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Fibrin and collagen as well as their combinations play an important biological role in tissue regeneration and are widely employed in surgery as fleeces or sealants and in bioengineering as tissue scaffolds. Earlier studies demonstrated that fibrin-collagen composite networks displayed improved tensile mechanical properties compared to the isolated protein matrices. Unlike previous studies, here unconfined compression was applied to a fibrin-collagen filamentous polymer composite matrix to study its structural and mechanical responses to compressive deformation. Combining collagen with fibrin resulted in formation of a composite hydrogel exhibiting synergistic mechanical properties compared to the isolated fibrin and collagen matrices. Specifically, the composite matrix revealed a one order of magnitude increase in the shear storage modulus at compressive strains>0.8 in response to compression compared to the mechanical features of individual components. These material enhancements were attributed to the observed structural alterations, such as network density changes, an increase in connectivity along with criss-crossing, and bundling of fibers. In addition, the compressed composite collagen/fibrin networks revealed a non-linear transformation of their viscoelastic properties with softening and stiffening regimes. These transitions were shown to depend on protein concentrations. Namely, a decrease in protein content drastically affected the mechanical response of the networks to compression by shifting the onset of stiffening to higher degrees of compression. Since both natural and artificially composed extracellular matrices experience compression in various (patho)physiological conditions, our results provide new insights into the structural biomechanics of the polymeric composite matrix that can help to create fibrin-collagen sealants, sponges, and tissue scaffolds with tunable and predictable mechanical properties.

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.)

Background: Leukocyte-platelet aggregates comprise a pathogenic link between hemostasis and immunity, but the prerequisites and mechanisms of their formation remain not understood., Aims: To quantify the formation, composition, and morphology of leukocyte-platelet aggregates in vitro under the influence of various cellular activators., Methods: Phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS), thrombin receptor-activating peptide (TRAP-6), and adenosine diphosphate (ADP) were used as cellular activators. Flow cytometry was utilized to identify and quantify aggregates in whole human blood and platelet-rich plasma. Cell types and cellular aggregates were identified using fluorescently labeled antibodies against the appropriate cellular markers, and cell activation was assessed by the expression of appropriate surface markers. For confocal fluorescent microscopy, cell membranes and nuclei were labeled. Neutrophil-platelet aggregates were studied using scanning electron microscopy., Results: In the presence of PMA, ADP or TRAP-6, about 17-38 % of neutrophils and 61-77 % of monocytes formed aggregates with platelets in whole blood, whereas LPS did not induce platelet aggregation with either neutrophils or monocytes due the inability to activate platelets. Similar results were obtained when isolated neutrophils were added to platelet-rich plasma. All the cell types involved in the heterotypic aggregation expressed molecular markers of activation. Fluorescent and electron microscopy of the aggregates showed that the predominant platelet/leukocyte ratios were 1:1 and 2:1., Conclusions: Formation of leukocyte-platelet aggregates depends on the nature of the cellular activator and the spectrum of its cell-activating ability. An indispensable condition for formation of leukocyte-platelet aggregates is activation of all cell types including platelets, which is the restrictive step., 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 Elsevier Ltd. All rights reserved.)

Background: Deep vein thrombosis is a common vascular event that can result in debilitating morbidity and even death due to pulmonary embolism. Clinically, patients with faster resolution of a venous thrombus have improved prognosis, but the detailed structural information regarding changes that occur in a resolving thrombus over time is lacking., Objectives: To define the spatial-morphologic characteristics of venous thrombus formation, propagation, and resolution at the submicron level over time., Methods: Using a murine model of stasis-induced deep vein thrombosis along with scanning electron microscopy and immunohistology, we determine the specific structural, compositional, and morphologic characteristics of venous thrombi formed after 4 days and identify the changes that take place during resolution by day 7. Comparison is made with the structure and composition of venous thrombi formed in mice genetically deficient in plasminogen activator inhibitor type 1., Results: As venous thrombus resolution progresses, fibrin exists in different structural forms, and there are dynamic cellular changes in the compositions of leukocytes, platelet aggregates, and red blood cells. Intrathrombus microvesicles are present that are not evident by histology, and red blood cells in the form of polyhedrocytes are an indicator of clot contraction. Structural evidence of fibrinolysis is observed early during thrombogenesis and is accelerated by plasminogen activator inhibitor type 1 deficiency., Conclusion: The results reveal unique, detailed ultrastructural and compositional insights along with documentation of the dynamic changes that occur during accelerated resolution that are not evident by standard pathologic procedures and can be applied to inform diagnosis and effectiveness of thrombolytic treatments to improve patient outcomes., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Published by Elsevier Inc.)

Background: The platelet-driven contraction or retraction of blood clots has been utilized to obtain blood serum for laboratory studies, but now, in vitro clot contraction assays are used in research laboratories and clinics to assess platelet functionality. The static final extent of clot contraction measured using a clot size or expelled serum volume can be supplemented substantially with a dynamic analysis., Objectives: To provide a step-by-step protocol for a relatively simple and affordable new automated methodology to follow the kinetics of blood clot contraction, which allows for simultaneous measurements of various samples at a time and requires only a fluorescence plate reader., Methods: The kinetics of clot contraction in whole blood was assessed by continuously detecting the fluorescence intensity of fluorescein isothiocyanate-albumin added to a blood sample before clotting and expelled into the serum during clot shrinkage., Results: The clots are formed and fluorescence is measured in the wells of a black multiwell plate using a standard plate fluorescent reader. The specificity of this technique for clot contraction has been demonstrated by the strong inhibitory effects of blebbistatin, latrunculin A, and abciximab. To validate the new technique, increased fluorescence intensity in the contracting clots was measured in parallel with a visual decrease in clot size performed with the same blood samples., Conclusion: The resulting clot contraction dynamics based on the expulsion of fluorescein isothiocyanate-albumin can be quantified using a number of kinetic parameters as well as a phase kinetics analysis. The advantages and drawbacks of the new technique are discussed., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

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.

