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John W. Weisel
Department of Cell and Developmental Biology University of Pennsylvania Perelman School of Medicine Philadelphia Pennsylvania USA

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Journal article
Published: 11 August 2021 in Scientific Reports
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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.

ACS Style

Rustem I. Litvinov; Natalia G. Evtugina; Alina D. Peshkova; Svetlana I. Safiullina; Izabella A. Andrianova; Alina I. Khabirova; Chandrasekaran Nagaswami; Rafael R. Khismatullin; Svetlana S. Sannikova; John W. Weisel. Altered platelet and coagulation function in moderate-to-severe COVID-19. Scientific Reports 2021, 11, 1 .

AMA Style

Rustem I. Litvinov, Natalia G. Evtugina, Alina D. Peshkova, Svetlana I. Safiullina, Izabella A. Andrianova, Alina I. Khabirova, Chandrasekaran Nagaswami, Rafael R. Khismatullin, Svetlana S. Sannikova, John W. Weisel. Altered platelet and coagulation function in moderate-to-severe COVID-19. Scientific Reports. 2021; 11 ():1.

Chicago/Turabian Style

Rustem I. Litvinov; Natalia G. Evtugina; Alina D. Peshkova; Svetlana I. Safiullina; Izabella A. Andrianova; Alina I. Khabirova; Chandrasekaran Nagaswami; Rafael R. Khismatullin; Svetlana S. Sannikova; John W. Weisel. 2021. "Altered platelet and coagulation function in moderate-to-severe COVID-19." Scientific Reports 11, no. : 1.

Original article
Published: 07 July 2021 in Journal of Thrombosis and Haemostasis
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Background Blood clot contraction, volume shrinkage of the clot, is driven by platelet contraction and accompanied by compaction of the erythrocytes and their gradual shape change from biconcave to polyhedral, with the resulting cells named polyhedrocytes. Objectives Here, we examined the role of erythrocyte rigidity on clot contraction and erythrocyte shape transformation. Methods We used an optical tracking methodology that allowed us to quantify changes in contracting clot size over time. Results and conclusions Erythrocyte rigidity has been shown to be increased in sickle cell disease (SCD), and in our experiments erythrocytes from SCD patients were 4-fold stiffer than those from healthy subjects. On average, the final extent of clot contraction was reduced by 53% in the clots from the blood of patients with SCD compared to healthy individuals, and there was significantly less polyhedrocyte formation. To test if this reduction in clot contraction was due to the increase in erythrocyte rigidity, we used stiffening of erythrocytes via chemical cross-linking (glutaraldehyde), rigidifying Wrightb antibodies (Wrb), and naturally more rigid llama ovalocytes. Results revealed that stiffening erythrocytes result in impaired clot contraction and fewer polyhedrocytes. These results demonstrate the role of erythrocyte rigidity in the contraction of blood clots and suggest that the impaired clot contraction/shrinkage in SCD is due to the reduced erythrocyte deformability, which may be an underappreciated mechanism that aggravates obstructiveness of erythrocyte-rich (micro)thrombi in SCD.

ACS Style

Valerie Tutwiler; Rustem I. Litvinov; Anna Protopopova; Chandrasekaran Nagaswami; Carlos Villa; Eric Woods; Osheiza Abdulmalik; Don L. Siegel; J. Eric Russell; Vladimir R. Muzykantov; Wilbur A. Lam; David R. Myers; John W. Weisel. Pathologically stiff erythrocytes impede contraction of blood clots. Journal of Thrombosis and Haemostasis 2021, 19, 1990 -2001.

AMA Style

Valerie Tutwiler, Rustem I. Litvinov, Anna Protopopova, Chandrasekaran Nagaswami, Carlos Villa, Eric Woods, Osheiza Abdulmalik, Don L. Siegel, J. Eric Russell, Vladimir R. Muzykantov, Wilbur A. Lam, David R. Myers, John W. Weisel. Pathologically stiff erythrocytes impede contraction of blood clots. Journal of Thrombosis and Haemostasis. 2021; 19 (8):1990-2001.

Chicago/Turabian Style

Valerie Tutwiler; Rustem I. Litvinov; Anna Protopopova; Chandrasekaran Nagaswami; Carlos Villa; Eric Woods; Osheiza Abdulmalik; Don L. Siegel; J. Eric Russell; Vladimir R. Muzykantov; Wilbur A. Lam; David R. Myers; John W. Weisel. 2021. "Pathologically stiff erythrocytes impede contraction of blood clots." Journal of Thrombosis and Haemostasis 19, no. 8: 1990-2001.

Journal article
Published: 05 July 2021 in Acta Biomaterialia
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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. 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.

ACS Style

Valerie Tutwiler; Farkhad Maksudov; Rustem I. Litvinov; John W. Weisel; Valeri Barsegov. Strength and deformability of fibrin clots: Biomechanics, thermodynamics, and mechanisms of rupture. Acta Biomaterialia 2021, 131, 355 -369.

