Summary. The serotonin release assay (SRA) tests for antibodies responsible for heparin-induced thrombocytopenia (HIT). By definition, SRA-positive antibodies cause platelet serotonin release in vitro, in the presence of low concentrations of heparin, but not with excess heparin. Many SRA-positive sera activate platelets in the presence of saline without drug, either as a result of residual heparin in the specimen, or because of intrinsic features of the HIT antibodies. The present experiments show that neither exhaustive heparinase treatment, nor chromatographic removal of heparin abrogates the spontaneous platelet activation caused by these HIT antibodies. This is the first study to systematically demonstrate that in vitro activity of HIT antibodies can be independent of heparin. In addition, T-gel chromatography demonstrated differences among fractions of enzyme-linked-immunosorbent assay (ELISA)-positive HIT antibodies within individual specimens. Certain ELISA-positive fractions had SRA activity while others did not, and the SRA activity was not proportional to HIT antibody ELISA titer. These data suggest that antibodies formed as a result of heparin treatment are heterogeneous, and that some can contribute to the pathogenesis of HIT even when heparin is no longer present.
This study characterized heparin isolated from tuna skins. Glycosaminoglycans were isolated from tuna skin after digestion using anion exchange resin. Heparin was eluted from the resin by sodium chloride gradient and was further fractionated by acetone fractionation. Anticoagulant activity was determined using the activated partial thromboplastin time and Heptest assays. Potency was determined using amidolytic antifactor IIa and antifactor Xa assays. The presence of heparin in the extracted tuna skin glycosaminoglycans was confirmed using (13)C-nuclear magnetic resonance. The activated partial thromboplastin time and Heptest clotting times were doubled at concentrations of about 4 and 1 microg/mL, respectively. The clotting time prolongation and antiprotease activity induced by tuna heparin was readily neutralized by 25 microg/mL protamine sulfate. These results demonstrate that biologically active heparin with properties similar to clinical grade heparin can be derived from tuna skin, a raw material with otherwise relatively little economic value.
Background: The Serotonin Release Assay (SRA) is frequently used to test for the presence of heparin-platelet factor 4 (H:PF4) antibodies as part of a diagnosis of Heparin-Induced Thrombocytopenia (HIT). In this test, immune complexes formed when antibodies bind to soluble or membrane-bound H:PF4 complexes, activate platelets via FcγIIa receptors. A HIT-positive SRA demonstrates donor platelet activation when test sera are incubated with low concentrations (0.1–1.0 U/ml) of heparin, but not when the incubation includes excess, neutralizing levels (10–100 U/ml) of heparin. Platelet activation at both low and high heparin is designated an indeterminate result. This heparin-independent platelet activation can be caused by immune complexes unrelated to HIT, by other anti-platelet antibodies or by platelet agonists. The present studies were conducted to study the occurrence of anti-platelet antibodies in specimens with marginal or inconclusive SRA activity. Methods: The study included two populations of specimens that had been previously tested by both SRA and H:PF4 antibody ELISA (GTI, Brookfield, WI). Group I included 44 specimens that tested SRA positive in spite of the absence of measurable H:PF4 antibodies. Most were relatively weak in the SRA: 51% ± 3.4 % (S.E.) serotonin release with 0.1 U/ml heparin. Group II consisted of 18 specimens that gave an indeterminate SRA response: heparin-independent platelet activation. Of these, 5 were positive for H:PF4 antibodies and 13 were negative. All specimens were analysed by PakPlus ELISA screening (GTI, Brookfield, WI) to determine if antibodies to HLA Class I and/or to common platelet specific glycoproteins (GPs) were present. Results: In Group I, 19 of the 44 (43%) specimens tested positive for one or more anti-platelet antibody. 18 of the 19 (95%) had either anti-GP IIb/IIIa (n=10)(53%) and/or anti- HLA Class I (n=11)(58%) antibodies. One specimen had antibodies to GP Ib/IX and to GP IV. The remaining 25 (57%) specimens tested negative. In Group II, 13 of the 18 (72%) SRA-indeterminate specimens had detectable anti-platelet antibodies. All but one of the 13 (92%) included antibody to HLA Class I. The anti-glycoprotein antibodies were less frequent in this group: anti-GP IIb/IIIa (n=2)(15%), GP Ia/IIa (n=3)(23%), GP Ib/IX (n=2)(15%) or GP IV (n=3)(23%). Conclusion: Non-H:PF4 anti-platelet antibodies, especially antibodies to GP IIb/IIIa or to HLA Class I, are not uncommon in sera referred for SRA testing. Specimens containing anti-platelet antibodies can give a positive or an indeterminate response in the two-point SRA. Specimens without H:PF4 antibodies that test positive in the SRA should be scrutinized for anti-platelet antibodies as an alternative to the diagnosis of HIT. Also, an indeterminate SRA should not be considered a negative test result. Anti-platelet antibodies alone can cause a non-heparin dependent platelet activation: however, their presence may also mask a positive, heparin-dependent, SRA response to H:PF4 antibodies. Finally, it is not uncommon for specimens to test positive by the 2-point SRA in the absence of antibodies to H:PF4 or to other platelet antigens. The mechanism for this response, and its significance to diagnosis of HIT, requires further investigation.
