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BackgroundThe haemostatic balance is an equilibrium of pro- and anticoagulant factors that work synergistically to prevent bleeding and thrombosis. As thrombin is the central enzyme in the coagulation pathway, it is desirable to measure thrombin generation (TG) in order to detect possible bleeding or thrombotic phenotypes, as well as to investigate the capacity of drugs affecting the formation of thrombin. By investigating the underlying processes of TG (i.e., prothrombin conversion and inactivation), additional information is collected about the dynamics of thrombin formation.ObjectivesTo obtain reference values for thrombin dynamics (TD) analysis in 112 healthy donors using an automated system for TG.MethodsTG was measured on the ST Genesia, fibrinogen on the Start, anti-thrombin (AT) on the STA R Max and α2Macroglobulin (α2M) with an in-house chromogenic assay.ResultsTG was measured using STG-BleedScreen, STG-ThromboScreen and STG-DrugScreen. The TG data was used as an input for TD analysis, in combination with plasma levels of AT, α2M and fibrinogen that were 113% (108–118%), 2.6 μM (2.2 μM−3.1 μM) and 2.9 g/L (2.6–3.2 g/L), respectively. The maximum rate of the prothrombinase complex (PCmax) and the total amount of prothrombin converted (PCtot) increased with increasing tissue factor (TF) concentration. PCtot increased from 902 to 988 nM, whereas PCmax increased from 172 to 508 nM/min. Thrombin (T)-AT and T-α2M complexes also increased with increasing TF concentration (i.e., from 860 to 955 nM and from 28 to 33 nm, respectively). PCtot, T-AT and T-α2M complex formation were strongly inhibited by addition of thrombomodulin (−44%, −43%, and −48%, respectively), whereas PCmax was affected less (−24%). PCtot, PCmax, T-AT, and T-α2M were higher in women using oral contraceptives (OC) compared to men/women without OC, and inhibition by thrombomodulin was also significantly less in women on OC (p < 0.05).ConclusionsTG measured on the ST Genesia can be used as an input for TD analysis. The data obtained can be used as reference values for future clinical studies as the balance between prothrombin conversion and thrombin inactivation has shown to be useful in several clinical settings.

Background: Heparins are sulphated glycosaminoglycans mainly used as an anticoagulant therapy to prevent venousthromboembolism. However, anticoagulants as heparinoids are known to impact coagulation tests by increasing clottingtimes or reducing thrombin generation in the test sample. Therefore, to ensure an unbiased evaluation of the patientcoagulability status, neutralization of the anticoagulant effect of heparinoids is necessary. This can be achieved by usingneutralizing agents such as hexadimethrine bromide, also known as polybrene, or Hepa-remove® (5-Diagnostics,Switzerland), a newly developed synthetic tripeptide.Aims: This study aimed to assess the capacity of polybrene and Hepa-remove® to neutralize the anticoagulant effect ofenoxaparin on thrombin generation assay (TGA) and to evaluate their impact on this test.Method: Low molecular weight heparin was spiked in a normal pooled plasma composed of 50 healthy individuals at differentconcentrations, 0 [=sterile saline solution], 0.2, 0.4 and 0.6 UI/mL. TGA was assessed on the ST Genesia system using STGThromboScreen-TM (Stago, France) as triggering reagent. Polybrene and Hepa-remove® were added into the triggeringreagent upon reconstitution in order to obtain final plasma concentrations of 0.025mg/mL and 0.166mg/mL, respectively.Analyses were performed in duplicate and assessed in three independent runs. The impact of these neutralizing agents onTG was assessed by comparing plasma (without heparin) treated with and without Polybrene or Hepa-remove®. Theneutralizing capacity was measured as the percentages of change from baseline in which the plasma without heparin treatedwith Polybrene or Hepa-remove® was considered as the baseline condition.Results: No TGA parameter was impacted by the presence of polybrene at the concentration tested in this study. On theopposite, Hepa-remove® significantly reduces he Lag Time (LT) and the Time-to-peak (Ttp) (p-value< 0.05). Polybrene permitsto fully neutralize the effect of enoxaparin. The residual Endogenous Thrombin Potential (ETP), mean Velocity Rate Index(mVRI) and Peak Height (PH) after treatment by polybrene were increased by 5.1%, 8.6% and 6.6% compared to baseline TGon NPP spiked with saline, even at the highest enoxaparin concentration tested (i.e., 0.6 IU/mL). The LT decreased by 6.4%.On the other hand, the Ttp decreased by 3.9% which was significant compared to the baseline (p-value = 0.048). The Heparemove® permitted the recovery of the baseline ETP, mVRI and PH values regardless of the heparin concentration. However,as it impacted the LT and Ttp even in absence of heparin, it does not permit to recover the baseline value of these parametersin absence of enoxaparin.Conclusion: This study showed that polybrene had no impact on TG under our assay conditions; while the Hepa-remove®impacted the LT and Ttp. Regarding their neutralizing capacity, polybrene was able to fully neutralize enoxaparin up to aconcentration of 0.6 UI/mL with no impact on TG. The Hepa-remove® showed acceptable neutralizing capacity, but impactedsome TG parameters independently to its neutralizing capacity on enoxaparin.

The distribution of total fibrinolytic activity in seminal plasma, as well as specific tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and plasminogen activator inhibitor (PAI), has been studied using antigen and activity techniques in 170 ejaculates of men attending for assessment because of infertility without genital urinary pathology. Among these 170 patients, 18 showed oligoasthenoteratospermia, 28 showed azoospermia, and 124 showed normozoospermia. The seminal values were 50 times higher (262 to 289 ng/mL in antigen and 179 to 199 x 10 (3) IU/L for activity) than values in blood for t-PA and 15 times higher than values in blood for u-PA (18.4 to 26 ng/mL and 1.5 to 2.4 IU/mL, respectively). There was no correlation between the two levels in antigen or activity, but a higher concentration was observed in all first fractions from split ejaculates measurements. Moreover, t-PA was significantly lower in semen with abnormal liquefaction compared with semen exhibiting normal liquefaction. Zymography confirms the active forms. PAI was absent or at the detection limit for normozoospermia, whereas patients with oligoasthenoteratospermia or azoospermia showed high PAI antigen and activity levels. These data demonstrate that seminal PA activity may be related to sperm fertilizing capacity.