AMA Style

Valerie Tutwiler, Farkhad Maksudov, Rustem I. Litvinov, John W. Weisel, Valeri Barsegov. Strength and deformability of fibrin clots: Biomechanics, thermodynamics, and mechanisms of rupture. Acta Biomaterialia. 2021; 131 ():355-369.

Chicago/Turabian Style

Valerie Tutwiler; Farkhad Maksudov; Rustem I. Litvinov; John W. Weisel; Valeri Barsegov. 2021. "Strength and deformability of fibrin clots: Biomechanics, thermodynamics, and mechanisms of rupture." Acta Biomaterialia 131, no. : 355-369.

Journal article
Published: 01 June 2021 in Metabolites
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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.

ACS Style

Rustem Litvinov; Alina Peshkova; Giang Le Minh; Nail Khaertdinov; Natalia Evtugina; Guzel Sitdikova; John Weisel. Effects of Hyperhomocysteinemia on the Platelet-Driven Contraction of Blood Clots. Metabolites 2021, 11, 354 .

AMA Style

Rustem Litvinov, Alina Peshkova, Giang Le Minh, Nail Khaertdinov, Natalia Evtugina, Guzel Sitdikova, John Weisel. Effects of Hyperhomocysteinemia on the Platelet-Driven Contraction of Blood Clots. Metabolites. 2021; 11 (6):354.

Chicago/Turabian Style

Rustem Litvinov; Alina Peshkova; Giang Le Minh; Nail Khaertdinov; Natalia Evtugina; Guzel Sitdikova; John Weisel. 2021. "Effects of Hyperhomocysteinemia on the Platelet-Driven Contraction of Blood Clots." Metabolites 11, no. 6: 354.

Obituary
Published: 15 April 2021 in Metabolites
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Note: In lieu of an abstract, this is an excerpt from the first page. Prof

ACS Style

Leona Mášová-Chrastinová; John W. Weisel. Obituary for Professor Dr. Jan Evangelista Dyr. Metabolites 2021, 11, 243 .

AMA Style

Leona Mášová-Chrastinová, John W. Weisel. Obituary for Professor Dr. Jan Evangelista Dyr. Metabolites. 2021; 11 (4):243.

Chicago/Turabian Style

Leona Mášová-Chrastinová; John W. Weisel. 2021. "Obituary for Professor Dr. Jan Evangelista Dyr." Metabolites 11, no. 4: 243.

Review
Published: 20 January 2021 in Viruses
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Although ACE2 (angiotensin converting enzyme 2) is considered the primary receptor for CoV-2 cell entry, recent reports suggest that alternative pathways may contribute. This paper considers the hypothesis that viral binding to cell-surface integrins may contribute to the high infectivity and widespread extra-pulmonary impacts of the SARS-CoV-2 virus. This potential is suggested on the basis of the emergence of an RGD (arginine-glycine-aspartate) sequence in the receptor-binding domain of the spike protein. RGD is a motif commonly used by viruses to bind cell-surface integrins. Numerous signaling pathways are mediated by integrins and virion binding could lead to dysregulation of these pathways, with consequent tissue damage. Integrins on the surfaces of pneumocytes, endothelial cells and platelets may be vulnerable to CoV-2 virion binding. For instance, binding of intact virions to integrins on alveolar cells could enhance viral entry. Binding of virions to integrins on endothelial cells could activate angiogenic cell signaling pathways; dysregulate integrin-mediated signaling pathways controlling developmental processes; and precipitate endothelial activation to initiate blood clotting. Such a procoagulant state, perhaps together with enhancement of platelet aggregation through virions binding to integrins on platelets, could amplify the production of microthrombi that pose the threat of pulmonary thrombosis and embolism, strokes and other thrombotic consequences. The susceptibility of different tissues to virion–integrin interactions may be modulated by a host of factors, including the conformation of relevant integrins and the impact of the tissue microenvironment on spike protein conformation. Patient-specific differences in these factors may contribute to the high variability of clinical presentation. There is danger that the emergence of receptor-binding domain mutations that increase infectivity may also enhance access of the RGD motif for integrin binding, resulting in viral strains with ACE2 independent routes of cell entry and novel integrin-mediated biological and clinical impacts. The highly infectious variant, B.1.1.7 (or VUI 202012/01), includes a receptor-binding domain amino acid replacement, N501Y, that could potentially provide the RGD motif with enhanced access to cell-surface integrins, with consequent clinical impacts.

ACS Style

Lee Makowski; William Olson-Sidford; John W.-Weisel. Biological and Clinical Consequences of Integrin Binding via a Rogue RGD Motif in the SARS CoV-2 Spike Protein. Viruses 2021, 13, 146 .

AMA Style

Lee Makowski, William Olson-Sidford, John W.-Weisel. Biological and Clinical Consequences of Integrin Binding via a Rogue RGD Motif in the SARS CoV-2 Spike Protein. Viruses. 2021; 13 (2):146.

