Plasminogen missense variants and their involvement in cardiovascular and inflammatory disease.

Human plasminogen (PLG), the zymogen of the fibrinolytic protease, plasmin, is a polymorphic protein with two widely distributed codominant alleles, PLG/Asp453 and PLG/Asn453. About 15 other missense or non-synonymous single nucleotide polymorphisms (nsSNPs) of PLG show major, yet different, relative abundances in world populations. Although the existence of these relatively abundant allelic variants is generally acknowledged, they are often overlooked or assumed to be non-pathogenic. In fact, at least half of those major variants are classified as having conflicting pathogenicity, and it is unclear if they contribute to different molecular phenotypes. From those, PLG/K19E and PLG/A601T are examples of two relatively abundant PLG variants that have been associated with PLG deficiencies (PD), but their pathogenic mechanisms are unclear. On the other hand, approximately 50 rare and ultra-rare PLG missense variants have been reported to cause PD as homozygous or compound heterozygous variants, often leading to a debilitating disease known as ligneous conjunctivitis. The true abundance of PD-associated nsSNPs is unknown since they can remain undetected in heterozygous carriers. However, PD variants may also contribute to other diseases. Recently, the ultra-rare autosomal dominant PLG/K311E has been found to be causative of hereditary angioedema (HAE) with normal C1 inhibitor. Two other rare pathogenic PLG missense variants, PLG/R153G and PLG/V709E, appear to affect platelet function and lead to HAE, respectively. Herein, PLG missense variants that are abundant and/or clinically relevant due to association with disease are examined along with their world distribution. Proposed molecular mechanisms are discussed when known or can be reasonably assumed.

Streptococcus co-opts a conformational lock in human plasminogen to facilitate streptokinase cleavage and bacterial virulence.

Virulent strains of Streptococcus pyogenes (gram-positive group A Streptococcus pyogenes [GAS]) recruit host single-chain human plasminogen (hPg) to the cell surface-where in the case of Pattern D strains of GAS, hPg binds directly to the cells through a surface receptor, plasminogen-binding group A streptococcal M-protein (PAM). The coinherited Pattern D GAS-secreted streptokinase (SK2b) then accelerates cleavage of hPg at the R561-V562 peptide bond, resulting in the disulfide-linked two-chain protease, human plasmin (hPm). hPm localizes on the bacterial surface, assisting bacterial dissemination via proteolysis of host defense proteins. Studies using isolated domains from PAM and hPg revealed that the A-domain of PAM binds to the hPg kringle-2 module (K2hPg), but how this relates to the function of the full-length proteins is unclear. Herein, we use intact proteins to show that the lysine-binding site of K2hPg is a major determinant of the activation-resistant T-conformation of hPg. The binding of PAM to the lysine-binding site of K2hPg relaxes the conformation of hPg, leading to a greatly enhanced activation rate of hPg by SK2b. Domain swapping between hPg and mouse Pg emphasizes the importance of the Pg latent heavy chain (residues 1-561) in PAM binding and shows that while SK2b binds to both hPg and mouse Pg, the activation properties of streptokinase are strictly attributed to the serine protease domain (residues 562-791) of hPg. Overall, these data show that native hPg is locked in an activation-resistant conformation that is relaxed upon its direct binding to PAM, allowing hPm to form and provide GAS cells with a proteolytic surface.

Levels of agreement in the measurements of carotid artery ultrasound across a regional vascular network.

INTRODUCTION: Duplex ultrasound is often the sole imaging modality used in diagnosing carotid artery disease. However, the reproducibility and repeatability of scientists in determining the peak systolic velocity and end diastolic velocity of the internal carotid artery and common carotid artery (CCA) is widely debated. STUDY AIM: To investigate intra- and inter-operator variability in diagnostic ultrasound of the carotid arteries across a centralised vascular network using a healthy test subject. To identify potential causes of variability and highlight areas for improvement. METHODS: Fifteen vascular scientists across four hospital Trusts in the Bristol, Bath and Weston vascular network measured the peak systolic velocity and end diastolic velocity of the internal carotid artery and common carotid artery in a subject using a single portable ultrasound machine. A double blind assessment of spectral Doppler images was performed by two vascular clinical scientists for optimal caliper placement, spectral gain and angle correction. Results were compared for intra- and inter-operator variability. RESULTS: Initial quality assessment of the Doppler images revealed that three out of 15 scientists produced suboptimal results. Box plot analysis of the common carotid artery and internal carotid artery for each scientist revealed significant variance (ANOVA p < 0.05). However, a Levene's test revealed no single operator who consistently produced highly variable results (p = 0.569). CONCLUSION: This study highlights the difficulty in obtaining consistent velocity measurements from a subject. Despite the variability in absolute peak systolic velocity and end diastolic velocity, scientists were generally consistent in obtaining an optimal spectral Doppler trace. Some issues with consistency were, however, identified which were subsequently addressed.

