Inactivation of factor XIa in human plasma assessed by measuring factor XIa-protease inhibitor complexes: major role for C1-inhibitor

From experiments with purified proteins, it has been concluded that factor XIa (FXIa) is inhibited in plasma mainly by alpha 1-antitrypsin (a1AT), followed by antithrombin III (ATIII), C1-inhibitor (C1Inh), and alpha 2-antiplasmin (a2AP). However, the validity of this concept has never been studied in plasma. We established the relative contribution of different inhibitors to the inactivation of FXIa in human plasma, using enzyme-linked immunosorbent assays (ELISAs) for the quantification of complexes of FXIa with a1AT, C1Inh, a2AP, and ATIII. We found that 47% of FXIa added to plasma formed complexes with C1Inh, 24.5% with a2AP, 23.5% with a1AT, and 5% with ATIII. The distribution of FXIa between these inhibitors in plasma was independent of whether FXIa was added to plasma, or was activated endogenously by kaolin, celite, or glass. However, in the presence of heparin (1 or 50 U/mL), C1Inh appeared to be the major inhibitor of FXIa, followed by ATIII. Furthermore, at lower temperatures, less FXIa-C1Inh and FXIa-a1AT complexes but more FXIa-a2AP complexes were formed. These data demonstrate that the contribution of the different inhibitors to inactivation of FXIa in plasma may vary, but C1Inh is the principal inhibitor under most conditions.

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Inactivation of Factor XIa in Vivo: Studies in Chimpanzees and in Humans

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650621 ◽

1996 ◽

Vol 76 (04) ◽

pp. 549-555 ◽

Cited By ~ 16

Author(s):

Walter A Wuillemin ◽

C Erik Hack ◽

Wim K Bleeker ◽

Bart J Biemond ◽

Marcel Levi ◽

...

Keyword(s):

Antithrombin Iii ◽

Life Time ◽

In Vivo Studies ◽

Clinical Samples ◽

Plasma Samples ◽

C1 Inhibitor ◽

Elimination Kinetics ◽

Factor Xia ◽

Best Parameter

SummaryC1-inhibitor (C1Inh), antithrombin III (ATIII), α1-antitrypsin (a1AT), and α2-antiplasmin (a2AP) are known inhibitors of factor XIa (FXIa). However, their precise contribution to FXIa inactivation in vivo is not known. We investigated FXIa inactivation in chimpanzees and assessed the contribution of these inhibitors to FXIa inactivation in patients with presumed FXI activation.Chimpanzees were infused with FXIa and the various FXIa-FXIa inhibitor complexes formed were measured. Most of FXIa was complexed to C1Inh (68%), followed by a2AP (13%), a1AT (10%), and ATIII (9%). Analysis of the plasma elimination kinetics revealed a half-life time of clearance (t1/2) for the FXIa-FXIa inhibitor complexes of 95 to 104 min, except for FXIa-a1AT, which had a t1/2 of 349 min. Due to this long t1/2, FXIa-a1AT complexes were predicted to show the highest levels in plasma samples from patients with activation of FXI. This was indeed shown in patients with disseminated intravascular coagulation, recent myocardial infarction or unstable angina pectoris. We conclude from this study that in vivo C1Inh is the predominant inhibitor of FXIa, but that FXIa-a1 AT complexes due to their relatively long t1/2 may be the best parameter to assess FXI activation in clinical samples.

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Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV-2–Priming Protease TMPRSS2

Pathogens and Immunity ◽

10.20411/pai.v6i1.408 ◽

2021 ◽

Vol 6 (1) ◽

pp. 55-74

Author(s):

Nurit P Azouz ◽

Andrea Klingler ◽

Victoria Callahan ◽

Ivan Akhrymuk ◽

Katarina Elez ◽

...

Keyword(s):

Viral Load ◽

Protease Inhibitor ◽

Serine Protease ◽

Inhibitory Activity ◽

Serine Proteases ◽

Serine Protease Inhibitor ◽

Host Cells ◽

S Protein ◽

Relative Contribution ◽

Alpha 1 Antitrypsin

Background: Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry. Methods: We developed a cell-based assay to identify TMPRSS2 inhibitors. Inhibitory activity was established in SARS-CoV-2 viral load systems. Results: We identified the human extracellular serine protease inhibitor (serpin) alpha 1 antitrypsin (A1AT) as a novel TMPRSS2 inhibitor. Structural modeling revealed that A1AT docked to an extracellular domain of TMPRSS2 in a conformation that is suitable for catalysis, resembling similar serine protease inhibitor complexes. Inhibitory activity of A1AT was established in a SARS-CoV-2 viral load system. Notably, plasma A1AT levels were associated with COVID-19 disease severity. Conclusions: Our data support the key role of extracellular serine proteases in SARS CoV-2 infections and indicate that treatment with serpins, particularly the FDA-approved drug A1AT, may be effective in limiting SARS-CoV-2 dissemination by affecting the surface of the host cells.

