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Adam Cuker,
Ann H Rux,
Jillian L Hinds,
May Dela Cruz,
Serge V Yarovoi,
Isola A M Brown,
Wei Yang,
Barbara A Konkle, Gowthami M Arepally,
Stephen P Watson,
Douglas B Cines,
Bruce S Sachais
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ABSTRACT: Laboratory testing for heparin-induced thrombocytopenia (HIT) has important shortcomings. Immunoassays fail to discriminate platelet-activating from non-pathogenic antibodies. Specific functional assays are often impracticable due to the need for platelets and radioisotope. We describe two assays that may overcome these limitations. The KKO-inhibition test (KKO-I) measures the effect of plasma on binding of the HIT-like monoclonal antibody KKO to PF4/heparin. DT40-luciferase (DT40-luc) is a functional test comprised of a B-cell line expressing FcγRIIa coupled to a luciferase reporter. We compared these assays to polyspecific and IgG-specific PF4/heparin ELISAs in samples from 58 patients with suspected HIT and circulating anti-PF4/heparin antibodies. HIT was defined as a 4Ts score ≥4 and positive 14C-serotonin release assay. HIT-positive plasma demonstrated greater mean inhibition of KKO binding than HIT-negative plasma (78.9% vs. 26.0%, p<0.0001) and induced greater luciferase activity (3.14-fold basal vs. 0.96-fold basal, p<0.0001). The area under the ROC curve (AUC) was greater for KKO-I (0.93) than for the polyspecific (0.82, p=0.020) and IgG-specific ELISA (0.76, p=0.0044) and for DT40-luc (0.89) than for the IgG-specific ELISA (p=0.046). KKO-I and DT40-luc showed better discrimination than two commercially available immunoassays, are simple to perform, and hold promise for improving the specificity and feasibility of HIT laboratory testing.
Blood 02/2013; · 9.90 Impact Factor
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ABSTRACT: Protamine is routinely used to reverse heparin anticoagulation during cardiopulmonary bypass (CPB). Heparin (H) interacts with protamine (PRT) to form ultra-large complexes that are immunogenic in mice. We hypothesized that patients exposed to PRT and H during CPB will develop antibodies to PRT/H complexes that are capable of platelet activation. Specimens from a recently completed prospective clinical trial (HIT 5801 study; n=500) of CPB patients were examined for PRT/H antibodies at baseline, 3-7 days, and 30 days after CPB. PRT/H antibody features were characterized and correlated with adverse cardiovascular outcomes. We found a high incidence of PRT/H antibody formation (29%) in patients undergoing cardiac surgery. PRT/H antibodies were of high titer (mean titer 1:14,744), showed heparin-dependent binding and activated platelets in the presence of protamine. PRT/H antibodies showed no cross-reactivity to platelet factor 4/heparin complexes, but were cross-reactive with protamine-containing insulin preparations. Complications of thrombocytopenia, thrombotic events or long-term cardiovascular events were not seen in the absence of circulating antigen. These studies show that antibodies to PRT/H occur commonly after cardiac bypass surgery, share a number of serologic features with HIT antibodies, including platelet activation, and may pose health risks to patients requiring drug re-exposure.
Blood 02/2013; · 9.90 Impact Factor
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Bruce S Sachais,
Rustem I Litvinov,
Serge V Yarovoi,
Lubica Rauova,
Jillian L Hinds,
Ann H Rux, Gowthami M Arepally,
Mortimer Poncz,
Adam Cuker,
John W Weisel,
Douglas B Cines
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ABSTRACT: Rapid laboratory assessment of heparin-induced thrombocytopenia (HIT) is important for disease recognition and management. The utility of contemporary immunoassays to detect antiplatelet factor 4 (PF4)/heparin antibodies is hindered by detection of antibodies unassociated with disease. To begin to distinguish properties of pathogenic anti-PF4/heparin antibodies, we compared isotype-matched monoclonal antibodies that bind to different epitopes: KKO causes thrombocytopenia in an in vivo model of HIT, whereas RTO does not. KKO binding to PF4 and heparin is specifically inhibited by human HIT antibodies that activate platelets, whereas inhibition of RTO binding is not differentially affected. Heparin increased the avidity of KKO binding to PF4 without affecting RTO, but it did not increase total binding or binding to nontetrameric PF4(K50E). Single-molecule forced unbinding demonstrated KKO was 8-fold more reactive toward PF4 tetramers and formed stronger complexes than RTO, but not to PF4(K50E) dimers. KKO, but not RTO, promoted oligomerization of PF4 but not PF4(K50E). This study reveals differences in the properties of anti-PF4 antibodies that cause thrombocytopenia not revealed by ELISA that correlate with oligomerization of PF4 and sustained high-avidity interactions that may simulate transient antibody-antigen interactions in vivo. These differences suggest the potential importance of epitope specificity in the pathogenesis of HIT.
