Design and characterization of α1-antitrypsin variants for treatment of contact system–driven thromboinflammation

Blood ◽  
2019 ◽  
Vol 134 (19) ◽  
pp. 1658-1669 ◽  
Author(s):  
Steven de Maat ◽  
Wariya Sanrattana ◽  
Reiner K. Mailer ◽  
Naomi M. J. Parr ◽  
Martin Hessing ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Therapeutic Strategies ◽  
Contact System ◽  
Amino Acid Substitutions ◽  
Contact Activation ◽  
Elastase Inhibitor ◽  
Hemostatic System ◽  
Simple Amino Acid

Targeting natural anticoagulant proteins as a means to rebalance the hemostatic system is an emerging trend in the development of innovative therapeutic strategies for hemophilia. These 2 articles develop these concepts in the areas of hemostasis and contact activation. In the study by Aymonnier and colleagues, simple amino acid substitutions converted a serpin elastase inhibitor, α1-antitrypsin (α1AT), into a potent antithrombin, activated protein C inhibitor, or anti-PKa/FXIIa inhibitor. In the study by de Maat and colleagues, redesign of α1AT strongly altered its inhibitory behavior and enables it to be used for the treatment of contact system–mediated thrombosis and inflammation.

scholarly journals Targeting protease nexin-1, a natural anticoagulant serpin, to control bleeding and improve hemostasis in hemophilia

Blood ◽  
2019 ◽  
Vol 134 (19) ◽  
pp. 1632-1644 ◽  
Author(s):  
Karen Aymonnier ◽  
Charlotte Kawecki ◽  
Laurence Venisse ◽  
Yacine Boulaftali ◽  
Olivier D. Christophe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein C ◽  
Activated Protein C ◽  
Therapeutic Strategies ◽  
Amino Acid Substitutions ◽  
Contact Activation ◽  
Elastase Inhibitor ◽  
Hemostatic System ◽  
Protease Nexin ◽  
Simple Amino Acid

Targeting natural anticoagulant proteins as a means to rebalance the hemostatic system is an emerging trend in the development of innovative therapeutic strategies for hemophilia. These 2 articles develop these concepts in the areas of hemostasis and contact activation. In the study by Aymonnier and colleagues, simple amino acid substitutions converted a serpin elastase inhibitor, α1-antitrypsin (α1AT), into a potent antithrombin, activated protein C inhibitor, or anti-PKa/FXIIa inhibitor. In the study by de Maat and colleagues, redesign of α1AT strongly altered its inhibitory behavior and enables it to be used for the treatment of contact system–mediated thrombosis and inflammation.

Characterization of a cDNA for Rhesus Monkey Protein C Inhibitor – Evidence for N-Terminal Involvement in Heparin Stimulation

1995 ◽  
Vol 74 (04) ◽  
pp. 1079-1087 ◽  
Author(s):  
Klaus-P Radtke ◽  
José A Fernández ◽  
Bruno O Villoutreix ◽  
Judith S Greengard ◽  
John H Griffin
Keyword(s):  
Rhesus Monkey ◽  
Protein C ◽  
Activated Protein C ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Heparin Binding ◽  
Reactive Center ◽  
Protein C Inhibitor ◽  
Polymerase Chain

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

scholarly journals Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 642-647 ◽  
Author(s):  
K Murakami ◽  
K Okajima ◽  
M Uchiba ◽  
M Johno ◽  
T Nakagaki ◽  
...  
Keyword(s):  
Vascular Permeability ◽  
Vascular Injury ◽  
Active Site ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Myeloperoxidase Activity ◽  
Granulocyte Elastase ◽  
Pulmonary Vascular Permeability ◽  
Elastase Inhibitor

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS- induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.

scholarly journals Coagulation and fibrinolytic activities in 2 siblings with β2-glycoprotein I deficiency

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1594-1595
Author(s):  
Rie Takeuchi ◽  
Tatsuya Atsumi ◽  
Masahiro Ieko ◽  
Hiroyuki Takeya ◽  
Shinsuke Yasuda ◽  
...  
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Healthy Woman ◽  
Contact Activation ◽  
Glycoprotein I ◽  
Normal Ranges

β2-Glycoprotein I (β2GPI) is a major antigen for antiphospholipid antibodies, and its multiple in vitro functions have been reported. This glycoprotein not only down-regulates thrombin formation by inhibiting contact activation or prothrombinase activity, but also up-regulates coagulation by reducing protein C anticoagulant activity. However, the in vivo roles of β2GPI remain obscure. Coagulation and fibrinolytic characteristics were investigated in individuals with β2GPI deficiency. An apparently healthy woman and her brother are homozygotes for β2GPI deficiency. In these patients, Russell viper venom time was shortened (40.4 seconds; normal range, 47.8 ± 4.95 seconds), but all markers of thrombin generation and fibrin turnover were within normal ranges. Exogenous activated protein C adequately prolonged the clotting time of the β2GPI-deficient plasma, and euglobulin lysis time was also normal. Thus, elevated thrombin generation, enhancement of activated protein C response, and an altered fibrinolytic system were not found in congenitally β2GPI-deficient plasma.