A recently introduced series of antithrombotic agents brings the novel characteristic of dual anti-coagulant and anti-platelet actions in one molecule. These low molecular weight, synthetic, serine protease inhibitors, depending on structural modifications, have variable ratios of both anti-thrombin and anti-platelet activities. Studies have shown that these agents produce stronger antithrombotic actions relative to single targeted therapeutic agents (O. Iqbal #P0521 and D. Hoppensteadt #OR335, ISTH meeting Sydney, Australia August 2005). Heparin-induced thrombocytopenia (HIT) is an adverse effect of heparin in which both thrombin generation and platelet activation augment hypercoagulable and inflammatory states leading to a high probability of developing severe thrombosis. Current guidelines for patients who have HIT recommend the use of a direct thrombin inhibitor (DTI) to prevent or treat associated thrombosis. Clinical trial data, as well as practice outcomes, show that DTI treatment alone is not sufficient to overcome the pathology and resultant thrombosis in all HIT patients. Thus, more optimal treatment options are needed. A focus of treatment on inhibiting both platelet and coagulation activation is logical based on the pathophysiology of HIT. This study was undertaken to determine if the novel CanAm series of agents may have a role in the management of patients with HIT. Eight agents (MC45301, MC45308, CA207, CA216, CA234, CA247, CA250, CA254) with varying ratios of anticoagulant/anti-platelet activities were studied using the 14C-serotonin release assay (SRA) and flow cytometry for the detection of platelet P-selectin expression and platelet microparticle generation. The DTI argatroban, the FXa inhibitor fondaparinux, and the platelet GPIIb/IIIa receptor antagonist eptifibatide were included for comparison. Both cross-reactivity to HIT antibodies and amelioration of HIT antibody-induced platelet activation were assessed. In the absence of heparin, at 1-100 μg/ml none of the CanAm agents caused platelet activation in the presence of serum from patients with HIT (n=12) ruling out cross-reactivity with HIT antibodies. None of the comparator drugs showed cross reactivity with HIT antibodies. In the presence of heparin (0.1 U/ml) and serum from patients with HIT (n=12), the CanAm agents were able to inhibit all platelet activation responses at concentrations of 10–100 μg/ml. In comparison, the DTI and FXa inhibitor were not able to inhibit the HIT antibody/heparin induced platelet activation with any HIT serum. The GPIIb/IIIa inhibitor, however, showed a concentration-dependent inhibition of the platelet activities with complete blockade at 1 μg/ml, suggesting the importance of platelet activation inhibition for the treatment of HIT. Thus, compared to mono-therapeutic agents such as DTIs and fondaparinux, the dual-acting CanAm agents not only lack cross reactivity with HIT antibodies, but they have the added ability to block the antibody-induced platelet activation that occurs during an acute episode of HIT. A dual-acting anticoagulant/anti-platelet drug may, therefore, be of more value than single targeted anti-thrombin drugs for the management of HIT and associated thrombosis.
Heparin-induced thrombocytopenia (HIT) represents a disease spectrum triggered by an immune response to heparin. The most dramatic clinical expression of HIT is HIT antibody-driven thrombosis. Direct thrombin inhibitors (DTIs) are a promising new class of drugs for treatment of the acute phase of HIT; however, they have a narrow safety/efficacy window (high bleeding risk) and morbidity/mortality have not been eliminated. In addition, the high probability of developing thrombosis in HIT combined with extreme mortality, has led to a bias for prophylactic treatment. Thus, there remains a clinical need to identify optimal treatment options for patients with HIT. A new concept is to use an agent that can effectively compete with heparin in a manner that prevents the 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/heparin induced platelet activation. The test agent was added to a mixture of known-reactive platelets and pre-formed immune complexes (heparin, PF4, HIT antibodies), and SRA and flow cytometry were performed. Due to the inherent biological variability of HIT antibodies, sera from four different patients (clinically diagnosed as HIT; SRA positive) were used. Two concentrations of heparin which reflect typical prevention (0.1 U/ml) and treatment (0.5 U/ml) clinical doses were used. The 2-O, 3-O desulfated heparin produced an amelioration of HIT antibody/heparin induced platelet activation as demonstrated in the SRA by inhibition of 14C serotonin release from activated platelets, and in flow cytometric analysis by inhibition of platelet microparticle formation and platelet cell surface P-selectin expression. Significant amelioration activity was initiated at 6.25 μg/ml 2-O, 3-O desulfated heparin 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 new treatment approach blocks platelet activation caused by HIT antibody/heparin (not a characteristic of DTIs), we propose that a non-anticoagulant glycosaminoglycan (GAG) may be useful in improving the clinical management of patients with HIT. The concept of amelioration differs from all previous options for the clinical management of patients with HIT including the use of danaparoid, fondaparinux, DTIs and other drugs that target thrombin/thrombin generation inhibition. Although not directed at platelet activation inhibition, this type of GAG effects an inhibition of HIT antibody mediated platelet activation, which is the source of the pathophysiology of HIT. The data of this study suggest that this 2-O, 3-O desulfated heparin may be effective as either an adjunct or sole treatment of an ongoing HIT pathology, or as a preventive measure in patients who will be exposed to heparin.
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