The Experts below are selected from a list of 261 Experts worldwide ranked by ideXlab platform
Frank C Church - One of the best experts on this subject based on the ideXlab platform.
-
Essential Thrombin residues for inhibition by protein C inhibitor with the cofactors heparin and thrombomodulin
Journal of Thrombosis and Haemostasis, 2007Co-Authors: Yolanda M Fortenberry, Herbert C. Whinna, Timothy Myles, Lawrence L K Leung, Frank C ChurchAbstract:BACKGROUND: Protein C inhibitor (PCI) and antiThrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including Thrombin. OBJECTIVE: Fifty-five Ala-scanned Recombinant Thrombin mutants were used to determine Thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT. RESULTS: Residues around the active site (Tyr50 and Glu202) and the sodium-binding site (Glu229 and Arg233) were required for Thrombin inhibition by PCI with and without cofactors. Exosite-2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin-accelerated inhibition of Thrombin by PCI. Exosite-1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of Thrombin-TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the approximately 150-fold enhanced rate of Thrombin inhibition by PCI. Using the aforementioned Thrombin exosite-2 mutants that were essential for heparin-catalyzed PCI-Thrombin inhibition reactions we found no change in PCI inhibition rates for Thrombin-TM. CONCLUSIONS: Collectively, these results show that (i) similar Thrombin exosite-2 residues are critical for the heparin-catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their Thrombin-TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of Thrombin-TM.
-
Essential Thrombin residues for inhibition by protein C inhibitor with the cofactors heparin and thrombomodulin
Journal of thrombosis and haemostasis : JTH, 2007Co-Authors: Yolanda M Fortenberry, Herbert C. Whinna, Timothy Myles, Lawrence L K Leung, Scott Cooper, Frank C ChurchAbstract:Summary. Background: Protein C inhibitor (PCI) and antiThrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including Thrombin. Objective: Fifty-five Ala-scanned Recombinant Thrombin mutants were used to determine Thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT. Results: Residues around the active site (Tyr50 and Glu202) and the sodium-binding site (Glu229 and Arg233) were required for Thrombin inhibition by PCI with and without cofactors. Exosite-2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin-accelerated inhibition of Thrombin by PCI. Exosite-1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of Thrombin–TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the ∼150-fold enhanced rate of Thrombin inhibition by PCI. Using the aforementioned Thrombin exosite-2 mutants that were essential for heparin-catalyzed PCI–Thrombin inhibition reactions we found no change in PCI inhibition rates for Thrombin–TM. Conclusions: Collectively, these results show that (i) similar Thrombin exosite-2 residues are critical for the heparin-catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their Thrombin–TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of Thrombin–TM.
-
Essential Thrombin Residues for Inhibition by Plasminogen Activator Inhibitor-1 in the Absence and Presence of Heparin and Vitronectin.
Blood, 2004Co-Authors: Yolanda M Fortenberry, Timothy Myles, Lawrence L K Leung, Jill C. Rau, Lauren C. Cranford, Frank C ChurchAbstract:The serine protease inhibitor (serpin), plasminogen activator inhibitor-1 (PAI-1) rapidly inactivates tissue plasminogen activator (tPA) and urokinase plasminogen activator to prevent plasminogen activation to plasmin. Although PAI-1 is a major regulator of fibrinolysis, PAI-1 also has a role in regulating coagulation due to its ability to inhibit Thrombin. Previous studies have shown that replacing the 39-loop of Thrombin with the 39-loop of tPA increases the rate of Thrombin inhibition by PAI-1 suggesting that the 39-loop of Thrombin is responsible for the relatively slow rate of inhibition by PAI-1 compared to tPA. Nevertheless, the role of other Thrombin residues in the Thrombin-PAI interaction [in the absence and presence of heparin and vitronectin (VN)] has not been fully investigated. We used 55 Recombinant Thrombin mutants in which solvent accessible residues are replaced with alanine to determine their effect on Thrombin-PAI-1, Thrombin-PAI-1-heparin and Thrombin-PAI-1-VN interactions. Results from this study identified Thrombin residues that either increased or decreased Thrombin inhibition by PAI-1 relative to wild-type Thrombin. First, we confirmed that Glu25 (E25A, located in the 39-loop) had an enhanced rate of inhibition by PAI-1 in the presence and absence of heparin and vitronectin. Also, Thrombin residues, Asn216/Asn217 (N216A/N217A, located in the 203–206 loop) and Lys145/Thr147/Trp148 (K145A/T147A/W148A, located in the autolysis loop), showed increased rates of Thrombin inhibition by PAI-1. These results suggest that these three Thrombin regions contribute to the slow rate of Thrombin inhibition by PAI-1. Second, we identified two anion-binding exosite-1 Thrombin mutants, R68A and to a lesser extent Y71A, that showed decreased rates of inhibition by PAI-1 compared to wild-type Thrombin. Consistent with this finding, there is a decrease in inhibition of γ-Thrombin (α-Thrombin proteolyzed in exosite-1) by PAI-1. These results suggest that Arg68 and Tyr71 of Thrombin exosite-1 are potential PAI-1 interacting residues since there is a decrease in inhibition in the absence and presence of heparin and VN. Third, we identified four anion-binding exosite-2 Thrombin mutants (R89A/R93A/E94A, R98, R178A/R180A/D183A, R245A/K248A/Q251) that are resistant to PAI-1-heparin accelerated inhibition compared to wild-type Thrombin, which implies that the Thrombin residues important for antiThrombin-heparin inhibition are also involved in the PAI-1-heparin inhibition reaction. By contrast, these exosite-2 Thrombin mutants are not as resistant to VN-accelerated PAI-1 inhibition, which indicates that exosite-2 is more important for heparin interaction than for VN interaction. Lastly, active site Thrombin mutants (W50A, D51A, E202A) and the sodium binding site Thrombin mutants (E233A, R233A) were very resistant to PAI-1 inhibition in the absence and presence of heparin and VN. Considering that Thrombin, PAI-1 and VN are localized in atherosclerotic arterial vessel wall, our results illustrate the importance of various Thrombin domains for PAI-1 inhibition with and without heparin and VN.
