Liposomes bearing fibrinogen could potentially interfere with platelet interaction and procoagulant activity

M Rosa Hernández¹, Patricia Urbán², Elisenda Casals³, Joan Estelrich³, Ginés Escolar¹, Ana M Galán<sup>1

1</sup>Department of Hemotherapy and Hemostasis, Hospital Clinic, CDB, IDIBAPS, UB, Barcelona, Spain; ²Nanobioengineering Group, Institute for Bioengineering of Catalonia, Barcelona, Spain; ³Department of Physical Chemistry, Faculty of Pharmacy, University of Barcelona, Spain

Background: The contribution of fibrinogen (FBN) to hemostasis acting on platelet aggregation and clot formation is well established. It has been suggested that FBN-coated liposomes could be useful in restoring hemostasis. In the present study, we evaluated the modifications induced by multilamellar raw liposomes (MLV) or fibrinogen-coated liposomes (MLV-FBN) on hemostatic parameters.
Materials and methods: Different experimental settings using whole blood or thrombocytopenic blood were used. Thromboelastometry, aggregation studies, platelet function analyzer (PFA-100^®) tests and studies under flow conditions were applied to detect the effect of MLV-FBN on hemostatic parameters.
Results: The presence of MLV-FBN in whole blood modified its viscoelastic properties, prolonging clot formation time (CFT) (226.5 ± 26.1 mm versus 124.1 ± 9.4 mm; P < 0.01) but reducing clot firmness (45.4 ± 1.8 mm versus 35.5 ± 2.3 mm; P < 0.05). Under thrombocytopenic conditions, FIBTEM analysis revealed that MLV-FBN shortened clotting time (CT) compared to MLV (153.3 ± 2.8 s versus 128.0 ± 4.6 s; P < 0.05). Addition of either liposome decreased fibrin formation on the subendothelium (MLV 8.1% ± 4.7% and MLV-FBN 0.8% ± 0.5% versus control 36.4% ± 6.7%; P < 0.01), whereas only MLV-FBN significantly reduced fibrin deposition in thrombocytopenic blood (14.4% ± 6.3% versus control 34.5% ± 5.2%; P < 0.05). MLV-FBN inhibited aggregation induced by arachidonic acid (52.1% ± 8.1% versus 88.0% ± 2.1% in control; P < 0.01) and ristocetin (40.3% ± 8.8% versus 94.3% ± 1.1%; P < 0.005), but it did not modify closure times in PFA-100^® studies. In perfusion experiments using whole blood, MLV and MLV-FBN decreased the covered surface (13.25% ± 2.4% and 9.85% ± 2.41%, respectively, versus control 22.0% ± 2.0%; P < 0.01) and the percentage of large aggregates (8.4% ± 2.3% and 3.3% ± 1.01%, respectively, versus control 14.6% ± 1.8%; P < 0.01).
Conclusion: Our results reveal that, in addition to the main contribution of fibrinogen to hemostasis, MLV-FBN inhibits platelet-mediated hemostasis and coagulation mechanisms.

Keywords: thrombocytopenia, hemostasis, fibrinogen, liposomes, procoagulant activity, fibrin