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

Blood ◽  
2011 ◽  
Vol 117 (4) ◽  
pp. 1370-1378 ◽  
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.

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

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3345-3352 ◽  
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.

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.

Polyphosphates form antigenic complexes with platelet factor 4 (PF4) and enhance PF4-binding to bacteria

2015 ◽  
Vol 114 (12) ◽  
pp. 1189-1198 ◽  
Author(s):  
Sven Brandt ◽  
Krystin Krauel ◽  
Miriam Jaax ◽  
Thomas Renné ◽  
Christiane A. Helm ◽  
...  
Keyword(s):  
Host Defense ◽  
Defense Mechanisms ◽  
Lipid A ◽  
Defense Mechanism ◽  
Platelet Factor 4 ◽  
Short Chain ◽  
Titration Calorimetry ◽  
Inorganic Polymers ◽  
Platelet Factor ◽  
Heparin Complexes

SummaryShort chain polyphosphates (polyP) are pro-coagulant and pro-inflammatory platelet released inorganic polymers. The platelet chemokine platelet factor 4 (PF4) binds to lipid A on bacteria, inducing an antibody mediated host defense mechanism, which can be misdirected against PF4/heparin complexes leading to the adverse drug reaction heparin-induced thrombocytopenia (HIT). Here, we demonstrate that PF4 complex formation with soluble short chain polyP contributes to host defense mechanisms. Circular dichroism spectroscopy and isothermal titration calorimetry revealed that PF4 changed its structure upon binding to polyP in a similar way as seen in PF4/heparin complexes. Consequently, PF4/polyP complexes exposed neoepitopes to which human anti-PF4/heparin antibodies bound. PolyP enhanced binding of PF4 to Escherichia coli, hereby facilitating bacterial opsonisation and, in the presence of human anti-PF4/polyanion antibodies, phagocytosis. Our study indicates a role of polyP in enhancing PF4-mediated defense mechanisms of innate immunity.

scholarly journals Anti–platelet factor 4/heparin antibodies in orthopedic surgery patients receiving antithrombotic prophylaxis with fondaparinux or enoxaparin

Blood ◽  
2005 ◽  
Vol 106 (12) ◽  
pp. 3791-3796 ◽  
Author(s):  
Theodore E. Warkentin ◽  
Richard J. Cook ◽  
Victor J. Marder ◽  
Jo-Ann I. Sheppard ◽  
Jane C. Moore ◽  
...  
Keyword(s):  
Cross Reactivity ◽  
Platelet Factor 4 ◽  
Low Molecular Weight ◽  
Igg Antibodies ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Antithrombotic Prophylaxis ◽  
High Concentrations ◽  
To Receive

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating IgG antibodies that recognize platelet factor 4 (PF4) bound to heparin. Immunogenicity of heparins differs in that unfractionated heparin (UFH) induces more anti–PF4/heparin antibodies than low-molecular-weight heparin (LMWH) and UFH also causes more HIT. Fondaparinux, a synthetic anticoagulant modeled after the antithrombin-binding pentasaccharide, is believed to be nonimmunogenic. We tested 2726 patients for anti–PF4/heparin antibodies after they were randomized to receive antithrombotic prophylaxis with fondaparinux or LMWH (enoxaparin) following hip or knee surgery. We also evaluated in vitro cross-reactivity of the IgG antibodies generated against PF4 in the presence of UFH, LMWH, danaparoid, or fondaparinux. We found that anti–PF4/heparin antibodies were generated at similar frequencies in patients treated with fondaparinux or enoxaparin. Although antibodies reacted equally well in vitro against PF4/UFH and PF4/LMWH, and sometimes weakly against PF4/danaparoid, none reacted against PF4/fondaparinux, including even those sera obtained from patients who formed antibodies during fondaparinux treatment. At high concentrations, however, fondaparinux inhibited binding of HIT antibodies to PF4/polysaccharide, indicating that PF4/fondaparinux interactions occur. No patient developed HIT. We conclude that despite similar immunogenicity of fondaparinux and LMWH, PF4/fondaparinux, but not PF4/LMWH, is recognized poorly by the antibodies generated, suggesting that the risk of HIT with fondaparinux likely is very low.