-
molecular mapping of the Thrombin heparin cofactor ii complex
Journal of Biological Chemistry, 2004Co-Authors: Yolanda M Fortenberry, Herbert C. Whinna, Holly R Gentry, Timothy Myles, Lawrence L K Leung, Frank C ChurchAbstract:Abstract We used 55 Ala-scanned Recombinant Thrombin molecules to define residues important for inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) in the absence and presence of glycosaminoglycans. We verified the importance of numerous basic residues in anion-binding exosite-1 (exosite-1) and found 4 additional residues, Gln24, Lys65, His66, and Tyr71 (using the Thrombin numbering system), that were resistant to HCII inhibition with and without glycosaminoglycans. Inhibition rate constants for these exosite-1 (Q24A, K65A, H66A, Y71A) Thrombin mutants (0.02-0.38 × 108 m-1 min-1 for HCII-heparin when compared with 2.36 × 108 m-1 min-1 with wild-type Thrombin and 0.03-0.53 × 108 m-1 min-1 for HCII-dermatan sulfate when compared with 5.23 × 108 m-1 min-1 with wild-type Thrombin) confirmed that the structural integrity of Thrombin exosite-1 is critical for optimal HCII-Thrombin interactions in the presence of glycosaminoglycans. However, our results are also consistent for HCII-glycosaminoglycan-Thrombin ternary complex formation. Ten residues surrounding the active site of Thrombin were implicated in HCII interactions. Four mutants (Asp51, Lys52, Lys145/Thr147/Trp148, Asp234) showed normal increased rates of inhibition by HCII-glycosaminoglycans, whereas four mutants (Trp50, Glu202, Glu229, Arg233) remained resistant to inhibition by HCII with glycosaminoglycans. Using 11 exosite-2 Thrombin mutants with 20 different mutated residues, we saw no major perturbations of HCII-glycosaminoglycan inhibition reactions. Collectively, our results support a “double bridge” mechanism for HCII inhibition of Thrombin in the presence of glycosaminoglycans, which relies in part on ternary complex formation but is primarily dominated by an allosteric process involving contact of the “hirudin-like” domain of HCII with Thrombin exosite-1.
-
Inhibition of a Thrombin anion-binding exosite-2 mutant by the glycosaminoglycan-dependent serpins protein C inhibitor and heparin cofactor II
Thrombosis Research, 2002Co-Authors: Scott Cooper, Alireza R. Rezaie, Charles T. Esmon, Frank C ChurchAbstract:Abstract AntiThrombin (ATIII), heparin cofactor II (HCII) and protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) are serine protease inhibitors (serpins) whose Thrombin inhibition activity is accelerated in the presence of glycosaminoglycans. We compared the inhibition properties of PCI and HCII to ATIII using R93A/R97A/R101A Thrombin, an anion-binding exosite-2 (exosite-2) mutant that has greatly reduced heparin-binding properties. Heparin-enhanced PCI inhibition of R93A/R97A/R101A Thrombin was only ∼2-fold compared to 40-fold enhancement with wild-type Recombinant Thrombin. Thrombomodulin (TM) (with or without the chondroitin sulfate moiety) accelerated PCI inhibition of both wild-type and R93A/R97A/R101A Thrombins. HCII achieved the same maximum activity in the presence of heparin with both wild-type and R93A/R97A/R101A Thrombins; however, the optimum heparin concentration was 20 times greater than the reaction with wild-type Thrombin, indicative of a decrease in heparin affinity. Dermatan sulfate (DSO4)-catalyzed HCII Thrombin inhibition was unchanged in R93A/R97A/R101A Thrombin compared to wild-type Recombinant Thrombin. These results suggest that PCI is similar to ATIII and depends upon ternary complex formation with heparin and these specific Thrombin exosite-2 residues to accelerate Thrombin inhibition. In contrast, HCII does not require Arg93, Arg97 and Arg101 of Thrombin exosite-2 and further supports the hypothesis that HCII uses an allosteric process following glycosaminoglycan binding to inhibit Thrombin.
