Cellular Clotting Research Articles

Systemic coagulopathy promotes host lethality in a new Drosophila tumor model.

Malignant tumors trigger a complex network of inflammatory and wound repair responses, prompting Dvorak's characterization of tumors as "wounds that never heal."1 Some of these responses lead to profound defects in blood clotting, such as disseminated intravascular coagulopathy (DIC), which correlate with poor prognoses.2,3,4 Here, we demonstrate that a new tumor model in Drosophila provokes phenotypes that resemble coagulopathies observed in patients. Fly ovarian tumors overproduce multiple secreted components of the clotting cascade and trigger hypercoagulation of fly blood (hemolymph). Hypercoagulation occurs shortly after tumor induction and is transient; it is followed by a hypocoagulative state that is defective in wound healing. Cellular clotting regulators accumulate on the tumor over time and are depleted from the body, suggesting that hypocoagulation is caused by exhaustion of host clotting components. We show that rescuing coagulopathy by depleting a tumor-produced clotting factor improves survival of tumor-bearing flies, despite the fact that flies have an open (non-vascular) circulatory system. As clinical studies suggest that lethality in patients with high serum levels of clotting components can be independent of thrombotic events,5,6 our work establishes a platform for identifying alternative mechanisms by which tumor-driven coagulopathy triggers early mortality. Moreover, it opens up exploration of other conserved mechanisms of host responses to chronic wounds.

Insect hemolymph coagulation: Kinetics of classically and non-classically secreted clotting factors

In most insects, hemolymph coagulation, which is analogous to mammalian blood clotting, involves close collaboration between humoral and cellular components. To gain insights into the secretion of cellular clotting factors, we created tagged versions of three different clotting factors. Our focus was on factors which are released in a non-classical manner and to characterize them in comparison to a protein that is classically released, namely Glutactin (Glt). Transglutaminase-A (Tg) and Prophenoloxidase 2 (PPO2), both of which lack signal peptide sequences, have been previously demonstrated to be released from plasmatocytes and crystal cells (CCs) respectively, the two hemocyte classes in naïve larvae. We found that at the molecular level, Tg secretion resembles the release of tissue transglutaminase in mammals. Specifically, Drosophila Tg is associated with vesicular membranes and remains membrane-bound after release, in contrast to Glt, which we found localizes to a different class of vesicles and is integrated into clot fibers. PPO2 on the other hand, is set free from CCs through cytolysis. We confirm that PPO2 is a central component of the cytosolic crystals and find that the distribution of PPO2 appears to vary across crystals and cells. We propose a tentative scheme for the secretory events during early and late hemolymph coagulation.

Haemostatic and immune role of cellular clotting in the sipunculan Themiste petricola

Sipunculans, a small phylum of coelomated marine worms closely related to polychaete annelids, lack a true circulatory system. We have previously shown that the sipunculan Themiste petricola can form a cellular clot, without congealing, of cell-free coelomic fluid. The clot is formed by the aggregation of large granular leukocytes (LGLs) and may serve not only haemostatic but immune functions, since dissimilar particles may become entrapped within it. We have now evaluated the capacity of a massive clot, induced in vitro by sea water contact, to stop coelomic fluid flow. We have further studied smaller clots induced on glass-slides either with or without the presence of bacteria placed for entrapment within the clot. The fate of clotting LGLs is cell death while forming a cohesive mass, although cytoplasmic and nuclear remnants are shed from the clot. These remnants and any bacteria that avoid clot entrapment or are detached from the clot are engulfed by non-clotting cells that include small granular leukocytes (SGLs) and large hyaline amebocytes (LHAs). Both cell types can be found other than in the clot but SGLs also occur around the clot edges heavily loaded with engulfed material. The cytoskeletal arrangement of SGLs evaluated with phalloidin-rhodamine correspond to motile cells and contrast with that of clotting LGLs that form a massive network of F-actin. Thus, the complementary roles between clotting LGLs and non-clotting SGLs and LHAs act a central immune strategy of Themiste petricola to deal with body wall injury and pathogen intrusion into the coelomic cavity.