Despite rapid technological advancement in factor and nonfactor products in the prevention and treatment of bleeding in haemophilia patients, it is imperative that we acknowledge gaps in our understanding of how hemostasis is achieved. The authors will briefly review three unresolved issues in persons with haemophilia (PwH) focusing on the forgotten function that red blood cells play in hemostasis, the critical role of extravascular (outside circulation) FIX in hemostasis in the context of unmodified and extended half-life FIX products and finally on the role that skeletal muscle myosin plays in prothrombinase assembly and subsequent thrombin generation that could mitigate breakthrough muscle hematomas., (© 2024 John Wiley & Sons Ltd.)

Background: Blood clots are primarily composed of red blood cells (RBCs), platelets/thrombocytes, and fibrin. Despite the similarities observed between mammals and zebrafish, the composition of fish thrombi is not as well known., Objectives: To analyze the formation of zebrafish blood clots ex vivo and arterial and venous thrombi in vivo., Methods: Transgenic zebrafish lines and laser-mediated endothelial injury were used to determine the relative ratio of RBCs and thrombocytes in clots. Scanning electron and confocal microscopy provided high-resolution images of the structure of adult and larval clots. Adult and larval thrombocyte spreading on fibrinogen was evaluated ex vivo., Results: RBCs were present in arterial and venous thrombi, making up the majority of cells in both circulations. However, bloodless mutant fish demonstrated that fibrin clots can form in vivo in the absence of blood cells. Scanning electron and confocal microscopy showed that larval and adult zebrafish thrombi and mammalian thrombi look surprisingly similar externally and internally, even though the former have nucleated RBCs and thrombocytes. Although adult thrombocytes spread on fibrinogen, we found that larval cells do not fully activate without the addition of plasma from adult fish, suggesting a developmental deficiency of a plasma activating factor. Finally, mutants lacking αIIbβ3 demonstrated that this integrin mediates thrombocyte spreading on fibrinogen., Conclusion: Our data showed strong conservation of arterial and venous and clot/thrombus formation across species, including developmental regulation of thrombocyte function. This correlation supports the possibility that mammals also do not absolutely require circulating cells to form fibrin clots in vivo., Competing Interests: Declaration of competing interests J.A.S. has been a consultant for Sanofi, Takeda, Genentech, CSL Behring, and HEMA Biologics. M.H. is a consultant and equity holder for Veralox Therapeutics and Cereno Scientific. M.S.G., A.R.D., C.N., K.K., C.M., R.I.L., R.F., R.J.F., M.N.-A., J.W.W., and R.A., have no conflicts of interest to declare., (Copyright © 2023. Published by Elsevier Inc.)

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.

Abstract: Nonmuscle cell contractility is an essential feature underlying diverse cellular processes such as motility, morphogenesis, division and genome replication, intracellular transport, and secretion. Blood clot contraction is a well-studied process driven by contracting platelets. Megakaryocytes (MKs), which are the precursors to platelets, can be found in bone marrow and lungs. Although they express many of the same proteins and structures found in platelets, little is known about their ability to engage with extracellular proteins such as fibrin and contract. Here, we have measured the ability of MKs to compress plasma clots. Megakaryocytes derived from human induced pluripotent stem cells (iPSCs) were suspended in human platelet-free blood plasma and stimulated with thrombin. Using real-time macroscale optical tracking, confocal microscopy, and biomechanical measurements, we found that activated iPSC-derived MKs (iMKs) caused macroscopic volumetric clot shrinkage, as well as densification and stiffening of the fibrin network via fibrin-attached plasma membrane protrusions undergoing extension-retraction cycles that cause shortening and bending of fibrin fibers. Contraction induced by iMKs involved 2 kinetic phases with distinct rates and durations. It was suppressed by inhibitors of nonmuscle myosin IIA, actin polymerization, and integrin αIIbβ3-fibrin interactions, indicating that the molecular mechanisms of iMK contractility were similar or identical to those in activated platelets. Our findings provide new insights into MK biomechanics and suggest that iMKs can be used as a model system to study platelet contractility. Physiologically, the ability of MKs to contract plasma clots may play a role in the mechanical remodeling of intravascular blood clots and thrombi., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

The multiple roles of red blood cells (RBCs) are often neglected as contributors in hemostasis and thrombosis. Proactive opportunities to increase RBC numbers, either acutely or subacutely in the case of iron deficiency, are critical as RBCs are the cellular elements that initiate hemostasis together with platelets and stabilize fibrin and clot structure. RBCs also possess several functional properties to assist hemostasis: releasing platelet agonists, promoting shear force-induced von Willebrand factor unfolding, procoagulant capacity, and binding to fibrin. Additionally, blood clot contraction is important to compress RBCs to form a tightly packed array of polyhedrocytes, making an impermeable seal for hemostasis. All these functions are important for patients having intrinsically poor capacity to cease bleeds (ie, hemostatic disorders) but, conversely, can also play a role in thrombosis if these RBC-mediated reactions overshoot. One acquired example of bleeding with anemia is in patients treated with anticoagulants and/or antithrombotic medication because upon initiation of these drugs, baseline anemia doubles the risk of bleeding complications and mortality. Also, anemia is a risk factor for reoccurring gastrointestinal and urogenital bleeds, pregnancy, and delivery complications. This review summarizes the clinically relevant properties and profiles of RBCs at various steps of platelet adhesion, aggregation, thrombin generation, and fibrin formation, including both structural and functional elements. Regarding patient blood management guidelines, they support minimizing transfusions, but this approach does not deal with severe inherited and acquired bleeding disorders where a poor hemostatic propensity is exacerbated by limited RBC availability, for which future guidance will be needed., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.)

