Heparin-induced thrombocytopenia: an autoimmune disorder regulated through dynamic autoantigen assembly/disassembly

Heparin‐induced thrombocytopenia (HIT) is the most common drug‐induced, antibody‐mediated cause of thrombocytopenia and thrombosis. HIT is caused by IgG antibodies that bind to epitopes on platelet factor 4 (PF4) released from activated platelets that develop when it forms complexes with heparin. Anti‐PF4/antibodies develop in over 50% of patients undergoing surgery involving cardiopulmonary bypass (CPB), an incidence 20‐fold higher than HIT. Why might this occur? Binding of HIT IgG occurs only over a narrow molar ratio of reactants, being optimal at 1 mol PF4 tetramer to 1 mol unfractionated heparin (UFH). At these ratios, PF4 and UFH form ultralarge (>670 kD) complexes that bind multiple IgG molecules/complex, are highly antigenic, and promote platelet activation. Low molecular weight heparin (LMWH), which is less antigenic, forms ultralarge complexes less efficiently and largely at supratherapeutic concentrations. In transgenic mice that vary in expression of human PF4 on their platelets, antigenic complexes form between PF4 and endogenous chondroitin sulfate. Binding of HIT IgG to platelets and induction of thrombocytopenia in vivo is proportional to PF4 expression. Heparin prolongs the duration and exacerbates the severity of the thrombocytopenia. High doses of heparin, as used in CPB, or protamine, which competes with PF4 for heparin, disrupts antigen formation and prevents thrombocytopenia induced by HIT antibody. These studies may help explain the disparity between the incidence of antibody formation and clinical disease and may help identify patients at risk for HIT (high platelet PF4). They also demonstrate that this autoimmune disease can be modulated at the level of autoantigen formation and point to rational means to intervene proximal to thrombin generation. J. Clin. Apheresis. 22:, 2007 © 2007 Wiley‐Liss, Inc.     

Antibodies from patients with heparin-induced thrombocytopenia/thrombosis are specific for platelet factor 4 complexed with heparin or bound to endothelial cells.

Heparin-induced thrombocytopenia/thrombosis (HITP) is thought to be mediated by immunoglobulins that activate platelets in the presence of pharmacologic concentrations of heparin, but the molecular basis for this relatively common and often serious complication of heparin therapy has not been established. We found that plasma from each of 12 patients with HITP contained high titer (> or = 1:200) antibodies that reacted with immobilized complexes of heparin and platelet factor 4 (PF4), a heparin-binding protein contained in platelet alpha-granules. Recombinant human PF4 behaved similarly to PF4 isolated from platelets in this assay system. Complexes formed at an apparent heparin/PF4 molecular ratio of approximately 1:2 (fresh heparin) and approximately 1:12 (outdated heparin) were most effective in binding antibody. Immune complexes consisting of PF4, heparin, and antibody reacted with resting platelets; this interaction was inhibited by a monoclonal antibody specific for the Fc gamma RII receptor and by excess heparin. Human umbilical vein endothelial cells, known to express heparin-like glycosaminoglycan molecules on their surface, were recognized by antibody in the presence of PF4 alone; this reaction was inhibited by excess heparin, but not by anti-Fc gamma RII. Antibodies reactive with heparin/PF4 were not found in normal plasma, but IgG and IgM antibodies were detected at dilutions of 1:10 (IgG) and 1:50 (IgM) in 3 of 50 patients (6%) with other types of immune thrombocytopenia. These findings indicate that antibodies associated with HITP react with PF4 complexed with heparin in solution or with glycosaminoglycan molecules on the surface of endothelial cells and provide the basis for a new hypothesis to explain the development of thrombocytopenia with thrombosis or disseminated intravascular coagulation in patients sensitive to heparin.     

