Semi-automated thrombin dynamics applying the ST Genesia thrombin generation assay

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

Experimental Bearing Capacity Determination of Bonded Rock Bolts

Import 26/02/2015Diplomová práce se zabývá únosností tmelených horninových svorníků a jejím experimentální stanovením. Tato práce analyzuje a poukazuje na možné způsoby porušení, které vznikají zatížením v těsné blízkosti svorníkové tyče a to především v prstenci tmele na kontaktech svorník – tmel a tmel – hornina. V teoretické části byl navržen způsob měření a stanovení pevnostních a deformačních charakteristik tahem zatížených tmelených svorníků napodobujících zatížení v kořenové délce svorníkové výztuže. V praktické části byly pak navržené postupy uskutečněny sérií laboratorních zkoušek. Výstupem z provedených zkoušek je pracovně-deformační charakteristika tmelené svorníkové výztuže zvoleného testovaného materiálu.The thesis focuses on the bearing capacity of bonded rock bolts and experimental determination of this capacity. Possible ways of failure, which is caused by the load near the rock bolt, especially in the circular ring of the grout and between the rock bolt - the grout and between the grout - the rock, are analysed. The theoretical part includes design of measuring and assessment of strength and deformation characteristics on drawn rock bolts as it simulates the load in the root length of bolt reinforcement. This theory was applied in the laboratory tests and presents the practical part of the thesis. In conclusion, the load - deformation characteristics of bonded rock bolt reinforcement made from chosen material are stated.224 - Katedra geotechniky a podzemního stavitelstvívýborn

Deciphering the coagulation profile through the dynamics of thrombin activity

Thrombosis has proven to be extremely difficult to predict. Measuring the generation of thrombin is a very sensitive method to detect changes in the hemostatic system. We developed a method based on the generation of thrombin to further fingerprint hemostasis, which we have named thrombin dynamics. Via this method we are able to exactly measure the prothrombin conversion and thrombin inactivation, and any change in the coagulation cascade will be reflected in these two processes. In the current study we analyzed the importance of the members of the pr

Platelet-Associated Matrix Metalloproteinases Regulate Thrombus Formation and Exert Local Collagenolytic Activity

Objective Platelets are increasingly implicated in processes beyond hemostasis and thrombosis, such as vascular remodeling. Members of the matrix metalloproteinase (MMP) family not only remodel the extracellular matrix but also modulate platelet function. Here, we made a systematic comparison of the roles of MMP family members in acute thrombus formation under flow conditions and assessed platelet-dependent collagenolytic activity over time. Approach and Results Pharmacological inhibition of MMP-1 or MMP-2 (human) or deficiency in MMP-2 (mouse) suppressed collagen-dependent platelet activation and thrombus formation under flow, whereas MMP-9 inhibition/deficiency stimulated these processes. The absence of MMP-3 was without effect. Interestingly, MMP-14 inhibition led to the formation of larger thrombi, which occurred independently of its capacity to activate MMP-2. Platelet thrombi exerted local collagenolytic activity capable of cleaving immobilized dye-quenched collagen and fibrillar collagen fibers within hours, with loss of the majority of the platelet adhesive properties of collagen as a consequence. This collagenolytic activity was redundantly mediated by platelet-associated MMP-1, MMP-2, MMP-9, and MMP-14 but occurred independently of platelet -granule release (Nbeal2(-/-) mice). The latter was in line with subcellular localization experiments, which indicated a granular distribution of MMP-1 and MMP-2 in platelets, distinct from -granules. Whereas MMP-9 protein could not be detected inside platelets, activated platelets did bind plasma-derived MMP-9 to their plasma membrane. Overall, platelet MMP activity was predominantly membrane-associated and influenced by platelet activation status. Conclusions Platelet-associated MMP-1, MMP-2, MMP-9, and MMP-14 differentially modulate acute thrombus formation and at later time points limit thrombus formation by exerting collagenolytic activity

Plasma fibrinogen levels and all-cause and cause-specific mortality in an Italian adult population: results from the Moli-sani study