Blood 05/2012; 120(5):1137-42. · 9.90 Impact Factor
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ABSTRACT: The immune response in heparin-induced thrombocytopenia is initiated by and directed to large multimolecular complexes of platelet factor 4 (PF4) and heparin (H). We have previously shown that PF4:H multimolecular complexes assemble through electrostatic interactions and, once formed, are highly immunogenic in vivo. Based on these observations, we hypothesized that other positively charged proteins would exhibit similar biologic interactions with H. To test this hypothesis, we selected 2 unrelated positively charged proteins, protamine (PRT) and lysozyme, and studied H-dependent interactions using in vitro and in vivo techniques. Our studies indicate that PRT/H and lysozyme/H, like PF4/H, show H-dependent binding over a range of H concentrations and that formation of complexes occurs at distinct stoichiometric ratios. We show that protein/H complexes are capable of eliciting high-titer antigen-specific antibodies in a murine immunization model and that PRT/H antibodies occur in patients undergoing cardiopulmonary bypass surgery. Finally, our studies indicate that protein/H complexes, but not uncomplexed protein, directly activate dendritic cells in vitro leading to interleukin-12 release. Taken together, these studies indicate that H significantly alters the biophysical and biologic properties of positively charged compounds through formation of multimolecular complexes that lead to dendritic cell activation and trigger immune responses in vivo.
Blood 12/2010; 116(26):6046-53. · 9.90 Impact Factor
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ABSTRACT: Platelet factor 4 (PF4)/heparin antibody, typically associated with heparin therapy, is reported in some heparin-naive people. Seroprevalence in the general population, however, remains unclear. We prospectively evaluated PF4/heparin antibody in approximately 4,000 blood bank donors using a commercial enzyme-linked immunosorbent assay for initial and then repeated (confirmatory) testing. Antibody was detected initially in 249 (6.6%; 95% confidence interval [CI], 5.8%-7.4%) of 3,795 donors and repeatedly in 163 (4.3%; 95% CI, 3.7%-5.0%) of 3,789 evaluable donors. "Unconfirmed" positives were mostly (93%) low positives (optical density [OD] = 0.40-0.59). Of 163 repeatedly positive samples, 116 (71.2%) were low positives, and 124 (76.1%) exhibited heparin-dependent binding. Predominant isotypes of intermediate to high seropositive samples (OD >0.6) were IgG (20/39 [51%]), IgM (9/39 [23%]), and indeterminate (10/39 [26%]). The marked background seroprevalence of PF4/heparin antibody (4.3%-6.6%) with the preponderance of low (and frequently nonreproducible) positives in blood donors suggests the need for further assay calibration, categorization of antibody level, and studies evaluating clinical relevance of "naturally occurring" PF4/heparin antibodies.
