Management of the thrombotic risk associated with COVID-19: guidance for the hemostasis laboratory.

Coronavirus disease 2019 (COVID-19) is associated with extreme inflammatory response, disordered hemostasis and high thrombotic risk. A high incidence of thromboembolic events has been reported despite thromboprophylaxis, raising the question of a more effective anticoagulation. First-line hemostasis tests such as activated partial thromboplastin time, prothrombin time, fibrinogen and D-dimers are proposed for assessing thrombotic risk and monitoring hemostasis, but are vulnerable to many drawbacks affecting their reliability and clinical relevance. Specialized hemostasis-related tests (soluble fibrin complexes, tests assessing fibrinolytic capacity, viscoelastic tests, thrombin generation) may have an interest to assess the thrombotic risk associated with COVID-19. Another challenge for the hemostasis laboratory is the monitoring of heparin treatment, especially unfractionated heparin in the setting of an extreme inflammatory response. This review aimed at evaluating the role of hemostasis tests in the management of COVID-19 and discussing their main limitations.

A flowcytometric analysis to efficiently quantify multiple innate immune cells and T Cell subsets in human blood.

The balance of inflammation and immunosuppression driven by changed ratios in diverse myeloid and T cell subsets, as well as their state of activation and ability to migrate to lymphoid compartments or inflammatory sites, has emerged as a highly active area of study across clinical trials of vaccines and therapies against cancer, trauma, as well as autoimmune and infectious diseases. There is a need for effective protocols which maximally use the possibilities offered by modern flow cytometers to characterize such immune cell changes in peripheral blood using small volumes of human blood. Additionally, longitudinal clinical studies often use cryopreserved samples, which can impact flow cytometric results. To efficiently gauge both the innate and the adaptive immune response, two novel 15-color antibody panels to identify key myeloid and T cell subsets and their functional potential were established. This approach was used to compare cellular immune profiles in fresh whole blood and in matched cryopreserved peripheral blood mononuclear cells (PBMCs). Cocktail I was designed to identify and characterize myeloid cell populations including dendritic cells (DCs), monocytic monocyte-derived suppressor cells (MO-MDSC), and monocytes, determining further core aspects of their state of maturity, T cell stimulatory (or inhibitory) potential, and migration capability. Cocktail II was used for phenotyping diverse T cells subsets, and their key migration and functional regulatory capabilities. The two 15-color antibody panels for the evaluation of both immune-stimulating and immunosuppressive processes presented herein allowed for efficient evaluation of the balance of immune activation versus immunosuppression across key blood cells, with good resolution for all 15 markers stained for in each panel. Gating strategies for the myeloid and T cells are presented to further support specific subset identification. This protocol was shown to be reproducible across donors and useful to study both RBC-lysed whole blood and cryopreserved PBMCs. © 2017 International Society for Advancement of Cytometry.

Scanning laser-induced endothelial injury: a standardized and reproducible thrombosis model for intravital microscopy.

Vascular injury models are indispensable for studying thrombotic processes in vivo. Amongst the available methods for inducing thrombosis, laser-induced endothelial injury (LIEI) has several unique advantages. However, a lack of methodological standardization and expensive instrumentation remain significant problems decreasing reproducibility and impeding the adoption of LIEI in the wider scientific community. In this, study, we developed a standardized protocol for scanning laser-induced endothelial injury (scanning-LIEI) of murine mesenteric veins using the intrinsic 405 nm laser of a conventional laser scanning confocal microscope. We show that our model produces thrombi with prominent core-shell architectures and minimal radiation-related fluorescence artefacts. In comparison with previous methods, the scanning-LIEI model exhibits reduced experimental variability, enabling the demonstration of dose-response effects for anti-thrombotic drugs using small animal cohorts. Scanning-LIEI using the intrinsic 405 nm laser of a confocal laser scanning microscope represents a new method to induce standardized vascular injury with improved reproducibility of thrombus formation. The reduced need for instrument customisation and user experience means that this model could be more readily adopted in the research community.

