Complex formation with nucleic acids and aptamers alters the antigenic properties of platelet factor 4

Key PointsPF4 binds to nucleic acids and thereby exposes the epitope to which anti-PF4/heparin antibodies bind. PF4/aptamer complexes can induce an immune response resembling heparin-induced thrombocytopenia.

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Influence of Protein Tyrosine Phosphatase Polymorphisms on the Development of Antibodies to Platelet Factor 4 and the Risk of Heparin-Induced Thrombocytopenia.

Blood ◽

10.1182/blood.v116.21.3683.3683 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3683-3683

Author(s):

Jerôme Rollin ◽

Claire Pouplard ◽

Dorothee Leroux ◽

Marc-Antoine May ◽

Yves Gruel

Keyword(s):

Immune Response ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Critical Role ◽

Platelet Factor 4 ◽

Control Group ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Protein Tyrosine ◽

Significant Difference

Abstract Abstract 3683 Introduction. Heparin-induced thrombocytopenia (HIT) results from an atypical immune response to platelet factor 4/heparin complexes (PF4/H), with rapid synthesis of platelet-activating IgG antibodies that activate platelets via FcgRIIa receptors. The reasons explaining why only a subset of patients treated with heparin develop IgG to PF4/H complexes, and why most patients who synthesize these antibodies do not develop HIT, have not been fully defined. The immune response in HIT involves both B and T cells, and protein tyrosine kinases (PTKs) and phosphatases (PTPs) are crucial for regulating antigen receptor-induced lymphocyte activation. Moreover, some PTPs such as CD148 and low-molecular-weight PTP (LMW-PTP) could also have a critical role in platelet activation. Dysregulation of the equilibrium between PTK and PTP function could therefore have pathologic consequences and influence the pathogenesis of HIT. Aim of the study. To investigate an association between polymorphisms affecting genes encoding 4 different PTPs i.e. CD45 (PTPRC), CD148 (PTPRJ), LYP (PTPN22) and LMW-PTP (ACP1) and the development of heparin-dependent antibodies to PF4 and HIT. Patients and methods. A cohort of 89 patients with definite HIT (positive PF4-specific ELISA and positive serotonin release assay) and two control groups were studied. The first control group (Abneg) consisted of 179 patients who had undergone cardiopulmonary bypass (CBP) with high doses of heparin and who did not develop Abs to PF4 post-operatively. The second control group (Abpos) consisted of 160 patients who had also undergone cardiac surgery with CPB and heparin, who had all developed significant levels of PF4-specific antibodies but without HIT. Genotypes of PTPRC 77C/G (rs17612648), PTPN22 1858C/T (rs2476601), PTPRJ 2965 C/G (rs4752904) and PTPRJ 1176 A/C (rs1566734) were studied by a PCR-HRM method using the LightCycler 480 (Roche). In addition, the ACP1 A, B, C alleles were defined by combining the analysis of T/C transition at codon 43 of exon 3 (rs11553742) and T/C transition at codon 41 of exon 4 (rs11553746). Results. The frequency of PTPRC 77G and PTPN22 1858T alleles was not different in HIT patients and controls, whether they had developed antibodies to PF4 or not. The third PTP gene analyzed was ACP1, in which three alleles (A, B and C) were previously associated with the synthesis of distinct active LMW-PTP isoforms exhibiting different catalytic properties. The percentage of subjects in our study carrying the AC, BB and BC genotypes was significantly higher in the HIT and the Abpos groups than in patients without antibodies to PF4 after CPB (Abneg). In addition, the ACP1 A allele was less frequent in patients with antibodies to PF4, whether they had developed HIT (25%) or not (27.5% in Abpos controls), than in Abneg subjects (37%). The AC, BB and BC genotypes (associated in Caucasians with the highest LMW-PTP enzyme activity) therefore appeared to increase the risk of antibody formation in heparin-treated patients (OR 1.8; 95% CI 1.2–2.6, p=0.004 after comparing Abpos + HIT vs. Abneg). We also evaluated 2 SNPs affecting PTPRJ encoding CD148. No significant difference was found concerning the 2965 C/G polymorphism, but the frequency of PTPRJ 1176 AC and CC genotypes was significantly lower in the HIT (17%) than in the Abneg and Abpos groups (35%, p=0.003 and 29.5%, p=0.041, respectively). The C allele therefore appeared to provide a significant protection from the risk of HIT (OR 0.52; 95%CI 0.29–0.94, p=0.041) in patients with antibodies to PF4. Discussion-Conclusion. Recent studies have demonstrated that CD148 is a positive regulator of platelet activation by maintaining a pool of active SFKs in platelets. This non-synonym PTPRJ 1176 A/C SNP is associated with a Q276P substitution inducing a torsional stress of a fibronectin domain that is critical for the activity of CD148 and may influence the pathogenic effects of HIT Abs. This study supports the hypothesis that PTPs such as LMW-PTP and CD148 influence the immune response to heparin and the risk of HIT in patients with antibodies to PF4. Disclosures: No relevant conflicts of interest to declare.

