We employ prothrombin activation catalyzed by the prothrombinase complex as a paradigm for the enzyme complexes of coagulation to investigate the molecular bases for enzymic function. An emphasis is placed on addressing major unanswered questions on how membrane-dependent function is achieved. A series of approaches derive from unexpected ideas developed with a snake venom variant of factor V that is structurally similar to human factor V but fails to bind membranes. A combination of binding, rapid kinetics, thermodynamic approaches and structural studies with gain-in-function variants will address the hypothesis that membrane binding by factor Va arises from flexibility in the C1 and C2 domains that allows the protein to adopt a high affinity membrane-binding conformation. We will also investigate the hypothesis that impaired membrane binding arising from altered malleability in these domains explains the functional defect in the original patient identified with factor V deficiency.
The second aim draws from the surprising ability to prepare recombinant variants of human Xa with a large gain in membrane-independent function. These variants are capable of binding human Va with nanomolar affinity in solution with a greatly improved ability to catalyze thrombin formation in the absence of membranes. We will employ these species to separately address mechanisms by which membranes facilitate prothrombinase assembly and modulate its action on prothrombin in a way that was not previously considered possible.
The third aim will pursue physical studies to test the hypothesis that the peculiarities ascribed to prothrombinase function on cell surfaces relative to synthetic membranes result from the physical constraints associated with the exposure of limiting binding sites for the vitamin K-dependent proteins on activated platelets and endothelial cells that can be approximated by synthetic membranes with low phosphatidylserine content.
The final aim proposes to employ new reagents and a series of factor X variants to address the contribution of exosite-dependent recognition of factor X by the VIIIa/IXa complex by kinetic and binding studies. These strategies are based on novel concepts derived from an expanded understanding of substrate recognition by prothrombinase. The proposed approaches will shed new light on important yet poorly understood facets of the biochemistry and biology of enzyme function relevant to normal hemostasis, in disease states and for therapeutic targeting of this reaction in thrombotic and vascular disease.
Excessive blood clotting is a major cause of heart attacks and strokes in the United States. Our research brings new concepts and methodologies to bear on understanding basic molecular mechanisms underlying the blood clotting reactions. Our findings will reveal novel strategies for interfering with the formation of life-threatening blood clots in a wide range of human diseases.
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| Kamikubo, Yuichi; Mendolicchio, G Loredana; Zampolli, Antonella et al. (2017) Selective factor VIII activation by the tissue factor-factor VIIa-factor Xa complex. Blood 130:1661-1670 |
| Zhao, Liang; Thorsheim, Chelsea L; Suzuki, Aae et al. (2017) Phosphatidylinositol transfer protein-? in platelets is inconsequential for thrombosis yet is utilized for tumor metastasis. Nat Commun 8:1216 |
| Bradford, Harlan N; Krishnaswamy, Sriram (2016) The Fragment 1 Region of Prothrombin Facilitates the Favored Binding of Fragment 12 to Zymogen and Enforces Zymogen-like Character in the Proteinase. J Biol Chem 291:11114-23 |
