Objective—: Protein S is a vitamin K–dependent plasma protein that functions in the feedback regulation of thrombin generation. Our goal was to determine how protein S regulates the intrinsic pathway of blood coagulation. Methods and Results—: We used plasma, including platelet-rich plasma, and in vitro methods to determine how the intrinsic pathway of blood coagulation is regulated by protein S. We obtained the following results: (1) activated partial thromboplastin time assays with protein S–supplemented plasma confirmed that protein S prolongs clotting time; (2) a modified activated partial thromboplastin time assay with factor IX (fIX)–deficient plasma confirmed that protein S affects fIX-initiated clotting; (3) a fIXa/factor VIIIa (fVIIIa)–mediated thrombin generation assay with either platelet-rich plasma or factor-deficient plasma, initiated with a limiting amount of tissue factor, was regulated by protein S; (4) in the presence of phosphatidylserine vesicles, protein S inhibited fIXa in the absence and presence of fVIIIa; and (5) protein S altered only the K _M for factor X activation by fIXa in the absence of fVIIIa and both k _cat and K _M in the presence of fVIIIa. Conclusion—: From our findings, it can be concluded that protein S inhibits fIXa in the presence or absence of fVIIIa in an activated protein C–independent way.

The serpinopathies are a group of inherited disorders that share as their molecular basis the misfolding and polymerization of serpins, an important class of protease inhibitors. Depending on the identity of the serpin, conditions arising from polymerization include emphysema, thrombosis, and dementia. The structure of serpin polymers is thus of considerable medical interest. Wild-type α₁-antitrypsin will form polymers upon incubation at moderate temperatures and has been widely used as a model system for studying serpin polymerization. Using hydrogen/deuterium exchange and mass spectrometry, we have obtained molecular level structural information on the α₁-antitrypsin polymer. We found that the flexible reactive center loop becomes strongly protected upon polymerization. We also found significant increases in protection in the center of β-sheet A and in helix F. These results support a model in which linkage between serpins is achieved through insertion of the reactive center loop of one serpin into β-sheet A of another. We have also examined the heat-induced conformational changes preceding polymerization. We found that polymerization is preceded by significant destabilization of β-sheet C. On the basis of our results, we propose a mechanism for polymerization in which β-strand 1C is displaced from the rest of β-sheet C through a binary serpin/serpin interaction. Displacement of strand 1C triggers further conformational changes, including the opening of β-sheet A, and allows for subsequent polymerization.