Project 1 Abstract 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. X-ray crystallography and hydrogen-deuterium exchange will be used complementarily to investigate the structural basis for how the activation of factor V imparts Xa binding properties to factor Va and the related interaction of TFPI with factor V/Va variants.
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 protein binding sites (phospholipid or otherwise) on activated platelets and endothelial cells.
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.

Public Health Relevance

Project 1 Narrative Excessive blood clotting is a major cause of heart attacks and strokes in the United States and also underlies ~25% of cancer-related death. Our research brings new concepts and methodologies to bear on understanding basic molecular mechanisms underlying the blood clotting reactions. Our findings will address important but unresolved aspects of function to yield novel strategies for interfering with the formation of life-threatening blood clots in human disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
1P01HL139420-01
Application #
9416297
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Program Officer
Warren, Ronald Q
Project Start
Project End
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104