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Embolization is a leading cause of mortality, yet we know little about clot rupture mechanics. Fibrin provides the main structural and mechanical stability to blood clots. Previous studies have shown that altering the concentration of coagulation activators (thrombin or tissue factor (TF)) has a significant impact on fibrin structure and viscoelastic properties, but their effects on rupture properties are mostly unknown. Toughness, which corresponds to the ability to resist rupture, is independent of viscoelastic properties. We used varying TF concentrations to alter the structure and toughness of human plasma clots. We performed single-edge notch rupture tests to examine fibrin toughness under a constant strain rate and we assessed viscoelastic mechanics using rheology. We utilized fluorescent confocal and scanning electron microscopy (SEM) to quantify the fibrin network structure under varying TF concentrations. Our results revealed that increased TF concentration resulted in increased number of fibrin fibers with a reduction in network pore size, thinner and shorter fibrin fibers. Increasing TF concentration yielded a maximum toughness at mid-TF concentration, such that fibrin diameter and number of fibers underlie a complex role in influencing the rupture resistance of blood clots, resulting in a nonmonotonic relationship between TF and toughness. A simple mechanical model, built on our findings from our Fluctuating Spring (FS) computational model, adopted to estimate the fracture toughness (critical energy release rate) as a function of TF predicts trends that are in good agreement with experiments. The differences in mechanical responses point to the importance of studying the structure-function relationships of fibrin networks, which may be predictive of the tendency for embolization. STATEMENT OF SIGNIFICANCE: Fibrin, a naturally occurring biomaterial, is the main mechanical and structural scaffold of blood clots that provides the necessary strength and stability to the clot, ensuring effective stemming of bleeding. The rupture of blood clots can result in the blockage of downstream vessels thereby blocking blood flow and oxygen supply. The fibrin network structure has been shown to influence the viscoelastic mechanical properties of clots, but has not been explored for fracture mechanics. Here, we modulate the fibrin network structure by varying the concentration of Tissue Factor (TF). Interestingly, the association between TF concentration and maximum toughness of the clots is non-monotonic. The variations in mechanical responses highlight the importance of studying the structure-function relationships of fibrin networks, as these may predict the tendency for embolization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Polymeric fibrin provides the structural and mechanical stability of a blood clot. Fibrin fibers are rod-like and create a network mesh that holds blood cells. When a clot has performed its physiological function in wound healing and preventing excessive blood loss, it must be resolved by the enzymatic degradation of fibrin, otherwise known as fibrinolysis. If a blood clot forms when or where it is not needed, as occurs in ischemic strokes and myocardial infarctions, the blood clot (thrombus) can obstruct blood flow to downstream organs. Obstructive thrombi must be degraded or removed to prevent further complications. If a clot is not degraded on its own, lytic agents (i.e., tissue plasminogen activator, tPA) are given exogenously to induce fibrinolysis. Here, we fluorescently labeled both fibrin and tPA to visualize degradation at the edge of the clot. The fibers with bound tPA were looped or coiled while the fibers farther into the clot remain straight and stable displaying the diffusion of tPA and depth of lysis. This image provides (1) a new method to monitor fibrinolysis with a commercially available chamber with convenient inlets and (2) the visualization of tPA-bound fibrin and the behavior of fibers during degradation. Future work could utilize this technique to study tPA molecule and fibrin interactions, lysis front degradation, and fibrin fiber linearity to understand the mechanisms of intermolecular dynamics dependent on network structure. An enhanced insight into this process can aid in the development of optimized therapeutics to target stubborn clots., Competing Interests: The authors have no conflicts to disclose., (© 2024 Author(s).)

Intravascular blood clots are subject to hydrodynamic shear and other forces that cause clot deformation and rupture (embolization). A portion of the ruptured clot can block blood flow in downstream vessels. The mechanical stability of blood clots is determined primarily by the 3D polymeric fibrin network that forms a gel. Previous studies have primarily focused on the rupture of blood plasma clots under tensile loading (Mode I), our current study investigates the rupture of fibrin induced by shear loading (Mode II), dominating under physiological conditions induced by blood flow. Using experimental and theoretical approaches, we show that fracture toughness, i.e. the critical energy release rate, is relatively independent of the type of loading and is therefore a fundamental property of the gel. Ultrastructural studies and finite element simulations demonstrate that cracks propagate perpendicular to the direction of maximum stretch at the crack tip. These observations indicate that locally, the mechanism of rupture is predominantly tensile. Knowledge gained from this study will aid in the development of methods for prediction/prevention of thrombotic embolization.

