The analysis of molecular masses of the products were examined by polyacrylamide gel electrophoresis at 100 V for ~50 min., by comparison with the electrophoretic mobility of the standard low-molecular-weight compounds dextran sulfate (~8 kDa), chondroitin-4-sulfate (~40 kDa), chondroitin-6-sulfate (~60 kDa) and/or UHEP (~14 kDa).
Unfractionated heparin (UHEP) with 193 international units per mg (IU [mg.sup.-1]) of polysaccharide was used as standard.
For agarose analysis, Am-1 and Am-2 exhibited single, homogeneous and coincident metachromatic bands, while Am-3 presented a very clear band on gel; in addition, all of them had higher mobility than UHEP due to a different structural conformation and charge/mass ratio between SPs (Dietrich & Dietrich, 1976; Fidelis et al., 2014).
The highest anti-clotting potencies were recorded for the 0.75 M NaCl fractions (3.07 (Am.E-1) -3.06 (Am.E-3) IU [mg.sup.-1], respectively) when only at a high concentration of SPs (1 mg mL-1) was capable of doubling the normal APTT values in relation to a 193 IU [mg.sup.-1] UHEP standard.
All isolated polymers of these species had specific heparin cofactor II-dependent thrombin inhibition, but their abilities to inhibition thrombin are not as strong as that of UHEP. Therefore, SPs from Chlorophyta activate heparin cofactor II primarily by a mechanism different from displacement and template mechanisms.
Also, it was observed its property to induce 2-fold more potency than UHEP the stimulating of the synthesis of an antithrombotic SP by endothelial cells.
racemosa fractions (F I and F II): 0, 4.1, 41.6 or 83.3 [micro]g [well-plate.sup.-1] or UHEP: 2 or 4 [micro]g [well-plate.sup.-1]) + 60 [micro]L of 20 mM Ca[Cl.sub.2]:0.33 mM specific chromogenic substrate S2238 (10:50 ratio, v:v).
racemosa SPs (extract/fractions) and standards (glycosaminoglycans chondrotin sulfate, UHEP or dextran sulfate) in complex mixtures, agarose/polyacrylamide techniques associated with sequential toluidine blue/Stains-All staining were carried out as a function of charge density and molecular size, respectively, as already reported for glycosaminoglycans extracted from animal tissues (Volpi & Maccari, 2002; Salles et al., 2017) (Figure 1).
TG assay was performed in a microplate format, containing: 10 [micro]L of cephalin (contact-activator system) + 30 [micro]L 0.02 M Tris HCl/PEG-buffer (pH 7.4) + 10 [micro]L SPs (Cc-SP1: 0, 4.1, 8.3, 41.6 or 83.3 [micro]g well-[plate.sup.-1] or UHEP: 2 [micro]g well-[plate.sup.-1]) + 60 [micro]L 20 mm Ca[Cl.sub.2]/0.33 mm chromogenic substrate S2238 (10:50 ratio, v [v.sup.-1]).
Although polydispersion naturally occurs in SPs from seaweeds (Pomin, 2012, Fidelis et al., 2014, Mourao, 2015, Rodrigues et al., 2017), the current study contrasted with that previously performed, where Cc-SP1 did not appear on agarose gel (Rodrigues et al., 2011b) allowing us to identify here at least two major sulfated components (SC-1 and SC-2) in this fraction with profiles of migration compared to condroitin-4-sulfate and UHEP, respectively, when they were used as standards (Volpi & Maccari, 2002, Salles et al., 2017).
UHEP (193 IU [mg.sup.-1]) (fourth International Standard (85 [502.sup.-1])) from the National Institute for Biological Standards and Control (Potters Bar, UK) (Nader el al., 2001) was used as reference.
TG assay was performed in a microplate format, containing: 10 [micro]L of cephalin (contact-activator system) + 30 [micro]L of 0.02 M Tris HCl/PEG-buffer (pH 7.4) + 10 [micro]L of SPs (Am-2 [native]: 0, 4.1, 8.3, 41.6 or 83.3 [micro]g well-plate-1 [hydrolysates-1 in 10 mL of solution], or UHEP: 2 jig well-plate-1) + 60 [micro]L of 20 mM Ca[Cl.sub.2] 0.33-1 mM chromogenic substrate S2238 (10:50 ratio, v v-1).