Analysis of Prothrombotic Effects of two Human Monoclonal IgG Antiphospholipid Antibodies of Apparently Similar Specificity

Karl J. Lackner; Christine von Landenberg; Stefan Barlage; Gerd Schmitz

doi:10.1055/s-0037-1613867

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2000; 83(04): 583-588
DOI: 10.1055/s-0037-1613867

Commentary

Schattauer GmbH

Analysis of Prothrombotic Effects of two Human Monoclonal IgG Antiphospholipid Antibodies of Apparently Similar Specificity

Karl J. Lackner

¹From the Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany

,

Christine von Landenberg

¹From the Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany

,

Stefan Barlage

¹From the Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany

,

Gerd Schmitz

¹From the Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany

› Author Affiliations

Abstract

Summary

Two human monoclonal antiphospholipid antibodies (APA) of the IgG type, HL-5B and RR-7F have been generated from a patient with primary antiphospholipid syndrome and recurrent cerebral microemboli (H.L.) and from a patient with SLE without evidence of recurrent thrombosis (R.R.). Both monoclonal APA have similar characteristics in ELISA tests. To further analyse the prothrombotic potential, their effect on human monocytes and platelets, and bovine aortic endothelial cells (BAEC) was investigated. Monocytes were isolated from buffy coats by standard techniques. They were incubated either with the respective monoclonal APA in different concentrations, or a control monoclonal IgG of the same subtype, or plasma of the patients, from whom the antibodies were isolated. Incubation with LPS served as positive control. BAEC were grown to confluence, and then incubated with the appropriate agonists. Procoagulant activity (PCA) was determined by a single stage clotting assay. PCA expression after incubation is given as the ratio of the coagulation times observed with media only divided by that observed with the agonist. A PCA ratio >1 indicates the induction of PCA by the agonist. At 1 µg/ml HL-5B yielded a PCA ratio of 1.63 ± 0.16 while RR-7F induced no significant rise to 1.06 ± 0.18. Dose response curves showed that RR-7F can induce PCA at higher concentrations. However, its effect is approx. 1/50 of HL-5B based on equimolar antibody concentration. Further analysis indicates that the majority of the PCA induced by monoclonal APA can be inhibited by a specific tissue factor antibody. Neither monoclonal antibody induced PCA in BAEC. Sera from both patients were able to induce PCA in monocytes. However, the PCA ratio of serum from H.L. was higher (1.78) than that of R.R. (1.44). Neither monoclonal APA had an effect on platelets as determined by flow cytometric analysis of CD62P, CD41, CD42b expression and fibrinogen binding with and without previous activation with 5 µM ADP or 15 µM TRAP-6. Similarly, there were no differences in platelet aggregation to different stimuli including submaximal activation. In summary, these data provide further evidence that induction of tissue factor in monocytes is one of the procoagulant effects of APA. Furthermore, the binding specificity of APA is perhaps not suited to predict the biological effects of the antibodies.

Keywords

Antiphospholipid antibodies - tissue factor - monocytes - endothelial cells - platelets

Full Text

References

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