We modify a three-dimensional multiscale model of fibrinolysis to study the effect of plasmin-mediated degradation of fibrin on tissue plasminogen activator (tPA) diffusion and fibrinolysis. We propose that tPA is released from a fibrin fiber by simple kinetic unbinding, as well as by "forced unbinding," which occurs when plasmin degrades fibrin to which tPA is bound. We show that, if tPA is bound to a small-enough piece of fibrin that it can diffuse into the clot, then plasmin can increase the effective diffusion of tPA. If tPA is bound to larger fibrin degradation products (FDPs) that can only diffuse along the clot, then plasmin can decrease the effective diffusion of tPA. We find that lysis rates are fastest when tPA is bound to fibrin that can diffuse into the clot, and slowest when tPA is bound to FDPs that can only diffuse along the clot. Laboratory experiments confirm that FDPs can diffuse into a clot, and they support the model hypothesis that forced unbinding of tPA results in a mix of FDPs, such that tPA bound to FDPs can diffuse both into and along the clot. Regardless of how tPA is released from a fiber, a tPA mutant with a smaller dissociation constant results in slower lysis (because tPA binds strongly to fibrin), and a tPA mutant with a larger dissociation constant results in faster lysis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Altered properties of fibrin clots have been associated with bleeding and thrombotic disorders, including hemophilia or trauma and heart attack or stroke. Clotting factors, such as thrombin and tissue factor, or blood plasma proteins, such as fibrinogen, play critical roles in fibrin network polymerization. The concentrations and combinations of these proteins affect the structure and stability of clots, which can lead to downstream complications. The present work includes clots made from plasma and purified fibrinogen and shows how varying fibrinogen and activation factor concentrations affect the fibrin properties under both conditions. We used a combination of scanning electron microscopy, confocal microscopy, and turbidimetry to analyze clot/fiber structure and polymerization. We quantified the structural and polymerization features and found similar trends with increasing/decreasing fibrinogen and thrombin concentrations for both purified fibrinogen and plasma clots. Using our compiled results, we were able to generate multiple linear regressions that predict structural and polymerization features using various fibrinogen and clotting agent concentrations. This study provides an analysis of structural and polymerization features of clots made with purified fibrinogen or plasma at various fibrinogen and clotting agent concentrations. Our results could be utilized to aid in interpreting results, designing future experiments, or developing relevant mathematical models.

Blood clots, which are composed of blood cells and a stabilizing mesh of fibrin fibers, are critical in cessation of bleeding following injury. However, their action is transient and after performing their physiological function they must be resolved through a process known as fibrinolysis. Internal fibrinolysis is the degradation of fibrin by the endogenous or innate presence of lytic enzymes in the bloodstream; under healthy conditions, this process regulates hemostasis and prevents bleeding or clotting. Fibrin-bound tissue plasminogen activator (tPA) converts nearby plasminogen into active plasmin, which is bound to the fibrin network, breaking it down into fibrin degradation products and releasing the entrapped blood cells. It is poorly understood how changes in the fibrin structure and lytic protein ratios influence the biochemical regulation and behavior of internal fibrinolysis. We used turbidity kinetic tracking and microscopy paired with mathematical modeling to study fibrin structure and lytic protein ratios that restrict internal fibrinolysis. Analysis of simulations and experiments indicate that fibrinolysis is driven by pore expansion of the fibrin network. We show that this effect is strongly influenced by the ratio of fibrin:tPAwhen compared to absolute tPA concentration. Thus, it is essential to consider relative protein concentrations when studying internal fibrinolysis both experimentally and in the clinic. An improved understanding of effective internal lysis can aid in development of better therapeutics for the treatment of bleeding and thrombosis., (© 2024. The Author(s).)