Chicago/Turabian Style

Lee Makowski; William Olson-Sidford; John W.-Weisel. 2021. "Biological and Clinical Consequences of Integrin Binding via a Rogue RGD Motif in the SARS CoV-2 Spike Protein." Viruses 13, no. 2: 146.

State of the art isth 2020
Published: 06 January 2021 in Research and Practice in Thrombosis and Haemostasis
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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.

ACS Style

John W. Weisel; Rustem I. Litvinov Md. Visualizing thrombosis to improve thrombus resolution. Research and Practice in Thrombosis and Haemostasis 2021, 5, 38 -50.

AMA Style

John W. Weisel, Rustem I. Litvinov Md. Visualizing thrombosis to improve thrombus resolution. Research and Practice in Thrombosis and Haemostasis. 2021; 5 (1):38-50.

Chicago/Turabian Style

John W. Weisel; Rustem I. Litvinov Md. 2021. "Visualizing thrombosis to improve thrombus resolution." Research and Practice in Thrombosis and Haemostasis 5, no. 1: 38-50.

Journal article
Published: 11 December 2020 in International Journal of Molecular Sciences
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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.

ACS Style

Alina Peshkova; Tatiana Evdokimova; Timur Sibgatullin; Fazoil Ataullakhanov; Rustem Litvinov; John Weisel. Accelerated Spatial Fibrin Growth and Impaired Contraction of Blood Clots in Patients with Rheumatoid Arthritis. International Journal of Molecular Sciences 2020, 21, 9434 .

AMA Style

Alina Peshkova, Tatiana Evdokimova, Timur Sibgatullin, Fazoil Ataullakhanov, Rustem Litvinov, John Weisel. Accelerated Spatial Fibrin Growth and Impaired Contraction of Blood Clots in Patients with Rheumatoid Arthritis. International Journal of Molecular Sciences. 2020; 21 (24):9434.

Chicago/Turabian Style

Alina Peshkova; Tatiana Evdokimova; Timur Sibgatullin; Fazoil Ataullakhanov; Rustem Litvinov; John Weisel. 2020. "Accelerated Spatial Fibrin Growth and Impaired Contraction of Blood Clots in Patients with Rheumatoid Arthritis." International Journal of Molecular Sciences 21, no. 24: 9434.

Research article
Published: 01 December 2020 in Stroke
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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.

ACS Style

Rafael R. Khismatullin; Chandrasekaran Nagaswami; Asia Z. Shakirova; Adéla Vrtková; Václav Procházka; Jaromír Gumulec; Jiří Mačák; Rustem I. Litvinov; John W. Weisel. Quantitative Morphology of Cerebral Thrombi Related to Intravital Contraction and Clinical Features of Ischemic Stroke. Stroke 2020, 51, 3640 -3650.

AMA Style

Rafael R. Khismatullin, Chandrasekaran Nagaswami, Asia Z. Shakirova, Adéla Vrtková, Václav Procházka, Jaromír Gumulec, Jiří Mačák, Rustem I. Litvinov, John W. Weisel. Quantitative Morphology of Cerebral Thrombi Related to Intravital Contraction and Clinical Features of Ischemic Stroke. Stroke. 2020; 51 (12):3640-3650.

Chicago/Turabian Style

Rafael R. Khismatullin; Chandrasekaran Nagaswami; Asia Z. Shakirova; Adéla Vrtková; Václav Procházka; Jaromír Gumulec; Jiří Mačák; Rustem I. Litvinov; John W. Weisel. 2020. "Quantitative Morphology of Cerebral Thrombi Related to Intravital Contraction and Clinical Features of Ischemic Stroke." Stroke 51, no. 12: 3640-3650.

Research article
Published: 26 August 2020 in Science Advances
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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/m2. 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.

ACS Style

Valerie Tutwiler; Jaspreet Singh; Rustem I. Litvinov; John L. Bassani; Prashant K. Purohit; John W. Weisel. Rupture of blood clots: Mechanics and pathophysiology. Science Advances 2020, 6, eabc0496 .

AMA Style

Valerie Tutwiler, Jaspreet Singh, Rustem I. Litvinov, John L. Bassani, Prashant K. Purohit, John W. Weisel. Rupture of blood clots: Mechanics and pathophysiology. Science Advances. 2020; 6 (35):eabc0496.

Chicago/Turabian Style

Valerie Tutwiler; Jaspreet Singh; Rustem I. Litvinov; John L. Bassani; Prashant K. Purohit; John W. Weisel. 2020. "Rupture of blood clots: Mechanics and pathophysiology." Science Advances 6, no. 35: eabc0496.

Paper
Published: 17 August 2020 in Soft Matter
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The blood clotting protein fibrin has extraordinary elastomeric properties due to its hierarchical structure. SAXS is combined with computational molecular modeling, providing insight in fibrin elasticity and guidelines for designing new polymers.

