Claire Pouplard

Antibodies to Platelet Factor 4–Heparin After Cardiopulmonary Bypass in Patients Anticoagulated With Unfractionated Heparin or a Low-Molecular-Weight Heparin Clinical Implications for Heparin-Induced Thrombocytopenia

BACKGROUND Cardiopulmonary bypass (CPB) induces platelet activation with release of platelet factor 4 (PF4), and patients are exposed to high doses of heparin (H). We investigated whether this contributes to the development of antibodies to H-PF4 and heparin-induced thrombocytopenia (HIT). METHODS AND RESULTS CPB was performed with unfractionated heparin (UFH) in 328 patients. After surgery, patients received UFH (calcium heparin, 200 IU. kg-1. d-1) (group 1, n=157) or low-molecular-weight heparin (LMWH, Dalteparin, 5000 IU once daily) (group 2, n=171). Eight days after surgery, antibodies to H-PF4 were present in 83 patients (25.3%), 46 in group 1 and 37 in group 2 (P=0.12). Most patients (61%) had IgG1 to H-PF4, but only 8 samples with antibodies induced platelet activation with positive results on serotonin release assay. HIT occurred in 6 patients in group 1, but no thrombocytopenia was observed in subjects receiving LMWH, although 2 had high levels of antibodies with positive serotonin release assay results. When antibodies to H-PF4 were present, mean platelet counts were lower only in patients with FcgammaRIIA R/R131 platelets. CONCLUSIONS These results provide evidence that the development of antibodies to H-PF4 after CPB performed with UFH is not influenced by the postoperative heparin treatment. The antibodies associated with high risk of HIT are mainly IgG1, which is present at high titers in the plasma of patients continuously treated with UFH.

Changes in platelet count after cardiac surgery can effectively predict the development of pathogenic heparin-dependent antibodies

Cardiopulmonary bypass (CPB) induces the release of platelet factor 4 (PF4) and patients are at risk of heparin‐induced thrombocytopenia (HIT). This study was aimed to determine whether an abnormal evolution in platelet count (PC) after CPB is predictive of the development of HIT antibodies. Two abnormal PC patterns were defined: pattern P1, characterized by a decrease in PC following previous correction of thrombocytopenia occurring during CPB, and pattern P2, defined as a persistent low PC in the days following CPB. PC was evaluated for 10 d in 305 consecutive patients before and after CPB. Serotonin release assay (SRA) was carried out between days 8 and 10 to detect pathogenic heparin‐dependent antibodies. Moreover, antibodies to heparin–PF4 (H–PF4) complexes were assayed by enzyme‐linked immunosorbent assay. PC evolution after CPB was normal in 300 patients although antibodies to H–PF4 were frequently present (53·4%). Changes in PC were abnormal in five patients with pattern P1 (n = 4) or P2 (n = 1). As SRA was positive in four of the five cases, the positive predictive value of abnormal PC pattern for pathogenic HIT antibodies was 80%. Careful follow‐up of PC after CPB makes it possible to predict with high specificity (99%) for those patients who develop pathogenic HIT antibodies.

Biological and clinical features of low-molecular-weight heparin-induced thrombocytopenia

Summary. Heparin‐induced thrombocytopenia (HIT) is a common adverse effect of unfractionated heparin (UFH) therapy. In contrast, only a few patients have been reported with HIT following low‐molecular‐weight heparin (LMWH) therapy (LMW‐HIT). To define the clinical and biological characteristics of LMW‐HIT, 180 patients treated for suspected HIT at 15 French centres were investigated. Clinical history was recorded and HIT was confirmed in 59 patients with positive serotonin release assay results: 57 of them had high levels of antibodies (Abs) to heparin–platelet factor 4 complexes (H/PF4) and two had Abs to interleukin 8. Eleven patients were treated exclusively with LMWH (LMW‐HIT) and 48 with UFH either alone (UF‐HIT, n = 34) or combined with LMWH (UF/LMW‐HIT, n = 14). The LMW‐HIT and UF‐HIT groups were similar with respect to sex, age, platelet count before heparin therapy, frequency of bleeding and occurrence of disseminated intravascular coagulation. The interval to onset of HIT was longer in LMW‐HIT patients compared with UF‐HIT patients (P = 0·03). Severe thrombocytopenia (platelets < 15 × 109/l) was more frequent in the LMW‐HIT group (P = 0·04). Thrombosis occurred in three of 11 LMW‐HIT patients, i.e. as frequently as in UF‐HIT patients. LMW‐HIT is potentially severe and may be observed after longer heparin treatment compared with UF‐HIT. It is highly recommended, therefore, that platelet counts be monitored carefully whenever LMWH is administered.