Diabetic retinopathy affects more than 100 million people worldwide and is projected to increase by 50% within 20 years. Increased blood glucose leads to the formation of advanced glycation end products (AGEs), which cause cellular and molecular dysfunction across neurovascular systems. These molecules initiate the slow breakdown of the retinal vasculature and the inner blood retinal barrier (iBRB), resulting in ischemia and abnormal angiogenesis. This project examined the impact of AGEs in altering the morphology of healthy cells that comprise the iBRB, as well as the effects of AGEs on thrombi formation, in vitro. Our results illustrate that AGEs significantly alter cellular areas and increase the formation of blood clots via elevated levels of tissue factor. Likewise, AGEs upregulate the expression of cell receptors (RAGE) on both endothelial and glial cells, a hallmark biomarker of inflammation in diabetic cells. Examining the effects of AGEs stimulation on cellular functions that work to diminish iBRB integrity will greatly help to advance therapies that target vision loss in adults.

Fibrin provides the main structural integrity and mechanical strength to blood clots. Failure of fibrin clots can result in life-threating complications, such as stroke or pulmonary embolism. The dependence of rupture resistance of fibrin networks (uncracked and cracked) on fibrin(ogen) concentrations in the (patho)physiological 1-5 g L -1 range is explored by performing the ultrastructural studies and theoretical analysis of the experimental stress-strain profiles available from mechanical tensile loading assays. Fibrin fibers in the uncracked network stretched evenly, whereas, in the cracked network, fibers around the crack tip showed greater deformation. Unlike fibrin fibers in cracked networks formed at the lower 1-2.7 g L -1 fibrinogen concentrations, fibers formed at the higher 2.7-5 g L -1 concentrations align and stretch simultaneously. Cracked fibrin networks formed in higher fibrinogen solutions are tougher yet less extensible. Statistical modeling revealed that the characteristic strain for fiber alignment, crack size, and fracture toughness of fibrin networks control their rupture resistance. The results obtained provide a structural and biomechanical basis to quantitatively understand the material properties of blood plasma clots and to illuminate the mechanisms of their rupture., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

The mechanical stability of blood clots necessary for their functions is provided by fibrin, a fibrous gel. Rupture of clots leads to life-threatening thrombotic embolization, which is little understood. Here, we combine experiments and simulations to determine the toughness of plasma clots as a function of fibrin content and correlate toughness with fibrin network structure characterized by confocal and scanning electron microscopy. We develop fibrin constitutive laws that scale with fibrin concentration and capture the force-stretch response of cracked clot specimens using only a few material parameters. Toughness is calculated from the path-independent J * integral that includes dissipative effects due to fluid flow and uses only the constitutive model and overall stretch at crack propagation as input. We show that internal fluid motion, which is not directly measurable, contributes significantly to clot toughness, with its effect increasing as fibrin content increases, because the reduced gel porosity at higher density results in greater expense of energy in fluid motion. Increasing fibrin content (1→10mg/mL) results in a significant increase in clot toughness (3→15 N/m) in accordance with a power law relation reminiscent of cellular solids and elastomeric gels. These results provide a basis for understanding and predicting the tendency for thrombotic embolization. STATEMENT OF SIGNIFICANCE: Fibrin, a naturally occurring biomaterial, is the major determinant of the structural and mechanical integrity of blood clots. We determined that increasing the fibrin content in clots, as in some thrombi and fibrin-based anti-bleeding sealants, results in an increase in clot toughness. Toughness corresponds to the ability to resist rupturing in the presence of a defect. We couple bulk mechanical testing, microstructural measurements, and finite element modeling to capture the force-stretch response of fibrin clots and compute toughness. We show that increased fibrin content in clots reduces porosity and limits fluid motion and that fluid motion drastically alters the clot toughness. These results provide a fundamental understanding of blood clot rupture and could help in rational design of fibrin-containing biomaterials., Competing Interests: Declaration of Competing Interest The authors have no competing interests to disclose, (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In response to vessel injury (or other pathological conditions), the hemostatic process is activated, resulting in a fibrous, cellular-rich structure commonly referred to as a blood clot. Succeeding the clot's function in wound healing, it must be resolved. This illustrated review focuses on fibrinolysis-the degradation of blood clots or thrombi. Fibrin is the main mechanical and structural component of a blood clot, which encases the cellular components of the clot, including platelets and red blood cells. Fibrinolysis is the proteolytic degradation of the fibrin network that results in the release of the cellular components into the bloodstream. In the case of thrombosis, fibrinolysis is required for restoration of blood flow, which is accomplished clinically through exogenously delivered lytic factors in a process called external lysis. Fibrinolysis is regulated by plasminogen activators (tissue-type and urokinase-type) that convert plasminogen into plasmin to initiate fiber lysis and lytic inhibitors that impede this lysis (plasminogen activator inhibitors, alpha 2-antiplasmin, and thrombin activatable fibrinolysis inhibitor). Furthermore, the network structure has been shown to regulate lysis: thinner fibers and coarser clots lyse faster than thicker fibers and finer clots. Clot contraction, a result of platelets pulling on fibers, results in densely packed red blood cells (polyhedrocytes), reduced permeability to fibrinolytic factors, and increased fiber tension. Extensive research in the field has allowed for critical advancements leading to improved thrombolytic agents. In this review, we summarize the state of the field, highlight gaps in knowledge, and propose future research questions., (© 2023 The Author(s).)