ACS Style

Karin A. Jansen; Artem Zhmurov; Bart E. Vos; Giuseppe Portale; Daniel Hermida-Merino; Rustem I. Litvinov; Valerie Tutwiler; Nicholas A. Kurniawan; Wim Bras; John W. Weisel; Valeri Barsegov; Gijsje H. Koenderink. Molecular packing structure of fibrin fibers resolved by X-ray scattering and molecular modeling. Soft Matter 2020, 16, 8272 -8283.

AMA Style

Karin A. Jansen, Artem Zhmurov, Bart E. Vos, Giuseppe Portale, Daniel Hermida-Merino, Rustem I. Litvinov, Valerie Tutwiler, Nicholas A. Kurniawan, Wim Bras, John W. Weisel, Valeri Barsegov, Gijsje H. Koenderink. Molecular packing structure of fibrin fibers resolved by X-ray scattering and molecular modeling. Soft Matter. 2020; 16 (35):8272-8283.

Chicago/Turabian Style

Karin A. Jansen; Artem Zhmurov; Bart E. Vos; Giuseppe Portale; Daniel Hermida-Merino; Rustem I. Litvinov; Valerie Tutwiler; Nicholas A. Kurniawan; Wim Bras; John W. Weisel; Valeri Barsegov; Gijsje H. Koenderink. 2020. "Molecular packing structure of fibrin fibers resolved by X-ray scattering and molecular modeling." Soft Matter 16, no. 35: 8272-8283.

Journal article
Published: 16 August 2020 in Colloids and Surfaces B: Biointerfaces
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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.

ACS Style

Dmitry V. Klinov; Anna D. Protopopova; Dmitry S. Andrianov; Rustem I. Litvinov; John W. Weisel. An Improved Substrate for Superior Imaging of Individual Biomacromolecules with Atomic Force Microscopy. Colloids and Surfaces B: Biointerfaces 2020, 196, 111321 .

AMA Style

Dmitry V. Klinov, Anna D. Protopopova, Dmitry S. Andrianov, Rustem I. Litvinov, John W. Weisel. An Improved Substrate for Superior Imaging of Individual Biomacromolecules with Atomic Force Microscopy. Colloids and Surfaces B: Biointerfaces. 2020; 196 ():111321.

Chicago/Turabian Style

Dmitry V. Klinov; Anna D. Protopopova; Dmitry S. Andrianov; Rustem I. Litvinov; John W. Weisel. 2020. "An Improved Substrate for Superior Imaging of Individual Biomacromolecules with Atomic Force Microscopy." Colloids and Surfaces B: Biointerfaces 196, no. : 111321.

Journal article
Published: 07 April 2020 in International Journal of Molecular Sciences
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Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by thrombocytopenia and a high risk for venous or arterial thrombosis. HIT is caused by antibodies that recognize complexes of platelet factor 4 and heparin. The pathogenic mechanisms of this condition are not fully understood. In this study, we used flow cytometry, fluorimetry, and Western blot analysis to study the direct effects of pathogenic immune complexes containing platelet factor 4 on human platelets isolated by gel-filtration. HIT-like pathogenic immune complexes initially caused pronounced activation of platelets detected by an increased expression of phosphatidylserine and P-selectin. This activation was mediated either directly through the FcγRIIA receptors or indirectly via protease-activated receptor 1 (PAR1) receptors due to thrombin generated on or near the surface of activated platelets. The immune activation was later followed by the biochemical signs of cell death, such as mitochondrial membrane depolarization, up-regulation of Bax, down-regulation of Bcl-XL, and moderate activation of procaspase 3 and increased calpain activity. The results show that platelet activation under the action of HIT-like immune complexes is accompanied by their death through complex apoptotic and calpain-dependent non-apoptotic pathways that may underlie the low platelet count in HIT.

ACS Style

Elmira R. Mordakhanova; Tatiana A. Nevzorova; Gulnaz Synbulatova; Lubica Rauova; John W. Weisel; Rustem I. Litvinov. Platelet Activation in Heparin-Induced Thrombocytopenia is Followed by Platelet Death via Complex Apoptotic and Non-Apoptotic Pathways. International Journal of Molecular Sciences 2020, 21, 2556 .

AMA Style

Elmira R. Mordakhanova, Tatiana A. Nevzorova, Gulnaz Synbulatova, Lubica Rauova, John W. Weisel, Rustem I. Litvinov. Platelet Activation in Heparin-Induced Thrombocytopenia is Followed by Platelet Death via Complex Apoptotic and Non-Apoptotic Pathways. International Journal of Molecular Sciences. 2020; 21 (7):2556.

Chicago/Turabian Style

Elmira R. Mordakhanova; Tatiana A. Nevzorova; Gulnaz Synbulatova; Lubica Rauova; John W. Weisel; Rustem I. Litvinov. 2020. "Platelet Activation in Heparin-Induced Thrombocytopenia is Followed by Platelet Death via Complex Apoptotic and Non-Apoptotic Pathways." International Journal of Molecular Sciences 21, no. 7: 2556.

