Mechanisms for Inhibition of the Generation of Thrombin Activity by Sulfated Polysaccharides

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Abstract
Three mechanisms by which sulfated polysaccharides act as anticoagulants and possibly as antithrombotic agents have been described. These are the two heparin cofactor-dependent mechanisms involving the catalysis of the inhibition of various proteases of coagulation by either antithrombin III or heparin cofactor II. The third is a heparin cofactor-independent mechanism involving the inhibition of formation of prothrombinase and tenase complexes. Four sulfated polysaccharides previously shown to have anticoagulant and antithrombotic effects were assessed to determine which of the three mechanisms operate in the expression of their anticoagulant effects. To do this, [125I]prothrombin was added to undiluted human plasma, and the inhibition of [125I]prothrombin activation, or the catalysis of the formation of thrombin-inhibitor complexes was determined in plasma containing one of the four sulfated polysaccharides. Prothrombin activation was demonstrated by the formation of [125I]prothrombin fragment 1.2 and [125I]thrombin. The effect of the thrombin-specific inhibitor, D-Phe-L-Pro-L-ArgCH2Cl (PPACK), on prothrombin activation was also investigated to determine the role of thrombin-dependent feedback reactions on efficient prothrombin activation. Use of PPACK with sulfated polysaccharides also facilitated estimation of the role of the heparin cofactor-independent effects of sulfated polysaccharides on prothrombin activation. Three concentrations of each of the sulfated polysaccharides were used: 0.66, 6.6, and 66 micrograms/ml of plasma. PPACK (1.0 X 10(-6)M) completely inhibited both intrinsic and extrinsic prothrombin activation. The inhibition of prothrombin activation caused by PPACK was abolished when thrombin was added to the plasma before PPACK. These observations indicate that the presence of trace thrombin activity is critical for efficient prothrombin activation by both the intrinsic and extrinsic pathways. All three concentrations of standard heparin completely inhibited the intrinsic activation of prothrombin. This inhibition was only partially abolished when thrombin was added to the plasma before heparin, indicating that heparin inhibits prothrombin activation both by catalyzing the inhibition of thrombin activity and by a heparin cofactor-independent mechanism. Heparan sulfate did not inhibit intrinsic prothrombin activation but catalyzed the inhibition of the thrombin generated by the formation of thrombin-antithrombin III complex. Dematan sulfate inhibited intrinsic prothrombin activation only at the highest concentration. At the two lower concentrations, dermatan sulfate catalyzed formation of thrombin-heparin cofactor II.(ABSTRACT TRUNCATED AT 400 WORDS)