Visualization of the dynamics of fibrin clot growth 1 molecule at a time by total internal reflection fluorescence microscopy.

Individual fluorescently labeled fibrin(ogen) molecules and their assembly to make a clot were observed by total internal reflection fluorescence microscopy (TIRFM). We used the bleaching of the fluorescent labels to determine the number of active fluorophores attached nonspecifically to each molecule. From the total intensity of bleaching steps, as single-molecule signature events, and the distribution of active labeling, we developed a new single-molecule intensity calibration, which accounts for all molecules, including those "not seen." Live observation of fibrin polymerization in TIRFM by diffusive mixing of thrombin and plasma revealed the real-time growth kinetics of individual fibrin fibers quantitatively at the molecular level. Some fibers thickened in time to thousands of molecules across, equivalent to hundreds of nanometers in diameter, whereas others reached an early stationary state at smaller diameters. This new approach to determine the molecular dynamics of fiber growth provides information important for understanding clotting mechanisms and the associated clinical implications.

Flow-dependent channel formation in clots by an erythrocyte-bound fibrinolytic agent.

Studies in animal models have shown that plasminogen activators bound to erythrocytes (RBC-PA) have an extended lifetime in the circulation and are safer than free PAs. RBC-PAs incorporate into nascent thrombi, which are focally lysed from within, an attractive thromboprophylactic option. In static systems, RBC-PAs cleave surrounding fibrin fibers, forming pores larger than the cells themselves, and move around the pore edges, enlarging them until eventual clot dissolution. We hypothesized that under flow in blood vessels, RBC-PAs form functional patent channels before clot dissolution. Here we used perfusion chambers to study clot lysis by RBC-PAs under static versus arterial and venous flow conditions. We found that flow decelerates bulk clot lysis but quickly generates patent channels filled with passing RBCs, via pore enlargement and merging in the direction of flow. Formation of such channels by RBC-PAs may help rescue ischemic tissue before bulk dissolution of potentially occlusive clots.

Fibrin network structure and clot mechanical properties are altered by incorporation of erythrocytes.

Although many in vitro fibrin studies are performed with plasma, in vivo clots and thrombi contain erythrocytes, or red blood cells (RBCs). To determine the effects of RBCs on fibrin clot structure and mechanical properties, we compared plasma clots without RBCs to those prepared with low (2 vol%), intermediate (5-10 vol%), or high (> or =20 vol%) numbers of RBCs. By confocal microscopy, we found that low RBC concentrations had little effect on clot structure. Intermediate RBC concentrations caused heterogeneity in the fiber network with pockets of densely packed fibers alongside regions with few fibers. With high levels of RBCs, fibers arranged more uniformly but loosely around the cells. Scanning electron micrographs demonstrated an uneven distribution of RBCs throughout the clot and a significant increase in fiber diameter upon RBC incorporation. While permeability was not affected by RBC addition, at 20% or higher RBCs, the ratio of viscous modulus (G'') to elastic modulus (G') increased significantly over that of a clot without any RBCs. RBCs triggered variability in the fibrin network structure, individual fiber characteristics, and overall clot viscoelasticity compared to the absence of cells. These results are important for understanding in vivo clots and thrombi.

The presence of gamma' chain impairs fibrin polymerization.

INTRODUCTION: A fraction of fibrinogen molecules contain an alternatively spliced variant chain called gamma'. Plasma levels of this variant have been associated with both myocardial infarction and venous thrombosis. Because clot structure has been associated with cardiovascular risk, we examined the effect of gamma' chain on clot structure. MATERIALS AND METHODS: We expressed three fibrinogen variants in Chinese hamster ovary (CHO) cells: gamma/gamma homodimer, gamma/gamma' heterodimer, and gamma'/gamma' homodimer. We observed thrombin-catalyzed fibrinopeptide release by HPLC, fibrin polymerization by turbidity, and clot structure by scanning electron microscopy. We characterized post-translational modifications by mass spectrometry. RESULTS: Fibrinopeptide A was released at the same rate for all three fibrinogens, while fibrinopeptide B was released faster from the gamma'/gamma' homodimer. The rise in turbidity was slower and final absorbance was lower during polymerization of gamma'-containing fibrinogens than for gamma/gamma fibrinogen. Micrographs showed that gamma'/gamma' fibrin clots are composed of very thin fibers, while the diameter of gamma/gamma' fibers is similar to gamma/gamma fibers. Further, the fiber networks formed from gamma'-containing samples were non-uniform. Mass spectrometry showed heterogeneous addition of N-glycans and tyrosine sulfation in the gamma' chain. CONCLUSIONS: The presence of gamma' chains slows lateral aggregation and alters fibrin structure. We suggest these changes are likely due to charge-charge repulsion, such that polymerization of the gamma'/gamma' homodimer is more impaired than the heterodimer since these repulsions are partially offset by incorporation of gamma chains in the gamma/gamma' heterodimer.