Journal article
Published: 20 March 2020 in Scientific Reports
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Although arterial and venous thromboembolic disorders are among the most frequent causes of mortality and morbidity, there has been little description of how the composition of thrombi and emboli depends on their vascular origin and age. We quantified the structure and composition of arterial and venous thrombi and pulmonary emboli using high-resolution scanning electron microscopy. Arterial thrombi contained a surprisingly large amount of fibrin, in addition to platelets. The composition of pulmonary emboli mirrored the most distal part of venous thrombi from which they originated, which differed from the structure of the body and head of the same thrombi. All thrombi and emboli contained few biconcave red blood cells but many polyhedrocytes or related forms of compressed red blood cells, demonstrating that these structures are a signature of clot contraction in vivo. Polyhedrocytes and intermediate forms comprised the major constituents of venous thrombi and pulmonary emboli. The structures within all of the thrombi and emboli were very tightly packed, in contrast to clots formed in vitro. There are distinctive, reproducible differences among arterial and venous thrombi and emboli related to their origin, destination and duration, which may have clinical implications for the understanding and treatment of thrombotic disorders.

ACS Style

Irina N. Chernysh; Chandrasekaran Nagaswami; Sofia Kosolapova; Alina D. Peshkova; Adam Cuker; Douglas B. Cines; Carolyn L. Cambor; Rustem I. Litvinov; John W. Weisel. The distinctive structure and composition of arterial and venous thrombi and pulmonary emboli. Scientific Reports 2020, 10, 1 -12.

AMA Style

Irina N. Chernysh, Chandrasekaran Nagaswami, Sofia Kosolapova, Alina D. Peshkova, Adam Cuker, Douglas B. Cines, Carolyn L. Cambor, Rustem I. Litvinov, John W. Weisel. The distinctive structure and composition of arterial and venous thrombi and pulmonary emboli. Scientific Reports. 2020; 10 (1):1-12.

Chicago/Turabian Style

Irina N. Chernysh; Chandrasekaran Nagaswami; Sofia Kosolapova; Alina D. Peshkova; Adam Cuker; Douglas B. Cines; Carolyn L. Cambor; Rustem I. Litvinov; John W. Weisel. 2020. "The distinctive structure and composition of arterial and venous thrombi and pulmonary emboli." Scientific Reports 10, no. 1: 1-12.

Journal article
Published: 19 February 2020 in Journal of the Mechanical Behavior of Biomedical Materials
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Pulmonary embolism occurs when blood flow to a part of the lungs is blocked by a venous thrombus that has traveled from the lower limbs. Little is known about the mechanical behavior of emboli under compressive forces from the surrounding musculature and blood pressure. We measured the stress-strain responses of human pulmonary emboli under cyclic compression, and showed that emboli exhibit a hysteretic stress-strain curve. The fibrin fibers and red blood cells (RBCs) are damaged during the compression process, causing irreversible changes in the structure of the emboli. We showed using electron and confocal microscopy that bundling of fibrin fibers occurs due to compression, and damage is accumulated as more cycles are applied. The stress-strain curves depend on embolus structure, such that variations in composition give quantitatively different responses. Emboli with a high fibrin component demonstrate higher normal stress compared to emboli that have a high RBC component. We compared the compression response of emboli to that of whole blood clots containing various volume fractions of RBCs, and found that RBCs rupture at a certain critical stress. We describe the hysteretic response characteristic of foams, using a model of phase transitions in which the compressed foam is segregated into coexisting rarefied and densified phases whose fractions change during compression. Our model takes account of the rupture of RBCs in the compressed emboli and stresses due to fluid flow through their small pores. Our results can help in classifying emboli as rich in fibrin or rich in red blood cells, and can help in understanding what responses to expect when stresses are applied to thrombi in vivo.

ACS Style

Irina N. Chernysh; Russell Spiewak; Carolyn L. Cambor; Prashant K. Purohit; John W. Weisel. Structure, mechanical properties, and modeling of cyclically compressed pulmonary emboli. Journal of the Mechanical Behavior of Biomedical Materials 2020, 105, 103699 .

AMA Style

Irina N. Chernysh, Russell Spiewak, Carolyn L. Cambor, Prashant K. Purohit, John W. Weisel. Structure, mechanical properties, and modeling of cyclically compressed pulmonary emboli. Journal of the Mechanical Behavior of Biomedical Materials. 2020; 105 ():103699.

Chicago/Turabian Style

Irina N. Chernysh; Russell Spiewak; Carolyn L. Cambor; Prashant K. Purohit; John W. Weisel. 2020. "Structure, mechanical properties, and modeling of cyclically compressed pulmonary emboli." Journal of the Mechanical Behavior of Biomedical Materials 105, no. : 103699.

