Study On Pharmacological Properties Of The Thrombolytic Candidate FGFC1 In Dissolving Cerebral Thrombus

Cerebrovascular embolism can be recanalized by thrombolytic therapy,the use of thrombolytic drugs to stimulate the body’s fibrinolytic activity to enhance the dissolution of cerebral blood clots.The principle of commonly used thrombolytic drugs is to catalyze the conversion of fibrinogen into fibrinolytic enzymes,which dissolve fibrin and lead to the loss of stable structure of cerebral thrombus and thus be dissolved,representing drugs such as urokinase and alteplase,etc.These are thrombolytic biomolecular drugs,which have good cerebrovascular recanalization after thrombolysis but have the risk of inducing cerebral hemorrhage.Small-molecule thrombolytic drugs can overcome or abate the side effects of cerebral hemorrhage caused by thrombolytic biomolecule drugs,which act on plasminogen（PLG）,plasminogen activator inhibitor-1（PAI-1）,thrombin-activatable fibrinolysis inhibitor（TAFI）,plasminogen activator（PA）,and other potential thrombolytic targets to convert PLG into Plasmin（PL）.The smoother fibrinolytic response induced by small molecule thrombolytic drugs or lead compounds will reduce the risk of cerebral hemorrhage,etc.The small molecule thrombolytic lead compound marine bisindole compound Fungi fibrinolytic compound 1（FGFC1）will be further investigated for its fibrinolytic properties and pharmacological properties in animal models of cerebral thrombosis.Cerebral thrombosis was induced in rats by jugular vein injection of fluorescein isothiocyanate-fibrin（FITC-fibrin）at a dose of 1 m L/kg.Low-field MRI thrombus imaging and tissue sectioning were performed to evaluate the thrombus formation in the rats after induction.The results indicated that FITC-fibrin induced cerebral thrombosis in rats,as evident vascular embolism was observed in paraffin sections and fluorescent bright spots were evident in frozen sections,which were still present at 24 h.After the animal model of cerebral thrombosis was successfully established,FGFC1 was injected into the jugular vein at three doses of 2.5 mg/kg,5 mg/kg,and10 mg/kg for high-dose treatment（HT）,medium-dose treatment（MT）,and low-dose treatment（LT）groups,respectively;the low-field MRI of rat brain tissue at 24 h was50-100,50-150,and 100-200 density units for HT,MT,LT groups,respectively.The results of HE staining of paraffin sections of rat brain tissue at 24 h in the HT,MT,LT groups,showed that the walls of cerebral blood vessels were thin and there was no obvious embolism,and there were no obvious fluorescent bright spots in frozen tissue sections,similar to the results of control and positive control rats.The plasma fluorescence intensity of rats with cerebral thrombus was 1503.33-1519.00.The plasma fluorescence intensity of rats in HT,MT,LT groups decreased to 511.33,210.67 and 100.33 within 4 h,respectively.The results indicated that FITC-fibrin was degraded and excreted after FGFC1 injection.The levels of plasma D-dimer（D-D）and fibrin degradation products（FDP）were detected in rats,and the levels of D-D rose to about 4 ng/m L after the formation of cerebral thrombosis model in rats,and the levels of D-D in HT,MT,LT groups rose rapidly to 4.5-6 ng/m L after 2 h of administration.After the formation of cerebral thrombosis model in rats,the FDP level rose to about 450 ng/m L,and the HT,MT,LT groups had a rapid rise to500-600 ng/m L after 2 h of administration,and then fell back to 350-400 ng/m L at 4 h.The results of the changes in D-D levels and FDP levels indicated that FGFC1promoted fibrin degradation and accelerated the lysis of cerebral thrombus;the activity of the fibrinolytic system in rats was measured by detecting plasma PLG,tissue plasminogen activator（t-PA）,PAI-1,and TAFI levels in rat plasma after the formation of rat cerebral thrombosis model,and PLG,t-PA,PAI-1,and TAFI levels in rat plasma after the formation of rat cerebral thrombosis model were around 100IU/L,5 ng/m L,38 ng/m L and 32 ng/m L,and the levels of each index changed to around 80 IU/L,25 ng/m L,32 ng/m L and 28 ng/m L in the HT,MT,LT groups after 2h of administration,and returned to levels similar to those of the control and positive control groups after 4 h.The results indicate that FGFC1 stimulates activation of the fibrinolytic system in the body,and its potential targets are PLG,PA,PAI-1 and TAFI receptors;the activity of the coagulation system in rats was measured by detecting plasma fibrinogen（FIB）,prothrombin time（PT）,thrombin time（TT）and activated partial thromboplastin time（APTT）levels were measured in rats after the formation of cerebral thrombosis model,and the plasma FIB,PT,TT and APTT levels were about 200 ng/m L,13.5 s,9.3 s and 23.2 s in HT,MT,LT groups.The levels of each index changed to about 150 ng/m L,17.0 s,11.6 s and 27.8 s after 2 h of administration in the HT,MT,LT groups,and returned to levels similar to those of the control and positive control groups after 4 h.The results indicate that FGFC1inhibits the activity of the coagulation system and prevents the aggravation of cerebral thrombosis;the levels of Thromboxane B₂（TXB₂）in rats were measured,after the formation of rat cerebral thrombosis model,TXB₂level in rat plasma rose to about1200 ng/m L,and TXB₂levels in HT,MT,LT groups decreased to 1000-1100 ng/m L after 2 h of administration,and fell back to 900-1000 ng/m L after 4 h.The results were similar to those of positive control group,but still differed from those of control group.It indicates that FGFC1 restores TXB₂to normal level faster and prostacyclin and thromboxane A₂to balance faster,impeding cerebral thrombosis.The study showed that FGFC1 acts on potential thrombolytic targets such as PLG,PAI-1,TAFI and PA,accelerates fibrinogen activation and produces fibrinolytic pharmacological effects on rat cerebral thrombosis,and the results provide a basis for clinical trials of FGFC1.