a Gla/EGF-1 interaction in this conformational effect. In addition, the binding of mAb 231-7 to FVIIa EGF-1 stimulated the amidolytic activity of free FVIIa. Taken together, these results imply an allosteric interaction between the FVIIa active site and the EGF-1 domain that is sensitive to variation in active site occupant structure. Thus, these present studies indicate that the conformational change associated with FVII activation and active site occupation involves the EGF-1 domain and suggest potential functional consequences of these changes.

Activation of a recombinant human factor VII structural analogue alters its affinity of binding to tissue factor

A competitive enzyme‐linked immunoadsorbent assay (ELISA) technique has been developed to facilitate quantitative analysis of the earliest step in the initiation of the extrinsic pathway of coagulation, i.e., complex formation of factor VII/VIIa with tissue factor. The ELISA measures the binding of biotinylated human plasma factor VII to relipidated recombinant human tissue factor. Quantitation of the relative affinity (expressed as IC50) of any factor VII molecular population or structural analogue for tissue factor can be determined by competitive binding. Subnanomolar concentrations of both wild‐type recombinant human factor VII (rFVII) and rFVII(R152Q), a mutation at the FVII activation site, competed effectively with biotinylated plasma‐derived factor VII in binding to tissue factor. In contrast, the affinity of rFVII(R79Q), a mutation in the first epidermal growth factor‐like domain, was 12‐fold lower. Following activation of rFVII(R79Q), its affinity for tissue factor and enzymatic activity increased 4‐fold and 6‐fold, respectively. For wild‐type rFVII, enzymatic activity rose significantly following activation. However, its affinity for tissue factor was unchanged. We conclude that both the activation state of factor VII and the mutation of amino‐acid residues within the first epidermal growth factor‐like domain may alter the affinity of factor VII for tissue factor.

The immunodepletion of factor VII from human plasma using a monoclonal antibody

Summary. A murine hybridoma cell line which secretes monoclonal antibody to factor VII has been prepared to facilitate the immunodepletion of this clotting factor from plasma. Specific monoclonal antibody was purified from mouse ascites tumours by protein A‐Sepharose chromatography and shown to be of the IgG1 immunoglobulin subclass. On immunoblotting, this antibody reacted with a single protein band identical to purified factor VII. The purified monoclonal antibody was coupled to Sepharose 4B and was used to immunodeplete factor VII from pooled normal human plasma. The prothrombin time of plasma immunodepleted in this way was 35 s compared to 12 s for the starting plasma. Specific factor assays of the immunodepleted plasma showed factor VII activity to be less than 1% while the levels of the other clotting factors were unchanged. The immunodepleted plasma was equivalent to severe congenital factor VII deficient plasma as a substrate for factor VII assays. Bound factor VII could be eluted from the immunoaffinity column with citrate buffer, pH 6·0, with good recovery.

Consumption of plasma factor VII in a rabbit model of non-overt disseminated intravascular coagulation.

INTRODUCTION We have recently described an experimental animal model of non-overt disseminated intravascular coagulation (DIC) in the rabbit in which the induction of tissue factor (TF) mRNA and TF antigen expression in peripheral blood leukocytes (PBL) was demonstrated to occur within 2 h of administration of low-dose endotoxin [Hematol. J. 2 (2001) 188]. In the present study, we demonstrate that the leukocyte TF expressed has procoagulant activity leading to a rapid decline in the concentration of factor VII (FVII) in rabbit plasma. METHODS Total plasma FVII antigen and FVIIa were quantitated by rabbit FVII-specific immunoassay and FVIIa-specific clotting assays, respectively. Plasma samples from either saline-injected rabbits or rabbits administered a single bolus of 10 microg/kg Salmonella lipopolysaccharide were compared over a 24-h period. RESULTS Total plasma FVII antigen decreased progressively post-endotoxin injection, reaching 71% of the baseline concentration at 8 h (p<0.001, n=18), and remained low (78%) at 24 h post-injection (p<0.01, n=16), returning to normal by 48 h. Plasma FVIIa levels increased to 120% within 2 h of endotoxin injection, fell to 73% of the baseline concentration at 8 h (p<0.05, n=18) and returned to normal by 24 h post-endotoxin administration. Procoagulant activity of rabbit peripheral blood leukocytes was enhanced at 2 h (p<0.01, n=6) and 4 h (p<0.05, n=6) post-endotoxin injection. The prothrombin time (PT) was increased by <3 s, and thrombin-antithrombin (TAT) complex formation was not significantly increased in the plasma of endotoxin-treated rabbits. No significant changes in total plasma FVII antigen, FVIIa or leukocyte procoagulant activity were observed in rabbits treated with saline. CONCLUSIONS We conclude that the activation of FVII to FVIIa and rapid consumption of total FVII/FVIIa occur very early and likely are integral events linked to the initiation and propagation of non-overt DIC induced by endotoxin.

Identification of Immunoglobulin Class and Subclass of House Monoclonal Antibodies to Human Cell Surface Antigens by Mixed Hemadsorption Assay

A microassay is described to determine immunoglobulin Ig class or IgG subclass of mouse monoclonal antibodies by mixed hemadsorption assay. Monoclonal antibody bound to adherent target cells is reacted with serial dilutions of a panel of class or subclass specific rabbit anti-mouse Ig antisera and binding of the latter is traced by anti-rabbit globulin-coated indicator erythrocytes. The class or subclass of the bound monoclonal antibody is revealed by preferential binding of the corresponding rabbit antibody. Unlike gel immunodiffusion analysis, the mixed hemadsorption assay may be performed with unconcentrated hybridoma culture supernatants.

