Dynamic distribution of ERK,p38 and JNK during the development of pancreatic ductal adenocarcinoma

We recently discovered that oncogenic c-kit is highly expressed concomitantly with the development of pancreatic ductal adenocarcinoma (PDAC). Since oncogenic c-kit may activate major pathways of protein tyrosine phosphorylation, we decided to investigate this issue in the major protein phosphorylation cascades. In normal pancreas labeling with antiphosphorylated ERK1/2 (pERK1/2) antibody was mainly confined to islets of Langerhans in close overlapping with insulin containing cells. Phosphorylated p38 (pp38) showed a similar pattern of distribution, while only weak labeling was evident for pJNK and no labeling of pMEK was observed. As expected, general ERK1/2 (gERK1/2), general p38 (gp38), general JNK (gJNK) as well as general MEK (gMEK) were all evident in islets of Langerhans and in the exocrine tissue. In early development of PDAC, pERK1/2 and pp38 retained their localization in islets of Langerhans. Intensive staining of pERK1/2 was also evident in the cancerous ducts, while the labeling with antibodies to pp38 was more moderate. While pJNK staining in islets of Langerhans was weak, with no labeling in the cancerous ducts, antibodies to gJNK revealed intensive staining suggesting the weak staining of pJNK is not due to the lack of the enzyme. In a more advanced stage of PDAC the carcinomas were clearly stained with pERK1/2 and pp38, while moderate staining with pJNK was also evident. In liver metastases, the cancer cells were heavily labeled with all three phospho-MAPKs. It should be noted that the localization of all three kinases was mainly in the cell nuclei. In the more advanced stage of PDAC, heavy labeling was evident using antibodies to gERK1/2, gp38, gJNK and gMEK. However, no labeling to pMEK was evident in parallel sections. Our data suggest that both in normal and cancerous pancreas, most of the MAPK activities are located in islets of Langerhans and cancerous ducts. It is suggested that using inhibitors to protein kinases may attenuate the progression of the disease.     

Ontogeny and innervation of taste buds in mouse palatal gustatory epithelium

We investigated the relationship between mouse taste bud development and innervation of the soft palate. We employed scanning electron microscopy and immunohistochemistry using antibodies against protein gene product 9.5 and peripherin to detect sensory nerves, and cytokeratin 8 and α-gustducin to stain palatal taste buds. At E14, nerve fibers were observed along the medial border of the palatal shelves that tracked toward the epithelium. At E15.5, primordial stages of taste buds in the basal lamina of the soft palate first appeared. At E16, the taste buds became large spherical masses of columnar cells scattered in the soft palate basal lamina. At E17, the morphology and also the location of taste buds changed. At E18–19, some taste buds acquired a more elongated shape with a short neck, extending a variable distance from the soft palate basal lamina toward the surface epithelium. At E18, mature taste buds with taste pores and perigemmal nerve fibers were observed on the surface epithelium of the soft palate. The expression of α-gustducin was demonstrated at postnatal day 1 and the number of pored taste buds increased with age and they became pear-shaped at 8 weeks. The percent of pored fungiform-like papillae at birth was 58.3% of the whole palate; this increased to 83.8% at postnatal day 8 and reached a maximum of 95.7% at 12 weeks. The innervation of the soft palate was classified into three types of plexuses in relation to taste buds: basal nerve plexus, intragemmal and perigemmal nerve fibers. This study reveals that the nerve fibers preceded the development of taste buds in the palate of mice, and therefore the nerve fibers have roles in the initial induction of taste buds in the soft palate.     

Localization of Na+–K+-ATPase α/β, Na+–K+–2Cl-cotransporter 1 and aquaporin-5 in human eccrine sweat glands

In order to evaluate the function of the repaired or regenerated eccrine sweat glands, we must first localize the proteins involved in sweat secretion and absorption in normal human eccrine sweat glands. In our studies, the cellular localization of Na⁺–K⁺-ATPase α/β, Na⁺–K⁺–2Cl-cotransporter 1 (NKCC1) and aquaporin-5 (AQP5) in eccrine sweat glands were detected by immunoperoxidase labeling. The results showed that Na⁺–K⁺-ATPase α was immunolocalized in the cell membrane of the basal layer and suprabasal layer cells of the epidermis, the basolateral membrane of the secretory coils, and the cell membrane of the outer cells and the basolateral membrane of the luminal cells of the ducts. The localization of Na⁺–K⁺-ATPase β in the secretory coils was the same as Na⁺–K⁺-ATPase α, but Na⁺–K⁺-ATPase β labeling was absent in the straight ducts and epidermis. NKCC1 labeling was seen only in the basolateral membrane of the secretory coils. AQP5 was strongly localized in the apical membrane and weakly localized in the cytoplasm of secretory epithelial cells. The different distribution of these proteins in eccrine sweat glands was related to their functions in sweat secretion and absorption.     