Affinity purification of heparin‐dependent antibodies to platelet factor 4 developed in heparin‐induced thrombocytopenia: biological characteristics and effects on platelet activation

Antibodies to heparin platelet factor 4 (H‐PF4) complexes were purified from the plasma of three patients with heparin‐induced thrombocytopenia (HIT) using affinity chromatography. From each plasma, the largest amount of antibodies was eluted with 2 m NaCl at pH 7·5 (peak 1) and the remainder was obtained using 0·1 m glycine/0·5 m NaCl at pH 2·5 (peak 2). In an enzyme‐linked immunosorbent assay (ELISA), we then showed that each patient had developed antibodies to PF4 displaying different characteristics. In patient 1, peak 1 IgG reacted almost exclusively with H‐PF4 complexes, whereas peak 2 IgG had similar reactivity with PF4 whether or not heparin was present. Patient 2 expressed a mixture of IgA, IgM and IgG and both fractions bound to PF4 alone or to H‐PF4 complexes. Finally, IgG in patient 3 only bound to H‐PF4 and was unreactive with PF4 alone. Using [14C]‐serotonin release assays, the antibodies developed in the three patients and exhibiting the strongest ability to activate platelets with heparin were those having the highest affinity to H‐PF4. These results strongly support the hypothesis that HIT antibodies to PF4 are heterogeneous regarding their affinity and specificity for target antigens and this may greatly influence their ability to activate platelets and their pathogenicity.

Differences in specificity of heparin-dependent antibodies developed in heparin-induced thrombocytopenia and consequences on cross-reactivity with danaparoid sodium

Heparin‐induced thrombocytopenia (HIT) is frequently associated with antibodies (Abs) to heparin–PF4 complexes (H‐PF4). In order to investigate whether there are variations in specificity of Abs, we studied 63 samples from patients with suspected HIT. Two groups of samples were separated after comparing their reactivity against H‐PF4 or recombinant PF4 (r‐PF4) using ELISA. In group Ab1 (n = 46), Abs only or mainly bound to H‐PF4 complexes and thus most of the epitopes recognized probably involved both heparin and PF4. In group Ab2 (n = 17), Abs exhibited similar reactivity to r‐PF4 and H‐PF4, and the antigens recognized were possibly neoepitopes mainly expressed by modified PF4 and by H‐PF4 complexes. Platelet activation tests were positive with 56 samples containing high titres of Abs to H‐PF4. Most samples (n = 59) contained IgG antibodies, often associated with IgA antibodies which were more frequently found in group Ab2, and/or IgM. With unfractionated heparin treatment, HIT was associated with Ab1 or Ab2 antibodies, whereas only Ab1 antibodies were detected after low‐molecular‐weight heparin (LMWH). Furthermore, cross‐reactivity with danaparoid sodium was present only in group Ab1 and mainly involved LMWH‐treated patients.

Effectiveness of a new immuno-assay for the diagnosis of heparin-induced thrombocytopenia and improved specificity when detecting IgG antibodies

The diagnosis of heparin-induced thrombocytopenia (HIT) is based on clinical criteria and biological assays. Most immunoassays detect antibodies (either IgG alone or additionally IgA and IgM) against PF4 immobilised in wells of microtiter plates with stoichiometric concentrations of polyanion (heparin or polyvinylsulfonate). We studied whether diagnostic sensitivity and/or specificity for HIT could be improved using a novel assay in which unfractionated heparin is immobilised alone to the microwells, with PF4 (and, potentially, other heparin-dependent antigen proteins) provided by adding platelet lysate during the procedure. Samples from 101 patients with suspected HIT and from 101 controls (including 50 with antiphospholipid antibodies) were tested. The global assay (Zymutest HIA IgG/A/M, Hyphen BioMed) was positive for 39 of 40 patients with definite HIT (positive PF4-specific ELISA and positive serotonin release assay). It was positive in only two of the 101 control patients studied and also in 14 of the 61 patients with suspected HIT for whom the disease was excluded (specificity (sp): 77%). On the other hand, Zymutest HIA IgG, an IgG-specific assay, was positive in only six patients without HIT (Sp: 90%). Heparin-dependent IgG antibodies were present at higher levels in patients with definite HIT than in those for whom the diagnosis of HIT was ruled out. A single ELISA that detects IgG antibodies is more effective for the diagnosis of HIT in clinical practice. These results also support the hypothesis that heparin-dependent antibodies of IgG class have a major role in the pathogenesis of HIT.