Prothrombotic factors enhance heparin-induced thrombocytopenia and thrombosis in vivo in a mouse model

BACKGROUND: Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common cause of life- and limb-threatening thrombosis. The development of antibodies that react with complexes of heparin and platelet factor 4 (PF4) is fundamental to the development of the disease. However, anti-PF4/heparin antibodies are far more common than is HIT/T and there is less understanding of the factors that contribute to thrombosis in only a subset of patients. OBJECTIVES: Both qualitative and quantitative differences in multiple factors (e.g. antibodies, heparin and platelets) may influence the clinical course of patients who develop anti-PF4/heparin antibodies. We examined the hypothesis that host-specific factors, such as comorbid prothrombotic conditions, would exacerbate the pathologic effects of anti-PF4/heparin antibodies. METHODS AND RESULTS: A mouse model transgenic for human Fcgamma RIIa and PF4 and null for mouse PF4 was used to study the influence of prothrombotic conditions on the effects of anti-PF4/heparin antibodies in vivo. To simulate a prothrombotic milieu, mice were fed a hypercholesterolemic diet (HD). HD-fed mice had elevated plasma cholesterol, increased platelet reactivity and increased endothelial activation relative to mice fed a standard diet (SD). Age- and sex-matched mice from each diet group were treated with an anti-PF4/heparin antibody and heparin. HD-fed mice developed more severe thrombocytopenia than similarly treated SD-fed mice. Mice with moderate to severe thrombocytopenia had elevated plasma levels of thrombin-antithrombin complexes, indicative of increased thrombin generation in vivo. Platelet-fibrin thrombi were observed in multiple organs of HD-fed mice that developed severe thrombocytopenia. CONCLUSIONS: Host-specific factors, such as prothrombotic changes in platelet reactivity and/or endothelial activation, may influence the development of thrombosis in a subset of patients who develop anti-PF4/heparin antibodies.     

Heparin‐induced thrombocytopenia – therapeutic concentrations of danaparoid,unlike fondaparinux and direct thrombin inhibitors,inhibit formation of platelet factor 4–heparin complexes

Summary. Background: Treatment of heparin‐induced thrombocytopenia (HIT), a disorder in which anti‐platelet factor 4 (PF4)–heparin antibodies cause platelet activation and hypercoagulability, requires alternative (non‐heparin) anticoagulation. Treatment options include direct thrombin inhibitors [lepirudin and argatroban (approved), and bivalirudin], danaparoid (approved) (mixture of anticoagulant glycosaminoglycans), or fondaparinux (synthetic heparin‐mimicking pentasaccharide). PF4–heparin complexes form at optimal stoichiometric ratios. Objectives: To compare the effects of these various non‐heparin anticoagulants in disrupting the formation of PF4–heparin complexes, and PF4‐containing immune complexes. Patients/methods: Sera were obtained from patients with serologically confirmed HIT. The effects of the alternative anticoagulants on PF4 and PF4–heparin complex interactions with platelets, as well as HIT antibody binding and platelet activation, were investigated. Results: Danaparoid at very low concentrations increased PF4 binding to platelets. In therapeutic concentrations, however, it decreased PF4 binding to platelets (P = 0.0004), displaced PF4–heparin complexes from platelets (P = 0.0033) and PF4 from the surface of a PF4‐transfected HEK‐293 EBNA cell line expressing the PF4 receptor CXCR3‐B (P = 0.0408), reduced PF4–heparin complex size (P = 0.025), inhibited HIT antibody binding to PF4–heparin complexes (P = 0.001), and prevented platelet activation by HIT antibodies (P = 0.046). Although fondaparinux also interfered with PF4 binding to platelets, HIT antibody binding to PF4–heparin complexes, and activation of platelets by HIT antibodies, these effects occurred only at supratherapeutic concentrations. The direct thrombin inhibitors had no effect at any concentrations. Conclusions: Danaparoid uniquely interferes with the pathogenesis of HIT by disrupting PF4‐containing immune complexes at therapeutic dose concentrations. It is possible that these effects contribute to its therapeutic efficacy.     