Whole blood clot optical clearing for nondestructive 3D imaging and quantitative analysis.

A technological revolution in both light and electron microscopy imaging now allows unprecedented views of clotting, especially in animal models of hemostasis and thrombosis. However, our understanding of three-dimensional high-resolution clot structure remains incomplete since most of our recent knowledge has come from studies of relatively small clots or thrombi, due to the optical impenetrability of clots beyond a few cell layers in depth. Here, we developed an optimized optical clearing method termed cCLOT that renders large whole blood clots transparent and allows confocal imaging as deep as one millimeter inside the clot. We have tested this method by investigating the 3D structure of clots made from reconstituted pre-labeled blood components yielding new information about the effects of clot contraction on erythrocytes. Although it has been shown recently that erythrocytes are compressed to form polyhedrocytes during clot contraction, observations of this phenomenon have been impeded by the inability to easily image inside clots. As an efficient and non-destructive method, cCLOT represents a powerful research tool in studying blood clot structure and mechanisms controlling clot morphology. Additionally, cCLOT optical clearing has the potential to facilitate imaging of ex vivo clots and thrombi derived from healthy or pathological conditions.

Human axillary skin condition is improved following incorporation of glycerol into the stratum corneum from an antiperspirant formulation.

The study objectives were to demonstrate that glycerol, when topically applied from a roll-on antiperspirant formulation, can be delivered directly to human skin ex vivo and the axillary stratum corneum (SC) in vivo, and to assess whether it improves the quality of the axillary skin barrier. Ex vivo human skin absorption of glycerol was measured following application of a roll-on antiperspirant formulation containing 4% 13C3-glycerol. Skin distribution of 13C3-glycerol over 24 h was assessed using gas chromatography-mass spectrometry. In vivo axillary SC penetration was measured by confocal Raman spectroscopy and multivariate curve-resolution software 1 h after topical application of a roll-on antiperspirant formulation containing 8% deuterated glycerol (d5-glycerol). A clinical study was conducted to determine the efficacy of a roll-on antiperspirant formulation containing 4% glycerol in reducing shaving-induced visual irritation and in increasing axillary-skin hydration. Ex vivo skin absorption studies indicated that the formulation delivered 13C3-glycerol into the SC at all timepoints over the 24-h period. In vivo Raman measurements (1 h after application) demonstrated that d5-glycerol was detectable to a depth of at least 10 µm in the axillary SC. Application of 4% glycerol from a roll-on antiperspirant formulation to the axilla was associated with significantly less visible irritation and greater skin hydration than observed with the control (glycerol-free) product. These studies demonstrate that glycerol, incorporated in a roll-on antiperspirant formulation, is delivered directly and rapidly to all depths of the axillary SC, and results in improvements in visible irritation and hydration in the axilla.

Visualisation of 4-D colour and power Doppler data.

Mechanically scanned, cardiac-gated, 4-D colour Doppler and power Doppler data were acquired and rendered using multiplanar slice display, surface-fitting (S-F) and direct volume rendering (DVR), with interactive viewpoint control or tool command language (Tcl)-scripted generation of animation sequences across the cardiac cycle. S-F applied the marching cubes algorithm within a YCbCr colour space to extract time-varying Doppler regions; supplementally generated surfaces enclosed all regions across the full cardiac cycle, partially to alleviate the problem of incomplete vessel filling. DVR used integrated intensity, maximum intensity and front-to-back projection; the latter applied an opacity function to the Cb, Cr and Y colour channels to control the appearance of the blood flow signal relative to the background B-mode image. Our most detailed display was a hybrid approach that combined multiplanar slicing and DVR.