Coagulation and fibrinolytic activity in patients with acute cerebral infarction

Objective To measure the concentration of D-dimer (DD), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen (PLG) activity in plasma and cerebrospinal fluid in patients with acute cerebral infarction and to investigate their clinical significance.Methods The concentrations of D-dimer, t-PA, and PAI-1 in plasma and cerebrospinal fluid in patients were measured by enzyme-linked immunosorbent assay (ELISA). The PLG biological activity was detected using the chromophore method. The results were compared with those of the controls.Results The concentrations of D-dimer, t-PA and PAI-1 in cerebrospinal fluid and plasma in patients with acute cerebral infarction were much higher than those of normal subjects (P<0.01). Conversely, the level of PLG activity was significantly lower in the patients than in the controls (P<0.01).Conclusion Hypercoagulability and secondary hyperfibrinolysis exist in patients with acute cerebral infarction.

Fibrinolytic Evaluation of Compounds Isolated from a Marine Fungus Stachybotrys Iongispora FG216

As a part of our continuing work to discover bioactive leading molecules from marine microorganism, ethyl acetate fraction of organic extract of the train Stachybotrys longispora FG216 showed fibrinolytic activity in our primary screen. The bioassay-guided purification of the active fractions resulted in isolation of a new isoindolone, FGFC2 （1） （FGFC2, Fungi fibrinolytic compound 2）, together with two known compounds, LL-Zl272β （2） and ergosterol （3）. The structure of compound 1 was elucidated by the spectral analysis of 1D （^1H, ^13C） NMR, 2D （COSY, HSQC, and HMBC） and ESI-MS. Three compounds were evaluated for fibrinolytic activities in vitro. Compared to FGFC1 （EC50=47 μmol/L） as a reference drug, compound 1 and ergosterol （3） showed moderate fibrinolytic activities in vitro with EC50 values of 108.16 and 156.30 μmol/L, respectively. LL-Z127213 （2） had no fibrinolytic activity.

Preparation of pegylated lumbrokinase and an evaluation of its thrombolytic activity both in vitro and in vivo

Lumbrokinase(LK)is a group of serine proteases with strong fibrinolytic and thrombolytic activities.In clinical practice,LK can only be administered orally because of its antigenicity,iinmunogenicity and potential to produce anaphylactic reactions after injection.However,many useful drugs such as interferon,insulin,erythropoietin and interleukin have been modified with polyethylene glycol(PEG)to prepare injectable formulations.In this study,LK was modified with methoxy PEG succinimidyl carbonate(mPEG-SC)with molecular weights of 5000,10,000 and 20,000 and the pegylatcd products were isolated and purified using the Akta protein purification system.The extent of pegylation was detennined by HPLC.Fibrinolytic activities of pegylatcd and unmodified LK were measured both in vitro against urokinase on fibrin plates and in vivo using a mouse carageenan black tail model.Optimal pegylation was obtainal using mPEG-SC_(5000) in a buffer pH 8.0 with a reaction time of 5 h,reaction temperature of 0℃ and LK:mPEG-SC molar ratio of 1:25.The results show that mPEG modified LK has strong fibrinolytic and thrombolytic activities both in vitro and in vivo.It is suggested that the pegylatcd LK is a promising injectable thrombolytic agent for the treatment of thrombotic diseases in clinical practice.

Identification and Fibrinolytic Evaluation of an Isoindolone Derivative Isolated from a Rare Marine Fungus Stachybotrys Iongispora FG216

An isoindolone derivative, Fungi fibrinolytic compound （R）-2,5-bis（（2R,3R）-2-（（E）-4,8-dimethylnona-3,7-dien- 1-y1）-3,5-dihydroxy-2-methy--7-oxo-3,4,7,9-tetrahydr pyrano[2,3-e]isoindol-8（2H）-yl）pentanoic acid （FGFC1, Fungi fibrinolytic compound 1）, was isolated from a rare marine microorganism strain Stachybotrys longispora FG216. The structure of FGFC1 was elucidated by 1H NMR, 13C NMR, IR, and MS data; moreover, it was also evaluated for fibrinolytic activity in vitro and in vivo. The results showed that 0.1--0.4 mmol/L of FGFC1 could stimulate generation of plasmin activity （increased by 2.05--11.44 folds） by measuring Glu-plasminogen and Lys-plasminogen activation in vitro. The experiment of fluorescein isothiocyanate （FITC）-fibrinogen degradation indicated that the effect of FGFCI on fibrinolytic activity was mediated by plasminogen and scuPA. In addition, FGFC 1 （10 mg/kg） could dissolve most of pulmonary thrombus of Wistar rat in vivo. It is possible that FGFC 1 is a potential thrombolytic agent in the future.