Background: Piezo1 is a mechanosensitive cationic channel that boosts intracellular [Ca 2+ ] i . Compression of red blood cells (RBCs) during platelet-driven contraction of blood clots may cause the activation of Piezo1., Objectives: To establish relationships between Piezo1 activity and blood clot contraction., Methods: Effects of a Piezo1 agonist, Yoda1, and antagonist, GsMTx-4, on clot contraction in vitro were studied in human blood containing physiological [Ca 2+ ]. Clot contraction was induced by exogenous thrombin. Activation of Piezo1 was assessed by Ca 2+ influx in RBCs and with other functional and morphologic features., Results: Piezo1 channels in compressed RBCs are activated naturally during blood clot contraction and induce an upsurge in the intracellular [Ca 2+ ] i , followed by phosphatidylserine exposure. Adding the Piezo1 agonist Yoda1 to whole blood increased the extent of clot contraction due to Ca 2+ -dependent volumetric shrinkage of RBCs and increased platelet contractility due to their hyperactivation by the enhanced generation of endogenous thrombin on activated RBCs. Addition of rivaroxaban, the inhibitor of thrombin formation, or elimination of Ca 2+ from the extracellular space abrogated the stimulating effect of Yoda1 on clot contraction. The Piezo1 antagonist, GsMTx-4, caused a decrease in the extent of clot contraction relative to the control both in whole blood and in platelet-rich plasma. Activated Piezo1 in compressed and deformed RBCs amplified the platelet contractility as a positive feedback mechanism during clot contraction., Conclusion: The results obtained demonstrate that the Piezo1 channel expressed on RBCs comprises a mechanochemical modulator of blood clotting that may be considered a potential therapeutic target to correct hemostatic disorders., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

While blood clot formation has been relatively well studied, little is known about the mechanisms underlying the subsequent structural and mechanical clot remodeling called contraction or retraction. Impairment of the clot contraction process is associated with both life-threatening bleeding and thrombotic conditions, such as ischemic stroke, venous thromboembolism, and others. Recently, blood clot contraction was observed to be hindered in patients with COVID-19. A three-dimensional multiscale computational model is developed and used to quantify biomechanical mechanisms of the kinetics of clot contraction driven by platelet-fibrin pulling interactions. These results provide important biological insights into contraction of platelet filopodia, the mechanically active thin protrusions of the plasma membrane, described previously as performing mostly a sensory function. The biomechanical mechanisms and modeling approach described can potentially apply to studying other systems in which cells are embedded in a filamentous network and exert forces on the extracellular matrix modulated by the substrate stiffness., (© 2023. Springer Nature Limited.)

Aortic dissection occurs when a weakened portion of the intima tears, and a separation of layers propagates along the aortic wall to form a false lumen filled with active blood flow or intramural thrombus. The unpredictable nature of aortic dissection formation and need for immediate intervention leaves limited serial human image data to study the formation and morphological changes that follow dissection. We used volumetric ultrasound examination, histology, and scanning electron microscopy (SEM) to examine intramural thrombi at well-defined timepoints after dissection occurs in apolipoprotein E-deficient mice infused with angiotensin II (n = 71). Stratification of red blood cell (RBC) morphologies (biconcave, intermediate biconcave, intermediate polyhedrocyte, and polyhedrocyte) in the thrombi with scanning electron microscopy (n = 5) was used to determine degree of thrombus deposition/contraction. Very few biconcave RBCs (1.2 ± 0.6%) were in the thrombi, and greater amounts of intermediate biconcave RBCs (25.8 ± 6.7%) were located in the descending thoracic portion of the dissection while more polyhedrocytes (14.6 ± 5.1%) and fibrin (42.3 ± 4.5%; P < .05) were found in the distal suprarenal aorta. Thrombus deposition likely plays some role in patient outcomes, and this multimodality technique can help investigate thrombus deposition and characteristics in experimental animal models and human tissue samples., (© 2023 by the Society for Vascular Surgery. Published by Elsevier Inc.)

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.)

A State of the Art lecture titled "Blood Clot Contraction: Mechanisms, Pathophysiology, and Disease" was presented at the International Society on Thrombosis and Haemostasis (ISTH) Congress in 2022. This was a systematic description of blood clot contraction or retraction, driven by activated platelets and causing compaction of the fibrin network along with compression of the embedded erythrocytes. The consequences of clot contraction include redistribution of the fibrin-platelet meshwork toward the periphery of the clot and condensation of erythrocytes in the core, followed by their deformation from the biconcave shape into polyhedral cells (polyhedrocytes). These structural signatures of contraction have been found in ex vivo thrombi derived from various locations, which indicated that clots undergo intravital contraction within the blood vessels. In hemostatic clots, tightly packed polyhedrocytes make a nearly impermeable seal that stems bleeding and is impaired in hemorrhagic disorders. In thrombosis, contraction facilitates the local blood flow by decreasing thrombus obstructiveness, reducing permeability, and changing susceptibility to fibrinolytic enzymes. However, in (pro)thrombotic conditions, continuous background platelet activation is followed by platelet exhaustion, refractoriness, and impaired intravital clot contraction, which is associated with weaker thrombi predisposed to embolization. Therefore, assays that detect imperfect in vitro clot contraction have potential diagnostic and prognostic values for imminent or ongoing thrombosis and thrombotic embolism. Collectively, the contraction of blood clots and thrombi is an underappreciated and understudied process that has a pathogenic and clinical significance in bleeding and thrombosis of various etiologies. Finally, we have summarized relevant new data on this topic presented during the 2022 ISTH Congress., (© 2022 The Author(s).)

