scholarly journals Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa

2006 ◽  
Vol 396 (2) ◽  
pp. 355-362 ◽  
Author(s):  
Fatbardha Varfaj ◽  
Julie Neuberg ◽  
P. Vincent Jenkins ◽  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Catalytic Efficiency ◽  
Wild Type ◽  
High Concentration ◽  
Subunit Dissociation ◽  
Factor Viiia ◽  
Km Value ◽  
Type Factor

APC (activated Protein C) inactivates human Factor VIIIa following cleavage at residues Arg336 and Arg562 within the A1 and A2 subunits respectively. The role of the P1 arginine in APC-catalysed inactivation of Factor VIIIa was examined by employing recombinant Factor VIIIa molecules where residues 336 and 562 were replaced with alanine and/or glutamine. Stably expressed Factor VIII proteins were activated by thrombin and resultant Factor VIIIa was reacted at high concentration with APC to minimize cofactor inactivation due to A2 subunit dissociation. APC cleaved wild-type Factor VIIIa at the A1 site with a rate ∼25-fold greater than that for the A2 site. A1 mutants R336A and R336Q were inactivated ∼9-fold slower than wild-type Factor VIIIa, whereas the A2 mutant R562A was inactivated ∼2-fold slower. No cleavage at the mutated sites was observed. Taken together, these results suggested that cleavage at the A1 site was the dominant mechanism for Factor VIIIa inactivation catalysed by the proteinase. On the basis of cleavage at Arg336, a Km value for wild-type Factor VIIIa of 102 nM was determined, and this value was significantly greater than Ki values (∼9–18 nM) obtained for an R336Q/R562Q Factor VIIIa. Furthermore, evaluation of a series of cluster mutants in the C-terminal region of the A1 subunit revealed a role for acidic residues in segment 341–345 in the APC-catalysed proteolysis of Arg336. Thus, while P1 residues contribute to catalytic efficiency, residues removed from these sites make a primary contribution to the overall binding of APC to Factor VIIIa.

Anticoagulant Dysfunction of Human Arg352Trp-Activated Protein C Caused by Defective Factor Va Inactivation

2001 ◽  
Vol 85 (02) ◽  
pp. 274-279 ◽  
Author(s):  
Claudia Rintelen ◽  
Subramanian Yegneswaran ◽  
John Griffin
Keyword(s):  
Venous Thrombosis ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Catalytic Efficiency ◽  
Protein S ◽  
Wild Type ◽  
Factor Va ◽  
Wild Type Protein ◽  
Factor Viiia

SummaryThe dysfunctional mutant R352W-protein C was found in two patients with venous thrombosis. The mutant R352A-protein C was constructed to define the contribution of charge/size of the residue at 352 on protein C (chymotrypsin numbering 187). Compared with wild type-protein C, R352W-protein C showed no difference in activation by thrombin·thrombomodulin or α-thrombin. However, R352W-activated protein C (APC) anticoagulant activity (aPTT assay) was reduced to ~65%. Although the catalytic efficiency of R352W-APC towards the oligopeptide substrate S-2366 was unperturbed, factor Va and R506Q-factor Va were not efficiently inactivated by R352W-APC compared with wild type-APC. R352A-APC showed reduced anticoagulant activity and reduced efficiency in factor Va inactivation and in factor VIIIa-inactivation in the presence of protein S. These observations suggest that the dysfunction of R352W-APC in factor Va inactivation may be one of the mechanisms leading to venous thrombosis in affected patients and that R352 plays an important role in the physiological functioning of APC.

Exosite Interactions Contribute to the Activated Protein C-Catalyzed Inactivation of Factor VIIIa.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1739-1739
Author(s):  
Fatbardha Varfaj ◽  
Julie Neuberg ◽  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Cleavage Site ◽  
Protein C ◽  
Human Factor ◽  
Activated Protein C ◽  
Q Factor ◽  
Wild Type ◽  
Factor Viiia ◽  
Human Factor Viii ◽  
Type Factor

