Progress Curve Analysis of the Kinetics with Which Blood Coagulation Factor XIa Is Inhibited by Protease Nexin-2

Scandura, Joseph M.; Zhang, Y.; Nostrand, William E. Van; Walsh, Peter N.

doi:10.1021/bi9612576

Cited by 46 publications

(90 citation statements)

References 51 publications

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“…Thus, it has been observed that normal human plasma contains very little (Ͻ60 pM) PN2 (i.e. concentrations well below the reported K i value) (300 -500 pM) for FXIa inhibition by PN2 (19,20,(22)(23)(24). However, the protein is secreted from platelet ␣-granules in sufficient quantities (2-30 nM) to result in rapid and complete inhibition of FXIa in solution (20,23).…”

Section: Discussionmentioning

confidence: 99%

“…The WT PN2KPI was characterized by a prolonged transient phase for inhibition of FXIac, typical of slow, tight binding Kunitz inhibitors (20). Increasing concentrations of either WT PN2KPI or mutant PN2KPI domains, incubated with 0.1 nM FXIac, resulted in plots of fractional amidolytic activity versus the inhibitor concentration (Figs.…”

Section: Purification and Characterization Of Pn2kpi Wt And Mutantmentioning

confidence: 98%

“…Upon platelet activation by physiological stimulators, PN2 is secreted from ␣-granules into plasma and inhibits FXIa (22)(23)(24), suggesting a role for this protein in blood coagulation. PN2 is a slow, tight binding inhibitor of FXIa with K i of ϳ300 -500 pM (18,20,22). The KPI domain of PN2 is 57 amino acids in length (Glu 289 -Ile 345 in the 751-amino acid isoform of PN2) and is known to contain the entire FXIa inhibitory function of PN2 (19 -21, 28).…”

mentioning

confidence: 99%

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Structural and Mutational Analyses of the Molecular Interactions between the Catalytic Domain of Factor XIa and the Kunitz Protease Inhibitor Domain of Protease Nexin 2

Navaneetham

Jin²,

Pandey³

et al. 2005

Journal of Biological Chemistry

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Factor XIa (FXIa) is a serine protease important for initiating the intrinsic pathway of blood coagulation. Protease nexin 2 (PN2) is a Kunitz-type protease inhibitor secreted by activated platelets and a physiologically important inhibitor of FXIa. Inhibition of FXIa by PN2 requires interactions between the two proteins that are confined to the catalytic domain of the enzyme and the Kunitz protease inhibitor (KPI) domain of PN2. Recombinant PN2KPI and a mutant form of the FXI catalytic domain (FXIac) were expressed in yeast, purified to homogeneity, co-crystallized, and the structure of the complex was solved at 2.6 Å (Protein Data Bank code 1ZJD). In this complex, PN2KPI has a characteristic, disulfide-stabilized double loop structure that fits into the FXIac active site. To determine the contributions of residues within PN2KPI to its inhibitory activity, selected point mutations in PN2KPI Coagulation factor XI (FXI) 4 is a unique homodimeric coagulation protein, present in human plasma at a concentration of ϳ30 nM, that is essential for normal hemostasis, as evidenced by the fact that FXI deficiency is associated with a hemorrhagic disorder (1). The zymogen FXI can bind to receptors (2-4), consisting of the glycoprotein Ib-IX-V complex (5) on the plasma membranes of activated human platelets, where it can be incorporated into platelet membrane microdomains (i.e. lipid rafts) (6) for efficient activation by thrombin or by FXIIa (7-10). The resulting enzyme, FXIa, can then activate the vitamin K-dependent protein, FIX, to initiate the consolidation phase of blood coagulation (1).A variety of important control mechanisms exist for regulating the activity of coagulation proteases in plasma. Several members of the serpin family have been proposed as physiological regulators of FXIa activity in plasma, including C1 inhibitor (11, 12), ␣-1-protease inhibitor (13, 14), antithrombin III (15), ␣-2-antiplasmin (16), and protease nexin 1 (17). However, a platelet secretory protein and member of the class of Kunitz-type inhibitors, protease nexin 2 (PN2), has recently been shown to be a much more potent and physiologically relevant FXIa inhibitor based on detailed kinetics studies (18 -23). PN2 is a ϳ120-kDa isoform of the Alzheimer ␤-amyloid protein precursor (APP) that contains a Kunitz-type serine protease inhibitor (PN2KPI) domain (22). Platelets are an important source of several isoforms of APP, including the APP 751 isoform of PN2 (23, 24). Full-length APP is membraneassociated (25) and is processed by proteases in platelets (26, 27). Upon platelet activation by physiological stimulators, PN2 is secreted from ␣-granules into plasma and inhibits FXIa (22-24), suggesting a role for this protein in blood coagulation. PN2 is a slow, tight binding inhibitor of FXIa with K i of ϳ300 -500 pM (18,20,22). The KPI domain of PN2 is 57 amino acids in length (Glu 289 -Ile 345 in the 751-amino acid isoform of PN2) and is known to contain the entire FXIa inhibitory function of PN2 (19 -21, 28). Similarly, all of the information r...