Mutations in the MYH9 gene result in macrothrombocytopenia often associated with hemorrhages. Here, we studied the function and structure of platelets in three family members with a heterozygous mutation R1933X in the MYH9 gene, characteristic of closely related disorders known as the May-Hegglin anomaly and Sebastian syndrome. The examination included complete blood count, blood smear microscopy, platelet flow cytometry (expression of P-selectin and active integrin αIIbβ3 before and after activation), the kinetics of platelet-driven contraction (retraction) of blood clots, as well as scanning/transmission electron microscopy of platelets. Despite severe thrombocytopenia ranging (36-86) × 10 9 /l, none of the patients had hemorrhages at the time of examination, although they had a history of heavy menstruation, spontaneous ecchymosis, and postpartum hemorrhage. Flow cytometry showed background platelet activation, revealed by overexpression of P-selectin and active αIIbβ3 integrin above normal levels. After TRAP-induced stimulation, the fractions of platelets expressing P-selectin in the proband and her sister were below normal response, indicating partial platelet refractoriness. The initiation of clot contraction was delayed. Electron microscopy revealed giant platelets with multiple filopodia and fusion of α-granules with dilated open canalicular system, containing filamentous and vesicular inclusions. The novel concept implies that the R1933X mutation in the MYH9 gene is associated not only with thrombocytopenia, but also with qualitative structural and functional defects in platelets. Platelet dysfunction includes impaired contractility, which can disrupt the compaction of hemostatic clots, making the clots weak and permeable, therefore predisposing patients with MYH9 gene mutations to the hemorrhagic phenotype., (© 2022. The Author(s).)

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.)

Although much has been established concerning the overall structure and function of fibrinogen, much less has been known about its two αC regions, each consisting of an αC-connector and an αC-domain, but new information has been accumulating. This review summarizes the state of our current knowledge of the structure and interactions of fibrinogen's αC regions. A series of studies with isolated αC regions and their fragments demonstrated that the αC-domain forms compact ordered structures consisting of N- and C-terminal subdomains including β sheets and suggested that the αC-connector has a poly(L-proline) type II structure. Functionally, the αC-domains interact intramolecularly with each other and with the central region of the molecule, first demonstrated by electron microscopy and then quantified by optical trap force spectroscopy. Upon conversion of fibrinogen into fibrin, the αC-domains switch from intra- to intermolecular interactions to form ordered αC polymers. The formation of αC polymers occurs mainly through the homophilic interaction between the N-terminal subdomains; interaction between the C-terminal subdomains and the αC-connectors also contributes to this process. Considerable evidence supports the idea that the αC-regions accelerate fibrin polymerization and affect the final structure of fibrin clots. The interactions between αC-regions are important for the mechanical properties of clots, increasing their stiffness and extensibility. Conversion of fibrinogen into fibrin results in exposure of multiple binding sites in its αC regions, providing interaction of fibrin with different proteins and cell types during hemostasis and wound healing. This heretofore mysterious part of the fibrinogen molecule is finally giving up its secrets., Competing Interests: None declared., (Thieme. All rights reserved.)

Autoimmune diseases, including systemic lupus erythematosus (SLE), have a high risk of thrombotic and hemorrhagic complications associated with altered platelet functionality. We studied platelets from the blood of SLE patients and their reactivity. The surface expression of phosphatidylserine, P-selectin, and active integrin αIIbβ3 were measured using flow cytometry before and after platelet stimulation. Soluble P-selectin was measured in plasma. The kinetics of platelet-driven clot contraction was studied, as well as scanning and transmission electron microscopy of unstimulated platelets. Elevated levels of membrane-associated phosphatidylserine and platelet-attached and soluble P-selectin correlated directly with the titers of IgG, anti-dsDNA-antibodies, and circulating immune complexes. Morphologically, platelets in SLE lost their resting discoid shape, formed membrane protrusions and aggregates, and had a rough plasma membrane. The signs of platelet activation were associated paradoxically with reduced reactivity to a physiological stimulus and impaired contractility that revealed platelet exhaustion and refractoriness. Platelet activation has multiple pro-coagulant effects, and the inability to fully contract (retract) blood clots can be either a hemorrhagic or pro-thrombotic mechanism related to altered clot permeability, sensitivity of clots to fibrinolysis, obstructiveness, and embologenicity. Therefore, chronic immune platelet activation followed by secondary platelet dysfunction comprise an understudied pathogenic mechanism that supports hemostatic disorders in autoimmune diseases, such as SLE.

Although septins have been well-studied in nucleated cells, their role in anucleate blood platelets remains obscure. Here, we elucidate the contribution of septins to human platelet structure and functionality. We show that Septin-2 and Septin-9 are predominantly distributed at the periphery of resting platelets and co-localize strongly with microtubules. Activation of platelets by thrombin causes clustering of septins and impairs their association with microtubules. Inhibition of septin dynamics with forchlorfenuron (FCF) reduces thrombin-induced densification of septins and lessens their colocalization with microtubules in resting and activated platelets. Exposure to FCF alters platelet shape, suggesting that septins stabilize platelet cytoskeleton. FCF suppresses platelet integrin αIIbβ3 activation, promotes phosphatidylserine exposure on activated platelets, and induces P-selectin expression on resting platelets, suggesting septin involvement in these processes. Inhibition of septin dynamics substantially reduces platelet contractility and abrogates their spreading on fibrinogen-coated surfaces. Overall, septins strongly contribute to platelet structure, activation and biomechanics., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Fibrin deformation and interaction of fibrin with other blood components play critical roles in hemostasis and thrombosis. In this review, computational and mathematical biomechanical models of fibrin network deformation and contraction at different spatio-temporal scales as well as challenges in developing and calibrating multiscale models are discussed. There are long standing challenges. For instance, applicability of models to identify and test potential mechanisms of the biomechanical processes mediating interactions between platelets and fiber networks in blood clot stretching and contraction needs to be examined carefully. How the structural and mechanical properties of major blood clot components influences biomechanical responses of the entire clot subjected to external forces, such as blood flow or vessel wall deformations needs to be investigated thoroughly., Competing Interests: Conflict of interest statement The authors declare no conflict of interest.

