Platelet kinetics in the pulmonary microcirculation in vivo assessed by intravital microscopy

Growing evidence supports the substantial pathophysiological impact of platelets on the development of acute lung injury. Methods for studying these cellular mechanisms in vivo are not present yet. The aim of this study was to develop a model enabling the quantitative analysis of platelet kinetics and platelet-endothelium interaction within consecutive segments of the pulmonary microcirculation in vivo. New Zealand White rabbits were anesthetized and ventilated. Autologous platelets were separated from blood and labeled ex vivo with rhodamine 6G. After implantation of a thoracic window, microhemodynamics and kinetics of platelets were investigated by intravital microscopy. Velocities of red blood cells (RBCs) and platelets were measured in arterioles, capillaries and venules, and the number of platelets adhering to the microvascular endothelium was counted. Kinetics of unstimulated platelets was compared with kinetics of thrombin-activated platelets. Velocity of unstimulated platelets was comparable to RBC velocity in all vessel segments. Unstimulated platelets passed the pulmonary microcirculation without substantial platelet-endothelial interaction. In contrast, velocity of activated platelets was decreased in all vascular segments indicating platelet margination and temporal platelet-endothelium interaction. Thrombin-activated platelets adhered to arteriolar endothelium; in capillaries and venules adherence of platelets was increased 8-fold and 13-fold, respectively. In conclusion, using intravital microscopy platelet kinetics were directly analyzed in the pulmonary microcirculation in vivo for the first time. In contrast to leukocytes, no substantial platelet-endothelium interaction occurs in the pulmonary microcirculation without any further stimulus. In response to platelet activation, molecular mechanisms enable adhesion of platelets in arterioles and venules as well as retention of platelets within capillaries. Copyright (C) 2002 S. Karger AG, Basel

Islets of Langerhans Are Protected from Inflammatory Cell Recruitment during Reperfusion of Rat Pancreas Grafts

Background: Ischemia/reperfusion (I/R) injury plays a pivotal role in the development of graft pancreatitis, with ischemia time representing one of its crucial factors. However, it is unclear, whether exocrine and endocrine tissue experience similar inflammatory responses during pancreas transplantation (PTx). This study evaluated inflammatory susceptibilities of islets of Langerhans (ILH) and exocrine tissue after different preservation periods during early reperfusion. Methods: PTx was performed in rats following 2 h (2h-I) or 18 h (18h-I) preservation. Leukocyte-endothelial cell interactions (LEI) were analyzed in venules of acinar tissue and ILH in vivo over 2 h reperfusion. Nontransplanted animals served as controls. Tissue samples were analyzed by histomorphometry. Results: In exocrine venules leukocyte rolling predominated in the 2h-I group. In the 18h-I group, additionally, high numbers of adherent leukocytes were found. Histology revealed significant edema formation and leukocyte extravasation in the 18h-I group. Notably, LEI in postcapillary venules of ILH were significantly lower. Leukocyte rolling was only moderately enhanced and few leukocytes were found adherent. Histology revealed minor leukocyte extravasation. Conclusion: Ischemia time contributes decisively to the extent of the I/R-injury in PTx. However, ILH have a significantly lower susceptibility towards I/R, even when inflammatory reactions in adjacent exocrine tissue are evident. Copyright (C) 2010 S. Karger AG, Base

Platelet migration and bacterial trapping assay under flow

Blood platelets are critical for hemostasis and thrombosis, but also play diverse roles during immune responses. We have recently reported that platelets migrate at sites of infection in vitro and in vivo. Importantly, platelets use their ability to migrate to collect and bundle fibrin (ogen)-bound bacteria accomplishing efficient intravascular bacterial trapping. Here, we describe a method that allows analyzing platelet migration in vitro, focusing on their ability to collect bacteria and trap bacteria under flow

Proof of Concept: Measuring Aortic Annulus Resistance by Means of Pressure-Volume Curves During Balloon Inflation to Guide Transcatheter Aortic Valve Implantation