Histological studies of neuroprotective effects of Curcuma longa Linn. on neuronal loss induced by dexamethasone treatment in the rat hippocampus

Long term exposure to dexamethasone (Dx) is associated with brain damage especially in the hippocampus via the oxidative stress pathway. Previously, an ethanolic extract from Curcuma longa Linn. (CL) containing the curcumin constituent has been reported to produce antioxidant effects. However, its neuroprotective property on brain histology has remained unexplored. This study has examined the effects of a CL extract on the densities of cresyl violet positive neurons and glial fibrillary acidic protein immunoreactive (GFAP-ir) astrocytes in the hippocampus of Dx treated male rats. It showed that 21days of Dx treatment (0.5 mg/kg, i.p. once daily) significantly reduced the densities of cresyl violet positive neurons in the sub-areas CA1, CA3 and the dentate gyrus, but not in the CA2 area. However, CL pretreatment (100 mg/kg, p.o.) was found to significantly restore neuronal densities in the CA1 and dentate gyrus. In addition, Dx treatment also significantly decreased the densities of the GFAP-ir astrocytes in the sub-areas CA1, CA3 and the dentate gyrus. However, CL pretreatment (100 mg/kg, p.o.) failed to protect the loss of astrocytes in these sub-areas. These findings confirm the neuroprotective effects of the CL extract and indicate that the cause of astrocyte loss might be partially reduced by a non-oxidative mechanism. Moreover, the detection of neuronal and glial densities was suitable method to study brain damage and the effects of treatment.     

Effect of mobile telephony on blood-brain barrier permeability in the fetal mouse brain

AIMS: To study the effect of mobile telephone exposure on blood-brain barrier (BBB) permeability in the immature brain. METHODS: Using a purpose-designed exposure system at 900 MHz, pregnant mice were given a single, far-field, whole body exposure at a specific absorption rate of 4 W/kg for 60 min/day from day 1 to day 19 of gestation. Pregnant control mice were sham-exposed or freely mobile in a cage without further restraint and a positive control group with cadmium-induced BBB damage was also included. Immediately prior to parturition on gestational day 19, fetal heads were collected, fixed in Bouin's fixative and paraffin embedded. Disruption of BBB integrity was detected immunohistochemically using endogenous albumin as a vascular tracer in cerebral cortex, thalamus, basal ganglia, hippocampus, cerebellum, midbrain and medulla. RESULTS: No albumin extravasation was found in exposed or control brains. CONCLUSION: In this animal model, whole of gestation exposure to global system for mobile communication-like radiofrequency fields did not produce any increase in vascular permeability in the fetal brain regions studied using endogenous albumin as a light microscopic immunohistochemical marker.     

Expression of the immediate early gene, c-fos, in fetal brain after whole of gestation exposure of pregnant mice to global system for mobile communication microwaves

AIMS: To study immediate early gene, c-fos, expression as a marker of neural stress after whole of gestation exposure of the fetal mouse brain to mobile telephone-type radiofrequency fields. METHODS: Using a purpose-designed exposure system at 900 MHz, pregnant mice were given a single, far-field, whole body exposure at a specific absorption rate of 4 W/kg for 60 min/day from day 1 to day 19 of gestation. Pregnant control mice were sham-exposed or freely mobile in a cage without further restraint. Immediately prior to parturition on gestational day 19, fetal heads were collected, fixed in 4% paraformaldehyde and paraffin embedded. Any stress response in the brain was detected by c-fos immunohistochemistry in the cerebral cortex, basal ganglia, thalamus, hippocampus, midbrain, cerebellum and medulla. RESULTS: c-fos expression was of limited, but consistent, neuroanatomical distribution and there was no difference in immunoreactivity between exposed and control brains. CONCLUSION: In this animal model, no stress response was detected in the fetal brain using c-fos immunohistochemistry after whole of gestation exposure to mobile telephony.     