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Section: Discussionmentioning

confidence: 99%

Section: Purification and Characterization Of Pn2kpi Wt And Mutantmentioning

confidence: 98%

mentioning

confidence: 99%

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Structural and Mutational Analyses of the Molecular Interactions between the Catalytic Domain of Factor XIa and the Kunitz Protease Inhibitor Domain of Protease Nexin 2

Navaneetham

Jin²,

Pandey³

et al. 2005

Journal of Biological Chemistry

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“…However, since APP processing changes during platelet aggregation, it is possible that the local concentration of A␤ rises transiently when platelets are activated. 18 Thus, the steady state level of A␤ in serum may not be an accurate indication of the exposure of platelets to A␤. Many agents, such as thrombin or acetylcholine are known to alter A␤ processing.…”

Section: Discussionmentioning

confidence: 99%

β-Amyloid augments platelet aggregation: reduced activity of familial angiopathy-associated mutants

Wolozin

Maheshwari²,

Jones³

et al. 1998

Mol Psychiatry

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The ␤-amyloid (A␤) peptide is present both in serum and in platelets, however it is unclear whether A␤ plays a role in platelet function. We have now investigated the effects of soluble A␤ on platelet function and have found that low levels (0.1-1 nM) of soluble A␤ augment ADP-dependent platelet aggregation and translocation of focal adhesion kinase to the platelet cytoskeleton. Addition of A␤ to gel-filtered platelets along with concentrations of adenosine diphosphate (ADP) producing submaximal aggregation responses increased the aggregation response by over 2-fold depending on the ADP:A␤ ratios. The structure activity requirements for A␤ activity showed intriguing constraints. Only full length A␤ has significant activity. Truncated A␤ peptides, such as A␤ 1-16 or A␤ 25-35 , or reverse A␤ 40-1 all show little or no activity. We also examined the activity of mutant A␤ peptides, corresponding with the APP 692A→G and APP 693E→Q (at A␤21 and A␤22, respectively) which are found in familial Alzheimer's disease and hereditary cerebral hemorrhagic amyloidosis, Dutch type (HCHWA-D), and found that these peptides showed little or no activity. These results suggest that A␤ interacts with platelets in a highly specific manner and may play a role in regulating platelet function.

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“…where S is the substrate concentration and K m is the Michaelis constant of FXIa for S-2366 previously determined to be ϳ0.25 mM (29).…”

Section: Fxi Mutant Constructs-mentioning

confidence: 99%

The Role of Factor XIa (FXIa) Catalytic Domain Exosite Residues in Substrate Catalysis and Inhibition by the Kunitz Protease Inhibitor Domain of Protease Nexin 2

Miller

Navaneetham

et al. 2011

Journal of Biological Chemistry

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Progress Curve Analysis of the Kinetics with Which Blood Coagulation Factor XIa Is Inhibited by Protease Nexin-2

Cited by 46 publications

References 51 publications

Structural and Mutational Analyses of the Molecular Interactions between the Catalytic Domain of Factor XIa and the Kunitz Protease Inhibitor Domain of Protease Nexin 2

Structural and Mutational Analyses of the Molecular Interactions between the Catalytic Domain of Factor XIa and the Kunitz Protease Inhibitor Domain of Protease Nexin 2

β-Amyloid augments platelet aggregation: reduced activity of familial angiopathy-associated mutants

The Role of Factor XIa (FXIa) Catalytic Domain Exosite Residues in Substrate Catalysis and Inhibition by the Kunitz Protease Inhibitor Domain of Protease Nexin 2

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