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.

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.

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.

scholarly journals The functional significance of the autolysis loop in protein C and activated protein C

2005 ◽  
Vol 94 (07) ◽  
pp. 60-68 ◽  
Author(s):  
Likui Yang ◽  
Chandrashekhara Manithody ◽  
Alireza R. Rezaie
Keyword(s):  
Half Life ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Factor X ◽  
Binding Studies ◽  
Factor Va ◽  
Direct Binding ◽  
Anticoagulant Function

SummaryThe autolysis loop of activated protein C (APC) is five residues longer than the autolysis loop of other vitamin K-dependent coagulation proteases. To investigate the role of this loop in the zymogenic and anticoagulant properties of the molecule, a protein C mutant was constructed in which the autolysis loop of the protein was replaced with the corresponding loop of factor X. The protein C mutant was activated by thrombin with ~5-fold higher rate in the presence of Ca2+. Both kinetics and direct binding studies revealed that the Ca2+ affinity of the mutant has been impaired ∼3-fold. The result of a factorVa degradation assay revealed that the anticoagulant function of the mutant has been improved 4–5-fold in the absence but not in the presence of protein S. The improvement was due to a better recognition of both the P1-Arg506 and P1-Arg306 cleavage sites by the mutant protease. However, the plasma half-life of the mutant was markedly shortened due to faster inactivation by plasma serpins. These results suggest that the autolysis loop of protein C is critical for the Ca2+-dependence of activation by thrombin. Moreover, a longer autolysis loop in APC is not optimal for interaction with factor Va in the absence of protein S, but it contributes to the lack of serpin reactivity and longer half-life of the protease in plasma.

Protective role of activated protein C in lung and airway remodeling

2004 ◽  
Vol 32 (Supplement) ◽  
pp. S262-S265 ◽  
Author(s):  
Koji Suzuki ◽  
Esteban Cesar Gabazza ◽  
Tatsuya Hayashi ◽  
Haruhiko Kamada ◽  
Yukihiko Adachi ◽  
...  
Keyword(s):  
Protein C ◽  
Airway Remodeling ◽  
Activated Protein C ◽  
Protective Role

Sepsis and the Role of Activated Protein C

2004 ◽  
Vol 24 (6) ◽  
pp. 40-45
Author(s):  
Janice Tazbir
Keyword(s):  
Protein C ◽  
Activated Protein C

scholarly journals Role of coagulation pathways and treatment with activated protein C in hyperoxic lung injury

Thorax ◽  
2009 ◽  
Vol 64 (2) ◽  
pp. 114-120 ◽  
Author(s):  
M R Looney ◽  
C T Esmon ◽  
M A Matthay
Keyword(s):  
Lung Injury ◽  
Protein C ◽  
Activated Protein C ◽  
Hyperoxic Lung Injury

Antiphospholipid Antibodies with LAC Activity That Bind beta2-Glycoprotein I Cause Increased Resistance Against Activated Protein C.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3622-3622
Author(s):  
Bas de Laat ◽  
Sander B. Meijer ◽  
Carel M. Eckmann ◽  
M. van Schagen ◽  
Koen Mertens ◽  
...  
Keyword(s):  
Antiphospholipid Antibodies ◽  
Protein C ◽  
Activated Protein C ◽  
Plasma Samples ◽  
Surface Plasmon Resonance Analysis ◽  
Resonance Analysis ◽  
Igm Antibodies ◽  
Apc Resistance ◽  
Glycoprotein I ◽  
Direct Binding

Abstract Background: The antiphospholipid syndrome is characterized by the occurrence of vascular thrombosis combined with the presence of antiphospholipid antibodies (aPL) in plasma of patients. Recently it was published that aPL with lupus anticoagulant activity (LAC), caused by anti-beta2-glycoprotein I (beta2GPI) antibodies, highly correlate with a history of thrombosis. aPL-related resistance against activated protein C (APC) is one of the proposed mechanism responsible for thrombosis. We investigated a possible correlation between a beta2GPI-dependent LAC and increased APC-resistance in a population of 22 plasma samples with LAC activity. Methods: Twenty-two LAC-positive plasma samples were tested for beta2GPI-dependence (titration of cardiolipin into an APTT-based assay), increased APC-resistance, anti-beta2GPI IgG/IgM antibodies, anti-prothrombin IgG/IgM antibodies and anti-protein C IgG/IgM antibodies. In addition, a monoclonal anti-beta2GPI antibody and patient-purified IgG (both with LAC activity) were diluted in plasma with/without protein C and tested for occurrence of a beta2GPI-dependent LAC (normalization of clotting time by the addition of cardiolipin). To study aPL-induced APC-resistance in more detail, surface plasmon resonance analysis was used to investigate binding between APC and beta2GPI in the presence/absence of a mouse-derived monoclonal anti-beta2GPI antibody. Results: Eleven plasma samples that displayed a beta2GPI-dependent LAC also showed increased APC resistance. In contrast, only 1 of the 11 plasma samples with a beta2GPI-independent LAC displayed increased APC-resistance. None of the other serological parameters (antibodies against beta2-glycoprotein I, prothrombin or protein C) displayed the same association with increased APC resistance as a beta2-glycoprotein I dependent LAC. Furthermore, we found a linear correlation between the potency of a beta2GPI-dependent LAC and the level of APC-resistance. When a monoclonal anti-beta2GPI antibody and a patient-purified IgG were tested for a beta2GPI-dependent LAC, both antibodies did not display a beta2GPI-dependent LAC when diluted in protein C deficient plasma. In literature it has been proposed that direct binding of beta2GPI to APC results in a decreased activity of APC. By using surface plasmon resonance analysis, we found that beta2GPI displayed a higher affinity for coated APC in the presence of the monoclonal anti-beta2GPI antibody (4 nM) compared to beta2GPI alone (400 nM). Conclusion: The results of this study indicate that by adding cardiolipin into an APTT-based clotting assay, one can detect beta2GPI-dependent LAC based on increased resistance against APC. Increased resistance against activated protein C might result from direct binding of beta2GPI to activated protein C. In conclusion, our observations indicate a direct correlation between a major clinical symptom of APS (thrombosis), a diagnostic assay (beta2GPI-dependent LAC) and a potential mechanism responsible for thrombosis in the antiphospholipid syndrome (increased APC-resistance).

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