<b><i>In vitro</i> inactivation of thrombin generation by polysulfated fractions isolated from the tropical coenocytic green seaweed <i>Caulerpa racemosa</i> (Caulerpaceae, Bryopsidales)

Abstract

ABSTRACT. Pharmacological efficacy of Caulerpa racemosa (Chlorophyta) sulfated polysaccharidic (SPs) fractions (F I→III) on models of coagulation and inflammation has been demonstrated, but not their effects on thrombin generation (TG). This study examined fractions for composition and physicalchemical characteristics and in vitro inactivation of TG by F I and F II in 60-fold diluted human plasma using continuous method. Papain-extraction yield of 0.7% revealed F I→III by DEAE-cellulose chromatography, with differences among the relative proportions of sulfate (17.37-24.00%), total sugars (30.03-48.34%) and absence of proteins. Charge density patterns and molecular sizes > 100 kDa of the fractions were verified by both agarose/polyacrylamide analyses, respectively. These electrophoreses combined with toluidine blue/Stains-All also indicated nonSPs. Anticoagulant effects of 4.76 (F I), 12.00 (F II) and 2.32 (F II) IU mg ), without changes in prothrombin time. Diluted plasma treated with F I and F II reduced concentration-dependent and sulfation pattern TG by both intrinsic and extrinsic pathways, with 50% inactivation by intrinsic pathway of F II even at 4.1 μg. Heparin abolished TG at least 4-fold lower. Therefore, C. racemosa produces SPs with TG inhibition.

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

“…Fractions Cc-SP2 and Cc-SP3 primarily showed here electrophoretic mobilities compared to UHEP. These observations suggested that C. cupressoides produced other polysaccharides that were not detected after staining with toluidine blue alone (Volpi & Maccari, 2002;Salles et al, 2017;Rodrigues et al, 2017a;Rodrigues et al, 2017b).…”

“…This anticoagulant action of Cc-SP2 was revealed to be 15 ± 0.3 IU mg -1 ( Figure 3A), whereas UHEP (193 IU mg -1 ) still altered the APTT test when a minimum concentration of 2.5 μg mL -1 (42.15 ± 0.60 s) was used (Salles et al, 2017;Rodrigues et al, 2017b) as inhibitor of the contact-pathway (p < 0.01) (Pomin, 2012;Mourão, 2015). As also expected, fractions Cc-SP1 and Cc-SP3 had no effect on APTT, when a high concentration of SPs (1 mg mL -1 ) was tested in vitro (p > 0.05) (data not shown) (Rodrigues et al, 2011a).…”

“…Chemical determination of sulfate, total sugars and proteins of the fractions were carried out as previously described (Rodrigues et al, 2011a). Visualization of SPs on agarose (Dietrich & Dietrich, 1976) / polyacrylamide gel by sequential staining with toluidine blue and stains-all (Volpi & Maccari, 2002;Rodrigues et al, 2017b) was conducted by comparison with the electrophoretic mobility of standard compounds dextran sulfate (ca. 8 kDa), chondroitin-4-sulfate (ca.…”

“…Since fraction Cc-SP1 was weakly visualized on the gel by staining with toluidine blue (Figure 2Aa), it was intended to more accurately analyze this SPs preparation using stains-all as another cationic dye for detection of glycans (Volpi & Maccari, 2002;Rodrigues et al, 2017b;Salles et al, 2017). Figure 2Bb allowed an improved examination of standards and fractions, especially Cc-SP1 that clearly exhibited two polysaccharide components stained distinctly on gel, named C1 and C2, where the first one co-migrated as UHEP and the second close to CS because ulvan resemble glycosaminoglycans containing uronic acid and sulfate (Wang et al, 2014).…”

“…TG-based coagulation tests would offer relevant information of plasma alternative anticoagulants to HEP in prevention of thrombosis in vitro (Nishino, Fukuda, Nagumo, Fujihara, & Kaji, 1999;Glauser et al, 2009;Rodrigues et al, 2016;Salles et al, 2017) or predicting hypercoagulability (Zhang et al, 2014;Barcellos et al, 2018). Little evidence of green seaweeds SPs as inhibitors of TG has been demonstrated as a more refined step for the rational development of functional agents from these polymers (Barcellos et al, 2018;Rodrigues, Benevides, Tovar, & Mourão, 2017b).…”