Introduction: Non-compressible intra-abdominal hemorrhage (NCIAH) is a major cause of preventable death on the battlefield and in civilian trauma. Currently, it can only be definitively managed with surgery, as there are limited strategies for controlling ongoing NCIAH in the prehospital environment. We hypothesized that a self-propelling thrombin-containing powder (SPTP) could increase survival in a swine model of NCIAH when delivered percutaneously into the closed abdomen using an engineered spray system., Materials and Methods: Nineteen swine underwent surgical laparotomy followed by a Grade V liver injury that created massive hemorrhage, before closing the abdomen with sutures. Animals either received treatment with standard of care fluid resuscitation (n=9) or the SPTP spray system (n=10), which consisted of a spray device and a 14 Fr catheter. Using the spray system, SPTP was delivered into a hemoperitoneum identified using a focused assessment with sonography in trauma (FAST) exam. Lactated Ringer's solution was administered to all animals to maintain a mean arterial pressure (MAP) of >50 mmHg. The primary outcome was percentage of animals surviving at three hours following injury., Results: In the swine model of NCIAH, a greater percentage of animals receiving SPTP survived to three hours, although differences were not significant. The SPTP spray system increased the median survival of animals from 1.6 hr in the fluid resuscitation group to 4.3 hr. The SPTP spray system delivered a total mass of 18.5 ± 1.0 g of SPTP. The mean change in intra-abdominal pressure following SPTP delivery was 5.2 ± 1.8 mmHg (mean ± SEM). The intervention time was 6.7 ± 1.7 min. No adverse effects related to the SPTP formulation or the spray system were observed. SPTP was especially beneficial in animals that had either severely elevated lactate concentrations or low mean arterial pressure of <35 mmHg shortly after injury., Conclusions: This demonstrates proof-of-concept for use of a new minimally invasive procedure for managing NCIAH, which could extend survival time to enable patients to reach definitive surgical care., Competing Interests: Declaration of Conflicts of Interest and Sources of Funding C.J.K., M.F.C., N.A., and J.R.B. are involved in commercialization activities for self-propelling hemostatic powder. A.B. is an active serving member of the Canadian Armed Forces., (Crown Copyright © 2022. Published by Elsevier Ltd. All rights reserved.)

Background and Aim: Portal vein thrombosis (PVT) is a common complication of cirrhosis. The exact pathophysiology remains largely unknown, and treatment with anticoagulants does not lead to recanalization of the portal vein in all patients. A better insight into the structure and composition of portal vein thrombi may assist in developing strategies for the prevention and treatment of PVT., Approach and Results: Sixteen prospectively and 63 retrospectively collected nonmalignant portal vein thrombi from patients with cirrhosis who underwent liver transplantation were included. Histology, immunohistochemistry, and scanning electron microscopy were used to assess structure and composition of the thrombi. Most recent CT scans were reanalyzed for thrombus characteristics. Clinical characteristics were related to histological and radiological findings. All samples showed a thickened, fibrotic tunica intima. Fibrin-rich thrombi were present on top of the fibrotic intima in 9/16 prospective cases and in 21/63 retrospective cases. A minority of the fibrotic areas stained focally positive for fibrin/fibrinogen (16% of cases), von Willebrand factor (VWF; 10%), and CD61 (platelets, 21%), while most of the fibrin-rich areas stained positive for those markers (fibrin/fibrinogen, 100%; VWF, 77%; CD61, 100%). No associations were found between clinical characteristics including estimated thrombus age and use of anticoagulants and presence of fibrin-rich thrombi., Conclusion: We demonstrate that PVT in patients with cirrhosis consists of intimal fibrosis with an additional fibrin-rich thrombus in only one-third of cases. We hypothesize that our observations may explain why not all portal vein thrombi in patients with cirrhosis recanalize by anticoagulant therapy., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Blood clots and thrombi undergo platelet-driven contraction/retraction followed by structural rearrangements. We have established quantitative relationships between the composition of blood clots and extent of contraction to determine intravital contraction of thrombi and emboli based on their content. The composition of human blood clots and thrombi was quantified using histology and scanning electron microscopy. Contracting blood clots were segregated into the gradually shrinking outer layer that contains a fibrin-platelet mesh and the expanding inner portion with compacted red blood cells (RBCs). At 10% contraction, biconcave RBCs were partially compressed into polyhedral RBCs, which became dominant at 20% contraction and higher. The polyhedral/biconcave RBC ratio and the extent of contraction displayed an exponential relationship, which was used to determine the extent of intravital contraction of ex vivo thrombi, ranging from 30% to 50%. In venous thrombi, the extent of contraction decreased gradually from the older (head) to the younger (body, tail) parts. In pulmonary emboli, the extent of contraction was significantly lower than in the venous head but was similar to the body and tail, suggesting that the emboli originate from the younger portion(s) of venous thrombi. The extent of contraction in arterial cerebral thrombi was significantly higher than in the younger parts of venous thrombi (body, tail) and pulmonary emboli but was indistinguishable from the older part (head). A novel tool, named the "contraction ruler," has been developed to use the composition of ex vivo thrombi to assess the extent of their intravital contraction, which contributes to the pathophysiology of thromboembolism., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

We describe the internal structure, spatial organization and dynamic formation of coronary artery thrombi from ST-segment elevation myocardial infarction patients. Scanning electron microscopy (SEM) revealed significant differences among four groups of patients (<2 hours; 2-6 hours; 6-12 hours, and >12 hours) related to the time of ischemia. Coronary artery thrombi from patients presenting less than 2 hours after the infarction were almost entirely composed of platelets, with small amounts of fibrin and red blood cells. In contrast, thrombi from late presenters (>12 hours) consisted of mainly platelets at the distal end, where clotting was initiated, with almost no platelets at the proximal end, while the red blood cell content went from low at the initiating end to more than 90% at the proximal end. Furthermore, fibrin was present mainly on the outside of the thrombi and older thrombi contained thicker fibers. The red blood cells in late thrombi were compressed to a close-packed, tessellated array of polyhedral structures, called polyhedrocytes. Moreover, there was redistribution from the originally homogeneous composition to fibrin and platelets to the outside, with polyhedrocytes on the interior. The presence of polyhedrocytes and the redistribution of components are signs of in vivo clot contraction (or retraction). These results suggest why later thrombi are resistant to fibrinolytic agents and other treatment modalities, since the close-packed polyhedrocytes form a nearly impermeable seal. Furthermore, it is of particular clinical significance that these findings suggest specific disparate therapies that will be most effective at different stages of thrombus development., Competing Interests: None declared., (Thieme. All rights reserved.)