Plasminogen activator inhibitor 1 and 2 are tumor necrosis factor/cachectin-responsive genes.

Human rTNF/Cachectin was shown to stimulate gene transcription of plasminogen activator inhibitor (PA1)-1 and PAI-2, and simultaneously suppress constitutive gene expression of tissue-type plasminogen activator (t-PA) in human fibrosarcoma cells. We propose that a TNF-mediated reprogramming of gene transcription induces, in appropriate target cells, an anti-fibrinolytic state, which may cooperate with the induction of procoagulant activity (tissue factor) to stabilize the fibrin deposits commonly found in inflamed tissue. PAI genes also provide a model system for a study of the molecular pathways underlying TNF-mediated signal transduction.

Data on the modulatory effects of a single bolus dexamethasone on the surface marker expression of various leucocyte subsets.

Dexamethasone is frequently administered to surgical patients for anti-emetic prophylaxis. We have examined the immunomodulatory effects of a single bolus of dexamethasone on circulating peripheral blood mononuclear cells (PBMCs) in the same 10 healthy male volunteers, previously used in our investigation on early in vivo effects of a single anti-emetic dose of dexamethasone on innate immune cell gene expression and activation [1]. Blood samples were drawn at baseline, 2 h, 4 h and 24 h. Immune cell phenotypes were examined with flow cytometry. In this data article the expression strength of markers involved in immune activation and immunosuppression as well as maturation, migration, cell death and responsiveness to signalling on monocyte and cDC subsets, as well as NK cells, CD4+ and CD8+ T cells and regulatory T cells (Treg) are presented. These data improve our understanding of the immunomodulatory effects of the glucocorticoid dexamethasone in-vivo, which may be important for the optimisation of treatment regimens as well as the evaluation of new indications for glucocorticoid treatment.

Glucocorticoid-modulated gene expression of tissue- and urinary-type plasminogen activator and plasminogen activator inhibitor 1 and 2.

Constitutive gene expression of four components of plasminogen activating enzyme system, urinary and tissue-type plasminogen activator (u-PA and t-PA), plasminogen activator inhibitor 1 (PAI-1) and PAI-2 in HT-1080 human fibrosarcoma cells, was modulated by the synthetic glucocorticoid dexamethasone (Dex, 10(-7) M). More than 90% of u-PA, t-PA and PAI-1 antigen was found in conditioned medium, whereas PAI-2 was mainly cell associated. In 48-h culture supernatants (expressed per 10(6) cells) PAI-1 antigen increased from 350 to 3,300 ng and t-PA from 19 to 38 ng. u-PA and PAI-2 in the same samples decreased from 380 to 46 ng and from 3.5 to 1.8 ng, respectively. Northern blot hybridization and nuclear "Run-on" transcription assays demonstrated that the increase of t-PA and PAI-1 and the decrease of u-PA were associated with equivalent changes of gene template activity. Modulation of u-PA, t-PA and PAI-1 gene expression by Dex was completely blocked by the glucocorticoid antagonist RU 38486, suggesting that all effects were mediated through the glucocorticoid receptor. Cycloheximide, an inhibitor of protein biosynthesis induced a rapid transient increase of t-PA, u-PA and PAI-1 mRNA and a sustained increase of PAI-2 mRNA, but blocked the more long term effects of Dex, suggesting that both constitutive and hormonally regulated maintenance of mRNA steady state levels required protein biosynthesis.

Mouse L cells expressing human prourokinase-type plasminogen activator: effects on extracellular matrix degradation and invasion.