American Journal of Clinical Pathology 11/2010; 134(5):774-80. · 2.60 Impact Factor
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ABSTRACT: The diagnosis of heparin-induced thrombocytopenia (HIT) requires detection of antibodies to the heparin/platelet factor 4 (PF4) complexes via enzyme-linked immunosorbent assay. Addition of excess heparin to the sample decreases the optical density by 50% or more and confirms the presence of these antibodies. One hundred fifteen patients with anti-heparin/PF4 antibodies detected by enzyme-linked immunosorbent assay were classified as clinically HIT-positive or HIT-negative, followed by confirmation with excess heparin. A multivariate logistic regression model was fitted to estimate relationships between patient characteristics, laboratory findings, and clinical HIT status. This model was validated on an independent sample of 97 patients with anti-heparin/PF4 antibodies. No relationship between age, race, or sex and clinical HIT status was found. Maximal optical density and confirmatory positive status independently predicted HIT in multivariate analysis. Predictive accuracy on the training set (c-index 0.78, Brier score 0.17) was maintained when the algorithm was applied to the independent validation population (c-index 0.80, Brier score 0.20). This study quantifies the clinical utility of the confirmatory test to diagnose HIT. On the basis of data from the heparin/PF4 enzyme-linked immunosorbent assay and confirmatory assays, a predictive computer algorithm could distinguish patients likely to have HIT from those who do not.
Blood 09/2010; 116(10):1761-6. · 9.90 Impact Factor
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ABSTRACT: Aprotinin, a nonspecific serine protease inhibitor, has been used to decrease bleeding and reduce the systemic inflammatory response after cardiopulmonary bypass (CPB). Studies have variably linked aprotinin administration with both improved as well as adverse cerebral consequences after cardiac surgery. We designed this study to determine whether an antiinflammatory dose of aprotinin could improve the histologic and functional neurologic outcome in a rat model of focal cerebral ischemia during CPB.
After surgical preparation, the animals were randomized into 2 groups: an aprotinin group (60,000 kIU/kg IV) and a control group (0.9% NaCl IV). Normothermic CPB was performed for 60 minutes during which time a partial overlapping 60 minutes of right middle cerebral artery occlusion was induced. Cytokines (tumor necrosis factor-alpha, interleukin [IL]-1beta, IL-6, and IL-10) were measured at baseline, the end of CPB, then 2 and 24 hours after CPB. On postoperative day 3, the animals underwent functional neurologic testing and histologic assessment of cerebral infarct volume.
There was a reduction in systemic inflammation in the aprotinin group compared with the control group, demonstrated by lower levels of IL-1beta (P = 0.035) and IL-6 (P = 0.047). The aprotinin group also had a better functional neurologic performance (median [interquartile range]: aprotinin 27 [8] vs control 32 [6]; P = 0.042). However, there was no difference in cerebral infarct volume (aprotinin 306 [27] mm(3) vs control 297 [52] mm(3); P = 0.599).
In this experimental model of stroke occurring during CPB, aprotinin decreased the systemic inflammatory response to CPB. Although there was no difference in the cerebral infarct volume, there was a small improvement in the short-term functional neurologic outcome in the aprotinin group.
Anesthesia and analgesia 07/2010; 111(1):38-45. · 3.08 Impact Factor
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ABSTRACT: Heparin-induced thrombocytopenia (HIT) is an immune-mediated hypercoagulable disorder caused by antibodies to platelet factor 4 (PF4) and heparin. HIT develops in temporal association with heparin therapy and manifests either as an unexplained thrombocytopenia or thrombocytopenia complicated by thrombosis. The propensity for thrombosis distinguishes HIT from other common drug-induced thrombocytopenias. Diagnosing HIT in hospitalized patients is often challenging because of the frequency of heparin use, occurrence of thrombocytopenia from other causes, and development of asymptomatic PF4/heparin antibodies in patients treated with heparin. This review summarizes our current understanding of the pathogenesis, clinical features, diagnostic criteria, and management approaches in HIT.
