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

Lytic and mechanical stability of clots composed of fibrin and blood vessel wall components.

Background Proteases expressed in atherosclerotic plaque lesions generate collagen fragments, release glycosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]) and expose extracellular matrix (ECM) proteins (e.g. decorin) at sites of fibrin formation. Objective Here we address the effect of these vessel wall components on the lysis of fibrin by the tissue plasminogen activator (tPA)/plasminogen system and on the mechanical stability of clots. Methods and results MMP-8-digested collagen fragments, isolated CS, DS, glycosylated decorin and its core protein were used to prepare mixed matrices with fibrin (additives present at a 50-fold lower mass concentration than fibrinogen). Scanning electron microscopy (SEM) showed that the presence of ECM components resulted in a coarse fibrin structure, most pronounced for glycosylated decorin causing an increase in the median fiber diameter from 85 to 187 nm. Rheological measurements indicated that these structural alterations were coupled to decreased shear resistance (1.8-fold lower shear stress needed for gel/fluid transition of the clots containing glycosylated decorin) and rigidity (reduction of the storage modulus from 54.3 to 33.2 Pa). The lytic susceptibility of the modified fibrin structures was increased. The time to 50% lysis by plasmin was reduced approximately 2-fold for all investigated ECM components (apart from the core protein of decorin which produced a moderate reduction of the lysis time by 25%), whereas fibrin-dependent plasminogen activation by tPA was inhibited by up to 30%. Conclusion ECM components compromise the chemical and mechanical stability of fibrin as a result of changes in its ultrastructure

Multi-scale strain-stiffening of semiflexible bundle networks

Bundles of polymer filaments are responsible for the rich and unique mechanical behaviors of many biomaterials, including cells and extracellular matrices. In fibrin biopolymers, whose nonlinear elastic properties are crucial for normal blood clotting, protofibrils self-assemble and bundle to form networks of semiflexible fibers. Here we show that the extraordinary strain-stiffening response of fibrin networks is a direct reflection of the hierarchical architecture of the fibrin fibers. We measure the rheology of networks of unbundled protofibrils and find excellent agreement with an affine model of extensible wormlike polymers. By direct comparison with these data, we show that physiological fibrin networks composed of thick fibers can be modeled as networks of tight protofibril bundles. We demonstrate that the tightness of coupling between protofibrils in the fibers can be tuned by the degree of enzymatic intermolecular crosslinking by the coagulation Factor XIII. Furthermore, at high stress, the protofibrils contribute independently to the network elasticity, which may reflect a decoupling of the tight bundle structure. The hierarchical architecture of fibrin fibers can thus account for the nonlinearity and enormous elastic resilience characteristic of blood clots.Comment: 27 pages including 8 figures and Supplementary Dat

Fibrin association at hybrid biointerfaces made of clot-binding peptides and polythiophene

The properties as biointerfaces of electroactive conducting polymer-peptide biocomposites formed by poly(3,4-ethylenedioxythiophene) (PEDOT) and CREKA or CR(NMe)EKA peptide sequences (where Glu has been replaced by N-methyl-Glu in the latter) have been compared. CREKA is a linear pentapeptide that recognizes clotted plasma proteins and selectively homes to tumors, while CR(NMe)EKA is an engineer to improve such properties by altering peptide-fibrin interactions. Differences between PEDOT-CREKA and PEDOT-CR(NMe)EKA reflect dissemblance in the organization of the peptides into the polymeric matrix. Both peptides affect fibrinogen thrombin-catalyzed polymerization causing the immediate formation of fibrin, whereas in the absence of thrombin this phenomenon is only observed for CR(NMe)EKA. Consistently, the fibrin-adsorption capacity is higher for PEDOT-CR(NMe)EKA than for PEDOT-CREKA, even though in both cases adsorbed fibrin exhibits round-like morphologies rather than the characteristic fibrous structure. PEDOT-peptide films coated with fibrin are selective in terms of cell adhesion, promoting the attachment of metastatic cells with respect to normal cells.Peer ReviewedPostprint (author's final draft

Fibrin D-dimer, markers of coagulation activation and the risk of major ischaemic heart disease in the Caerphilly Study