Atrial fibrillation is associated with impaired cognitive function and hippocampal atrophy: silent cerebral ischaemia vs. Alzheimer's disease?

Atrial fibrillation (AF) is the most common cause of thromboembolic stroke. Several studies have shown an activation of the plasmatic and cellular clotting system in patients with AF.1,2 Interestingly, these changes occur within hours after initiation of AF. In order to predict the individual risk for AF-related thromboembolic events, the CHADS2-score has been developed.1 It takes into account that especially elderly patients, patients with diabetes, hypertension, heart failure, and/or previous stroke are at greatest risk. Depending on the CHADS2-score, the yearly risk of stroke may vary between 1.9% (CHADS2-score of 0) and 18.2% (CHADS2-score of 6). The pathophysiological basis of how these clinical risk factors affect cardiac thrombogenesis or predict future thromboembolic events is complex. According to the Virchow triad there are three determinants of thrombus formation: circulatory stasis, hypercoagulable state, and endothelial injury at the site of thrombus initiation. Clinical co-morbidities such as diabetes, hypertension, or heart failure influence all three factors of Virchow's triad. Increased expression of adhesion molecules at the atrial endocardium promotes the adhesion of leukocytes, leading to the formation of granulocyte–platelet conjugates at the endocardium.3 This process appears to be intensified in heart failure. Diabetes and hypertension further increase inflammatory responses. Thus, small granulocyte–platelet conjugates or larger thrombi may develop in fibrillating atria, causing silent cerebral ischaemia or manifest stroke.2,4,5 In addition to thromboembolic events, cerebral hypoperfusion induced by cardiac beat-to-beat variability is considered as an alternative pathophysiological mechanism for silent cerebral ischaemia during AF.6 Thus, it appears very reasonable to believe that AF may cause … *Corresponding author. Tel: +49 391 6713225, Fax: +49 391 6713202, Email: andreas.goette{at}medizin.uni-magdeburg.de

Cellular clot formation in a sipunculan worm: Entrapment of foreign particles, cell death and identification of a PGRP-related protein

Clotting in animals having open or closed circulatory system comprises humoral and cellular mechanisms. Sipunculans are a small phylum of non-segmented marine worms that do not have a true circulatory system. These worms can form a cellular clot without transforming cell-free coelomic fluid into an insoluble mass. The clot may also contribute to immune response by entrapping foreign particles. We evaluated the formation of a cellular clot ex vivo in the sipunculan Themiste petricola after activation through glass contact and sea water, the ability to entrap magnetic beads and non-pathogen cyanobacteria particles within the clot, and the presence of a peptidoglycan recognition protein S (PGRP-S) antigen in cells forming the clot. Our results showed that the clot was formed by homotypic aggregation of large granular leukocytes (LGLs), a subtype of cells found in the coelomic fluid. Aggregated LGLs served to entrap magnetic beads and non-pathogen cyanobacteria particles, and PGRP-S antigen was detected both in non-activated LGLs and in activated homotypic aggregates through immunofluorescence, Western blot and flow cytometry. Cellular clots were found to be positive to Annexin V-FITC labelling. Complete disintegration of cytoplasm with shedding of microparticles, nuclear isolation and degradation were also observed by light microscopy and flow cytometry. In conclusion, cellular clot formation in Themiste petricola may serve both haemostatic and immune functions entailing rapid activation changes in LGLs, entrapment of foreign particles within a homotypic aggregate, and further cell death.

Delayed, reduced or inhibited thrombin production reduces platelet contractile force and results in weaker clot formation.

