Using fractions at different concentrations (4.1 [right arrow] 83.3 [micro]g well-[plate.sup.-1]) (Rodrigues et al., 2016; Salles et al., 2017; Rodrigues et al., 2017b), our results showed that sulfate differentially interfered with the TG response in 60-fold diluted human plasma activated by cephalin (intrinsic coagulation pathway) as recorded continually at 37[degrees]C for 60 min., in comparison with UHEP used as a reference.
This in vitro inhibition degree of the fractions on the intrinsic coagulation pathway of TG stimulated by cephalin did not occur on the same elution order, where fraction Cc-SP3 (eluted at 1 M NaCl) with high sulfation exhibited almost equal inhibitory effects compared with the fraction Cc-SP1, which revealed a comparatively lower sulfation.
As low-molecular-weight fragments are more promising as new antithrombotic drugs (Glauser et al., 2009; Rodrigues et al., 2016), our study was also directed to examine the fraction Cc-SP2 on the in vitro TG system without cephalin as contact-activator and continually analyze regarding its possible stimulatory response, as illustrated in Figure 4.
However, the coagulation effect of platelets was dramatically reduced, as kaolin cephalin clotting time lengthened from 39,3 [+ or -] 1,2s to 51,8 [+ or -] 0,3s (P<0.001).
Up to this period the data shortened from 120,0 [+ or -] 2,3s to 67,5 [+ or -] 1,1s (P<0.001), plasma recalcification time, with a 89,3 [+ or -] 1,0s to 23,3 [+ or -] 0,7s (P<0.001) and kaolin with a 39,3 [+ or -] 1,2s to 24,1[+ or -]0,7s (P<0.001) kaolin cephalin clotting time.
Healthy animals Indications (control) Platelet quantity (x[10.sup.9]/l) 367,8 [+ or -] 10,4 Platelet adhesion (%) 33,8 [+ or -] 1,7 Platelet aggregation (s) 22,0 [+ or -] 0,7 Clotting time Lee-White (min) in 5,1 [+ or -] 0,53 non-siliconized tube in siliconized tube 7,1 [+ or -] 0,7 Starting clotting (s) 483,8 [+ or -] 21,7 Duration of blood clotting (s) 253,0 [+ or -] 2,8 Closing clotting (s) 720,1 [+ or -] 21,9 Blood viscosity (unit of activity) 2,6 [+ or -] 0,5 The density of blood (units of activity) 0,02 [+ or -] 0,5 Plasma recalcification time (s) 120,0 [+ or -] 2,3 Plasma kaolin time (s) 89,3 [+ or -] 1,0 Kaolin cephalin time (s) 39,3 [+ or -] 1,2 ACT (s) for 6 min.
(2016) in a microplate format, containing: 10 [micro]L cephalin (contact-activator system) or thromboplastin (830 [micro]g [well-plate.sup.-1], factor tissue-activator system) + 30 [micro]L 0.02 M Tris HCl/PEG-buffer (pH 7.4) + 10 [micro]L SPs (C.
racemosa SPs (F I and F II) reduced dependently TG induced by cephalin or thromboplastin (data not shown).
racemosa fractions on TG by the coagulation reaction of the intrinsic pathway stimulated by cephalin (contact-activation) could perhaps be revealed as serpins-dependent thrombin inhibitors (Rodrigues et al., 2013; Wang et al., 2014) at differential inactivation patterns to modulate thrombosis in vitro (Nishino et al., 1999; Mourao et al., 2001) because antithrombin-dependent anticoagulants do not enhance TG in plasma and have ability to attenuate thrombin activity (Furugohri, Sugiyama, Morishima & Shibano, 2011).