Interrogating a Compound Library in Search of an Inhibitor for TREM-like Transcript-1 to Fibrinogen Binding.

Cardiovascular disease (CVD) remains one of leading causes of death worldwide. Aberrant platelet function mediate fibrin(ogen) rich thrombi that lead to occlusive thrombi associated with mortality. The receptor, TREM-like transcript-1 (TLT-1), stored in the platelet a-granules and released upon platelet activation, binds fibrinogen and von Willebrand factor. Once it is released from platelets TLT-1 is a potential therapeutic target to prevent the thrombosis associated with CVD. Here we design an assay to screen a compound library of small molecules inhibitors. HEK-293 cells stably transfected with a full length human treml-1 construct were used to screen library of 800 compounds, for inhibition of TLT-1 to fibrinogen binding in an attachment assay using crystal violet staining. The possible cytotoxicity of the best compounds was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide MTT and calcein AM staining assays. Here we demonstrate that the addition of TLT-1 to HEK-293 cells increases cell adhesion by more than 2-fold. We identified ~80 compounds that inhibit binding by more than 80%. We further tested the top compounds and confirmed that reduction of hTLT-1 to fibrinogen bound in the top compounds was not caused by cytotoxicity, as per colorimetric and fluorescent viability assays. Four compounds were identified as potential small molecule inhibitors one of which, BM-8372, demonstrated significant effect in platelet aggregation assays. Significance Statement TLT-1 is a key platelet receptor that binds fibrinogen and mediates clot formation The developed assay successfully screens 800 small molecules, pinpointing ~80 potent inhibitors that reduce TLT-1 binding by over 80%. Importantly, the study rigorously rules out cytotoxicity concerns, affirming the therapeutic potential of the identified compounds. By elucidating TLT-1's role and presenting promising inhibitors, this research offers a significant stride toward developing novel strategies to combat CVD-related thrombosis.

The Characterization and Evaluation of the Soluble Triggering Receptor Expressed on Myeloid Cells-like Transcript-1 in Stable Coronary Artery Disease.

Platelets play crucial roles in the development and progression of coronary artery disease (CAD). The triggering receptor expressed in myeloid cells-like transcript-1 (TLT-1) is stored in platelet α granules, and activated platelets release a soluble fragment (sTLT-1). We set out to better characterize the constituent amino acids of sTLT-1 and to evaluate sTLT-1 for use as a biomarker in patients with stable CAD. We evaluated sTLT-1 release using immunoprecipitation and mass spectrometry and employed statistical methods to retrospectively correlate sTLT-1 concentrations, utilizing ELISA in plasma samples from 1510 patients with documented stable CAD. We identified TLT-1 residues to 133 in platelet releasates. ADAM17 cuts TLT-1, suggesting that S136 is the C-terminal amino acid in sTLT-1. Our results revealed that for CAD patients, sTLT-1 levels did not differ significantly according to primary outcomes of death or major cardiac event; however, patients with left ventricular (LV) dysfunction had significantly lower plasma sTLT-1 levels as compared to those with normal LV function (981.62 ± 1141 pg/mL vs. 1247.48 ± 1589 pg/mL; p = 0.003). When patients were stratified based on sTLT-1 peak frequency distribution (544 pg/mL), a significant association with congestive heart failure was identified (OR = 2.94; 1.040-8.282; p = 0.042), which could be explained by LV dysfunction.

Human megakaryocytes possess intrinsic antiviral immunity through regulated induction of IFITM3.

Evolving evidence indicates that platelets and megakaryocytes (MKs) have unexpected activities in inflammation and infection; whether viral infections upregulate biologically active, antiviral immune genes in platelets and MKs is unknown, however. We examined antiviral immune genes in these cells in dengue and influenza infections, viruses that are global public health threats. Using complementary biochemical, pharmacological, and genetic approaches, we examined the regulation and function of interferon-induced transmembrane protein 3 (IFITM3), an antiviral immune effector gene not previously studied in human platelets and MKs. IFITM3 was markedly upregulated in platelets isolated from patients during clinical influenza and dengue virus (DENV) infections. Lower IFITM3 expression in platelets correlated with increased illness severity and mortality in patients. Administering a live, attenuated DENV vaccine to healthy subjects significantly increased platelet IFITM3 expression. Infecting human MKs with DENV selectively increased type I interferons and IFITM3. Overexpression of IFITM3 in MKs was sufficient to prevent DENV infection. In naturally occurring, genetic loss-of-function studies, MKs from healthy subjects harboring a homozygous mutation in IFITM3 (rs12252-C, a common single-nucleotide polymorphism in areas of the world where DENV is endemic) were significantly more susceptible to DENV infection. DENV-induced MK secretion of interferons prevented infection of bystander MKs and hematopoietic stem cells. Thus, viral infections upregulate IFITM3 in human platelets and MKs, and IFITM3 expression is associated with adverse clinical outcomes. These observations establish, for the first time, that human MKs possess antiviral functions, preventing DENV infection of MKs and hematopoietic stem cells after local immune signaling.

