Effects of Activation Peptide Bond Cleavage and Fragment 2 Interactions on the Pathway of Exosite I Expression during Activation of Human Prethrombin 1 to Thrombin

Anderson, Patricia J.; Nesset, Anna; Bock, Paul E.

doi:10.1074/jbc.m306917200

Cited by 24 publications

(48 citation statements)

References 55 publications

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“…The equilibrium dissociation constants for interactions of thrombin or P2 with F2 or F12 are in agreement with those determined in careful studies with human proteins (8,9,19). We speculate that the somewhat higher affinity we report for the binding of F12 to P2 could reflect the fact that P2 prepared by preparative proteolysis of II A195 or generated in situ with II Q320 is not proteolyzed at Arg 284 and therefore possesses an authentic NH 2 terminus.…”

Section: Discussionsupporting

confidence: 88%

“…These findings are generally consistent with the interpretation that thrombin is readily and rapidly released from the membrane surface upon its formation regardless of the proteolysis of F12 to F1 and F2 through cleavages mediated by thrombin. In agreement with recent observations (8,9), these findings also imply that potential interactions between thrombin and the F2 domain within F12 are relatively weak and play a minor role in retaining thrombin on the membrane surface at the site of its formation.…”

Section: Membrane-bound Species During Prothrombin Activationsupporting

confidence: 93%

“…Similar experiments with IIa A195 revealed a ϳ20-fold lower affinity of the proteinase for F12 that could completely be accounted for by a weaker interaction between IIa A195 and F2 (Table 1). Consistent with recent findings (8,9,19), the interaction between P2 A195 and F2 was also weak and comparable with the affinities observed with IIa A195 (Table 1). The thermodynamic constants are consistent with a large ordering effect observed in the interaction of P2 with F12 that is substantially reduced with IIa A195 or the binding of F2 to either species.…”

Section: Membrane-bound Species During Prothrombin Activationsupporting

confidence: 92%

“…F2 and by extension, F12, bind to residues within the anion binding exosite II region in thrombin and P2 (10). The reduction in affinity for F12 that accompanies the conversion of the zymogen to the proteinase mirrors the increase in affinity for hirugen, a prototypic ligand for anion binding exosite I (9,19). Thus, the conversion of zymogen to proteinase is accompanied by reciprocal changes of approximately equal magnitude in liganding at these two exosites.…”

Section: Discussionmentioning

confidence: 99%

“…A series of more recent and careful studies with human prothrombin derivatives, conducted at physiological pH and ionic strength (or NaCl), now indicate that the interactions between F2 and thrombin or P2 are 1000-fold (or more) weaker than initially reported in the bovine system (8,9). Based on the possible concentrations of the various species expected during the activation of prothrombin, these findings suggest that noncovalent interactions with the F2 domain either on its own or within F12 probably play a minor role in retaining thrombin on the membrane surface or in modulating the function of thrombin in solution.…”

mentioning

confidence: 99%

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Fate of Membrane-bound Reactants and Products during the Activation of Human Prothrombin by Prothrombinase