W. Allan Alexander - One of the best experts on this subject based on the ideXlab platform.
-
Safety and Immunogenicity Observations Pooled from Eight Clinical Trials of Recombinant Human Thrombin
Journal of the American College of Surgeons, 2009Co-Authors: Jeffrey L. Ballard, Fred A Weaver, William C. Chapman, Neil Singla, W. Allan AlexanderAbstract:Background We evaluated safety and immunogenicity observations pooled from 8 clinical trials of Recombinant human Thrombin (rThrombin), an active topical hemostatic agent. Study Design Recombinant Thrombin was applied with an absorbable gelatin sponge or spray applicator during a surgical procedure (day 1). Adverse events and laboratory parameters were monitored until study end (day 29). Immunogenicity was evaluated after study completion on plasma samples collected at baseline and on day 29. Results Studies included 583 rThrombin-treated patients (median age, 59 years; 54% men). Surgical procedures included: spinal, 33% of patients; hepatic resection, 14%; peripheral arterial bypass, 23%; arteriovenous graft formation for hemodialysis access, 18%; and skin graft after burn wound excision, 12%. Adverse events reported for ≥ 10% patients included incision site pain, procedural pain, nausea, constipation, pyrexia, anemia, insomnia, vomiting, and pruritus. Five of 552 patients developed antibodies to rThrombin (0.9%; 95% CI, 0.3 to 2.1; day 29); antibodies did not neutralize the biologic activity of native human Thrombin. At baseline, 12 patients had pre-existing, antibodies recognizing rThrombin (12 of 552; 2.2%; 95% CI, 1.1 to 3.8); these patients had no previous exposure to rThrombin and their antibody titer did not increase ≥ 1.0 unit (≥ 10-fold) at day 29. Conclusions Results from 8 clinical trials collectively demonstrated that rThrombin is well tolerated in numerous surgical settings when used as a topical adjunct to hemostasis. Adverse events and changes in laboratory parameters were consistent with commonly reported postoperative events. Less than 1% of patients developed antibodies to rThrombin; the antibodies did not neutralize native human Thrombin.
-
Topical Recombinant Thrombin at a concentration of 1000 IU/mL reliably shortens in vivo TTH and delivers durable hemostasis in the presence of heparin anticoagulation and clopidogrel platelet inhibition in a rabbit model of vascular bleeding
Annals of Surgical Innovation and Research, 2009Co-Authors: Steven D Hughes, Paul D Bishop, Richard Garcia, Tracy Zhang, W. Allan AlexanderAbstract:Background This study was designed to evaluate the effect of Recombinant human Thrombin (rThrombin) concentration on time to hemostasis (TTH), clot durability, and clot strength in settings that replicate the heparinization and platelet inhibition often found in surgical populations. Methods A modified, anticoagulated rabbit arteriovenous shunt preparation was selected to model vascular anastomotic bleeding. Rabbits were treated with heparin or heparin + clopidogrel and TTH was measured after applying a range of topical rThrombin concentrations or placebo, in combination with absorbable gelatin sponge, USP. Treatments (placebo, rThrombin) were randomly assigned and the investigator was blinded to treatment. TTH was evaluated with the Kaplan-Meier method. After hemostasis was achieved, clot burst assessment was performed for heparin + clopidogrel treated animals. Clot viscoelastic strength and kinetics were measured in ex-vivo samples using thromboelastography (TEG) methods. Results TTH decreased with increasing concentrations of rThrombin in heparin-treated animals and was shorter after treatment with 1000 IU/mL rThrombin (73 seconds) than with 125 IU/mL rThrombin (78 seconds; p = 0.007). TTH also decreased with increasing concentrations of rThrombin in heparin + clopidogrel treated animals; again it was significantly shorter after treatment with 1000 IU/mL rThrombin (71 seconds) than with 125 IU/mL rThrombin (177 seconds; p
-
Topical Recombinant Thrombin at a concentration of 1000 IU/mL reliably shortens in vivo TTH and delivers durable hemostasis in the presence of heparin anticoagulation and clopidogrel platelet inhibition in a rabbit model of vascular bleeding.
Annals of surgical innovation and research, 2009Co-Authors: Steven D Hughes, Paul D Bishop, Tracy Zhang, Richard M. Garcia, W. Allan AlexanderAbstract:Background This study was designed to evaluate the effect of Recombinant human Thrombin (rThrombin) concentration on time to hemostasis (TTH), clot durability, and clot strength in settings that replicate the heparinization and platelet inhibition often found in surgical populations.