Abstract Activated Protein C (APC) is an anticoagulant serine protease that proteolytically inactivates cofactors Va and VIIIa. Cleavage of human factor VIIIa occurs at Arg336 and Arg562 located within the A1 and A2 subunits, respectively. While cleavages are not ordered, the former site appears to represent a preferred cleavage site. Efficient catalysis requires binding of APC to a phospholipid surface and to the A3-C1-C2 subunit of factor VIIIa. The latter observation suggests that APC likely binds substrate via an exosite(s) thereby contributing to substrate specificity. A study was undertaken to evaluate contributions of substrate docking at the active site and exosite tethering to the APC catalytic mechanism. Recombinant, human factor VIII mutants where P1 Arg residues at 336 and 562 were substituted with Ala or Gln were constructed and stably expressed. Purified factor VIII was converted to factor VIIIa by thrombin and used as substrate to elucidate the mechanism of cleavages. Proteins mutated at Arg336 were also mutated at Lys338 because the latter residue may serve as an alternative APC cleavage site when residue 336 is mutated. Rates of inactivation of wild type and mutant factor VIIIa molecules and rates of cleavage at Arg336 and Arg562 by APC were monitored in the presence and absence of protein S. The R336A/K338A mutant was inactivated and cleaved at the 336 site approximately 20-fold slower than the wild type, whereas the R336Q/K338Q mutant was completely resistant to cleavage at the 336 site. These results indicate that residues other than Arg may be tolerated at the P1 site, whereas Gln yields a cleavage-resistant substrate. Indeed, the R336Q/K338Q/R562Q (triple Q) mutant was resistant to cleavage at both P1 sites. Furthermore, mutations retarding cleavage at residue 336 showed a dramatic decrease in rates of inactivation suggesting that cleavage at this site correlated with the inactivation of factor VIIIa. The importance of exosite interactions was explored by inhibition experiments examining the inactivation of wild type factor VIIIa in the presence of triple Q mutant factor VIIIa. Wild type factor VIIIa inactivation rates decreased as the proportion of triple Q factor VIIIa increased, indicating that the P1 mutant factor VIIIa effectively sequestered APC from the native substrate. Evaluation of inactivation rates suggested that APC possessed an ~8-fold greater affinity for the triple Q FVIIIa than the wild type factor VIIIa. Consistent with that observation, the Ki for triple Q factor VIIIa (29.5 ± 3.6 nM) was ~5-fold less than the Km for wild type factor VIIIa (133 ± 27 nM). Taken together, these results indicate that mutations in the P1 site that prevent cleavage may also retard dissociation of the enzyme-substrate complex. Overall, results from this study suggest that exosite interactions make a primary contribution to substrate affinity during APC-catalyzed inactivation of factor VIIIa.

Role of the A+ Helix in Heparin Binding to Protein C Inhibitor

1996 ◽  
Vol 75 (05) ◽  
pp. 760-766 ◽  
Author(s):  
Marc G L M Elisen ◽  
Machiel H H Maseland ◽  
Frank C Church ◽  
Bonno N Bouma ◽  
Joost C M Meijers
Keyword(s):  
Protein C ◽  
Electrostatic Interactions ◽  
Deletion Mutant ◽  
Activated Protein C ◽  
Structural Features ◽  
Heparin Binding ◽  
Wild Type ◽  
Protein C Inhibitor ◽  
Positively Charged

SummaryInteractions between proteins and heparin(-like) structures involve electrostatic forces and structural features. Based on charge distributions in the linear sequence of protein C inhibitor (PCI), two positively charged regions of PCI were proposed as possible candidates for this interaction. The first region, the A+ helix, is located at the N-terminus (residues 1-11), whereas the second region, the H helix, is positioned between residues 264 and 280 of PCI. Competition experiments with synthetic peptides based on the sequence of these regions demonstrated that the H helix has the highest affinity for heparin. In contrast to previous observations we found that the A+ helix peptide competed for the interaction of PCI with heparin, but its affinity was much lower than that of the H helix peptide.Recombinant PCI was also used to investigate the role of the A+ helix in heparin binding. Full-length (wild-type) rPCI as well as an A+ helix deletion mutant of PCI (rPCI-Δ2-l 1) were expressed in baby hamster kidney cells and both had normal inhibition activity with activated protein C and thrombin. The interaction of the recombinant PCIs with heparin was investigated and compared to plasma PCI. The A+ helix deletion mutant showed a decreased affinity for heparin in inhibition reactions with activated protein C and thrombin, but had similar association constants compared to wild-type rPCI. The synthetic A+ helix peptide competed with rPCI-Δ2-11 for binding to heparin. This indicated that the interaction between PCI and heparin is fairly non-specific and that the interaction is primarily based on electrostatic interactions.In summary, our data suggest that the H helix of PCI is the main heparin binding region of PCI, but the A+ helix increases the overall affinity for the PCI-heparin interaction by contributing a second positively charged region to the surface of PCI.

