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Adults with chronic relapsing ITP present a difficult therapeutic challenge. The ongoing antibody- mediated platelet destruction in this group might be expected to be associated with increased expression of platelet surface membrane activation antigens.
We have studied a group of 10 patients with refractory ITP and 35 healthy controls. Using an immediate, sensitive, unfixed, whole blood, flow cytometric method to detect platelet surface P-selectin and GP53, we have detected markedly increased platelet activation in the ITP group compared with the controls (P-selectin; patient median 24.5% v control median 2.0%, GP53 median 6.5% v 2.1%, P < 0.01 for both).
Five patients underwent protein A immunoadsorption therapy. The effect of protein A immunoadsorption on platelet activation before, during and after 18 treatments in these patients was studied and patients were followed-up to assess clinical outcome. Platelet-associated immunoglobulin measurements were made before and at the end of six treatments.
Platelet activation decreased after immunoadsorption. P-selectin expression fell significantly; pre- and post-treatment median values differed by 15.5%, P < 0.01, for GP53 the difference was 2.5.%, P = NS. A reduction in both platelet-associated IgG (median reduction of 11.8 ng/106 platelets, P = 0.08) and IgM (7.6 ng/106 platelets, P = 0.06) was recorded.
ITP in adults is frequently resistant to first- and second-line therapy (steroids and intravenous immunoglobulin) ( Newland et al, 1983 ; Manoharan, 1991) and patients may proceed to splenectomy. Splenectomy, although useful ( McMillan et al, 1972 ), is not universally successful, and some patients, having responded, relapse. For these patients the next treatment options are often cytotoxic or immunosuppressive therapies ( Andersen, 1994) which are associated with significant side-effects. Such patients may respond to immunoadsorption with protein A columns ( Snyder et al, 1992 ).
In ITP the patients' platelets are frequently coated with immunoglobulin, some of which is directed against specific platelet surface antigens such as GPIIb/IIIa ( Brighton et al, 1996 ), although the majority is non-specifically bound to the platelet membrane. This immunoglobulin may be derived from within the platelet alpha granules ( George et al, 1985 ). The binding of immunoglobulin and immune complexes to the platelet surface has been associated with platelet activation ( Yanabu et al, 1991 ). Platelet activation is associated with alterations in platelet size and shape, alterations in transmembrane molecules, such as GPIIb/IIIa and the release of intraplatelet substances to the cell membrane and plasma. Two such molecules, normally held intracellularly in alpha and lysosomal granules respectively, are P-selectin (CD62P) ( McEver & Martin, 1984; Hsu-Lin et al, 1984 ) and GP53 (CD63) ( Nieuwenhuis et al, 1987 ). These molecules are expressed solely on the surface of activated platelets and are useful markers of platelet activation. P selectin is an adhesion molecule which functions as a ligand for platelet–neutrophil binding ( Larsen et al, 1989 ). The function of GP53 is currently unknown.
We have previously reported a strong positive correlation between P-selectin and platelet-associated IgG (PAIgG) ( Morris et al, 1991 ). Platelet activation and immunoglobulin binding appear to be closely related. Platelet destruction is likely to result from this bound autoantibody or from immune complexes binding to platelet Fc receptors. Platelets, opsonized in this way, are destroyed in liver, spleen and bone marrow ( Imbach, 1995).
We hypothesized that sufficient immunoglobulins might be removed by protein A immunoadsorption to reduce platelet activation. This might lead in turn to longer platelet survival and translate to improvement in the platelet count.
To investigate these hypotheses we examined the platelets of 10 adults with severe refractory ITP. We studied platelet activation flow cytometrically. One previous study reported elevated P-selectin in a minority (17%) of children with ITP ( Semple et al, 1996 ) and this is the first study to report elevated P-selectin and GP53 in adults with ITP.
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These results support the conclusion that platelet activation is markedly increased in autoimmune thrombocytopenic purpura. The presence of platelet activation in ITP is not unexpected. Indeed, the results of this study support those of other workers whose data associated platelet surface immunoglobulin with platelet activation, measured by a variety of techniques. Immunoglobulin binding to the platelet surface has been linked to alpha granule release ( George, 1990; George et al, 1985 ), and F(ab′)2 fragments have also been associated with activation ( Sugiyama et al, 1987 ). The binding of platelet-specific immunoglobulin to GPIIb/IIIa has been shown to cause platelet aggregation and granule release in ITP ( Yanabu et al, 1991 ). Platelet activation increases Fcγ RII expression ( McCrae et al, 1990 ) and some platelet activation may be mediated via Fcγ RII ( Worthington et al, 1990 ).
Therefore we propose a positive feedback loop promoting platelet activation and enhancing platelet destruction. Activated platelets may be more susceptible to immune destruction due to increased Fcγ expression ( McCrae et al, 1990 ). Immune complex binding to Fc receptors is associated with increased platelet destruction which can be blocked by infusion of Fcγ fragments in vivo ( Debre et al, 1993 ).