肝素／血小板因子4抗体与肝素诱导的血小板减少症

肝素诱导的血小板减少症（heparin—induced thrombocytopenia，HIT）是肝素治疗引起的严重并发症，可导致血栓形成和栓塞。HIT的发病机制主要与肝素／血小板因子4抗体介导的免疫反应有关，IgG类是主要的致病抗体，能与肝素和血小板因子4结合形成复合物，引起血小板凝集和凝血反应增强，同时抗体还通过作用于血管内皮细胞和单核细胞参与HIT的形成。抗体相关的实验室检测包括功能性血小板试验和免疫学试验，临床表现结合实验室检测有助于本病的早期诊断和治疗，但是在检测方面目前尚没有理想的方法。本文就AHPF4抗体、HIT发病机制、临床实验室检测和免疫学试验检测等问题进行了综述。     

Intravenous immunoglobulin as an adjunct therapy in persisting heparin-induced thrombocytopenia

Heparin induced thrombocytopenia (HIT) is a serious adverse drug reaction caused by transient antibodies against platelet factor 4 (PF4)/heparin complexes, resulting in platelet activation and potentially fatal arterial and/or venous thrombosis. Most cases of HIT respond to cessation of heparin and administration of an alternative non-heparin anticoagulant, but there are cases of persisting HIT, defined as thrombocytopenia due to platelet activation/consumption for greater than seven days despite standard therapy. These patients remain at high risk for thrombotic events, which may result in limb-loss and mortality. Intravenous immunoglobulin (IVIg) has been proposed as an adjunct therapy for these refractory cases based on its ability to saturate FcγRIIa receptors on platelets, thus preventing HIT antibody binding and platelet activation. We describe 2 cases of persisting HIT (strongly positive antigen and functional assays, and persisting thrombocytopenia >7 days) with rapid clinical response to IVIg. We performed in-vitro experiments to support IVIg response. Healthy donor platelets (1?×?10e6) were treated with PF4 (3.75?μg/mL) for 20?min followed by 1-hour incubation with patients’ sera. Platelet activation with and without addition of IVIg (levels equivalent to those reached in a patient after treatment with 2?gm/Kg) was evaluated in the PF4-dependent P-selectin expression assay (PEA). A significantly decreased platelet activation was demonstrated after the addition of IVIg to both patient samples, which correlated well with the rapid clinical response that each patient experienced. Thus, our study supports the use of IVIg as an adjunct therapy for persisting HIT.     

Testing for heparin-induced thrombocytopenia antibodies

Heparin-induced thrombocytopenia (HIT) has a distinct clinical profile and unique pathogenesis. It is caused by platelet-activating IgG antibodies that recognize multimolecular complexes of platelet factor 4 (PF4) bound to heparin or certain other polyanions. Although an immune response to PF4/heparin associated with heparin treatment is very common, clinical HIT occurs only among the minority of patients whose antibodies are capable of strongly activating platelets. This explains why certain platelet activation assays and anti-PF4/polyanion immunoassays have high sensitivity for HIT and why diagnostic specificity is highest for those assays that preferentially detect pathogenic antibodies, such as the washed platelet activation assays or immunoassays that detect only IgG antibodies. Negative results obtained in a solid-phase PF4/polyanion immunoassay generally exclude HIT (high negative predictive value), especially in a setting of a low pretest probability. In addition, because the magnitude of a positive test result correlates with greater likelihood of HIT, a Bayesian diagnostic approach that combines pretest probability and the magnitude of a positive test result is recommended. Recent studies suggest that presence of anti-PF4/polyanion antibodies in certain clinical settings confers an adverse prognosis, even without clinically evident HIT. Whether such antibodies impart "forme fruste" HIT or are simply a surrogate marker for a non-HIT adverse risk factor such as inflammation is unresolved.     