A cosmid (cos pUK0322) harboring the complete human urokinase-type plasminogen activator (u-PA) gene and Geneticin resistance as a selectable marker was isolated from a human genomic library and characterized. After transfection of cos pUK0322 into mouse L cells and selection, several plasminogen activator (PA)-expressing clones were obtained and one (LuPA) was chosen for additional study. The PA expressed was identical to human pro-u-PA in enzymatic, electrophoretic, and antigenic properties. The expression of PA was stable over 50 population doublings. The regulation of the transfected gene was studied by treatment of the cells with various hormones and other effectors. Expression of PA activity was inhibited fivefold by dexamethasone and stimulated two- to threefold by agonists of the adenylate cyclase dependent pathway of signal transduction, such as dibutyryl cyclic AMP and cholera and pertussis toxins. The modulation of PA activity was associated with corresponding changes in mRNA steady-state levels. The phenotypic changes associated with pro-u-PA expression were analyzed in vitro by degradation of 3H-labeled extracellular matrix (ECM), invasion of a matrigel basement membrane analogue, and by light and electron microscopy. LuPA cells and reference HT-1080 fibrosarcoma cells, in contrast to control Lneo cells transfected with the neomycin resistance gene, degraded the ECM and invaded the matrigel basement membrane. Matrix degradation correlated with the modulation of pro-u-PA gene expression as it was inhibited by dexamethasone and promoted by dibutyryl cyclic AMP. Inhibition of PA or plasmin using anti-u-PA IgG or aprotinin prevented ECM degradation and invasion. These results demonstrate that u-PA expression alone is sufficient to confer to a cell an experimental invasive phenotype.

Fibrinolysis, inflammation, and regulation of the plasminogen activating system.

The maintenance of a given physiological process demands a coordinated and spatially regulated pattern of gene regulation. This applies to genes encoding components of enzyme cascades, including those of the plasminogen activating system. This family of proteases is vital to fibrinolysis and dysregulation of the expression pattern of one or more of these proteins in response to inflammatory events can impact on hemostasis. Gene regulation occurs on many levels, and it is apparent that the genes encoding the plasminogen activator (fibrinolytic) proteins are subject to both direct transcriptional control and significant post-transcriptional mechanisms. It is now clear that perturbation of these genes at either of these levels can dramatically alter expression levels and have a direct impact on the host's response to a variety of physiological and pharmacological challenges. Inflammatory processes are well known to impact on the fibrinolytic system and to promote thrombosis, cancer and diabetes. This review discusses how inflammatory and other signals affect the transcriptional and post-transcriptional expression patterns of this system, and how this modulates fibrinolysis in vivo.

Plasminogen activator inhibitor 2: regulation of gene transcription during phorbol ester-mediated differentiation of U-937 human histiocytic lymphoma cells.

We have isolated and sequenced two cDNA clones coding for plasminogen activator inhibitor 2 (PAI-2). The cDNA was used to study the regulation of PAI-2 gene transcription by the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate in the human histiocytic lymphoma cell line U-937. The tumor promoter caused a transient, 50-fold increase of PAI-2 gene transcription.

Fibrinolysis: from blood to the brain.

We all know about classical fibrinolysis, how plasminogen activation by either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) promotes fibrin breakdown, and how this process was harnessed for the therapeutic removal of blood clots. While this is still perfectly true and still applicable to thromboembolic conditions today, another dimension to this system came to light over two decades ago that implicated the plasminogen activating system in a context far removed from the dissolution of blood clots. This unsuspected area related to brain biology where t-PA was linked to a plethora of activities in the CNS, some of which do not necessarily require plasmin generation. Indeed, t-PA either directly or via plasmin, has been shown to not only have key roles in modulating astrocytes, neurons, microglia, and pericytes, but also to have profound effects in a number of CNS conditions, including ischaemic stroke, severe traumatic brain injury and also in neurodegenerative disorders. While compelling insights have been obtained from various animal models, the clinical relevance of aberrant expression of these components in the CNS, although strongly implied, are only just emerging. This review will cover these areas and will also discuss how the use of thrombolytic agents and anti-fibrinolytic drugs may potentially have impacts outside of their clinical intention, particularly in the CNS.

Passenger mutations and aberrant gene expression in congenic tissue plasminogen activator-deficient mouse strains.