scholarly journals The role of P4-P3’ residues flanking Arg336 in facilitating activated protein C-catalyzed cleavage and inactivation of factor VIIIa

2011 ◽  
Vol 128 (5) ◽  
pp. 470-476 ◽  
Author(s):  
Jennifer P. DeAngelis ◽  
Fatbardha Varfaj ◽  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor Viiia

Genetic Absence of Thrombin Receptor Par4 Overcomes the Obligate Requirement of EPCR in Mouse Placenta

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 425-425
Author(s):  
Michelle M Storage ◽  
Jianzhong An ◽  
Helena Liang ◽  
Qiuhui Yang ◽  
Mark Zogg ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Fold Increase ◽  
Thrombin Receptor ◽  
Mutant Form ◽  
Wild Type ◽  
Placental Development ◽  
Transgenic Expression ◽  
Placental Failure

Abstract Introduction: Murine models suggest that the Thrombomodulin-Protein C system plays a critical role in placentation and the maintenance of pregnancy. Severe Protein C deficiency in the mother results in pregnancy failure in early gestation. Thrombomodulin (Thbd) or the Endothelial Protein C Receptor (EPCR/ProcR) gene deletions result in embryonic death, secondary to developmental and functional abnormalities of the placenta. These molecules play multiple roles in coagulation and inflammation. The mechanisms governing their role in placental development and maintenance of placental function remain to be fully understood. The objective of this work is to identify the critical functions of EPCR and Thbd that are required for placental development. Both Thbd and EPCR augment activated protein C generation, albeit to different extents. We have examined if reduced activated Protein C generation mediates placental abnormalities of EPCR- and Thbd-null mice. Activation of thrombin receptors expressed on platelets and trophoblast cells can also contribute to placental failure. We examined the role of thrombin receptor Par4 in placental failure of EPCR-null mice. Methods: To assess the role of a PC generation in placental phenotype of Thbd- and EPCR-null mice, we used a transgene to express a hyperactivatable form of murine protein C (hMPC) under the control of transthyretin promoter. Thrombin cleaves this mutant form of Protein C 30-fold more efficiently than wild type protein C, without requiring the cofactor function of thrombomodulin. Wild type mice expressing hMPC show 2-fold increase in PC and 3-fold increase in aPC levels. hMPC expression in PC-null mice restores their ability to carry pregnancies. Breeding strategies were used to generate hMPCtg ProcR+/- or hMPCtg Thbd+/- female mice. These were mated to ProcR+/- or Thbd+/- males, respectively, and survival of ProcR-/- and Thbd-/- embryos was analyzed. Similar genetic strategy was used to analyze the role of thrombin receptor Par4 in the demise of EPCR-null embryos. Placental phenotypes and embryonic survival was compared with experiments in which the mother was continuously infused with LMWH using a subcutaneous osmotic pump. Results: As previously reported, EPCR-null mice die before 10.5 days post coitum (dpc) (ProcR+/- intercrosses, out of 41 live embryos none were ProcR-/-, 10 were expected, 21 aborted not genotyped, 7 pregnancies analyzed at 11.5 dpc) and none are found at wean (out of 30 live pups none were ProcR-/-, 8 were expected, 5 litters analyzed). Transgenic expression of hMPC in the mother resulted in some live ProcR-/- embryos at 11.5 dpc (4 ProcR-/- out of 41 live embryos, 10 were expected, 15 aborted not genotyped, 7 pregnancies at 11.5 dpc) and pups at wean (2 ProcR-/- out of 28 live, 7 litters analyzed). Despite transgenic hMPC expression ProcR-/- embryos and pups were underrepresented (P=0.007, chi square GOF test). Surviving ProcR-/- embryos showed normal placental histology grossly comparable to littermate controls. Expression of hMPC in the mother did not ameliorate fetal death of Thbd-null mice (out of 38 live embryos none were Thbd-/-, 10 expected, 16 aborted not genotyped, 7 pregnancies at 9.5 dpc). Continuous infusion of LMWH also resulted in some live ProcR-/- embryos at 11.5 dpc (3 ProcR-/- out of 19 live embryos, 5 expected, 11 aborted not genotyped, 3 pregnancies analyzed), but two were growth retarded and all 3 placentae showed markedly reduced placental labyrinth formation. In contrast to transgenic expression of hMPC and treatment with LMWH, when Par4-/- ProcR+/- animals were intercrossed, ProcR-/- animals were born at an expected Mendelian frequency (7 ProcR-/- out of 35 live pups, 9 expected, 7 litters analyzed). Conclusions: Our results show that transgenic expression of hMPC allows normal placental development and rescues a fraction of EPCR-null embryos. Thus, placental defect of EPCR-null mice is in part mediated by reduced generation of aPC on placental cells. In contrast to the transgenic expression of hMPC and LMWH treatment, genetic absence of Par4 completely overcame the placental defect and allowed development of EPCR-null embryos. Further studies will clarify contributions of maternal versus fetal Par4 in this phenomenon. Disclosures No relevant conflicts of interest to declare.

