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

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649823 ◽

1995 ◽

Vol 74 (02) ◽

pp. 804-805 ◽

Cited By ~ 8

Author(s):

Philippe Nguyên ◽

Chantal Droullé ◽

Gérard Potron

Keyword(s):

Platelet Aggregation ◽

Platelet Factor 4 ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Heparin Complexes

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Pathophysiology of Heparin-Induced Thrombocytopenia

Archives of Pathology & Laboratory Medicine ◽

10.5858/2000-124-1657-pohit ◽

2000 ◽

Vol 124 (11) ◽

pp. 1657-1666 ◽

Cited By ~ 4

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.

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Comparison of Clinical Outcomes in Patients with Heparin-Induced Thrombocytopenia Treated with Direct Thrombin Inhibitors.

Blood ◽

10.1182/blood.v104.11.3942.3942 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3942-3942 ◽

Cited By ~ 2

Author(s):

John L. Francis ◽

Ranjeev Sandhu ◽

Alane Drexler ◽

John Dougherty

Keyword(s):

Length Of Stay ◽

Clinical Outcomes ◽

Clinical Syndrome ◽

Thrombin Inhibitors ◽

Direct Thrombin Inhibitors ◽

Efficacy And Safety ◽

Platelet Factor ◽

Platelet Recovery ◽

Heparin Induced Thrombocytopenia ◽

All Cause Mortality

Abstract Heparin-induced thrombocytopenia (HIT) is a clinical syndrome that has been reported to occur in 3–5% of patients treated with unfractionated heparin. If untreated, 36–50% of patients diagnosed with HIT develop life or limb-threatening thromboses. The occurrence of thrombosis, the most common serious complication of HIT, results in rates of amputation and death of 10–20% and 20–30% respectively. Thus, the desired clinical outcomes in patients with HIT are the prevention of thromboembolic complications, limb amputation, and death. The three direct thrombin inhibitors available in the U.S. are lepirudin, argatroban and bivalirudin. Lepirudin and argatroban have both been shown in clinical trials to significantly decrease the incidence of these complications in patients with HIT. Our institution also has experience with bivalirudin as treatment for HIT. We therefore sought to confirm whether all-cause mortality, length of stay, bleeding rate, time to platelet recovery, absolute change in platelet count following therapy, and percentage of therapeutic APTTs differed among patients treated with these agents at our institution. Data were collected by retrospective chart reviews, and from the Florida Hospital pharmacy computer system. Length of stay was calculated as the time to hospital discharge following the finding of a positive heparin-platelet factor 4 antibody test. For the purposes of comparison, the therapeutic range for APTT was taken as 50–90 seconds. As shown in the table, there were no statistically significant differences in any of the endpoints, when the efficacy and safety of the three direct thrombin inhibitors were compared. It therefore appears that within our institution, each of the direct thrombin inhibitors are equally efficacious and safe in treating the clinical syndrome of HIT, with similar outcomes with respect to length of stay, recovery of platelet counts, incidence of bleeding, and overall mortality. We conclude that selection of a direct thrombin inhibitor can be guided by the patient’s clinical status and organ function instead of efficacy and safety considerations. Drug n All-cause mortality (%) Length of stay (days) Bleeding (%) Time to platelet recovery (days) Therapeutic APTT (%) Lepirudin 7 28.5 11.0 28.5 5.3 70 Argatroban 20 30.0 15.1 20.0 5.2 65 Bivalirudin 24 25.0 18.7 25.0 5.4 74 P-value NS NS NS NS NS

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651670 ◽

1995 ◽

Vol 73 (01) ◽

pp. 021-028 ◽

Cited By ~ 189

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.

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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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Incidence of Isolated Heparin-Induced Thrombocytopenia and the Risk of Developing Subsequent Thrombosis Using the New IgG Specific Anti-PF4/Heparin ELISA

Blood ◽

10.1182/blood.v116.21.1448.1448 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1448-1448

Author(s):

Sofyan M. Radaideh ◽

Eugene P. Frenkel ◽

Ravindra Sarode ◽

Yu-Min Shen

Keyword(s):

Conflicts Of Interest ◽

Clinical Findings ◽

Thrombin Inhibitors ◽

Direct Thrombin Inhibitors ◽

High Dose ◽

Elisa Assay ◽

Initial Manifestation ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Number Of Patients