Incomplete gamma carboxylation of human coagulation factor VII: differential effects on tissue factor binding and enzymatic activity

The integrity of the γ‐carboxylic glutamic acid (GLA) residues of coagulation factor VII are thought to be essential for both the interaction of factor VII with its cell‐surface lipoprotein receptor tissue factor and for the activated protein to manifest its serine protease activity. During the course of transiently expressing recombinant human factor VII in monkey COS cells it was noted that the factor VII synthesized in the absence of added vitamin K had < 20% of expected procoagulant activity yet retained 65% of its binding activity to recombinant human tissue factor. Similar results were obtained when vitamin K was omitted from human 293 cell cultures permanently expressing recombinant factor VII. In contrast, both transient and permanent expression of factor VII in human 293 cell cultures containing physiological concentrations of vitamin K resulted in the synthesis of fully functional factor VII. Furthermore, factor VII in plasma samples from 24 patients undergoing warfarin therapy bound quantitatively to tissue factor whereas factor VII procoagulant activity averaged 65% of normal. Thus, data from both in vitro and in vivo situations indicated that factor VII molecules with suboptimal GLA content retained most of their ability to bind tissue factor but exhibited reduced procoagulant activity.

Interspecies exchange mutagenesis of the first epidermal growth factor-like domain of human factor VII

Summary.  The first epidermal growth factor‐like (EGF1) domain of human factor VII (FVII) is essential for binding to tissue factor (TF). We hypothesized that the previously observed increased coagulant activity of rabbit plasma (i.e. FVII) with human TF might be explained by the five non‐conserved amino acids in the rabbit vs. the human FVII EGF1 domain. Accordingly, we ‘rabbitized’ the human FVII EGF1 domain either by exchanging the entire EGF1 domain creating human FVII(rabEGF1) or by the single amino acid substitutions S53N, K62E, P74A, A75D and T83K. After transient expression in HEK293 cells, the recombinant FVII (rFVII) mutant proteins were analyzed for biological activity and binding affinity to human TF by competitive enzyme‐linked immunosorbent assay (ELISA). Biological activity of the unpurified rFVII mutant proteins was either depressed or statistically unchanged vs. rFVII(WT). However, three of six rFVII mutant proteins had increased affinity for human TF in the rank order rFVII(rabEGF1) (3.3‐fold) > rFVII(K62E) (2.9‐fold) > rFVII(A75D) (1.7‐fold). The mutant protein rFVII(K62E) was then permanently expressed and purified. Fully activated, purified rFVIIa(K62E) had a twofold greater clotting activity and 2.8‐fold greater direct FVIIa amidolytic activity when compared with rFVIIa(WT). Quantitation of the affinity of TF binding by surface plasmon resonance indicated that the KD of purified rFVII(K62E) for human soluble TF (sTF) was 1.5 nm compared with 7.5 nm for rFVII(WT), i.e. fivefold greater affinity. We conclude that substitution of selected amino acid residues of the FVII EGF1 domain facilitated the creation of human rFVII chimeric proteins with both enhanced biological activity and increased affinity for TF.

The direct binding of human factor VII in plasma to recombinant human tissue factor.

The extrinsic pathway of coagulation is initiated when zymogen factor VII binds to its cell surface receptor tissue factor. Recently recombinant human tissue factor has become available and therefore in this study the direct binding of human factor VII in plasma to recombinant tissue factor was explored. Factor VII binding was quantitated by a standard ELISA protocol using monospecific polyclonal rabbit anti-human factor VII as the primary antibody and goat anti-rabbit IgG conjugated to alkaline phosphatase as the second antibody. Both the oxidation state of the recombinant tissue factor and calcium ion concentration were found to be critical for the efficient binding of factor VII. A linear relationship was observed between absorbance and factor VII concentration when normal pooled human plasma was diluted in the range 1:25 to 1:1000 (factor VII concentration 0.5-20 ng/ml). Evidence is provided to show that binding is both specific for human coagulation factor VII and can be utilized to detect factor VII molecular variants with impaired tissue factor binding.

Human FVII(K62E) does not exhibit enhanced binding to tissue factor

In 2005, in this journal [1], we described a mutant species of human factor (F)VII in which the first epidermal growth factor-like domain (EGF1) was mutated at residue 62 (K62E). This species of FVII, FVII(K62E), was reported by us to have a 5-fold increased affinity for tissue factor (TF), compared with wild-type FVII[FVII(WT)]. As a result of the relatively low concentrations of FVII utilized in these experiments, the antigen concentration of both FVII(WT) and mutant FVII(K62E) was quantitated using a sandwich ELISA, employing a polyclonal antibody raised in sheep to rFVIIa(WT). Subsequent to the publication of these results, milligram amounts of FVII(K62E) have been expressed in and purified from HEK293 cells to facilitate in vivo animal experimentation. Recently, however, it became apparent that the FVII ELISA used for our study [1], substantially underestimated the concentration of FVII(K62E) compared with that obtained for total FVII(K62E) protein determined by absorption at A280nm. As illustrated in Fig. 1, equal concentrations of FVII(WT) and FVII(K62E) determined by A280nm, resulted in markedly differing slopes in our FVII ELISA. Thus calculation of FVII antigen concentration (for example at 0.5 optical density units) resulted in a greater than 4-fold difference in protein concentration values between FVII(WT) and FVII(K62E). This property of FVII(K62E) results in an underestimate of the amount of FVII(K62E) in a particular solution when determined by ELISA, thus overestimating the procoagulant and TFbinding activities of this protein species at a given concentration. As both the calculated specific enzyme activity and TF-binding are dependent on an accurate determination of the protein concentration of FVII(K62E) antigen, we now feel that our previously reported results are incorrect [1,2]. Similar results to those shown in Fig. 1 (B. J. Clarke, M. A. Blajchman, unpublished) have recently been generated using FVII(K62E) synthesized in both the HEK293 and CHO cell lines.