Introduction: Trauma induced coagulopathy (TIC) is common after severe trauma, increasing transfusion requirements and mortality among patients. TIC has several phenotypes, with primary hyperfibrinolysis being among the most lethal. We aimed to investigate the contribution of hypercoagulation, hemodilution, and fibrinolytic activation to the hyperfibrinolytic phenotype of TIC, by examining fibrin formation in a plasma-based model of TIC. We hypothesized that instabilities arising from TIC will be due primarily to increased fibrinolytic activation rather than hemodilution or tissue factor (TF) induced hypercoagulation., Methods: The influence of TF, hemodilution, fibrinogen consumption, tissue plasminogen activator (tPA), and the antifibrinolytic tranexamic acid (TXA) on plasma clot formation and structure were examined using rheometry, optical properties, and confocal microscopy. These were then compared to plasma samples from trauma patients at risk of developing TIC., Results: Combining TF-induced clot formation, 15 % hemodilution, fibrinogen consumption, and tPA-induced fibrinolysis, the clot characteristics and hyperfibrinolysis were consistent with primary hyperfibrinolysis. TF primarily increased fibrin polymerization rates and reduced fiber length. Hemodilution decreased clot optical density but had no significant effect on mechanical clot stiffness. TPA addition induced primary clot lysis as observed mechanically and optically. TXA restored mechanical clot formation but did not restore clot structure to control levels. Patients at risk of TIC showed increased clot formation, and lysis like that of our simulated model., Conclusions: This simulated TIC plasma model demonstrated that fibrinolytic activation is a primary driver of instability during TIC and that clot mechanics can be restored, but clot structure remains altered with TXA treatment., Competing Interests: Declaration of competing interest The authors have no relevant competing interests to disclose., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Thrombosis, resulting in occlusive blood clots, blocks blood flow to downstream organs and causes life-threatening conditions such as heart attacks and strokes. The administration of tissue plasminogen activator (t-PA), which drives the enzymatic degradation (fibrinolysis) of these blood clots, is a treatment for thrombotic conditions, but the use of these therapeutics is often limited due to the time-dependent nature of treatment and their limited success. We have shown that clot contraction, which is altered in prothrombotic conditions, influences the efficacy of fibrinolysis. Clot contraction results in the volume shrinkage of blood clots, with the redistribution and densification of fibrin and platelets on the exterior of the clot and red blood cells in the interior. Understanding how these key structural changes influence fibrinolysis can lead to improved diagnostics and patient care. We used a combination of mathematical modeling and experimental methodologies to characterize the process of exogenous delivery of t-PA (external fibrinolysis). A three-dimensional (3D) stochastic, multiscale model of external fibrinolysis was used to determine how the structural changes that occur during the process of clot contraction influence the mechanism(s) of fibrinolysis. Experiments were performed based on modeling predictions using pooled human plasma and the external delivery of t-PA to initiate lysis. Analysis of fibrinolysis simulations and experiments indicate that fibrin densification makes the most significant contribution to the rate of fibrinolysis compared with the distribution of components and degree of compaction (p < 0.0001). This result suggests the possibility of a certain fibrin density threshold above which t-PA effective diffusion is limited. From a clinical perspective, this information can be used to improve on current therapeutics by optimizing timing and delivery of lysis agents., Competing Interests: Declaration of interests The authors state that they have no conflict of interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Blood clots form at the site of vascular injury to seal the wound and prevent bleeding. Clots are in tension as they perform their biological functions and withstand hydrodynamic forces of blood flow, vessel wall fluctuations, extravascular muscle contraction and other forces. There are several mechanisms that generate tension in a blood clot, of which the most well-known is the contraction/retraction caused by activated platelets. Here we show through experiments and modeling that clot tension is generated by the polymerization of fibrin. Our mathematical model is built on the hypothesis that the shape of fibrin monomers having two-fold symmetry and off-axis binding sites is ultimately the source of inherent tension in individual fibers and the clot. As the diameter of a fiber grows during polymerization the fibrin monomers must suffer axial twisting deformation so