Robert Wagenvoord

The inhibition of blood coagulation by heparins of different molecular weight is caused by a common functional motif—the C‐domain

Summary.  Background: Heparins in clinical use differ considerably as to mode of preparation, molecular weight distribution and pharmacodynamic properties. Objectives: Find a common basis for their anticoagulant action. Methods: In 50 fractions of virtually single molecular weight (Mr), prepared from unfractionated heparin (UFH) and four low‐molecular‐weight heparins (LMWH), we determined: (i) the molar concentration of material (HAM) containing the antithrombin binding pentasaccharide (A‐domain); (ii) the specific catalytic activity in thrombin and factor Xa inactivation; (iii) the capacity to inhibit thrombin generation (TG) and prolong the activated partial thromboplastin time (APTT). We also calculated the molar concentration of A‐domain with 12 sugar units at its non‐reducing end, i.e. the structure that carries antithrombin activity (C‐domain). Results: The antithrombin activity and the effects on TG and APTT are primarily determined by the concentration of C‐domain and independent of the source material (UFH or LMWH) or Mr. High Mr fractions (>15 000) are less active, probably through interaction with non‐antithrombin plasma proteins. Anti‐factor Xa activity is proportional to the concentration of A‐domain, it is Ca2+‐ and Mr‐dependent and does not determine the effect on TG and APTT. Conclusion: For any type of heparin, the capacity to inhibit the coagulation process in plasma is primarily determined by the concentration of C‐domain, i.e. the AT‐binding pentasaccharide with 12 or more sugar units at its non‐reducing end.

The balance of pro- and anticoagulant processes underlying thrombin generation

The generation of thrombin in time is the combined effect of the processes of prothrombin conversion and thrombin inactivation. Measurement of prothrombin consumption used to provide valuable information on hemostatic disorders, but is no longer used, due to its elaborate nature.

A new regulatory function of activated factor V: inhibition of the activation by tissue factor/factor VII(a) of factor X.

We observed that minute amounts of thrombin or the enzyme Russells viper venom activating factor V (RVV‐V) added to plasma strongly diminish the potential of that plasma to generate thrombin after being triggered by tissue factor.

A reduction of prothrombin conversion by cardiac surgery with cardiopulmonary bypass shifts the haemostatic balance towards bleeding

Cardiac surgery with cardiopulmonary bypass (CPB) is associated with blood loss and post-surgery thrombotic complications. The process of thrombin generation is disturbed during surgery with CPB because of haemodilution, coagulation factor consumption and heparin administration. We aimed to investigate the changes in thrombin generation during cardiac surgery and its underlying pro- and anticoagulant processes, and to explore the clinical consequences of these changes using in silico experimentation. Plasma was obtained from 29 patients undergoing surgery with CPB before heparinisation, after heparinisation, after haemodilution, and after protamine administration. Thrombin generation was measured and prothrombin conversion and thrombin inactivation were quantified. In silico experimentation was used to investigate the reaction of patients to the administration of procoagulant factors and/or anticoagulant factors. Surgery with CPB causes significant coagulation factor consumption and a reduction of thrombin generation. The total amount of prothrombin converted and the rate of prothrombin conversion decreased during surgery. As the surgery progressed, the relative contribution of α2-macroglobulin-dependent thrombin inhibition increased, at the expense of antithrombin-dependent inhibition. In silico restoration of post-surgical prothrombin conversion to pre-surgical levels increased thrombin generation excessively, whereas co-administration of antithrombin resulted in the normalisation of post-surgical thrombin generation. Thrombin generation is reduced during surgery with cardiopulmonary bypass because of a balance shift between prothrombin conversion and thrombin inactivation. According to in silico predictions of thrombin generation, this new balance increases the risk of thrombotic complications with prothrombin complex concentrate administration, but not if antithrombin is co-administered.

Comment on the use of computational models to study the effect of apixaban and rivaroxaban on thrombin generation

Comment on the use of computational models to study the effect of apixaban and rivaroxaban on thrombin generation -

Computational modelling of clot development in patient-specific cerebral aneurysm cases : rebuttal

See also Ngoepe MN, Ventikos Y. Computational modelling of clot development in patient-specific cerebral aneurysm cases. J Thromb Haemost 2016; 14: 262–72 and Ngoepe MN, Ventikos Y. Computational modeling of clot development in patient-specific cerebral aneurysm cases: reply. This issue, pp 397–8 and Kremers R, de Laat B, Wagenvoord R, Hemker C. Computational modelling of clot development in patientspecific cerebral aneurysm cases: rebuttal. This issue, pp 399.

C0305: A2Macroglobulin does Not Compensate for Antithrombin Deficiency in Severe Liver Cirrhosis

Background: In liver cirrhosis antithrombin (AT) decreases with the severity of the disease and may approach the level of heterozygous AT deficiency (50%). Alpha2Macroglobulin (a2M) may increase up to fourfold. We investigated in how far this makes up for the decrease of AT. Methods: In healthy controls (n = 32) and liver cirrhosis patients (n = 29) we measured AT and a2M with a chromogenic assay and fibrinogen according to Clauss. The overall inhibitory capacity of plasmas (AT plus a2M) was calculated from the descending leg of a thrombin generation (TG) curve after prothrombin conversion was over (after 3 min and at 50 pM tissue factor). Thrombin decay by a2M was calculated from the velocity of a2M-thrombin formation. A computer simulation of thrombin decay, based on ordinary differential equations, was validated by finding superposition of simulated and observed thrombin decay curves within the limits of experimental accuracy both in patients and controls. Results: Mean AT and a2M concentrations in healthy controls were 2.01uM (SD=0.36uM) and 3.34uM (SD=0.89uM), respectively. In liver cirrhosis patients, the average AT level was decreased (1.62uM; p=0.002) and the average a2M level was increased (5.05uM; p=0.001). In accordance, thrombin decay by a2M in patients is 180% of normal (p = 0.001), whereas AT-dependent thrombin decay is 88% of control (p = 0.009). Overall thrombin decay in cirrhosis patients was 95% of control (p = 0.345), so a2M increase compensates for AT decrease. However, in the subset of patients with AT lower than 75% of normal, the rate of thrombin decay is abnormally low (76% of normal, p = 0.001). Computer simulation of thrombin decay in this subset allowed to calculate that that the elevation in a2M levels increases the over-all thrombin decay rate significantly (10%, p = 0.016), but not enough to completely restore physiological thrombin decay rates (76%, p = 0.001). Conclusions: In liver cirrhosis thrombin inactivation by AT is substantially reduced but a simultaneous increase in a2M levels normalizes thrombin decay in patients with moderate AT loss (more than 75% of normal). In patients with severe AT deficiency (less than 75% of normal) complete compensation is no longer achieved.