News and Publications

The Antithrombotic Protein C Pathway

J.H. Griffin, B.N. Bouma,* H. Deguchi, J.A. Fernández, A.J. Gale, T.M. Hackeng, M.J. Heeb, K. Kojima, Y. Kojima, R. Macko,** M. Manco-Johnson,*** Y. Montejano, J. Petäjä,**** C. Rintelen, X. Xu, S. Yegneswaran

* University Hospital Utrecht, Utrecht, the Netherlands
** University of Maryland, Baltimore, MD
*** University of Colorado Health Sciences Center, Denver, CO
**** University of Helsinki, Helsinki, Finland

Thrombosis is caused by a failure of normal physiologic mechanisms that control coagulation. In blood vessels, a triad normally regulates blood coagulation: the endothelium, blood flow, and blood constituents, including cells and plasma components. Coagulation pathways, fibrinolysis pathways, and anticoagulant mechanisms must act in concert to achieve the normal hemostatic balance. Our research studies focus on the coagulation and anticoagulant protein C pathways and potential molecular defects in these pathways associated with increased risks of thrombosis.

Activated protein C (APC) is generated in vivo from its zymogen form by thrombin bound to thrombomodulin on endothelial surfaces. Like the famous French red wine paradox, there is a "thrombin paradox." Because infusions of low levels of thrombin generate APC, but high levels cause extensive coagulation, too much thrombin, like too much red wine, is harmful to blood vessels, whereas a little bit of either is much better than none. Using our assay for APC in circulating blood, which can detect 40 pg of APC per milliliter, we showed that in healthy subjects, the concentration of APC in circulating plasma is 2.2 ng/ml and that an inverse relationship exists between fibrinopeptide A, a direct marker of thrombin activity, and circulating APC. These findings support the hypothesis that APC in basal physiologic states downregulates thrombin levels.

To test the hypothesis that cerebral ischemia causes generation of APC, we measured APC in blood samples collected simultaneously from the internal jugular vein and the radial artery before and during cross-clamping of the carotid artery and upon deocclusion in 8 awake patients undergoing routine endarterectomy. We found that the concentration of APC in the blood in the internal jugular vein increased by 28% exclusively during carotid occlusion and then decreased to baseline levels with deocclusion. No significant changes occurred in the concentrations of APC in blood in the radial artery. These findings indicate that the change in APC was localized to the cerebral circulation during ischemia and suggest a protective role for endogenous generation of APC during cerebrovascular occlusion.

To test the hypothesis that reperfusion of blood through the heart and lungs after cardiopulmonary bypass surgery generates APC, we measured APC in blood samples collected from the coronary sinus and the aorta at various times before, during, and after surgery. We found that APC levels increased linearly with time of reperfusion. These results show that APC is generated during reperfusion of ischemic vascular beds and suggest a protective role for generation of APC. We also divided the bypass patients into 2 groups on the basis of the level of APC observed at 10 minutes after reperfusion and compared cardiac function observed at 24 hours after surgery. We found that the group with higher APC generation had significantly better cardiac function and pulmonary artery pressure than did the group with lower APC generation. These results in patients undergoing carotid endarterectomy or bypass surgery suggest that treatment with APC might protect ischemic cerebral, coronary, or pulmonary vascular beds.

At the final stage of blood coagulation, thrombin is formed from prothrombin by the action of a complex containing factors Xa and Va. Inactivation of factor Va by APC is associated with cleavages at Arg306, Arg506, and Arg679; the cleavage at Arg306 causes the major loss of activity. To study functional roles of the Arg306 region, we synthesized overlapping 15mer peptides representing the sequence of factor Va residues 271--345 and screened the peptides for anticoagulant activities.

The peptide containing residues 311--325 noncompetitively inhibited prothrombin activation by factor Xa, but only in the presence of factor Va. Fluorescence studies showed that this peptide bound to fluorescence-labeled Glu--Gly--Arg--factor Xa in solution with a K_d of 70 µM. Diisopropylphosphoryl--factor Xa and factor Xa, but not factor VII/VIIa or prothrombin, bound to immobilized residues 311--325 with relatively high affinity. These results support the hypothesis that residues 311-325, which are positioned between the A1 and A2 domains of factor Va and most likely are exposed to solvent, contribute to the binding of factor Xa by factor Va. On the basis of this hypothesis, we suggest that cleavage by APC at Arg306 in factor Va not only severs the covalent connection between the A1 and A2 domains but also disrupts the environment and structure of residues 311--325, thereby downregulating the binding of factor Xa to factor Va.