that they remain in register to form the half-staggered arrangement characteristic of fibrin protofibrils. This deformation results in a pre-strain that causes fiber and network tension. Our results for the pre-strain in single fibrin fibers is in agreement with experiments that measured it by cutting fibers and measuring their relaxed length. We connect the mechanics of a fiber to that of the network using the 8-chain model of polymer elasticity. By combining this with a continuum model of swellable elastomers we can compute the evolution of tension in a constrained fibrin gel. The temporal evolution and tensile stresses predicted by this model are in qualitative agreement with experimental measurements of the inherent tension of fibrin clots polymerized between two fixed rheometer plates. These experiments also revealed that increasing thrombin concentration leads to increasing internal tension in the fibrin network. Our model may be extended to account for other mechanisms that generate pre-strains in individual fibers and cause tension in three-dimensional proteinaceous polymeric networks., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Fibrin is the major determinant of the mechanical stability and integrity of blood clots and thrombi. To explore the rupture of blood clots, emulating thrombus breakage, we stretched fibrin gels with single-edge cracks of varying size. Ultrastructural alterations of the fibrin network correlated with three regimes of stress vs. strain profiles: the weakly non-linear regime due to alignment of fibrin fibers; linear regime owing to further alignment and stretching of fibers; and the rupture regime for large deformations reaching the critical strain and stress, at which irreversible breakage of fibers ahead of the crack tip occurs. To interpret the stress-strain curves, we developed a new Fluctuating Spring model, which maps the fibrin alignment at the characteristic strain, network stretching with the Young modulus, and simultaneous cooperative rupture of coupled fibrin fibers into a theoretical framework to obtain the closed-form expressions for the strain-dependent stress profiles. Cracks render network rupture stochastic, and the free energy change for fiber deformation and rupture decreases with the crack length, making network rupture more spontaneous. By contrast, mechanical cooperativity due to the presence of inter-fiber contacts strengthens fibrin networks. The results obtained provide a fundamental understanding of blood clot breakage that underlies thrombotic embolization. STATEMENT OF SIGNIFICANCE: Fibrin, a naturally occurring biomaterial, is the major determinant of mechanical stability and integrity of blood clots and obstructive thrombi. We tested mechanically fibrin gels with single-edge cracks and followed ultrastructural alterations of the fibrin network. Rupture of fibrin gel involves initial alignment and elastic stretching of fibers followed by their eventual rupture for deformations reaching the critical level. To interpret the stress-strain curves, we developed Fluctuating Spring model, which showed that cracks render rupture of fibrin networks more spontaneous; yet, coupled fibrin fibers reinforce cracked fibrin networks. The results obtained provide fundamental understanding of blood clot breakage that underlies thrombotic embolization. Fluctuating Spring model can be applied to other protein networks with cracks and to interpret the stress-strain profiles., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Fibrin is the major extracellular component of blood clots and a proteinaceous hydrogel used as a versatile biomaterial. Fibrin forms branched networks built of laterally associated double-stranded protofibrils. This multiscale hierarchical structure is crucial for the extraordinary mechanical resilience of blood clots, yet the structural basis of clot mechanical properties remains largely unclear due, in part, to the unresolved molecular packing of fibrin fibers. Here the packing structure of fibrin fibers is quantitatively assessed by combining Small Angle X-ray Scattering (SAXS) measurements of fibrin reconstituted under a wide range of conditions with computational molecular modeling of fibrin protofibrils. The number, positions, and intensities of the Bragg peaks observed in the SAXS experiments were reproduced computationally based on the all-atom molecular structure of reconstructed fibrin protofibrils. Specifically, the model correctly predicts the intensities of the reflections of the 22.5 nm axial repeat, corresponding to the half-staggered longitudinal arrangement of fibrin molecules. In addition, the SAXS measurements showed that protofibrils within fibrin fibers have a partially ordered lateral arrangement with a characteristic transverse repeat distance of 13 nm, irrespective of the fiber thickness. These findings provide fundamental insights into the molecular structure of fibrin clots that underlies their biological and physical properties.

