Comparison between Platelet Factor 4/Heparin Complexes ELISA and Platelet Aggregation Test in Heparin-induced Thrombocytopenia

SummaryAs heparin-PF4 (H-PF4) complexes are the target for antibodies associated to heparin-induced thrombocytopenia (HIT), an ELISA has been developed and optimised for testing antibodies binding to H-PF4. This test was consistently negative in 50 healthy subjects (A492 <0.3) and 35 patients with other causes of thrombocytopenia (A492 <0.5). In contrast, 43 out of 44 HIT patients showed antibodies to H-PF4 (A492 = 1.70 ± 0.81) including 5 patients with a negative platelet aggregation test. In one patient with HIT, antibodies to H-PF4 were already present at day 7, whereas platelet counts dropped ≤ 100 × 109/l only at days 11–12. Surprisingly, among 41 patients under heparin for >7 days, 5 showed antibodies to H-PF4, without HIT. These findings underline the major interest of this ELISA for the early diagnosis of HIT. We also showed that LMWH as well as other sulphated polysaccharides can bind to HIT antibodies in the presence of PF4 and that their reactivity is dependent on the molecular weight and the sulphation grade. The mechanism for HIT involves platelet PF4 receptors which bind the macromolecular H-PF4 complexes formed in the presence of a well defined heparin/PF4 ratio.

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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 ◽

10.1182/blood-2011-05-353391 ◽

2012 ◽

Vol 119 (5) ◽

pp. 1248-1255 ◽

Cited By ~ 69

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.

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

Journal of Thrombosis and Haemostasis ◽

10.1111/j.1538-7836.2008.03171.x ◽

2008 ◽

Vol 6 (12) ◽

pp. 2160-2167 ◽

Cited By ~ 47

Author(s):

K. KRAUEL ◽

B. FÜRLL ◽

T. E. WARKENTIN ◽

W. WEITSCHIES ◽

T. KOHLMANN ◽

...

Keyword(s):

Platelet Factor 4 ◽

Thrombin Inhibitors ◽

Direct Thrombin Inhibitors ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Heparin Complexes

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Platelet factor 4 binds to bacteria, inducing antibodies cross-reacting with the major antigen in heparin-induced thrombocytopenia

Blood ◽

10.1182/blood-2010-08-301424 ◽

2011 ◽

Vol 117 (4) ◽

pp. 1370-1378 ◽

Cited By ~ 137

Author(s):

Krystin Krauel ◽

Christian Pötschke ◽

Claudia Weber ◽

Wolfram Kessler ◽

Birgitt Fürll ◽

...

Keyword(s):

Bacterial Infections ◽

Defense Mechanism ◽

Platelet Factor 4 ◽

Cross Sectional ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Host Defense Mechanism ◽

Heparin Complexes ◽

Major Antigen

AbstractA clinically important adverse drug reaction, heparin-induced thrombocytopenia (HIT), is induced by antibodies specific for complexes of the chemokine platelet factor 4 (PF4) and the polyanion heparin. Even heparin-naive patients can generate anti-PF4/heparin IgG as early as day 4 of heparin treatment, suggesting preimmunization by antigens mimicking PF4/heparin complexes. These antibodies probably result from bacterial infections, as (1) PF4 bound charge-dependently to various bacteria, (2) human heparin-induced anti-PF4/heparin antibodies cross-reacted with PF4-coated Staphylococcus aureus and Escherichia coli, and (3) mice developed anti-PF4/heparin antibodies during polymicrobial sepsis without heparin application. Thus, after binding to bacteria, the endogenous protein PF4 induces antibodies with specificity for PF4/polyanion complexes. These can target a large variety of PF4-coated bacteria and enhance bacterial phagocytosis in vitro. The same antigenic epitopes are expressed when pharmacologic heparin binds to platelets augmenting formation of PF4 complexes. Boosting of preformed B cells by PF4/heparin complexes could explain the early occurrence of IgG antibodies in HIT. We also found a continuous, rather than dichotomous, distribution of anti-PF4/heparin IgM and IgG serum concentrations in a cross-sectional population study (n = 4029), indicating frequent preimmunization to modified PF4. PF4 may have a role in bacterial defense, and HIT is probably a misdirected antibacterial host defense mechanism.

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Platelet factor 4 binding to lipid A of Gram-negative bacteria exposes PF4/heparin-like epitopes

Blood ◽

10.1182/blood-2012-06-434985 ◽

2012 ◽

Vol 120 (16) ◽

pp. 3345-3352 ◽

Cited By ~ 67

Author(s):

Krystin Krauel ◽

Claudia Weber ◽

Sven Brandt ◽

Ulrich Zähringer ◽

Uwe Mamat ◽

...

Keyword(s):

Lipid A ◽

Defense Mechanism ◽

Binding Activity ◽

Platelet Factor 4 ◽

Gram Negative Bacteria ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Gram Negative ◽

Host Defense Mechanism ◽

Heparin Complexes

AbstractThe positively charged chemokine platelet factor 4 (PF4) forms immunogenic complexes with heparin and other polyanions. Resulting antibodies can induce the adverse drug effect heparin-induced thrombocytopenia. PF4 also binds to bacteria, thereby exposing the same neoantigen(s) as with heparin. In this study, we identified the negatively charged lipopolysaccharide (LPS) as the PF4 binding structure on Gram-negative bacteria. We demonstrate by flow cytometry that mutant bacteria with progressively truncated LPS structures show increasingly enhanced PF4 binding activity. PF4 bound strongest to mutants lacking the O-antigen and core structure of LPS, but still exposing lipid A on their surfaces. Strikingly, PF4 bound more efficiently to bisphosphorylated lipid A than to monophosphorylated lipid A, suggesting that phosphate residues of lipid A mediate PF4 binding. Interactions of PF4 with Gram-negative bacteria, where only the lipid A part of LPS is exposed, induce epitopes on PF4 resembling those on PF4/heparin complexes as shown by binding of human anti-PF4/heparin antibodies. As both the lipid A on the surface of Gram-negative bacteria and the amino acids of PF4 contributing to polyanion binding are highly conserved, our results further support the hypothesis that neoepitope formation on PF4 after binding to bacteria is an ancient host defense mechanism.

