Molecular characteristics of polysulfated fractions from the green seaweed <i>Caulerpa cupressoides</i> and actions on thrombin generation <i>in vitro

Abstract

Caulerpa cupressoides produces sulfated polysaccharides (Cc-SPs) with serpin-dependent anticoagulant effect, but their actions on thrombin generation (TG) are unknown. This study aimed to partially characterize Cc-SPs and examine their potential as modulators of TG. Infrared analysis characterized extract containing three ulvan fractions (Cc-SP1, -SP2 and -SP3) separated by DEAEcellulose chromatography, with differences in the relative proportions of sulfate (10.99-18.38%) and total sugars (46.59-51.12%), without presenting proteins. Charge density patterns and nonSPs varying from 8 to > 100 kDa on agarose and polyacrylamide gel electrophoresis by sequential staining with toluidine blue and stains-all were also confirmed by gel permeation chromatography. The molecular weight of Cc-SP2 was not altered after treatment with 0.4 M HCl up to 5 h. Only Cc-SP2 altered the activated partial thromboplastin time (15 ± 0.3 IU) vs. heparin (193 IU) and abolished at high concentrations (> 4.1 μg) TG by intrinsic pathway in 60-fold diluted human plasma, while at 4.1 μg attenuated TG by 33.87% delaying the lag phase (32 min.) vs. control (28 min.). Cc-SP2 induced concentration-dependent TG in system without cephalin. Heparin abolished TG at 4.15-fold lower amount, but did not stimulate TG. Therefore, Cc-SPs express dual effects on thrombosis in vitro.

Sulfated agaran with 4,6-pyruvated form from red seaweed Acanthophora muscoides attenuates thrombin formation: in vitro and ex vivo studies

“…This result appeared on gel electrophoresis, after the use of Stains-all alone, in brown color, suggesting uronic acid-containing glycans in A. muscoides and purity of the agarocolloid confirmed by its structure as identified on FT-IR and NMR analyses (Quinderé et al, 2014;Vanderlei et al, 2016;Rodrigues et al, 2021) and by comparisons with other authors who found agar polysaccharide in Acanthophora (Duarte et al, 2004) and other seaweed species (Ferreira et al, 2012;Recalde et al, 2016). Similar to our study, polysaccharides isolated from animal (Volpi & Maccari, 2002;Andrade et al, 2017), plant (Souza et al, 2015) and seaweed (Rodrigues et al, 2017b and2019) were also characterized by their specific colors, reinforcing the use of Stains-all alone as a more precise strategy to also reveal acidic polysaccharides in red seaweeds than in combination with toluidine blue (Rodrigues et al, 2021). Additionally, HMBC analysis showed a doublet signal due to nondecoupling of the pulse sequence relatives to a sulfated galactan pyruvate form of six-membered cyclic ketal, including O-4 and O-6 positions (4,6-O-(1'carboxy)-ethylidene cyclic ketals), since that this structural feature has not been frequently found in agarophytes (Cardozo et al, 2007;Ferreira et al, 2012;Mourão, 2015).…”

“…Technology, v. 43, e55043, 2021 Cephalin or thromboplastin-devoid TG allowed to examine whether SP-Am would stimulate 60-fold diluted plasma, and suggested that, in the concentration range tested, the modestly TG-induced algal polysaccharide as FXII-speculated procoagulant effect previously related to low molecular mass accordingly, while UHEP did no activate FXII (Quinderé et al, 2014). This was also postulated for a SPs fraction from the green seaweed Caulerpa cupressoides, but 100% TG inducing at highest dose (Rodrigues et al, 2019). Mansour et al (2017) revealed that the oversulfated fucosylated chondroitin sulfate from the sea cucumber Holothuria polii body wall exhibited unusual procoagulant effect by means of calibrated automated thrombography.…”

“…On the contrary, the used doses of SP-Am were relatively lower than in the PT test (Barcellos et al, 2018), by which none allowed to detect slight differences due to its low lack, since it has fast kinetic to reveal thrombin formed (Arata et al, 2015), while TG assay had more precision to measure total thrombin (Castoldi & Rosing, 2011;Duarte et al, 2017), thus showing the anticoagulant dynamic of SP-Am compared with other studies (Nishino et al, 1999;Wu et al, 2014;Rodrigues et al, 2017a and2019;Salles et al, 2017). UHEP abolished TG at an amount 2-fold higher than that required to intrinsically inactivate TG (Rodrigues et al, 2019;Salles et al, 2017), in which UHEP had affinity for plasma antithrombin (Rodrigues et al, 2021); whereas in the PT assay, it did not alter the normal values, as reported by Rodrigues et al (2016b). The intrinsic inhibitory effect from all the polysaccharides was more important than the extrinsic one, similarly to brown seaweed E. kurome (Nishino et al, 1999) and freshwater fish O. niloticus (Salles et al, 2017) SPs.…”

“…Desulfation of thepyridinium salt of the SP-Am with dimethylsulfoxide wascarried out at 80°C for 4h (Nagasawa, Inoue, & Kamata, 1977). Its purity and molecular mass distribution was checked by electrophoresis on agarose (Dietrich & Dietrich, 1976) and polyacrylamide gels (Rodrigues et al, 2016b), respectively, associated with toluidine blue or Stains-all to reveal complex polysaccharides (Volpi & Maccari, 2002;Andrade, Oliveira, Tovar, Mourão, & Vilanova, 2017;Rodrigues et al, 2019) by comparison with the mobility of standard glycosaminoglycans dextran sulfate (̴ 8 kDa), chondroitin-4-sulfate (̴ 40 kDa), chondroitin-6-sulfate (̴ 60 kDa), heparan sulfate and/or dermatan sulfate (Rodrigues et al, 2021). The identity of the polymer (5 mg) was verified by Fourier Transform Infrared (FT-IR) spectroscopy in KBr using a Shimadzu IR spectrophotometer to characterize agar polysaccharide (Vanderlei et al, 2016;Rodrigues et al, 2021) and NMR spectroscopy ( 1 H/ 13 C HMBC) in 0.6 mL 99.9% deuterium oxide (Cambridge Isotope Laboratory, Cambridge, MA) using Bruker DRX 800 MHz (1024 × 256 points, with a 60 ms delay for evolution of long-range couplings and set with no decoupling during acquisition time) with trimethylsilyl propionic acid as a standard to characterize the SP pyruvate form (Quinderé et al, 2014), respectively, and spectrum processed using the SpinWorks 3.1.8 software package (USA).…”

“…This study was based on Rodrigues et al (2019) using a microplate format, without cephalin. The test was conducted as follow: 40 µL 0.02 M Tris-HCl/PEG buffer (pH 7.4) + 10 μL SP-Am (0, 4.1, 41.6 or 83.3 µg wellplate -1 ; UHEP: 2 µg.well-plate -1 ) + 60 μL 20 mM CaCl 2 /0.33 mM chromogenic substrate S-2238 (10:50 ratio, v:v).…”