Platelet and monocyte antigenic complexes in the pathogenesis of heparin-induced thrombocytopenia (HIT)

Summary.  Heparin-induced thrombocytopenia (HIT) is an iatrogenic disorder that occurs in a small subset of patients receiving heparin. Twenty-five per cent (or higher) of affected patients develop limb or life-threatening thrombosis. The effectiveness of therapy is incomplete and may be complicated by bleeding. HIT is caused by antibodies that recognize the platelet chemokine, Platelet Factor 4 (PF4), complexed to heparin or to cellular glycosaminoglycans (GAGs). However, antibodies with the same apparent specificity are found in many more patients without clinical disease and the reason why so few develop HIT is uncertain. We propose that HIT antibodies recognize cell surface PF4/GAG complexes on intravascular cells, including platelets and monocytes that are dynamic and mutable. Heparin removes cell surface-bound PF4 in most individuals, but removal is incomplete in those with high pre-exposure surface-bound PF4 levels. Such individuals retain critically localized cellular antigenic complexes at the time antibodies develop and are at risk to develop HIT. This article reviews the scientific basis for this model and its clinical implications.     

Vaccine-induced immune thrombotic thrombocytopenia (VITT): Update on diagnosis and management considering different resources

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but severe immunological reaction to the non-replicable adenoviral vector-based COVID-19 vaccines. Extreme activation of platelets and the coagulation system leads to a high risk of death from venous or arterial thrombosis or secondary hemorrhage. Public and clinician awareness has reduced mortality of VITT by nearly 90%. The World Health Organization provided a guideline in July 2021 on diagnosis and management of VITT (also called thrombosis with thrombocytopenia syndrome, or TTS). Since July 2021, new, clinically relevant information has become available. This update has been summarized by the authors in an informal process with recommendations for low resource environments. We provide new available evidence on VITT to empower clinicians to recognize VITT early, then effectively diagnose and treat the disorder to reduce morbidity and mortality. We strongly encourage production of clear management pathways for primary care settings and hospital settings.     

Autoimmune heparin‐induced thrombocytopenia

Summary

Autoimmune heparin‐induced thrombocytopenia (aHIT) indicates the presence in patients of anti‐platelet factor 4 (PF4)–polyanion antibodies that are able to activate platelets strongly even in the absence of heparin (heparin‐independent platelet activation). Nevertheless, as seen with serum obtained from patients with otherwise typical heparin‐induced thrombocytopenia (HIT), serum‐induced platelet activation is inhibited at high heparin concentrations (10–100 IU mL^?1 heparin). Furthermore, upon serial dilution, aHIT serum will usually show heparin‐dependent platelet activation. Clinical syndromes associated with aHIT include: delayed‐onset HIT, persisting HIT, spontaneous HIT syndrome, fondaparinux‐associated HIT, heparin ‘flush’‐induced HIT, and severe HIT (platelet count of < 20 × 10⁹ L^?1) with associated disseminated intravascular coagulation (DIC). Recent studies have implicated anti‐PF4 antibodies that are able to bridge two PF4 tetramers even in the absence of heparin, probably facilitated by non‐heparin platelet‐associated polyanions (chondroitin sulfate and polyphosphates); nascent PF4–aHIT‐IgG complexes recruit additional heparin‐dependent HIT antibodies, leading to the formation of large multimolecular immune complexes and marked platelet activation. aHIT can persist for several weeks, and serial fibrin, D‐dimer, and fibrinogen levels, rather than the platelet count, may be helpful for monitoring treatment response. Although standard anticoagulant therapy for HIT ought to be effective, published experience indicates frequent failure of activated partial thromboplastin time (APTT)‐adjusted anticoagulants (argatroban, bivalirudin), probably because of underdosing in the setting of HIT‐associated DIC, known as ‘APTT confounding’. Thus, non‐APTT‐adjusted therapies with drugs such as danaparoid and fondaparinux, or even direct oral anticoagulants, such as rivaroxaban or apixaban, are suggested therapies, especially for long‐term management of persisting HIT. In addition, emerging data indicate that high‐dose intravenous immunoglobulin can interrupt HIT antibody‐induced platelet activation, leading to rapid platelet count recovery.