Screening for lipid disorders

Lipid disorders, also known as dyslipidaemias, are abnormalities of lipoprotein metabolism and include elevations of total cholesterol, low density lipoprotein (LDL) cholesterol, and triglyceride, and reductions in high density lipoprotein (HDL) cholesterol, and can be acquired or familial in nature. Dyslipidaemia is a major risk factor for coronary heart disease and cardiovascular disease (CVD), which is the leading cause of morbidity and mortality in Australia. Dyslipidaemia is defined by laboratory testing and using statistically determined criteria. Although the benefits of detecting and treating dyslipidaemia in patients with known CVD is clear, controversy remains regarding screening asymptomatic individuals who are not known to be at increased cardiovascular risk. This review examines the role of screening in the detection and treatment of individuals with lipid disorders.     

Electromagnetic field and brain development

Rapid advances in technology involve increased exposures to radio-frequency/microwave radiation from mobile phones and other wireless transmitting devices. As cell phones are held close to the head during talking and often stored next to the reproductive organs, studies are mostly focused on the brain. In fact, more research is especially needed to investigate electromagnetic field (EMF)’s effects on the central nervous system (CNS). Several studies clearly demonstrate that EMF emitted by cell phones could affect a range of body systems and functions. Recent work has demonstrated that EMF inhibit the formation and differentiation of neural stem cells during embryonic development and also affect reproductive and neurological health of adults that have undergone prenatal exposure. The aim of this review is to discuss the developing CNS and explain potential impacts of EMF on this system.     

Cytotoxicity evaluation and antioxidant enzyme expression related to heavy metals found in tuna by-products meal: An in vitro study in human and rat liver cell lines

Heavy metals can accumulate in organisms via various pathways, including respiration, adsorption and ingestion. They are known to generate free radicals and induce oxidative and/or nitrosative stress with depletion of anti-oxidants. Tuna by-product meal (TBM) is rich in proteins and can, therefore, offer an attractive protein source for animals. This study was undertaken to assess the effects of metals present in TBM, namely cadmium (Cd), lead (Pb), and mercury (Hg), separately or in combination with oxidative stress, on cell viability. Three cell models: rat liver FTO2B, human hepatoma HepG2, and human hepatic WRL-68, were used. Cell viability was determined following exposure to various concentrations of the metals. Two antioxidant genes, catalase (CAT) and superoxide dismutase (SOD), were measured to obtain a better understanding of oxidative stress-associated gene expression. Among the metals present in TBM, only Cd at a concentration of 30 μM was noted to exhibit cytotoxic effects. This cytotoxicity was even more pronounced after co-stimulation with H₂O₂, used to mimic systemic oxidative stress. At non-toxic concentrations, Hg and Pb were noted to aggravate oxidative stress toxicity. The results further revealed that exposure to Cd, Pb, and a co-stimulation of H₂O₂ with Hg resulted in the increased expression of antioxidant gene SOD. A risk assessment of toxic contaminants in TBM indicated that food safety objectives should consider the human health impacts of foods derived from animals fed on contaminated meal and that much care should be taken when TBM is used in animal diet.     

The centrally projecting Edinger–Westphal nucleus—I: Efferents in the rat brain

The oculomotor accessory nucleus, often referred to as the Edinger–Westphal nucleus [EW], was first identified in the 17th century. Although its most well known function is the control of pupil diameter, some controversy has arisen regarding the exact location of these preganglionic neurons. Currently, the EW is thought to consist of two different parts. The first part [termed the preganglionic EW—EWpg], which controls lens accommodation, choroidal blood flow and pupillary constriction, primarily consists of cholinergic cells that project to the ciliary ganglion. The second part [termed the centrally projecting EW—EWcp], which is involved in non-ocular functions such as feeding behavior, stress responses, addiction and pain, consists of peptidergic neurons that project to the brainstem, the spinal cord and prosencephalic regions. However, in the literature, we found few reports related to either ascending or descending projections from the EWcp that are compatible with its currently described functions. Therefore, the objective of the present study was to systematically investigate the ascending and descending projections of the EW in the rat brain. We injected the anterograde tracer biotinylated dextran amine into the EW or the retrograde tracer cholera toxin subunit B into multiple EW targets as controls. Additionally, we investigated the potential EW-mediated innervation of neuronal populations with known neurochemical signatures, such as melanin-concentrating hormone in the lateral hypothalamic area [LHA] and corticotropin-releasing factor in the central nucleus of the amygdala [CeM]. We observed anterogradely labeled fibers in the LHA, the reuniens thalamic nucleus, the oval part of the bed nucleus of the stria terminalis, the medial part of the central nucleus of the amygdala, and the zona incerta. We confirmed our EW–LHA and EW–CeM connections using retrograde tracers. We also observed moderate EW-mediated innervation of the paraventricular nucleus of the hypothalamus and the posterior hypothalamus. Our findings provide anatomical bases for previously unrecognized roles of the EW in the modulation of several physiologic systems.     

