Cover Feature: Microscale Parallel Synthesis of Acylated Aminotriazoles Enabling the Development of Factor XIIa and Thrombin Inhibitors (ChemMedChem 24/2021)

SummaryThe activated partial thromboplastin time test (aPTT) represents one of the most commonly used diagnostic tools in order to monitor patients undergoing heparin therapy. Expression of aPTT coagulation time in seconds represents common practice in order to evaluate the integrity of the coagulation cascade. The prolongation of the aPTT thus can indicate whether or not the heparin level is likely to be within therapeutic range. Unfortunately aPTT results are highly variable depending on patient properties, manufacturer, different reagents and instruments among others but most importantly aPTT’s dose response curve to heparin often lacks linearity. Furthermore, aPTT assays are insensitive to drugs such as, for example, low molecular weight heparin (LMWH) and direct factor Xa (FXa) inhibitors among others. On the other hand, the protrombinase-induced clotting time assay (PiCT®) has been show to be a reliable functional assay sensitive to all heparinoids as well as direct thrombin inhibitors (DTIs). So far, the commercially available PiCT assay (Pefakit®-PiCT®, DSM Nutritional Products Ltd. Branch Pentapharm, Basel, Switzerland) is designed to express results in terms of units with the help of specific calibrators, while aPTT results are most commonly expressed as coagulation time in seconds. In this report, we describe the results of a pilot study indicating that the Pefakit PiCT UC assay is superior to the aPTT for the efficient monitoring of patients undergoing UFH therapy; it is also suitable to determine and quantitate the effect of LMWH therapy. This indicates a distinct benefit when using this new approach over the use of aPPT for heparin monitoring.

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Platelet-Bound Prekallikrein Promotes Pro-Urokinase-Induced Clot Lysis: A Mechanism for Targeting the Factor XII Dependent Intrinsic Pathway of Fibrinolysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642441 ◽

1994 ◽

Vol 71 (03) ◽

pp. 347-352 ◽

Cited By ~ 36

Author(s):

Jean-Pierre Loza ◽

Victor Gurewich ◽

Michael Johnstone ◽

Ralph Pannell

Keyword(s):

Platelet Rich Plasma ◽

Specific Inhibitor ◽

Specific Effect ◽

Clot Lysis ◽

Factor Xii ◽

Intrinsic Pathway ◽

Clot Retraction ◽

Enzymatic Mechanism ◽

Low Concentrations ◽

Factor Xiia

SummaryClots formed from platelet rich plasma were found to be lysed more readily by low concentrations of pro-urokinase (pro-UK) than clots formed from platelet poor plasma. This was not a non-specific effect since the reverse occurred with tissue plasminogen activator. A mechanical explanation due to platelet-mediated clot retraction was excluded by experiments in which retraction was inhibited with cyto-chalasin B. Therefore, a platelet-mediated enzymatic mechanism was postulated to explain the promotion of fibrinolysis. Casein autography of isolated platelets revealed a ≈ 90 kDa band of activity which comigrated with plasma prekallikrein (PK)/kallikrein, a known activator of pro-UK. Furthermore, treatment of platelets with plasma PK activator (PPA), consisting essentially of factor XIIa, induced activation of pro-UK and of chromomgenic substrate for kallikrein (S-2302). This activity corresponded to approximately 40-200 pM kallikrein per 10 8 washed and gel filtered platelets per ml. The activation of pro-UK by PPA-pretreated platelets was dose-dependent and inhibited by soybean trypsin inhibitor but not by bdellin, a specific inhibitor of plasmin, nor by the corn inhibitor of factor XIIa. Kinetic analysis of pro-UK activation by kallikrein showed promotion of the reaction by platelets. The KM of the reaction was reduced by platelets by ≈ 7-fold, while the kcat was essentially unchanged. In conclusion, PK was shown to be tightly associated with platelets where it can be activated by factor XIIa during clotting. The activation of pro-UK by platelet-bound kallikrein provides an explanation for the observed platelet mediated promotion of pro-UK-induced clot lysis. Since pro-UK and plasminogen have also been shown to be associated with platelets, the present findings suggest a mechanism by which the factor Xlla-dependent intrinsic pathway of fibrinolysis can be localized and targeted to a thrombus.

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Comparison of the Anticoagulant and Antithrombotic Effects of Synthetic Thrombin and Factor Xa Inhibitors

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645198 ◽

1990 ◽

Vol 63 (02) ◽

pp. 220-223 ◽

Cited By ~ 19

Author(s):

J Hauptmann ◽

B Kaiser ◽

G Nowak ◽

J Stürzebecher ◽

F Markwardt

Keyword(s):

Factor Xa ◽

Thrombin Inhibitors ◽

Factor Xa Inhibitors ◽

Venous Stasis ◽

Clotting Time ◽

Thrombosis Model ◽

Activity Factor ◽

Antithrombotic Effects

SummaryThe anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors.In vivo, in a venous stasis thrombosis model and a thromboplastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar IQ value for the respective enzyme were not effective at equimolar dosageThe results are discussed in the light of the different prelequisiles and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.

