Protease Nexin II Interactions with Coagulation Factor XIa Are Contained within the Kunitz Protease Inhibitor Domain of Protease Nexin II and the Factor XIa Catalytic Domain†

Biochemistry ◽  
2000 ◽  
Vol 39 (16) ◽  
pp. 4769-4777 ◽  
Author(s):  
Karen O. Badellino ◽  
Peter N. Walsh
Keyword(s):  
Protease Inhibitor ◽  
Catalytic Domain ◽  
Coagulation Factor ◽  
Kunitz Protease Inhibitor ◽  
Factor Xia ◽  
Protease Nexin ◽  
Kunitz Protease Inhibitor Domain

scholarly journals 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

2011 ◽  
Vol 286 (36) ◽  
pp. 31904-31914 ◽  
Author(s):  
Ya-Chi Su ◽  
Tara N. Miller ◽  
Duraiswamy Navaneetham ◽  
Robert T. Schoonmaker ◽  
Dipali Sinha ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Catalytic Domain ◽  
Kunitz Protease Inhibitor ◽  
Factor Xia ◽  
Protease Nexin ◽  
Kunitz Protease Inhibitor Domain

scholarly journals 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

2005 ◽  
Vol 280 (43) ◽  
pp. 36165-36175 ◽  
Author(s):  
Duraiswamy Navaneetham ◽  
Lei Jin ◽  
Pramod Pandey ◽  
James E. Strickler ◽  
Robert E. Babine ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Catalytic Domain ◽  
Point Mutations ◽  
Protein Data Bank Code ◽  
Mutant Form ◽  
Wild Type ◽  
Site Mutation ◽  
Kunitz Protease Inhibitor ◽  
Factor Xia ◽  
Protease Nexin

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 loop 1 11TGPCRAMISR20 and loop 2 34FYGGC38 were tested for their ability to inhibit FXIa. The P1 site mutation R15A completely abolished its ability to inhibit FXIa. IC50 values for the wild type protein and the remaining mutants were as follows: PN2KPI WT, 1.28 nm; P13A, 5.92 nm; M17A, 1.62 nm; S19A, 1.86 nm; R20A, 5.67 nm; F34A, 9.85 nm. The IC50 values for the M17A and S19A mutants were not significantly different from those obtained with wild type PN2KPI. These functional studies and activated partial thromboplastin time analysis validate predictions made from the PN2KPI-FXIac co-crystal structure and implicate PN2KPI residues, in descending order of importance, Arg15, Phe34, Pro13, and Arg20 in FXIa inhibition by PN2KPI.

Chemical Footprinting Reveals Conformational Changes Following Activation of Factor XI

2018 ◽  
Vol 118 (02) ◽  
pp. 340-350 ◽  
Author(s):  
Ingrid Stroo ◽  
J. Marquart ◽  
Kamran Bakhtiari ◽  
Tom Plug ◽  
Alexander Meijer ◽  
...  
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Catalytic Domain ◽  
Active Form ◽  
Coagulation Factor ◽  
Lysine Residue ◽  
Factor Ix ◽  
Factor Xi ◽  
Lysine Residues ◽  
Factor Xia

AbstractCoagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

Synthesis and characterization of the Kunitz protease-inhibitor domain of the β-amyloid precursor protein

Gene ◽  
1991 ◽  
Vol 98 (2) ◽  
pp. 225-230 ◽  
Author(s):  
James Schilling ◽  
Yu Wang ◽  
Ken Lau ◽  
Leanne Smith ◽  
Barbara Cordell
Keyword(s):  
Protease Inhibitor ◽  
Amyloid Precursor Protein ◽  
Precursor Protein ◽  
Synthesis And Characterization ◽  
Kunitz Protease Inhibitor ◽  
Β Amyloid ◽  
Kunitz Protease Inhibitor Domain

Structural and Mutational Analysis of Canonical Loop Residues In Protease Nexin 2 Involved In Factor XIa and Trypsin Inhibition.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1147-1147
Author(s):  
Duraiswamy Navaneetham ◽  
Dipali Sinha ◽  
Peter N. Walsh
Keyword(s):  
Inhibitory Activity ◽  
Significant Contribution ◽  
Catalytic Domain ◽  
Hydrophobic Interactions ◽  
Conflicts Of Interest ◽  
Side Chain ◽  
Trypsin Inhibition ◽  
Factor Xia ◽  
Protease Nexin ◽  
Loop 2

