Fibrin is the main component of blood clots. The mechanical properties of fibrin are therefore of critical importance in successful hemostasis. One of the divalent cations released by platelets during hemostasis is Zn2+; however, its effect on the network structure of fibrin gels and on the resultant mechanical properties remains poorly understood. Here, by combining mechanical measurements with three-dimensional confocal microscopy imaging, we show that Zn2+can tune the fibrin network structure and alter its mechanical properties. In the presence of Zn2+, fibrin protofibrils form large bundles that cause a coarsening of the fibrin network due to an increase in fiber diameter and reduction of the total fiber length. We further show that the protofibrils in these bundles are loosely coupled to one another, which results in a decrease of the elastic modulus with increasing Zn2+concentrations. We explore the elastic properties of these networks at both low and high stress: At low stress, the elasticity originates from pulling the thermal slack out of the network, and this is consistent with the thermal bending of the fibers. By contrast, at high stress, the elasticity exhibits a common master curve consistent with the stretching of individual protofibrils. These results show that the mechanics of a fibrin network are closely correlated with its microscopic structure and inform our understanding of the structure and physical mechanisms leading to defective or excessive clot stiffness.
The adaptive mechanical properties of soft and fibrous biological materials are relevant to their functionality. The emergence of the macroscopic response of these materials to external stress and intrinsic cell traction from local deformations of their structural components is not well understood. Here, we investigate the nonlinear elastic behavior of blood clots by combining microscopy, rheology, and an elastic network model that incorporates the stretching, bending, and buckling of constituent fibrin fibers. By inhibiting fibrin cross-linking in blood clots, we observe an anomalous softening regime in the macroscopic shear response as well as a reduction in platelet-induced clot contractility. Our model explains these observations from two independent macroscopic measurements in a unified manner, through a single mechanical parameter, the bending stiffness of individual fibers. Supported by experimental evidence, our mechanics-based model provides a framework for predicting and comprehending the nonlinear elastic behavior of blood clots and other active biopolymer networks in general.
more » « less- PAR ID:
- 10527269
- Publisher / Repository:
- American Association for the Advancement of Science
- Date Published:
- Journal Name:
- Science Advances
- Volume:
- 10
- Issue:
- 2
- ISSN:
- 2375-2548
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
-
more » « less
Abstract The maintenance of hemostasis to ensure vascular integrity is dependent upon the rapid conversion of zymogen species of the coagulation cascade to their enzymatically active forms. This process culminates in the generation of the serine protease thrombin and polymerization of fibrin to prevent vascular leak at sites of endothelial cell injury or loss of cellular junctions. Thrombin generation can be initiated by the extrinsic pathway of coagulation through exposure of blood to tissue factor at sites of vascular damage, or alternatively by the coagulation factor (F) XII activated by foreign surfaces with negative charges, such as glass, through the contact activation pathway. Here, we used transient particle tracking microrheology to investigate the mechanical properties of fibrin in response to thrombin generation downstream of both coagulation pathways. We found that the structural heterogeneity of fibrin formation was dependent on the reaction kinetics of thrombin generation. Pharmacological inhibition of FXII activity prolonged the time to form fibrin and increased the degree of heterogeneity of fibrin, resulting in fibrin clots with reduced mechanical properties. Taken together, this study demonstrates a dependency of the physical biology of fibrin formation on activation of the contact pathway of coagulation.
-
Fibrin is the fibrous protein network that comprises blood clots; it is uniquely capable of bearing very large tensile strains (up to 200%) due to multiscale force accommodation mechanisms. Fibrin is also a biochemical scaffold for numerous enzymes and blood factors. The biomechanics and biochemistry of fibrin have been independently studied. However, comparatively little is known about how fibrin biomechanics and biochemistry are coupled: how does fibrin deformation influence its biochemistry? In this study, we show that mechanically induced protein structural changes in fibrin affect fibrin biochemistry. We find that tensile deformation of fibrin leads to molecular structural transitions of α-helices to β-sheets, which reduced binding of tissue plasminogen activator (tPA), an enzyme that initiates fibrin lysis. Moreover, binding of tPA and Thioflavin T, a commonly used β-sheet marker, were mutually exclusive, further demonstrating the mechano-chemical control of fibrin biochemistry. Finally, we demonstrate that structural changes in fibrin suppressed the biological activity of platelets on mechanically strained fibrin due to reduced α IIb β 3 integrin binding. Our work shows that mechanical strain regulates fibrin molecular structure and biological activity in an elegant mechano-chemical feedback loop, which possibly extends to other fibrous biopolymers.more » « less
-
more » « less
Abstract Studying and quantifying the mechanics of blood clots is essential to better diagnosis and prognosis of, as well as therapy for, thromboembolic pathologies such as strokes, heart attacks, and pulmonary embolisms. Unfortunately, mechanically testing blood clots is complicated by their softness and fragility, thus making the use of classic mounting techniques, such as clamping, challenging. This is particularly true for mechanical testing under large deformation. Here, we describe protocols for creating in vitro blood clots and securely mounting these samples on mechanical test equipment. To this end, we line 3D‐printed molds with a hook‐and‐loop fabric that, after coagulation, provides a secure interface between the sample and device mount. In summary, our molding and mounting protocols are ideal for performing large‐deformation mechanical testing, with samples that can withstand substantial deformation without delaminating from the apparatus. © 2021 Wiley Periodicals LLC.
Basic Protocol 1 : Cube‐shaped blood clot preparationBasic Protocol 2 : Sheet‐shaped blood clot preparation -
Thromboembolism – that is, clot formation and the subsequent fragmentation of clot – is a leading cause of death worldwide. Clots’ mechanical properties are critical determinants of both the embolization process and the pathophysiological consequences thereof. Thus, understanding and quantifying the mechanical properties of clots is important to our ability to treat and prevent thromboembolic disease. However, assessing these properties from in vivo clots is experimentally challenging. Therefore, we and others have turned to studying in vitro clot mimics instead. Unfortunately, there are significant discrepancies in the reported properties of these clot mimics, which have been hypothesized to arise from differences in experimental techniques and blood sources. The goal of our current work is therefore to compare the mechanical behavior of clots made from the two most common sources, human and bovine blood, using the same experimental techniques. To this end, we tested clots under pure shear with and without initial cracks, under cyclic loading, and under stress relaxation. Based on these data, we computed and compared stiffness, strength, work-to-rupture, fracture toughness, relaxation time constants, and prestrain. While clots from both sources behaved qualitatively similarly, they differed quantitatively in almost every metric. We also correlated each mechanical metric to measures of blood composition. Thereby, we traced this inter-species variability in clot mechanics back to significant differences in hematocrit, but not platelet count. Thus, our work suggests that the results of past studies that have used bovine blood to make in vitro mimics – without adjusting blood composition – should be interpreted carefully. Future studies about the mechanical properties of blood clots should focus on human blood alone.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1126/sciadv.adh1265