Biosynthesis of collagen I,II, RUNX2 and lubricin at different time points of chondrogenic differentiation in a 3D in vitro model of human mesenchymal stem cells derived from adipose tissue

The first aim of the study was to identify the most appropriate time for differentiation of adipose tissue derived mesenchymal stem cells (MSCs) to chondrocytes, through the self-assembly process. For this purpose, the expression of some chondrocyte markers, such as collagen type I, collagen type II, RUNX2 and lubricin was investigated at different times (7, 14, 21 and 28 days) of chondrogenic differentiation of MSCs, by using immunohistochemistry and Western blot analysis. The second aim of the study was to demonstrate that the expression of lubricin, such as the expression of collagen type II, could be a possible biomarker for the detection of chondrocytes well-being and viability in the natural self-assembling constructs, called ‘cell pellets’. Histology (hematoxylin and eosin) and histochemistry (alcian blue staining) methods were used to assess the chondrogenic differentiation of MSCs. The results showed that after 21 days the differentiated chondrocytes, when compared with MSCs cultured without chondrogenic medium (CD44, CD90 and CD105 positive; CD45, CD14 and CD34 negative), were able to produce significant quantities of collagen type I, collagen type II, and lubricin, suggesting hyaline cartilage formation. During the differentiation phase, the cells showed a reduced expression of RUNX2, a protein expressed by osteoblasts. Our studies demonstrated that 21 days is the optimum time for the implantation of chondrocytes differentiated from adipose tissue-derived MSCs. This information could be useful for the future development of cell-based repair therapies for degenerative diseases of articular cartilage.     

Effect of low estrogen on neurons in the preoptic area of hypothalamus of ovariectomized rats

The purpose of this study was to investigate the difference in neuronal activity in the preoptic area of the hypothalamus (POAH) under low estrogen condition induced by ovariectomy. One hundred and twenty sham-operated (SHAM) and ovariectomized (OVX) rats were placed in different temperatures for 2 h. Twelve rats from each group were stimulated by 4 °C, 10 °C, 25 °C, 33 °C and 38 °C, respectively. c-Fos expression in the POAH was detected by immunohistochemistry. Following exposure to warm and cold stimuli, there were markedly lower c-Fos-positive cell densities in the OVX group compared with the SHAM group in the median preoptic nucleus (MnPO) at 4 °C, 10 °C, 33 °C and 38 °C, in the medial preoptic area (MPA) at 25 °C and 38 °C, in the ventromedial preoptic nucleus (VMPO) at 4 °C, 10 °C and 38 °C and in the ventrolateral preoptic nucleus (VLPO) at 4 °C and 38 °C. Both temperature and surgery had an impact on c-Fos expression by two-way ANOVA method except in the lateral preoptic area (LPO). c-Fos expression differed within different nuclei of the two groups in the same and different temperature stimuli. This indicated that the temperature-sensitive nuclei in the POAH exhibited lower and different activities during temperature stimuli following ovariectomy, which possibly resulted in abnormal thermoregulation and menopausal symptoms.     

Nuclear organization of some immunohistochemically identifiable neural systems in two species of the Euarchontoglires: A Lagomorph,Lepus capensis,and a Scandentia,Tupaia belangeri