UNLABELLED: Essentials C57BL/6J-tissue plasminogen activator (tPA)-deficient mice are widely used to study tPA function. Congenic C57BL/6J-tPA-deficient mice harbor large 129-derived chromosomal segments. The 129-derived chromosomal segments contain gene mutations that may confound data interpretation. Passenger mutation-free isogenic tPA-deficient mice were generated for study of tPA function. SUMMARY: Background The ability to generate defined null mutations in mice revolutionized the analysis of gene function in mammals. However, gene-deficient mice generated by using 129-derived embryonic stem cells may carry large segments of 129 DNA, even when extensively backcrossed to reference strains, such as C57BL/6J, and this may confound interpretation of experiments performed in these mice. Tissue plasminogen activator (tPA), encoded by the PLAT gene, is a fibrinolytic serine protease that is widely expressed in the brain. A number of neurological abnormalities have been reported in tPA-deficient mice. Objectives To study genetic contamination of tPA-deficient mice. Materials and methods Whole genome expression array analysis, RNAseq expression profiling, low- and high-density single nucleotide polymorphism (SNP) analysis, bioinformatics and genome editing were used to analyze gene expression in tPA-deficient mouse brains. Results and conclusions Genes differentially expressed in the brain of Plat(-/-) mice from two independent colonies highly backcrossed onto the C57BL/6J strain clustered near Plat on chromosome 8. SNP analysis attributed this anomaly to about 20 Mbp of DNA flanking Plat being of 129 origin in both strains. Bioinformatic analysis of these 129-derived chromosomal segments identified a significant number of mutations in genes co-segregating with the targeted Plat allele, including several potential null mutations. Using zinc finger nuclease technology, we generated novel 'passenger mutation'-free isogenic C57BL/6J-Plat(-/-) and FVB/NJ-Plat(-/-) mouse strains by introducing an 11 bp deletion into the exon encoding the signal peptide. These novel mouse strains will be a useful community resource for further exploration of tPA function in physiological and pathological processes.

Valproic acid selectively increases vascular endothelial tissue-type plasminogen activator production and reduces thrombus formation in the mouse.

Essentials Stimulating endogenous fibrinolysis could be a novel antithrombotic strategy. The effect of valproic acid on endothelial tissue plasminogen activator in mice was investigated. Valproic acid increased tissue plasminogen activator expression in vascular endothelium. Valproic acid reduced fibrin deposition and thrombus formation after vascular injury. SUMMARY: Background The endogenous fibrinolytic system has rarely been considered as a target to prevent thrombotic disease. Tissue-type plasminogen activator (t-PA) production is potently increased by histone deacetylase (HDAC) inhibitors in endothelial cells in vitro, but whether this translates into increased vascular t-PA production and an enhanced fibrinolytic capacity in vivo is unknown. Objectives To determine whether the HDAC inhibitor valproic acid (VPA) stimulates production of t-PA in the vasculature of mice, and whether VPA pretreatment affects fibrin deposition and clot formation after mechanical vessel injury. Methods Mice were injected with VPA twice daily for up to 5 days. t-PA mRNA, and antigen expression in the mouse aorta and the circulating levels of t-PA were determined. Fibrin and thrombus dynamics after mechanical vessel injury were monitored with intravital confocal microscopy. Potential effects of VPA on platelets and coagulation were investigated. Results and Conclusions We found that VPA treatment increased vascular t-PA production in vivo and, importantly, that VPA administration was associated with reduced fibrin accumulation and smaller thrombi in response to vascular injury, but still was not associated with an increased risk of bleeding. Furthermore, we observed that higher concentrations of VPA were required to stimulate t-PA production in the brain than in the vasculature. Thus, this study shows that VPA can be dosed to selectively manipulate the fibrinolytic system in the vascular compartment and reduce thrombus formation in vivo.

The novel anti-tumour agent oxamflatin differentially regulates urokinase and plasminogen activator inhibitor type 2 expression and inhibits urokinase-mediated proteolytic activity.