Fibrin is the three-dimensional mechanical scaffold of protective blood clots that stop bleeding and pathological thrombi that obstruct blood vessels. Fibrin must be mechanically tough to withstand rupture, after which life-threatening pieces (thrombotic emboli) are carried downstream by blood flow. Despite multiple studies on fibrin viscoelasticity, mechanisms of fibrin rupture remain unknown. Here, we examined mechanically and structurally the strain-driven rupture of human blood plasma-derived fibrin clots where clotting was triggered with tissue factor. Toughness, i.e., resistance to rupture, quantified by the critical energy release rate (a measure of the propensity for clot embolization) of physiologically relevant fibrin gels was determined to be 7.6 ± 0.45 J/m 2 . Finite element (FE) simulations using fibrin material models that account for forced protein unfolding independently supported this measured toughness and showed that breaking of fibers ahead the crack at a critical stretch is the mechanism of rupture of blood clots, including thrombotic embolization., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Essentials Clot contraction influences the rate of fibrinolysis in vitro. Internal fibrinolysis is enhanced ∼2-fold in contracted vs. uncontracted blood clots. External fibrinolysis is ∼4-fold slower in contracted vs. uncontracted blood clots. Contraction can modulate lytic resistance and potentially the clinical outcome of thrombosis. SUMMARY: Background Fibrinolysis involves dissolution of polymeric fibrin networks that is required to restore blood flow through vessels obstructed by thrombi. The efficiency of lysis depends in part on the susceptibility of fibrin to enzymatic digestion, which is governed by the structure and spatial organization of fibrin fibers. How platelet-driven clot contraction affects the efficacy of fibrinolysis has received relatively little study. Objective Here, we examined the effects of clot contraction on the rate of internal fibrinolysis emanating from within the clot to simulate (patho)physiological conditions and external fibrinolysis initiated from the clot exterior to simulate therapeutic thrombolysis. Methods Clot contraction was prevented by inhibiting platelet myosin IIa activity, actin polymerization or platelet-fibrin(ogen) binding. Internal fibrinolysis was measured by optical tracking of clot size. External fibrinolysis was determined by the release of radioactive fibrin degradation products. Results and Conclusions Clot contraction enhanced the rate of internal fibrinolysis ∼2-fold. In contrast, external fibrinolysis was ~4-fold slower in contracted clots. This dichotomy in the susceptibility of contracted and uncontracted clots to internal vs. external lysis suggests that the rate of lysis is dependent upon the interplay between accessibility of fibrin fibers to fibrinolytic agents, including clot permeability, and the spatial proximity of the fibrin fibers that modulate the effects of the fibrinolytic enzymes. Understanding how compaction of blood clots influences clot lysis might have important implications for prevention and treatment of thrombotic disorders., (© 2018 International Society on Thrombosis and Haemostasis.)