Blood clot contraction is driven by traction forces generated by the platelet cytoskeleton that are transmitted to fibrin fibers via the integrin αIIbβ3. Here we show that clot contraction is impaired by inhibitors of the platelet cytosolic protease calpain. We used subtiligase-mediated labeling of amino termini and mass spectrometry to identify proteolytically cleaved platelet proteins involved in clot contraction. Of 32 calpain-cleaved proteins after TRAP stimulation, 14 were cytoskeletal, most prominently talin and vinculin. A complex of talin and vinculin constitutes a mechanosensitive clutch connecting integrins bound to the extracellular matrix with the actin cytoskeleton. Accordingly, we focused on talin and vinculin. Talin is composed of an N-terminal head domain and a C-terminal rod domain organized into a series of 4- and 5-helix bundles. The bundles contain 11 vinculin binding sites (VBSs), each of which is an α-helix packed into a bundle interior and requiring structural rearrangement to initiate vinculin binding. We detected 8 calpain-mediated cleavages in talin, 2 previously identified in unstructured regions and 6 in α-helical regions in proximity to a VBS. There is evidence in vitro that applying mechanical force across talin enables vinculin binding to the talin rod. However, we found that inhibiting platelet cytoskeletal contraction had no effect on talin cleavage, indicating that talin cleavage by calpain in platelets does not require cytoskeleton-generated tensile force. Therefore, it is likely that calpain acts in the later stages of clot retraction through focal adhesion disassembly., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Infection by SARS-CoV-2 produces significant pulmonary pathology including endothelial damage with resultant thrombotic events. While pathologic features were described, there are limited data on the relationship of these changes to the inflammatory response and the production of thromboses., Objective: To investigate pathology of COVID-19-related immunothrombosis., Patients/methods: Tissue samples from lung, kidney, brain and heart that were collected from 45 patients who died of COVID-19. Histopathological examination was performed after H&E and Picro-Mallory staining in combination with (immuno)fluorescence to visualize neutrophil extracellular traps. Ultrastructural alterations in lungs were studied with scanning and transmission electron microscopy., Results: Inflammatory changes and thrombosis were substantially more pronounced in the lung than in the kidney, heart, and brain. The most common pathologic finding was diffuse alveolar damage. In addition, most lung samples showed thrombi in vessels. The cause of death in single cases was massive pulmonary embolism. Ultrastructural examination revealed neutrophils attached to endothelium, perhaps as a step towards transendothelial migration. In addition, platelets were identified in the midst of fibrin as individual procoagulant balloon-like cells. Ultrastructural examination demonstrated numerous virion-like particles., Conclusions: Studying (ultra)structural features of the autopsy lung samples from patients with COVID-19 has provided evidence for a pathogenic link between inflammation and thrombosis. The major features in the lungs of COVID-19 patients comprised primary inflammatory thrombosis associated with diffuse alveolar damage. The lungs had pronounced circulatory changes with inflammation-dependent intravascular blood clotting, whereas heart, brain, and kidneys had predominantly degenerative changes that were distinct from the lung pathology., (© 2021 International Society on Thrombosis and Haemostasis.)

Scanning Electron Microscopy (SEM) is a powerful, high-resolution imaging technique widely used to analyze the structure of fibrin networks. Currently, structural features, such as fiber diameter, length, density, and porosity, are mostly analyzed manually, which is tedious and may introduce user bias. A reliable, automated structural image analysis method would mitigate these drawbacks. We evaluated the performance of DiameterJ (an ImageJ plug-in) for analyzing fibrin fiber diameter by comparing automated DiameterJ outputs with manual diameter measurements in four SEM data sets with different imaging parameters. We also investigated correlations between biophysical fibrin clot properties and diameter, and between clot permeability and DiameterJ-determined clot porosity. Several of the 24 DiameterJ algorithms returned diameter values that highly correlated with and closely matched the values of the manual measurements. However, optimal performance was dependent on the pixel size of the images-best results were obtained for images with a pixel size of 8-10 nm (13-16 pixels/fiber). Larger or smaller pixels resulted in an over- or underestimation of diameter values, respectively. The correlation between clot permeability and DiameterJ-determined clot porosity was modest, likely because it is difficult to establish the correct image depth of field in this analysis. In conclusion, several DiameterJ algorithms (M6, M5, T3) perform well for diameter determination from SEM images, given the appropriate imaging conditions (13-16 pixels/fiber). Determining fibrin clot porosity via DiameterJ is challenging.

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.)

To reveal if coagulopathies relate to the course of COVID-19, we examined 255 patients with moderate and severe COVID-19, receiving anticoagulants and immunosuppressive drugs. Coagulopathy manifested predominantly as hypercoagulability that correlated directly with systemic inflammation, disease severity, comorbidities, and mortality risk. The prolonged clotting tests in about ¼ of cases were associated with high levels of C-reactive protein and antiphospholipid antibodies, which impeded coagulation in vitro. Contraction of blood clots was hindered in about ½ of patients, especially in severe and fatal cases, and correlated directly with prothrombotic parameters. A decrease in platelet contractility was due to moderate thrombocytopenia in combination with platelet dysfunction. Clots with impaired contraction were porous, had a low content of compressed polyhedral erythrocytes (polyhedrocytes) and an even distribution of fibrin, suggesting that the uncompacted intravital clots are more obstructive but patients could also be prone to bleeding. The absence of consumption coagulopathy suggests the predominance of local and/or regional microthrombosis rather than disseminated intravascular coagulation. The results obtained (i) confirm the importance of hemostatic disorders in COVID-19 and their relation to systemic inflammation; (ii) justify monitoring of hemostasis, including the kinetics of blood clot contraction; (iii) substantiate the active prophylaxis of thrombotic complications in COVID-19., (© 2021. The Author(s).)

