The "normal" factor VIII concentration in plasma.

INTRODUCTION: The quantitation of factor (F)VIII by activity-based assays is influenced by the method, procedure, the quality and properties of reagents used and concentrations of other plasma proteins, including von Willebrand factor (VWF). OBJECTIVE: To compare FVIII concentrations measured by activity-based assays with those obtained by an immunoassay and to establish the influence of plasma dilution on the FVIII clotting activity (FVIIIc). METHODS: The APTT, a chromogenic assay (Coatest) and two in-house immunoassays were used. Albumin-free recombinant FVIII was used as the calibrator in all assays. RESULTS: For a group of 44 healthy individuals (HI), the mean value observed for FVIII antigen (FVIIIag; 1.22+/-0.56 nM; S.D.) is substantially higher than that for FVIIIc (0.65+/-0.29 nM) and the chromogenic assay (FVIIIch; 0.50+/-0.23 nM). A positive correlation between FVIIIag and VWFag with R(2)=0.20 was observed. Since plasma VWF has an inhibitory effect on FVIIIc, we evaluated the influence of plasma dilutions on FVIIIc in HI (n=105). At a 4-fold dilution, estimates of FVIIIc by clotting assay were much lower than FVIIIag (0.77+/-0.31 vs. 1.14+/-0.48 nM). At 10- and 25-fold dilutions, the estimated FVIIIc increased to 0.87+/-0.36 and 0.94+/-0.44 nM, respectively. CONCLUSIONS: 1) In plasma, FVIIIag is higher than FVIIIc and FVIIIch; and 2) Real FVIII concentrations in plasma can be estimated by measuring FVIIIag.

Quantitation of anti-factor VIII antibodies in human plasma.

The presence of antibodies (Abs) in hemophilia A patients can potentially influence the therapeutic qualities of factor VIII (fVIII) administration. Much work has been focused on the presence of inhibitory antibodies, whereas the quantitation of noninhibitory anti-fVIII antibodies has been largely undetermined. Our objective was to develop a sensitive and specific fluorescence-based immunoassay (FLI) for the quantitation of anti-fVIIIAbs in human plasma. Affinity-purified human anti-fVIIIAb, isolated from a hemophilia A subject, was used as a calibrator with a detectability limit of 40 (+/-1.5) pM. The calibrator and the human plasma anti-fVIIIAb were captured on recombinant fVIII (rfVIII)- coupled microspheres and probed with mouse anti-human Ig-R-phycoerythrin. Plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were compared with 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1 BU/mL (94.6 +/- 0.8 nM), 11 BU/mL (214.3 +/- 7.1 nM), 106 BU/mL (2209.4 +/- 84.9 nM), 140 BU/mL (2417.7 +/- 3.8 nM) as measured by the Nijmegen method. We also describe the validation of a mouse anti-human fVIIIAb as a surrogate calibrator. Four healthy individuals (3%) showed detectable anti-fVIIIAb in the range of 0.6 to 6.2 nM, whereas 13 (33%) of the 39 inhibitor-free hemophilia A subjects were positive for anti-fVIIIAb in the range of 0.5 to 20 nM. The method may be useful for therapeutic management of hemophilia A patients.

Intracellular and surface distribution of monocyte tissue factor: application to intersubject variability.

OBJECTIVE: The high and low responder phenomenon describes individual differences in lipopolysaccharide (LPS)-induced monocyte tissue factor (TF) activity. We characterized patterns of intracellular accumulation, externalization, and shedding of TF in response to LPS in mononuclear cells (MNCs) from high responders (HRs) and low responders (LRs). METHODS AND RESULTS: After 2 hours of LPS stimulation of whole blood, flow cytometry analyses revealed a larger population of TF-positive monocytes in HRs (32.0+/-3.5%) versus LRs (11.2+/-1.2%; P< or =0.05), along with a stronger mean fluorescence intensity of TF signal in HRs (7.1+/-0.5 arbitrary units [AU]) compared with LRs (5.4+/-0.4 AU; P< or =0.05). The LPS-treated blood of the HR group contained 2-fold more TF-positive microparticles than LRs. In-cell Western assay demonstrated higher intracellular accumulation of TF in mononuclear cells (MNCs) from LRs because LPS induced a 3.7-fold increase of total TF levels in LRs versus a 1.5-fold increase in HRs. In contrast, in response to LPS stimulation, MNCs from HRs exhibited a 4-fold induction of surface TF, whereas MNCs from LRs only had a minor increase in surface TF levels. CONCLUSIONS: The higher availability of surface TF antigen on MNCs from HRs and TF-containing microparticles might make these individuals more susceptible to hypercoagulation.

Tissue factor activity in whole blood.

Tissue factor (TF) is an integral membrane protein essential for hemostasis. During the past several years, a number of studies have suggested that physiologically active TF circulates in blood at concentrations greater than 30 pM either as a component of blood cells and microparticles or as a soluble plasma protein. In our studies using contact pathway-inhibited blood or plasma containing activated platelets, typically no clot is observed for 20 minutes in the absence of exogenous TF. An inhibitory anti-TF antibody also has no effect on the clotting time in the absence of exogenous TF. The addition of TF to whole blood at a concentration as low as 16 to 20 fM results in pronounced acceleration of clot formation. The presence of potential platelet TF activity was evaluated using ionophore-treated platelets and employing functional and immunoassays. No detectable TF activity or antigen was observed on quiescent or ionophore-stimulated platelets. Similarly, no TF antigen was detected on mononuclear cells in nonstimulated whole blood, whereas in lipopolysaccharide (LPS)-stimulated blood a significant fraction of monocytes express TF. Our data indicate that the concentration of physiologically active TF in non-cytokine-stimulated blood from healthy individuals cannot exceed and is probably lower than 20 fM.

