Jolanta Krudysz-Amblo

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.

Carbohydrates and Activity of Natural and Recombinant Tissue Factor

The effect of glycosylation on tissue factor (TF) activity was evaluated, and site-specific glycosylation of full-length recombinant TF (rTF) and that of natural TF from human placenta (pTF) were studied by liquid chromatography-tandem mass spectrometry. The amidolytic activity of the TF·factor VIIa (FVIIa) complex toward a fluorogenic substrate showed that the catalytic efficiency (Vmax) of the complex increased in the order rTF1–243 (Escherichia coli) < rTF1–263 (Sf9 insect cells) < pTF for the glycosylated and deglycosylated forms. Substrate hydrolysis was unaltered by deglycosylation. In FXase, the Km of FX for rTF1–263-FVIIa remained unchanged after deglycosylation, whereas the kcat decreased slightly. A pronounced decrease, 4-fold, in kcat was observed for pTF·FVIIa upon deglycosylation, whereas the Km was minimally altered. The parameters of FX activation by both rTF1–263D-FVIIa and pTFD-FVIIa were identical and similar to those for rTF1–243-FVIIa. In conclusion, carbohydrates significantly influence the activity of TF proteins. Carbohydrate analysis revealed glycosylation on asparagines 11, 124, and 137 in both rTF1–263 and pTF. The carbohydrates of rTF1–263 contain high mannose, hybrid, and fucosylated glycans. Natural pTF contains no high mannose glycans but is modified with hybrid, highly fucosylated, and sialylated sugars.

Alloantibodies to factor VIII in haemophilia

Summary.  Up to one‐third of haemophilia A patients develop factor VIII (FVIII) alloantibodies (inhibitors). The Bethesda assay detects inhibitors but is relatively insensitive. Recently, a new fluorescence‐based immunoassay (FLI) was developed for antibody detection. The aim of this study was to assess the prevalence of inhibitors as measured by FLI. Assays of FVIII, FVIII inhibitor by Bethesda assay with Nijmegen modification, and FVIII inhibitor by FLI were performed on adult patients with haemophilia A. Data were complete for 46 patients (median age 39), of whom 72% were severe, 7% moderate and 22% mild. The Bethesda assay was positive in only two patients (4%), while FLI was positive in 23 of 46 patients (50%), with values ranging from 0.4 to 33.7 nm (median 3.5 nm). FLI titres exceeded 7.0 nm in 19.5% of patients, all but one of whom had severe haemophilia. FLI antibody‐positive patients were less likely to be HIV positive (30% vs. 70%, P = 0.02). The use of a prophylaxis regimen was associated with a lower incidence of antibody; only two of 23 patients with detectable antibody and none of those with antibody >7 nm were on a prophylaxis regimen, while nine of 23 patients without antibody were on prophylaxis, (P = 0.03). There was no difference in inhibitor presence in patients using recombinant versus plasma‐derived factor. Antibodies detected by FLI are frequent in patients with haemophilia A, but are less common in those who are HIV positive or are receiving regular FVIII prophylaxis.

Platelet tissue factor is not expressed transiently after platelet activation.

To the editor: Blood coagulation is initiated by the binding of plasma factor VII/VIIa to cell surface–expressed tissue factor (TF). Normally, TF expression is confined to subendothelial tissues like fibroblasts[1][1] and pericytes[2][2] to limit blood coagulation to sites of vascular injury.

Differences in the fractional abundances of carbohydrates of natural and recombinant human tissue factor

