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 FcγRIIA R/R 131 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.

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Heparin-induced thrombocytopenia (HIT)is due primarily to IgG antibodies specific to platelet factor 4/heparin complexes (PF4/Hs) that activate platelets via Fc␥RIIA. 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 Fc␥RIIA 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 Fc␥RIIA 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 C␥2, 2 major signaling proteins downstream from Fc␥RIIA. 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. (Blood. 2012; 120(6):1309-1316) IntroductionHeparin-induced thrombocytopenia (HIT) results from an atypical immune response to platelet factor 4/heparin complexes (PF4/Hs), with rapid synthesis of platelet-activating IgG antibodies that activate platelets via Fc␥RIIA receptors. 1 The risk of HIT is probably dependent on the intensity of this immune response, because the likelihood of developing thrombocytopenia and thrombotic complications has been related to the plasma levels of IgG antibodies to PF4. 2,3 However, the reasons explaining why only a subset of patients treated with heparin and who develop significant levels of IgG to PF4/Hs will present with HIT remain to be fully defined.Protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) are crucial for the regulation of signaling pathways involved in the control of several cellular processes, including immune responses and platelet activation. Dysregulation of the equilibrium between PTK and PTP function can therefore have pathologic consequences. 4,5 Among PTKs, Src family kinases (SFKs) have an important role in regulating immune responses and platelet adhesion, activation, and aggregation. 6 SFKs are essential in the platelet Fc␥RIIA-dependent signaling pathway, and it was recently demonstrated that dasatinib, a SFK inhibitor, prevents platelet activation induced by HIT antibodies. 7 In addition, SFKs are involved in collagen-dependent platelet activation via glycoprotein VI (GPVI). 8 After collagen/GPVI interaction or Fc␥RIIA cross-linking, SFKs induce phosphorylation of conserved tyrosine residues within the immunoreceptor tyrosine-based activation motif, providing a docking site for the tyrosine...

Introduction/Objectives Extracorporeal membrane oxygenation (ECMO) provides circulatory support in patients with severe heart failure, but the frequent use of unfractionated heparin exposes patients to high risk of heparin-induced thrombocytopenia (HIT). We prospectively evaluated the development and clinical impact of platelet factor 4 (PF4)-specific antibodies (Abs) during ECMO and whether specific biological characteristics could predict HIT. Materials and Methods From 2014 to 2018, we studied 57 adults who underwent an ECMO for at least 5 days. The plasma samples collected daily were tested for PF4-specific Abs using immunoassays to detect immunoglobulin (Ig) G, A, and M isotypes or only IgG. Serotonin release assay was performed without and with PF4 to detect pathogenic Abs. Results Twenty-nine patients (50%) were positive for PF4-specific Abs (IgG, A, M), with IgG in 17/57 (30%) and 16 of them (94%) were immunized within 10 days. PF4-specific IgG Abs did not affect the clinical or biological course of most patients. HIT was suspected in only two patients with ECMO circuit dysfunction and unexpected platelet count decrease after day 5. High levels of PF4-specific IgG were detected in both patients, and HIT was confirmed by a serotonin release assay, which was also more sensitive when exogenous PF4 was present. Conclusion PF4-specific Abs are common during ECMO but are mostly non-pathogenic and not associated with a less favorable prognosis. However, an abnormal platelet count evolution, in particular if associated with ECMO circuit dysfunction, should prompt the search for pathogenic PF4-specific IgG.

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Data of 401 patients who underwent mitral valve replacement with the Carpentier-Edwards Perimount bioprosthesis between 1984 and 2009 were evaluated. Their mean age was 68.1 ± 10.4 years (range, 22-90 years) and 54.9% were female. The most common etiology was degenerative disease (33.2%) and 62.1% of patients had mitral insufficiency. Follow-up was 3,178 patient-years, and 96.8% complete; the mean follow-up was 8.9 ± 3.1 years. Overall survival at 25 years was 10.2% ± 3%. Late mortality was 2.48% per patient-year, and valve-related deaths occurred at 1.62% per patient-year. The actuarial freedom from reoperation due to structural valve deterioration at 20 years was 24.3% ± 2% for degenerative disease and 15% ± 1.4% for non-degenerative disease. For degenerative valve disease, the freedom from structural valve deterioration at 18-years was 39% ± 1% for recipients <60-years old and 66% ± 2% for those ≥60-years old. Our data confirm the excellent durability and low mortality associated with the Carpentier-Edwards Perimount for mitral valve replacement. The rate of calcification of the valve was unrelated to degenerative valve disease, but our findings suggest that this prosthesis gives better results in recipients ≥60-years old than in younger patients.