Kamath

Krishnaswamy

2008

Journal of Biological Chemistry

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Membrane binding by prothrombin, mediated by its N-terminal fragment 1 (F1) domain, plays an essential role in its proteolytic activation by prothrombinase. Thrombin is produced in two cleavage reactions. One at Arg 320 yields the proteinase meizothrombin that retains membrane binding properties. The second, at Arg 271 , yields thrombin and severs covalent linkage with the N-terminal fragment 1.2 (F12) region. Covalent linkage with the membrane binding domain is also lost when prethrombin 2 (P2) and F12 are produced following initial cleavage at Arg 271 . We show that at the physiological concentration of prothrombin, thrombin formation results in rapid release of the proteinase into solution. Product release from the surface can be explained by the weak interaction between the proteinase and F12 domains. In contrast, the zymogen intermediate P2, formed following cleavage at Arg 271 , accumulates on the surface because of a ϳ20-fold higher affinity for F12. By kinetic studies, we show that this enhanced binding adequately explains the ability of unexpectedly low concentrations of F12 to greatly enhance the conversion of P2 to thrombin. Thus, the utilization of all three possible substrate species by prothrombinase is regulated by their ability to bind membranes regardless of whether covalent linkage to the F12 region is maintained. The product, thrombin, interacts with sufficiently poor affinity with F12 so that it is rapidly released from its site of production to participate in its numerous hemostatic functions.Thrombin, the key effector serine proteinase of the blood coagulation cascade, is produced by specific and limited proteolysis of the zymogen, prothrombin. The physiologically relevant catalyst for this reaction is the prothrombinase complex consisting of the serine proteinase, factor Xa, and the cofactor, factor Va, assembled on membranes in the presence of Ca 2ϩ(1). In addition to facilitating the assembly of the enzyme complex, membranes containing acidic or amino phospholipids play an important role in mediating the delivery of prothrombin to the membrane-bound enzyme (1, 2). This arises from the ability of prothrombin to bind to these membranes through the fragment 1 (F1) 2 domain present at its N terminus (1, 3, 4). Thrombin, derived from the C-terminal half of prothrombin, is produced as a result of cleavages 3 following Arg 271 and Arg 320(1, 3, 5). Cleavage at Arg 320 converts the zymogen to a proteinase, whereas cleavage at Arg 271 severs covalent linkage with the N-terminal fragment 1.2 (F12) domain harboring the membrane binding site (Scheme 1). Covalent linkage of the C-terminal domain with F12 is also lost in the zymogen intermediate, prethrombin 2 (P2), produced following cleavage only at Arg 271 (Scheme 1). In contrast, meizothrombin (mIIa), produced following cleavage only at Arg 320 is covalently linked to the membrane binding domain through a disulfide bond (Scheme 1). Accordingly, both prothrombin and mIIa are established to bind to membranes, and this binding interaction impacts the...

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Section: Discussionsupporting

confidence: 88%

Section: Membrane-bound Species During Prothrombin Activationsupporting

confidence: 93%

Section: Membrane-bound Species During Prothrombin Activationsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Fate of Membrane-bound Reactants and Products during the Activation of Human Prothrombin by Prothrombinase

Kamath

Krishnaswamy

2008

Journal of Biological Chemistry

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Modes and consequences of thrombin's interaction with fibrin

Fredenburgh

Stafford

Pospisil

et al. 2004

Biophysical Chemistry

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Lupus-Derived Antiprothrombin Autoantibodies from a V Gene Phage Display Library Are Specific for the Kringle 2 Domain of Prothrombin

Dasgupta

Planque

et al. 2004

Biochemistry

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Autoantibodies to prothrombin are common in patients with systemic lupus erythematosus. Although their presence is a risk factor for thrombosis, neither their origin nor their precise role in inducing the procoagulant state is known. We have developed a phage-display antibody library from patients with systemic lupus erythematosus with antiprothrombin antibodies, and we have selected two single-chain Fv antibody fragments (ScFvs) by panning on a prothrombin-coated surface. In prothrombin activation assays using purified components, these antibodies promoted prothrombin activation. These ScFvs, termed AN78 and AN129, bound to immobilized prothrombin in a concentration-dependent specific manner but not to other anionic phospholipid binding proteins such as beta2-glycoprotein I or annexin V. Phosphatidylserine-bound prothrombin, but not soluble prothrombin, inhibited the binding suggesting that the epitope is available only on immobilized prothrombin. To localize the epitope, prothrombin was treated with thrombin or factor Xa and various prothrombin activation fragments were subsequently isolated and tested in ELISA with the ScFvs. Both AN78 and AN129 bound to prethrombin I (the fragment lacking the Gla domain and the first kringle domain), to fragment 1.2 (containing Gla and the two kringle domains only) and to fragment 2 but not to thrombin, thus localizing the cognate epitope to the kringle 2 domain in prothrombin. Analysis of the cDNA sequences of these antibodies show clustered mutational patterns in the complementarity determining region, suggesting that variable domains are the products of antigen-driven B cell clonal maturation.

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Effects of Activation Peptide Bond Cleavage and Fragment 2 Interactions on the Pathway of Exosite I Expression during Activation of Human Prethrombin 1 to Thrombin

Cited by 24 publications

References 55 publications

Fate of Membrane-bound Reactants and Products during the Activation of Human Prothrombin by Prothrombinase

Fate of Membrane-bound Reactants and Products during the Activation of Human Prothrombin by Prothrombinase

Modes and consequences of thrombin's interaction with fibrin

Lupus-Derived Antiprothrombin Autoantibodies from a V Gene Phage Display Library Are Specific for the Kringle 2 Domain of Prothrombin

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