Behnaz Parhami-Seren

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≤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≤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.

Immunologic quantitation of tissue factors

Summary.  The large number of conflicting reports on the presence and concentration of circulating tissue factor (TF) in blood generates uncertainties regarding its relevance to hemostasis and association with specific diseases. We believe that the source of these controversies lies in part in the assays used for TF quantitation. We have developed a highly sensitive and specific double monoclonal antibody fluorescence‐based immunoassay and integrated it into the Luminex Multi‐Analyte Platform. This assay, which uses physiologically relevant standard and appropriate specificity controls, measures TF antigen in recombinant products and natural sources including placenta, plasma, cell lysates and cell membranes. Comparisons of reactivity patterns of various full‐length and truncated TFs on an equimolar basis revealed quantitative differences in the immune recognition of TFs by our antibodies in the order of TF 1‐263 > 1‐242 > 1‐218 > placental TF. Despite this differential recognition, all TF species are quantifiable at concentrations  2 pM. Using a calibration curve constructed with recombinant TF 1‐263 and plasma from healthy individuals (n = 91), we observed the concentration of TF antigen in plasma to be substantially lower than that generally reported in the literature: TF antigen in plasma of 72 individuals (79%) was below 2 pM (quantitative limit of our assay); TF antigen levels between 2.0 and 5.0 pM could be detected in six individuals (7%); and in 14% (13 plasmas), the non‐specific signal was higher than the specific signal, and thus TF levels could not be determined. These differential recognition patterns affect TF quantitation in plasma and should be considered when evaluating plasma TF‐like antigen concentrations.

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.

Potency and mass of factor VIII in FVIII products.

Summary.  Several factor (F) VIII products of different origin and structure are being used for haemophilia A treatment worldwide. The assessment of FVIII concentration in these products is done using activity assays, which are dependent upon the assay and its modifications. To evaluate FVIII products for potency and for FVIII concentration and specific activity, three activity‐based assays [activated partial thromboplastin time (APTT), intrinsic FXase and synthetic coagulation proteome] and two immunoassays (ELISA and western blotting) were used in this study with albumin‐free full‐length recombinant (r) FVIII as a standard. In all activity assays, products A and B (both contain full‐length rFVIII) at 1 U mL−1 showed potency similar to that of the 0.7 nm (1 U mL−1) rFVIII standard. Product E (contains truncated rFVIII) was less potent in the APTT (83% of standard) and product C (contains plasma FVIII) was less potent in FXase assays (66%). The ELISA immunoassay revealed that the specific activity of FVIII proteins in products A–C and E varied over a wide range (3900–13 200 U mg−1) and was higher for most lots when compared with the standard (5000 U mg−1), whereas the specific activity of product D (contains plasma FVIII) was lower than expected (3200–4800 U mg−1). (i) FVIII potency estimated in different assays gives dissimilar results; (ii) the specific activity of FVIII in various FVIII products is different and inconsistent. Thus, the administration of an equal FVIII potency in units means the administration of different amounts of FVIII protein, which may partly explain apparent discrepancies in product performance.

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.

The influence of von Willebrand factor on factor VIII activity measurements

Summary.  Background: It has been reported by multiple laboratories that the quantitation of factor (F)VIII by activity‐based assays is influenced by the method, procedure and the quality of reagents used in the assays. Objective: To evaluate the influence of von Willebrand factor (VWF) on FVIII activity in vitro. Methods: The activated partial thromboplastin time (APTT) and synthetic coagulation proteome assays were used. Citrated FVIII/VWF‐depleted substrate plasma (SP) (both antigens < 0.5%) was used in all APTT assays. Results: The concentration of FVIII antigen in pooled plasma from healthy donors [normal plasma (NP)] was 1.5 nm. The SP reconstituted with 1.5 nm recombinant (r)FVIII clotted in 23.8 ± 0.2 s (standard deviation). The addition of 10 μg mL−1 VWF to the SP increased the clotting time to 28.7 ± 0.1 s; that is, the activity of rFVIII (FVIIIc) decreased to 50%. This inhibitory effect of VWF decreased with decreasing rFVIII concentration in SP, and became negligible at rFVIII ≤ 10% (150 pm). The FVIIIc of 1.5 nm FVIII in two products formulated with VWF (Immunate and Hemofil M) was not affected by the addition of exogenous VWF to the SP, whereas the FVIIIc of the B‐domainless rFVIII product ReFacto was decreased to 50% by the addition of VWF. In the synthetic coagulation proteome triggered with 5 pm tissue factor, VWF had no effect on thrombin generation. Conclusions: VWF has an inhibitory effect on the measurement of FVIII clotting activity. This effect depends upon the structure and formulation of the FVIII product.

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.

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.

Structural Analysis of Mutants of High-Affinity and Low-Affinity p-Azophenylarsonate-Specific Antibodies Generated by Alanine Scanning of Heavy Chain Complementarity-Determining Region 2

Alanine scanning was used to determine the affinity contributions of 10 side chain amino acids (residues at position 50–60 inclusive) of H chain complementarity-determining region 2 (HCDR2) of the somatically mutated high-affinity anti-p-azophenylarsonate Ab, 36–71. Each mutated H chain gene was expressed in the context of mutated (36–71L) and the unmutated (36–65L) L chains to also assess the contribution of L chain mutations to affinity. Combined data from fluorescence quenching, direct binding, inhibition, and capture assays indicated that mutating H:Tyr50 and H:Tyr57 to Ala in the 36–71 H chain results in significant loss of binding with both mutated (36–71L) or unmutated (36–65L) L chain, although the decrease was more pronounced when unmutated L chain was used. All other HCDR2 mutations in 36–71 had minimal effect on Ab affinity when expressed with 36–71 L chain. However, in the context of unmutated L chain, of H:Gly54 to Ala resulted in significant loss of binding, while Abs containing Asn52 to Ala, Pro53 to Ala, or Ile58 to Ala mutation exhibited 4.3- to 7.1-fold reduced affinities. When alanine scanning was performed instead on certain HCDR2 residues of the germline-encoded (unmutated) 36–65 Ab and expressed with unmutated L chain as Fab in bacteria, these mutants exhibited affinities similar to or slightly higher than the wild-type 36–65. These findings indicate an important role of certain HCDR2 side chain residues on Ab affinity and the constraints imposed by L chain mutations in maintaining Ag binding.

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.