Platelet activation in the presence of neutral protamine Hagedorn insulin: a new feature of antibodies against protamine/heparin complexes

Abstract

T. Platelet activation in the presence of neutral protamine Hagedorn insulin: a new feature of antibodies against protamine/heparin complexes.J Thromb Haemost 2017; 15: 176-84. Essentials• Protamine (PRT) is used to stabilize insulin in neutral protamine Hagedorn (NPH) insulin.• The interaction between NPH-insulin, anti-PRT/heparin antibodies and platelets was investigated.• Anti-PRT/heparin antibodies activate platelets in presence of NPH-insulin dependent on heparin.• Cross-reactivity seems to have no major effect on the clinical outcome of medical patients.Summary. Background: Protamine (PRT) is used to stabilize insulin in neutral protamine Hagedorn (NPH) insulin, a commonly used therapeutic agent for diabetes mellitus. Immunization against PRT/heparin complexes is common in diabetic patients. Objectives: To investigate the impact of NPH-insulin on the interaction between anti-PRT/heparin antibodies and platelets. Methods: The interaction between NPH-insulin and anti-PRT/heparin antibodies was tested using in-house enzyme immunoassays. The ability of anti-PRT/heparin antibodies to activate platelets in the presence of NPH-insulin (and heparin) was investigated using flow cytometry. Results: Twenty-one out of 80 sera containing anti-PRT/heparin IgG showed binding to NPH-insulin. Anti-PRT/heparin IgG from immunized patients bound to platelets in the presence of NPH-insulin, but not in the presence of native insulin. Anti-PRT/heparin antibodies induced P-selectin expression in the presence of NPH-insulin in a heparin-dependent way (median mean fluorescence intensity in the presence of NPH-insulin: 55, 95% confidence interval [CI] 18.7-100.5 vs. NPH-insulin and heparin: 204, 95% CI 106.5-372.8). The clinical relevance of platelet-activating anti-PRT/heparin antibodies was assessed by investigating a multicenter study cohort of 332 acutely ill medical patients who received heparin. None of the 21 patients with anti-PRT/heparin IgG developed thrombocytopenia or thromboembolic complications. Conclusions: Anti-PRT/heparin antibodies activate platelets in the presence of NPH-insulin in a heparin-dependent way. However, results from our preliminary study indicate no major impact of these antibodies on the clinical outcome in medical patients receiving heparin, particularly on thromboembolic complications.

Blood donor‐derived buffy coat to produce platelets in vitro

“…The expression of CD62P was determined by flow cytometry using a FITC‐labelled anti‐CD62P antibody (CLB‐Thromb/6, Beckman Coulter). Fresh PLTs, used as positive control, were isolated from human blood anticoagulated with acid‐citrate dextrose (ACD, Vacutainer, Becton Dickinson) and resuspended in Tyrode‐buffer, as previously described .…”

Serologic characterization of anti-protamine/heparin and anti-PF4/heparin antibodies

“…Pouplard et al 1 found that ;29% (17 of 59) of patients with anti-PRT/heparin antibodies had a positive serotonin release assay in the presence of PRT alone. Recently, Zollner et al 19 noted that 21 (;29%) of 80 sera containing anti-PRT/heparin bound to platelets in the presence of neutral protamine hagedorn insulin, but not in the presence of insulin alone, and that heparin dissociated antibody binding from platelets, implying that PRT was bound to cellular GAGs.…”

Małopłytkowość indukowana protaminą — nowy problem czy inny typ małopłytkowości poheparynowej?

“…Aż 10% hospitalizowanych wykazało obecność PIT abs, mimo nieodnotowanej ekspozycji na protaminę podczas zabiegów kardiochirurgicznych. PIT abs w sposób zależny od heparyny aktywują płytki krwi w obecności insuliny NPH [28]. Lee i wsp.…”