Methodologic Variations in the Determinations of Anti-Platelet Factor 4-Heparin Antibodies in Patients Suspected of Having Heparin Induced Thrombocytopenia: Diagnostic and Prognostic Implications.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3206-3206
Author(s):  
Jawed Fareed ◽  
Margaret Prechel ◽  
Michelle Kujawski ◽  
He Zhu ◽  
Jeanine Walenga ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Medical Center ◽  
Sandwich Elisa ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Diagnostic Efficacy ◽  
Diagnostic Reliability ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Two Samples

Abstract It is now widely believed that the pathogenesis of heparin induced thrombocytopenia (HIT) is largely mediated via the generation of anti-platelet factor 4-heparin antibodies (APFA) in patients who are treated with heparin and related drugs. In addition antibodies to other heparin binding proteins such as neutrophil activating peptide-2 and interleukins also contribute to this syndrome and which are not detectable by the methods based on heparin platelet factor 4 capture probes. Currently, several immunologic methods mostly utilizing sandwich ELISA techniques to measure APFA in sera of patients with HIT syndrome are available. These methods utilize different capture probes including heparin platelet factor 4 complex (ASSERACHROM® HPIA; Diagnostica Stago, France), platelet factor 4 complexed to polyvinyl sulfonate (PF4 Enhanced; GTI, USA) and heparin protamine suflate along with platelet lysates as a source of PF4 (ZYMUTEST HIA IgGAM; Hyphen Biomedical, France). These different capture probes for the APFA have different affinity for the APFAs. Moreover, these capture probes can also bind to certain other heparin binding proteins. To compare these three methods, samples (n = 100) were selected from banked sera that had been referred to Loyola University Medical Center for the quantitation of HIT antibodies and 14C Serotonin Release Assay (SRA). All of these specimens were initially positive in the GTI PF4 Enhanced ELISA for the presence of anti-heparin platelet factor 4 antibodies with a broad range of antibody titers absorbance range (0.4 – 2.5). For the head to head comparison all of these samples were assayed using the three different methods. In the GTI method the reassayed samples showed 93 out of 100 positive (93%), in the HPIA test 79 out of 100 (79%) and in the ZYMUTEST 56 out of 100 (56%) were positive. Interestingly, the correlation coefficients also showed marked variation (GTI vs ZYMUTEST r2 = 0.38; GTI vs Stago r2 = 0.49; ZYMUTEST vs Stago r2 = 0.67). The prevalence of positive samples was not consistent in the three tests used. This data clearly shows that each of the different ELISA methods exhibit different performance characteristics in binding to not only the APFA but other proteins, which may contribute to the higher false positive prevalence. Moreover, the positive/negative cutoff limits in these assays are arbitrarily set without due consideration of the antibody titer, which can also account for the observed differences. More interestingly, all of the samples positive in the SRA (n = 14) were positive in each of the three methods used with the exception of two samples, which were positive in the GTI, ASSERACHROM® HPIA and negative in the ZYMUTEST. These results clearly indicate the differences in the diagnostic efficacy of various commercially available tests and warrant clinical field trials to validate their reliability in the monitoring of APFA. Furthermore, the diagnostic reliability of these tests can be further improved by immunoglobulin subtyping and utilizing other capture probes with platelet factor 4 and related proteins, which complex with heparins.

Functional Microparticles Are up Regulated in Patients with Anti-Heparin/Platelet Factor 4 Antibodies: A Potential Mechanism of Thrombogenesis in the Heparin-Induced Thrombocytopenia Syndrome.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2105-2105
Author(s):  
Josephine Cunanan ◽  
Michelle Kujawski ◽  
He Zhu ◽  
Margaret Prechel ◽  
Jeanine Walenga ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Medical Center ◽  
Annexin V ◽  
Platelet Factor 4 ◽  
Cellular Damage ◽  
Clinical Samples ◽  
Limiting Factor ◽  
Therapeutic Effects ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia

Abstract Heparin-induced thrombocytopenia (HIT) is one of the most catastrophic adverse effects of heparin therapy, representing a complex syndrome involving immunopathologic and hemostatic disorders. Vascular and blood cellular damage results in the generation of microparticles (MP). These MP are formed from stress conditions/cellular disruption and apoptosis. Cellular MP mediated pathophysiologic responses include platelet activation, up regulation of adhesion molecules, monocyte activation, up regulation of tissue factor and endothelial dysfunction. Several methods based on flow cytometric and other immunologic probes have been used to measure MP in the HIT syndrome. Recently, a functional method based on the complexation of MP with annexin V promoting the generation of factor Xa and thrombin has become available (Hyphen Biomedical, Neuville-Oise, France). To validate the hypothesis that functional MP are elevated in the HIT syndrome, this method was utilized for the quantitation of MP in sera ELISA positive for anti-heparin/platelet factor 4 (HIT) antibodies. Specimens (n = 53) were selected from archived samples that had been referred to Loyola University Medical Center for the laboratory diagnosis of HIT by quantitating anti-heparin/PF4 antibodies by ELISA and by evaluating HIT antibody induced platelet activation using the 14C Serotonin Release Assay (SRA). All selected specimens were positive for HIT antibodies in the GTI PF4 Enhanced ELISA with a broad range of antibody titers (absorbance range of 0.4 – 2.5). Eleven of these specimens were positive in the SRA. In addition, serial samples from HIT patients treated with argatroban (from the ARG-911 clinical study) were included (n = 23). The normal samples represented control sera obtained from healthy human volunteers (n = 25) and processed in the same manner as the clinical samples. Test samples were added to microtiter plates coated with streptavidin and biotinylated annexin V. MP present in the test sample bound to annexin V via exposed surface phospholipids. Following incubation and washing steps, a FXa – FVa mixture containing calcium and prothrombin was added. The assay was optimized so that MP associated phospholipid was the limiting factor for the generation of thrombin. In normal non-HIT sera, the MP levels ranged 5.6 – 10.1 nM (6.1 ± 2.8 nM). The pre-treatment, baseline levels of circulating MP in the suspected HIT patients ranged from 4.2 – 26.8 nM (15.8 ± 7.3 nM). Interestingly, SRA positive/ELISA positive samples had relatively higher levels of MP (19.9 ± 7.7 nM; range 11.5 – 29.8 nM) than SRA negative/ELISA positive samples (14.2± 4.6; range 6.8–21.2). In the ARG-911 study, sequential blood samples exhibited MP levels at the baseline ranging from 8.2 – 38.6 nM (21.8 ± 10.8 nM), whereas after 3 days of argatroban treatment were reduced to 5.1 – 19.2 nM (12.6 ± 6.3). The results of these studies suggest that circulating functional MP are increased in patients with ELISA positive HIT antibodies. Anticoagulation with such direct thrombin agents as argatroban effectively decreases the circulating functional MP levels. Since the elevated MP levels may mediate thrombin and FXa generation, the therapeutic effects of these drugs in HIT may be related to the decreased activation of coagulation and related thrombogenic processes.

Defining an Antigenic Epitope on Platelet Factor 4 Associated With Heparin-Induced Thrombocytopenia

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3250-3259 ◽  
Author(s):  
L. Ziporen ◽  
Z.Q. Li ◽  
K.S. Park ◽  
P. Sabnekar ◽  
W.Y. Liu ◽  
...  
Keyword(s):  
Antigenic Site ◽  
Cysteine Residue ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Antigenic Epitope ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
The Third ◽  
Lysine Residues ◽  
Heparin Complexes