Bovine Protein C Inhibitor Has a Unique Reactive Site and Can Transiently Inhibit Plasmin

2000 ◽  
Vol 83 (02) ◽  
pp. 262-267 ◽  
Author(s):  
Hiroyuki Yuasa ◽  
Hitoshi Tanaka ◽  
Tatsuya Hayashi ◽  
Toshiaki Wakita ◽  
Hideaki Nakamura ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Peptide Bond ◽  
Serine Protease Inhibitor ◽  
Mature Protein ◽  
Reactive Site ◽  
Plasmin Activity ◽  
Amino Acid Sequence Homology ◽  
Hemostatic System ◽  
Protein C Inhibitor

SummaryProtein C inhibitor (PCI) regulates the anticoagulant protein C pathway by neutralizing activated protein C and thrombin-thrombomodulin complex in the human hemostatic system. In this study, we cloned a full-length bovine PCI cDNA encoding a putative 19-residue signal peptide and a 385-residue mature protein; this showed 70.6%, 70.6%, 57.5% and 59.6% amino acid sequence homology with the human, rhesus monkey, rat and mouse PCIs, respectively. Bovine PCI mRNA (2.1 kb in size) was expressed strongly in the liver, and moderately in the kidney and testis, but not in other tissues tested. Bovine PCI has a putative reactive site peptide bond, Lys-Ser, that is different from the reactive site sequence (Arg-Ser) of other species’ PCI. We found that bovine PCI transiently inhibits bovine plasmin, but not human plasmin. Western blot analysis showed that the reactive site of bovine PCI is cleaved during the course of complex formation with bovine plasmin; degraded PCI is released from the complex gradually concomitant with the recovery of plasmin activity. These findings suggest that bovine PCI plays a role not only in the protein C pathway but also in the fibrinolytic activity of bovine hemostatic system. Abbreviations: PCI, protein C inhibitor, Serpin, serine protease inhibitor, APC, activated protein C, TM, thrombomodulin.

scholarly journals Characterization of an autosomal dominant bleeding disorder caused by a thrombomodulin mutation

Blood ◽  
2015 ◽  
Vol 125 (9) ◽  
pp. 1497-1501 ◽  
Author(s):  
Yesim Dargaud ◽  
Jean Yves Scoazec ◽  
Simone J. H. Wielders ◽  
Christine Trzeciak ◽  
Tilman M. Hackeng ◽  
...  
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Autosomal Dominant ◽  
Activated Protein C ◽  
Bleeding Disorder ◽  
Soluble Thrombomodulin ◽  
Factor Va ◽  
Factor Viiia ◽  
Key Points

Key Points The THBD c.1611C>A mutation (p.Cys537Stop) causes extremely high soluble thrombomodulin levels resulting in trauma-related bleeding. Soluble thrombomodulin acts by enhancing activated protein C generation and by impairing factor Va, factor VIIIa, and thrombin generation.

Dysfunctional Activated Protein C (PC Cádiz) in a Patient with Thrombotic Disease

1987 ◽  
Vol 57 (02) ◽  
pp. 183-186 ◽  
Author(s):  
N Sala ◽  
M Borrell ◽  
K A Bauer ◽  
S Viganò-D’Angelo ◽  
J Fontcuberta ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Hepatic Function ◽  
C Protein ◽  
Serine Protease Activity ◽  
Recurrent Venous Thrombosis ◽  
And Control ◽  
Normal Controls ◽  
Protein C Activity

SummaryThe partial characterization of a dysfunctional protein C (PC), provisionally named “PC Cádiz”, in a 45-year-old male patient suffering from recurrent venous thrombosis is described. The only defect found in laboratory assays for haemostasis and hepatic function was a half normal level of both amidolytic and anticoagulant protein C activity, measured by different functional assays that use thrombin-thrombomodulin complex and a snake venom to activate protein C. Protein C antigen was always found to be within normal levels. Two young daughters of the propositus were found to have the same defect. Double-crossed immunoelectrophoresis, performed in the presence and absence of Ca2+ in the first dimension, showed no clear differences between patient and control PC. PC adsorption to barium salts was also found to be normal. Measurement of the PC activation peptide in the barium citrate eluates after PC activation showed no significant differences between patient and 10 normal controls, the concentration of this peptide being very similar to that of PC zymogen in the same eluates before PC activation. These results indicate that this abnormal PC is able to be normally activated by thrombin-thrombomodulin complex but does not exhibit serine protease activity, probably due to a defect in the PC molecule near the active site center.

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