Margaret A. Howard

Factor-VIII-related antigen in platelets

Abstract Using quantitative immunoelectrophoresis against rabbit anti-human factor-VIII antibody, normal human platelets are shown to contain factor-VIII-related antigen (FVIII RAg) in amounts equivalent to 5–15% of that in whole platelet-rich-plasma. Platelet FVIII RAg is firmly bound to the membrane fraction and does not appear to exchange with plasma FVIII RAg. The platelets of von Willebrands disease and thrombasthenia are deficient in FVIII RAg, but the giant platelets of the Bernard-Soulier syndrome contain greater than normal amounts.

Studies of natural anticoagulant proteins and anticardiolipin antibodies in patients with the lupus anticoagulant

Components of the natural anticoagulant system (NAS) and anticardiolipin antibodies were examined in 21 patients with lupus anticoagulant (LA), 13 of whom had past histories of thrombotic episodes. No relationship could be shown between the antigenic levels of protein C and S (PC, PS) and a history of thrombosis. Inhibition of the anticoagulant activity of activated protein C (APC) was observed using plasma from 20/21 patients when phospholipid vesicles were used as the surface for the coagulation reaction. This effect was not affected by the addition of PS. When platelet membranes were employed only 2/21 patients demonstrated inhibition of APC. Under the latter condition, PS functional activity was inhibited in 7/21 patients, six of whom had a past history of thrombosis.

Inhibition and reversal of ristocetin - induced platelet aggregation.

Abstract Ristocetin-induced aggregation of a suspension of washed normal platelets in the presence of factor VIII was inhibited or reversed by the addition of rabbit anti-human factor VIII, by heparin, or by changing the pH of the solution, while urea was without effect. It is suggested that ristocetin-induced platelet aggregates are held together by electrostatic forces, and that the factor-VIII molecule is necessary for the maintenance of the weak inter-platelet bonds, as well as for their initiation.

Causes for the discrepancies in the measurements of factor VIII antigen

Abstract Previous authors have noted that discrepancies occur for factor VIII-related antigen (VIII-Ag) levels when different techniques are used for its measurement. However no systematic study has been undertaken to identify the causes for these discrepancies. Factor VIII-related antigen (VIII-Ag) has been quantitated by either electroimmunoassay (EIA), solid or liquid phase radioimmunometric assay (S-IRMA or L-IRMA respectively). In general a close correlation existed between levels of VIII-Ag measured by these techniques. Values for VIII-Ag in serum, cryosupernatant and the plasma of haemophiliacs who have developed antibodies to factor VIII(FVIII) were found to be lower when measured by S-IRMA than by EIA; the L-IRMA gave intermediate values. Wherever a lack of correlation existed between the assays the VIII-Ag was found to have an increased electrophoretic mobility on radio 2-dimensional immunoelectrophoresis (radio 2-DIEP). Measurement of VIII-Ag of column fractions from a purification of FVIII concentrate confirmed that the S-IRMA failed to detect small VIII-Ag forms. It could be demonstrated that the antibody used for IRMA showed selection of the antibody towards large VIII-Ag forms. This selection together with the ‘two antibody sandwich’ technique used in S-IRMA may explain loss of reactivity towards the small VIII-Ag forms of FVIII.

Quantitation of binding of factor VIII antigen to concanavalin A.

Summary. This study establishes a convenient method for screening plasma samples for abnormalities of the carbohydrate content of the factor VIII (FVIII) molecule. A radioimmuno‐electrophoretic technique has been developed to quantitate the percentage binding of FVIII‐related antigen (VIII‐Ag) to the lectin concanavalin A (Con A). Plasma samples were electrophoresed through a strip of agarose containing Con A into agarose containing a mixture of unlabelled anti‐FVIII and 125I‐anti‐FVIII where precipitant lines formed, the height of which was dependent upon the degree of VIII‐Ag binding to Con A in the first gel. Using this system reduced binding of VIII‐Ag to Con A was found in the plasma of 12 patients with moderate classical von Willebrands disease (vWd), while the Con A binding of six haemophilia A patients fell within the normal range. The VIII‐Ag in normal cryoprecipitate showed increased % binding to Con A while the VIII‐Ag remaining in the cryo‐supernate demonstrated reduced Con A % binding.

Analysis of Platelet von Willebrand Factor Antigen

Platelet lysates were obtained from suspensions of normal washed platelets by freeze-thawing or Triton X-100 lysis. The resultant platelet lysates contained 0.34 +/- 0.15 U/10(9) platelets (n = 8) of von Willebrand factor antigen (vWf:Ag) as determined by radioelectroimmunoassay using a monospecific antibody to vWf:Ag. The vWf:Ag level was higher in platelet lysates prepared from freshly drawn blood than from outdated platelet packs. Platelet lysates from patients with severe von Willebrands disease type I (n = 2) did not contain detectable vWf:Ag. When normal platelet lysates were analyzed by radiocrossed immunoelectrophoresis in agarose using a monospecific polyclonal antibody to plasma vWf:Ag, two immunochemically identical precipitin peaks were seen. One of the platelet vWf:Ag peaks corresponded in its electrophoretic mobility to plasma vWf:Ag, while the other peak, i.e. platelet vWf:Ag-peak II, migrated to a more anodal position. The presence of the platelet vWf:Ag-peak II suggests structural differences between plasma and platelet vWf:Ag and illustrates previously unrecognized heterogeneity of platelet vWf:Ag.

CROSSED AFFINITY ELECTROPHOSRESIS OF FACTOR VIII RELATED ANTIGEN

Thus my results disagree with the data of Wolos & Davey but support the earlier work of Halper et al (1979) who quantitated HLA-DR by absorption experiments in addition to fluorescence microscopy using well-defined antisera. The “anti-‘la-like’ ” serum used by Wolos & Davey was not characterized by them. The original characterization of this antiserum (Billing et al, 19 76,19 77) showed that it contained anti-HLA-DR activity but not that this was its only activity. Indeed, it would be astonishing if an antiserum raised against papain fragments of spleen membranes was completely specific for one molecule on the surface of B lymphocytes without absorption. Wolos & Davey did not present any direct quantitative evidence to support their further claim that HLA-A, -B, -C (serum defined) antigens may be decreased on the surface of CLL B cells. Using rabbit antisera raised against purified pz-microglobulin (a constituent chain of all HLA-A, -B and -C molecules) in indirect immunofluorescence microscopy, I have detected no difference in the surface density of these antigens on CLL compared with normal cells. Accurate comparison of cell surface molecule density in heterogeneous cell populations can only be accomplished satisfactorily by objective quantitation (e.g. Fluorescence Activated Cell Sorter analysis) and not by techniques such as immunofluorescence microscopy which are at best only semi-quantitative and can be quite subjective depending upon the design of the experiment.