Journal article
Published: 12 November 2019 in Journal of Autoimmunity
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Systemic lupus erythematosus (SLE) is associated with a high risk of venous and arterial thrombosis, not necessarily associated with prothrombotic antiphospholipid antibodies (Abs). Alternatively, thrombosis may be due to an increased titer of anti-dsDNA Abs that presumably promote thrombosis via direct platelet activation. Here, we investigated effects of purified anti-dsDNA Abs from the blood of SLE patients, alone or in a complex with dsDNA, on isolated normal human platelets. We showed that anti-dsDNA Abs and anti-dsDNA Ab/dsDNA complexes induced strong platelet activation assessed by enhanced P-selectin expression and dramatic morphological and ultrastructural changes. Electron microscopy revealed a significantly higher percentage of platelets that lost their discoid shape, formed multiple filopodia and had a shrunken body when treated with anti-dsDNA Abs or anti-dsDNA Ab/dsDNA complexes compared with control samples. In addition, these platelets activated with anti-dsDNA Ab/dsDNA complexes typically contained a reduced number of secretory α-granules that grouped in the middle and often merged into a solid electron dense area. Many activated platelets released plasma membrane-derived microvesicles and/or fell apart into subcellular cytoplasmic fragments. Confocal microscopy revealed that platelets treated with anti-dsDNA Ab/dsDNA complex had a heterogeneous distribution of septin2 compared with the homogeneous distribution in control platelets. Structural perturbations were concomitant with mitochondrial depolarization and a decreased content of platelet ATP, indicating energetic exhaustion. Most of the biochemical and morphological changes in platelets induced by anti-dsDNA Abs and anti-dsDNA Ab/dsDNA complexes were prevented by pre-treatment with a monoclonal mAb against FcγRIIA. The aggregate of data indicates that anti-dsDNA Abs alone or in a complex with dsDNA strongly affect platelets via the FcγRIIA receptor. The immune activation of platelets with antinuclear Abs may comprise a prothrombotic mechanism underlying a high risk of thrombotic complications in patients with SLE.

ACS Style

Izabella A. Andrianova; Anastasiya A. Ponomareva; Elmira R. Mordakhanova; Giang Le Minh; Amina G. Daminova; Tatiana A. Nevzorova; Lubica Rauova; Rustem I. Litvinov; John W. Weisel. In systemic lupus erythematosus anti-dsDNA antibodies can promote thrombosis through direct platelet activation. Journal of Autoimmunity 2019, 107, 102355 .

AMA Style

Izabella A. Andrianova, Anastasiya A. Ponomareva, Elmira R. Mordakhanova, Giang Le Minh, Amina G. Daminova, Tatiana A. Nevzorova, Lubica Rauova, Rustem I. Litvinov, John W. Weisel. In systemic lupus erythematosus anti-dsDNA antibodies can promote thrombosis through direct platelet activation. Journal of Autoimmunity. 2019; 107 ():102355.

Chicago/Turabian Style

Izabella A. Andrianova; Anastasiya A. Ponomareva; Elmira R. Mordakhanova; Giang Le Minh; Amina G. Daminova; Tatiana A. Nevzorova; Lubica Rauova; Rustem I. Litvinov; John W. Weisel. 2019. "In systemic lupus erythematosus anti-dsDNA antibodies can promote thrombosis through direct platelet activation." Journal of Autoimmunity 107, no. : 102355.

Preprint
Published: 08 October 2019
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Fibrin is an elastomeric protein forming highly extensible fiber networks that provide the scaffold of blood clots. Here we reveal the molecular mechanisms that explain the large extensibility of fibrin networks by performingin situsmall angle X-ray scattering measurements while applying a shear deformation. We simultaneously measure shear-induced alignment of the fibers and changes in their axially ordered molecular packing structure. We show that fibrin networks exhibit distinct structural responses that set in consecutively as the shear strain is increased. They exhibit an entropic response at small strains (25% strain) and finally changes in the fiber packing structure at high strain (>100%). Stretching reduces the fiber packing order and slightly increases the axial periodicity, indicative of molecular unfolding. However, the axial periodicity changes only by 0.7%, much less than the 80% length increase of the fibers, indicating that fiber elongation mainly stems from uncoiling of the natively disordered αC-peptide linkers that laterally bond the molecules. Upon removal of the load, the network structure returns to the original isotropic state, but the fiber structure becomes more ordered and adopts a smaller packing periodicity compared to the original state. We conclude that the hierarchical packing structure of fibrin fibers, with built-in disorder, makes the fibers extensible and allows for mechanical annealing. Our results provide a basis for interpreting the molecular basis of haemostatic and thrombotic disorders associated with clotting and provide inspiration to design resilient bio-mimicking materials.

ACS Style

Bart Eduard Vos; Cristina Martinez-Torres; Federica Burla; John W. Weisel; Gijsje H. Koenderink; Gijsje H. Koendernik. Revealing the molecular origins of fibrin’s elastomeric properties by in situ X-ray scattering. 2019, 797464 .

AMA Style

Bart Eduard Vos, Cristina Martinez-Torres, Federica Burla, John W. Weisel, Gijsje H. Koenderink, Gijsje H. Koendernik. Revealing the molecular origins of fibrin’s elastomeric properties by in situ X-ray scattering. . 2019; ():797464.