-
A Phase 3b, Open-Label, Single-Group Immunogenicity and Safety Study of Topical Recombinant Thrombin in Surgical Hemostasis
Journal of the American College of Surgeons, 2009Co-Authors: Neil Singla, Kenneth L. Renkens, Jeffrey L. Ballard, Gregory L. Moneta, C. Duane Randleman, W. Allan AlexanderAbstract:Background The immunogenicity and safety of Recombinant human Thrombin (rThrombin) were evaluated in this phase 3b, open-label, single-group, multisite study of 209 adult vascular and spinal operation patients at high risk for preexisting anti-bovine Thrombin product antibodies. Study Design Patients received rThrombin applied as a topical hemostat during a surgical procedure (day 1). Immunogenicity samples were collected at baseline and approximately 1 month after operation (day 29) and were analyzed after study participation. Results Mean patient age was 61.5 years; median number of previous surgical procedures was 5.0 (range, 1 to 25). Operation types included spinal (n = 89 of 209 [43%]), arterial reconstruction (including peripheral arterial bypass; n=75 of 209 [36%]), and arteriovenous vascular access procedures (n = 45 of 209 [22%]). All patients had confirmed or highly likely previous bovine Thrombin exposure; at baseline, 15.6% of patients (n = 32 of 205) had preexisting anti-bovine Thrombin antibodies. Of 200 patients with complete immunogenicity evaluations, 31 had preexisting anti-bovine Thrombin antibodies (15.5%), and 4 had preexisting anti-rThrombin product antibodies (2.0%). None of the 200 patients became antibody positive for rThrombin antibodies on day 29 (seroconversion or ≥ 10-fold increase in titer). Adverse events and laboratory results were consistent with a surgical population with substantial comorbidities. Patients with preexisting antibodies to bovine Thrombin were older (p = 0.04) and had undergone more surgical procedures previously (p Conclusions Results of this study confirm the low immunogenicity of rThrombin and suggest that rThrombin can be used safely as an aid to hemostasis in patients with or without preexisting anti-bovine Thrombin antibodies. A sizeable proportion of this vascular and spinal operation patient population (15.6%) had preexisting anti-bovine Thrombin antibodies; these patients are at risk for immune responses after reexposure to bovine Thrombin.
-
Favorable Immunologic Response Profile to Recombinant Thrombin Application in Surgery: An Integrated Analysis of 8 Clinical Trials
Blood, 2008Co-Authors: William C. Chapman, Kenneth L. Renkens, Richard L. Gamelli, Carlton Randleman, Jeffrey L. Ballard, W. Allan AlexanderAbstract:Recombinant Thrombin (rThrombin) is an active topical hemostat approved by the FDA as an aid to hemostasis. Treatment with Thrombin derived from bovine sources has occasionally been associated with adverse events which appear to be related to the formation of antibodies against bovine Thrombin and/or Factor V. The safety and immunogenicity of rThrombin have been evaluated in 8 clinical trials: 1 Phase 1 study, 5 Phase 2 studies, 1 randomized, double-blind Phase 3 study, and 1 open-label, single-group Phase 3b study. Pooled results from these trials provide a consistent and clinically relevant summary of safety information on the use of rThrombin in surgery. These trials utilized standardized data collection methods; more than 100 investigators from over 60 institutions participated. With 514 subjects treated with rThrombin in clinical trials to date (cut-off date July 2008), these results represent the largest safety database available for any commercially-available Thrombin product. Standard safety measures were collected prior to surgery (baseline) through Day 29 post-surgery. Immunogenicity was evaluated at baseline and Day 29 using enzyme-linked immunosorbent assays and a time-to-clot assay to measure neutralizing potential of any detected anti-rThrombin antibodies. Of the subjects for whom data are currently available (N=514), 54% were male and 46% were female. Mean age was 57.0 years (SD, 15.7; range, 12–89 years). The following surgery types were represented: spine, 32% of patients; liver, 14%; peripheral arterial bypass, 23%; arteriovenous graft, 18%; burn, 12%. The median dose of rThrombin was 10,000 IU (range, 1000–50,000 IU). Topical application of rThrombin was well tolerated by study subjects. Common adverse events (AEs; reported by ≥10% of subjects) were similar regardless of surgery type and were consistent with the post-surgical setting. Common AEs included incision site complication, procedural pain, nausea, constipation, pyrexia, anemia, vomiting, insomnia, incision site pain, and pruritus. No single serious adverse event type occurred in >1% of patients. Twelve of 514 subjects (2.3%) had anti-rThrombin product antibodies at baseline, a finding that is consistent with reports of antibodies to other Recombinant forms of endogenous proteins. None of the 12 subjects with anti-rThrombin product antibodies at baseline had a change in titer ≥1.0 unit at Day 29. Among subjects who did not have anti-rThrombin product antibodies at baseline, 5/502 (1.0%) had detectable antibodies at Day 29. In total, 5/514 subjects (1.0%) were antibody positive at Day 29. None of the antibodies neutralized native human Thrombin. These clinical studies, enrolling 514 subjects to date, collectively demonstrate that rThrombin is well-tolerated and has a favorable immunologic response profile when used in numerous surgical settings.
Srinath Samudrala - One of the best experts on this subject based on the ideXlab platform.
-
Risk of bleeding in surgical patients treated with topical bovine Thrombin sealants: a review of the literature.
