Quantitative structural mechanobiology of platelet-driven blood clot contraction.

Blood clot contraction plays an important role in prevention of bleeding and in thrombotic disorders. Here, we unveil and quantify the structural mechanisms of clot contraction at the level of single platelets. A key elementary step of contraction is sequential extension-retraction of platelet filopodia attached to fibrin fibers. In contrast to other cell-matrix systems in which cells migrate along fibers, the "hand-over-hand" longitudinal pulling causes shortening and bending of platelet-attached fibers, resulting in formation of fiber kinks. When attached to multiple fibers, platelets densify the fibrin network by pulling on fibers transversely to their longitudinal axes. Single platelets and aggregates use actomyosin contractile machinery and integrin-mediated adhesion to remodel the extracellular matrix, inducing compaction of fibrin into bundled agglomerates tightly associated with activated platelets. The revealed platelet-driven mechanisms of blood clot contraction demonstrate an important new biological application of cell motility principles

Foam-like compression behavior of fibrin networks

The rheological properties of fibrin networks have been of long-standing interest. As such there is a wealth of studies of their shear and tensile responses, but their compressive behavior remains unexplored. Here, by characterization of the network structure with synchronous measurement of the fibrin storage and loss moduli at increasing degrees of compression, we show that the compressive behavior of fibrin networks is similar to that of cellular solids. A non-linear stress-strain response of fibrin consists of three regimes: 1) an initial linear regime, in which most fibers are straight, 2) a plateau regime, in which more and more fibers buckle and collapse, and 3) a markedly non-linear regime, in which network densification occurs {{by bending of buckled fibers}} and inter-fiber contacts. Importantly, the spatially non-uniform network deformation included formation of a moving "compression front" along the axis of strain, which segregated the fibrin network into compartments with different fiber densities and structure. The Young's modulus of the linear phase depends quadratically on the fibrin volume fraction while that in the densified phase depends cubically on it. The viscoelastic plateau regime corresponds to a mixture of these two phases in which the fractions of the two phases change during compression. We model this regime using a continuum theory of phase transitions and analytically predict the storage and loss moduli which are in good agreement with the experimental data. Our work shows that fibrin networks are a member of a broad class of natural cellular materials which includes cancellous bone, wood and cork

Model predictions of deformation, embolization and permeability of partially obstructive blood clots under variable shear flow

Thromboembolism, one of the leading causes of morbidity and mortality worldwide, is characterized by formation of obstructive intravascular clots (thrombi) and their mechanical breakage (embolization). A novel two-dimensional multi-phase computational model is introduced that describes active interactions between the main components of the clot, including platelets and fibrin, to study the impact of various physiologically relevant blood shear flow conditions on deformation and embolization of a partially obstructive clot with variable permeability. Simulations provide new insights into mechanisms underlying clot stability and embolization that cannot be studied experimentally at this time. In particular, model simulations, calibrated using experimental intravital imaging of an established arteriolar clot, show that flow-induced changes in size, shape and internal structure of the clot are largely determined by two shear-dependent mechanisms: reversible attachment of platelets to the exterior of the clot and removal of large clot pieces. Model simulations predict that blood clots with higher permeability are more prone to embolization with enhanced disintegration under increasing shear rate. In contrast, less permeable clots are more resistant to rupture due to shear rate-dependent clot stiffening originating from enhanced platelet adhesion and aggregation. These results can be used in future to predict risk of thromboembolism based on the data about composition, permeability and deformability of a clot under specific local haemodynamic conditions

Mechanism of Fibrin(ogen) Forced Unfolding

SummaryFibrinogen, upon enzymatic conversion to monomeric fibrin, provides the building blocks for fibrin polymer, the scaffold of blood clots and thrombi. Little has been known about the force-induced unfolding of fibrin(ogen), even though it is the foundation for the mechanical and rheological properties of fibrin, which are essential for hemostasis. We determined mechanisms and mapped the free energy landscape of the elongation of fibrin(ogen) monomers and oligomers through combined experimental and theoretical studies of the nanomechanical properties of fibrin(ogen), using atomic force microscopy-based single-molecule unfolding and simulations in the experimentally relevant timescale. We have found that mechanical unraveling of fibrin(ogen) is determined by the combined molecular transitions that couple stepwise unfolding of the γ chain nodules and reversible extension-contraction of the α-helical coiled-coil connectors. These findings provide important characteristics of the fibrin(ogen) nanomechanics necessary to understand the molecular origins of fibrin viscoelasticity at the fiber and whole clot levels

The presence of γ′ chain impairs fibrin polymerization

A fraction of fibrinogen molecules contain an alternatively spliced variant chain called γ’. 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 γ’ chain on clot structure

Abnormal clot microstructure formed in blood containing HIT-like antibodies

IntroductionThrombosis is a severe and frequent complication of heparin-induced thrombocytopenia (HIT). However, there is currently no knowledge of the effects of HIT-like antibodies on the resulting microstructure of the formed clot, despite such information being linked to thrombotic events. We evaluate the effect of the addition of pathogenic HIT-like antibodies to blood on the resulting microstructure of the formed clot.Materials and methodsPathogenic HIT-like antibodies (KKO) and control antibodies (RTO) were added to samples of whole blood containing Unfractionated Heparin and Platelet Factor 4. The formed clot microstructure was investigated by rheological measurements (fractal dimension; df) and scanning electron microscopy (SEM), and platelet activation was measured by flow cytometry.Results and conclusionsOur results revealed striking effects of KKO on clot microstructure. A significant difference in df was found between samples containing KKO (df = 1.80) versus RTO (df = 1.74; p < 0.0001). This increase in df was often associated with an increase in activated platelets. SEM images of the clots formed with KKO showed a network consisting of a highly branched and compact arrangement of thin fibrin fibres, typically found in thrombotic disease. This is the first study to identify significant changes in clot microstructure formed in blood containing HIT-like antibodies. These observed alterations in clot microstructure can be potentially exploited as a much-needed biomarker for the detection, management and monitoring of HIT-associated thrombosis