Antibodies to Macromolecular Platelet Factor 4-Heparin Complexes in Heparin-induced Thrombocytopenia: a Study of 44 Cases

1995 ◽  
Vol 73 (01) ◽  
pp. 021-028 ◽  
Author(s):  
J Amiral ◽  
F Bridey ◽  
M Wolf ◽  
C Boyer-Neumann ◽  
E Fressinaud ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Early Diagnosis ◽  
Healthy Subjects ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Sulphated Polysaccharides ◽  
Major Interest ◽  
Heparin Complexes

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.

scholarly journals Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3232-3239 ◽  
Author(s):  
JG Kelton ◽  
JW Smith ◽  
TE Warkentin ◽  
CP Hayward ◽  
GA Denomme ◽  
...  
Keyword(s):  
Minimum Degree ◽  
Equilibrium Dialysis ◽  
Fluid Phase ◽  
Platelet Factor 4 ◽  
Minimum Size ◽  
Sulfated Polysaccharides ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Heparin Induced Thrombocytopenia

Abstract Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.

Heparin-Induced Thrombocytopenia Antibodies Inhibit PF4-Dependent Enhancement of Activated Protein C Formation by Binding to Antigenic Complexes Formed with the Chondroitin Sulfate Side-Chain of Thrombomodulin

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 721-721
Author(s):  
M. Anna Kowalska ◽  
Lubica Rauova ◽  
Vincent Hayes ◽  
Douglas B. Cines ◽  
Daniel W. Bougie ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Protein C ◽  
Conflicts Of Interest ◽  
Specific Binding ◽  
Activated Protein C ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Heparin Complexes

Abstract Abstract 721 Previous studies have shown that platelet factor 4 (PF4) increases activated protein C (aPC) generation both in vitro and in vivo. PF4 increase of aPC generation by thrombin (IIa) and thrombomodulin (TM) complex followed a bell-shaped curve when tested in solution, on human TM expressing HEK293 and on endothelial cells. PF4 failed to enhance aPC in the presence of chondroitin sulfate (CS)-free TM. These results were consistent with PF4 binding to the CS on the TM glycosaminoglycan (GAG) domain and forming complexes that are similar to PF4/GAG antigenic complexes seen in heparin-induced thrombocytopenia (HIT). We tested the hypothesis that PF4 forms a HIT-like antigenic complex with the TM-CS using the HIT-like monoclonal antibody KKO. KKO abolished the potentiating effects of PF4 on aPC formation measured with TM in solution or with a TM-expressing cell line. To further address the nature of complexes formed between PF4 and TM, we used a mutant of PF4, PF4T38Q, which forms complexes with GAGs that are not recognized by KKO and a subgroup of HIT antibodies. Similar to PF4, PF4T38Q potentiated TM-dependent aPC generation in a bell-shaped manner, but this potentiation was not blocked by KKO. Moreover, KKO did not have any effect when PF4 was replaced with protamine sulfate (PS), which can also form macromolecular complexes with heparin/GAGs and can also enhance aPC generation. We also tested HIT antibodies isolated from patients that developed HIT with thrombocytopenia and thromboembolism developing >4 days after the last exposure to heparin. Patient IgGs specific for PF4/GAG complex were purified using PF4 bound to heparin columns. Specific binding of antibodies to PF4/heparin complexes was checked by ELISA. Complex-specific antibodies were then tested in an aPC generation assay in the presence IIa and TM and near peak concentration of PF4 and compared to a control human IgG. Three of four patient‘s antibodies significantly inhibited the increase in aPC generation in the presence of PF4. These studies provide evidence that HIT-like PF4/GAG complexes develop naturally in vivo. In this case, the ability of HIT or HIT-like antibodies to specifically inhibit the PF4-dependent increase in aPC formation may contribute to the prothrombotic state in HIT. Disclosures: No relevant conflicts of interest to declare.

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