Although it is well established that transfusion of platelets in cases of severe bleeding reduces mortality, the availability of platelets is hampered by harsh restrictions on shelf life due to elevated risks of microbial contamination and functional losses with room temperature-stored platelets (RTP) kept at 22°C. In contrast, many recent studies have shown that 4°C cold-stored platelets (CSP) are able to overcome these shortcomings leading to the recent Food and Drug Administration licensure for 14-day stored CSP when conventional platelets are unavailable. This work expands the evidence supporting superiority of CSP function by assaying the less explored platelet-mediated clot retraction of RTP and CSP in either autologous plasma (AP) or platelet additive solution (PAS) for up to 21 days. The results demonstrate that CSP have better preservation of contractile function, exhibiting retraction for up to 21 days in both AP and PAS and forming highly ordered fibrin scaffolds similar to those of fresh platelets. In contrast, RTP stored in AP showed impaired contractile function by Day 5 with no retraction after 10 days, whereas PAS-stored RTP retained contractile function for up to 21 days. Collectively, these findings support extended storage of CSP and suggest that storage in PAS can mitigate functional losses in RTP., (© 2021 AABB. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

The fluorescent reporters commonly used to visualize proteins can perturb both protein structure and function. Recently, we found that 4-cyanotryptophan (4CN-Trp), a blue fluorescent amino acid, is suitable for one-photon imaging applications. Here, we demonstrate its utility in two-photon fluorescence microscopy by using it to image integrins on cell surfaces. Specifically, we used solid-phase peptide synthesis to generate CHAMP peptides labeled with 4-cyanoindole (4CNI) at their N-termini to image integrins on cell surfaces. CHAMP (computed helical anti-membrane protein) peptides spontaneously insert into membrane bilayers to target integrin transmembrane domains and cause integrin activation. We found that 4CNI labeling did not perturb the ability of CHAMP peptides to insert into membranes, bind to integrins, or cause integrin activation. We then used two-photon fluorescence microscopy to image 4CNI-containing integrins on the surface of platelets. Compared to a 4CNI-labeled scrambled peptide that uniformly decorated cell surfaces, 4CNI-labeled CHAMP peptides were present in discrete blue foci. To confirm that these foci represented CN peptide-containing integrins, we co-stained platelets with integrin-specific fluorescent monoclonal antibodies and found that CN peptide and antibody fluorescence coincided. Because 4CNI can readily be biosynthetically incorporated into proteins with little if any effect on protein structure and function, it provides a facile way to directly monitor protein behavior and protein-protein interactions in cellular environments. In addition, these results clearly demonstrate that the two-photon excitation cross section of 4CN-Trp is sufficiently large to make it a useful two-photon fluorescence reporter for biological applications.

Hyperhomocysteinemia (HHcy) is associated with thrombosis, but the mechanistic links between them are not understood. We studied effects of homocysteine (Hcy) on clot contraction in vitro and in a rat model of HHcy. Incubation of blood with exogenous Hcy for 1 min enhanced clot contraction, while 15-min incubation led to a dose-dependent suppression of contraction. These effects were likely due to direct Hcy-induced platelet activation followed by exhaustion, as revealed by an increase in fibrinogen-binding capacity and P-selectin expression determined by flow cytometry. In the blood of rats with HHcy, clot contraction was enhanced at moderately elevated Hcy levels (10-50 μM), while at higher Hcy levels (>50 μM), the onset of clot contraction was delayed. HHcy was associated with thrombocytosis combined with a reduced erythrocyte count and hypofibrinogenemia. These data suggest that in HHcy, platelets get activated directly and indirectly, leading to enhanced clot contraction that is facilitated by the reduced content and resilience of fibrin and erythrocytes in the clot. The excessive platelet activation can lead to exhaustion and impaired contractility, which makes clots larger and more obstructive. In conclusion, HHcy modulates blood clot contraction, which may comprise an underappreciated pro- or antithrombotic mechanism.

The severity, course, and outcomes of thrombosis are determined mainly by the size and location of the thrombus, but studying thrombus structure and composition has been an important but challenging task. The substantial progress in determination of thrombus morphology has become possible due to new intravital imaging methodologies in combination with mechanical thrombectomy, which allows extraction of a fresh thrombus from a patient followed by microscopy. Thrombi have been found to contain various structural forms of fibrin along with platelet aggregates, leukocytes, and red blood cells, many of which acquire a polyhedral shape (polyhedrocytes) as a result of intravital platelet-driven contraction. The relative volume fractions of thrombus components and their structural forms vary substantially, depending on the clinical and pathogenic characteristics. This review summarizes recent research that describes quantitative and qualitative morphologic characteristics of arterial and venous thrombi that are relevant for the pathogenesis, prophylaxis, diagnosis, and treatment of thrombosis., (© 2021 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Fibrinogen is a large glycoprotein, synthesized primarily in the liver. With a normal plasma concentration of 1.5-3.5 g/L, fibrinogen is the most abundant blood coagulation factor. The final stage of blood clot formation is the conversion of soluble fibrinogen to insoluble fibrin, the polymeric scaffold for blood clots that stop bleeding (a protective reaction called hemostasis) or obstruct blood vessels (pathological thrombosis). Fibrin is a viscoelastic polymer and the structural and mechanical properties of the fibrin scaffold determine its effectiveness in hemostasis and the development and outcome of thrombotic complications. Fibrin polymerization comprises a number of consecutive reactions, each affecting the ultimate 3D porous network structure. The physical properties of fibrin clots are determined by structural features at the individual fibrin molecule, fibrin fiber, network, and whole clot levels and are among the most important functional characteristics, enabling the blood clot to withstand arterial blood flow, platelet-driven clot contraction, and other dynamic forces. This chapter describes the molecular structure of fibrinogen, the conversion of fibrinogen to fibrin, the mechanical properties of fibrin as well as its structural origins and lastly provides evidence for the role of altered fibrin clot properties in both thrombosis and bleeding.