The enigmatic nature of the triggering receptor expressed in myeloid cells -1 (TLT- 1).

Receptors are important pharmacological targets on cells. The Triggering Receptor Expressed on Myeloid Cells (TREM) - Like Transcript - 1 is an abundant, yet little understood, platelet receptor. It is a single Ig domain containing receptor isolated in the α-granules of resting platelets and brought to the platelet surface upon activation. On platelets, the integrin αIIbß3 is the major receptor having roughly 80,000 copies. αIIbß3 is a heterodimeric multidomain structure that mediates platelet aggregation through its interaction with the plasma protein fibrinogen. Anti-platelet drugs have successfully targeted αIIbß3 to control thrombosis. Like αIIbß3, TLT-1 also binds fibrinogen, making its role in platelet function somewhat obscure. In this review, we highlight the known structural features of TLT-1 and present the challenges of understanding TLT-1 function. In our analysis of the dynamics of the platelet surface after activation we propose a model in which TLT-1 supports αIIbß3 function as a mechanoreceptor that may direct platelets toward immune function.

Platelet biology of the rapidly failing lung.

Acute respiratory distress syndrome (ARDS) is characterized by a rapid-onset respiratory failure with a mortality rate of approximately 40%. This physiologic inflammatory process is mediated by disruption of the alveolar-vascular interface, leading to pulmonary oedema and impaired oxygen exchange, which often warrants mechanical ventilation to increase survival in the acute setting. One of the least understood aspects of ARDS is the role of the platelets in this process. Platelets, which protect vascular integrity, play a pivotal role in the progression and resolution of ARDS. The recent substantiation of the age-old theory that megakaryocytes are found in the lungs has rejuvenated interest in and raised new questions about the importance of platelets for pulmonary function. In addition to primary haemostasis, platelets provide a myriad of inflammatory functions that are poised to aid the innate immune system. This review focuses on the evidence for regulatory roles of platelets in pulmonary inflammation, with an emphasis on two receptors, CLEC-2 and TLT-1. Studies of these receptors identify novel pathways through which platelets may regulate vascular integrity and inflammation in the lungs, thereby influencing the development of ARDS.

Platelet-derived TLT-1 is a prognostic indicator in ALI/ARDS and prevents tissue damage in the lungs in a mouse model.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) affect >200 000 individuals yearly with a 40% mortality rate. Although platelets are implicated in the progression of ALI/ARDS, their exact role remains undefined. Triggering receptor expressed in myeloid cells (TREM)-like transcript 1 (TLT-1) is found on platelets, binds fibrinogen, and mediates clot formation. We hypothesized that platelets use TLT-1 to manage the progression of ALI/ARDS. Here we retrospectively measure plasma levels of soluble TLT-1 (sTLT-1) from the ARDS Network clinical trial and show that patients whose sTLT-1 levels were >1200 pg/mL had nearly twice the mortality risk as those with <1200 pg/mL (P < .001). After correcting for confounding factors such as creatinine levels, Acute Physiology And Chronic Health Evaluation III scores, age, platelet counts, and ventilation volume, sTLT-1 remains significant, suggesting that sTLT-1 is an independent prognostic factor (P < .0001). These data point to a role for TLT-1 during the progression of ALI/ARDS. We use a murine lipopolysaccharide-induced ALI model and demonstrate increased alveolar bleeding, aberrant neutrophil transmigration and accumulation associated with decreased fibrinogen deposition, and increased pulmonary tissue damage in the absence of TLT-1. The loss of TLT-1 resulted in an increased proportion of platelet-neutrophil conjugates (43.73 ± 24.75% vs 8.92 ± 2.4% in wild-type mice), which correlated with increased neutrophil death. Infusion of sTLT-1 restores normal fibrinogen deposition and reduces pulmonary hemorrhage by 40% (P ≤ .001) and tissue damage by 25% (P ≤ .001) in vivo. Our findings suggest that TLT-1 uses fibrinogen to govern the transition between inflammation and hemostasis and facilitate controlled leukocyte transmigration during the progression of ARDS.

