Single platelet and megakaryocyte morpho-dynamics uncovered by multicolor reporter mouse strains in vitro and in vivo

Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Credriven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of megakaryocytes in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to WT controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and megakaryocyte biology on a single cell level

Procoagulant platelet sentinels prevent inflammatory bleeding through GPIIBIIIA and GPVI

Impairment of vascular integrity is a hallmark of inflammatory diseases. We recently reported that single immune-responsive platelets migrate and reposition themselves to sites of vascular injury to prevent bleeding. However, it remains unclear how single platelets preserve vascular integrity once encountering endothelial breaches. Here we demonstrate by intravital microscopy combined with genetic mouse models that procoagulant activation (PA) of single platelets and subsequent recruitment of the coagulation cascade are crucial for the prevention of inflammatory bleeding. Using a novel lactadherin-based compound, we detect phosphatidylserine (PS)-positive procoagulant platelets in the inflamed vasculature. We identify exposed collagen as the central trigger arresting platelets and initiating subsequent PA in a CypD- and TMEM16F-dependent manner both in vivo and in vitro. Platelet PA promotes binding of the prothrombinase complex to the platelet membrane, greatly enhancing thrombin activity and resulting in fibrin formation. PA of migrating platelets is initiated by costimulation via integrin αIIbβ3 (GPIIBIIIA)/Gα13-mediated outside-in signaling and glycoprotein VI signaling, leading to an above-threshold intracellular calcium release. This effectively targets the coagulation cascade to breaches of vascular integrity identified by patrolling platelets. Platelet-specific genetic loss of either CypD or TMEM16F as well as combined blockade of platelet GPIIBIIIA and glycoprotein VI reduce platelet PA in vivo and aggravate pulmonary inflammatory hemorrhage. Our findings illustrate a novel role of procoagulant platelets in the prevention of inflammatory bleeding and provide evidence that PA of patrolling platelet sentinels effectively targets and confines activation of coagulation to breaches of vascular integrity

A novel role of sphingosine 1-phosphate receptor S1pr1 in mouse thrombopoiesis

Millions of platelets are produced each hour by bone marrow (BM) megakaryocytes (MKs). MKs extend transendothelial proplatelet (PP) extensions into BM sinusoids and shed new platelets into the blood. The mechanisms that control platelet generation remain incompletely understood. Using conditional mutants and intravital multiphoton microscopy, we show here that the lipid mediator sphingosine 1-phosphate (S1P) serves as a critical directional cue guiding the elongation of megakaryocytic PP extensions from the interstitium into BM sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. Collectively, our findings uncover a novel function of the S1P-S1pr1 axis as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia

Neutrophil “plucking” on megakaryocytes drives platelet production and boosts cardiovascular disease

Intravascular neutrophils and platelets collaborate in maintaining host integrity, but their interaction can also trigger thrombotic complications. We report here that cooperation between neutrophil and platelet lineages extends to the earliest stages of platelet formation by megakaryocytes in the bone marrow. Using intravital microscopy, we show that neutrophils "plucked" intravascular megakaryocyte extensions, termed proplatelets, to control platelet production. Following CXCR4-CXCL12-dependent migration towards perisinusoidal megakaryocytes, plucking neutrophils actively pulled on proplatelets and triggered myosin light chain and extracellular-signal-regulated kinase activation through reactive oxygen species. By these mechanisms, neutrophils accelerate proplatelet growth and facilitate continuous release of platelets in steady state. Following myocardial infarction, plucking neutrophils drove excessive release of young, reticulated platelets and boosted the risk of recurrent ischemia. Ablation of neutrophil plucking normalized thrombopoiesis and reduced recurrent thrombosis after myocardial infarction and thrombus burden in venous thrombosis. We establish neutrophil plucking as a target to reduce thromboischemic events

Treatment of Peritoneal Carcinomatosis by Targeted Delivery of the Radio-Labeled Tumor Homing Peptide 213Bi-DTPA-[F3]2 into the Nucleus of Tumor Cells