Clot retraction is a thrombin-dependent, platelet-mediated contraction of the cellular clot mass. In this study, the effects of delayed, deficient and inhibited thrombin generation on the development of platelet contractile force (PCF) and clot elastic modulus (CEM) were measured. When normal citrated whole blood is clotted by the addition of exogenous thrombin (1 U/ml) and calcium (10 mmol/l), PCF and CEM start to develop within the first minute and begin to level off by 1200 s. If identical samples are clotted with batroxobin (0.21 microg/ml) and calcium (10 mmol/l), both PCF and CEM development are delayed approximately 5 min. After 1200 s of clotting, however, values in the batroxobin system approach those seen with exogenous thrombin. If the added calcium concentration is held constant at 10 mmol/l, increasing the exogenous thrombin concentration from 0 to 0.5 U/ml results in increased PCF and CEM values. Above 0.5 U thrombin, the effect plateaus. At exogenous calcium of 10 mmol/l, increasing batroxobin concentrations (0-0.210 microg/ml) caused a 75% increase in PCF and a 55% increase in CEM. The increase in CEM reached a plateau above 0.05 microg batroxobin/ml. The effects of varying calcium concentrations were evaluated at constant batroxobin (0.21 microg/ml) and thrombin (1 U/ml) concentrations. With thrombin, PCF and CEM increased by > 700% as CaCl2 increased from 0 to 5 mmol/l. Above 5 mmol/l, no additional increases occurred. With batroxobin, PCF did not develop at CaCl2 concentrations < or = 2.5 mmol/l. Above 2.5 mmol/l CaCl2, PCF values increased and at 10 mmol/l CaCl2 were equal to those seen with thrombin. CEM in batroxobin-mediated clots peaked at 10 mmol/l CaCl2 but were 40% less than the values found in thrombin-mediated clots. When the thrombin inhibitor P-PACK was added to the batroxobin system, dose-dependent decreases in PCF and CEM were noted. At 120 micromol/l, P-PACK totally suppressed PCF. PCF in blood from a factor VIII-deficient patient varied significantly when clotted with batroxobin versus thrombin. PCF development in factor VIII-deficient blood was normal with thrombin but is delayed and depressed with batroxobin. PCF values in factor VIII-deficient blood did not reach the thrombin value after 1200 s of clotting, and CEM was significantly less. These results confirm that PCF development is thrombin dependent and that delay or reduction of PCF development results in structurally weaker clots.

The effect of chitosan (poly-N-acetyl glucosamine) on lingual hemostasis in heparinized rabbits

Purpose: The purpose of this study was to evaluate the effect of chitosan on lingual hemostasis in rabbits whose coagulation pathway had been impaired by administration of intravenous heparin. Materials and Methods: Bleeding times were measured for bilateral (15 mm × 2 mm) tongue incisions in 10 New Zealand white rabbits. Using a randomized, blinded experimental design, one incision in each animal was treated with chitosan, and the other was treated with the control vehicle without chitosan. Activated coagulation times and extraoral bleeding times were measured for each animal before, during, and after heparinization. Results: Intravenous infusion of heparin more than tripled the mean activated coagulation time and increased mean systemic bleeding time by 40%. In this heparinized animal model, lingual incisions receiving the experimental substance showed a 43% improvement in bleeding time as compared with lingual incisions receiving the control solution ( P ≤.001). Chitosan treatment brought bleeding time of the lingual incision for heparinized animals within the normal range. Scanning electron microscopic evaluation of the incisions treated with chitosan showed an altered red blood cell morphology and an unusual affinity between erythrocytes. Conclusions: Topical application of chitosan to lingual incisions effectively decreased intraoral bleeding time in a therapeutically anticoagulated (heparinized) rabbit model. Chitosan facilitated lingual hemostasis, possibly through interaction with erythrocytes, linking them together to establish a cellular clot or hemostatic plug.

Relationships between plasma and hemocyte proteins in Decapoda

1. 1. Hematocyte lysates from lobster (Homarus vulgaris) and crayfish (Astacus leptodactylus) were prepared under pyrogen-free conditions in the presence of N-ethyl-maleimide and then acidified. 2. 2. In both lobster and crayfish lysates, one hemocyanin-related fraction and at least one hemagglutinin fraction were delected. 3. 3. Plasma coagulogen was not found inside the cells, except in the presence of cysteine hydrochloride. When present, it appeared to be linked to a pink cellular agglutinin. 4. 4. Activation of cellular clotting by lipopolysaccharides or β-1,3 glucans showed that most hemocyte proteins are involved in the coagulation process. A factor which dissolved in acidic medium and moved towards the cathode was observed, as well as urea-soluble proteins. The first factor might be a Limulus-like cellular coagulogen. 5. 5. The relationships between the humoral defense factors plasma coagulogen, agglutinins, phenoloxidase, and opsonins are discussed.