Chicago/Turabian Style

Bart Eduard Vos; Cristina Martinez-Torres; Federica Burla; John W. Weisel; Gijsje H. Koenderink; Gijsje H. Koendernik. 2019. "Revealing the molecular origins of fibrin’s elastomeric properties by in situ X-ray scattering." , no. : 797464.

Original research article
Published: 16 July 2019 in Frontiers in Immunology
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Among complications of systemic lupus erythematosus (SLE), thrombotic events are relatively common and contribute significantly to the morbidity and mortality rates. An increased risk of thrombosis in various diseases has been shown to be associated with the lytic stability and mechanical stiffness of the fibrin clot determined by its structure. Here we studied alterations of the fibrin clot properties in relation to disease severity in SLE patients. Plasma clots from 28 SLE patients were characterized by the kinetics of formation and fibrinolytic dissolution (using dynamic turbidimetry), the network and fiber ultrastructure (scanning electron microscopy), viscoelasticity (shear rheometry), and the rate and degree of crosslinking (Western blotting) correlated with the disease activity, blood composition, and compared to clotting of pooled normal human plasma. Clots made from plasma of SLE patients were lysed faster with exogenous t-PA than control clots from normal plasma without a significant difference between those from active (SLEDAI>4) and inactive (SLEDAI<4) SLE patients. Clots from the blood of patients with active SLE were characterized by significantly slower onset, but faster rate of fibrin polymerization and a higher optical density due to thicker fibers compared to those from inactive SLE and control pooled normal plasma. The rheological parameters of the clots (storage and loss moduli) were significantly increased in the active SLE patients along with enhanced fibrin crosslinking and hyperfibrinogenemia. The structural and rheological alterations displayed a strong positive correlation with high fibrinogen levels and other laboratory markers of immune inflammation. In conclusion, changes in the blood composition associated with active systemic inflammation in SLE cause significant alterations in the lytic resistance of fibrin clots associated with changes in polymerization kinetics, viscoelastic properties, and structure. The formation of more rigid prothrombotic fibrin clots in the plasma of SLE patients is likely due to the inflammatory hyperfibrinogenemia and greater extent of crosslinking. However, the higher susceptibility of the SLE clots to fibrinolysis may be a protective and/or compensatory mechanism that reduces the risk of thrombotic complications and improves patient outcomes.

ACS Style

Rustem I. Litvinov; Rosa M. Nabiullina; Laily D. Zubairova; Mileusha A. Shakurova; Izabella A. Andrianova; John W. Weisel. Lytic Susceptibility, Structure, and Mechanical Properties of Fibrin in Systemic Lupus Erythematosus. Frontiers in Immunology 2019, 10, 1626 .

AMA Style

Rustem I. Litvinov, Rosa M. Nabiullina, Laily D. Zubairova, Mileusha A. Shakurova, Izabella A. Andrianova, John W. Weisel. Lytic Susceptibility, Structure, and Mechanical Properties of Fibrin in Systemic Lupus Erythematosus. Frontiers in Immunology. 2019; 10 ():1626.

Chicago/Turabian Style

Rustem I. Litvinov; Rosa M. Nabiullina; Laily D. Zubairova; Mileusha A. Shakurova; Izabella A. Andrianova; John W. Weisel. 2019. "Lytic Susceptibility, Structure, and Mechanical Properties of Fibrin in Systemic Lupus Erythematosus." Frontiers in Immunology 10, no. : 1626.

Journal article
Published: 24 June 2019 in Cell Death Discovery
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Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy sometimes associated with thrombosis. The hallmark of HIT is antibodies to the heparin/platelet factor 4 (PF4) complex that cause thrombocytopenia and thrombosis through platelet activation. Despite the clinical importance, the molecular mechanisms and late consequences of immune platelet activation are not fully understood. Here, we studied immediate and delayed effects of the complexes formed by human PF4 and HIT-like monoclonal mouse anti-human-PF4/heparin IgG antibodies (named KKO) on isolated human platelets in vitro. Direct platelet-activating effect of the KKO/PF4 complexes was corroborated by the overexpression of phosphatidylserine (PS) and P-selectin on the platelet surface. The immune platelet activation was accompanied by a decrease of the mitochondrial transmembrane potential (ΔΨm), concurrent with a significant gradual reduction of the ATP content in platelets, indicating disruption of energy metabolism. A combination of PS expression and mitochondrial depolarization induced by the PF4-containing immune complexes observed in a substantial fraction of platelets was considered as a sign of ongoing platelet death, as opposed to a subpopulation of activated live platelets with PS on the plasma membrane but normal ΔΨm. Both activated and dying platelets treated with KKO/PF4 formed procoagulant extracellular microvesicles bearing PS on their surface. Scanning and transmission electron microscopy revealed dramatic morphological changes of KKO/PF4-treated platelets, including their fragmentation, another indicator of cell death. Most of the effects of KKO/PF4 were prevented by an anti-FcγRII monoclonal antibody IV.3. The adverse functional and structural changes in platelets induced by the KKO/PF4 complexes were associated with strong time-dependent activation of calpain, but only trace cleavage of caspase 3. The results indicate that the pathogenic PF4-containing HIT-like immune complexes induce direct prothrombotic platelet activation via FcγRIIA receptors followed by non-apoptotic calpain-dependent death of platelets, which can be an important mechanism of thrombocytopenia during HIT development.

