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We describe the common structural basis for the anticoagulant action of the many different heparins available to the clinician. From different types of heparin we prepared fractions of virtually single molecular weight. We determined the molar concentration of material (HAM) containing the antithrombin (AT) binding pentasaccharide (A-domain), the specific catalytic activity in thrombin- and factor Xa inactivation and the capacity to inhibit thrombin generation (TG). We also calculated the molar concentration of A-domain with 12 sugar units at its non-reducing end, i.e. the structure that carries anti-thrombin activity (Choay- or C-domain). The anti-thrombin activity and the effects on TG are determined by the concentration of C-domain and independent of the source material or Mr. High Mr fractions (> 15,000) are less active, probably through interaction with non-AT plasma proteins. Anti-factor Xa activity is not indicative of anticoagulant potency but is a sensitive indicator of Mr and therefore predicts favourable pharmacokinetic properties: long half-life in the circulation and high bioavailability.

The main threat from a beginning thrombus is that it tends to grow, and hence become occlusive and/or embolise. Although the progressive nature of thrombi has been recognised since a long time, the mechanisms behind thrombus growth remain only partially resolved. In order to investigate in what ways thrombi can themselves become foci of further thrombin -and hence fibrin-formation, we studied the effect of fibrin clots on thrombin generation in platelet poor--and platelet rich plasma (PPP and PRP). The thrombin always adsorbed on a natural fibrin clot is not inactivated by plasmatic antithrombins and could be shown to retain its ability to enhance further thrombin formation by activation of clotting factors V and VIII as well as of blood platelets. To our surprise, fibrin clots without any active thrombin adsorbed, because they were obtained by a snake-venom enzyme or because thrombin had been inhibited, retained their capacity to activate blood platelets and make them procoagulant. The activation could be shown to be due to a rearrangement of cell-membrane phospholipids, by which the procoagulant species (phosphatidyl serine and phosphatidyl ethanolamine) became available at the outer cell surface. The platelet membrane receptor involved could be recognised as glycoprotein Ib, interacting with fibrin through the plasma protein von Willebrand factor (vWf). In fact it appeared that vWf is indispensable for the generation of thrombin in PRP, with or without added clot. This assigns a new and hitherto unknown role to vWf. Our results also show that fibrin is far from being the inert end-product of coagulation but is a potent activator of blood platelets and by this action may foster thrombin generation and hence further fibrin production. We surmise this mechanism to be instrumental in the progression of thrombotic processes.

Thrombosis, either venous and arterial, is the most important cause of death in our society. Thrombin formation is a pivotal step in the pathogenesis of both forms of thrombosis. Drugs like heparin and vitamin K antagonists act because they inhibit thrombin formation. Better drugs than conventional heparin and oral anticoagulants are however urgently needed. They can be found on basis of a better understanding of thrombin formation. The mechanism of thrombin formation is explained. The mode in which conventional heparin and the newer, low molecular weight heparins inhibit this process is discussed. It is shown that, contrary to current belief, low molecular weight heparins inhibit primarily via their action on thrombin and that the inhibition of factor Xa is of less importance. Thrombin inhibition has a direct action but also acts indirectly via its influence on thrombin mediated feedback loops. It is argued that rather than anti-factor Xa activity or prolongation of some type of coagulation time, the time-concentration integral of thrombin (thrombin potential) in clotting plasma should be used as a yardstick for the efficiency of a thrombin inhibiting drug.