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Investigation of a platelet factor 4 polymorphism on the immune response in patients with heparin-induced thrombocytopenia

Platelets ◽

10.1080/09537100075751 ◽

2000 ◽

Vol 11 (1) ◽

pp. 23-27 ◽

Cited By ~ 12

Author(s):

Peter Horsewood, John G. Kelton

Keyword(s):

Immune Response ◽

Platelet Factor 4 ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia

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Disruption of PF4/H multimolecular complex formation with a minimally anticoagulant heparin (ODSH)

Thrombosis and Haemostasis ◽

10.1160/th11-11-0795 ◽

2012 ◽

Vol 107 (04) ◽

pp. 717-725 ◽

Cited By ~ 21

Author(s):

Manali V. Joglekar ◽

Pedro M. Diez ◽

Stephen Marcus ◽

Rui Qi ◽

Benjamin Espinasse ◽

...

Keyword(s):

Immune Response ◽

Complex Formation ◽

Unfractionated Heparin ◽

Electrostatic Interactions ◽

Essential Role ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Immune Mediated ◽

Novel Strategy ◽

American Society

SummaryRecent studies have shown that ultra-large complexes (ULCs) of platelet factor 4 (PF4) and heparin (H) play an essential role in the pathogenesis of heparin-induced thrombocytopenia (HIT), an immune-mediated disorder caused by PF4/H antibodies. Because antigenic PF4/H ULCs assemble through non-specific electrostatic interactions, we reasoned that disruption of charge-based interactions can modulate the immune response to antigen. We tested a minimally anticoagulant compound (2-O, 3-O desulfated heparin, ODSH) with preserved charge to disrupt PF4/H complex formation and immunogenicity. We show that ODSH disrupts complexes when added to pre-formed PF4/H ULCs and prevents ULC formation when incubated simultaneously with PF4 and UFH. In other studies, we show that excess ODSH reduces HIT antibody (Ab) binding in immunoassays and that PF4/ODSH complexes do not cross-react with HIT Abs. When ODSH and unfractionated heparin (UFH) are mixed at equimolar concentrations, we show that there is a negligible effect on amount of protamine required for heparin neutralisation and reduced immunogenicity of PF4/UFH in the presence of ODSH. Taken together, these studies suggest that ODSH can be used concurrently with UFH to disrupt PF4/H charge interactions and provides a novel strategy to reduce antibody mediated complications in HIT.Presented in part at the 52nd American Society of Hematology Annual Meeting and Exposition, December 6th, 2010, Orlando, Florida, USA.

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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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Platelet-Activating Anti-Platelet Factor 4/Polyanion Antibodies without Preceding Heparin Therapy: A Transient Autoimmune Disorder Resembling Heparin-Induced Thrombocytopenia (“Spontaneous HIT”).

Blood ◽

10.1182/blood.v108.11.1047.1047 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1047-1047 ◽

Cited By ~ 1

Author(s):

Theodore E. Warkentin ◽

Richard M. Jay ◽

Michael Makris ◽

John G. Kelton

Keyword(s):

Immune Response ◽

Platelet Count ◽

Systemic Reaction ◽

Autoimmune Disorder ◽

Serotonin Release ◽

Acute Illness ◽

Platelet Factor 4 ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Heparin Administration

Abstract Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder with autoimmune features: its target antigen is a “self” protein, platelet factor 4 (PF4), that is conformationally modified in the presence of heparin or certain other polyanions. A central dogma in HIT is that the antibodies are invariably triggered by treatment with heparin. We report four patients who developed an acute illness strongly resembling immune HIT despite the absence of any preceding heparin administration [Table 1]. Various inflammatory or invasive events were documented during the two-week period prior to each patient’s acute illness, suggesting that factors other than heparin can trigger an immune response against PF4/polyanion complexes. Following onset of their HIT-like illness, three of the patients received unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) to treat proven or suspected thrombosis, which precipitated abrupt platelet count falls in all. Serum from all four patients contained antibodies serologically indistinguishable from those causing HIT, namely platelet-activating immunoglobulin G (IgG) recognizing PF4 bound to heparin or polyvinyl sulfonate [Table 2]. Two patients died from thrombosis; in the two survivors, antibodies became undetectable during follow-up, consistent with the transient nature of the anti-PF4/heparin immune response reported in HIT. Conclusion: These four patient cases suggest that on rare occasions a transient prothrombotic autoimmune disorder strongly resembling immune HIT can occur, “spontaneous HIT”. Table 1. Clinical features: 4 patients with “spontaneous HIT” Case Age M/F Platelet count nadir (× 10^9^/L) Sequelae Event(s) in preceding two weeks * UFH or LMWH were only given for proven or suspected thrombosis after “spontaneous HIT” disorder was established; DIC = disseminated intravascular coagulation 1 69 M 17 (no heparin given) Limb artery thromboses (amputations); pulmonary embolism; DIC; fatal myocardial infarction Ampicillin (sore tooth); corticosteroid injection into wrist 2 40 F 175 (pre-UFH*); 59 (post-UFH*) Stroke; limb artery thrombosis (amputation) Incision & draining procedures (recurrent groin cyst) 3 69 F 33 (pre-UFH*); 11 (post-UFH*) Bilateral adrenal infarction; digital infarcts; deep-vein thrombosis; DIC Knee replacement surgery 4 24 F 301 (pre-LMWH*); 62 (post-LMWH*) Acute systemic reaction after LMWH Pneumonia Table 2. Serologic features: 4 patients with “spontaneous HIT” Case Maximum serotonin release (0.1-0.3 U/mL UFH) (N<10%) Serotonin release (Fc receptor-blocking monoclonal antibody) Serotonin release (100 U/mL UFH) Anti-PF4/H ELISA (IgG) (N<0.450 OD U)* Anti-PF4/polyanion ELISA, GTI (N<0.400 OD U)* * >80% inhibition in presence of 100 U/mL UFH was seen with all 4 blood samples and in both ELISAs (not shown) 1 99% 1% 0% 2.028 2.722 2 95% 0% 0% 2.909 2.284 3 100% 0% 0% 1.888 2.950 4 80% 0% 0% 1.866 2.622