Cell surface, urokinase (u-PA)-mediated, plasminogen activation has recently been recognised as a process integral to extracellular matrix degradation. The primary inhibitor of u-PA activity in the extracellular matrix is plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor. The malignant metastatic phenotype is associated with excessive and uncontrolled, tumour cell-associated, u-PA-mediated, extracellular matrix degradation. Inhibition of the malignant metastatic phenotype via induction of PAI-2 expression and/or inhibition of u-PA expression may represent a novel means via which the metastatic phenotype can be arrested. Agents capable of inducing PAI-2 and/or inhibiting u-PA activity may restrict u-PA-mediated tumour cell proteolysis and facilitate in the development of therapeutic strategies to combat malignant disease. We have identified the hydroxamic acid derivative oxamflatin, previously noted to revert the malignant phenotype in K-ras-transformed NIH-3T3 cells, as capable of upregulating PAI-2 and simultaneously suppressing u-PA expression in two different cell systems. In addition, zymographic analysis indicated that oxamflatin treatment results in a significant reduction in u-PA proteolytic activity in both HT-1080 fibrosarcoma and U-937 histiocytic lymphoma cells. We postulate that oxamflatin represents a novel means by which induction of PAI-2 and concomitant inhibition of u-PA gene and protein expression can be achieved and may be of benefit in inhibiting the malignant metastatic phenotype.

Cyclic AMP-dependent and -independent effects on tissue-type plasminogen activator activity in osteogenic sarcoma cells; evidence from phosphodiesterase inhibition and parathyroid hormone antagonists.

The plasminogen activator (PA) in clonal osteogenic sarcoma cells of rat origin (UMR 106-01 and UMR 106-06) and in osteoblast-rich rat calvarial cells has been characterized using specific antibodies to be tissue-type PA (tPA). An Mr value of 75,000 by SDS-polyacrylamide gel electrophoresis and fibrin autoradiography supports this characterization. There was also evidence for an Mr 105,000 component, which could be due to a proteinase-inhibitor complex. The mechanism of regulation of this tPA activity has been studied in the clonal osteogenic sarcoma cells. Parathyroid hormone (PTH) and prostaglandin E2, which increase cyclic AMP production in the sarcoma cells, also increased tPA activity. The sensitivity and magnitude of the tPA response to PTH and prostaglandin E2 were increased by simultaneous treatment with isobutylmethylxanthine (IBMX) at drug concentrations which had little effect themselves on tPA activity. In UMR 106-06 cells, which unlike UMR 106-01 cells show a cyclic AMP response to calcitonin, tPA activity was also increased in response to calcitonin, and the effect was enhanced by IBMX. 1,25-Dihydroxyvitamin D-3 also increased tPA activity in the cells, but this response was not modified by IBMX. Synthetic peptide antagonists of PTH-responsive adenylate cyclase, [34Tyr]-hPTH (3-34) amide and [34Tyr]-hPTH (5-34) amide, inhibited the PTH-induced increase in tPA activity over the same concentration range at which they inhibited cyclic AMP production, but the antagonist peptides had no effect on the tPA responses to prostaglandin E2, calcitonin or 1,25-dihydroxyvitamin D-3. These data indicate that cyclic AMP mediates the actions of PTH, prostaglandin E2 and calcitonin in increasing tPA activity in the clonal osteogenic sarcoma cells. 1,25-Dihydroxyvitamin D-3, on the other hand, increases tPA activity through a mechanism independent of cyclic AMP.

Cell-specific regulation of plasminogen activator inhibitor 1 and tissue type plasminogen activator release by human kidney mesangial cells.