Amyloid Beta Peptide Is Released during Thrombosis in the Skin.

While it is known that amyloid beta (Aß) deposits are found in different tissues of both Alzheimer's disease (AD) patients and healthy individuals, there remain questions about the physiological role of these deposits, the origin of the Aß peptide, and the mechanisms of its localization to the tissues. Using immunostaining with specific antibodies, as well as enzyme-linked immunosorbent assay, this study demonstrated Aß40 peptide accumulation in the skin during local experimental photothrombosis in mice. Specifically, Aß peptide accumulation was concentrated near the dermal blood vessels in thrombotic skin. It was also studied whether the released peptide affects microorganisms. Application of Aß40 (4 µM) to the external membrane of yeast cells significantly increased membrane conductance with no visible effect on mouse host cells. The results suggest that Aß release in the skin is related to skin injury and thrombosis, and occurs along with clotting whenever skin is damaged. These results support the proposition that Aß release during thrombosis serves as part of a natural defense against infection.

Whole Blood Aggregometry in Mice.

Aggregometry plays a crucial role in both clinical diagnostics and research within hematology, serving as a fundamental tool for understanding platelet function and its implications in physiological and pathological processes. In research, aggregometry provides insights into platelet aggregation dynamics and aids in understanding the underlying mechanisms of hemostasis, thrombosis, and related disorders. Light transmission aggregometry (LTA) and lumi-aggregometry, as well as whole blood aggregometry, are commonly employed methods. While LTA and lumi-aggregometry allow for specific platelet function assessment under controlled conditions, whole blood aggregometry provides a more physiologically relevant approach by evaluating platelet aggregation within the context of whole blood. Although both methodologies offer unique advantages, whole blood aggregometry allows for preservation of the native cellular environment, simplicity, and potential for better clinical correlation. In a clinical setting, with human blood samples, protocols are established for both LTA and whole blood aggregometry as they are frequently used diagnostic tools. A protocol for LTA and lumi-aggregometry in murine models has been described; however, to date, there is no standardized protocol for whole blood aggregometry in murine models accessible to hematology researchers. This article aims to outline a simple, basic protocol for murine whole blood aggregometry, offering an alternative method to the commonly used LTA aggregometry in research settings. Standardizing whole blood aggregometry protocols in murine models could enhance experimental reliability and facilitate translational research efforts in hematology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Whole blood aggregometry in mice Support Protocol: Phenylhydrazine-induced anemia in wild-type mice Basic Protocol 2: Hematocrit percentage in mice.

Affordable Method for Hematocrit Determination in Murine Models.

Hematocrit (Hct) is a powerful tool often used in a clinical setting for the diagnosis of blood conditions such as anemia. It is also used in the research field as a hematological parameter in both human and mouse models. Measuring Hct, however, involves the use of expensive standardized equipment (such as a CritSpin™ Microhematocrit Centrifuge). Here, we describe a novel, simple, and affordable method to determine the Hct in untreated wild-type (WT) mice and phenylhydrazine (PHZ)-induced anemic mice with reasonable accuracy, using a benchtop centrifuge commonly available in laboratories. Hct of murine samples processed with a benchtop centrifuge, when compared to the standardized method CritSpin™, showed comparable results. This approach for determining Hct of murine can prove useful to research laboratories that cannot afford specialized equipment for Hct studies. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Affordable Method for Hematocrit Determination in Murine Models Basic Protocol 2: Murine Sample Validation Support Protocol: Phenylhydrazine-induced anemia in wild-type (WT) mice.

High Levels of Triggering Receptor Expressed in Myeloid Cells-Like Transcript-1 Positive, but Not Glycoprotein 1b+, Microparticles Are Associated With Poor Outcomes in Acute Respiratory Distress Syndrome.