The present study describes the organization of the nuclei of the cholinergic, catecholaminergic, serotonergic and orexinergic systems in the brains of two members of Euarchontoglires, Lepus capensis and Tupaia belangeri. The aim of the present study was to investigate the nuclear complement of these neural systems in comparison to previous studies on Euarchontoglires and generally with other mammalian species. Brains were coronally sectioned and immunohistochemically stained with antibodies against choline acetyltransferase, tyrosine hydroxylase, serotonin and orexin-A. The majority of nuclei revealed in the current study were similar between the species investigated and to mammals generally, but certain differences in the nuclear complement highlight potential phylogenetic interrelationships within the Euarchontoglires and across mammals. In the northern tree shrew the nucleus of the trapezoid body contained neurons immunoreactive to the choline acetyltransferase antibody with some of these neurons extending into the lamellae within the superior olivary nuclear complex (SON). The cholinergic nature of the neurons of this nucleus, and the extension of cholinergic neurons into the SON, has not been noted in any mammal studied to date. In addition, cholinergic neurons forming the medullary tegmental field were also present in the northern tree shrew. Regarding the catecholaminergic system, the cape hare presented with the rodent specific rostral dorsal midline medullary nucleus (C3), and the northern tree shrew lacked both the ventral and dorsal divisions of the anterior hypothalamic group (A15v and A15d). Both species were lacking the primate/megachiropteran specific compact portion of the locus coeruleus complex (A6c). The nuclei of the serotonergic and orexinergic systems of both species were similar to those seen across most Eutherian mammals. Our results lend support to the monophyly of the Glires, and more broadly suggest that the megachiropterans are more closely related to the primates than are any other members of Euarchontoglires studied to date.     

Epidemiology of respiratory viruses in bronchoalveolar lavage samples in a tertiary hospital

BackgroundThe prevalence of respiratory viruses in adults is largely underexplored, as most studies focus on children. Additionally, in severely ill or immunocompromised adults, where respiratory infections are mostly attributed to bacteria and fungi; respiratory viruses can lead to severe complications.ObjectivesTo evaluate the epidemiology of respiratory viruses in bronchoalveolar lavage fluid (BAL) specimens from patients with lower respiratory tract disease. The study population consisted of different groups including immunocompetent patients (control patients), solid organ transplant recipients, patients with haematological malignancies and other immunocompromised adults.Study designA total of 134 BAL fluid specimens collected during 2009–2011 were retrospectively assessed with the new commercial multiplex real-time PCR FTD Respiratory 21 Plus^®, targeting 18 different viruses and 2 atypical bacterial pathogens.ResultsViral or atypical bacterial pathogens were detected in 29.1% of BAL fluid specimens. Coronaviruses were most prevalent (13.4%), followed by rhinoviruses (5.2%), RSV (4.5%) and bocaviruses (3.7%). Comparing the total number of viruses detected, a statistically significant difference was observed between the control group and patients with haematological malignancies (27.5% vs. 57.1%, p < 0.05).ConclusionIn conclusion, our study highlights the high prevalence of respiratory viruses in BAL fluid specimens from adult patients with lower respiratory tract disease. The methods to be used should be sensitive and cover a wide range of potential pathogens. The specific patient population can also influence the detection rates of respiratory viruses.     

Orexinergic innervation of urocortin1 and cocaine and amphetamine regulated transcript neurons in the midbrain centrally projecting Edinger–Westphal nucleus

Orexin is a neuropeptide that has been implicated in several processes, such as induction of appetite, arousal and alertness and sleep/wake regulation. Multiple lines of evidence also suggest that orexin is involved in the stress response. When orexin is administered intracerebroventricular it activates the hypothalamic pituitary adrenal (HPA)-axis, which is the main regulator of the stress response. The HPA-axis is not the only player in the stress response evidence suggests that urocortin 1 (Ucn1), a member of the corticotropin releasing factor (CRF) neuropeptide family, also plays an important role in the stress response adaptation. Ucn1 is primarily synthetized in the centrally projecting Edinger–Westphal nucleus (EWcp), which also receives dense innervation by orexin terminals. In this study we tested the hypothesis that orexin would directly shape the response of EWcp-Ucn1 neurons to acute cold stress. To test this hypothesis, we first assessed whether orexinergic axon terminals would innervate EWcp-Ucn1/CART neurons, and next we exposed orexin deficient (orexin-KO) male mice and their male wild-type (WT) littermates to acute cold stress for 2 h. We also assessed stress-associated changes in plasma corticosterone (CORT), as well as the activation of Ucn1/CART neurons in the EWcp nucleus. We found that orexin immunoreactive axon terminals were juxtaposed to EWcp-Ucn1/CART neurons, which also expressed orexin receptor 1 mRNA. Furthermore, acute stress strongly activated the EWcp-Ucn1/CART neurons and increased plasma CORT in both WT littermates and orexin-KO mice, however no genotype effect was found on these indices. Taken together our data show that orexin in general is not involved in the animal's acute stress response (plasma CORT) and it does not play a direct role in shaping the response of EWcp-Ucn1 neurons to acute stress either.     