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Monocytes Are a Particularly Favorable Target for Surface Platelet Factor 4 (PF4) Antigenic Complex Formation in Heparin-Induced Thrombocytopenia: New Insights into the Thrombotic Risk in HIT

Blood ◽

10.1182/blood.v112.11.271.271 ◽

2008 ◽

Vol 112 (11) ◽

pp. 271-271

Author(s):

Lubica Rauova ◽

Gowthami M Arepally ◽

Douglas B. Cines ◽

Mortimer Poncz

Keyword(s):

Complex Formation ◽

Platelet Factor 4 ◽

Drug Induced ◽

Platelet Factor ◽

Platelet Surface ◽

Thrombotic Risk ◽

Heparin Induced Thrombocytopenia ◽

Antigenic Complex

Abstract Monocytes are a Favored Target for Surface Platelet Factor 4 (PF4) Antigenic Complex Formation in Heparin-Induced Thrombocytopenia: New Insights into the Thrombotic Risk in HIT Lubica Rauova, Gowthami Arepally, Douglas Cines and Mortimer Poncz HIT is a drug-induced autoimmune thrombocytopenia caused by antibodies to heparin/PF4 complexes that predispose to thrombotic complications. The studies described below examine how monocytes (Mo) may contribute to the thrombotic risk. We demonstrated previously that glycosaminoglycans (GAG) on the surface of platelets bind PF4, forming complexes that are recognized by HIT antibody, leading to platelet activation via the platelet FcγRIIA receptor in vitro and thrombocytopenia/thrombosis in vivo. However, heparin not only induces antibodies to develop against the PF4/GAG surface antigenic complexes, but also rapidly removes the same PF4/GAG complexes from the platelet surface, which may limit the likelihood of developing HIT and help limit its duration. This led us to study the involvement of Mo, which are a rich potential source of tissue factor and are known to be activated in HIT. Moreover, unlike platelets, which are coated with GAG composed almost entirely of chrondroitin sulfate (CS), Mo also express heparan sulfate, which has the capacity to bind PF4 with greater avidity and be resistant to the effect of plasma heparin. We found that Mo bind PF4 with greater avidity than platelets and higher concentrations of UFH are needed to remove PF4/GAG complexes and reduce the binding of a HIT monoclonal antibody KKO. In contrast to platelets, dissociation of PF4/GAG complexes from monocytes requires heparinases in addition to chondroitinases. In addition, macrophages GAG undergo hypersulfation during inflammation. Because clinical studies have shown inflammation predisposes to HIT, we examined the binding of KKO to unstimulated and bacterial lipopolysaccharide (LPS, E. coli serotype 011) stimulated cultured macrophages. Macrophages were derived from primary human Mo or murine bone marrow, cultured in the presence of M-CSF and stimulated with 0–500 ng/mL of LPS for 72 hrs. LPS increased KKO binding in the presence of PF4 2.7±0.7-fold compared to unstimulated cells (p<0.002) and the stimulated cells required ~2-fold higher concentrations of heparin to remove surface PF4/GAG complexes. Addition of [35S]sulfate during the last 24 hrs of incubation lead to a 4.1±0.1-fold increase in the incorporation of 35S into surface GAG after LPS stimulation (p<0.0001). These results provide important insights into the potential role of Mo in the prothrombotic sequelae of HIT. Compared to platelets, Mo are relatively resistant to “antigen down-regulation” by heparin and are more likely to bind anti-PF4/GAG HIT antibodies and become activated. The relative resistance of Mo to the dissociation PF4/GAG complexes from the cell surface also suggest a role in the development of Delayed-Onset HIT after heparin withdrawal.

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