A Quantitative and Mechanistic Assessment of Activated Thrombin-Activatable Fibrinolysis Inhibitor and its Role in Pathological Bleeding and Thrombosis

The coagulation and fibrinolytic systems are linked by the thrombin-thrombomodulin complex which regulates each system through activation of protein C and TAFI, respectively. We have used novel assays and techniques to study the enzymology and biochemistry of TAFI and TAFIa, to measure TAFI activation in hemophilia A and protein C deficiency and to determine if enhancing TAFI activation can improve hemostasis in hemophilic plasma and whole blood. We show that TAFIa not TAFI attenuates fibrinolysis in vitro and this is supported by a relatively high catalytic efficiency (16.41μM-1s-1) of plasminogen binding site removal from fibrin degradation products (FDPs) by TAFIa. Since the catalytic efficiency of TAFIa in removing these sites is ~60-fold higher than that for inflammatory mediators such as bradykinin it is likely that FDPs are a physiological substrate of TAFIa. The high catalytic efficiency is primarily a result of a low Km which can be explained by a novel mechanism where TAFIa forms a binary complex with plasminogen and is recruited to the surface of FDPs. The low Km also suggests that TAFIa would effectively cleave lysines from FDPs during the early stages of fibrinolysis (i.e. at low concentrations of FDPs). Since individuals with hemophilia suffer from premature fibrinolysis as a result of insufficient TAFI activation we quantified TAFI activation in whole blood from hemophilic subjects. Both the rate of activation and the area under the TAFI activation time course (termed TAFIa potential) was determined to be reduced in hemophilia A and the TAFIa potential was significantly and inversely correlated with the clinical bleeding iii phenotype. Using a novel therapeutic strategy, we used soluble thrombomodulin to increase TAFI activation which improved the clot lysis time in factor VIII deficient human plasma and hemophilic dog plasma as well as hemophilic dog blood. Finally, we briefly show in a biochemical case study that TAFI activation is enhanced in protein C deficiency and when afflicted individuals are placed on Warfarin anticoagulant therapy, TAFI activation is reduced. Since TAFIa stabilizes blood clots, this suggests that reducing TAFI activation or inhibiting TAFIa may help restore blood flow in vessels with pathological thrombosis.

Robotic Outcome Assessment in a Non-Human Primate Model of Middle Cerebral Artery Stroke

Stroke is a prevalent disorder with immense socioeconomic impact. A variety of chronic neurological deficits result from stroke. In particular, sensorimotor deficits are a significant barrier to achieving post-stroke independence. Unfortunately, the majority of pre-clinical studies that show improved outcomes in animal stroke models have failed in clinical trials. Pre-clinical studies using non-human primate (NHP) stroke models prior to initiating human trials are a potential step to improving translation from animal studies to clinical trials. Robotic assessment tools represent a quantitative, reliable, and reproducible means to assess reaching behaviour following stroke in both humans and NHPs. We investigated the use of robotic technology to assess sensorimotor impairments in NHPs following middle cerebral artery occlusion (MCAO). Two cynomolgus macaques underwent transient MCAO for 90 minutes. Approximately 1.5 years following the procedure these NHPs and two non-stroke control monkeys were trained in a reaching task with both arms in the KINARM exoskeleton. This robot permits elbow and shoulder movements in the horizontal plane. The task required NHPs to make reaching movements from a centrally positioned start target to 1 of 8 peripheral targets uniformly distributed around the first target. We analyzed four movement parameters: reaction time, movement time (MT), initial direction error (IDE), and number of speed maxima to characterize sensorimotor deficiencies. We hypothesized reduced performance in these attributes during a neurobehavioural task with the paretic limb of NHPs following MCAO compared to controls. Reaching movements in the non-affected limbs of control and experimental NHPs showed bell-shaped velocity profiles. In contrast, the reaching movements with the affected limbs were highly variable. We found distinctive patterns in MT, IDE, and number of speed peaks between control and experimental monkeys and between limbs of NHPs with MCAO. NHPs with MCAO demonstrated more speed peaks, longer MTs, and greater IDE in their paretic limb compared to controls. These initial results qualitatively match human stroke subjects’ performance, suggesting that robotic neurobehavioural assessment in NHPs with stroke is feasible and could have translational relevance in subsequent human studies. Further studies will be necessary to replicate and expand on these preliminary findings.

The relationship of E. coli and total coliform culture to the quantitative molecular detection of various fecal indicators, sources, and pathogens in private drinking water wells

Water remains a predominant vector for human enteric pathogens not just for developing countries but also developed nations, where numerous infectious disease outbreaks, linked to the contamination of drinking water have been documented. Private drinking water wells are a source of drinking water that is largely unstudied even though a significant percentage of the population in Ontario relies on wells as their primary water source. As there exists little to no systematic surveillance for enteric infections or outbreaks related to well water sources, these individuals may be at higher risk of waterborne infectious diseases. The relationships between various fecal indicators in the water of private drinking water wells, including E. coli, Total Coliforms (TC) and Bacteroides, and enteric pathogens, including Campylobacter jejuni, Salmonella spp., and Shiga toxin producing E. coli, were studied. Convenience private well water samples collected from various regions of interest during the summer of 2014 underwent membrane filtration and culture to determine quantities of E. coli and TC colony forming units. 289 E. coli positive and 230 TC-only waters were successfully analyzed by individual qPCR assays for the aforementioned enteric pathogens. Microbial source tracking methods targeted to specific Bacteroides were used to determine the source of fecal contamination as either human or bovine. The source of fecal contamination varied by geographic region and is thought to be due to such things as differences in septic tank density and underlying geology, among others. Fecal indicators, E. coli and Bacteroides, were significantly correlated. E. coli as measured by qPCR was more strongly correlated to both total and human-specific Bacteroides genetic markers than culturable E. coli. Lastly, 1.9% of samples showed molecular evidence of contamination with enteric pathogens. Although low, this finding is significant given the limited volume of water available for testing, and suggests a potential health risk to consumers. Knowing the extent of contamination, as well as the biologic source, can better inform risk assessment and the development of potential intervention strategies for private well water in specific regions of Ontario.