Nuclear organization of cholinergic,catecholaminergic, serotonergic and orexinergic systems in the brain of the Tasmanian devil (Sarcophilus harrisii)

This study investigated the nuclear organization of four immunohistochemically identifiable neural systems (cholinergic, catecholaminergic, serotonergic and orexinergic) within the brains of three male Tasmanian devils (Sarcophilus harrisii), which had a mean brain mass of 11.6 g. We found that the nuclei generally observed for these systems in other mammalian brains were present in the brain of the Tasmanian devil. Despite this, specific differences in the nuclear organization of the cholinergic, catecholaminergic and serotonergic systems appear to carry a phylogenetic signal. In the cholinergic system, only the dorsal hypothalamic cholinergic nucleus could be observed, while an extra dorsal subdivision of the laterodorsal tegmental nucleus and cholinergic neurons within the gelatinous layer of the caudal spinal trigeminal nucleus were observed. Within the catecholaminergic system the A4 nucleus of the locus coeruleus complex was absent, as was the caudal ventrolateral serotonergic group of the serotonergic system. The organization of the orexinergic system was similar to that seen in many mammals previously studied. Overall, while showing strong similarities to the organization of these systems in other mammals, the specific differences observed in the Tasmanian devil reveal either order specific, or class specific, features of these systems. Further studies will reveal the extent of change in the nuclear organization of these systems in marsupials and how these potential changes may affect functionality.     

TRARESA: a tissue microarray-based hospital system for biomarker validation and discovery

Formalin fixed and paraffin embedded tissue (FFPE) collections in pathology departments are the largest resource for retrospective biomedical research studies. Based on the literature analysis of FFPE related research, as well as our own technical validation, we present the Translational Research Arrays (TRARESA), a tissue microarray centred, hospital based, translational research conceptual framework for both validation and/or discovery of novel biomarkers. TRARESA incorporates the analysis of protein, DNA and RNA in the same samples, correlating with clinical and pathological parameters from each case, and allowing (a) the confirmation of new biomarkers, disease hypotheses and drug targets, and (b) the postulation of novel hypotheses on disease mechanisms and drug targets based on known biomarkers. While presenting TRARESA, we illustrate the use of such a comprehensive approach. The conceptualisation of the role of FFPE-based studies in translational research allows the utilisation of this commodity, and adds to the hypothesis-generating armamentarium of existing high-throughput technologies.     

ShcC proteins: Brain aging and beyond

To date, most studies of Shc family of signaling adaptor proteins have been focused on the near-ubiquitously expressed ShcA, indicating its relevance to age-related diseases and longevity. Although the role of the neuronal ShcC protein is much less investigated, accumulated evidence suggests its importance for neuroprotection against such aging-associated conditions as brain ischemia and oxidative stress. Here, we summarize more than decade of studies on the ShcC expression and function in normal brain, age-related brain pathologies and immune disorders with a focus on the interactions of ShcC with signaling proteins/pathways, and the possible implications of these interactions for changes associated with aging.     

MASP-1 of the complement system promotes clotting via prothrombin activation

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity, and it has been shown to activate coagulation factors. Here we studied the effects of MASP-1 on clot formation in whole blood (WB) and platelet-poor plasma (PPP) by thrombelastography and further elucidated the underlying mechanism. Cleavage of prothrombin by MASP-1 was investigated by SDS-PAGE and N-terminal sequencing of cleavage products. Addition of MASP-1 or thrombin to WB and PPP shortened the clotting time and clot formation time significantly compared to recalcified-only samples. The combination of MASP-1 and thrombin had additive effects. In a purified system, MASP-1 was able to induce clotting only in presence of prothrombin. Analysis of MASP-1-digested prothrombin confirmed that MASP-1 cleaves prothrombin at three cleavage sites. In conclusion, we have shown that MASP-1 is able to induce and promote clot formation measured in a global setting using the technique of thrombelastography. We further confirmed that MASP-1-induced clotting is dependent on prothrombin. Finally, we have demonstrated that MASP-1 cleaves prothrombin and identified its cleavage sites, suggesting that MASP-1 gives rise to an alternative active form of thrombin by cleaving at the cleavage site R393.