Contribution of the 60 Loop To Substrate and Inhibitor Specificity of Thrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1734-1734 ◽  
Author(s):  
Alireza R. Rezaie
Keyword(s):  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Catalytic Efficiency ◽  
Inhibitor Specificity ◽  
Active Site Pocket ◽  
Catalytic Function ◽  
Target Molecules

Relative to chymotrypsin, the 60-loop of thrombin contains 8–9 insertion residues which are believed to be partly responsible for the restricted substrate and inhibitor specificity of thrombin. Previous deletion of 3–4 residues of this loop (des-PPW and des-YPPW) dramatically impaired the activity of thrombin toward antithrombin, protein C and fibrinogen, implicating a key role for the productive interaction of these residues with the target macromolecules. To further investigate the role of this loop, we expressed a mutant of thrombin in which all 8 insertion residues (Tyr-Pro-Pro-Trp-Asp-Lys-Asn-Phe) of the 60-loop were deleted (des-60-loop). In contrast to the partially deleted loop mutants, we discovered that des-60-loop thrombin cleaved small synthetic substrates, clotted purified fibrinogen, and activated protein C with a near normal catalytic efficiency; however, its activity toward cofactors V and VIII was impaired ~2–4-fold. Further studies revealed that the reactivity of des-60-loop with antithrombin is not impaired, but rather improved ~2-fold. Remarkably, the mutant could also activate prothrombin to thrombin. These results suggest that the 60-loop plays a key role in regulating the specificity of thrombin by shielding the active-site pocket; however, its productive interaction with the target molecules may not be as critical for the catalytic function of thrombin as has been speculated in previous reports.

P2 and P2′ Residues Are Important for the Efficient Proteolysis of Factor VIIIa at Arg336 Catalyzed by Activated Protein C.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2689-2689
Author(s):  
Fatbardha Varfaj ◽  
Jennifer DeAngelis ◽  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Protein C ◽  
Specific Activity ◽  
Activated Protein C ◽  
Cleavage Rate ◽  
Cleavage Efficiency ◽  
Factor Viiia ◽  
Fold Reduction ◽  
Sds Page ◽  
Recombinant Fviii

Abstract Activated Protein C (APC) is an anticoagulant serine protease that proteolytically inactivates the coagulation cofactors, factors (F) Va and VIIIa. FVIIIa is a non-covalent heterotrimer consisting of A1, A2 and A3-C1-C2 subunits. APC-catalyzed inactivation of FVIIIa results from proteolysis at the P1 residues Arg336 and Arg562 within the A1 and A2 subunits, respectively, with cleavage at Arg336 representing the dominant reaction. We recently showed that replacement of the P4-P3′ residues surrounding the Arg336 site with the corresponding residues flanking Arg562 resulted in an ∼100-fold reduction in cleavage rate at Arg336 (Varfaj et al, J. Biol. Chem.2007). Comparison of the P4-P3′ residues at the slow-reacting site in FVIIIa (Arg562) with the corresponding residues at the fast-reacting site in FVa (Arg506) revealed that these sequences differ primarily at the P2 and P2′ positions. This observation suggested an important contribution by these residues to cleavage efficiency. The role of the P2 and P2′ residues in the proteolysis of FVIIIa by APC was investigated by preparing recombinant FVIII proteins possessing mutations at Leu335 (P2) and Lys338 (P2′) flanking the P1 Arg336. B-domainless FVIII proteins were stably expressed in BHK cells and purified. Leu335Arg and Leu335Gln mutants were constructed based upon the P2 residues preceding Arg506 in FVa and Arg562 in FVIIIa, respectively. The Lys338Ile mutant was based upon the P2′ residue following Arg506 in FVa. Specific activity values for all FVIII variants were similar to wild type (WT) FVIII. APC-catalyzed inactivation rates for FVIIIa were determined using a FXa generation assay, and rates for proteolysis at the scissile bonds within the A1 and A2 subunits were determined by SDS-PAGE and Western blotting. Rates for APC-catalyzed cleavage of the A1 subunit for the FVIIIa variants Leu335Arg and Leu335Gln were reduced ∼2- and ∼4-fold, respectively, as compared to WT. However, the rate of cleavage of the A1 subunit in the Lys338Ile FVIIIa variant was enhanced ∼3-fold compared to WT. Cleavage rates at Arg562 in all the variants were unaffected by mutation at either residue 335 or 338. These relative values for rates of proteolysis paralleled the observed rates for inactivation of the FVIIIa forms. Overall, these results suggest that both P2 and P2′ residues are important in the efficient proteolysis at Arg336 in FVIIIa. Furthermore, Leu appears more optimal than either Arg or Gln at the P2 position, whereas Ile is preferred over Lys at the P2′ position in this macromolecular substrate for APC.