Polyhedral erythrocytes, named polyhedrocytes, are formed in contracted blood clots and thrombi, as a result of compression by activated contractile platelets pulling on fibrin. This deformation was shown to be mechanical in nature and polyhedrocytes were characterized using light and electron microscopy. Through three-dimensional reconstruction, we quantified the geometry of biconcave, intermediate, and polyhedral erythrocytes within contracting blood clots. During compression, erythrocytes became less oblate and more prolate than the biconcave cells and largely corresponded to convex, irregular polyhedra with a total number of faces ranging from 10 to 16. Faces were polygons with 3 to 6 sides. The majority of the faces were quadrilaterals, though not all sides were straight and not all faces were flat. There were no changes in the surface area or volume. These results describe the gradual natural deformation of erythrocytes as a part of compaction into a tightly packed array that is an important but understudied component of mature blood clots and thrombi.

Platelets express ≥2 members of the regulators of G protein signaling (RGS) family. Here, we have focused on the most abundant, RGS10, examining its impact on the hemostatic response in vivo and the mechanisms involved. We have previously shown that the hemostatic thrombi formed in response to penetrating injuries consist of a core of fully activated densely packed platelets overlaid by a shell of less-activated platelets responding to adenosine 5'-diphosphate (ADP) and thromboxane A 2 (TxA 2 ). Hemostatic thrombi formed in RGS10 -/- mice were larger than in controls, with the increase due to expansion of the shell but not the core. Clot retraction was slower, and average packing density was reduced. Deleting RGS10 had agonist-specific effects on signaling. There was a leftward shift in the dose/response curve for the thrombin receptor (PAR4) agonist peptide AYPGKF but no increase in the maximum response. This contrasted with ADP and TxA 2 , both of which evoked considerably greater maximum responses in RGS10 -/- platelets with enhanced G q - and G i -mediated signaling. Shape change, which is G 13 -mediated, was unaffected. Finally, we found that free RGS10 levels in platelets are actively regulated. In resting platelets, RGS10 was bound to 2 scaffold proteins: spinophilin and 14-3-3γ. Platelet activation caused an increase in free RGS10, as did the endothelium-derived platelet antagonist prostacyclin. Collectively, these observations show that RGS10 serves as an actively regulated node on the platelet signaling network, helping to produce smaller and more densely packed hemostatic thrombi with a greater proportion of fully activated platelets., (© 2018 by The American Society of Hematology.)

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies to platelet factor 4 (PF4)/heparin complexes. PF4 released from platelets binds to surface glycosaminoglycans on hematopoietic and vascular cells that are heterogenous in composition and differ in affinity for PF4. PF4 binds to monocytes with higher affinity than to platelets, and depletion of monocytes exacerbates thrombocytopenia in a murine HIT model. Here we show that the expression of PF4 on platelets and development of thrombocytopenia are modulated by the (re)distribution of PF4 among hematopoietic and endothelial cell surfaces. Binding of PF4 to platelets in whole blood in vitro varies inversely with the white cell count, likely because of the greater affinity of monocytes for PF4. In mice, monocyte depletion increased binding of PF4 to platelets by two- to three-fold. Induction of HIT in mice caused a transient >80-fold increase in binding of HIT antibody to monocytes vs 3.5-fold increase to platelets and rapid transient monocytopenia. Normalization of monocyte counts preceded the return in platelet counts. Exposure of blood to endothelial cells also depletes PF4 from platelet surfaces. These studies demonstrate a dynamic interchange of surface-bound PF4 among hematopoetic and vascular cells that may limit thrombocytopenia at the expense of promoting prothrombotic processes in HIT., (© 2018 by The American Society of Hematology.)

Contraction (retraction) of the blood clot is a part of the clotting process driven by activated platelets attached to fibrin that can potentially modulate the obstructiveness and integrity of thrombi. The aim of this work was to reveal the pathogenic importance of contraction of clots and thrombi in venous thromboembolism (VTE). We investigated the kinetics of clot contraction in the blood of 55 patients with VTE. In addition, we studied the ultrastructure of ex vivo venous thrombi as well as the morphology and functionality of isolated platelets. Thrombi from VTE patients contained compressed polyhedral erythrocytes, a marker for clot contraction in vivo. The extent and rate of contraction were reduced by twofold in clots from the blood of VTE patients compared with healthy controls. The contraction of clots from the blood of patients with pulmonary embolism was significantly impaired compared with that of those with isolated venous thrombosis, suggesting that less compacted thrombi are prone to embolization. The reduced ability of clots to contract correlated with continuous platelet activation followed by their partial refractoriness. Morphologically, 75% of platelets from VTE patients were spontaneously activated (with filopodia) compared with only 21% from healthy controls. At the same time, platelets from VTE patients showed a 1.4-fold reduction in activation markers expressed in response to chemical activation when compared with healthy individuals. The results obtained suggest that the impaired contraction of thrombi is an underappreciated pathogenic mechanism in VTE that may regulate the obstructiveness and embologenicity of venous thrombi.

Platelet-driven reduction in blood clot volume (clot contraction or retraction) has been implicated to play a role in hemostasis and thrombosis. Although these processes are often linked with inflammation, the role of inflammatory cells in contraction of blood clots and thrombi has not been investigated. The aim of this work was to study the influence of activated monocytes on clot contraction. The effects of monocytes were evaluated using a quantitative optical tracking methodology to follow volume changes in a blood clot formed in vitro. When a physiologically relevant number of isolated human monocytes pre-activated with phorbol-12-myristate-13-acetate (PMA) were added back into whole blood, the extent and rate of clot contraction were increased compared to addition of non-activated cells. Inhibition of tissue factor expression or its inactivation on the surface of PMA-treated monocytes reduced the extent and rate of clot contraction back to control levels with non-activated monocytes. On the contrary, addition of tissue factor enhanced clot contraction, mimicking the effects of tissue factor expressed on the activated monocytes. These data suggest that the inflammatory cells through their expression of tissue factor can directly affect hemostasis and thrombosis by modulating the size and density of intra- and extravascular clots and thrombi.