Patient safety in surgery, 2008Co-Authors: Matthew W. Reynolds, John Clark, Sheila Crean, Srinath SamudralaAbstract:Background One of the most anticipated, but potentially serious complications during or after surgery are bleeding events. Among the many potential factors associated with bleeding complications in surgery, the use of bovine Thrombin has been anecdotally identified as a possible cause of increased bleeding risk. Most of these reports of bleeding events in association with the use of topical bovine Thrombin have been limited to case reports lacking clear cause and effect relationship determination. Recent studies have failed to establish significant differences in the rates of bleeding events between those treated with bovine Thrombin and those treated with either human or Recombinant Thrombin.
-
Risk of bleeding in surgical patients treated with topical bovine Thrombin sealants: a review of the literature
Patient Safety in Surgery, 2008Co-Authors: Matthew W. Reynolds, John Clark, Sheila Crean, Srinath SamudralaAbstract:Background One of the most anticipated, but potentially serious complications during or after surgery are bleeding events. Among the many potential factors associated with bleeding complications in surgery, the use of bovine Thrombin has been anecdotally identified as a possible cause of increased bleeding risk. Most of these reports of bleeding events in association with the use of topical bovine Thrombin have been limited to case reports lacking clear cause and effect relationship determination. Recent studies have failed to establish significant differences in the rates of bleeding events between those treated with bovine Thrombin and those treated with either human or Recombinant Thrombin. Methods We conducted a search of MEDLINE for the most recent past 10 years (1997–2007) and identified all published studies that reported a study of surgical patients with a clear objective to examine the risk of bleeding events in surgical patients. We also specifically noted the reporting of any topical bovine Thrombin used during surgical procedures. We aimed to examine whether there were any differences in the risk of bleeds in general surgical populations as compared to those studies that reported exposure to topical bovine Thrombin. Results We identified 21 clinical studies that addressed the risk of bleeding in surgery. Of these, 5 studies analyzed the use of bovine Thrombin sealants in surgical patients. There were no standardized definitions for bleeding events employed across these studies. The rates of bleeds in the general surgery studies ranged from 0.1%–20.2%, with most studies reporting rates between 2.6%–4%. The rates of bleeding events ranged from 0.0%–13% in the bovine Thrombin studies with most studies reporting between a 2%–3% rate. Conclusion The risk of bleeds was not clearly different in those studies reporting use of bovine Thrombin in all patients compared to the other surgical populations studied. A well-designed and well-controlled study is needed to accurately examine the bleeding risks in surgical patients treated and unexposed to topical bovine Thrombin, and to evaluate the independent risk associated with topical bovine Thrombin as well as other risk factors.
Harold A. Scheraga - One of the best experts on this subject based on the ideXlab platform.
-
Thrombin Specificity: Further Evidence for the Importance of the β-Insertion Loop and Trp^96. Implications of the Hydrophobic Interaction Between Trp^96 and Pro^60B Pro^60C for the Activity of Thrombin
Journal of Protein Chemistry, 1998Co-Authors: Elsie E. Dibella, Harold A. ScheragaAbstract:A number of Thrombin mutants have been constructed to investigate the role of Trp^96 and the β-insertion loop for the specificity of Thrombin. Thrombin(60D) consists of the replacement of the β-insertion loop (14 amino acid residues from 59 to 63, including a 9-residue insertion at position 60) with the corresponding four residues in trypsin, Tyr-Lys-Ser-Gly; Thrombin(GGG) is a smaller loop mutation in which the residues Tyr^60APro^60BPro^60CTrp^60D Asp^60ELys^60F of the β-insertion loop were replaced by Gly-Gly-Gly; Thrombin(96S) consists of a point mutation Trp^96→Ser; and Thrombin(GGG/96S) is the double mutant incorporating both changes. Thrombin(96S) clots fibrinogen ~3 times more slowly than Thrombin, with the two β-insertion loop mutants, Thrombin(GGG) and Thrombin(GGG/96S), reacting ~3000- and 1300-fold more slowly, respectively. The specificity constant k _cat/ K _m for the cleavage of fibrinopeptide A and fibrinopeptide B by Thrombin(96S) was 2.6 and 0.35 μM^−1 s^−1 respectively, compared to 10 and 2.5 μM^−1 s^−1 for wild-type Recombinant Thrombin, respectively. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine- p -nitroaniline. The Michaelis constant K _m increased ~6-fold for Thrombin(96S) and >200-fold for Thrombin(GGG) and Thrombin(GGG/96S) when compared to wild-type Recombinant Thrombin, while the catalytic constant k _cat remained approximately the same. All mutants were more susceptible to inhibition by BPTI than wild-type Recombinant Thrombin. Clearly, the β-insertion loop is important for Thrombin activity. But the mutation of Trp^96→Ser can compensate somewhat for the loss of binding at the β-insertion loop. The deletion of the hydrophobic interaction between Trp^96 and Pro^60BPro^60C appears to decrease the stability of the β-insertion loop, thereby causing a decrease in binding efficiency.
-
Thrombin specificity: further evidence for the importance of the beta-insertion loop and Trp96. Implications of the hydrophobic interaction between Trp96 and Pro60B Pro60C for the activity of Thrombin.