Transmissible Disease, Probably Viral in Origin, Affecting the Amebocytes of the European Shore Crab, Carcinus maenas

A transmissible infection of the shore crab ("crabe enragée"), Carcinus maenas, was discovered in one of about 700 of this species collected by the Station Biologique, Roscoff, France. The infection has been transferred by subinoculation through four serial passages in crabs collected at Roscoff and through six more passages in C. maenas collected at Woods Hole, Mass. The agent causes abnormal cellular clotting, a drop in the peripheral amebocyte count, clumping of amebocytes in the peripheral tissue and blood, and abnormal behavior of the amebocytes of the infected animal on glass. Mortality over a period of some weeks is negligible. Electron microscopy shows characteristic virus particles, about 55 to 125 nm in size, in the infected amebocytes. The agent is present in large amounts in the whole blood and serum, is filterable, heat labile, and has been preserved in the frozen state. Final identification will depend upon purification, growth in tissue culture, and further electron microscopy.

The coelomic elements of sea urchins ( Strongylocentrotus) I. The normal coelomocytes; Their morphology and dynamics in hanging drops

The cells found in the coelom (coelomocytes) of Strongylocentrotus purpuratus, the purple sea urchin, and Strongylocentrotus franciscanus, the red sea urchin, can be maintained for long periods in hanging drops. The morphological changes, clotting mechanisms, and in vitro reactions undergone by the various coelomocyte types can be followed with ease. The four cell types present are phagocytic leukocytes, vibratile cells, red spherule cells, and colorless spherule cells. On contact with a surface, the phagocytic leukocytes change from bladder amoebocytes to flattened syncytial cells. Within the liquid they form permanent aggregations composed of cells which do not lose the plasma membranes to form plasmodia. Other cell types are caught passively in the aggregations. Apparently only the phagocytic leukocyte is involved in cellular clotting, and extracellular gelling of the coelomic fluid was not observed. A special lysing cell type which initiates clotting was not found despite suggestions in the literature that cell aggregation in echinoids is dependent upon such a mechanism. Under adverse conditions small aggregations of cells will form plasmodia but in large clots only the surface cells form a syncytial membrane while plasma membranes of the interior cells appear to remain intact. It has been though that the function of vibratile cells is to mix the fluid and cells of the capacious and nonmuscular echinoid coelom. In hanging drops, vibratile cells are not efficient in mixing the fluid or in moving cells from one place to another. However, both in vivo and in vitro they will release a portion of a mucoid compound present in their cytoplasm when stressed by intrusion of fluid from the gut into the coelom or hanging drop. The temporary semigel thus formed may operate in limiting spread of gut fluid or other foreign material through the coelom.

Clottable Protein in Limulus: Its Localization and Kinetics of Its Coagulation by Endotoxin

SummaryWhen blood is withdrawn from Limulus, the horseshoe crab, a cellular clot composed of amebocytes quickly forms. Amebocytes are the only type of cell in Limulus blood. During coagulation, Limulus amebocytes undergo morphological changes that are strikingly similar to those seen when mammalian platelets undergo aggregation and viscous metamorphosis. The clottable protein in Limulus blood is derived entirely from amebocytes, and gelation does not require extracellular factors. Cell free Limulus plasma is incoagulable. The cellular localization of clottable protein in Limulus blood provides a precedent for the presence of fibrinogen in mammalian platelets.The clottable protein in lysates of amebocytes has a spectral absorption pattern with a maximum at 270-275 mμ, appears to have a low sedimentation coefficient, is stable at –20° C for 1 week, and is destroyed by heating at 56° C for 30 min. The protein gels upon exposure to endotoxin; and the rate of gelation is related to the concentration of endotoxin but is independent of the concentration of protein in the range tested. The kinetics of this reaction are consistent with the concept that an enzymatic system mediates the conversion of the cellular protein into a gel by endotoxin. Increase in light scattering during the reaction detects as little as 0.004 μg of E. coli endotoxin/ml. This conversion will perhaps provide insight into one of the biological activities of endotoxins.