Abstract Abstract 1448 BACKGROUND: The new IgG specific ELISA for anti-platelet factor 4 (PF4)/heparin antibody is recently introduced for the diagnosis of heparin-induced thrombocytopenia (HIT). It is as sensitive but more specific than the poly-specific IgG, M, A ELISA. About 50% of patients testing positive for the anti-PF4/heparin antibody have thrombocytopenia alone as initial manifestation of HIT without thrombosis (isolated HIT). These patients are thought to be at high risk (≂f30%) for developing thrombosis within next 28 days. The incidence of isolated HIT using IgG-specific ELISA is unknown. OBJECTIVES: Evaluate the incidence of isolated HIT using the IgG specific anti-PF4/heparin ELISA and the risk of developing subsequent thrombosis in these patients. PATIENTS AND METHODS: We performed a retrospective observational study of patients being tested for the anti-PF4/heparin antibody at our institution from December 2008 to May 2010 using the IgG-specific anti-PF4/heparin ELISA with a two-step assay using high dose heparin in the second step to demonstrate heparin specificity. A positive test was defined as optical density (OD) >0.4 with >50% inhibition. RESULTS: 319 patients were tested during the study period. 23 (7.2%) patients were diagnosed with HIT based on clinical findings (4T score) and ELISA. 16/23 (70%) had thrombosis at diagnosis whereas 7/ 23 (30%) had isolated HIT. All 7 patients with isolated HIT had follow up data for at least 3 months after diagnosis. Only 2/7 were treated with direct thrombin inhibitors (DTI) and 4/7 were treated with warfarin for at least 1 month. While only 3 of the 7 isolated HIT patients had compression ultrasonography to rule out occult lower extremity thrombus, none of these 7 patients (including 4 with an OD of >1.0) developed symptoms or signs of thrombosis in the subsequent 3 months after the diagnosis of isolated HIT. CONCLUSION: The incidence of isolate HIT in this study (7.2%) is significantly lower than previously reported by others and our own historical patients (57.4%, Altuntus et al, Euro J of Haematology 2008) using the IgG, M, A poly -specific anti-PF4/heparin ELISA (z value 2.092, p=0.036). It is possible that many patients previously thought to have HIT by the poly-specific anti-PF4/heparin ELISA assay were false positive. Using the IgG-specific anti-PF4/heparin ELISA not only improved the specificity of the ELISA assay, it also reduced the number of patients with isolated HIT significantly who also seem to have lower risk of developing subsequent thrombosis. Disclosures: No relevant conflicts of interest to declare.

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Heparin-induced thrombocytopenia in cardiac surgery and critically ill patients

Thrombosis and Haemostasis ◽

10.1160/th16-03-0230 ◽

2016 ◽

Vol 116 (11) ◽

pp. 843-851 ◽

Cited By ~ 26

Author(s):

Sixten Selleng ◽

Kathleen Selleng

Keyword(s):

Cardiac Surgery ◽

Clinical Symptoms ◽

Therapeutic Option ◽

Thrombin Inhibitors ◽

Ventricular Assist Devices ◽

Direct Thrombin Inhibitors ◽

Impaired Liver Function ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Off Pump

SummaryThrombocytopenia as well as anti-platelet factor 4/heparin (PF4/H) antibodies are common in cardiac surgery patients and those treated in the intensive care unit. In contrast, heparin-induced thrombocytopenia (HIT) is uncommon in these populations (∼1 % and ∼0.5 %, respectively). A stepwise approach where testing for anti-PF4/H antibodies is performed only in patients with typical clinical symptoms of HIT improves diagnostic specificity of the laboratory assays without losing sensitivity, thereby helping to avoid overdiagnosis and resulting HIT overtreatment. Short-term re-exposure to heparin, especially given intraoperatively for cardiovascular surgery, is a reasonable therapeutic option in patients with a history of HIT who subsequently test negative for HIT antibodies. Organ failure(s), enhanced bleeding risks, and other characteristics require special considerations regarding non-heparin anticoagulation: Argatroban is the alternative anticoagulant with pharmacokinetics independent of renal function, but it has a prolonged half-life in case of impaired liver function. For bivalirudin, protocols during cardiopulmonary bypass surgery are established, and it is suitable for patients with liver insufficiency. A major issue of direct thrombin inhibitors are false high activated partial thromboplastin time values in patients with comorbidities affecting prothrombin, which can result in systematic underdosing of the drugs. This is not the case for danaparoid and fondaparinux, which can be monitored by anti-factor Xa assays, but have long half-lives and no suitable antidote. This review includes also information on management of on- and off-pump cardiac surgery, ventricular assist devices, percutaneous interventions, continuous renal replacement therapy, and extracorporeal membrane oxygenation in patients with HIT.

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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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