Journal of protein chemistry, 1998Co-Authors: Elsie E. Dibella, Harold A. ScheragaAbstract:A number of Thrombin mutants have been constructed to investigate the role of Trp96 and the β-insertion loop for the specificity of Thrombin. Thrombin(60D) consists of the replacement of the β-insertion loop (14 amino acid residues from 59 to 63, including a 9-residue insertion at position 60) with the corresponding four residues in trypsin, Tyr-Lys-Ser-Gly; Thrombin(GGG) is a smaller loop mutation in which the residues Tyr60APro60BPro60CTrp60D Asp60ELys60F of the β-insertion loop were replaced by Gly-Gly-Gly; Thrombin(96S) consists of a point mutation Trp96→Ser; and Thrombin(GGG/96S) is the double mutant incorporating both changes. Thrombin(96S) clots fibrinogen ~3 times more slowly than Thrombin, with the two β-insertion loop mutants, Thrombin(GGG) and Thrombin(GGG/96S), reacting ~3000- and 1300-fold more slowly, respectively. The specificity constant kcat/Km for the cleavage of fibrinopeptide A and fibrinopeptide B by Thrombin(96S) was 2.6 and 0.35 μM−1 s−1 respectively, compared to 10 and 2.5 μM−1 s−1 for wild-type Recombinant Thrombin, respectively. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline. The Michaelis constant Km increased ~6-fold for Thrombin(96S) and >200-fold for Thrombin(GGG) and Thrombin(GGG/96S) when compared to wild-type Recombinant Thrombin, while the catalytic constant kcat remained approximately the same. All mutants were more susceptible to inhibition by BPTI than wild-type Recombinant Thrombin. Clearly, the β-insertion loop is important for Thrombin activity. But the mutation of Trp96→Ser can compensate somewhat for the loss of binding at the β-insertion loop. The deletion of the hydrophobic interaction between Trp96 and Pro60BPro60C appears to decrease the stability of the β-insertion loop, thereby causing a decrease in binding efficiency.
-
The Role of the Insertion Loop around Tryptophan 148 in the Activity of Thrombin
Biochemistry, 1996Co-Authors: Elsie E. Dibella, Harold A. ScheragaAbstract:Thrombin has trypsin-like specificity for Arg-Xaa and Lys-Xaa peptide bonds; however, it is much more specific than trypsin, cleaving far fewer peptide bonds in macromolecular substrates. To probe the nature of the specificity of Thrombin, a mutant has been constructed in which the Trp148 loop of Thrombin has been replaced with the same loop of bovine trypsin. This mutant was expressed in Escherichia coli as preThrombin-2(148) using a T7 expression system previously described for wild-type preThrombin-2 [DiBella et al. (1995) J. Biol. Chem. 270, 163−169]. After refolding and purification, preThrombin-2(148) was activated to Thrombin(148) with Echis carinatus snake venom. The kcat/Km for the release of fibrinopeptide A from fibrinogen was 4.5 ± 0.5 μM-1 s-1 for Thrombin(148), which was ∼20% of that of Recombinant Thrombin (25 ± 2.0 μM-1 s-1). Thrombin(148) was inhibited less well by hirudin with a Ki of 500 pM compared to a value of 12 pM determined for Recombinant Thrombin. The mutant Thrombin was also co...
-
Expression and Folding of Recombinant Bovine PreThrombin-2 and Its Activation to Thrombin
The Journal of biological chemistry, 1995Co-Authors: Elsie E. Dibella, Muriel C. Maurer, Harold A. ScheragaAbstract:Bovine preThrombin-2 has been produced in Escherichia coli using a T7 expression system. The expressed preThrombin-2 formed intracellular inclusion bodies which were solubilized by reversible sulfonation of the cysteines in the presence of 7 M guanidine hydrochloride. Sulfonated preThrombin-2 was refolded in the presence of 4 M guanidine hydrochloride, using oxidized and reduced glutathione as the redox couple. The folded protein was purified by heparin affinity chromatography and activated to Thrombin with Echis carinatus snake venom. The resulting Thrombin was also purified by heparin affinity chromatography. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline by Recombinant Thrombin (kcat = 123 ± 10 s−1 and Km = 2.91 ± 0.3 μM). These values are in good agreement with those determined for wild-type Thrombin (kcat = 97 ± 8 s−1 and Km = 2.71 ± 0.25 μM). From the Thrombin-mediated release of fibrinopeptide A from fibrinogen, kcat/Kmwas found to be the same for Recombinant (17.3 ± 1.2 μM−1 s−1) and wild-type (16.7 ± 2.0 μM−1 s−1) Thrombin. These results, taken together with circular dichroism spectra and the elution position of preThrombin-2 from a heparin affinity resin, indicate that preThrombin-2 was folded into a conformation similar to that of the wild-type protein. In addition, since E. coli produces deglycosylated enzymes, these findings suggest that the carbohydrate on the B chain of wild-type Thrombin does not affect the amidolytic and fibrinolytic activities of Thrombin. Finally, this expression system can be used to prepare mutants of preThrombin-2 for future structure-function studies involving Thrombin and its substrates; some preliminary results of this type are presented here.
