Abstract
Optimal strength and stability of blood clots are keys to hemostasis and in prevention of hemorrhagic or thrombotic complications. Clots are biocomposite materials composed of fibrin network enmeshing platelets and other blood cells. We have previously shown that the storage temperature of platelets significantly impacts clot structure and stiffness. The objective of this work is to delineate the relationship between morphological characteristics and mechanical response of clot networks. We examined scanning electron microscope images of clots prepared from fresh apheresis platelets, and from apheresis platelets stored for 5 days at room temperature or at 4 °C, suspended in pooled plasma. Principal component analysis of nine different morphometric parameters revealed that a single principal component (PC1) can distinguish the effect of platelet storage on clot ultrastructure. Finite element analysis of clot response to uniaxial strain was used to map the spatially heterogeneous distribution of strain energy density for each clot. At modest deformations (25% strain), a single principal component (PC2) was able to predict these heterogeneities as quantified by variability in strain energy density distribution and in linear elastic stiffness, respectively. We have identified structural parameters that are primary regulators of stress distribution, and the observations provide insights into the importance of spatial heterogeneity on hemostasis and thrombosis.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ayres CE, Jha BS, Meredith H, Bowman JR, Bowlin GL, Henderson SC, Simpson DG (2008) Measuring fiber alignment in electrospun scaffolds: a user’s guide to the 2D fast Fourier transform approach. J Biomater Sci Polym Ed 19:603–621. https://doi.org/10.1163/156856208784089643
Ban E, Barocas VH, Shephard MS, Picu RC (2016) Softening in random networks of non-identical beams. J Mech Phys Solids 87:38–50. https://doi.org/10.1016/j.jmps.2015.11.001
Baradet TC, Haselgrove JC, Weisel JW (1995) Three-dimensional reconstruction of fibrin clot networks from stereoscopic intermediate voltage electron microscope images and analysis of branching. Biophys J 68:1551–1560. https://doi.org/10.1016/S0006-3495(95)80327-9
Berhan L, Sastry AM (2003) On modeling bonds in fused, porous networks: 3d simulations of fibrous–particulate joints. J Compos Mater 37:715–740. https://doi.org/10.1177/002199803029725
Brown AEX, Litvinov RI, Discher DE, Purohit PK, Weisel JW (2009) Multiscale mechanics of fibrin polymer: gel stretching with protein unfolding and loss of water. Science 325:741–744. https://doi.org/10.1126/science.1172484
Campbell RA, Overmyer KA, Bagnell CR, Wolberg AS (2008) Cellular procoagulant activity dictates clot structure and stability as a function of distance from the cell surface. Arterioscler Thromb Vasc Biol 28:2247–2254. https://doi.org/10.1161/ATVBAHA.108.176008
Campbell RA, Overmyer KA, Selzman CH, Sheridan BC, Wolberg AS (2009) Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability. Blood 114:4886–4896. https://doi.org/10.1182/blood-2009-06-228940
Campbell RA, Aleman M, Gray LD, Falvo MR, Wolberg AS (2010) Flow profoundly influences fibrin network structure: implications for fibrin formation and clot stability in haemostasis. Thromb Haemost 104:1281–1284. https://doi.org/10.1160/TH10-07-0442
Collet J-P, Shuman H, Ledger RE, Lee S, Weisel JW (2005) The elasticity of an individual fibrin fiber in a clot. Proc Natl Acad Sci USA 102:9133–9137. https://doi.org/10.1073/pnas.0504120102
Costalat V, Lobotesis K, Machi P, Mourand I, Maldonado I, Heroum C, Vendrell JF, Milhaud D, Riquelme C, Bonafé A, Arquizan C (2012) Prognostic factors related to clinical outcome following thrombectomy in ischemic stroke (RECOST study). 50 patients prospective study. Eur J Radiol 81:4075–4082. https://doi.org/10.1016/j.ejrad.2012.07.012
D’Amore A, Stella JA, Wagner WR, Sacks MS (2010) Characterization of the complete fiber network topology of planar fibrous tissues and scaffolds. Biomaterials 31:5345–5354. https://doi.org/10.1016/j.biomaterials.2010.03.052
D’Amore A, Amoroso N, Gottardi R, Hobson C, Carruthers C, Watkins S, Wagner WR, Sacks MS (2014) From single fiber to macro-level mechanics: a structural finite-element model for elastomeric fibrous biomaterials. J Mech Behav Biomed Mater 39:146–161. https://doi.org/10.1016/j.jmbbm.2014.07.016
Dulińska I, Targosz M, Strojny W, Lekka M, Czuba P, Balwierz W, Szymoński M (2006) Stiffness of normal and pathological erythrocytes studied by means of atomic force microscopy. J Biochem Biophys Methods 66:1–11. https://doi.org/10.1016/j.jbbm.2005.11.003
Duong H, Wu B, Tawil B (2009) Modulation of 3D fibrin matrix stiffness by intrinsic fibrinogen–thrombin compositions and by extrinsic cellular activity. Tissue Eng Part A 15:1865–1876. https://doi.org/10.1089/ten.tea.2008.0319
Eng W (2018) Nonlinear stiffness and viscoelasticity of inhibitor-treated blood clots by tensile testing. Master’s thesis, San Jose State University, San Jose, CA, USA