     

Laboratory diagnosis of heparin-associated thrombocytopenia and comparison of platelet aggregation test, heparin-induced platelet activation test, and platelet factor 4/heparin enzyme-linked immunosorbent assay

BACKGROUND : As clinical diagnosis of heparin-associated thrombocytopenia (HAT) is often difficult, confirmation by sensitive laboratory assays is desirable. STUDY DESIGN AND METHODS : The sensitivity of the heparin-induced platelet activation (HIPA) test and the platelet aggregation test (PAT) was prospectively compared by using the sera of 209 patients with the putative diagnosis of HAT. Both assays were performed concomitantly with platelets of the same four donors using a different combination of donors from day to day. Further, all sera were assessed with a platelet factor 4 (PF4)/heparin enzyme-linked immunosorbent assay (ELISA). RESULTS : Positive results were obtained with 33 percent of sera in the PF4/heparin ELISA, with 33.5 percent of sera in the HIPA test, and with 11.5 percent of sera in the PAT. The PF4/heparin ELISA and the HIPA test showed no difference in sensitivity (p = 0.27 by McNemar's test) and were more sensitive than PAT (p < 10(-8) by McNemar's test). However, they recognized different patient cohorts. Nine HIPA-indeterminate and 12 HIPA-negative sera were positive in the PF4/heparin ELISA. Eight of the nine indeterminate sera caused platelet activation with high heparin concentrations in the HIPA test. Eleven of the 12 negative sera contained no IgG, but 9 contained IgM and 2 contained IgA HAT antibodies. Four sera that were indeterminate in the PF4/heparin ELISA and 18 sera that were negative were positive in the HIPA test. None of the sera that were positive in the PAT was missed in the HIPA test, but two of those were negative in the PF4/heparin ELISA. All sera were assessed with four low-molecular-weight heparins and a low-molecular- weight heparinoid in the HIPA test with platelets from the same four donors. Low-molecular-weight heparin caused platelet activation with positive sera in 98 percent of tests, and the heparinoid did so in 10 percent; in a further 12.8 percent, crossreactivity to the low- molecular-weight heparinoid could not be excluded. CONCLUSION : The majority of HAT antibodies react with a PF4/heparin complex, but there is strong evidence that other antigens are involved in some patients. The HIPA test and the PF4/heparin ELISA are sensitive for diagnosing HAT, and they complement one another.     

Platelet chemokines and their receptors: what is their relevance to platelet storage and transfusion practice?

The role of platelets as inflammatory cells is demonstrated by the fact that they can release many growth factors and inflammatory mediators, including chemokines, when they are activated. The best known platelet chemokine family members are platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG), which are synthesized in megakaryocytes, stored as preformed proteins in alpha-granules and released from activated platelets. However, platelets also contain many other chemokines such as interleukin-8 (IL-8), growth-regulating oncogene-alpha(GRO-alpha), epithelial neutrophil-activating protein 78 (ENA-78), regulated on activation normal T expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), and monocyte chemotactic protein-3 (MCP-3). They also express chemokine receptors such as CCR4, CXCR4, CCR1 and CCR3. Platelet activation is a feature of many inflammatory diseases such as heparin-induced thrombocytopenia, acquired immunodeficiency syndrome, and congestive heart failure. Substantial amounts of PF4, beta-TG and RANTES are released from platelets on activation, which may occur during storage. Although very few data are available on the in vivo effects of transfused chemokines, it has been suggested that the high incidence of adverse reactions often observed after platelet transfusions may be attributed to the chemokines present in the plasma of stored platelet concentrates.     

