Antithrombotic Effects of Activated Protein C and Heparins in Deep Arterial Injury

Abstract: The investigations presented in this thesis have evaluated different strategies to prevent thrombosis in an experimental model of deep arterial injury in rats. Unfractionated heparin (UFH) is often used to prevent thrombosis in microvascular surgery and in other vascular interventions, but a major drawback of UFH is increased bleeding. In the first paper the low-molecular-weight heparin (LMWH) dalteparin prevented arterial thrombosis as effectively as UFH but with less bleeding. In the second paper increasing doses of plasma purified bovine activated protein C (bAPC), with or without co-administration of bovine protein S (bPS) demonstrated the in vivo significance of PS as a cofactor to APC, and that potent antithrombotic effect could be achieved by low doses of bAPC combined with bPS, without producing hemorrhagic side effects. Recent in vitro studies have shown enhanced anticoagulant effect of human activated protein C (hAPC) by selective mutagenesis of the Gla-domain. This mutant was combined with mutations in the protease domain of hAPC, in loop 148, that was changed to the four residues shorter corresponding loop in bAPC leading to even more enhanced anticoagulant activity due to higher catalytic activity against its natural substrates FVa and FVIIIa. In vitro clotting experiments in rat plasma showed distinct dose dependent anticoagulant effect, especially with the combined mutant in contrast to wild type (WT) hAPC that only slightly prolonged the clotting time. These results generated the hypothesis that the mutants given without hPS could be antithrombotic in our rat model. In the third paper, none of the APC variants had significant antithrombotic effect even though the ex vivo clotting times were clearly prolonged. In the fourth paper further in vitro experiments showed additional prolongation of the clotting time in rat plasma when either of the two mutants was combined with hPS, compared to almost no effect with WT-hAPC. This indicated that the mutants combined with hPS could be antithrombotic, but even though increased patency rates were noted with the combined mutant, there was no significant antithrombotic effect. These results stand in contrast to the strong antithrombotic effect demonstrated with bAPC combined with bPS. The reason for this species discrepancy is unknown.