OBJECTIVES: To identify triggering receptor expressed in myeloid cells-like transcript-1 positive (TLT-1+) microparticles (MPs) and evaluate if their presence is associated with clinical outcomes and/or disease severity in acute respiratory distress syndrome (ARDS). DESIGN: Retrospective cohort study. SETTING: ARDS Network clinical trials. PATIENTS: A total of 564 patients were diagnosed with ARDS. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Using flow cytometry, we demonstrated the presence of TLT-1+ platelet-derived microparticles (PMP) that bind fibrinogen in plasma samples from fresh donors. We retrospectively quantified TLT-1, glycoprotein (Gp) 1b, or αIIbßIIIa immunopositive microparticles in plasma samples from patients with ARDS enrolled in the ARMA, KARMA, and LARMA (Studies 01 and 03 lower versus higher tidal volume, ketoconazole treatment, and lisofylline treatment Clincial Trials) ARDS Network clinical trials and evaluated the relationship between these measures and clinical outcomes. No associations were found between Gp1b+ MPs and clinical outcomes for any of the cohorts. When stratified by quartile, associations were found for survival, ventilation-free breathing, and thrombocytopenia with αIIbßIIIa+ and TLT-1+ MPs (χ2p < 0.001). Notably, 63 of 64 patients in this study who failed to achieve unassisted breathing had TLT+ PMP in the 75th percentile. In all three cohorts, patients whose TLT+ MP counts were higher than the median had higher Acute Physiology and Chronic Health Evaluation III scores, were more likely to present with thrombocytopenia and were 3.7 times (p < 0.001) more likely to die than patients with lower TLT+ PMP after adjusting for other risk factors. CONCLUSIONS: Although both αIIbßIIIa+ and TLT+ microparticles (αIIbßIIIa, TLT-1) were associated with mortality, TLT-1+ MPs demonstrated stronger correlations with Acute Physiology and Chronic Health Evaluation III scores, unassisted breathing, and multiple system organ failure. These findings warrant further exploration of the mechanistic role of TLT-1+ PMP in ARDS or acute lung injury progression.

Clinical studies support a role for trem-like transcript-1 during the progression of sepsis.

Sepsis is a multi-factorial disease that kills an estimated 1,400 people a day worldwide. The Triggering Receptor Expressed in Myeloid (TREM) cells Like Transcript (TLT)-1 is a platelet receptor expressed on activated platelets. Translational studies of TLT-1 suggest that TLT-1 affects hemostatic and immunological parameters that lead to the formation of disseminated intravascular coagulation (DIC). Evaluation of mice suffering from endotoxic shock shows a dramatic increase of soluble TLT-1 (sTLT-1) in their blood. Accordingly, when we evaluated the blood of septic patients we find increased levels of sTLT-1 that correlate with the presence of DIC in humans. Based on current data we hypothesize that TLT-1 plays an important role in maintaining vascular integrity during sepsis; perhaps by modulation of both the immune and hemostatic systems, and that TLT-1 makes an attractive target not only for better understanding of sepsis, but also as a point of therapeutic intervention as well.

An exploratory study comparing students' science identity perceptions derived from a hands-on research and nonresearch-based summer learning experience.

Although multiple efforts have been initiated to increase students' science proficiency scores, most of the schools in the United States do not reach the expected student academic performance. This study addresses the impact of a one-week summer scientific learning experience on students that worked with experimental procedures and students that did not. We describe and evaluate these two different interventions to explore what components influence high school students' perception of their scientific competence, performance, and recognition, using science identity as an analytical lens. Science identity score was increased at the end of both interventions. Interestingly, science identity change index was higher for the group that did not work with experimental procedures. Although this group did not perform any hands-on experiments, they report, through reflexive diaries and interviews that working with CRISPR-Cas9 models, being in a research laboratory, and seeing the instrumentation made them feel like scientists. Regarding science competence, both groups report exponential learning gains, although the group that performed the experiments reports more difficulties. Both groups report that mentorship was key in their competence and performance development. These findings suggest that our one-week scientific learning programs influence participants' perception of scientific competence and performance and create an opportunity to develop further studies on short scientific learning experiences using models and active learning activities.

Defining the TLT-1 interactome from resting and activated human platelets.