ACS Style

Tatiana A. Nevzorova; Elmira R. Mordakhanova; Amina G. Daminova; Anastasia A. Ponomareva; Izabella A. Andrianova; Giang Le Minh; Lubica Rauova; Rustem I. Litvinov; John W. Weisel. Platelet factor 4-containing immune complexes induce platelet activation followed by calpain-dependent platelet death. Cell Death Discovery 2019, 5, 1 -12.

AMA Style

Tatiana A. Nevzorova, Elmira R. Mordakhanova, Amina G. Daminova, Anastasia A. Ponomareva, Izabella A. Andrianova, Giang Le Minh, Lubica Rauova, Rustem I. Litvinov, John W. Weisel. Platelet factor 4-containing immune complexes induce platelet activation followed by calpain-dependent platelet death. Cell Death Discovery. 2019; 5 (1):1-12.

Chicago/Turabian Style

Tatiana A. Nevzorova; Elmira R. Mordakhanova; Amina G. Daminova; Anastasia A. Ponomareva; Izabella A. Andrianova; Giang Le Minh; Lubica Rauova; Rustem I. Litvinov; John W. Weisel. 2019. "Platelet factor 4-containing immune complexes induce platelet activation followed by calpain-dependent platelet death." Cell Death Discovery 5, no. 1: 1-12.

Journal article
Published: 30 May 2019 in Acta Biomaterialia
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Fibrin is a viscoelastic proteinaceous polymer that determines the deformability and integrity of blood clots and fibrin-based biomaterials in response to biomechanical forces. Here, a previously unnoticed structural mechanism of fibrin clots' mechanical response to external tensile loads is tested using high-resolution confocal microscopy and recently developed three-dimensional computational model. This mechanism, underlying local strain-stiffening of individual fibers as well as global stiffening of the entire network, is based on previously neglected nascent cohesive pairwise interactions between individual fibers (crisscrossing) in fibrin networks formed under tensile load. Existence of fiber-fiber crisscrossings of reoriented fibers was confirmed using 3D imaging of experimentally obtained stretched fibrin clots. The computational model enabled us to study structural details and quantify mechanical effects of the fiber-fiber cohesive crisscrossing during stretching of fibrin gels at various spatial scales. The contribution of the fiber-fiber cohesive contacts to the elasticity of stretched fibrin networks was characterized by changes in individual fiber stiffness, the length, width, and alignment of fibers, as well as connectivity and density of the entire network. The results show that the nascent cohesive crisscrossing of fibers in stretched fibrin networks comprise an underappreciated important structural mechanism underlying the mechanical response of fibrin to (patho)physiological stresses that determine the course and outcomes of thrombotic and hemostatic disorders, such as heart attack and ischemic stroke. Fibrin is a viscoelastic proteinaceous polymer that determines the deformability and integrity of blood clots and fibrin-based biomaterials in response to biomechanical forces. In this paper, a novel structural mechanism of fibrin clots’ mechanical response to external tensile loads is tested using high-resolution confocal microscopy and newly developed computational model. This mechanism, underlying local strain-stiffening of individual fibers as well as global stiffening of the entire network, is based on previously neglected nascent cohesive pairwise interactions between individual fibers (crisscrossing) in fibrin networks formed under tensile load. Cohesive crisscrossing is an important structural mechanism that influences the mechanical response of blood clots and which can determine the outcomes of blood coagulation disorders, such as heart attacks and strokes.

ACS Style

Samuel Britton; Oleg Kim; Francesco Pancaldi; Zhiliang Xu; Rustem I. Litvinov; John W. Weisel; Mark Alber. Contribution of nascent cohesive fiber-fiber interactions to the non-linear elasticity of fibrin networks under tensile load. Acta Biomaterialia 2019, 94, 514 -523.

AMA Style

Samuel Britton, Oleg Kim, Francesco Pancaldi, Zhiliang Xu, Rustem I. Litvinov, John W. Weisel, Mark Alber. Contribution of nascent cohesive fiber-fiber interactions to the non-linear elasticity of fibrin networks under tensile load. Acta Biomaterialia. 2019; 94 ():514-523.

Chicago/Turabian Style

Samuel Britton; Oleg Kim; Francesco Pancaldi; Zhiliang Xu; Rustem I. Litvinov; John W. Weisel; Mark Alber. 2019. "Contribution of nascent cohesive fiber-fiber interactions to the non-linear elasticity of fibrin networks under tensile load." Acta Biomaterialia 94, no. : 514-523.