BACKGROUND Tissue factor (TF) is a single polypeptide integral membrane glycoprotein composed of 263 residues and is essential to life in its role as the initiator of blood coagulation. Previously we have shown that the activity of the natural placental TF (pTF) and the recombinant TF (rTF) from Sf9 insect cells is different (Krudysz-Amblo, J. et al (2010) J. Biol. Chem. 285, 3371-3382). METHODS In this study, using mass spectrometry, we show by quantitative analysis that the extent of glycosylation varies on each protein. RESULTS AND CONCLUSIONS Fractional abundance of each glycan composition at each of the three glycosylation sites reveals the most pronounced difference to be at asparagine (Asn) 11. This residue is located in the region of extensive TF-factor VIIa (FVIIa) interaction. Carbohydrate fractional abundance at Asn11 revealed that glycosylation in the natural placental TF is much more prevalent (~76%) than in the recombinant protein (~20%). The extent of glycosylation on Asn124 and Asn137 is similar in the two proteins, despite the pronounced differences in the carbohydrate composition. Additionally, 77% of rTF exists as TF des-1, 2 (missing the first two amino acids from the N-terminus). In contrast, only 31% of pTF is found in the des-1, 2 form. CONCLUSION These observations may attribute to the difference in the ability of TF-FVIIa complex to activate factor X (FX). GENERAL SIGNIFICANCE Structural and functional comparison of the recombinant and natural protein advances our understanding and knowledge on the biological activity of TF.

Posttranslational modifications and activity of natural and recombinant tissue factor.

Tissue factor is a membrane protein, which in a complex with factor VIIa initiates in vivo blood coagulation. Due to the scarcity of natural tissue factor protein, most studies have relied upon recombinant tissue factor forms. However, there have been only cursory experimental comparisons of natural and recombinant tissue factor proteins. Our preliminary data suggested that placental tissue factor in a complex with factor VIIa was more efficient activator of factor X than the recombinant protein. After deglycosylation, both forms of tissue factor showed almost an identical activity in the extrinsic factor Xase. Analyses using tryptic digestion and mass-spectrometry revealed that the levels of glycosylation and the composition of carbohydrates present in natural placental tissue factor were different than those in its recombinant counterpart. These data indicate that natural and recombinant tissue factor proteins differ in their posttranslational modifications and that these differences translate into different cofactor activity. Thus the use of recombinant tissue factor proteins for the quantitation of natural tissue factor is misleading.

Decryption of tissue factor

Tissue factor (TF) is a transmembrane protein which, in complex with factor (F)VIIa, initiates blood coagulation. Numerous studies have determined TF epitopes and individual amino acids which play an important role in the TF/FVIIa complex formation and its activity towards natural substrates. However the subject of cell-surface TF activity remains controversial. It has been almost commonly accepted that TF on the cell surface has low (if any) activity, i.e. is encrypted and requires specific conditions/reagents to become active, i.e. decrypted. One of the leading theories suggests that cell membrane lipid composition plays a crucial role in TF decryption, whereas another assigns the key role to the formation of the Cys(186)-Cys(209) disulfide bond. Despite a number of studies published from several laboratories, the role of this bond in the activity of the TF/FVIIa complex remains elusive and controversial. One of the causes of this controversy could be related to the lack of specificity of the reagents used for the cell treatment leading to possible alterations in other cell surface proteins and cell membrane environment. In conclusion, the influence of the Cys(186)-Cys(209) this bond on cell surface TF function remains unclear.

Product‐dependent anti‐factor VIII antibodies

The development of anti‐factor (F)VIII antibodies in haemophilia A (HA) subjects undergoing replacement therapy has been well documented. The correlation between antibody development and the FVIII product used for replacement therapy remains a subject of discussion. The aim of this study was to evaluate the presence of anti‐FVIII antibodies towards three commercial rFVIII products in 34 HA subjects’ plasmas. Antibodies were quantitated by a Multiplex Fluorescence Immunoassay. All plasmas contained anti‐FVIII antibodies at variable concentrations ranging from 50 nm to 570 μm. Eleven of the 20 HA subjects treated with one (r)FVIII product contained inhibitory anti‐FVIII antibodies (0.8‐3584 BU). The inhibitory antibody titre and the molar concentrations of total antibody were mildly correlated (r2 = 0.6). Pronounced differences in antibody recognition with the three rFVIII products were observed. For the group treated with Product ‘A’, the titre towards this product was 2.4‐fold higher than that observed with another full‐length rFVIII‐containing product (Product ‘B’) and almost four‐fold higher than that measured with a B domain‐less rFVIII product (Product ‘C’). For the group of 14 HA subjects treated with FVIII other than Product ‘A’, only one showed higher antibody titre when measured with this product. Our data suggest that the development of anti‐FVIII antibodies is biased towards the product used for treatment and that a significant fraction of antibodies bind to the B domain of FVIII.