THE EFFECT OF RT-PA ALONE AND IN COMBINATION WITH THROMBIN INHIBITORS IN A MODEL OF CEREBROVASCULAR THROMBOSIS IN THE RABBIT

Sustained accumulatin of 111 indium-labelled platelets is induced in the cerebral vascuiature or rabbits by bolus intracarotid (i. c. ) administration or thrombin (90 U/kg). Bolus i. c. injection of the fibrinolytic, recombiuant tissue plasminogen activator (rt-PA), 1 miu after thrombin,produced significant inbibition of the. platelet accumulation, albeit substantially less than that produced by 1 min Pretreatment. Hirulogm, PPACK and rt-PA alone had no direct effect on the hasalcirculating levels of 111 In-labedlled platelets in the pulmonary or cranial vasculature at the doses used.Hirulongm and PPACK did not enhance the ability or inrusion or a threshold dose of rt-PA to decrease an established cerebrovascular thromhosis, whereas Defibrotide plus rt-PA produced a significant reduction on accumulated plateiets. These results suggest that fiibrin deposition .plays the impor tant role in the induction and maintenance of the. sustained cerebral platelet accumulation induced bythrombin in this model and Defibrotide can .enhance the pro-fibrinolytic effect of rt-PA although thisProperty is not shared by direct thrombin inhibitors. The sustained platelet accumulation in the cranial vasculature in this model does not appear to be a result or continued generation or endogenousthrombin.

Thrombin Activatable Fibrinolysis Inhibitor in Preeclmapsia and Gestational Hypertension throughout the Gestation

To clarify the role of TAFI in hypertensive disorders in pregnancy, 22 subjects, including 10 with pre-eclampsia （PE） and 12 with gestational hypertension were examined for the levels of TAFI and thrombin-antithrombin （TAT） complex. Thirty normal pregnant women served as controls. ELISA was employed for the detection. The results showed that the TAFI antigen levels in normal pregnancy group, gestational hypertension group and PE group were （85.35±24.69）%, （99.65±18.27）%, （110.12±23.36）%; （97.06±21.40）%, （114.08±27.76）%, （125.49±24.70）%; （106.6±19.21）%, （129.2±25.07）%, （139.1±30.12）%, in the 1st, 2nd and 3rd trimester respectively. No significant differences were found between the normal pregnancy group and gestational hypertension group but significant difference existed between normal pregnancy group and PE group in each trimester （P〈0.05）. TAT complexes were significantly higher in patients with PE than that in controls （P〈0.05）, but no correlation was found between TAT and TAFI. It is concluded that TAFI may contributed to the impairment of fibrinolysis in the patients with PE and may serves as a sensitive indicator for PE, but it may not help in the diagnosis of the gestational hypertension.

Antithrombotic effects of bromophenol,an alga-derived thrombin inhibitor

Thrombin,the ultimate proteinase of the coagulation cascade,is an attractive target for the treatment of a variety of cardiovascular diseases.A bromophenol derivative named (+)-3-(2,3-dibromo-4,5-dihydroxy-phenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroiso-benzofuran 1,isolated from the brown alga Leathesia nana exhibited significant thrombin inhibitory activity.In this study,we investigated the inhibition of human thrombin in vitro with this bromophenol derivative,and its antithrombotic efficacy in vivo using the arteriovenous shunt model and the ferric chloride-induced arterial thrombosis model in rats.The results show that the bromophenol derivative is a potential inhibitor of thrombin (IC50=1.03 nmol/L).In antithrombotic experiments in vivo,the bromophenol derivative also shows good effect comparing with the control group.These data indicate that the bromophenol derivative is a potential drug for prophylaxis and the treatment of thrombotic diseases.