The triggering receptor expressed on myeloid cells (TREM) protein family forms a class of type I transmembrane proteins expressed in immune cells that play important roles in innate and adaptive immune responses. The TREM family member TREM-like transcript 1 (TLT-1, also TREML1) is expressed in megakaryocytes and packaged into platelet granules. TLT-1 binds fibrinogen and plays a role in bleeding initiated by inflammatory insults. Here, we describe a proteomics screen that maps the TLT-1 interactome in resting and activated human platelets. Several identified TLT-1 interactors are involved in cell adhesion and migration, as well as platelet activation. Select interactors, including ß3-integrin, RACK1, GRB2, and Rabs 5A, 7, and 11A, were additionally characterized in co-immunoprecipitation/immunoblotting experiments. Finally, several phosphorylation sites were found on immunoprecipitated TLT-1, including Thr280, a novel, regulated site on a conserved residue near the TLT-1 ITIM regulatory sequence. SIGNIFICANCE: Platelet function relies on the secretion of active molecules from intracellular vesicles, or granules, which contain soluble and membrane-bound proteins that are essential for platelet aggregation, coagulation reactions, and pathogen defense mechanisms. TLT-1 is sequestered in α-granules and transported to the plasma membrane, where it plays a unique role in hemostasis after inflammatory insults. Despite the known importance of TLT-1 in platelet biology, our knowledge of TLT-1 mechanistic signaling is limited. This study defines the TLT-1 interactome in resting and active human platelets, identifying several novel TLT-1 interactors, as well as TLT-1 phosphorylation sites, all with likely signaling implications in platelet aggregation dynamics.

Phenotypic heterogeneity in the Gray platelet syndrome extends to the expression of TREM family member, TLT-1.

The Gray platelet syndrome (GPS) is a rare inherited disorder linked to undefined molecular abnormalities that prevent the formation and maturation of alpha-granules. Here, we report studies on two patients from unrelated families that confirm phenotypic heterogeneity in the disease. First we used immunoelectron microscopy (I-EM) to confirm that TREM-like transcript-1 (TLT-1) is mostly localized to alpha-granule membranes of normal platelets. Then we performed Western blotting (WB) and flow cytometry with permeabilized platelets to show that TLT-1 is selectively reduced in the platelets of patient 1, previously noted to be deficient in glycoprotein (GP)VI (Nurden et al., Blood 2004; 104: 107-114). Yet both TLT-1 and GPVI were normally expressed in platelets of patient 2. Usual levels of JAM-C and claudin-5, also members of the immunoglobulin receptor family, were detected in platelets of both patients. In contrast, P-selectin was markedly decreased for patient 1 but not patient 2. Two metalloproteases, MMP-2 and MMP-9 were normally present. As predicted, platelets of patient 1 showed little labelling for TLT-1 in I-EM, whereas residual Fg was seen in small vesicular structures and P-selectin lining vacuoles or channels of what may be elements of the surface-connected canalicular system. Our results identify TLT-1 as a glycoprotein potentially targeted in platelets of GPS patients, while decreases in at least three membrane glycoproteins suggest that an unidentified proteolytic activity may contribute to the phenotype in some patients with this rare disease.

High Levels of Soluble Triggering Receptor Expressed on Myeloid Cells-Like Transcript (TLT)-1 Are Associated With Acute Respiratory Distress Syndrome.

We have previously demonstrated that elevated levels of soluble triggering receptor expressed on myeloid cells-like transcript 1 (sTLT-1) modulate sepsis-induced inflammation and positively correlate with disseminated intravascular coagulation (DIC). Here, we evaluate the clinical implications of plasma sTLT-1 in acute respiratory distress syndrome (ARDS), which is common in sepsis patients. Soluble TLT-1 levels in the plasma of ARDS patients (n = 20) were determined by slot blot analysis and were compared with clinical parameters to identify significant associations. For comparisons to ARDS, we also measured sTLT-1 levels in matched healthy controls (n = 20). Of the 20 plasma samples evaluated from patients with ARDS, 60% were diagnosed with sepsis and 40% were diagnosed with septic shock. The white blood cells (WBCs) of patients with ARDS were found to be significantly elevated over healthy controls with a mean of 13 k/µL over 6.2 k/µL, respectively. The mean plasma levels of sTLT-1 were 148.4 pg/mL ± 16.52 in the patient cohort and 92.45 pg/mL ± 17.12 in the control group ( P = .02). No statistically significant correlations were found between plasma levels of sTLT-1 and WBCs, sepsis, septic shock or acute physiologic, and chronic health evaluation II scores. A statistically significant inverse correlation (r2 = .25, P < .05) was found between plasma sTLT-1 and peripheral platelet counts in patients with ARDS. Increased levels of sTLT-1 in ARDS patients suggest that TLT-1 may mediate the pathobiology of ARDS. Moreover, our data are the first to demonstrate a specific platelet marker in the development of ARDS due to sepsis.

Association of soluble TREM-like transcript-1 with clinical features and patient reported outcomes in systemic lupus erythematosus.

