Barry G. Firkin

Relationship of Raised Platelet IgG in Thrombocytopenia to Total Platelet Protein Content

Summary. Platelet‐associated IgG (PA IgG) of washed platelets of 20 normal controls and 45 thrombocytopenic patients was measured by radioimmunoassay and related to total platelet protein content.

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.

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.

Quinine- and Quinidine-dependent Antiplatelet Antibodies: REQUIREMENT OF FACTOR VIII-RELATED ANTIGEN FOR PLATELET DAMAGE AND FOR IN VITRO TRANSFORMATION OF LYMPHOCYTES FROM PATIENTS WITH DRUG-INDUCED THROMBOCYTOPENIA

The requirement of Factor VIII-related antigen (VIIIR:Ag) for platelet damage by quinine-and quinidine-dependent antibodies was studied in platelet-rich plasma (PRP) of four patients with severe von Willebrands disease (vWd) (Factor VIII deficiency). Platelet factor 3 availability, platelet aggregation, and release of [(14)C]serotonin from labeled vWd-PRP by drug-dependent antibodies were significantly reduced in comparison with PRP from normal controls. Addition of purified VIIIR:Ag restored levels of platelet damage to that of normal PRP. In vWd-PRP, platelet damage by two human antiplatelet sera, not dependent on drugs, and by a rabbit antiplatelet serum did not differ from that in normal PRP. PRP from patients deficient in Factor VIII coagulant activity, Factor IX, or Factors II, VII, IX, and X behaved like normal PRP. The role of VIIIR:Ag in forming antigen able to transform lymphocytes of patients who had recovered from drug-induced thrombocytopenia was investigated by measuring incorporation of [methyl-(3)H]thymidine into DNA. When lymphocytes were cultured for 7 d, significantly less transformation occurred in response to platelets and the drug in the presence of vWd sera than in normal sera or sera deficient only in Factor VIII coagulant activity or Factor IX. Addition of purified VIIIR:Ag to vWd sera restored transformation to that obtained in normal sera. Nonspecific lymphocyte transformation by pokeweed mitogen was not affected by VIIIR:Ag. Thus VIIIR:Ag is involved both in platelet damage by drug-dependent antibodies and in the interaction between platelet and drug which produces an antigen able to transform sensitized lymphocytes.

Analysis of immunoglobulins that bind to platelets from serum of patients with immune thrombocytopenia: molecular weight distribution

The nature of platelet- bindable immunoglobulins (PB-Ig) in serum has been investigated. PB-IgG, -A and -M were measured by an ELISA using platelets coated on microtitre plates. This assay detected alloantibodies at high serum dilutions. In 32 patients with idiopathic thrombocytopenic purpura (ITP) or systemic lupus erythematosus (SLE) raised levels of at least one PB-Ig class were found in 18. To distinguish binding due to immune complexes, the molecular weight of PB-IgG was studied by gel filtration on Sepharose 4B. In sera from patients with ITP and SLE, PB-IgG with Mr of primarily 150 Kd was observed, compatible with monomeric IgG antiplatelet antibodies. Levels of PB-IgG in serum were not related to total serum IgG. In sera from the patients with SLE and some with ITP (most of whom had several of the features of SLE), PB-IgG with Mr of 200 Kd - greater than 1000 Kd was seen. In heat-aggregated preparations of normal IgG, PB-IgG with Mr up to 1000 Kd was also found. Rabbit IgG was able to block PB-IgG in fractions of high molecular weight in purified normal IgG, heat-aggregated normal IgG and in patient serum, but had no effect on the 150 Kd peak. In whole serum from patients who had high molecular weight PB-IgG, the inhibitory effects of rabbit IgG were much less than in isolated high molecular weight column fractions. Thus although the majority of PB-IgG is monomeric antiplatelet antibody, some PB-IgG with higher molecular weight, characteristic of immune complexes, occurs in sera of some patients with autoimmune thrombocytopenia and it makes a small contribution to PB-IgG levels measured in whole serum.

Effects of EACA on thrombin generation as measured by the chromagen S2238

In the presence of epsilon aminocaproic acid (EACA) thrombin generation in recalcified platelet rich plasma (PRP) was markedly stimulated, as measured by the cleavage of the synthetic substrate S2238. However, thrombin activity remaining after 30 minutes incubation was reduced when compared with control values. The residual activity was shown to be hirudin insensitive and to be associated with a species of higher molecular weight than free thrombin. These results suggested an inhibition of thrombin binding to the antithrombin, alpha 2-macroglobulin (alpha 2M). Preincubation of PRP with EACA reduced the concentration at which EACA elicited its dual effects. Similar results were obtained with the alpha 2M inhibitor, hydrazine. These experiments indicated that alpha 2M may play a more important role in regulating thrombin generation than has been previously recognized.

Impaired fibrinolysis in patients with thrombotic or haemostatic defects

In a retrospective study of the 47 patients examined for thrombotic defects, 34 had prolonged euglobulin lysis times (ELT) with 27 of these also having elevated tissue-plasminogen activator inhibitor (t-PAI) activity. Twenty three of the 57 patients examined for possible bleeding problems had prolonged ELT with 15 of this group also having raised levels of t-PAI. There was a statistically greater (P less than 0.005) incidence of elevated t-PAI in the thrombotic group. The incidence of 40% patients with prolonged ELT in the haemostasis group was surprising and perhaps represents some compensatory mechanism to combat bleeding. Thus in vitro tests showed that a reduced capacity for fibrinolysis was present in diametrically opposed groups.

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.