The effect of hirudin on antagonisting thrombin induced apoptosis of human microvascular endothelial cells

Abstract Purpose: To investigate whether hirudin exerts its antithrombin action to decrease the ratio of Human Microvascular Endothelial Cells (HMVECs) apoptosis. Methods: Human microvascular endothelial cells (HMVECs) cultured in the third and fifth generations were used. HMVECs were divided into normal group, thrombin group (T group), natrual hirudin group (H group), thrombin + natrual hirudin group (T + H group), AG490 group, thrombin + AG490 group (T + AG490 group), natrual hirudin + AG490 group (H + AG490 group), thrombin + natural hirudin + AG490 (T + H + AG490 group).Apart from the normal group, the other groups were exposed to the relevant drugs for 24 hours.HMVEC apoptosis was assessed by flow cytometric and double Immunofluorescence of phosphorylation of JAK (P-JAK2) and TUNEL assay. Results: Compared with the normal group, in thrombin group the HMVECs apoptosis rate were significantly increased (P<0.05).The results indicated that the index of apoptosis and the apoptosis rate were improved in cultures treated by natural hirudin (T + H group), relative to cultures with thrombin only (T group). We found that the index of apoptosis and the apoptosis rate in the AG490 + thrombin group were higher than that in the hirudin + thrombin group (P<0.05). Double Immunofluorescence of p-JAK2 and TUNEL assays showed that cells were double positive for P-JAK2 uptake and TUNEL detection liquid binding. Conclusion: The natural hirudin and JAK2/STATs signal inhibitor AG490 could block the effects of thrombin. Natural hirudin could attenuate HMVECs apoptosis via antagonizing thrombin and it is suggested that this effect may occur by blocking the JAK2/STATs signaling pathway and this signaling pathways appears to be not the only pathway.

Bivalirudin: a new promising direct antithrombin.

The deadliest manifestations of ischemic heart disease are initiated and propagated by intra-coronary thrombin generation. Thrombin is resistant to inactivation by heparin when it is bound to fibrin, fibrin degradation products or subendothelial collagen. Recognition of these limitations has led to development of a new class of antithrombin agents which directly target the active sites on the surface of thrombin molecule and are therefore designated as direct antithrombins. These agents do not need mediation of antithrombin III for their action and are not inhibited by platelet factor 4. This report focuses on bivalirudin, a new agent of promising impact on both interventional as well as non-interventional cardiology. It is a short acting anticoagulant which bivalently and directly inhibits thrombin (coagulation factor II). It binds the active (catalytic) site and the fibrinogen-binding site (exosite I). This provides high affinity and specificity for thrombin. Slow cleavage at the Arg3-Pro4 bond results in recovery of thrombin activity after discontinuation of bivalirudin. Bivalirudin inhibits both protease activated receptor 1 and 4 (PAR 1 and PAR 4) thereby effectively inhibiting acute thrombin mediated platelet aggregation. Clinical efficacy has been assessed and proved in over 20 published patient series focussing on patients with acute coronary syndrome with or without myocardial infarction, patients undergoing percutaneous coronary interventions, patients receiving various adjunctive anti-platelet medications, patients with heparin induced thrombocytopenia or patients undergoing cardiac surgery. In contrast to the well established unfractionated heparin, bivalirudin lacks the risk of heparin induced thrombocytopenia. It shows a tendency to lower bleeding risks without reduction of efficacy when compared with the two-pronged treatment with unfractionated heparin and glycoprotein IIb/IIIa inhibitors.