Background: The Clauss assay is widely used to quantify blood fibrinogen levels in clinical laboratories. However, by relying on thrombin as the main reagent, the Clauss assay is susceptible to interference from thrombin inhibitors, such as heparin or direct thrombin inhibitors. Here, we developed an innovative fibrinogen assay utilizing both recombinant batroxobin (rBat) and carboxymethyl chitosan (CMCS). Methods: Various biopolymers were tested to identify a suitable candidate that could enhance rBat-induced fibrin clot formation. Chromogenic substrate hydrolysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that CMCS potentiated rBat activity. Consequently, we formulated a novel fibrinogen assay reagent, ANYFIB.C, comprising rBat and CMCS. We compared ANYFIB.C fibrinogen with an established reagent (HemosIL fibrinogen-C) with 96 clinical samples using an ACL-TOP 700 coagulation analyzer. We also evaluated the interfering effects of thrombin inhibitors on fibrinogen measurements. Results: CMCS significantly enhanced the enzymatic activity of rBat and dose-dependently reduced plasma clotting times. ANYFIB.C fibrinogen levels were comparable with those of HemosIL fibrinogen-C, with the 95% confidence intervals of the Passing–Bablok regres-sion intercept and slope being −7.4797 to 6.0185 and 0.9581 to 1.0116, respectively. Nosignificant interference was observed with heparin concentrations up to 10 U/mL or dabigatran concentrations up to 600 µg/L in the ANYFIB.C fibrinogen assays. In contrast, the HemosIL fibrinogen-C reagent demonstrated inhibitory interference at dabigatran concentrations as low as 150 µg/L. Conclusions Our results suggest that ANYFIB.C (a mixture of CMCS and rBat) can be used to measure blood fibrinogen levels effectively and protect from thrombin inhibitor interference.

Antiplatelet aggregation, anticoagulant and lipid-lowering drugs are clinically widely used for secondary preventive purpose in the cardiovascular patients, but there is no primary preventive agents to prevent these diseases. With the aim of developing effective primary agents for cardiovascular diseases, we tried to formulate an optimized mixture of natural plants extract containing Theae sinensis, Camelliae sinensis, Vitis vinifera, Gingko folium and curcuminoids from Curcuma longa and to evaluate its anti-thrombotic and anti-hypercholesterolemic effects in vivo. The inhibitory effect of curcuminoids on vascular smooth muscle cell proliferation and migration were also investigated in vitro. in the animal experiments treated with hyperlipidemic diet, oral treatment of curcuminoids and natural plants extracts mixture (100 mg/kg) into male Sprague Dawley rats for 7 week simultaneously inhibited platelet aggregation as well as improved lipid profile in the blood. Compared to control group, both of curcuminoids-treated and mixture-treated groups revealed significantly decrease of total cholesterol (24.4%, 28.6%), free cholesterol (25.1%, 24.0%), cholesterol ester (14.6%, 29.0%), LDL-cholesterol (27.0%, 32.0%) and triglyceride (15.0%, 31.0%), respectively. However, both groups showed increase of HDL-cholesterol (46.6% and 51.5%) . In particular, atherogenic index of curcuminoids and mixture treatment group was significantly decreased to 47.0% and 56.0%, respectively. Furthermore, oral treatment of curcuminoids and mixture significantly inhibited collagen-induced platelet aggregation (21.1% and 29.1%, respectively), compared to control group. The anti-thrombotic values of mixture was almost similar to that of aspirin treatment (100 mg/kg) group. These results suggest that the oral treatment of curcuminoids-based natural plant extract mixture improved cardiovascular conditions in hyperlipidemic rats.
Animal Experimentation ; Animals ; Aspirin ; Camellia sinensis ; Cardiovascular Diseases ; Cardiovascular System* ; Cell Proliferation ; Cholesterol ; Curcuma ; Diet ; Ginkgo biloba ; Humans ; Male ; Muscle, Smooth, Vascular ; Plants ; Platelet Aggregation ; Rats* ; Rats, Sprague-Dawley ; Triglycerides ; Vitis