To cite this article: Rollin J, Pouplard C, Leroux D, May M-A, Gruel Y. Impact of polymorphisms affecting the ACP1 gene on levels of antibodies against platelet factor 4-heparin complexes. J Thromb Haemost 2013; 11: 1609-11.Heparin-induced thrombocytopenia (HIT) results from an atypical immune response to platelet factor 4 (PF4)-heparin complexes, with rapid synthesis of platelet-activating IgG antibodies that activate platelets via FccRIIa receptors [1]. The reasons why only a subset of patients treated with heparin develop antibodies to PF4-heparin complexes, and why most patients who synthesize these antibodies do not develop HIT, have not been fully defined. The immune response in HIT probably involves both B cells and T cells [2,3], and protein tyrosine kinases and protein tyrosine phosphatases (PTPs) are crucial for regulating receptor-induced lymphocyte activation and Fc receptors, including FccRIIa. Among PTPs, Lyp, encoded by PTPN22, plays a key role in regulating the Src family kinases (SKFs) in immune cells. Several studies have shown that carriers of the PTPN22 1858T allele are at higher risk for various autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and immune thrombocytopenia [4]. In addition, polymorphisms affecting ACP1, which encodes another PTP, named low molecular weight PTP (LMW-PTP), have also been associated with several human diseases, including autoimmune diseases [5]. On the other hand, FccRIIa-dependent platelet activation is a central event in HIT, and variations in regulatory elements of this process may also modify the risk of HIT. We recently confirmed this hypothesis by demonstrating that polymorphisms of CD148, a PTP receptor regulating SKFs, influence the platelet activation induced by HIT antibodies and risk of HIT in patients with significant levels of antibodies against PF4-heparin complexes [6]. Interestingly, Mancini et al. [7] had previously shown that LMW-PTP regulates antibody-induced phosphorylation of the immunoreceptor tyrosine-based activation motif of FccRIIa receptors in platelets. Given their central role in regulating SFKs and multiple signaling networks essential for immune cell function and/or platelet activation, we therefore looked for an association between functional polymorphisms in PTPN22 (1858C/T) or ACP1 (A, B and C alleles) and the development of antibodies against PF4-heparin complexes and HIT.The patient group comprised 89 individuals with definite HIT. In every case, the diagnosis of HIT was based on the clinical history and PF4-specific ELISA (IgG/A/M HAT; GTI, Brookfield, WI, USA; median A 405 nm value of 2.2; range, 0.76-4.0) combined with a serotonin release assay (SRA), and both tests always gave positive results. The first control group (Ab neg ) consisted of 179 patients who had undergone heart surgery with cardiopulmonary bypass and received heparin. All patients were negative for HIT antibodies between the 7th and 10th postoperative days (median A 405 nm value of 0.13; range, 0.04-0.38; cut-off value, 0.4)...

A new ELISA (Zymutest HIA®), based on incubation of diluted plasma with protamine/heparin (PRT/H) complexes without and with platelet factor 4 (PF4) provided by a platelet lysate, was used to detect heparin-dependent antibodies in a cohort of 232 cardiac surgery (CS) patients and in 47 patients with heparin-induced thrombocytopenia (HIT). Significant binding of IgG/A/M to PRT/H complexes was demonstrated in 59 CS patients (25.4%), with similar absorbances whether platelet lysate was added to the plasma or not, and significant reactivity to PF4/H in 29 of them. Antibodies to PRT or heparin alone were present in 15 and two of these patients, respectively. Importantly, antibodies to PRT/H were detected in only three of the 47 HIT patients, who had also undergone recent CS. The Zymutest HIA® was positive in another 41 CS patients (17%), but only or mainly when their plasma was tested with platelet lysate, with significant levels of antibodies to PF4/H in 40 of them without detectable reactivity to PRT or heparin alone. Slight antibody binding to PRT/H complexes was also measured in six of these 41 patients. Therefore, a total of 35 CS patients exhibited dual antibody reactivity towards PRT/H and PF4/H complexes. Serotonin release assay performed with PRT alone was positive in 17 CS patients with antibodies to PRT/H, but all had normal platelet count evolution without thrombosis postoperatively. In conclusion, antibodies to PRT/H are frequently present in CS patients postoperatively (25.4%), and can activate platelets in vitro, but their clinical impact remains questionable.