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Thrombin, Thrombin Inhibitors, and the Arterial Thrombotic Process

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646192 ◽

1993 ◽

Vol 70 (01) ◽

pp. 208-211 ◽

Cited By ~ 26

Author(s):

John M Maraganore

Keyword(s):

Thrombin Inhibitors

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A Collaborative Study to Establish an International Standard for Alpha-Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648464 ◽

1992 ◽

Vol 67 (04) ◽

pp. 424-427 ◽

Cited By ~ 3

Author(s):

P J Gaffney ◽

A B Heath ◽

J W Fenton II

Keyword(s):

Elevated Temperatures ◽

Collaborative Study ◽

World Health Organisation ◽

Significant Loss ◽

Thrombin Inhibitors ◽

World Health ◽

Expert Committee ◽

International Standard ◽

Assay Procedure ◽

And Control

SummarySince 1975 an International Standard for Thrombin of low purity has been used. While this standard was stable and of value for calibrating thrombins of unknown potency the need for a pure a-thrombin standard arose both for accurate calibration and for precise measurement of thrombin inhibitors, notably hirudin. An international collaborative study was undertaken to establish the potency and stability of an ampouled pure a-thrombin preparation. A potency of 97.5 international units (95% confidence limits 86.5-98.5) was established for the new a-thrombin standard (89/ 588) using a clotting-assay procedure. Stability data at various elevated temperatures indicated that the standard could be transported and stored with no significant loss of potency.Ampoules of lyophilised a-thrombin (coded 89/588) have been recommended as an International Standard for a-thrombin with an assigned potency of 100 international units per ampoule by the International Society for Thrombosis and Haemostasis (Thrombin and its Inhibitors Sub-Committee) in Barcelona, Spain in July 1990 while the Expert Committee on Biological Standardisation and Control of the World Health Organisation will consider its status at its next meeting in Geneva in 1991.

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Comparison of Progressive Antithrombin Activity and the Concentration of Three Thrombin Inhibitors in Nephrotic Syndrome

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653432 ◽

1981 ◽

Vol 46 (03) ◽

pp. 623-625 ◽

Cited By ~ 25

Author(s):

B Boneu ◽

F Bouissou ◽

M Abbal ◽

P Sie ◽

C Caranobe ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Antithrombin Iii ◽

Heparin Therapy ◽

Thrombin Inhibitors ◽

Compensatory Mechanism ◽

Dramatic Reduction ◽

Antithrombin Activity ◽

Α2 Macroglobulin ◽

At Iii ◽

A Prospective Study

SummaryIn order to compare the plasmatic progressive antithrombin activity to the concentration of three thrombin inhibitors, antithrombin III (AT III), α2 macroglobulin (α2, M), α1 anti-trypsin (α1 AT) in nephrotic syndrome, a prospective study was carried out on a group of 28 children affected with the disease. A dramatic reduction of the level of AT III and of α1 AT, two inhibitors of molecular weight close to that of albumin, was observed. The decreased level of AT III was counterbalanced by an increase in α2 M. This phenomenon accounts for the increased progressive antithrombin activity observed in all the affected children. It is suggested that the above compensatory mechanism explains the absence of thrombotic accidents in this series and that the benefit of heparin therapy is doubtful in these conditions.

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Inhibition of Thrombin Generation in Plasma by Inhibitors of Factor Xa

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657717 ◽

1997 ◽

Vol 78 (04) ◽

pp. 1215-1220 ◽

Cited By ~ 25

Author(s):

D Prasa ◽

L Svendsen ◽

J Stürzebecher

Keyword(s):

Thrombin Generation ◽

Factor Xa ◽

Thrombin Inhibitors ◽

Thrombin Inhibitor ◽

Thrombin Generation Assay ◽

Ic50 Values ◽

Fxa Inhibitor ◽

20 Nm ◽

Tick Anticoagulant Peptide ◽

Inhibition Of Thrombin

SummaryA series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations.The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.

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The Ability of Thrombin Inhibitors to Reduce the Thrombin Activity Generated in Plasma on Extrinsic and Intrinsic Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655996 ◽

1997 ◽

Vol 77 (03) ◽

pp. 498-503 ◽

Cited By ~ 26

Author(s):

D Prasa ◽

L Svendsen ◽

J Stürzebecher

Keyword(s):

Active Site ◽

Thrombin Generation ◽

Anion Binding ◽

Thrombin Inhibitors ◽

Coagulation System ◽

Thrombin Activity ◽

Continuous Registration ◽

High Concentrations ◽

20 Nm ◽

Generation Test

SummaryIn a thrombin generation test with continuous registration of thrombin activity in plasma we studied the ability of a variety of thrombin inhibitors of different type and mechanism of action to influence the activity of thrombin after activation of the coagulation system. Depending on the inhibitor, the peak of thrombin activity is delayed and/or reduced.By blocking the active site of generated thrombin inhibitors cause a concentration dependent reduction of the thrombin peak and inhibit feed-back reactions of thrombin resulting in a delay of thrombin generation. Highly potent synthetic active-site directed inhibitors (Ki ≤ 20 nM) reduce the thrombin activity formed in plasma after extrinsic or intrinsic activation with the same efficiency (IC50 0.1 - 0.6 μM) as hirudin. The delay and reduction of thrombin generation by inhibitors of the anion-binding exosite 1 of thrombin is only attributed to an inhibition of feed-back reactions of thrombin. For a 50% reduction of thrombin activity in plasma by this type of inhibitors relatively high concentrations were determined.

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