SRA Does Not Detect Potentially Relevant HIT Antibodies.
(2004)
Journal - ASH Annual Meeting Abstracts
Abstract :
Abstract
Even before the nature of the heparin:platelet factor 4 (H:PF4)antigenic target was identified, it was known that pathogenicactivity in sera/plasma from Heparin-Induced Thrombocytopenia(HIT) patients could be recovered in the immunoglobulin G (IgG)fraction. In in vitro diagnostic tests, blockade of plateletFcIIa receptors completely prevents platelet activation by HITsera. For these reasons, the IgG isotype is considered the predominantHIT pathogenic agent. H:PF4 ELISA techniques have made it possibleto distinguish between antibodies (Abs) of the IgG, IgA andIgM isotypes, and have shown that IgA and IgM Abs are more prevalentthan previously thought. In a group of 247 H:PF4 ELISA-positivespecimens studied at Loyola University Medical Center, 65% containedIgG, 53% had IgA, and 56% had an IgM component. The biologicalactivity and pathogenic relevance of non-IgG H:PF4 Abs is asyet unknown. These studies were undertaken to specifically testwhether H:PF4 IgA or IgM isotypes, in the absence of IgG, couldcause platelet activation in the Serotonin Release Assay (SRA),the reference standard activation assay for diagnosis of HIT.Four SRA positive specimens with multiple isotypes were fractionatedusing Protein G (Amersham Biosciences) chromatography to separateIgGs. Isotyping of the fractions demonstrated that the H:PF4IgA and IgM were present in the flow through peak; IgGs werein fractions eluted from the Protein G by low pH. Only fractionsthat bound to Protein G, ie IgG isotype, caused the characteristicSRA positive response of platelet activation in the presenceof low heparin (0.1 U/ml) and not in the presence of excessheparin (100 U/ml). Study of an SRA positive specimen that testednegative for the IgG isotype, showed that the SRA activity wasin fractions that bound to Protein G even though the antibodywas not detected in the ELISA. Another four SRA positive specimenwith multiple isotypes were fractionated using immobilized jacalinchromatography (Pierce, Rockford IL) to separate IgA Abs. Isotypingdemonstrated that IgG Abs did not bind to the column; IgA Abswere in fractions eluted from the jacalin column in the presenceof melibiose. Only the flow through H:PF4 IgG, and not the elutedIgA Abs, tested positive in the SRA. However, three of the isolatedH:PF4 IgAs elicited some degree of platelet serotonin releasein the absence of heparin only, without the characteristic 2-pointresponse to heparin. In conclusion, the platelet activationby SRA positive specimens with multiple isotypes is attributableto the IgG component only. Neither IgA nor IgM H:PF4 Abs aredetected in the 2-point SRA. While the SRA does not detect IgAor IgM Abs, these isotypes may still have pathogenic activity.Preliminary studies with H:PF4 IgA, indicate that these Abscan bind to platelets and cause serotonin release in the absenceof heparin. Until there is better understanding of in vivo biologicalactivity of non-IgG HIT isotypes, it should be noted that theSRA, the reference standard activation assay for HIT, may notdetect all physiologically relevant H:PF4 Abs.
Desulfated Heparin Blocks Platelet Activation Induced by HIT Antibodies / Heparin: A New Approach to Patient Management.
(2004)
Journal - ASH Annual Meeting Abstracts
Abstract :
Abstract
Heparin-induced thrombocytopenia (HIT) represents a diseasespectrum triggered by an immune response to heparin. The mostdramatic clinical expression of HIT is HIT antibody-driven thrombosis.Direct thrombin inhibitors (DTIs) are a promising new classof drugs for treatment of the acute phase of HIT; however, theyhave a narrow safety/efficacy window (high bleeding risk) andmorbidity/mortality have not been eliminated. In addition, thehigh probability of developing thrombosis in HIT combined withextreme mortality, has led to a bias for prophylactic treatment.Thus, there remains a clinical need to identify optimal treatmentoptions for patients with HIT. A new concept is to use an agentthat can effectively compete with heparin in a manner that preventsthe HIT antibody from inducing platelet activation, i.e., amelioration.We evaluated a 2-O, 3-O desulfated heparin (ParinGenix, Inc.;Tucson, AZ) to determine its ability to ameliorate HIT antibody/heparininduced platelet activation. The test agent was added to a mixtureof known-reactive platelets and pre-formed immune complexes(heparin, PF4, HIT antibodies), and SRA and flow cytometry wereperformed. Due to the inherent biological variability of HITantibodies, sera from four different patients (clinically diagnosedas HIT; SRA positive) were used. Two concentrations of heparinwhich reflect typical prevention (0.1 U/ml) and treatment (0.5U/ml) clinical doses were used. The 2-O, 3-O desulfated heparinproduced an amelioration of HIT antibody/heparin induced plateletactivation as demonstrated in the SRA by inhibition of 14C serotoninrelease from activated platelets, and in flow cytometric analysisby inhibition of platelet microparticle formation and plateletcell surface P-selectin expression. Significant ameliorationactivity was initiated at 6.25 µg/ml 2-O, 3-O desulfatedheparin and complete inhibition of the induced platelet activation(equal to the 100 U/ml heparin HIT assay ‘no response’)was achieved with 50 µg/ml of the agent. Since this newtreatment approach blocks platelet activation caused by HITantibody/heparin (not a characteristic of DTIs), we proposethat a non-anticoagulant glycosaminoglycan (GAG) may be usefulin improving the clinical management of patients with HIT. Theconcept of amelioration differs from all previous options forthe clinical management of patients with HIT including the useof danaparoid, fondaparinux, DTIs and other drugs that targetthrombin/thrombin generation inhibition. Although not directedat platelet activation inhibition, this type of GAG effectsan inhibition of HIT antibody mediated platelet activation,which is the source of the pathophysiology of HIT. The dataof this study suggest that this 2-O, 3-O desulfated heparinmay be effective as either an adjunct or sole treatment of anongoing HIT pathology, or as a preventive measure in patientswho will be exposed to heparin.