Activated protein C inhibits thrombus formation in a system with flowing blood M. L OZANO, G. E SCOLAR , H. P. S CHWARZ ,* R. HERNA ´ NDEZ , J. B OZZO AND A. O RDINAS Department of Haemotherapy and Haemostasis, Hospital Clı ´nic i Provincial, University of Barcelona, Barcelona, Spain, and *Immuno AG, Vienna, Austria Received 13 February 1996; accepted for publication 21 June 1996 Summary. We studied the effect of increasing concentrations of protein C (PC) and activated protein C (APC) on haemostasis in an in vitro thrombosis model. Blood from healthy donors was anticoagulated with citrate–phosphate– dextrose (final citrate concentration 19 mM) or a low molecular weight heparin (LMWH, 20 IU/ml). Enzymatically denuded rabbit aorta segments were exposed to flowing blood for 10 min in an annular perfusion chamber. PC and APC were added to the perfusate immediately prior to exposure. In citrated blood at a shear rate of 800/s, PC and APC induced a statistically significant decrease in platelet deposit at 16 g/ml and 32 g/ml. In perfusions performed with blood anticoagulated with LMWH, there was no effect on platelet deposition at 16 and 32 g/ml either at shear rates of 300/s or 800/s. Addition of PC showed no effect on fibrin deposition at a shear rate of 300/s; in contrast, a non- statistically significant 40% reduction was seen at a shear rate of 800/s, compared to controls. Addition of APC caused a 100% reduction in fibrin formation at 16 and 32 g/ml at both shear rates studied. PC and APC inhibited platelet deposition on the exposed subendothelial surface, in a dose- dependent manner. Effects of PC and APC on platelet function might be mediated through inhibition of thrombin generation at the platelet microenvironment. Keywords: activated protein C, thrombosis, platelet adhesion, perfusion, low molecular weight heparin. Protein C (PC) is a vitamin K dependent glycoprotein synthesized in the liver, which plays an important role in haemostasis as a thrombin inhibitor (Esmon, 1989). The physiological significance of PC has been demonstrated by the clinical observation of cases of severe thrombotic episodes in newborn children with homozygous PC deficiency, which can be fatal if not treated with an adequate substitutive treatment consisting of PC concentrates (Dreyfus et al, 1991). PC circulates in plasma in an inactive zymogen form. The formation of thrombin triggers the PC-activation system. Firstly, thrombin binds to thrombomodulin, a protein anchored to the surface of endothelial cells (Dittman & Majerus, 1990). This complex starts the rapid activation of PC. The activated protein C (APC) then binds to protein S at the surface of platelets or endothelial cells (Fulcher et al, 1984). In turn, this complex catalyses the proteolytic inactivation of factors Va and VIIIa (Kalafatis & Mann, 1993; Shen & Dahlback, 1994). Once the PC has been activated, the anticoagulant action of the APC is neutralized by the PC inhibitor or 1 -antitrypsin. APC inhibition is very slow in vivo, with an average life-span of 15–30 min (Heeb & Griffin, 1988). Local thrombin formation might be important, not only in coagulation but also in the regulation of platelet response (Fenton et al, 1991). Although the effects of PC on coagulation are well established, possible action on platelets has not been investigated in depth. The purpose of the present study was to assess, separately, the effects of PC and APC on platelet deposition, and on fibrin formation on subendothelium exposed to human flowing blood. For this purpose, citrate and a low molecular weight heparin (LMWH) were used as anticoagulant; citrate in the perfusates prevents fibrin formation, and, at the concentra- tions used, LMWH allows fibrin formation on perfused subendothelium when the coagulation cascade is triggered (Zwaginga et al, 1990; Lozano et al, 1994). To examine the possible mechanisms involved in the effects on platelets during perfusions, additional flow cytometry and coagula- tion studies were performed on the perfusates. British Journal of Haematology , 1996, 95, 179–183 179 1996 Blackwell Science Ltd Correspondence: Dr Miguel Lozano, Hospital Clı ´nic i Provincial, Department of Haemotherapy and Haemostasis, Villarroel 170, 08036 Barcelona, Spain.