In summary: initiating event — platelet alpha granule release — increased platelet associated IgG and IgM binding — increased Fcγ RII expression — further platelet activation — alpha granule release — platelet destruction.
Our hypothesis, substantiated by previous data ( Morris et al, 1991 ), predicts also that the most severely affected patients should have highest expression of platelet surface markers of activation. Our data also demonstrate this, showing a negative correlation between P-selectin and platelet count (r = −0.46, Fig 6). Although not statistically significant (most probably because of lack of power), this data confirms previous findings in larger numbers of patients ( Morris et al, 1991 ) which did show statistical significance.
The data on GP53 show that it too was expressed in increased amounts on the platelet surface in ITP. Protein A immunoadsorption also decreased the expression of this molecule, although the results were not significant. Both were sensitive markers of platelet activation, although the data on GP53 were more variable than that on P-selectin. We have previously shown that P-selectin and GP53 behave differently and that their expressions are not normally correlated ( Cahill et al, 1996 ).
Documenting platelet activation is difficult because measurement techniques may cause in vitro activation ( Cahill et al, 1993 ). Before this study was undertaken, an extensive analysis of analytical and pre-analytical variables was undertaken in our laboratory (see Methods). As a result, the risks of ex vivo activation were minimized by using the whole blood technique and rapid analysis.
Protein A binds the Fc fragment of IgG with an increased affinity for complexed IgG ( McDougal et al, 1979 ). The binding capacity of the columns used in this study (PROSORBAc (Imré)) is about 1 g of IgG per elution cycle. The small absorption capacity of the column and the variable volume of plasma processed with success (250–2000 ml with no apparent difference in outcome ( Snyder et al, 1992 )), make it unlikely that bulk removal of IgG and complexes is important. However, removal of small amounts of immunoglobulin may be enough to restore the idiotype/anti-idiotype control networks ( Berchtold et al, 1989 ), or to reduce platelet activation. The data demonstrate a reduction in both IgG and IgM bound to platelets which cannot be explained simply by immunoglobulin removal by the column since protein A does not bind IgM ( Goding, 1978). It may be that reductions in both PAIgG and PAIgM are brought about because of the reduction in platelet activation, reduction in alpha granule secretion and therefore reduction in granule-derived PAIgM, after column treatment. In addition, reduction in platelet activation may make it more likely that plasma immunoglobulins do not become platelet associated or surface bound.
Our data showed an overall decrease in platelet activation after protein A immunoadsorption which accompanied most (but not all) column treatments. It is possible that the most activated platelets remained bound to the columns, leaving the least activated in the circulation to be analysed. However, in the small surface area available, bound platelets were unlikely to significantly influence the characteristics of the circulating platelet mass. Attempts to remove column bound platelets for analysis would possibly result in significant spurious platelet activation ( Goodall, 1991).
In a few cases activation antigen expression appeared to increase. A number of explanations for this phenomenon include host factors on that day (e.g. acute reactions associated with protein A), chance and within-assay variation where the ‘increases’ are small. One patient (patient 5) had a moderate hypersensitivity reaction while undergoing immunoadsorption and his GP53 rose from 4.7% to 13.2% after treatment. P-selectin was unchanged. Another patient (patient 4) showed a rise in P-selectin (GP53 unchanged) from 17.6% to 30% on his fourth column treatment. Five other treatments in this patient were accompanied by a fall in expression. This patient also had hypersensitivity reactions to protein A immunoadsorption after a number of treatment episodes.
The failure of the protein A columns to produce clinical benefit was probably due to the failure of the therapy to reverse either the initiating cause of the platelet activation (usually attributed to immune dysregulation with or without a viral trigger) or the platelet destruction. Our data are consistent with a temporary beneficial effect on platelet surface membrane factors which if prolonged might, arguably, lead to remission.
Another view of platelet activation in ITP, for which there is no explicit evidence, might be that platelet activation is protective in ITP. Increased baseline platelet activation is noted in conditions associated with increased thrombosis, e.g. inflammatory bowel disease ( Collins et al, 1994 ), diabetes ( Tschoepe et al, 1995 ) and coronary artery disease ( Cahill et al, 1994 ). It could be postulated that in thrombocytopenic patients a degree of platelet activation may be haemostatically desirable in order to offset low platelet numbers and therefore it may be a physiological response to thrombocytopenia. It is observed clinically that patients with chronic thrombocytopenia bleed less at the same platelet count than those whose thrombocytopenia is acute. One observation which supports this hypothesis comes from a recent study of acute ITP in children which failed to demonstrate significant platelet activation (P-selectin expression) in this condition ( Semple et al, 1996 ). These questions cannot be resolved, however, on the basis of the data in this paper.
In conclusion, our data suggest that increased expression of platelet surface activation antigens is present in chronic ITP and that protein A immunoadsorption decreases platelet activation. This might provide an additional mechanism of action of the protein A columns, although we did not observe clinically significant benefits of this treatment.