This study assessed the basic working principle to measure aortic annulus resistance during balloon inflation for transcatheter aortic valve implantation (TAVI), by acquisition of pressure-volume curve for a guided semi-automatic implantation. A modular bench-system was used which allows the incremental inflation of valvuloplasty balloons by means of a stepper-motor driven linear axis with simultaneous recording of the pressure changes inside the system. Different porcine aortic xenografts were assessed by use of a non-compliant valvuloplasty balloon. In a second step transcatheter aortic stents were implanted inside target sized xenografts. The recorded pressure volume-curves showed that the system can accurately differentiate between different xenografts and assess the quality of the tissue rendering real-time analysis of pressure-volume curves during balloon-inflation possible, which has the potential to optimize the implantation procedure by direct adaptation to the patient specific anatomy and characteristics. Further investigations and development are warranted

Platelet dysfunction and inhibition of multiple electrode platelet aggregometry caused by penicillin

Beta-lactam antibiotics, e.g. penicillin, may inhibit platelet function and lead to reduced response in light transmission aggregometry and adhesion. However, influence on platelet function tests more commonly used in clinical practice, such as multiple electrode platelet aggregometry (MEA), have not been described so far. We report a case of a patient with local streptococcus infection. Treatment with penicillin resulted in mild bleeding tendency after 3 days. While coagulation parameters were normal, assessment of platelet function by MEA revealed strong platelet inhibition of both ADP and arachidonic acid induced platelet aggregation comparable to normal responders to antiplatelet therapy. Change of antibiotic regime resulted in recovery of platelet function. Thus, penicillin therapy may impact on platelet function and consecutively commonly used platelet function assays, e.g. MEA

Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation

The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased — and, under conditions of decreased platelet adhesion, PDI inhibition reduced — fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet–secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury

Mechanical Stability and Fibrinolytic Resistance of Clots Containing Fibrin, DNA, and Histones

Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones + DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nm dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator

Association of prehospital acetylsalicylic acid and heparin administration with favorable neurological outcome after out-of-hospital cardiac arrest: a matched cohort analysis of the German Resuscitation Registry

Purpose!#!To investigate the global burden of sepsis in hospitalized adults by updating and expanding a systematic review and meta-analysis and to compare findings with recent Institute for Health Metrics and Evaluation (IHME) sepsis estimates.!##!Methods!#!Thirteen electronic databases were searched for studies on population-level sepsis incidence defined according to clinical criteria (Sepsis-1, -2: severe sepsis criteria, or sepsis-3: sepsis criteria) or relevant ICD-codes. The search of the original systematic review was updated for studies published 05/2015-02/2019 and complemented by a search targeting low- or middle-income-country (LMIC) studies published 01/1979-02/2019. We performed a random-effects meta-analysis with incidence of hospital- and ICU-treated sepsis and proportion of deaths among these sepsis cases as outcomes.!##!Results!#!Of 4746 results, 28 met the inclusion criteria. 21 studies contributed data for the meta-analysis and were pooled with 30 studies from the original meta-analysis. Pooled incidence was 189 [95% CI 133, 267] hospital-treated sepsis cases per 100,000 person-years. An estimated 26.7% [22.9, 30.7] of sepsis patients died. Estimated incidence of ICU-treated sepsis was 58 [42, 81] per 100,000 person-years, of which 41.9% [95% CI 36.2, 47.7] died prior to hospital discharge. There was a considerably higher incidence of hospital-treated sepsis observed after 2008 (+ 46% compared to the overall time frame).!##!Conclusions!#!Compared to results from the IHME study, we found an approximately 50% lower incidence of hospital-treated sepsis. The majority of studies included were based on administrative data, thus limiting our ability to assess temporal trends and regional differences. The incidence of sepsis remains unknown for the vast majority of LMICs, highlighting the urgent need for improved epidemiological sepsis surveillance

Multiphoton In Vivo Microscopy of Embryonic Thrombopoiesis Reveals the Generation of Platelets through Budding.

Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK's origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model under control of the platelet factor 4 promoter (Pf4CreRosa26mTmG). Using this microscopy tool, we discovered that fetal liver MKs provide higher thrombopoietic activity than yolk sac MKs. Mechanistically, fetal platelets were released from MKs either by membrane buds or the formation of proplatelets, with the former constituting the key process. In E14.5 c-Myb-deficient embryos that lack definitive hematopoiesis, MK and platelet numbers were similar to wild-type embryos, indicating the independence of embryonic thrombopoiesis from definitive hematopoiesis at this stage of development. In summary, our novel MP-IVM protocol allows the characterization of thrombopoiesis with high spatio-temporal resolution in the mouse embryo and has identified membrane budding as the main mechanism of fetal platelet production