Partial Characterization of Thrombin Inhibitor(s) Derived from Salivary Glands of the Tick, <i>Hyalomma dromedarii</i>, and Related Anti-Cancer Potential

A long-term blood feeder, like the Hyalomma dromedarii tick, requires extended control over all hemostatic defense mechanisms generated by the host during feeding, including blood coagulation. To overcome this, ticks have evolved numerous molecules that target proteases in the blood coagulation cascade. New insights into the role of clotting factors in the development and progression of cancer have identified anticoagulant treatment as a potential therapeutic approach. In this context, the present work assessed the anticoagulation activities of crude and fractionated salivary gland extract (SGE) prepared from semi-fed H. dromedarii females. Additionally, the antitumor effects of the potent anti-thrombin fractions were determined against colon cancer (Caco-2) and normal skin (HFB4) cells. Crude SGE significantly prolonged clotting time in prothrombin time (PT), activated partial thromboplastin time (aPTT) and thrombin time (TT) assays and inhibited thrombin in FII-activity assay. Using anion-exchange chromatography, the fractions that strongly inhibited thrombin (3.A4 and 3.A5) were eluted. Both fractions prolonged the aPTT and TT clotting times and reduced the activity of FII significantly. The protein profiles of both fractions indicated the presence of a single polypeptide band of about 99 kDa. Regarding anti-cancer potential of the tested fractions, Caco-2 cells showed reduced viability with obvious morphological changes, induced apoptosis and a reduced level of vascular endothelial growth factor (VEGF). G2/M cell cycle arrest was observed only in 3.A5-treated cells. No cytotoxic effects were observed in HFB4 cells. These results demonstrated the potential of tick-derived anticoagulants, specifically thrombin inhibitors, as effective tools in colorectal cancer treatment. Further purification of the effector molecule(s) is required to fully characterize their structures and mechanisms of action.

A Review of an Unorthodox Argatroban Infusion Rate for Anticoagulation in a Patient Case

Argatroban is an intravenous DTI （direct synthetic thrombin inhibitor） that is not routinely used for anticoagulation; thus, expertise surrounding its use is very limited. Therefore, this case reviews an unusually high argatroban infusion rate, which was needed to prevent further emboli formation in a patient. In this case, a 61-year-old Caucasian male patient exhibited heparin resistance during an intraoperative vascular procedure as measured by activated clotting time and PTT （partial thromboplastin time）. The patient had multiple occlusions in his right lower extremities and underwent embolectomies of the right popliteal and posterior tibial arteries. The clinical pharmacist was consulted to manage the argatroban infusion once heparin was discontinued. The therapeutic window required a PTr of 1.5-3 times the patient baseline （35-75 s）. The patient was reported to be 89 kg with a baseline PTT of 24.7 s and INR （international normalized ratio） of 0.98. The starting dose ofargatroban was initiated by the pharmacist at 2 mcg/kg/min （10.7 mL/h） as the patient did not have hepatic failure or sepsis. The patient was maintained on argatroban in the therapeutic PTT window for more than 72 h; however, frequent and aggressive dose increases, to a final rate of 7.5 mcg/kg/min （40 mL/h）, were needed to maintain the therapeutic PTT level. From the case, the cause of heparin resistance still has not been determined despite a hematologic work-up; however, this patient required an unusually high infusion rate of argatroban to maintain a therapeutic PTT during the hospital course before being changed to an anticoagulation regimen for discharge.

Functional and binding studies of gallic acid showing platelet aggregation inhibitory effect as a thrombin inhibitor

Objective:This study was devoted to identifying natural thrombin inhibitors from traditional Chinese medicine(TCM)and evaluating its biological activity in vitro and binding characteristics.Methods:A combination strategy containing molecular docking,thrombin inhibition assay,surface plasmon resonance(SPR)and molecular dynamics simulation were applied to verify the study result.Results:Gallic acid was confirmed as a direct thrombin inhibitor with IC;of 9.07μmol/L and showed a significant inhibitory effect on thrombin induced platelet aggregation.SPR-based binding studies demonstrated that gallic acid interacted with thrombin with a KDvalue of 8.29μmol/L.Molecular dynamics and binding free energy analysis revealed that thrombin-gallic acid system attained equilibrium rapidly with very low fluctuations,the calculated binding free energies was-14.61 kcal/mol.Ala230,Glu232,Ser235,Gly258 and Gly260 were the main amino acid residues responsible for thrombin inhibition by gallic acid,providing a mechanistic basis for further optimization.Conclusion:This study proved that gallic acid is a direct thrombin inhibitor with platelet aggregation inhibitory effect,which could provide a basis for the follow-up research and development for novel thrombin inhibitors.