Blood clot contraction (retraction) is driven by platelet-generated forces propagated by the fibrin network and results in clot shrinkage and deformation of erythrocytes. To elucidate the mechanical nature of this process, we developed a model that combines an active contractile motor element with passive viscoelastic elements. Despite its importance for thrombosis and wound healing, clot contraction is poorly understood. This model predicts how clot contraction occurs due to active contractile platelets interacting with a viscoelastic material, rather than to the poroelastic nature of fibrin, and explains the observed dynamics of clot size, ultrastructure, and measured forces. Mechanically passive erythrocytes and fibrin are present in series and parallel to active contractile cells. This mechanical interplay induces compressive and tensile resistance, resulting in increased contractile force and a reduced extent of contraction in the presence of erythrocytes. This experimentally validated model provides the fundamental mechanical basis for understanding contraction of blood clots., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Objective: Obstructive thrombi or thrombotic emboli are the pathogenic basis of ischemic stroke. In vitro blood clots and in vivo thrombi can undergo platelet-driven contraction (retraction), resulting in volume shrinkage. Clot contraction can potentially reduce vessel occlusion and improve blood flow past emboli or thrombi. The aim of this work was to examine a potential pathogenic role of clot contraction in ischemic stroke., Approach and Results: We used a novel automated method that enabled us to quantify time of initiation and extent and rate of clot contraction in vitro. The main finding is that clot contraction from the blood of stroke patients was reduced compared with healthy subjects. Reduced clot contraction correlated with a lower platelet count and their dysfunction, higher levels of fibrinogen and hematocrit, leukocytosis, and other changes in blood composition that may affect platelet function and properties of blood clots. Platelets from stroke patents were spontaneously activated and displayed reduced responsiveness to additional stimulation. Clinical correlations with respect to severity and stroke pathogenesis suggest that the impaired clot contraction has the potential to be a pathogenic factor in ischemic stroke., Conclusions: The changeable ability of clots and thrombi to shrink in volume may be a novel unappreciated mechanism that aggravates or alleviates the course and outcomes of ischemic stroke. The clinical importance of clot or thrombus transformations in vivo and the diagnostic and prognostic value of this blood test for clot contraction need further exploration., (© 2016 American Heart Association, Inc.)

Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via fragment crystallizable (Fc)γRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcγRIIA, which activates spleen tyrosine kinase and leads to the generation of tissue factor (TF) and thrombin. The combination of direct platelet activation by HIT immune complexes through FcγRIIA and transactivation by monocyte-derived thrombin markedly increases Annexin V and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcγRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk., (© 2016 by The American Society of Hematology.)

Platelet-driven blood clot contraction (retraction) is thought to promote wound closure and secure hemostasis while preventing vascular occlusion. Notwithstanding its importance, clot contraction remains a poorly understood process, partially because of the lack of methodology to quantify its dynamics and requirements. We used a novel automated optical analyzer to continuously track in vitro changes in the size of contracting clots in whole blood and in variously reconstituted samples. Kinetics of contraction was complemented with dynamic rheometry to characterize the viscoelasticity of contracting clots. This combined approach enabled investigation of the coordinated mechanistic impact of platelets, including nonmuscle myosin II, red blood cells (RBCs), fibrin(ogen), factor XIIIa (FXIIIa), and thrombin on the kinetics and mechanics of the contraction process. Clot contraction is composed of 3 sequential phases, each characterized by a distinct rate constant. Thrombin, Ca(2+), the integrin αIIbβ3, myosin IIa, FXIIIa cross-linking, and platelet count all promote 1 or more phases of the clot contraction process. In contrast, RBCs impair contraction and reduce elasticity, while increasing the overall contractile stress generated by the platelet-fibrin meshwork. A better understanding of the mechanisms by which blood cells, fibrin(ogen), and platelet-fibrin interactions modulate clot contraction may generate novel approaches to reveal and to manage thrombosis and hemostatic disorders., (© 2016 by The American Society of Hematology.)