Human mesangial cells in culture synthesize and secrete plasminogen activator inhibitor 1 (PAI-1) and tissue-type plasminogen activator (t-PA). Phorbol myristate acetate (PMA), a known activator of protein kinase C, induces a three to four-fold increase in t-PA and PAI-1 release over a period of 24 h, whereas cell-associated t-PA and PAI-1 levels remain relatively stable. A similar effect is obtained with oleylacetyl glycerol, a more physiologic protein kinase C activator. The effect of PMA is suppressed in the presence of H7, an inhibitor of cellular protein kinases, and by cycloheximide and actinomycin D, indicating a requirement for de novo protein and RNA synthesis, respectively. Northern blot analysis of PMA-treated cells reveals a rapid and transient increase in PAI-1 mRNA reaching a maximum after 4-8 h, whereas increase in t-PA mRNA levels requires 24 h. Activation of protein kinase A by addition of 8-bromocyclic AMP (8-bromo cAMP) has no significant effect on PAI-1 release but inhibits the PMA-mediated increases in PAI-1 antigen and mRNA. Addition of 8-bromo cAMP alone does not affect t-PA release. When added to PMA-stimulated cells, 8-bromo cAMP inhibits t-PA release in a dose-dependent manner, but causes a superinduction of t-PA mRNA. 8-bromo cAMP also induces a decrease in PMA-stimulated intracellular t-PA release. Similar inhibition is observed after stimulation of endogenous adenylate cyclase with prostaglandin E1 or isoproterenol. This indicates that protein kinase A activation may inhibit PMA-stimulated t-PA release via a post-transcriptional effect, e.g. inhibition of protein synthesis or activation of protein degradation. In conclusion, hormones or mediators which activate protein kinase C can stimulate t-PA and PAI-1 synthesis in human mesangial cells. Protein kinase A activation has no effect on the basal release of PAI-1 and t-PA by human mesangial cells, and, in contrast to endothelial cells, it inhibits both PMA-stimulated PAI-1 and t-PA releases. This cell-specific regulation of t-PA and PAI-1 seems to be mediated by differential transcriptional and post transcriptional mechanisms.

In vivo and in vitro analysis of the human tissue-type plasminogen activator gene promoter in neuroblastomal cell lines: evidence for a functional upstream kappaB element.

Besides its well-established role in wound healing and fibrinolysis, tissue-type plasminogen activator (t-PA) has been shown to contribute to cognitive processes and memory formation within the central nervous system, and to promote glutamate receptor-mediated excitotoxicity. The t-PA gene is expressed and regulated in neuronal cells but the regulatory transcriptional processes directing this expression are still poorly characterized. We have used DNase I-hypersensitivity mapping and in vivo foot printing to identify putative regulatory elements and transcription factor binding sites in two human neuroblastomal (KELLY and SK-N-SH) and one human glioblastomal (SNB-19) cell lines. Hypersensitive sites were found in the proximal promoter region of all cell lines, and within the first exon for KELLY and SNB-19 cells. Mapping of methylation-protected residues in vivo detected a cluster of protected residues corresponding to a cAMP response element (CRE) and Sp1 sites in the proximal promoter previously shown to be essential for basal expression in other cell types. Protected residues were also found at other sites, notably a kappaB element at position bp -3081 to -3072 that was partly protected in KELLY and SNB-19 cells. Analysis of transfected reporter constructs in KELLY and SNB-19 cells confirmed that this particular element is functionally significant in the transactivation of the t-PA promoter in both cell types. This study defines, by in vivo and in vitro methods, a previously undescribed kappaB site in the t-PA gene promoter that influences t-PA expression in neuronal cells.

Regulation of human tissue-type plasminogen activator gene transcription by epidermal growth factor and 3',5'-cyclic adenosine monophosphate.

Epidermal growth factor (EGF) induces tissue-type plasminogen activator (t-PA) biosynthesis in HeLa cells. Based on nuclear run-on transcription assays, t-PA biosynthesis is modulated by EGF on the level of gene transcription. The effect of EGF is slow, requiring 4-8 h to induce t-PA gene transcription and up to 24 h to induce t-PA mRNA and antigen secretion. An additive response is observed when cells are treated with both phorbol 12-myristate 13-acetate and EGF, suggesting that the two pathways converge and act independently to implement their respective effects. cAMP has previously been shown to potentiate phorbol 12-myristate 13-acetate-mediated induction of t-PA biosynthesis in HeLa cells and in human endothelial cells. Akin to this observation, cAMP also potentiates the EGF-mediated increase in t-PA mRNA. Maximal levels of t-PA mRNA is seen in the presence of all three agonists. The regulation of t-PA by EGF alone and in the presence of either PMA or cAMP is consistent with a role of t-PA during growth and development, and further indicates a functional interplay between protein kinase C-, tyrosine kinase, - and cAMP-dependent signal transduction pathways during regulation of t-PA gene expression.