scholarly journals A thrombomodulin mutation that impairs activated protein C generation results in uncontrolled lung inflammation during murine tuberculosis

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2761-2768 ◽  
Author(s):  
Sebastiaan Weijer ◽  
Catharina W. Wieland ◽  
Sandrine Florquin ◽  
Tom van der Poll
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Inflammatory Response ◽  
Lung Inflammation ◽  
Protein C ◽  
Essential Role ◽  
Activated Protein C ◽  
Wild Type ◽  
Anti Inflammatory

AbstractThrombomodulin (TM) plays an essential role in the generation of activated protein C (APC), a mediator with both anticoagulant and anti-inflammatory properties, and is preferentially expressed in lungs. To investigate the role of TM in the coagulant and inflammatory response in the lung during tuberculosis, mice with a mutation in the TM gene (Thbd), which results in a minimal capacity for APC generation (TMpro/pro mice), were intranasally infected with live virulent Mycobacterium tuberculosis. Whereas pulmonary tuberculosis was not associated with activation of coagulation in either wild-type or TMpro/pro mice, 5 weeks after infection TMpro/pro mice displayed an uncontrolled inflammatory response in their lungs, as reflected by higher lung weights, a diminished ability to form well-shaped granulomas, elevated levels of proinflammatory cytokines, and concurrently reduced concentrations of anti-inflammatory cytokines. During a 36-week follow-up after infection with a lower dose of M tuberculosis, 35% of TMpro/pro mice died from week 28 onward versus none of the wild-type mice, and the surviving TMpro/pro mice displayed increased lung inflammation accompanied by higher mycobacterial loads in liver and spleen. These data suggest that a TM mutation that impairs APC generation results in uncontrolled lung inflammation during tuberculosis.

Deletion of the 60-loop provides new insights into the substrate and inhibitor specificity of thrombin

2005 ◽  
Vol 93 (06) ◽  
pp. 1047-1054 ◽  
Author(s):  
Likui Yang ◽  
Alireza Rezaie
Keyword(s):  
Mammalian Cells ◽  
Protein C ◽  
Activated Protein C ◽  
Catalytic Efficiency ◽  
Structural Data ◽  
Inhibitor Specificity ◽  
Direct Binding ◽  
Target Molecules ◽  
Inhibition Studies

SummaryStructural data have indicated that the 60-loop of thrombin with 8–9 insertion residues is responsible for the restricted substrate and inhibitor specificity of thrombin. However, previous deletion of 3–4 residues of this loop (des-PPW and des-YPPW) did not widen the specificity of thrombin, but further restricted it. The partial deletion of this loop also dramatically impaired the reactivity of thrombin with antithrombin (AT), protein C and fibrinogen, implicating a role for the productive interaction of the 60-loop with the target macromolecules. To further investigate the role of this loop, a mutant of thrombin was expressed in mammalian cells in which all 8 residues (Tyr-Pro-Pro-Trp-Asp-Lys-Asn-Phe) of the 60-loop were deleted (des-60-loop). In contrast to the partially deleted loop mutants, it was discovered that the des-60-loop mutant cleaved small synthetic substrates, clotted purified fibrinogen, and activated protein C with a near normal catalytic efficiency; however, its activity toward cofactors V and VIII was impaired ~2–4-fold. Direct binding and AT inhibition studies in the presence of heparin revealed that the affinity of heparin for interaction with exosite-2 of des-60-loop thrombin was impaired, though the reactivity of the mutant with AT and other plasma serpins was not impaired, but rather improved ~2-fold. These results suggest that the 60-loop plays a key role in regulating the specificity of thrombin by shielding the active-site pocket, but its productive interaction with the target molecules may not be as critical as has been speculated in previous reports.

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