We have previously reported that plasma fibrin D-dimer (a marker of turnover of cross-linked Fibrin) showed a strong and independent association with incident ischaemic heart disease (IHD) in the Caerphilly Study cohort of 1,998 men a-ed 49-65. To establish the specificity of this finding, we assayed plasma samples from this cohort with a more specific assay for fibrin D-dimer: this showed an association with incident IHD which was at least as strong and independent as that for the original assay (odds ratio, OR for top fifth compared to bottom fifth 3.79; 95% CI 1.77-8.10; p lt 0.0001). To establish potential causes of the increased fibrin turnover. we also assayed several potential markers of coagulation activation or thrombotic tendency (prothrombin fragment F1+2, thrombin- antithrombin complexes, factor VIIc, activated partial thromboplastin time [APTT] and activated protein C resistance): none of these variables were associated with incident IHD in this cohort. We suggest that further studies are required to establish the causes of increased cross-linked fibrin turnover, which is associated with incident IHD in the general population when measured by a specific assay

Impaired endogenous fibrinolysis at high shear using a point-of-care test in STEMI is associated with alterations in clot architecture

© The Author(s) 2019Impaired endogenous fibrinolysis is an adverse prognostic biomarker in acute coronary syndrome (ACS). Abnormally dense in vitro fibrin thrombi have been demonstrated in ACS patients and related to hypofibrinolysis using cumbersome, laboratory-based methods. We aimed to assess endogenous fibrinolysis using a point-of-care technique and relate this to clot architecture. From patients with ST-segment elevation myocardial infarction (STEMI), venous blood was drawn immediately on arrival to assess thrombotic status. Blood was assessed using the point-of-care Global Thrombosis Test which measures occlusive thrombus formation under high shear and subsequently endogenous fibrinolysis (lysis time, LT). Two samples per patient were run in parallel. In one channel, the measurement was allowed to proceed as normal. In the other, after occlusion, thrombus was extracted, washed, fixed in glutaraldehyde, dried, sputter-coated, and assessed using scanning electron microscope. Endogenous fibrinolysis was strongly associated fibrin fibre thickness (p = 0.0001). As LT increased (less efficient fibrinolysis), the fibrin network of the thrombus was significantly more compact and dense, with thinner fibrin fibres and smaller gaps. Fibrin fibre thickness correlated inversely with LT (r = - 0.89, p = 0.001). Adverse clot architecture in vitro is directly related to impaired endogenous fibrinolysis using a relatively new point-of-care technique in patients with STEMI. This may transform the relevance of fibrin clot architecture from an off-line laboratory association to being directly relevant to endogenous fibrinolysis at the patient bedside, which could be used as a near-patient test to guide prognosis and assess the effect of treatment.Peer reviewedFinal Published versio

Biomimetic implant surface functionalization with liquid L-PRF products: in vitro study

Abstract Objective: Platelet-rich fibrin (PRF) clots and membranes are autologous blood concentrates widely used in oral surgical procedures; less is known, however, about the liquid formulations of such products. The aim of this in vitro study is to assess the behavior of different implant surfaces when in contact with two liquid leucocyte- and platelet-rich fibrin (L-PRF) products. Methods: Six commercial pure titanium discs, of 9.5 mm diameter and 1.5 mm thickness, were used. Three of these samples had a micro/nano-rough surface; three were machined. Three different protocols were tested. Protocols involved the immersion of the samples in (1) a platelets, lymphocytes, and fibrinogen liquid concentrate (PLyF) for 10 minutes, (2) an exudate obtained from L-PRF clots rich in fibronectin and vitronectin for 5 minutes, and (3) the fibronectin/vitronectin exudate for 2 minutes followed by immersion in the PLyF concentrate for further 8 minutes. After these treatments, the samples were fixed and observed using a scanning electron microscope (SEM). Results: Under microscopic observation, (1) the samples treated with the PLyF concentrate revealed a dense fibrin network in direct contact with the implant surface and a significant number of formed elements of blood; (2) in the samples treated with the fibronectin/vitronectin exudates, only a small number of white and red blood cells were detectable; and (3) in samples exposed to the combined treatment, there was an apparent increase in the thickness of the fibrin layer. When compared to the machined surface, the micro/nano-rough samples showed an overall increased retention of fibrin, leading to a thicker coating. Conclusions: Liquid L-PRF products promote the formation of a dense fibrin clot on micro/nano-rough implant surfaces in vitro. The adjunctive treatment of surfaces with the fibronectin/vitronectin exudate could provide support to contact of the fibrin with the surface, though it is not essential for the clot formation. Further studies are necessary to better elucidate the properties and benefits of liquid L-PRF products

Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation

The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased — and, under conditions of decreased platelet adhesion, PDI inhibition reduced — fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet–secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury

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