Membrane binding by prothrombin mediates its constrained presentation to prothrombinase for cleavage.
Long-standing dogma proposes a profound contribution of membrane binding by prothrombin in determining the rate at which it is converted to thrombin by prothrombinase. We have examined the action of prothrombinase on full-length prothrombin variants lacking γ-carboxyglutamate modifications (desGla) with impaired membrane binding. We show an unexpectedly modest decrease in the rate of thrombin formation for desGla prothrombin but with a major effect on the pathway for substrate cleavage. Using desGla prothrombin variants in which the individual cleavage sites have been singly rendered uncleavable, we find that loss of membrane binding and other Gla-dependent functions in the substrate leads to a decrease in the rate of cleavage at Arg(320) and a surprising increase in the rate of cleavage at Arg(271). These compensating effects arise from a loss in the membrane component of exosite-dependent tethering of substrate to prothrombinase and a relaxation in the constrained presentation of the individual cleavage sites for active site docking and catalysis. Loss of constraint is evident as a switch in the pathway for prothrombin cleavage and the intermediate produced but without the expected profound decrease in rate. Extension of these findings to the action of prothrombinase assembled on platelets and endothelial cells on fully carboxylated prothrombin reveals new mechanistic insights into function on physiological membranes. Cell-dependent enzyme function is probably governed by a differential ability to support prothrombin binding and the variable accumulation of intermediates from the two possible pathways of prothrombin activation.
From the Research Institute, Children's Hospital of Philadelphia, and.
Harlan N
HN
Bradford
Steven J
SJ
Orcutt
Sriram
S
Krishnaswamy
D001777:Blood Coagulation; D001792:Blood Platelets / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D005165:Factor V / Q000737:chemistry; D015943:Factor Va / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D011485:Protein Binding; D011516:Prothrombin / Q000737:chemistry*; D013379:Substrate Specificity; D013925:Thromboplastin / Q000737:chemistry*
D015943:Factor Va; D005165:Factor V; D011516:Prothrombin; D013925:Thromboplastin; D015951:Factor Xa
Blood Coagulation Factors; Enzyme Kinetics; Membrane Enzymes; Prothrombin; Prothrombinase; Serine Protease; Zymogen Activation