Anionic surfaces with minimal charge density can restore burst coagulation of microparticle/exosome-depleted blood plasma

ANGEL, CONTRERAS-GARCIA; DESGAGNE, MAXIME; D'ELIA, NOELIA; CHARLES-HUBERT, LAFANTAISIE-FAVREAU; JUAN CARLOS, RUIZ; GEORGES-ETIENNE, RIVARD; WERTHEIMER, MICHEL; PAULA, MESSINA; CAROLINE, HOEMANN

Frontiers in Bioengineering and Biotechnology

Frontiers

Lugar: Lausanne; Año: 2016 vol. 4

Blood contains microparticles (MP) and exosomes derived from a variety of cell types including activated platelets.MPs are procoagulant because their surfaces can express tissue factor and/or calciumbinding sites for coagulation factors that cooperate to induce burst thrombin activation [1]. Plateletpoor plasma (PPP) contains low levels of MPs and can be induced to coagulate after contact with both hydrophilic and Hydrophobic surfaces [2]. In a standard thromboelastography (TEG) assay withplastic cups and pins, PPP undergoes spontaneous burst coagulation, as revealed in the TEG trace by a variable clotting time (10 to 26 minutes), a low Angle (~33º) and reproducible low clot tensile strength or Maximum Amplitude (MA ~28 mm) [3] (Fig. 1). PPP coagulates faster and more reproducibly in TEG cups and pins coated with anionic carboxylaterich nanolayers suggesting FXII activation [2], however the role of MPs is unclear. This study tested the hypothesis that MPs are necessary for PPP burst coagulation in plastic TEG cups, and that anionic surfaces are sufficient to restore burst coagulation of MPdepletedplasma.Materials and Methods: Plastic Cyrolite® TEG cups and pins (Hemoscope, IN, USA) were modified by plasmaenhanced chemical vapor deposition to make hydrophobic (PPHMDSO, poly(hexamethyldisiloxane) or variable anionic (LPPE:O, 0, 4.4% or 6.8% carboxylate functional group content) nanocoatings [3]. Coagulation in the TEG cup was initiated by combining i) human citrated PPP (Precision Biologics, NS, Canada), ii) MPdepleted plasma (by ultracentrifugation 150,000xg, 30 min) or iii) PPP 2xenriched for MP, with 20 μL of 200 mM CaCl2 containing 2.5 mg/mL 10 μm diameter beads (latex, borosilicate glass, carboxylated plastic), or 0.25 to25 mg/mL hydroxyapatite (HA) nanoparticles (2.03 Ca/P ratio) [4]. Bead Composition was analyzed by XPS. Statistical tests used ANOVA (Statistica).Results: Glass beads with 9% Si and 4% Ca content triggered the fastest PPP clotting time (p55º. HA nanoparticles accelerated PPP burst coagulation in a dosedependent manner (p=0.004). MPdepleted plasma failed to clot in plastic TEG cups (Fig. 1), and burst coagulation was Progressively restored by LPPE: O with 4.4 to 6.8% carboxylate content (p4.4% carboxylated surfaces and HA were effective MP biomimetics that restored burst coagulation. These results suggest that inorganic anionic surfaces can serve as functional binding sites for the calciumbinding gla domain of factors FIXa, FXa and prothrombin. This study is consistent with the notion that tenase and prothrombinase form sidebysideon the same anionic surface [1].Conclusions: A minimal 6.8% anionic charge group density is required to bring tenase and prothrombinase in close physical proximity for optimal cooperative behavior.