Rheumatoid arthritis (RA) is an autoimmune disease associated with thrombotic complications. To elucidate pathogenic mechanisms, hemostatic disorders in RA were correlated with other laboratory and clinical manifestations. Hemostasis was assessed using relatively new complementary tests, the spatial growth of a plasma clot (Thrombodynamics assay), and contraction of whole blood clots. Platelet functionality was assessed with flow cytometry that quantified the expression of P-selectin and the fibrinogen-binding capacity of platelets before and after activation with a thrombin receptor-activating peptide. Parameters of fibrin clot growth and the kinetics of contraction of blood clots were significantly altered in patients with RA compared to the control group. In Thrombodynamics measurements, an increase in the clot growth rate, size, and optical density of plasma clots altogether indicated chronic hypercoagulability. The rate and extent of blood clot contraction in patients with RA was significantly reduced and associated with platelet dysfunction revealed by an impaired response to activation. Changes in the parameters of clot growth and contraction correlated with the laboratory signs of systemic inflammation, including hyperfibrinogenemia. These results confirm the pathogenic role of hemostatic disorders in RA and support the validity of fibrin clot growth and the blood clot contraction assay as indicators of a (pro)thrombotic state.

High-resolution atomic force microscopy (AFM) of biomacromolecules is a valuable method for structural studies in biology. Traditionally, the surfaces used for AFM imaging of individual molecules are limited to mica, graphite, and glass. Because these substrates have certain shortcomings, new or modified surfaces that improve the quality of AFM imaging are highly desirable. Here, we describe an improved substrate for imaging of individual biomacromolecules with high-resolution AFM based on graphite surfaces coated by physical adsorption. We provide a detailed methodology, including the chemical structure, synthesis, characterization and the use of a substance that modifies the surface of freshly cleaved graphite, making it suitable for adsorption and AFM visualization of various biomacromolecules while minimizing spatial distortions. We illustrate the advantages of the modified graphite over regular surfaces with examples of high-resolution single-molecule imaging of proteins, polysaccharides, DNA and DNA-protein complexes. The proposed methodology is easy to use and helps to improve substantially AFM imaging of biomacromolecules of various natures, including flexible and/or unstructured sub-molecular regions that are not seen on other AFM substrates. The proposed technique has the potential to improve the use of AFM in structural biology for visualization and morphometric characterization of macromolecular objects., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background and Purpose: The purpose was to assess quantitatively and qualitatively the composition and structure of cerebral thrombi and correlate them with the signs of intravital clot contraction (retraction), as well as with etiology, severity, duration, and outcomes of acute ischemic stroke., Methods: We quantified high-resolution scanning electron micrographs of 41 cerebral thrombi for their detailed cellular and noncellular composition and analyzed histological images for the overall structure with the emphasis on red blood cell compression, fibrin age, and the signs of inflammation., Results: Cerebral thrombi were quite compact and had extremely low porosity. The prevailing cell type was polyhedral compressed erythrocytes (polyhedrocytes) in the core, and fibrin-platelet aggregates were concentrated at the periphery; both findings are indicative of intravital contraction of the thrombi. The content of polyhedrocytes directly correlated with the stroke severity. The prevalence of fibrin bundles was typical for more severe cases, while the content of fibrin sponge prevailed in cases with a more favorable course. The overall platelet content in cerebral thrombi was surprisingly small, while the higher content of platelet aggregates was a marker of stroke severity. Fibrillar types of fibrin prevailed in atherothrombogenic thrombi. Older fibrin prevailed in thrombi from the patients who received thrombolytics, and younger fibrin dominated in cardioembolic thrombi. Alternating layers of erythrocytes and fibrin mixed with platelets were common for thrombi from the patients with more favorable outcomes. Thrombi with a higher number of leukocytes were associated with fatal cases., Conclusions: Most cerebral thrombi undergo intravital clot contraction (retraction) that may be of underestimated clinical importance. Despite the high variability of the composition and structure of cerebral thrombi, the content of certain types of blood cells and fibrin structures combined with the morphological signs of intravital contraction correlate with the clinical course and outcomes of acute ischemic stroke.

Several biological materials are fibre networks infused with fluid, often referred to as fibrous gels. An important feature of these gels is that the fibres buckle under compression, causing a densification of the network that is accompanied by a reduction in volume and release of fluid. Displacement-controlled compression of fibrous gels has shown that the network can exist in a rarefied and a densified state over a range of stresses. Continuum chemo-elastic theories can be used to model the mechanical behaviour of these gels, but they suffer from the drawback that the stored energy function of the underlying network is based on neo-Hookean elasticity, which cannot account for the existence of multiple phases. Here we use a double-well stored energy function in a chemo-elastic model of gels to capture the existence of two phases of the network. We model cyclic compression/decompression experiments on fibrous gels and show that they exhibit propagating interfaces and hysteretic stress-strain curves that have been observed in experiments. We can capture features in the rate-dependent response of these fibrous gels without recourse to finite-element calculations. We also perform experiments to show that certain features in the stress-strain curves of fibrous gels predicted by our model can be found in the compression response of blood clots. Our methods may be extended to other tissues and synthetic gels that have a fibrous structure., Competing Interests: We declare we have no competing interests., (© 2020 The Author(s).)

Deep vein thrombosis (DVT) is a common but unpredictable complication of surgical interventions. To reveal an association between the blood clot contraction (retraction) and the incidence of postoperative venous thrombosis, 78 patients with brain tumors that were operated on were studied, of which 23 (29%) were diagnosed with postoperative DVT. A clot contraction assay, along with other hemostatic and hematologic tests, was performed 1-3 days before the surgery and on the 1st day and 5-7th days after the surgery. On the 1st postoperative day, clot contraction was significantly suppressed in patients who subsequently developed DVT, compared to the patients without DVT. Importantly, this difference was observed at least 5 days before DVT had developed. The weakening of contraction on the 1st postoperative day was more pronounced in the DVT patients with malignant versus benign brain tumors, atherosclerosis, hypertension, as well as in patients receiving steroids before and during the operation. These results indicate that impaired clot contraction in the postoperative period is associated with imminent DVT, suggesting that it is a prothrombotic risk factor and promotional mechanism. The clot contraction assay has a predictive value in assessing the threat of postoperative thrombosis in patients with benign and malignant brain tumors.