To clarify the role of charge in protein movement across the pulmonary endothelial barrier, we simultaneously measured the permeability-surface area product (PS) for native [isoelectric point (pI) 4.4-5.1] and cationic (pI 7.2-8.0) albumin in isolated rabbit lungs perfused with and without protamine sulfate. We focused our measurement on the initial (endothelial) barrier by using a technique that is based on the very rapid (3 min) uptake of tracer. This allowed us to distinguish the charge properties of the endothelium separate from other barriers. In control studies, PS was greater for cationic than for native albumin (8.67 ± 0.93 vs. 2.55 ± 0.20 x 10-2 ml · min-1 · g dry lung-1). In the presence of 1 mg/ml protamine sulfate, cationic albumin permeability was not different from control (7.34 ± 0.49 x 10-2 ml · min-1 · g dry lung-1), whereas PS for anionic albumin increased to 8.82 ± 1.32 x 10-2 ml · min-1 · g dry lung-1. Thus the protamine sulfate eliminated the difference between native and cationic albumin PS. This selective increase in anionic albumin permeability is presumably due to the cation, protamine sulfate, binding to the anionic charges on the endothelium and reducing the anionic charge-charge repulsion. If protamine sulfate had produced a general endothelial injury, the PS for both albumins would have increased. Our results suggest that the normal pulmonary endothelium is an anionic charge barrier restricting the transcapillary movement of negatively charged molecules.

La réponse immune sous héparine (H) est associée à la synthèse d’anticorps (Ac) d’isotype IgG dirigés contre le facteur plaquettaire 4 (FP4) modifié par l’héparine. Ces anticorps se fixent par leur fragment Fc aux récepteurs FcγRIIa des plaquettes et induisent une forte activation plaquettaire. Les héparines de bas poids moléculaire sont constituées d’un mélange hétérogène d’oligosaccharides (OS) dont la structure varie en fonction de leur nombre de sucres et de groupements sulfates. Nous avons montré que seuls les OS ayant dix groupements sulfates ou plus, peuvent modifier le FP4 et permettre la fixation des Ac héparine-dépendants. La chirurgie cardiaque est associée à une forte activation plaquettaire et les patients sont exposés à de fortes concentrations d’héparine qui est neutralisée en fin d’intervention par le sulfate de protamine (SP). Alors que 30 à 50 % d’entre eux développent des Ac anti H/FP4 nous avons montré que 25% développent également des Ac dirigés contre les complexes H/SP et que ces Ac sont capables in vitro d’induire une activation plaquettaire. Le rôle de ces Ac in vivo reste cependant discuté. / The immune response under heparin (H) treatment is associated with IgG antibodies (Abs) synthesis against heparin-modified Platelet Factor 4 (PF4). These Abs bind FcγRIIa receptors via their Fc fragment and promote strong platelet activation. Low Molecular Weight Heparins are complex mixtures of polysaccharide fragments. These oligosaccharides (OS) have a variable structure due to variations in the type of sugar units and the number of sulphate groups. We demonstrated that OS longer than 10 saccharides and with a large number of sulphate groups are likely able to modify PF4 and allow the binding of heparin-dependent Abs. Cardiac surgery is associated with strong platelet activation and high doses of unfractionated heparin are administered to patients during surgery, and then neutralized with protamine sulfate (SP) at the end of the intervention. 30 to 50% of patients develop anti H/PF4 Abs, but we demonstrated that 25% do synthethized anti H/SP Abs able to activate platelets in vitro. The pathogenic role of these Abs to H/SP in vivo is controversial.

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