OBJECTIVE: The soluble triggering receptor expressed on myeloid cells (TREM-1)-like transcript 1 (sTLT-1) has a modulatory effect on the activation of TREM-1. We compared plasma sTLT-1 levels between patients with systemic lupus erythematosus (SLE) and healthy individuals and determined the association between sTLT-1 levels and clinical features and patient-reported outcomes (PROs) among patients with lupus. METHODS: An unmatched case-control study was conducted in 46 patients with SLE and 28 healthy subjects. sTLT-1 plasma levels were determined using enzyme-linked immunosorbent assay. Demographic factors, SLE manifestations, comorbidities, pharmacologic profile, disease activity (per SLAM-R), damage accrual, and PROs (as per Lupus Patient-Reported Outcome [LupusPRO]) were studied. RESULTS: Patients with SLE were found to have lower sTLT-1 levels compared with healthy individuals (9.0±7.2 vs. 18.6±22.3 pg/mL, p=0.008). Among patients with SLE, higher sTLT-1 levels were found in those taking corticosteroids (11.1±8.8 vs. 6.9±4.6 pg/mL, p=0.014). Significant correlations were found for the cognition (r=-0.442, p=0.027) and desires/goals (r=0.435, p=0.030) domains of LupusPRO. A tendency was observed between sTLT-1 levels and the SLAM-R (r=-0.278, p=0.064) and the lupus symptoms (r=-0.388, p=0.055) and physical health (r=-0.382, p=0.060) domains of LupusPRO. CONCLUSION: Compared with healthy individuals, sTLT-1 levels were significantly lower in patients with SLE. Among patients with SLE, correlations were observed for some domains of LupusPRO. Given that sTLT-1 has anti-inflammatory properties, the deficiency of this protein could play an important role in the pathogenesis of SLE.

The Characterization of Monoclonal Antibodies to Mouse TLT-1 Suggests That TLT-1 Plays a Role in Wound Healing.

Platelets play a vital role in hemostasis and inflammation. The membrane receptor TREM-like transcript-1 (TLT-1) is involved in platelet aggregation, bleeding, and inflammation, and it is localized in the α-granules of platelets. Upon platelet activation, TLT-1 is released from α-granules both in its transmembrane form and as a soluble fragment (sTLT-1). Higher levels of sTLT-1 have been detected in the plasma of patients with acute inflammation or sepsis, suggesting an important role for TLT-1 during inflammation. However, the roles of TLT-1 in hemostasis and inflammation are not well understood. We are developing the mouse model of TLT-1 to mechanistically test clinical associations of TLT-1 in health and disease. To facilitate our studies, monoclonal murine TLT-1 (mTLT-1) antibodies were produced by the immunization of a rabbit using the negatively charged region of the mTLT-1 extracellular domain 122PPVPGPREGEEAEDEK139. In the present study, we demonstrate that two selected clones, 4.6 and 4.8, are suitable for the detection of mTLT-1 by western blot, immunoprecipitation, immunofluorescent staining, flow cytometry and inhibit platelet aggregation in aggregometry assays. In addition, we found that the topical administration of clone 4.8 delayed the wound healing process in an experimental burn model. These results suggest that TLT-1 plays an important role in wound healing and because both clones specifically detect mTLT-1, they are suitable to further develop TLT-1 based models of inflammation and hemostasis in vivo.

Platelets are responsible for the accumulation of ß-amyloid in blood clots inside and around blood vessels in mouse brain after thrombosis.

INTRODUCTION: Platelets contain beta-amyloid precursor protein (APP) as well as Aß peptide (Aß) that can be released upon activation. During thrombosis, platelets are concentrated in clots and activated. METHODS: We used in vivo fluorescent analysis and electron microscopy in mice to determine to what degree platelets are concentrated in clots. We used immunostaining to visualize Aß after photothrombosis in mouse brains. RESULTS: Both in vivo results and electron microscopy revealed that platelets were 300-500 times more concentrated in clots than in non-clotted blood. After thrombosis in control mice, but not in thrombocytopenic animals, Aß immunofluorescence was present inside blood vessels in the visual cortex and around capillaries in the entorhinal cortex. CONCLUSION: The increased concentration of platelets allows enhanced release of Aß during thrombosis, suggesting an additional source of Aß in the brains of Alzheimer's patients that may arise if frequent micro-thrombosis events occur in their brains.