Antithrombotic properties of Ixolaris, a potent inhibitor of the extrinsic pathway of the coagulation cascade.

Ixolaris is a two-Kunitz tick salivary gland protein identified in Ixodes scapularis that presents extensive sequence homology to TFPI. It binds to FXa or FX as scaffolds and inhibits tissue factor/FVIIa complex (extrinsic Xnase). Differently from TFPI, ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite, which may also results in decreased FXa activity into the prothrombinase complex. In this report, we show that recombinant (125)I-ixolaris interacts with rat and human FX in plasma and prolongs the prothrombin time (PT) and activated partial thromboplastin time (aPTT) in vitro. We have also investigated the effects of ixolaris in vivo, using a venous thrombosis model. Subcutaneous (s.c.) or intravenous (i.v.) administration of ixolaris in rats caused a dose-dependent reduction in thrombus formation, with complete inhibition attained at 20 microg/kg and 10 microg/kg, respectively. Antithrombotic effects were observed 3 h after s.c. administration of ixolaris and lasted for 24 h thereafter. Ex vivo experiments showed that ixolaris (up to 100 microg/kg) did not affect the aPTT, while the PT was increased by approximately 0.4-fold at the highest ixolaris concentration. Remarkably, effective antithrombotic doses of ixolaris (20 microg/kg) was not associated with bleeding which was significant only at higher doses of the anticoagulant (40 microg/kg). Our experiments demonstrate that ixolaris is an effective and possibly safe antithrombotic agent in vivo.

Dermatan sulfate is the predominant antithrombotic glycosaminoglycan in vessel walls: implications for a possible physiological function of heparin cofactor II.

The role of different glycosaminoglycan species from the vessel walls as physiological antithrombotic agents remains controversial. To further investigate this aspect we extracted glycosaminoglycans from human thoracic aorta and saphenous vein. The different species were highly purified and their anticoagulant and antithrombotic activities tested by in vitro and in vivo assays. We observed that dermatan sulfate is the major anticoagulant and antithrombotic among the vessel wall glycosaminoglycans while the bulk of heparan sulfate is a poorly sulfated glycosaminoglycan, devoid of anticoagulant and antithrombotic activities. Minor amounts of particular a heparan sulfate (< 5% of the total arterial glycosaminoglycans) with high anticoagulant activity were also observed, as assessed by its retention on an antithrombin-affinity column. Possibly, this anticoagulant heparan sulfate originates from the endothelial cells and may exert a significant physiological role due to its location in the interface between the vessel wall and the blood. In view of these results we discuss a possible balance between the two glycosaminoglycan-dependent anticoagulant pathways present in the vascular wall. One is based on antithrombin activation by the heparan sulfate expressed by the endothelial cells. The other, which may assume special relevance after vascular endothelial injury, is based on heparin cofactor II activation by the dermatan sulfate proteoglycans synthesized by cells from the subendothelial layer.