Annual review of medicine 01/2010; 61:77-90. · 9.94 Impact Factor
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Gowthami M Arepally
Blood 06/2009; 113(20):4825-6. · 9.90 Impact Factor
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British Journal of Haematology 06/2008; 142(4):671-3. · 4.94 Impact Factor
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ABSTRACT: Heparin-induced thrombocytopenia (HIT) is an antibody-mediated disorder that occurs with variable frequency in patients exposed to heparin. HIT antibodies preferentially recognize large macromolecular complexes formed between PF4 and heparin over a narrow range of molar ratios, but the biophysical properties of complexes that initiate antibody production are unknown. To identify structural determinants underlying PF4/heparin immunogenicity, we characterized the in vitro interactions of murine PF4 (mPF4) and heparin with respect to light absorption, size, and surface charge (zeta potential). We show that PF4/heparin macromolecular assembly occurs through colloidal interactions, wherein heparin facilitates the growth of complexes through charge neutralization. The size of PF4/heparin macromolecules is governed by the molar ratios of the reactants. Maximal complex size occurs at molar ratios of PF4/heparin at which surface charge is neutral. When mice are immunized with complexes that differ in size and/or zeta potential, antibody formation varies inversely with heparin concentration and is most robust in animals immunized with complexes displaying a net positive zeta-potential. These studies suggest that the clinical heterogeneity in the HIT immune response may be due in part to requirements for specific biophysical parameters of the PF4/heparin complexes that occur in settings of intense platelet activation and PF4 release.
Blood 01/2008; 110(13):4253-60. · 9.90 Impact Factor
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ABSTRACT: We report a case of Thrombotic Thrombocytopenic Purpura occurring as an allergic response to trimethoprim-sulfamethoxazole therapy (Bactrim, Septra) in a Jehovah's Witness patient. The patient presented with fulminant microangiopathic hemolytic anemia and thrombocytopenia within 48 hr of initiating therapy with trimethoprim-sulfamethoxazole. Other symptoms of drug hypersensitivity included nausea, vomiting, urticarial rash, and leukopenia. Because of her religious faith, the patient was supported without plasma therapy with use of intravenous immunoglobulin, steroids, rituximab, and erythropoietin.
American Journal of Hematology 08/2007; 82(7):679-81. · 4.67 Impact Factor
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ABSTRACT: Heparin-induced thrombocytopenia is a life threatening thrombotic disorder caused by antibodies to platelet factor 4 (PF4) and heparin. Commercial immunoassays are frequently used for the detection of PF4-heparin antibodies, and several studies have reported that higher antibody titers are more frequently associated with adverse events. It is not known if conditions involving sample preparation and/or storage affect the operational characteristics of PF4-heparin immunoassays. We compared the detection of PF4-heparin antibodies from 48 patient samples collected concordantly in serum separator tubes or tubes containing EDTA or sodium citrate. We also examined the effects of extended sample storage on whole blood collected in serum separator, EDTA, or citrate tubes at 4 degrees C for up to 96 hours on antibody detection. We noted that serum or plasma anticoagulated with sodium citrate or EDTA yielded comparable results. In addition, we could not demonstrate any significant sample deterioration after storage at 4 degrees C in any medium for up to 4 days. These findings suggest that PF4-heparin antibodies are largely insensitive to the effects of sample preparation and storage.
American Journal of Clinical Pathology 08/2007; 128(1):150-5. · 2.60 Impact Factor
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New England Journal of Medicine 09/2006; 355(8):809-17. · 53.30 Impact Factor
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ABSTRACT: Heparin-induced thrombocytopenia (HIT) antibodies recognize complexes between heparin and platelet factor 4 (PF4). Heparin and PF4 bind HIT antibodies only over a narrow molar ratio. We explored the involvement of platelet surface-bound PF4 as an antigen in the pathogenesis of experimental HIT. We show that cell-surface PF4 complexes are also antigenic only over a restricted concentration range of PF4. Heparin is not required for HIT antibody binding but shifts the concentration of PF4 needed for optimal surface antigenicity to higher levels. These data are supported by in vitro studies involving both human and murine platelets with exogenous recombinant human (h) PF4 and either an anti-PF4-heparin monoclonal antibody (KKO) or HIT immunoglobulin. Injection of KKO into transgenic mice expressing different levels of hPF4 demonstrates a correlation between the severity of the thrombocytopenia and platelet hPF4 expression. Therapeutic interventions in this model using high-dose heparin or protamine sulfate support the pathogenic role of surface PF4 antigenic complexes in the etiology of HIT. We believe that this focus on surface PF4 advances our understanding of the pathogenesis of HIT, suggests ways to identify patients at high risk to develop HIT upon heparin exposure, and offers new therapeutic strategies.
Blood 04/2006; 107(6):2346-53. · 9.90 Impact Factor
