Hysteresis-like binding of coagulation factors X/Xa to procoagulant activated platelets and phospholipids results from multistep association and membrane-dependent multimerization.

Binding of coagulation factors X (fX) and Xa (fXa) to activated platelets is required for the formation of membrane-dependent enzymatic complexes of intrinsic tenase and prothrombinase. We carried out an in-depth characterization of fX/fXa binding to phospholipids and gel-filtered, thrombin-activated platelets. Flow cytometry, surface plasmon resonance, and computational modeling were used to investigate interactions of fX/fXa with the membranes. Confocal microscopy was employed to study fXa binding to platelet thrombi formed in flowing whole blood under arterial conditions. Binding of fX/fXa to either vesicles or procoagulant platelets did not follow a traditional one-step reversible binding model. Their dissociation was a two-step process resulting in a plateau that was up to 10-fold greater than the saturation value observed in the association experiments. Computational modeling and experimental evidence suggested that this was caused by a combination of two-step association (mainly for fX) and multimerization on the membrane (mainly for fXa). Importantly, fX formed multimers with fXa, thereby improving its retention. The same binding/dissociation hysteresis was observed for annexin V known to form trimers on the membranes. Experiments with platelets from gray syndrome patients showed that alpha-granular factor Va provided an additional high-affinity binding site for fXa that did not affect the hysteresis. Confocal microscopy observation of fXa binding to platelet thrombi in a flow chamber and its wash-out confirmed that this phenomenon persisted under physiologically relevant conditions. This suggests its possible role of "locking" coagulation factors on the membrane and preventing their inhibition in plasma and removal from thrombi by flow.

Tubulin bond energies and microtubule biomechanics determined from nanoindentation in silico.

Microtubules, the primary components of the chromosome segregation machinery, are stabilized by longitudinal and lateral noncovalent bonds between the tubulin subunits. However, the thermodynamics of these bonds and the microtubule physicochemical properties are poorly understood. Here, we explore the biomechanics of microtubule polymers using multiscale computational modeling and nanoindentations in silico of a contiguous microtubule fragment. A close match between the simulated and experimental force-deformation spectra enabled us to correlate the microtubule biomechanics with dynamic structural transitions at the nanoscale. Our mechanical testing revealed that the compressed MT behaves as a system of rigid elements interconnected through a network of lateral and longitudinal elastic bonds. The initial regime of continuous elastic deformation of the microtubule is followed by the transition regime, during which the microtubule lattice undergoes discrete structural changes, which include first the reversible dissociation of lateral bonds followed by irreversible dissociation of the longitudinal bonds. We have determined the free energies of dissociation of the lateral (6.9 ± 0.4 kcal/mol) and longitudinal (14.9 ± 1.5 kcal/mol) tubulin-tubulin bonds. These values in conjunction with the large flexural rigidity of tubulin protofilaments obtained (18,000-26,000 pN·nm(2)) support the idea that the disassembling microtubule is capable of generating a large mechanical force to move chromosomes during cell division. Our computational modeling offers a comprehensive quantitative platform to link molecular tubulin characteristics with the physiological behavior of microtubules. The developed in silico nanoindentation method provides a powerful tool for the exploration of biomechanical properties of other cytoskeletal and multiprotein assemblies.

H2O2-Responsive Polymeric Micelles of Biodegradable Aliphatic Poly(carbonate)s as Promising Therapeutic Agents for Inflammatory Diseases.

Excessive amounts of reactive oxygen species (ROS) cause various biological damages and are involved in many diseases, such as cancer, inflammatory and thrombotic complications, and neurodegenerative diseases. Thus, ROS-responsive polymers with inherent ROS scavenging activity and biodegradability are extremely needed for the efficient treatment of ROS-related diseases. Here, this work fabricates the amphiphilic diblock copolymer PEG-b-PBC via ring-opening polymerization (ROP) of phenylboronic acid ester conjugated cyclic carbonate monomer. The copolymer easily forms micelles (BCM) and scavenges ROS rapidly. BCM not only releases the delivered drug but degrades to produce the small molecules p-hydroxybenzyl alcohol (HBA) with anti-inflammatory capability in the presence of H2O2. BCM can reduce the oxidative stress of human umbilical vein endothelial cells (HUVEC) and the levels of inflammatory factors secreted by macrophages, showing antioxidative and anti-inflammatory activity. Finally, BCM exerts a significant capability to reduce the complications of inflammation and thrombosis in vivo. The biodegradable aliphatic poly(carbonate)s have the potential to be used for drug delivery systems (DDS) for diseases induced by reactive oxygen species.

CLASP2 recognizes tubulins exposed at the microtubule plus-end in a nucleotide state-sensitive manner.

CLASPs (cytoplasmic linker-associated proteins) are ubiquitous stabilizers of microtubule dynamics, but their molecular targets at the microtubule plus-end are not understood. Using DNA origami-based reconstructions, we show that clusters of human CLASP2 form a load-bearing bond with terminal non-GTP tubulins at the stabilized microtubule tip. This activity relies on the unconventional TOG2 domain of CLASP2, which releases its high-affinity bond with non-GTP dimers upon their conversion into polymerization-competent GTP-tubulins. The ability of CLASP2 to recognize nucleotide-specific tubulin conformation and stabilize the catastrophe-promoting non-GTP tubulins intertwines with the previously underappreciated exchange between GDP and GTP at terminal tubulins. We propose that TOG2-dependent stabilization of sporadically occurring non-GTP tubulins represents a distinct molecular mechanism to suppress catastrophe at the freely assembling microtubule ends and to promote persistent tubulin assembly at the load-bearing tethered ends, such as at the kinetochores in dividing cells.

Force production by disassembling microtubules.

Microtubules (MTs) are important components of the eukaryotic cytoskeleton: they contribute to cell shape and movement, as well as to the motions of organelles including mitotic chromosomes. MTs bind motor enzymes that drive many such movements, but MT dynamics can also contribute to organelle motility. Each MT polymer is a store of chemical energy that can be used to do mechanical work, but how this energy is converted to motility remains unknown. Here we show, by conjugating glass microbeads to tubulin polymers through strong inert linkages, such as biotin-avidin, that depolymerizing MTs exert a brief tug on the beads, as measured with laser tweezers. Analysis of these interactions with a molecular-mechanical model of MT structure and force production shows that a single depolymerizing MT can generate about ten times the force that is developed by a motor enzyme; thus, this mechanism might be the primary driving force for chromosome motion. Because even the simple coupler used here slows MT disassembly, physiological couplers may modulate MT dynamics in vivo.

Hemophilia A and B are associated with abnormal spatial dynamics of clot growth.

To gain greater insight into the nature of the bleeding tendency in hemophilia, we compared the spatial dynamics of clotting in platelet-free plasma from healthy donors and from patients with severe hemophilia A or B (factor VIII:C or IX:C<1%). Clotting was initiated via the intrinsic or extrinsic pathway in a thin layer of nonstirred plasma by bringing it in contact with the glass or fibroblast monolayer surface. The results suggest that clot growth is a process consisting of two distinct phases, initiation and elongation. The clotting events on the activator surface and the preceding period free of visible signs of clotting are the initiation phase. In experiments with and without stirring alike, this phase is prolonged in hemophilic plasma activated by the intrinsic, but not the extrinsic pathway. Strikingly, both hemophilia A and B are associated with a significant deterioration in the elongation phase (clot thickening), irrespective of the activation pathway. The rate of clot growth in hemophilic plasma is significantly lower than normal and declines quickly. The resulting clots are thin, which may account for the bleeding disorder.