Timothy Myles - One of the best experts on this subject based on the ideXlab platform.
-
Essential Thrombin residues for inhibition by protein C inhibitor with the cofactors heparin and thrombomodulin
Journal of Thrombosis and Haemostasis, 2007Co-Authors: Yolanda M Fortenberry, Herbert C. Whinna, Timothy Myles, Lawrence L K Leung, Frank C ChurchAbstract:BACKGROUND: Protein C inhibitor (PCI) and antiThrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including Thrombin. OBJECTIVE: Fifty-five Ala-scanned Recombinant Thrombin mutants were used to determine Thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT. RESULTS: Residues around the active site (Tyr50 and Glu202) and the sodium-binding site (Glu229 and Arg233) were required for Thrombin inhibition by PCI with and without cofactors. Exosite-2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin-accelerated inhibition of Thrombin by PCI. Exosite-1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of Thrombin-TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the approximately 150-fold enhanced rate of Thrombin inhibition by PCI. Using the aforementioned Thrombin exosite-2 mutants that were essential for heparin-catalyzed PCI-Thrombin inhibition reactions we found no change in PCI inhibition rates for Thrombin-TM. CONCLUSIONS: Collectively, these results show that (i) similar Thrombin exosite-2 residues are critical for the heparin-catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their Thrombin-TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of Thrombin-TM.
-
Essential Thrombin residues for inhibition by protein C inhibitor with the cofactors heparin and thrombomodulin
Journal of thrombosis and haemostasis : JTH, 2007Co-Authors: Yolanda M Fortenberry, Herbert C. Whinna, Timothy Myles, Lawrence L K Leung, Scott Cooper, Frank C ChurchAbstract:Summary. Background: Protein C inhibitor (PCI) and antiThrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including Thrombin. Objective: Fifty-five Ala-scanned Recombinant Thrombin mutants were used to determine Thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT. Results: Residues around the active site (Tyr50 and Glu202) and the sodium-binding site (Glu229 and Arg233) were required for Thrombin inhibition by PCI with and without cofactors. Exosite-2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin-accelerated inhibition of Thrombin by PCI. Exosite-1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of Thrombin–TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the ∼150-fold enhanced rate of Thrombin inhibition by PCI. Using the aforementioned Thrombin exosite-2 mutants that were essential for heparin-catalyzed PCI–Thrombin inhibition reactions we found no change in PCI inhibition rates for Thrombin–TM. Conclusions: Collectively, these results show that (i) similar Thrombin exosite-2 residues are critical for the heparin-catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their Thrombin–TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of Thrombin–TM.
-
Glycoprotein Ibα–Mediated Platelet Adhesion and Aggregation to Immobilized Thrombin Under Conditions of Flow
Arteriosclerosis thrombosis and vascular biology, 2005Co-Authors: Cees Weeterings, Timothy Myles, Jelle Adelmeijer, Philip G. De Groot, Ton LismanAbstract:Objectives— Thrombin interacts with platelets via the protease-activated receptors (PARs) 1 and 4, and via glycoprotein Ibα (GPIbα). Recently, it was shown that platelets are able to adhere to immobilized Thrombin under static conditions via GPIbα. Methods and Results— Here, we show that platelets are also able to adhere to and form stable aggregates on immobilized Thrombin under conditions of flow. Adhesion and aggregation to Thrombin was dependent on the interaction with GPIbα, as addition of glycocalicin or an antibody blocking the interaction between Thrombin and GPIbα inhibited platelet adhesion. Additionally, platelet adhesion to Recombinant Thrombin mutants, which are unable to bind GPIbα, was severely suppressed. Furthermore, platelet adhesion to Thrombin was dependent on activation of PARs, and partly on granule secretion and thromboxane-A2 synthesis. Immobilization of Thrombin on a fibrin network resulted in substantially increased adhesion compared with fibrin alone. The adhesion to fibrin alone was completely abolished by addition of dRGDW, whereas fibrin-bound Thrombin still showed substantial platelet adhesion in the presence of dRGDW, indicating that fibrin-bound Thrombin is able to directly capture platelets under flow. Conclusion— These results indicate that platelets are able to adhere to Thrombin under flow conditions, which is dependent on the interaction with GPIbα.
-
Essential Thrombin Residues for Inhibition by Plasminogen Activator Inhibitor-1 in the Absence and Presence of Heparin and Vitronectin.