Epidemiological data on the association between fibrinogen levels and mortality are scarse and controversial. Longitudinal analyses were performed, separately by sex, on 17,689 individuals from the Moli-sani study [53% women, ≥35 years, free from cardiovascular disease (CVD) or cancer at enrolment], to evaluate the association between plasma fibrinogen and all-cause and cause-specific mortality. Over a median follow-up of 11.2 years, 1,058 deaths (34.7% CVD, 36.3% cancer) were ascertained. Both in the lowest (1.12-2.64 g/L) and highest (≥3.62 g/L) fibrinogen quintiles, women had an increased all-cause mortality hazard, when compared with third quintile (2.97-3.23 g/L). Dose-response analyses showed a U-shaped relationship in women (P overall <0.0001; P non-linear association <0.0001), but a positive linear association for all-cause mortality in men (P overall 0.0038; P non-linear association 0.76). Similar trends for a U-shaped association were observed for CVD mortality, while no association was observed with cancer deaths. A U-shaped association of fibrinogen levels with other-cause mortality was also found in both sexes. This study shows that not only higher but also lower fibrinogen levels represent hazard for mortality when compared to normal levels; U-shaped curves being prevalently observed in women

Population-wide persistent hemostatic changes after vaccination with ChAdOx1-S

Various vaccines were developed to reduce the spread of the Severe Acute Respiratory Syndrome Cov-2 (SARS-CoV-2) virus. Quickly after the start of vaccination, reports emerged that anti-SARS-CoV-2 vaccines, including ChAdOx1-S, could be associated with an increased risk of thrombosis. We investigated the hemostatic changes after ChAdOx1-S vaccination in 631 health care workers. Blood samples were collected 32 days on average after the second ChAdOx1-S vaccination, to evaluate hemostatic markers such as D-dimer, fibrinogen, α2-macroglobulin, FVIII and thrombin generation. Endothelial function was assessed by measuring Von Willebrand Factor (VWF) and active VWF. IL-6 and IL-10 were measured to study the activation of the immune system. Additionally, SARS-CoV-2 anti-nucleoside and anti-spike protein antibody titers were determined. Prothrombin and fibrinogen levels were significantly reduced after vaccination (−7.5% and −16.9%, p &lt; 0.0001). Significantly more vaccinated subjects were outside the normal range compared to controls for prothrombin (42.1% vs. 26.4%, p = 0.026) and antithrombin (23.9% vs. 3.6%, p = 0.0010). Thrombin generation indicated a more procoagulant profile, characterized by a significantly shortened lag time (−11.3%, p &lt; 0.0001) and time-to-peak (−13.0% and p &lt; 0.0001) and an increased peak height (32.6%, p = 0.0015) in vaccinated subjects compared to unvaccinated controls. Increased VWF (+39.5%, p &lt; 0.0001) and active VWF levels (+24.1 %, p &lt; 0.0001) pointed toward endothelial activation, and IL-10 levels were significantly increased (9.29 pg/mL vs. 2.43 pg/mL, p = 0.032). The persistent increase of IL-10 indicates that the immune system remains active after ChAdOx1-S vaccination. This could trigger a pathophysiological mechanism causing an increased thrombin generation profile and vascular endothelial activation, which could subsequently result in and increased risk of thrombotic events

Thrombin dynamics

Cardiovascular diseases (CVDs) are the number one cause of death globally. In 2012, 17.5 million people died of CVDs, of which 43% were caused by ischemic heart disease and 38% by stroke. Thrombosis plays a pivotal role in the development of ischemic heart disease and stroke. Therefore, the haemostatic system has been one of the main targets of prevent and clinical management of thrombosis related pathologies. Adequate diagnosis of a medical condition is pivotal in the fight against thrombosis and the development of a suitable diagnostic test that can accurately predict the thrombotic risk is essential in the development of an effective treatment strategy to treat thrombosis. In this thesis the thrombin dynamics method was introduced, a technique to investigate both the pro- and anticoagulant processes that underlie thrombin generation separately. This method was used to study how these two processes deviates in different patient groups such as patients with liver cirrhosis and patients who are being treated with anticoagulants