cytochalasin-d and Thrombosis

To investigate parameters causing canine thromboelastographic hypercoagulability and to investigate whether thromboelastography (TEG) with Cytochalasin D (Cyt D) added is related to parameters of platelet activity.. Prospective observational study on hemostatic and inflammatory parameters. Data were collected between November 2012 and July 2013.. University teaching hospital.. Twenty-eight dogs suffering from diseases predisposing to thrombosis and 19 clinically healthy dogs. Diseased dogs were enrolled if they fulfilled inclusion criteria regarding age, size, informed client consent, and obtained a diagnosis of a disease that has been associated with thrombosis or hypercoagulability.. None.. Parameters of coagulation and anticoagulation, fibrinolysis, and antifibrinolysis, platelet activity, inflammation, platelet count, and hematocrit were measured using CBC, TEG, platelet aggregation on multiplate, platelet activity on flow cytometry, and hemostatic and inflammatory markers on plasma and serum analyses. ANOVA and multilinear regression analyses indicated that especially hematocrit and the inflammatory parameters C-reactive protein and interleukin-8 showed best association with overall clot strength in diseased dogs with hypercoagulable TEG tracings. Ratios presumed to reflect platelet contribution to the TEG tracing obtained in TEG analyses with Cyt D were related especially with hematocrit and P-selectin expression of platelets measured after γ-Thrombin activation on flow cytometry.. Overall clot strength in TEG analyses of the hypercoagulable dogs included in the present study appears to be primarily associated with inflammation as well as hematocrit. Furthermore, the ratio between standard TEG analyses and TEG analyses with Cyt D may reflect some degree of platelet activity.

Topics: Animals; Blood Coagulation; Blood Coagulation Disorders; Blood Platelets; Case-Control Studies; Cytochalasin D; Dog Diseases; Dogs; Female; Hematocrit; Hemostasis; Inflammation; Male; Platelet Activation; Platelet Aggregation; Platelet Count; Platelet Function Tests; Prospective Studies; Thrombelastography; Thrombophilia; Thrombosis

Platelet activation plays a critical role in haemostasis and thrombosis. It is well-known that platelets generate contractile forces during activation. However, their mechanical material properties have rarely been investigated. Here, we use scanning ion conductance microscopy (SICM) to visualise morphological and mechanical properties of live human platelets at high spatial resolution. We found that their mean elastic modulus decreases during thrombin-induced activation by about a factor of two. We observed a similar softening of platelets during cytochalasin D-induced cytoskeleton depolymerisation. However, thrombin-induced temporal and spatial modulations of the elastic modulus were substantially different from cytochalasin D-mediated changes. We thereby provide new insights into the mechanics of haemostasis and establish SICM as a novel imaging platform for the ex vivo investigation of the mechanical properties of live platelets.

Topics: Blood Platelets; Cytochalasin D; Cytoskeleton; Diagnostic Imaging; Elastic Modulus; Hemostasis; Humans; Ions; Microscopy; Platelet Activation; Polymerization; Polymers; Stress, Mechanical; Thrombin; Thrombosis

Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic response. It remains unclear, however, how and why platelets generate these structures. We set out to determine the physiological relevance and cellular and molecular mechanisms underlying platelet membrane ballooning.. Using 4-dimensional live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloonlike structures with expansive macro/microvesiculate contact surfaces, by a process that we termed procoagulant spreading. We reveal that ballooning is mechanistically and structurally distinct from membrane blebbing and involves disruption to the platelet microtubule cytoskeleton and inflation through fluid entry. Unlike blebbing, procoagulant ballooning is irreversible and a consequence of Na(+), Cl(-), and water entry. Furthermore, membrane ballooning correlated with microparticle generation. Inhibition of Na(+), Cl(-), or water entry impaired ballooning, procoagulant spreading, and microparticle generation, and it also diminished local thrombin generation. Human Scott syndrome platelets, which lack expression of Ano-6, also showed a marked reduction in membrane ballooning, consistent with a role for chloride entry in the process. Finally, the blockade of water entry by acetazolamide attenuated ballooning in vitro and markedly suppressed thrombus formation in vivo in a mouse model of thrombosis.. Ballooning and procoagulant spreading of platelets are driven by fluid entry into the cells, and are important for the amplification of localized coagulation in thrombosis.

Topics: Acetazolamide; Actomyosin; Amides; Animals; Anoctamins; Blood Coagulation Disorders; Blood Platelets; Carotid Artery Thrombosis; Cell Adhesion; Cell Membrane; Cell Shape; Cell Size; Cell-Derived Microparticles; Chlorides; Collagen; Cytochalasin D; Heterocyclic Compounds, 4 or More Rings; Humans; Mice; Microtubules; Phospholipid Transfer Proteins; Pyridines; Sodium; Thrombin; Thrombosis; Water

Thromboelastography analysis providing a global assessment of coagulation is gaining new interest in clinical practice. MinimalTF triggered whole blood thromboelastography provides a valuable tool for studying the kinetics of clot formation (expressed by the parameters R, K and alpha-angle) and the physical characteristics of the clot, such as its firmness and the elastic modulus shear (expressed by the parameters maximal amplitude MA and G). We studied the influence of fibrin polymerization and platelet functional status on each parameter of thromboelastographic trace obtained by minimalTF activation inWB by employing increasing concentrations of a fibrin polymerization inhibitors (the tetrapeptide Gly-Pro-Arg-Pro-OH.AcOH; Pefabloc-FG) and an inhibitor of actin polymerization (Cytochalasin D). Pefabloc-FG at concentrations higher than 5 mg/ml prolonged the R and K times and decreased the alpha-angle in a concentration-dependent manner but it did not modify MA and G parameters. At the concentration of 5 mg/ml, Pefabloc-FG completely inhibited clot formation. Cytochalasin D had no effect on R time but decreased the alpha-angle, MA and G parameters by reaching a plateau at the concentration of 5 microM. The effect of cytochalasin D was more pronounced on MA and G than on the alpha-angle. A combination of both Pefabloc-FG (0.5 mg/ml) and cytochalasin D (50 microM) significantly decreased alpha-angle compared to control as well as their single effect. However, G value was dramatically reduced in the presence of cytochalasin D exposure, without any additional effect when both inhibitors were combined. This study confirms the importance of fibrin polymerisation on the kinetics of thrombus formation and demonstrates the close association between the quality of the thrombus and the functional status of platelets. Normal platelet contractile forces are of major importance for the maximum amplitude of TEG which is related to the strength and elastic modulus of the thrombus.

Topics: Biomechanical Phenomena; Blood Coagulation; Blood Platelets; Citric Acid; Cytochalasin D; Dose-Response Relationship, Drug; Fibrin; Humans; Oligopeptides; Sulfones; Thrombelastography; Thrombosis