Blood, 2004Co-Authors: Yolanda M Fortenberry, Timothy Myles, Lawrence L K Leung, Jill C. Rau, Lauren C. Cranford, Frank C ChurchAbstract:The serine protease inhibitor (serpin), plasminogen activator inhibitor-1 (PAI-1) rapidly inactivates tissue plasminogen activator (tPA) and urokinase plasminogen activator to prevent plasminogen activation to plasmin. Although PAI-1 is a major regulator of fibrinolysis, PAI-1 also has a role in regulating coagulation due to its ability to inhibit Thrombin. Previous studies have shown that replacing the 39-loop of Thrombin with the 39-loop of tPA increases the rate of Thrombin inhibition by PAI-1 suggesting that the 39-loop of Thrombin is responsible for the relatively slow rate of inhibition by PAI-1 compared to tPA. Nevertheless, the role of other Thrombin residues in the Thrombin-PAI interaction [in the absence and presence of heparin and vitronectin (VN)] has not been fully investigated. We used 55 Recombinant Thrombin mutants in which solvent accessible residues are replaced with alanine to determine their effect on Thrombin-PAI-1, Thrombin-PAI-1-heparin and Thrombin-PAI-1-VN interactions. Results from this study identified Thrombin residues that either increased or decreased Thrombin inhibition by PAI-1 relative to wild-type Thrombin. First, we confirmed that Glu25 (E25A, located in the 39-loop) had an enhanced rate of inhibition by PAI-1 in the presence and absence of heparin and vitronectin. Also, Thrombin residues, Asn216/Asn217 (N216A/N217A, located in the 203–206 loop) and Lys145/Thr147/Trp148 (K145A/T147A/W148A, located in the autolysis loop), showed increased rates of Thrombin inhibition by PAI-1. These results suggest that these three Thrombin regions contribute to the slow rate of Thrombin inhibition by PAI-1. Second, we identified two anion-binding exosite-1 Thrombin mutants, R68A and to a lesser extent Y71A, that showed decreased rates of inhibition by PAI-1 compared to wild-type Thrombin. Consistent with this finding, there is a decrease in inhibition of γ-Thrombin (α-Thrombin proteolyzed in exosite-1) by PAI-1. These results suggest that Arg68 and Tyr71 of Thrombin exosite-1 are potential PAI-1 interacting residues since there is a decrease in inhibition in the absence and presence of heparin and VN. Third, we identified four anion-binding exosite-2 Thrombin mutants (R89A/R93A/E94A, R98, R178A/R180A/D183A, R245A/K248A/Q251) that are resistant to PAI-1-heparin accelerated inhibition compared to wild-type Thrombin, which implies that the Thrombin residues important for antiThrombin-heparin inhibition are also involved in the PAI-1-heparin inhibition reaction. By contrast, these exosite-2 Thrombin mutants are not as resistant to VN-accelerated PAI-1 inhibition, which indicates that exosite-2 is more important for heparin interaction than for VN interaction. Lastly, active site Thrombin mutants (W50A, D51A, E202A) and the sodium binding site Thrombin mutants (E233A, R233A) were very resistant to PAI-1 inhibition in the absence and presence of heparin and VN. Considering that Thrombin, PAI-1 and VN are localized in atherosclerotic arterial vessel wall, our results illustrate the importance of various Thrombin domains for PAI-1 inhibition with and without heparin and VN.
-
molecular mapping of the Thrombin heparin cofactor ii complex
Journal of Biological Chemistry, 2004Co-Authors: Yolanda M Fortenberry, Herbert C. Whinna, Holly R Gentry, Timothy Myles, Lawrence L K Leung, Frank C ChurchAbstract:Abstract We used 55 Ala-scanned Recombinant Thrombin molecules to define residues important for inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) in the absence and presence of glycosaminoglycans. We verified the importance of numerous basic residues in anion-binding exosite-1 (exosite-1) and found 4 additional residues, Gln24, Lys65, His66, and Tyr71 (using the Thrombin numbering system), that were resistant to HCII inhibition with and without glycosaminoglycans. Inhibition rate constants for these exosite-1 (Q24A, K65A, H66A, Y71A) Thrombin mutants (0.02-0.38 × 108 m-1 min-1 for HCII-heparin when compared with 2.36 × 108 m-1 min-1 with wild-type Thrombin and 0.03-0.53 × 108 m-1 min-1 for HCII-dermatan sulfate when compared with 5.23 × 108 m-1 min-1 with wild-type Thrombin) confirmed that the structural integrity of Thrombin exosite-1 is critical for optimal HCII-Thrombin interactions in the presence of glycosaminoglycans. However, our results are also consistent for HCII-glycosaminoglycan-Thrombin ternary complex formation. Ten residues surrounding the active site of Thrombin were implicated in HCII interactions. Four mutants (Asp51, Lys52, Lys145/Thr147/Trp148, Asp234) showed normal increased rates of inhibition by HCII-glycosaminoglycans, whereas four mutants (Trp50, Glu202, Glu229, Arg233) remained resistant to inhibition by HCII with glycosaminoglycans. Using 11 exosite-2 Thrombin mutants with 20 different mutated residues, we saw no major perturbations of HCII-glycosaminoglycan inhibition reactions. Collectively, our results support a “double bridge” mechanism for HCII inhibition of Thrombin in the presence of glycosaminoglycans, which relies in part on ternary complex formation but is primarily dominated by an allosteric process involving contact of the “hirudin-like” domain of HCII with Thrombin exosite-1.