SummaryUltrasound accelerates fibrinolysis in vitro and in vivo, primarily through non-thermal mechanisms including cavitation. We have previously observed that ultrasound reversibly increases flow through fibrin gels, a property primarily determined by the structure of the fibrin matrix. Therefore, the effect of ultrasound on the ultrastructure of fibrin gels was examined using scanning electron microscopy. Non-cross- linked fibrin gels were fixed and prepared for microscopy before, during and after exposure to 1 MHz ultrasound, and quantitative analysis of fiber population density and diameter was performed. Gels exposed and fixed in the presence of ultrasound exhibited an increase in density of 65 ± 26% (mean ± SD) at 4 W/cm2 (p <0.000001) accompanied by a decrease in fiber diameter of 27 ± 9% (p <0.000001). Gels fixed 15 min following ultrasound exposure showed no significant change in either density or diameter compared to unexposed gels, indicating that the ultrasound-induced change in fiber structure was reversible. Factor XIII-crosslinked fibrin gels exhibited no change in population density or diameter when exposed to ultrasound. These results indicate that ultrasound exposure causes reversible disaggregation of uncrosslinked fibrin fibers into smaller fibers, an effect that may alter flow resistance and create additional binding sites for fibrinolytic components, improving fibrinolytic efficacy.

This work examines the affinity of alpha(4)beta(1)-integrin and whether affinity regulation by G protein-coupled receptor (GPCR) and chemokines receptors is compatible with cell adhesion mediated between alpha(4)-integrin and vascular cell adhesion molecule-1. We used flow cytometry to examine the binding of a fluorescent derivative of an LDV peptide (Chen, L. L., Whitty, A., Lobb, R. R., Adams, S. P., and Pepinsky, R. B. (1999) J. Biol. Chem. 274, 13167-13175) to several cell lines and leukocytes with alpha(4)-integrin ranging from about 2,000 to 100,000 sites/cell. The results support the idea that alpha(4)-integrins exhibit multiple affinities and that affinity changes are regulated by the dissociation rate and conformation. The affinity varies by 3 orders of magnitude with the affinity induced by binding mAb TS2/16 plus Mn(2+) > Mn(2+) ' TS2/16 > activation because of occupancy of GPCR or chemokines receptor > resting receptors. A significant fraction of the receptors respond to the activating process. The change in alpha(4)-integrin affinity and the corresponding change in off rates mediated by GPCR receptor activation are rapid and transient, and their duration depends on GPCR desensitization. The affinity changes mediated by IgE receptor or interleukin-5 receptor persist longer. It appears that the physiologically active state of the alpha(4)-integrin, determined by inside-out signaling, has similar affinity in several cell types.

The prototypical integrin receptor, alpha IIb beta 3, isolated from the membrane fraction of human blood platelets by solubilization in Triton X-100 (reduced) and affinity chromatography on lentil lectin-agarose, has been further purified by gel filtration chromatography in octyl glucoside to obtain the intact receptor complex in a form suitable for hydrodynamic measurements. The molecular weight [(6.0 +/- 0.2) x 10(3)] and Stokes radius (2.3 +/- 0.1 nm) of detergent micelles formed in 0.03 M octyl glucoside have been determined by classical light scattering intensity and dynamic light scattering measurements, respectively. An algorithm has been developed which explicitly considers the contribution of detergent micelles to the intensity autocorrelation function of particles suspended in detergent. This procedure has been validated with polystyrene particles of known radius, as well as with the soluble protein fibrinogen. Application of these procedures to dynamic light scattering data obtained with alpha IIb beta 3 resulted in a translational diffusion coefficient (Dto(20,w)) of (2.78 +/- 0.31) x 10(-7) cm2 s-1, corresponding to a Strokes radius (Rs) of 7.67 +/- 0.85 nm for the integrin/octyl glucoside complex. Light scattering intensity measurements gave a molecular weight of (2.26 +/- 0.22) x 10(5) for the polypeptide moiety of the complex, in excellent agreement with the 2.28 x 10(5) value calculated from primary structure data. As a spherical, hydrated alpha IIb beta 3 complex, with bound detergent, would exhibit a Stokes radius of approximately 5 nm, these data indicate considerable asymmetry in the solution conformation of alpha IIb beta 3.

SummaryUltrasound accelerates fibrinolysis in vitro and in animal models of thrombosis. Since transport of fibrinolytic enzymes into clots by permeation may be an important determinant of the rate of fibrinolysis, we examined the effect of ultrasound on permeation through fibrin gels in vitro. Gels of purified fibrin were prepared in plastic tubes, and the rate of pressure-mediated fluid permeation was measured. Exposure to 1 MHz ultrasound at 2 W/cm2 and a duty cycle of 5 msec on, 5 msec off resulted in a significant (p = .005) increase in flow through the gel of 29.0 ± 4.2% (SEM). The ultrasound-induced flow increase was intensity-dependent, increasing from 17.0 ± 1.2% at 1 W/cm2 to 30.1 ± 1.9% at 2.3 W/cm2. Increased flow was not due to heating, detachment of fibrin from the tube wall or fragmentation of the gel resulting in channeling. However, degassing the fluid by autoclaving significantly reduced the ultrasound-induced increase in flow. We conclude that exposure of fibrin gels to ultrasound increases pressure-mediated permeation. This effect may be related to cavitation-induced changes in fibrin gel structure, and could contribute to the accelerated fibrinolysis observed in an ultrasound field.

SummaryThe effects of GPIIb/IIIa blockade on clot retraction were studied utilizing an instrument which directly measures force produced by platelets. GPIIb/IIIa disruption by calcium chelation, and GPIIb/IIIa blockade by peptides and anti-GPIIb/IIIa antibodies were investigated. One mM EDTA suppressed ADP-induced platelet aggregation by 72% and reduced force developed at 1200 s by 33%. At 234 μM, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) suppressed platelet aggregation by 74%, reduced force at 1200 s by 45% and reduced gel elastic modulus by 19%. At 10 μM, the peptide D-Arg-Gly-L-Asp-L-Try (D-RGDW) completely suppressed platelet aggregation, reduced force development by 38% and reduced gel elastic modulus by 29%. At 0.133 μM, monoclonal anti-GPIIIa antibody (AP-3) reduced force development by 74% and reduced gel modulus by 60%. Murine antiGPIIb/IIIa antibodies 10E5 and 7E3 markedly suppressed force development. At 0.133 μM, 10E5 reduced force by 89% and reduced gel modulus by 67%. At 0.053 μM, 7E3 completely stopped force development and reduced gel modulus by 46%. Platelet aggregation was blocked by 0.027 μM 7E3. Selective GPIIb blockade by antibodies did not affect force development. None of the agents studied altered fibrin structure as monitored by effects on fibrin mass/length ratios. Suppression of platelet aggregation occurred at inhibitor concentrations substantially lower than those required to suppress force development. Complete suppression of platelet aggregation did not assure inhibition of clot retraction probably due to profound platelet activation by thrombin. These results indicate that inhibition of fibrin(ogen) binding to GPIIb/IIIa, either by disruption of GPIIb/IIIa or by competitive blockade, inhibits platelet mediated force development and results in clot structures which are substantially less resistant to deformation by outside forces.