Persistence Pays Off: Milvexian Emerges from the Industry’s Longstanding Search for Orally Bioavailable Factor XIa Inhibitors

SummaryThe activation of factor IX purified from human plasma has been studied. Factor XIa and kallikrein separately activated factor IX to factor IXa. In both cases factor IX a had an apparent molecular weight of about 42–45000 in sodium dodecyl sul-phate-polyacrylamide disc gel electrophoresis compared with a molecular weight of about 70000 for the native factor IX. The activation by XIa required Ca2+-ions whereas Ca2+-ions did not influence the activation by kallikrein. A mixture of tissue thromboplastin and factor VII or RusselPs-viper venom alone did not activate factor IX. Trypsin activated and plasmin inactivated factor IX.

Download Full-text

Amidolytic Detection of Prothrombin Activation Products after SDS-Gel Electrophoresis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646601 ◽

1989 ◽

Vol 61 (03) ◽

pp. 386-391 ◽

Cited By ~ 6

Author(s):

Guido Tans ◽

Truus Janssen-Claessen ◽

Jan Rosing

Keyword(s):

Gel Electrophoresis ◽

Activated Protein C ◽

Factor Xa ◽

Coagulation Factors ◽

Product Formation ◽

Chromogenic Substrate ◽

Nitrocellulose Membrane ◽

Activation Products ◽

Factor Xia ◽

Prothrombin Activation

SummaryIn this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation.The detection limit of the so-called “amidoblot” is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting.It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).

Download Full-text

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.

Download Full-text

Ultrasensitive Fluorogenic Substrates for Serine Proteases

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657714 ◽

1997 ◽

Vol 78 (04) ◽

pp. 1193-1201 ◽

Cited By ~ 28

Author(s):

Saulius Butenas ◽

Maria E DiLorenzo ◽

Kenneth G Mann

Keyword(s):

Serine Proteases ◽

Factor Viia ◽

Activated Protein C ◽

Factor Xa ◽

High Specificity ◽

High Rate ◽

Fluorogenic Substrates ◽

Type Plasminogen Activator ◽

Factor Xia ◽

Coagulation And Fibrinolysis

SummarySelective, sensitive assays for the quantitation of serine proteases involved in coagulation and fibrinolysis have been developed employing fluorogenic substrates containing a 6-amino-1-naphthalenesulfonamide leaving group (PNS-substrates). Over one hundred substrates were evaluated for hydrolysis by the serine proteases of blood coagulation and fibrinolysis, and substrate structure-efficiency correlations were examined. PNS-substrates which contain Lys in the P1 position are specific for Lys-plasmin and are either not hydrolyzed or hydrolyzed at a relatively low rate by factor Xa, thrombin, or urokinase-type plasminogen activator (uPA). These substrates allow quantitation of Lys-plasmin at concentrations as low as 1 pM. Eighteen of over 90 substrates tested for factor XIa are hydrolyzed by this enzyme at a relatively high rate reaching a kcat value of 170 s-1 and allowing quantitation of factor XIa at 10 fM. Eighteen of almost 90 PNS-substrates tested display high specificity for thrombin, some exceeding that for factor Xa by > 10,000-fold and > 100-fold for activated protein C (APC). Seven of these substrates have a over 100 s-1 and three of them have a KM below 1 μM. They allow the quantitation of thrombin at concentrations as low as 20 fM. For APC, uPA and the factor Vila/tissue factor complex, quantitation is feasible at 1 pM concentration. For factor Xa and factor VIIa the limits are 0.4 pM and 40 pM respectively. The PNS-substrates presented in this study may be employed for the development of direct and sensitive serine protease assays.

Download Full-text