Semi-Synthetic Sulfaminoheparosans Induce Tissue Factor Pathway Inhibitor Release by Human Umbelical Vein Endothelial Cells (HUVEC)

Abstract number: P1940

Gori¹ AM, Gazzini¹ A, Evangelisti¹ L, Attanasio¹ M, Manoni² M, Laudati² G, Seghi² F, Cesari¹ F, Abbate¹ R, Gensini¹ GF

¹¹Department of Medical and Surgical Critical Care, Thrombosis Centre, University of Florence; Azienda Ospedaliero-Universitaria Careggi, Florence, Italy ¹¹Department of Medical and Surgical Critical Care, Thrombosis Centre, University of Florence; Azienda Ospedaliero-Universitaria Careggi, Florence, Italy ²²INALCO RSM SpA Research Center, Montale (PI), Italy

Tissue factor pathway inhibitor (TFPI) is the main physiological inhibitor of tissue factor, the principal activator of coagulation. Both unfractionated heparin and low molecular weight heparins are able to release TFPI from the vascular endothelium. Semi-synthetic sulfaminoheparosan analogues (SAH) were obtained by chemical and enzymatic modifications of the K5 polysaccharide (E.coli strain 010 : K5 : H4) a polymer of the disaccharidic unit formed by D-glucuronic acid and N-acetylglucosamine. This structure is alike to N-acetylheparosan, the precursory polymer of heparin and of heparan sulphate. Aim of our study was to evaluate the effect of two different SAH with different molecular weight and anti-Xa/anti-IIa activities on TFPI release from human endothelial cells. We performed 10 independent experiments in which human endothelial cells (HUVEC), isolated from umbilical vein (2 × 10⁵ cells/well), were incubated for 1 hour at 37°C in the presence of 15, 10 and 5 µg/ml of SAH-A (8300 Da and 0.71 anti-Xa/anti-IIa ratio), SAH-B (6000 Da and 2.4 anti-Xa/anti-IIa ratio), and unfractionated heparin (UFH) (1 U anti-Xa/ml). Incubation of HUVEC for 1 hour in the presence of 15, 10 and 5 µg/ml SHDs caused a dose-dependent release of TFPI from HUVEC. The heparin analogue SAH-A with the highest molecular weight and lowest anti-Xa/anti IIa ratio, at 15 and 10 µg/ml induced higher amounts of released TFPI in the supernatants in comparison to UFH (1 IU anti-Xa /ml) (SAH-A 15 µg/ml: 3.3 ± 0.09 ng/10⁵ cells; 10 µg/ml: 2.27 ± 0.22 ng/10⁵ cells; UFH: 1.93 ± 0.23 ng/10⁵ cells). The addition of SAH-B (15 µg/ml) to HUVEC cells induced significant (P < 0.05) higher TFPI concentrations in comparison to that induced by UFH (2.44 ± 0.13 ng/10⁵ cells vs 1.93 ± 0.23 ng/10⁵ cells). Our results demonstrate that heparin-like semi-synthetic derivatives are able to induce TFPI release from human HUVEC in a dose-dependent manner. This effect seems to be related to the molecular weight of heparin analogues.