Abstract Heparin-induced thrombocytopenia (HIT) is a potentially serious complication of heparin therapy. Antibodies to platelet factor 4 (PF4)/heparin complexes have been implicated in the pathogenesis of this disorder, but the antigenic epitope(s) on the protein have not been defined. To address this issue, we studied the binding of HIT antibodies to a series of recombinant proteins containing either point mutations in PF4 or chimeras containing various domains of PF4 and the related protein, neutrophil activating peptide-2 (NAP-2). Serum samples from 50 patients with a positive 14C-serotonin release assay (14C-SRA) and a clinical diagnosis of HIT and 20 normal controls were studied. HIT antibodies reacted strongly with wild-type (WT) PF4/heparin complexes, but reacted little, if at all, with NAP-2/heparin complexes (optical density [OD]405 = 2.5 and 0.2, respectively). Alanine substitutions at three of the four lysine residues implicated in heparin binding, K62, K65, and K66, had little effect on recognition by HIT antibodies (OD405 = 2.2, 2.8, and 2.0, respectively), whereas an alanine substitution at position K61 led to reduced, but still significant binding (OD405 = 1.0). Similar studies involving chimeras between PF4 and NAP-2 localized a major antigenic site to the region between the third and fourth cysteine residues for more than half of the sera tested. This site appears to involve a series of amino acids immediately after the third cysteine residue beginning with P37. Thus our studies suggest that whereas the C-terminal lysine residues of PF4 are important for heparin binding, they do not comprise a critical antigenic site for most HIT antibodies. Rather, we propose that maintaining a region near the third cysteine residue of PF4, distal from the proposed heparin-binding domain, is required to form the epitope recognized by many HIT antibodies. © 1998 by The American Society of Hematology.

Defining an Antigenic Epitope on Platelet Factor 4 Associated With Heparin-Induced Thrombocytopenia

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3250-3259 ◽  
Author(s):  
L. Ziporen ◽  
Z.Q. Li ◽  
K.S. Park ◽  
P. Sabnekar ◽  
W.Y. Liu ◽  
...  
Keyword(s):  
Antigenic Site ◽  
Cysteine Residue ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Antigenic Epitope ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
The Third ◽  
Lysine Residues ◽  
Heparin Complexes

Heparin-induced thrombocytopenia (HIT) is a potentially serious complication of heparin therapy. Antibodies to platelet factor 4 (PF4)/heparin complexes have been implicated in the pathogenesis of this disorder, but the antigenic epitope(s) on the protein have not been defined. To address this issue, we studied the binding of HIT antibodies to a series of recombinant proteins containing either point mutations in PF4 or chimeras containing various domains of PF4 and the related protein, neutrophil activating peptide-2 (NAP-2). Serum samples from 50 patients with a positive 14C-serotonin release assay (14C-SRA) and a clinical diagnosis of HIT and 20 normal controls were studied. HIT antibodies reacted strongly with wild-type (WT) PF4/heparin complexes, but reacted little, if at all, with NAP-2/heparin complexes (optical density [OD]405 = 2.5 and 0.2, respectively). Alanine substitutions at three of the four lysine residues implicated in heparin binding, K62, K65, and K66, had little effect on recognition by HIT antibodies (OD405 = 2.2, 2.8, and 2.0, respectively), whereas an alanine substitution at position K61 led to reduced, but still significant binding (OD405 = 1.0). Similar studies involving chimeras between PF4 and NAP-2 localized a major antigenic site to the region between the third and fourth cysteine residues for more than half of the sera tested. This site appears to involve a series of amino acids immediately after the third cysteine residue beginning with P37. Thus our studies suggest that whereas the C-terminal lysine residues of PF4 are important for heparin binding, they do not comprise a critical antigenic site for most HIT antibodies. Rather, we propose that maintaining a region near the third cysteine residue of PF4, distal from the proposed heparin-binding domain, is required to form the epitope recognized by many HIT antibodies. © 1998 by The American Society of Hematology.

Prevalence, isotype, and functionality of antiheparin–platelet factor 4 antibodies in patients treated with heparin and clinically suspected for heparin-induced thrombocytopenia

2002 ◽  
Vol 105 (2) ◽  
pp. 117-123 ◽  
Author(s):  
Brian Untch ◽  
Sarfraz Ahmad ◽  
Walter P. Jeske ◽  
Harry L. Messmore ◽  
Debra A. Hoppensteadt ◽  
...  
Keyword(s):  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia

scholarly journals Heparin-induced thrombocytopenia: in vitro studies on the interaction of dabigatran, rivaroxaban, and low-sulfated heparin, with platelet factor 4 and anti-PF4/heparin antibodies