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Assessment of the Relative Contribution of Different Protease Inhibitors to the Inhibition of Plasmin In Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651570 ◽

1993 ◽

Vol 69 (02) ◽

pp. 141-146 ◽

Cited By ~ 43

Author(s):

Marcel Levi ◽

Dorina Roem ◽

Angela M Kamp ◽

Jan Paul de Boer ◽

C Erik Hack ◽

...

Keyword(s):

Monoclonal Antibody ◽

Protease Inhibitors ◽

Proteolytic Enzyme ◽

Antithrombin Iii ◽

C1 Inhibitor ◽

Relative Contribution ◽

Α2 Macroglobulin ◽

Diffuse Intravascular Coagulation

SummaryIt has been shown that the most important inhibitor of plasmin is α2-antiplasmin, however, other protease inhibitors are able to inhibit this proteolytic enzyme as well. The contribution of the various protease inhibitors to the inhibition of plasmin in vivo has never been quantitatively assessed.To assess the relative contribution of the different protease inhibitors on the inhibition of plasmin we developed a series of sensitive immunoassays for the detection of complexes between plasmin and the protease inhibitors α2-antiplasmin, α2-macroglobulin, antithrombin III, α1antitrypsin and C1-inhibitor, utilizing monoclonal antibodies that are specifically directed against complexed protease inhibitors and a monoclonal antibody against plasmin.It was confirmed that α2-antiplasmin is the most important inhibitor of plasmin in vivo, however, complexes of plasmin with α2-macroglobulin, antithrombin III, α1antitrypsin- and C1-inhibitor were also detected. Particularly during activation of fibrinolysis complexes between plasmin and inhibitors other than α2-antiplasmin were detected. It was observed that during different situations the inhibition profile of plasmin was not constant e.g. in patients with diffuse intravascular coagulation plasma levels of plasmin-α1-antitrypsin and plasmin-C1-inhibitor were increased whereas in plasma from patients who were treated with thrombolytic agents complexes of plasmin with α2-macroglobulin and with antithrombin III were significantly elevated.In conclusion, we confirmed the important role of α2-antiplasmin in the inhibition of plasmin, however, in situations in which fibrinolysis is activated other protease inhibitors also account for the inhibition of plasmin in vivo. Further investigations to assess the role of the various protease inhibitors in the fibrinolytic system can be assisted by the assays described in this study.

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Alpha-1-antitrypsin-Pittsburgh. A potent inhibitor of human plasma factor XIa, kallikrein, and factor XIIf.

Journal of Clinical Investigation ◽

10.1172/jci112346 ◽

1986 ◽

Vol 77 (2) ◽

pp. 631-634 ◽

Cited By ~ 57

Author(s):

C F Scott ◽

R W Carrell ◽

C B Glaser ◽

F Kueppers ◽

J H Lewis ◽

...

Keyword(s):

Human Plasma ◽

Potent Inhibitor ◽

Plasma Factor ◽

Factor Xia ◽

Alpha 1 Antitrypsin

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Potentially Thrombogenic Materials in Factor IX Concentrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647857 ◽

1975 ◽

Vol 33 (03) ◽

pp. 617-631 ◽

Cited By ~ 47

Author(s):

H. S Kingdon ◽

R. L Lundblad ◽

J. J Veltkamp ◽

D. L Aronson

Keyword(s):

Human Plasma ◽

Antithrombin Iii ◽

Factor Ix ◽

In Vitro Assays ◽

Substantial Agreement ◽

Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

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A Hitherto Undescribed Naturally Occurring Plasma Antagonist of Activated Factor X Inhibitor (Antithrombin III)

10.1055/s-0038-1665828 ◽

1979 ◽

Author(s):

E. T. Yin ◽

W. J. Salsgiver ◽

O. Tangen

Keyword(s):

Equilibrium State ◽

Human Plasma ◽

Antithrombin Iii ◽

Circumstantial Evidence ◽

Incubation Mixture ◽

Factor X ◽

Normal Human Plasma ◽

Plasma Component ◽

Naturally Occurring ◽

Normal Human

Circumstantial evidence suggested that normal human plasma contained a substance regulating the neutralization of F.Xa by F.Xa inhibitor(XaI), (Yin et.al.,Adv.Exper. Med. & Biol., 52 : 239, 1975, Plenum Press, N.Y.).This plasma component has now been isolated and partially purified in our laboratory, and tentatively designated as “Anti-XaI”.In experiments employing purified components, when Anti-XaI was incubated at 37°C with F.Xa, Xal and heparin for two minutes at pH7.5, the amount of F.Xa inhibited was inversely proportional to the Anti-XaI concentration. But, when the F.Xa was replaced by thrombin in the incubation mixture, the neutralization of thrombin clotting activity was undisturbed.Anti-XaI was found to be neither PF3 nor PF4.These and other data strongly suggest that the “Antithrombin III pathway” is more complex than currently believed to be. In circulating blood an equilibrium state must exist between Anti-XaI and XaI.Under certain conditions when the Anti-XaI activity is predominant the rate of F.Xa neutralization bv XaI then becomes slower than the activation of prothrombin to thrombin by F.Xa.

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