Blood

Thrombin generation is a pivotal part of the clot formation process, and analysing an individual's thrombin generation potential can help assess their bleeding or thrombosis risk. A method for continuous assessment of thrombin generation was first introduced by Hemker et al in 1993. At that time, TG assays (TGA) were time-consuming: measuring just one sample took even a skilled laboratory technician up to an hour. The efforts of Hemker and colleagues resulted in the calibrated automated thrombogram (CAT) which was introduced in 2003. CAT employs a slow-reacting fluorogenic substrate that enables the continuous measurement of TGA. Thrombin activity is calculated as a function of time by comparing the fluorescent signal from the thrombin-generating sample to that from a known stable level of thrombin activity, measured simultaneously in a control sample. The TGA parameters are derived from the TGA curve and include lag time (time to minimum thrombin formed [min]), peak height (the maximum amount of thrombin formed [nM]) and endogenous thrombin potential (ETP or area under the curve [nM.min]). Several studies have suggested that TGA could be a marker of thrombotic or haemorrhagic potential. 

TGA is a highly sensitive method for determining coagulation activation. Preanalytical conditions can influence the results, depending on the activator applied in the assay. In collaboration with the Department of Biochemistry at Maastricht University in the Netherlands, Coagulation Profile BV has compiled several standardised operating procedures to limit the activation of coagulation and platelets during the pre-analytical procedures of blood sample collection, plasma sample preparation, and performance of the assay. 

A limiting factor for applying the TGA in a diagnostic setting has been the lack of reference ranges in a larger population. For this purpose, Coagulation Profile BV has supported thrombin generation assessments in the Gutenberg Health Study (GHS), a prospective population-based study in the Rhein-Main area in Germany. In this study, thrombin generation was assessed in plasma samples from 5,000 subjects, thereby establishing reference ranges for the thrombin generation parameters. 

Using various autoanalysers for quantification of fibrin formation, either through light shift or mechanical forces, Coagulation Profile BV can offer a wide range of routine coagulation assays, including (but not limited to): 

Prothrombin Time (PT) Activated Partial Thromboplastin Time (aPTT) Coagulation Factor levels through PT or aPTT Fibrinogen Antithrombin Factor VIII Inhibitors (Bethesda assay) Factor IX Inhibitors (Bethesda assay) Low Factor IX levels Factor VIIa D-Dimer 

Please contact us for more details or to discuss customised assays based on specific needs. 

The body has a large number of regulatory mechanisms in place to prevent excessive coagulation from causing thrombosis. Activated coagulation factors are rapidly inactivated by various endogenous inhibitors, which are essential to maintaining the balance between coagulation and bleeding. 

C1-inhibitor
The C1-inhibitor (C1inh) belongs to the serpin family, and with a plasma concentration of 240 µg/mL it represents one of the most abundant protease inhibitors in the systemic circulation. C1inh is primarily a regulator of the complement system; it is also the most important inhibitor of Factor Xlla and, alongside a2-macroglobulin, the primary inhibitor of plasma kallikrein (Pka). C1inh is therefore considered a key regulator of the contact system of coagulation. 

⍺1-antitrypsin, ⍺2-antiplasmin, and antithrombin III (AT)
C1inh also controls the activity of Factor Xla together with ⍺1-antitrypsin (⍺1-AT) and ⍺2-antiplasmin (⍺2-AP), the main inhibitors of elastase released by leukocytes and plasmin, respectively, and antithrombin III (AT) – all three members of the serpin superfamily of serine protease inhibitors. It has been shown that C1inh is not only a major inhibitor of Factor Xlla and PKa but also of Factor Xla, as demonstrated by its contribution of 47% to the inactivation of Factor Xla in human plasma, compared with 24.5% from ⍺2AP, 23.5% from ⍺1AT, and 5% from AT. A similar pattern of contribution from these inhibitors to the inactivation of Factor Xla was observed in vivo, with C1inh being the predominant inhibitor. The observation that the Factor Xla-⍺1AT complex has the longest elimination half-life of the three Factor Xla-inhibitor complexes suggests that this marker may be the most appropriate for determining FXI activation status in patient cohorts. 

AT is the most important regulator of coagulation as it inhibits thrombin, Factor Xa, Factor IXa, and also to a lesser extent other proteases such as Factors Xlla, Xla and Vlla. The importance of AT as a regulator of coagulation is highlighted by the fact that individuals with congenital or acquired AT deficiency are predisposed to thrombotic diseases. 

Coagulation Profile BV offers a range of standardised and validated enzyme-linked immunosorbent assays (ELISAs) for quantifying enzyme-inhibitor complexes. Please contact us for more details or to discuss specific requirements. 

Inhibition of the tissue factor-factor VIIa-factor Xa complex is one of the main physiological mechanisms responsible for controlling coagulation. The tissue factor pathway inhibitor (TFPI) protein is characterised by three sequential Kunitz-type domains, the first and second of which are essential for binding and inhibiting Factors VIIa and Xa, respectively. The third Kunitz domain in TFPI is involved in lipoprotein binding; it has a heparin-binding site and is required for binding to its co-factor protein S. 

TFPI is mainly synthesised by endothelial cells, and its in vivo distribution is rather complex. Roughly 80% of all TFPI remains bound to the vascular endothelium. The remaining 20% of TFPI circulates in the blood. Around 5 to 10% of circulating TFPI is present in platelets; the remaining 90% occurs in two forms: 80% as truncated TFPI with reduced inhibitory properties and 10% as full-length TFPI, which is the most potent form of TFPI in terms of anticoagulant activity. 

Coagulation Profile BV offers a variety of assays for assessing the TFPI-Protein S anticoagulant pathway. Full-length TFPI antigen levels are quantified through an enzyme-linked immunosorbent assay (ELISA) developed and validated in house, whereas the functional TFPI-Protein S pathway is assessed through a modified thrombin generation-based assay. Please contact us for more details or to discuss specific requirements.