Blood ◽  
2012 ◽  
Vol 119 (5) ◽  
pp. 1248-1255 ◽  
Author(s):  
Krystin Krauel ◽  
Christine Hackbarth ◽  
Birgitt Fürll ◽  
Andreas Greinacher
Keyword(s):  
Factor Xa ◽  
Platelet Factor 4 ◽  
Thrombin Inhibitors ◽  
Direct Thrombin Inhibitors ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Alternative Anticoagulation ◽  
Heparin Complexes ◽  
History Of

Abstract Heparin is a widely used anticoagulant. Because of its negative charge, it forms complexes with positively charged platelet factor 4 (PF4). This can induce anti-PF4/heparin IgG Abs. Resulting immune complexes activate platelets, leading to the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). HIT requires treatment with alternative anticoagulants. Approved for HIT are 2 direct thrombin inhibitors (DTI; lepirudin, argatroban) and danaparoid. They are niche products with limitations. We assessed the effects of the DTI dabigatran, the direct factor Xa-inhibitor rivaroxaban, and of 2-O, 3-O desulfated heparin (ODSH; a partially desulfated heparin with minimal anticoagulant effects) on PF4/heparin complexes and the interaction of anti-PF4/heparin Abs with platelets. Neither dabigatran nor rivaroxaban had any effect on the interaction of PF4 or anti-PF4/heparin Abs with platelets. In contrast, ODSH inhibited PF4 binding to gel-filtered platelets, displaced PF4 from a PF4-transfected cell line, displaced PF4/heparin complexes from platelet surfaces, and inhibited anti-PF4/heparin Ab binding to PF4/heparin complexes and subsequent platelet activation. Dabigatran and rivaroxaban seem to be options for alternative anticoagulation in patients with a history of HIT. ODSH prevents formation of immunogenic PF4/heparin complexes, and, when given together with heparin, may have the potential to reduce the risk for HIT during treatment with heparin.

Pathophysiology of Heparin-Induced Thrombocytopenia

2000 ◽  
Vol 124 (11) ◽  
pp. 1657-1666 ◽  
Author(s):  
Fabrizio Fabris ◽  
Sarfraz Ahmad ◽  
Giuseppe Cella ◽  
Walter P. Jeske ◽  
Jeanine M. Walenga ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Serotonin Release ◽  
Platelet Factor 4 ◽  
Thrombin Inhibitors ◽  
Platelet Factor ◽  
Cellular Activation ◽  
Heparin Induced Thrombocytopenia ◽  
New Information ◽  
Study Selection ◽  
Release Assay

Abstract Objective.—This review of heparin-induced thrombocytopenia (HIT), the most frequent and dangerous side effect of heparin exposure, covers the epidemiology, pathophysiology, clinical presentation, diagnosis, and treatment of this disease syndrome. Data Sources and Study Selection.—Current consensus of opinion is given based on literature reports, as well as new information where available. A comprehensive analysis of the reasons for discrepancies in incidence numbers is given. The currently known mechanism is that HIT is mediated by an antibody to the complex of heparin–platelet factor 4, which binds to the Fc receptor on platelets. New evidence suggests a functional heterogeneity in the anti-heparin-platelet factor 4 antibodies generated to heparin, and a “superactive” heparin-platelet factor 4 antibody that does not require the presence of heparin to promote platelet activation or aggregation has been identified. Up-regulation of cell adhesion molecules and inflammatory markers, as well as preactivation of platelets/endothelial cells/leukocytes, are also considered to be related to the pathophysiology of HIT. Issues related to the specificity of currently available and new laboratory assays that support a clinical diagnosis are addressed in relation to the serotonin-release assay. Past experience with various anticoagulant treatments is reviewed with a focus on the recent successes of thrombin inhibitors and platelet GPIIb/IIIa inhibitors to combat the platelet activation and severe thrombotic episodes associated with HIT. Conclusions.—The pathophysiology of HIT is multifactorial. However, the primary factor in the mediation of the cellular activation is due to the generation of an antibody to the heparin-platelet factor 4 complex. This review is written as a reference for HIT research.

Sign in / Sign up

Export Citation Format

Share Document