Collagen And Thrombin, Two Of The Crucial Components Of Thrombus Formation
Kötet címe (évfolyam száma)
Haemostasis is the process that maintains the integrity of a closed, circulatory system after vascular damage. When the vessel wall is breached or the endothelium is disrupted, collagen and tissue factor become exposed to the flowing blood, thereby initiating formation of a thrombus. Exposed collagen triggers the accumulation and activation of platelets, which rapidly adhere to the collagen, continued by the activation and aggregation that amplified by the local generation of thrombin. These events occur concomitantly. Collagen is one of the most thrombotic surfaces on which thrombus is growing when endothelium is damaged. In vitro thrombosis models are used to study this process at static and different shear conditions. In order to optimize the collagen surface one of our aims was to find optimal condition to prepare collagen surfaces for VWF and platelet binding. The effects of pH, salt and ligand concentration and binding time were tested when human collagen type I and III matrices were prepared by adsorption. Surface-bound collagen and collagen-bound VWF measured by specific antibodies. Platelet adhesion was tested under flow conditions at arteria shear rate. Matrices and platelets were visualized by atomic force and scanning electron microscope. Our experiments provide evidences that collagen coating of surfaces for VWF binding and platelet adhesion studies is very variable from acid solution; neutralizing the acid and adding NaCl in physiological concentration facilitates formation of collagen fibril molecules in solution resulting in efficient coating of human type-I and type III collagens; and both bind normal VWF equally well. Thrombin is a coagulation protein that has many effects in the coagulation cascade, converts soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation and anticoagulant related reactions. Therefore thrombin is usually considered as the suitable target for anti-coagulants and antithrombotics. I studied the antithrombotic effect of aptamers (single-stranded DNA oligonucleotides) a new group of thrombin inhibitors. Bock et al. selected aptamers with a consensus sequence of 15 nucleotides (C15-mer). Our other aim was to examine different anti hemostatic/thrombotic activities of 4-thio-deoxyuridylate-containing analogs and compare its effect to C15-mer. Three different analogs (UC15-mer, aUC15-mer, vUC15-mer) of the original thrombin-inhibiting sequence, in which some of the thymidylate residues were replaced by 4-thio-deoxyuridylates. The inhibitory effect of modified aptamers was tested on thrombin-catalyzed fibrin clot formation and fibrinopeptide A release from fibrinogen, thrombin-induced platelet aggregation/secretion, and the formation of thrombus on human collagen type III, thrombin-treated fibrinogen surfaces or subendothelial matrix of human microvascular endothelial cells at shear condition. The analog with the sequence GG(s4dU)TGG(s4dU)G(s4dU)GGT(s4dU)GG (UC15-mer) showed a 2-3 fold increase in all of the above functions and our experimental approach allowed us to better understand the regulatory mechanism of aptamers on thrombus formation at shear conditions.