Polymorphisms of protein tyrosine phosphatase CD148 influence FcγRIIA-dependent platelet activation and the risk of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is due primarily to IgG antibodies specific to platelet factor 4/heparin complexes (PF4/Hs) that activate platelets via FcγRIIA. CD148 is a protein tyrosine phosphatase that regulates Src kinases and collagen-induced platelet activation. Three polymorphisms affecting CD148 (Q276P, R326Q, and D872E) were studied in HIT patients and 2 control groups, with or without antibodies to PF4/Hs. Heterozygote status for CD148 276P or 326Q alleles was less frequent in HIT patients, suggesting a protective effect of these polymorphisms. Aggregation tests performed with collagen, HIT plasma, and monoclonal antibodies cross-linking FcγRIIA showed consistent hyporesponsiveness of platelets expressing the 276P/326Q alleles. In addition, platelets expressing the 276P/326Q alleles exhibited a greater sensitivity to the Src family kinases inhibitor dasatinib in response to collagen or ALB6 cross-linking FcγRIIA receptors. Moreover, the activatory phosphorylation of Src family kinases was considerably delayed as well as the phosphorylation of Linker for activation of T cells and phospholipase Cγ2, 2 major signaling proteins downstream from FcγRIIA. In conclusion, this study shows that CD148 polymorphisms affect platelet activation and probably exert a protective effect on the risk of HIT in patients with antibodies to PF4/Hs.

Usefulness of pretest clinical score (4Ts) combined with immunoassay for the diagnosis of heparin-induced thrombocytopenia.

Purpose of review This review addresses the clinical and biological strategy to be applied for the diagnosis of heparin-induced thrombocytopenia. Recent findings Heparin-induced thrombocytopenia is a severe prothrombotic disease caused by immunoglobulin G antibodies, which bind to platelet factor 4 and activate platelets with subsequent increased thrombin generation. Venous and arterial thromboses are frequent, explaining why substituting heparin with a potent alternative anticoagulant (danaparoid, lepirudin, or argatroban) is necessary in every affected patient. However, the diagnosis of heparin-induced thrombocytopenia is often difficult and it has to be diagnosed on the basis of clinical criteria and reliable laboratory tests (immunoassays and platelet activation tests). The profiles of platelet count evolution suggestive of heparin-induced thrombocytopenia in cardiac surgery patients are now well defined. For other clinical settings, the usefulness of a scoring system, the ‘4Ts’, estimating the probability of heparin-induced thrombocytopenia before laboratory testing, combined with one immunoassay allowing specific detection of heparin-induced thrombocytopenia antibodies, has been recently documented. Summary Recent studies have demonstrated the usefulness of combining a pretest clinical score (4Ts) and biological assays to diagnose heparin-induced thrombocytopenia and the algorithms that have to be applied for clinical practice are now better defined.

Risk factors for unfavorable clinical outcome in patients with documented heparin-induced thrombocytopenia

BACKGROUND Prognostic factors for unfavorable clinical outcome in patients with heparin-induced thrombocytopenia (HIT) are largely unknown. DESIGN AND METHODS In this multicenter, retrospective, case-control study, all HIT patients were treated with danaparoid. Study cases were HIT patients with an unfavorable clinical outcome. Controls were HIT patients who were not study cases. Unfavorable clinical outcome was defined as the occurrence of at least one of the following clinical events: death within 60 days after HIT start date, or venous or arterial thromboembolism, amputation, major bleeding, or disseminated intra-vascular coagulation between 48 hours and 60 days after HIT start date. RESULTS Compared with controls (n=65), thrombotic episodes within 48 hours of HIT start date were more frequent (59.2% versus 32.3%; p=0.004), the median time between HIT start date and initiation of danaparoid infusion was longer (3.0 versus 1.0 days; p=0.001), and this treatment was more frequently underdosed (43.8% versus 18.8%; p=0.004) in study cases (n=49). Upon multivariate analysis, all these three parameters were significant predictive factors for unfavorable clinical outcome. The adjusted odds ratios [95% confidence interval] were 6.6 [2.5-17.3] for time between HIT start date and danaparoid initiation over 48 hours, 4.3 [1.5-12.0] for danaparoid underdosing, and 3.2 [1.3-8.0] for presence of a thromboembolic episode at HIT start date. CONCLUSIONS This study supports the recommendations concerning the management of HIT patients, namely discontinuation of all heparin administration once the diagnosis is suspected and prompt initiation of an alternative anticoagulant drug with a strict adherence to doses specifically recommended for these patients.

Characteristics, mechanisms of action, and epitope mapping of anti-factor VIII antibodies.

The development of anti-factor VIII (FVIII) antibodies (Abs), also called inhibitors, is currently one of the most serious complications arising during the treatment of hemophilia A patients. Improved prevention and eradication of these Abs remain a challenge both for clinicians and scientists. Numerous studies in the literature have reported on their epitope specificity, on their mechanism of FVIII inactivation, as well as on the methods used for their detection. In this review, we summarize the current knowledge on the nature (isotypes, kinetic properties), epitope properties, and mechanisms of action of anti-FVIII Abs. Furthermore, we present methods for detection and epitope characterization of anti-FVIII Abs with emphasis on the Luminex technique susceptible to facilitate the monitoring of changes in the epitope specificity of these Abs.