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

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

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

ST Genesia reference values of 117 healthy donors measured with STG-BleedScreen, STG-DrugScreen and STG-ThromboScreen reagents

INTRODUCTION: The ST Genesia is a benchtop, fully automated thrombin generation (TG) device. It is completely standardized and ensures a uniform heat distribution throughout the measurement. We aimed to determine reference values and to compare TG in men and women with and without the use of oral contraceptives (OCs). MATERIALS AND METHODS: Plasma from 117 healthy donors was measured on the ST Genesia with the available reagent kits: STG-BleedScreen, STG-DrugScreen, and STG-ThromboScreen. All kits include at least two quality controls and a reference plasma to normalize data. STG-ThromboScreen has a second trigger containing thrombomodulin (TM) to include the effect on the protein C pathway. Means were compared with one-way analysis of variance and reference ranges were established with 2.5th to 97.5th percentiles on absolute TG parameters. RESULTS: Mean age of the donors was 35 years (SD ± 12); 49.6% were men, 37.6% women without OCs, and 12.8% women with OCs. Men and women without OCs had, respectively, a mean peak height of 167 nM and 164 nM with STG-BleedScreen, 335 nM and 351 nM with STG-DrugScreen, and 192 nM and 198 nM with STG-ThromboScreen. Women taking OCs had a mean peak height of 263 nM, 473 nM, and 312nM, respectively (  < .05 compared to men/women without OCs). TM decreased endogenous thrombin potential by 54% in men, 47% in women without OCs, and only 25% in women with OCs (  < .05 compared to men/women without OCs). CONCLUSIONS: TG in men and women without OCs was similar; however, women taking OCs had significantly higher TG values, and the effect of TM was also less pronounced in these women

Reference values for thrombin dynamics in platelet rich plasma

Thrombin generation (TG) is a better determinant of the overall function of the hemostatic system than routinely used clotting time-based assays and can be studied more in detail by thrombin dynamics analysis. Platelet poor plasma is often used to measure TG, however, measuring the contribution of the platelets is also important as patients with a low platelet count or with dysfunctional platelets have an increased risk of developing bleeding. In this study, platelet rich plasma (PRP) was collected from 117 healthy individuals. PRP was measured undiluted and diluted to a varying platelet concentration of 10*109/L to 400*109/L. Prothrombin conversion and thrombin inactivation were calculated from the data obtained by the TG parameters and coagulation factor levels (antithrombin, α2Macroglobulin (α2M) and fibrinogen). Reference ranges of TG and thrombin dynamics in PRP of 117 healthy individuals were established. Peak, velocity index and the maximum rate of prothrombin conversion increased linearly with platelet count, but endogenous thrombin potential reached a maximum at 150*109/L as seen in a subset population (n = 20). More extensive analysis revealed that a platelet count below 50*109/L did not affect TG parameters (except for the ETP). Correlation analysis indicated that the platelet count mainly affected the rate of prothrombin conversion. Inhibition of thrombin by antithrombin and α2M increased with increasing TG, but the ratio of inhibition by antithrombin or α2M remained the same independently of the total thrombin formed. In conclusion, TG and thrombin dynamics were assessed in PRP of healthy donors to provide reference values for future TG studies in PRP. Increasing the platelet count mainly affected the rate of prothrombin conversion and TG, rather than the total amount of thrombin formed

Low paediatric thrombin generation is caused by an attenuation of prothrombin conversion

Thrombin generation (TG) is decreased in children. TG is determined by two underlying processes: the conversion of prothrombin to thrombin and the inactivation of thrombin. Therefore, lower TG capacity in children can either be caused by a reduction of prothrombin conversion, an increase of thrombin inactivation, or both. In 36 children and 8 adults, TG and the factors that determine thrombin inactivation (antithrombin, alpha(2)Macroglobulin (alpha M-2) and fibrinogen) were measured. Prothrombin conversion, thrombin inhibitor complex formation, and the overall thrombin decay capacity were determined. In silico modelling was performed to determine the contribution pro thrombin conversion and thrombin inactivation to deviant paediatric TG. Both the amount of prothrombin converted and the maximal pro thrombin conversion rate are significantly reduced in children as compared to adults. This is partly due to the prothrombin levels being lower and partly to a lower prothrombin conversion rate. The overall thrombin decay capacity is not significantly different in children, but alpha(2)Macroglobulin plays a more important role than it does in adults. In silico experiments demonstrate that reduced prothrombin conversion and to a lesser extent elevated alpha M-2 levels provide an explanation for low TG in children. Young age has a dual effect on prothrombin conversion. Lower plasma prothrombin levels result in decreased pro thrombin conversion but the rate of prothrombin conversion is also decreased, i.e. the development of prothrombinase is lower than in adults

A thrombin-driven neural net diagnoses the antiphospholipid syndrome without the need for interruption of anticoagulation

Thrombosis is an important manifestation of the antiphospholipid syndrome (APS). The thrombin generation (TG) test is a global hemostasis assay, and increased TG is associated with thrombosis. APS is currently diagnosed based on clinical and laboratory criteria, the latter defined as anti-cardiolipin, anti-beta 2-glycoprotein I antibodies, or lupus anticoagulant (LA). APS testing is often performed after a thrombotic episode and subsequent administration of anticoagulation, which might hamper the interpretation of clotting assays used for LA testing. We set out to develop an artificial neural network (NN) that can diagnose APS in patients who underwent vitamin K antagonist (VKA) treatment, based on TG test results. Five NNs were trained to diagnose APS in 48 VKA-treated patients with APS and 64 VKA-treated controls, using TG and thrombin dynamics parameters as inputs. The 2 bestperforming NNs were selected (accuracy, 96%; sensitivity, 96%-98%; and specificity, 95%97%) and further validated in an independent cohort of VKA-anticoagulated patients with APS (n = 33) and controls (n = 62). Independent clinical validation favored 1 of the 2 selected NNs, with a sensitivity of 88% and a specificity of 94% for the diagnosis of APS. In conclusion, the combined use of TG and NN methodology allowed for us to develop an NN that diagnoses APS with an accuracy of 92% in individuals with VKA anticoagulation (n = 95). After further clinical validation, the NN could serve as a screening and diagnostic tool for patients with thrombosis, especially because there is no need to interrupt anticoagulant therapy