Early-onset and persisting thrombocytopenia in post-cardiac surgery patients is rarely due to heparin-induced thrombocytopenia, even when antibody tests are positive

See also Gruel Y, Pouplard C. Post‐operative platelet count profile: the most reliable tool for identifying patients with true heparin‐induced thrombocypenia after cardiac surgery. This issue, pp 27–29. Summary. Background: The high frequency of thrombocytopenia in post‐cardiac surgery patients makes it challenging to diagnose heparin‐induced thrombocytopenia (HIT). Two platelet count profiles are reported as indicating possible HIT in these patients: profile 1 describes a platelet count fall that begins between postoperative days 5 and 10, whereas profile 2 denotes early‐onset thrombocytopenia that persists beyond day 5. Objectives: To examine how these platelet count profiles correlate with antibody status and HIT post‐cardiac surgery. Methods: We prospectively screened 581 cardiac surgery patients for heparin‐dependent antibodies by platelet factor 4 (PF4)–heparin immunoassay and platelet‐activation test, and performed daily platelet counts (until day 10) with 30‐day follow‐up. Results: All three patients with platelet count profile 1 tested positive for platelet‐activating anti‐PF4–heparin IgG antibodies [odds ratio (OR) 521.7, 95% confidence interval (CI) 3.9–34 000, P = 0.002], and were judged to have HIT. In contrast, none of 25 patients with early‐onset and persisting thrombocytopenia (profile 2) was judged to have HIT, including five patients testing positive for platelet‐activating anti‐PF4–heparin IgG antibodies. In these patients, the frequency of heparin‐dependent antibodies did not differ from that in non‐thrombocytopenic controls, either for anti‐PF4–heparin IgG (OR 1.7, 95% CI 0.7–4.1, P = 0.31) or for platelet‐activating antibodies (OR 1.9, 95% CI 0.6–5.7, P = 0.20). Multivariate analysis revealed that type of cardiac surgery, but not HIT antibody status, predicted early‐onset and persisting thrombocytopenia. Together, these findings show that HIT was uncommon in this study population [overall frequency, 3/581 (0.5%), 95% CI 0.1–1.5%]. Conclusions: Thrombocytopenia that begins between 5 and 10 days post‐cardiac surgery is highly predictive for HIT. In contrast, early‐onset and persisting thrombocytopenia is usually caused by non‐HIT factors with coinciding heparin‐dependent antibody seroconversion.     

Frequency of heparin/platelet factor 4-dependent platelet antibodies in patients undergoing angioplasty and stenting for cardiovascular disease and their role for on-clopidogrel platelet reactivity

Background  

The frequency of heparin-induced platelet antibodies (H/PF4 antibodies) following heparin exposure during percutaneous intervention with stent implantation is unknown. These antibodies may activate platelets and therefore contribute to high on-clopidogrel residual platelet reactivity (HRPR).     

Low-density lipoprotein apheresis reduces platelet factor 4 on the surface of platelets: a possible protective mechanism against heparin-induced thrombocytopenia and thrombosis

BACKGROUND: Heparin‐induced thrombocytopenia and thrombosis (HITT) is characterized by thrombocytopenia due to the formation of antibodies against heparin : platelet factor 4 (PF4) complexes. Despite the exposure to heparin during treatment and predisposition of patients with atherosclerosis to HITT, HITT in patients undergoing low‐density lipoprotein (LDL) apheresis is rare. We investigated the possibility that LDL apheresis decreases PF4 on platelet (PLT) surfaces and/or plasma HITT antibody levels, either of which would disfavor HITT. STUDY DESIGN AND METHODS: We enrolled 25 patients undergoing LDL apheresis at the Hospital of the University of Pennsylvania. Blood samples were drawn before and after treatment. Plasma samples were drawn proximal and distal to the LA‐15 treatment column. PF4, HITT antibodies, heparin levels, and P‐selectin were measured. RESULTS: No patient had clinical symptoms of HITT. The LA‐15 column was found to efficiently remove PF4. PF4 levels in peripheral blood plasma did not change significantly after LDL apheresis. However, PLT surface PF4 significantly decreased after treatment. HITT antibodies were found in only two patients and were nonfunctional. PLT surface P‐selectin did not change during treatment. CONCLUSIONS: We have demonstrated that LDL apheresis via dextran sulfate absorption removes plasma PF4 and reduces the amount of PF4 on the surface of circulating PLTs. Reduced surface PF4 may decrease antibody formation and/or recognition by HITT antibodies. These data provide a potential explanation for the near lack of HITT in hypercholesterolemic patients undergoing LDL apheresis. They also suggest the possibility that LDL apheresis using dextran sulfate adsorption may have therapeutic value in the treatment of HITT.     