Abstract Abstract 1147 Factor XIa (FXIa) activates FIX and is regulated by platelet-secreted protease nexin 2 (PN2) that contains a Kunitz-type protease inhibitor (KPI) domain. Trypsin is regulated by basic pancreatic trypsin inhibitor (BPTI). The primary and tertiary structures of trypsin and the catalytic domain of FXIa are highly homologous, and KPI and BPTI are nearly identical structurally. We have previously identified two loop structures (loops 1 and 2) in the KPI domain of PN2 that interact with residues in the FXIa catalytic domain. Based on the structure of the FXIa/KPI complex crystal structure, residues within loops 1 and 2 were mutated for experiments examining the inhibition of FXIa and trypsin. Results show that the loop-1-region P1 site residue Arg15 of PN2KPI plays a major role in FXIa inhibition by protruding into the S1 specificity pocket of FXIa. Ala mutation at this site renders PN2KPI non-inhibitory for both FXIa and trypsin. BPTI has Lys15 at the P1 site. BPTI inhibits both FXIa and trypsin significantly less effectively than PN2KPI. PN2KPI-R15K lost FXIa inhibitory activity, whereas BPTI-K15R substantially gained affinity for FXIa. Like FXIa, trypsin preferred BPTI-K15R showing a significant enhancement in affinity. Thus, a major determinant of the inhibitory activity of PN2KPI and BPTI against FXIa and trypsin is the P1 residue, with Arg being preferred over Lys for both inhibitors and both proteases. In addition, loop 1 residues Pro13 and Arg20 make important contributions to both FXIa and trypsin inhibition as demonstrated by significantly elevated Ki values for Ala mutations (P13A, and R20A) at these sites. In contrast, Ser19 makes no significant contribution to inhibition of either FXIa or trypsin whereas Met17 makes a significant contribution to the inhibition of trypsin, but not FXIa. In loop 2, only Phe34 is identified as a residue making significant contributions to the inhibition of both FXIa and trypsin, since the PN2KPI-F34A mutant displayed reduced inhibitory activity for both FXIa (6-fold) and for trypsin (3-fold). To rationalize these findings, we examined the crystal structures of the FXIa(catalytic domain)/PN2KPI complex, and the trypsin/BPTI complex. Structurally, the PN2KPI loop-1, P1-site residue Arg15 makes a complex primary interaction with Asp189 of both FXIa and trypsin. Disruption of this site by R15A mutation renders PN2KPI non-inhibitory because it preempts salt bridge interactions from two nitrogen atoms of the guanidinium group of Arg15 with Asp189 and Gly218 in FXIa. In addition, the Arg15 carbonyl oxygen forms hydrogen bonds with main-chain nitrogen atoms of one of the catalytic triad residues, Ser195, and with Gly193. The other important interaction in FXIa/PN2KPI or trypsin/BPTI is hydrophobic, between PN2KPI-Phe34 and FXIa-Tyr143 and between BPTI-Val34 and trypsin-Tyr151. This intermolecular interaction is further strengthened by an intramolecular interaction in which the side chain of Phe34 packs closely with the side chain of Met17 within PN2KPI, altogether forming a strong hydrophobic patch in FXIa-PN2KPI and trypsin-PN2KPI. PN2KPI-F34A disrupts both inter- and intramolecular hydrophobic interactions, leading to discernable reductions in affinity for both FXIa and trypsin. Despite occupying extreme positions in the autolysis loops, (143YRKLRDKI151 in FXIa and 143NTKSSGTSY151 in trypsin), Tyr143/151 residues still orient themselves in close proximity to Phe34. Thus, loop-1 residues of PN2KPI establish complex ionic interactions that play a major role, which is supplemented by the loop-2 residue, Phe34 (in PN2KPI) or Val34 (in BPTI), which establish hydrophobic interactions with residues in FXIa and trypsin leading to very high-affinity enzyme-inhibitor complexes. Disclosures: No relevant conflicts of interest to declare.

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

Biochemistry ◽  
1997 ◽  
Vol 36 (2) ◽  
pp. 412-420 ◽  
Author(s):  
Joseph M. Scandura ◽  
Yan Zhang ◽  
William E. Van Nostrand ◽  
Peter N. Walsh
Keyword(s):  
Blood Coagulation ◽  
Coagulation Factor ◽  
Curve Analysis ◽  
Progress Curve ◽  
Factor Xia ◽  
Protease Nexin

scholarly journals The Mechanism by Which Heparin Promotes the Inhibition of Coagulation Factor XIa by Protease Nexin-2

1997 ◽  
Vol 272 (42) ◽  
pp. 26139-26144 ◽  
Author(s):  
Yan Zhang ◽  
Joseph M. Scandura ◽  
William E. Van Nostrand ◽  
Peter N. Walsh
Keyword(s):  
Coagulation Factor ◽  
Factor Xia ◽  
Protease Nexin

P1-231: The Kunitz protease inhibitor domain controls homodimerization of the amyloid precursor protein

2010 ◽  
Vol 6 ◽  
pp. S241-S241
Author(s):  
Naouel Ben Khalifa ◽  
Jean-Christophe Renauld ◽  
Pierre J. Courtoy ◽  
Donatienne Tyteca ◽  
Jean-François Collet ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Amyloid Precursor Protein ◽  
Precursor Protein ◽  
Kunitz Protease Inhibitor ◽  
Kunitz Protease Inhibitor Domain

scholarly journals Protein Z–dependent protease inhibitor deficiency produces a more severe murine phenotype than protein Z deficiency

Blood ◽  
2008 ◽  
Vol 111 (10) ◽  
pp. 4973-4978 ◽  
Author(s):  
Jing Zhang ◽  
Yizheng Tu ◽  
Lan Lu ◽  
Nina Lasky ◽  
George J. Broze
Keyword(s):  
Protease Inhibitor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Plasma Vitamin ◽  
Severe Phenotype ◽  
Epinephrine Infusion ◽  
Protein Z ◽  
Thrombotic Risk ◽  
Factor Xia

Abstract Protein Z (PZ) is a plasma vitamin K–dependent protein that functions as a cofactor to dramatically enhance the inhibition of coagulation factor Xa by the serpin, protein Z–dependent protease inhibitor (ZPI). In vitro, ZPI not only inhibits factor Xa in a calcium ion–, phospholipid-, and PZ-dependent fashion, but also directly inhibits coagulation factor XIa. In murine gene-deletion models, PZ and ZPI deficiency enhances thrombosis following arterial injury and increases mortality from pulmonary thromboembolism following collagen/epinephrine infusion. On a factor VLeiden genetic background, ZPI deficiency produces a significantly more severe phenotype than PZ deficiency, implying that factor XIa inhibition by ZPI is physiologically relevant. The studies in mice suggest that human PZ and ZPI deficiency would be associated with a modest thrombotic risk with ZPI deficiency producing a more severe phenotype.

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