What drives "fibrinolysis"?

The timely removal of blood clots and fibrin deposits is essential in the regulation of haemostasis. This is achieved by the fibrinolytic system, an enzymatic process that regulates the activation of plasminogen into its proteolytic form, plasmin. This is a self-regulated event as the very presence of fibrin initiates plasminogen activation on the fibrin surface due to the presentation of exposed C-terminal lysine residues in fibrin that allow plasminogen to position itself via its lysine binding sites and to be more efficiently cleaved by tissue-type plasminogen activator (t-PA). Hence fibrin, the ultimate substrate of plasmin during fibrinolysis, is indeed an essential cofactor in the cascade. What has now come to light is that the fibrinolytic system is not solely designed to eliminate fibrin. Indeed, it is a broad acting system that processes a variety of proteins, including many in the brain where there is no fibrin. So what drives t-PA-mediated plasminogen activation when fibrin is not available? This review will describe the broadening role of the fibrinolytic system highlighting the importance of fibrin and other key proteins as facilitators during t-PA-mediated plasminogen activation.

Evaluation of gait impairment in mice subjected to craniotomy and traumatic brain injury.

Traumatic brain injury (TBI) represents a significant global health burden and causes long-lasting neuromotor deficits, particularly in individuals who sustain severe TBI. A better understanding of gait impairment after experimental TBI will provide valuable information for the recovery and rehabilitation of TBI survivors. Here we utilised the DigiGait system to perform kinematic gait analysis in mice subjected to brain injury induced by the controlled cortical impact (CCI) TBI model. Naïve mice, non-craniotomised and craniotomised mice were included as controls. The temporal and spatial profile of gait was mapped from 3h to 1-week post-TBI. Remarkably, there was a noticeable alteration in some aspects of gait in craniotomised sham mice from their pre-surgery baseline at various time-points over the testing period. This was not observed in naïve mice or non-craniotomised sham controls over the same time period. This finding indicates that the craniotomy procedure alone effects gait. When craniotomised mice were subjected to TBI, additional deleterious effects on gait function were observed, including forelimb stance and swing duration as well as left hindlimb swing and stride duration and frequency. Hence, mice subjected to CCI-induced TBI develop clear alterations in gait but part of this is attributable to the effect of craniotomy alone. This study also highlights the need to include both non-craniotomised and craniotomised sham mice as controls when undertaking the CCI-induced model of TBI, particularly when early time points are being evaluated.

Retinoic acid potentiates phorbol ester-mediated induction of urokinase and plasminogen activator inhibitor type 2 in human myeloid leukemic cell lines.

We investigated the interactive regulation of the plasminogen activators (PAs) and their inhibitors (PAIs) by all-trans-retinoic acid (RA) in the presence and absence of the phorbol ester, phorbol myristate acetate (PMA), in four developmentally distinct human myeloid leukemic cell lines. Treatment of HL-60, K562, THP-1, and U937 cells with PMA resulted in an induction of urokinase-type PA (u-PA), the u-PA receptor (u-PAR), and PAI types 1 and 2 (PAI-1 and PAI-2). The addition of RA alone failed to alter gene expression or antigen production of PAI-1, PAI-2, or u-PAR. However, RA potentiated PMA-mediated induction of PAI-2 mRNA in HL-60 and U937 cells and PAI-2 antigen in all four cell lines. The effect of PMA on u-PA mRNA was also potentiated by RA in HL-60 and U937 cells. A similar, but transient, effect was seen on u-PA antigen levels. Run-on transcription analysis confirmed that these effects were due at least in part to changes in gene template activity. Furthermore, RA did not potentiate the effects of PMA on either u-PAR or PAI-1. In fact, in U937 cells, RA inhibited PMA-induced PAI-1 antigen secretion by approximately 60%. It would seem that interactive regulation of these genes allows for greater diversity of control, which may, in turn, be required for localized control of plasminogen-dependent extracellular proteolysis generated by monocytes/macrophage during cell migration and tissue remodeling.