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The role of platelet factor 4 in platelet aggregation induced by the antibodies implicated in heparin-induced thrombocytopenia

Blood Coagulation & Fibrinolysis ◽

10.1097/00001721-200110000-00002 ◽

2001 ◽

Vol 12 (7) ◽

pp. 511-520 ◽

Cited By ~ 11

Author(s):

G. T. Gerotziafas ◽

I. Elalamy ◽

C. Lecrubier ◽

J. Lebrazi ◽

M. Mirshahi ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Factor 4 ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia

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

Blood ◽

10.1182/blood.v92.9.3250 ◽

1998 ◽

Vol 92 (9) ◽

pp. 3250-3259 ◽

Cited By ~ 86

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.

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Heparin-induced Platelet Aggregation Vs Platelet Factor 4 Enzyme-Linked Immunosorbent Assay in the Diagnosis of Heparin-induced Thrombocytopenia-Thrombosis

American Journal of Clinical Pathology ◽

10.1093/ajcp/108.1.78 ◽

1997 ◽

Vol 108 (1) ◽

pp. 78-82 ◽

Cited By ~ 24

Author(s):

Kelly A. Look ◽

Mervyn Sahud ◽

Sheila Flaherty ◽

James L. Zehnder

Keyword(s):

Platelet Aggregation ◽

Immunosorbent Assay ◽

Platelet Factor 4 ◽

Enzyme Linked Immunosorbent Assay ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia

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Anti-SARS-CoV-2 Spike Protein and Anti-Platelet Factor 4 Antibody Responses Induced by COVID-19 Disease and ChAdOx1 nCov-19 vaccination

10.21203/rs.3.rs-404769/v1 ◽

2021 ◽

Author(s):

Andreas Greinacher ◽

Kathleen Selleng ◽

Julia Mayerle ◽

Raghavendra Palankar ◽

Jan Wesche ◽

...

Keyword(s):

Platelet Activation ◽

Molecular Mimicry ◽

Cross Reactivity ◽

Platelet Factor 4 ◽

Antibody Responses ◽

Spike Protein ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Activation Assay ◽

Heparin Complexes

Abstract Background: Some recipients of ChAdOx1 nCoV-19 COVID-19 Vaccine AstraZeneca develop antibody-mediated vaccine-induced thrombotic thrombocytopenia (VITT), associated with cerebral venous and other unusual thrombosis resembling autoimmune heparin-induced thrombocytopenia. A prothrombotic predisposition is also observed in Covid-19. We explored whether antibodies against the SARS-CoV-2 spike protein induced by Covid-19 cross-react with platelet factor 4 (PF4/CXLC4), the protein targeted in both VITT and autoimmune heparin-induced thrombocytopenia.Methods: Immunogenic epitopes of PF4 and SARS-CoV-2 spike protein were compared via prediction tools and 3D modelling software (IMED, SIM, MacMYPOL). Sera from 222 PCR-confirmed Covid-19 patients from five European centers were tested by PF4/heparin ELISA, heparin-dependent and PF4-dependent platelet activation assays. Immunogenic reactivity of purified anti-PF4 and anti-PF4/heparin antibodies from patients with VITT were tested against recombinant SARS-CoV-2 spike protein. Results: Three motifs within the spike protein sequence share a potential immunogenic epitope with PF4. Nineteen of 222 (8.6%) Covid-19 patient sera tested positive in the IgG-specific PF4/heparin ELISA, none of which showed platelet activation in the heparin-dependent activation assay, including 10 (4.5%) of the 222 Covid-19 patients who developed thromboembolic complications. Purified anti-PF4 and anti-PF4/heparin antibodies from two VITT patients did not show cross-reactivity to recombinant SARS-CoV-2 spike protein. Conclusions: The antibody responses to PF4 in SARS-CoV-2 infection and after vaccination with COVID-19 Vaccine AstraZeneca differ. Antibodies against SARS-CoV-2 spike protein do not cross-react with PF4 or PF4/heparin complexes through molecular mimicry. These findings make it very unlikely that the intended vaccine-induced immune response against SARS-CoV-2 spike protein would itself induce VITT.

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Polyphosphate/platelet factor 4 complexes can mediate heparin-independent platelet activation in heparin-induced thrombocytopenia

Blood Advances ◽

10.1182/bloodadvances.2016000877 ◽

2016 ◽

Vol 1 (1) ◽

pp. 62-74 ◽

Cited By ~ 24

Author(s):

Douglas B. Cines ◽

Serge V. Yarovoi ◽

Sergei V. Zaitsev ◽

Tatiana Lebedeva ◽

Lubica Rauova ◽

...

Keyword(s):

Platelet Aggregation ◽

Cell Surface ◽

Chondroitin Sulfate ◽

Platelet Activation ◽

Platelet Factor 4 ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Activated Platelets ◽

Key Points

Key Points Polyphosphates form antigenic complexes with PF4 that are recognized by HIT antibodies. Polyphosphate/PF4 complexes released by activated platelets can mediate platelet aggregation by HIT antibodies in the absence of heparin or cell-surface chondroitin sulfate.

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