Characterization of antibodies directed against platelet cryptantigens detected during the immunological study of 356 consecutive patients with presumed autoimmune thrombocytopenia (AITP)

SUMMARY. Cryptic antigens are detected by anti-bodies present in a wide spectrum of patients with or without thrombocytopenia, and even in healthy individuals. They are produced for unknown reasons and do not react with antigens of native platelets, but only with altered platelets. Cryptantigen antibodies may not only result in spuriously low platelet counts, but also in 'falsely' positive tests for platelet antibodies. We report our experience in the characterization of the different types of antibodies directed against cryptantigens of platelets: EDTA-dependent antibodies, PFA-dependent antibodies, EDTA-PFA-dependent antibodies and cold agglutinins. These antibodies were detected in the course of the serological study of 37 patients from a group of 356 (10%) whose blood was sent to our laboratory for platelet antibody testing. Pseudothrombocytopenia was diagnosed in 24 cases. Twenty-one of these showed EDTA-dependent or EDTA-PFA-dependent platelet agglutination and three were due to the presence of cold agglutinins. In 13 patients the thrombocytopenia was genuine. Eleven of these presented EDTA-dependent or EDTA-PFA-dependent antibodies in their serum and in the two remaining cases PFA-dependent antibodies were found. Cryptantigen antibodies were also detected in 9 out of 228 (4%) blood donors who were used as healthy controls in the platelet immunofluorescence test. In the light of the results obtained we put forward some guidelines to detect the presence of these antibodies and establish an accurate serological and clinical diagnosis of the autoimmune thrombocytopenias.     

False-positive tests for heparin-induced thrombocytopenia in patients with antiphospholipid syndrome and systemic lupus erythematosus

Summary.  Background : Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy that can be associated with arterial or venous thrombosis and is caused by antibodies against platelet factor 4 (PF4)–heparin complex. Patients with antiphospholipid syndrome (APS) have been reported with positive tests for PF4–heparin complex antibodies by antigen assay. Whether such patients can be treated with heparin is a dilemma. Objectives : To determine the incidence and nature of the HIT immune reaction in patients with APS and/or systemic lupus erythematosus (SLE). Methods : Antibodies against PF4–heparin complex were assayed by particle gel immunoassay (PaGIA), or enzyme immunoassay (EIA) with or without an excess of heparin. EIA for PF4 alone was also performed. Functional assays for HIT, that is, heparin-induced platelet activation (HIPA) and heparin-induced platelet aggregation, were also performed. Results : In 32 of 42 patients (76.2%) with APS, APS and SLE, SLE, or SLE with antiphospholipid antibodies, EIA IgG or PaGIA for PF4–heparin complex antibodies were positive. Of these 32 samples, 26 (81.3%) tested positive for anti-PF4 antibodies. All 24 samples that were positive for PF4–heparin complex by EIA IgG were also positive for EIA IgG in the presence of heparin excess, and all were negative by the HIPA and heparin-induced platelet aggregation tests. Conclusion : A large proportion of patients with APS and/or SLE give false-positive HIT antigen test results that are presumably related to autoantibodies against PF4, which can be distinguished from true HIT antibodies by EIA for PF4–heparin complexes tested with heparin excess, and by functional assays.     