Structural correlates of a functional streptokinase antigenic epitope: serine 138 is an essential residue for antibody binding.

We determined the pattern of cross-reactivity of a panel of anti-streptokinase (SK) monoclonal antibodies (mAbs) with SK variants in order to map the antigenic and functional epitope of SK. Comparison of the pattern of cross-reactivity of the anti-SK mAb A4.3 with SK variants and sequence alignments of SK variants and native (n) SK suggested that mutation of Ser 138 to Lys results in loss of binding of mAb A4.3 to SK variants. However, this mutation does not affect formation of activator complex by these proteins. The epitope specificity of the mAb A4.3 was further confirmed by mutating Ser 138 to Lys in n SK. Monoclonal Ab A4.3 did not bind to mutant SK (Ser138Lys). Activator activity of mutant SK (Ser138Lys) was indistinguishable from that of n SK and recombinant n SK. Since addition of A4.3 mAb to an equimolar mixture of SK and human plasminogen inhibits activator complex formation, the sequences spanning position 138 are likely important for formation of streptokinase-plasminogen activator complex or processing of the plasminogen substrate.

Affinity panning of peptide libraries using anti-streptokinase monoclonal antibodies: selection of an inhibitor of plasmin(ogen) active site.

To select sequences complementary to their binding sites, two anti-streptokinase (SK) monoclonal antibodies (mAbs), A4.5 and A5.5, were used in biopanning of 15-mer and hexamer phage-displayed peptide libraries, respectively. mAb A4.5 inhibits the catalytic activity of streptokinase-plasminogen activator complex (SKPAC), the binding of plasminogen to SK and the binding of human anti-SK polyclonal Abs to SK. All clones selected from the 15-mer peptide library by mAb A4.5 had identical nucleotide and amino acid sequences, RSVYRCSPFVGCWFG. An 11-mer peptide (peptide A4.5, YRCSPFVGCWF) derived from this sequence inhibited the binding of mAb A4.5 and human anti-SK polyclonal Abs to SK as well as the catalytic activity of both SKPAC and plasmin. The binding of the second mAb (mAb A5.5) to SK is lost upon interaction of SK with plasminogen, suggesting that sequences selected by this mAb are likely associated with the C-terminal cleavage site of SK. Biopanning of a hexamer peptide library with mAb A5.5 selected the sequence RYLQDY that is homologous to residues 324-328, adjacent to one possible C-terminal cleavage site in SK. A 10-mer synthetic peptide (LDFRDLYDPR) corresponding to residues 321-330 in SK specifically inhibited the binding of mAb A5.5 to SK. The selection and characterization of these two peptides enhances our understanding of SK structure, maps an antigenic epitope, and identifies a peptide inhibitor of plasminogen activation.

Ouabain-binding protein(s) from human plasma.

Conservation of the binding site on mammalian Na+,K+-ATPase for cardiac glycosides and the importance of the Na+ pump in mammalian cellular physiology has stimulated the search for a mammalian analog of these plant compounds. One candidate, isolated from brain and blood, appears to be ouabain itself or a closely related isomer, the ouabain-like compound. Little is known about the circulating form. Because human steroid hormones circulate with carrier proteins, we produced a ouabain-specific monoclonal antibody (mAb 1-10) and used it to probe normal human plasma for ouabain-protein carrier complex. Ouabain-like biological activity was isolated in association with protein bands of 80, 50, and 25 kDa. These proteins appear to be human immunoglobulins or immunoglobulin-like because they are recognized by anti-human immunoglobulin antibodies, but not by anti-mouse immunoglobulin antibodies. The protein-containing fractions inhibit the binding of mAb 1-10 to immobilized ouabain, and with further purification on protein A, the immunoglobulin-like protein binds radioactive ouabain with an IC50 of 200 to 600 nmol/L, but binds digoxin with 100-fold less affinity, suggesting specificity for ouabain or its isomer. Active protein fractions after purification on C18 inhibit Na+ pump activity in human erythrocytes (IC50 approximately 4 nmol/L, ouabain equivalents), and this chromatography appears to dissociate the ouabain-like compound from the immunoglobulin protein(s). These immunoglobulin-like molecules may represent a subset of immunoglobulins (< or =0.5% of total protein A immunoglobulin) that function as a reservoir and delivery system for ouabain-like compounds in the modulation of human Na+, K+-ATPase in vivo.

Selection of high affinity p-azophenyarsonate Fabs from heavy-chain CDR2 insertion libraries.

The length of the heavy chain complementarity-determining region two (HCDR2) of the unmutated anti-p-azophenylarsonate (Ars) monoclonal antibody (36-65 mAb) was extended by three residues in order to test whether this insertion can provide additional contacts between the Ab and the antigen. Two libraries were generated using 36-65 heavy and light chain genes which were cloned as Fab in the phage-display vector pComb3. In the first library, three randomized amino acids were inserted between residues Gly 54 and Asn 55, which are the most solvent exposed residues in the HCDR2 loop. In the second library, in addition to the 3-mer randomized insertion, the flanking residues at positions 54 and 55 were also randomized to allow additional loop flexibility for binding to Ars. Solid-phase and solution phase affinity panning were used to select for clones that bind to Ars. Results indicate that diverse 3-mer HCDR2 insertions can be tolerated, and affinities 10-fold higher than germline encoded 36-65 Ab can be obtained. The sequence diversity of the insertion among the selected clones from both libraries suggests that the insertion increases contact between the Ab and the protein carrier rather than the hapten alone.