BACKGROUND: Alpha-particle emitting isotopes are effective novel tools in cancer therapy, but targeted delivery into tumors is a prerequisite of their application to avoid toxic side effects. Peritoneal carcinomatosis is a widespread dissemination of tumors throughout the peritoneal cavity. As peritoneal carcinomatosis is fatal in most cases, novel therapies are needed. F3 is a tumor homing peptide which is internalized into the nucleus of tumor cells upon binding to nucleolin on the cell surface. Therefore, F3 may be an appropriate carrier for alpha-particle emitting isotopes facilitating selective tumor therapies. PRINCIPAL FINDINGS: A dimer of the vascular tumor homing peptide F3 was chemically coupled to the alpha-emitter (213)Bi ((213)Bi-DTPA-[F3](2)). We found (213)Bi-DTPA-[F3](2) to accumulate in the nucleus of tumor cells in vitro and in intraperitoneally growing tumors in vivo. To study the anti-tumor activity of (213)Bi-DTPA-[F3](2) we treated mice bearing intraperitoneally growing xenograft tumors with (213)Bi-DTPA-[F3](2). In a tumor prevention study between the days 4-14 after inoculation of tumor cells 6x1.85 MBq (50 microCi) of (213)Bi-DTPA-[F3](2) were injected. In a tumor reduction study between the days 16-26 after inoculation of tumor cells 6x1.85 MBq of (213)Bi-DTPA-[F3](2) were injected. The survival time of the animals was increased from 51 to 93.5 days in the prevention study and from 57 days to 78 days in the tumor reduction study. No toxicity of the treatment was observed. In bio-distribution studies we found (213)Bi-DTPA-[F3](2) to accumulate in tumors but only low activities were found in control organs except for the kidneys, where (213)Bi-DTPA-[F3](2) is found due to renal excretion. CONCLUSIONS/SIGNIFICANCE: In conclusion we report that (213)Bi-DTPA-[F3](2) is a novel tool for the targeted delivery of alpha-emitters into the nucleus of tumor cells that effectively controls peritoneal carcinomatosis in preclinical models and may also be useful in oncology

Behavioral health risk factor profiles in general hospital patients: identifying the need for screening and brief intervention

Background Little is known about the clustering of behavioral health risk factors (HRFs), namely the occurrence of 16 specific combinations of tobacco smoking, at-risk alcohol use, overweight and physical inactivity in general hospital patients. Furthermore, social inequalities in HRFs, health and life expectancy are a major concern in public health. In order to establish the need for screening and intervention in general hospital care, the study aimed to determine the co-occurrence of HRFs in patients in four medical departments, and to investigate differences by gender, age and socio-economic characteristics. Methods Over 17 months, a systematic multiple HRF screening was conducted at one general hospital in northeastern Germany. In total, 6251 18–64 year old patients (92% of eligibles) participated. Proportions and confidence intervals were calculated for all 16 HRF profiles stratified by department, gender, age group, school education, and employment status. Results In total, 92.2% of the participants (58.6% male) reported ≥1 HRF, and 65.7% ≥2 HRFs. Men (71.2%), patients aged 35–49 (67.9%) and 50–64 years (69.5%), lower educated (79.0%), and unemployed (77.8%) patients had larger proportions of ≥2 HRFs than their counterparts. In all departments, the most common HRF profiles included overweight. HRF profiles that included alcohol and/ or smoking were more common in ear-nose-throat and trauma surgery than in internal medicine and general surgery patients. Men had higher rates concerning almost all HRF profiles including ≥2 HRFs and alcohol; women concerning profiles that included ≤2 HRFs and inactivity. In older patients, profiles with ≥2 HRFs including overweight; and in younger patients, profiles with smoking and/or alcohol were more common. In lower educated patients, profiles with ≥2 HRFs including inactivity; and in higher educated patients profiles with ≤2 HRFs including alcohol were more common. Compared to others, unemployed patients had higher rates of profiles with ≥3 HRFs including smoking. Conclusions Two in three patients require interventions targeting two or more HRFs. The findings help to develop screening and brief intervention for patients with specific health risk profiles, that can reach most patients, including those most in need and those most hard to reach, with socio-economically disadvantaged people in particular.Peer Reviewe

Estimating the Potential of Radiomics Features and Radiomics Signature from Pretherapeutic PSMA-PET-CT Scans and Clinical Data for Prediction of Overall Survival When Treated with 177Lu-PSMA

Prostate-specific membrane antigen (PSMA) positron emission tomography/computed tomography (PSMA-PET/CT) scans can facilitate diagnosis and treatment of prostate disease. Radiomics signature (RS) is widely used for the analysis of overall survival (OS) in cancer diseases. This study aims at investigating the role of radiomics features (RFs) and RS from pretherapeutic gallium-68 (68Ga)-PSMA-PET/CT findings and patient-specific clinical parameters to analyze overall survival of prostate cancer (PC) patients when treated with lutethium-177 (177Lu)-PSMA. A cohort of 83 patients with advanced PC was retrospectively analyzed. Average values of 73 RFs of 2070 malignant hotspots as well as 22 clinical parameters were analyzed for each patient. From the Cox proportional hazard model, the least absolute shrinkage and selection operator (LASSO) regularization method is used to select most relevant features (standardized uptake value (SUV)Min and kurtosis with the coefficients of 0.984 and −0.118, respectively) and to calculate the RS from the RFs. Kaplan–Meier (KM) estimator was used to analyze the potential of RFs and conventional clinical parameters, such as metabolic tumor volume (MTV) and standardized uptake value (SUV) for the prediction of survival. As a result, SUVMin, kurtosis, the calculated RS, SUVMean, as well as Hemoglobin (Hb)1, C-reactive protein (CRP)1, and ECOG1 (clinical parameters) achieved p-values less than 0.05, which suggest the potential of findings from 68Ga-PSMA-PET/CT scans as well as patient-specific clinical parameters for the prediction of OS for patients with advanced PC treated with 177Lu-PSMA therapy