The use of flow cytometry in the diagnosis of heparin-induced thrombocytopenia (HIT)

Heparin-induced thrombocytopenia (HIT) affects some of the patients exposed to heparin. It is mediated by antibodies that recognize neoepitopes on platelet factor 4 (PF4)/heparin complexes. A HIT diagnosis requires both clinical and laboratory evaluation and remains a challenge. Since many patients develop antibodies in response to heparin, but only a few of them generate anti-PF4/heparin antibodies capable of activating platelets which consequently cause clinical complications, the performance of serologic assays is not enough to diagnose HIT. Functional assays can identify pathogenic antibodies capable of platelet activation, but they are more demanding and their limited availability contributes to the problem of diagnosing HIT. Restricted laboratories usually collect sera of multiple patients to perform functional assays only once or twice a week; hence, a HIT diagnosis can take several days. The use of flow cytometry appears to be a promising alternative in the confirmation of pathogenic anti-PF4/heparin antibodies. Flow cytometric assays detect either activation markers on a healthy donor’s platelet surfaces or platelet derived microparticles formed after platelet incubation with a patient’s serum. Flow cytometers are readily available in many clinical laboratories, so this technology introduces the possibility of an earlier HIT diagnosis. The objective of this review was to collect findings on flow cytometric HIT confirmations to the present date, and to review the currently available flow cytometric assays used in the diagnosis of HIT.     

Determinants of donor platelet variability when testing for heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia is a common immune-mediated drug reaction that can be complicated by life-threatening arterial thrombosis. The diagnosis can be confirmed by demonstrating heparin-dependent release of radiolabeled serotonin from washed normal platelets in the presence of patient serum. However, certain serum samples from these patients produce 14C-serotonin release from some but not other normal donor platelets. We investigated this problem of donor platelet variability by studying the reactivities of 10 serum samples from patients with heparin-induced thrombocytopenia with platelets from 10 normal donors (100 serum and platelet combinations). We observed a marked variability in reactivity for patient serum and platelets from normal donors; this initially appeared random. However, closer examination indicated that the reactivities varied hierarchically. Because heparin-induced thrombocytopenia is caused by binding of heparin-dependent IgG to platelet Fc receptors, we examined whether platelet Fc receptor number or function explained the variability in platelet reactivity. We observed that platelet Fc receptor function, as measured by platelet release associated with heat-aggregated IgG, was highly correlated with platelet reactivity to heparin-induced thrombocytopenia serum samples. No significant correlation, however, was found between Fc receptor number and platelet response. Reaction of murine monoclonal antibodies that activate human platelets by means of the platelet Fc receptors was not predictive of platelet reactivity to heparin-induced thrombocytopenia serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative effects of two synthetic oligosaccharides on platelet activation induced by plasma from HIT patients

Summary.  Heparin-induced thrombocytopenia (HIT) is a serious secondary event encountered in the clinical use of heparin. HIT results from the consumption of platelets that are immunologically activated by antibodies directed against complexes formed by platelet factor 4 (PF4) and sulfated polysaccharides that activate platelet aggregation, leading to paradoxical, life-threatening thrombosis. There is strong evidence that the ability of heparin and related compounds to induce HIT is closely linked to the structure of the polysaccharide, and particularly to its negative charge and to the length of the molecule. To test this hypothesis, we synthesized two sulfated oligosaccharides: SanOrg123781, a 16-mer, presenting two terminal charged domains separated by a 7-mer neutral linker, and SR121903, a highly sulfated 17-mer. Both of them displayed strong anti-factor (F) Xa and anti-FIIa activities but their affinities for PF4 were markedly different. SR121903 displaced PF4-bound heparin, whereas SanOrg123781 did not, underlining the importance of the charge of the molecule for the interaction with PF4. Platelet studies, in the presence of HIT serum, showed that SR121903 induced the secretion of platelet-dense granules (measured by the release of serotonin) whereas SanOrg123781 did not, a result in accordance with an absence of affinity of this molecule for PF4. These results were confirmed by measurements of platelet activation by flow cytometry (measured by annexin V binding, CD62 detection and activation of the GpIIb–IIIa complexes). In conclusion, we have demonstrated the importance of the charge of the polysaccharides in the HIT-induced platelet reactions measured by diverse methods, of which some are described for this purpose for the first time.