Eng W, Kim M, Ramasubramanian A, Lee S-JJ (2018) A modular test platform for micromechanical tensile testing of soft biomaterials. Presented at the ASME 2018 international mechanical engineering congress and exposition, American Society of Mechanical Engineers, pp V003T04A030–V003T04A030. https://doi.org/10.1115/IMECE2018-87259
Heussinger C, Frey E (2007) Role of architecture in the elastic response of semiflexible polymer and fiber networks. Phys Rev E Stat Nonlinear Soft Matter Phys 75:011917. https://doi.org/10.1103/PhysRevE.75.011917
Höök P, Brito-Robinson T, Kim O, Narciso C, Goodson HV, Weisel JW, Alber MS, Zartman JJ (2017) Whole blood clot optical clearing for nondestructive 3D imaging and quantitative analysis. Biomed Opt Express 8:3671–3686. https://doi.org/10.1364/BOE.8.003671
Hotaling NA, Bharti K, Kriel H, Simon CG Jr (2015) DiameterJ: a validated open source nanofiber diameter measurement tool. Biomaterials 61:327–338. https://doi.org/10.1016/j.biomaterials.2015.05.015
Jansen KA, Licup AJ, Sharma A, Rens R, MacKintosh FC, Koenderink GH (2018) The role of network architecture in collagen mechanics. Biophys J 114:2665–2678. https://doi.org/10.1016/j.bpj.2018.04.043
Kim E, Kim OV, Machlus KR, Liu X, Kupaev T, Lioi J, Wolberg AS, Chen DZ, Rosen ED, Xu Z, Alber M (2011) Correlation between fibrin network structure and mechanical properties: an experimental and computational analysis. Soft Matter 7:4983–4992. https://doi.org/10.1039/C0SM01528H
Kim OV, Litvinov RI, Alber MS, Weisel JW (2017) Quantitative structural mechanobiology of platelet-driven blood clot contraction. Nat Commun 8:1274. https://doi.org/10.1038/s41467-017-00885-x
Krasokha N, Theisen W, Reese S, Mordasini P, Brekenfeld C, Gralla J, Slotboom J, Schrott G, Monstadt H (2010) Mechanical properties of blood clots—a new test method. Materialwissenschaft Werkstofftechnik 41:1019–1024
Kulachenko A, Uesaka T (2012) Direct simulations of fiber network deformation and failure. Mech Mater 51:1–14. https://doi.org/10.1016/j.mechmat.2012.03.010
Lai VK, Lake SP, Frey CR, Tranquillo RT, Barocas VH (2012) Mechanical behavior of collagen-fibrin co-gels reflects transition from series to parallel interactions with increasing collagen content. J Biomech Eng 134:011004. https://doi.org/10.1115/1.4005544
Lever J, Krzywinski M, Altman N (2017) Points of significance: principal component analysis. Nat Methods 14:641–642. https://doi.org/10.1038/nmeth.4346
Li W, Sigley J, Pieters M, Helms CC, Nagaswami C, Weisel JW, Guthold M (2016) Fibrin fiber stiffness is strongly affected by fiber diameter, but not by fibrinogen glycation. Biophys J 110:1400–1410. https://doi.org/10.1016/j.bpj.2016.02.021
Li W, Lucioni T, Li R, Bonin K, Cho SS, Guthold M (2017) Stretching single fibrin fibers hampers their lysis. Acta Biomater 60:264–274. https://doi.org/10.1016/j.actbio.2017.07.037
Lippi G, Favaloro EJ (2018) Venous and arterial thromboses: two sides of the same coin? Semin Thromb Hemost 44:239–248. https://doi.org/10.1055/s-0037-1607202
Liu W, Jawerth LM, Sparks EA, Falvo MR, Hantgan RR, Superfine R, Lord ST, Guthold M (2006) Fibrin fibers have extraordinary extensibility and elasticity. Science 313:634. https://doi.org/10.1126/science.1127317
Matsumoto T, Sasaki J-I, Alsberg E, Egusa H, Yatani H, Sohmura T (2007) Three-dimensional cell and tissue patterning in a strained fibrin gel system. PLoS ONE 2:e1211. https://doi.org/10.1371/journal.pone.0001211
Mehta BP, Nogueira RG (2012) Should clot composition affect choice of endovascular therapy? Neurology 79:S63–S67. https://doi.org/10.1212/WNL.0b013e3182695859
Moreno-Arotzena O, Meier J, del Amo C, García-Aznar J (2015) Characterization of fibrin and collagen gels for engineering wound healing models. Materials 8:1636–1651. https://doi.org/10.3390/ma8041636
Nair PM, Pandya SG, Dallo SF, Reddoch KM, Montgomery RK, Pidcoke HF, Cap AP, Ramasubramanian AK (2017) Platelets stored at 4 °C contribute to superior clot properties compared to current standard-of-care through fibrin-crosslinking. Br J Haematol 178:119–129. https://doi.org/10.1111/bjh.14751
Oval Profile Plot [WWW Document] (n.d.) https://imagej.nih.gov/ij/plugins/oval-profile.html. Accessed 1 Dec 2019
Parazzi V, Lazzari L, Rebulla P (2010) Platelet gel from cord blood: a novel tool for tissue engineering. Platelets 21:549–554. https://doi.org/10.3109/09537104.2010.514626
Peter Selinger: Potrace [WWW Document] (n.d.) http://potrace.sourceforge.net/. Accessed 1 Dec 2019
Picu RC (2011) Mechanics of random fiber networks—a review. Soft Matter 7:6768–6785. https://doi.org/10.1039/C1SM05022B
Pidcoke HF, Spinella PC, Ramasubramanian AK, Strandenes G, Hervig T, Ness PM, Cap AP (2014) Refrigerated platelets for the treatment of acute bleeding: a review of the literature and reexamination of current standards. Shock 41(Suppl 1):51–53. https://doi.org/10.1097/SHK.0000000000000078
Piechocka IK, Bacabac RG, Potters M, Mackintosh FC, Koenderink GH (2010) Structural hierarchy governs fibrin gel mechanics. Biophys J 98:2281–2289. https://doi.org/10.1016/j.bpj.2010.01.040
Reddoch KM, Pidcoke HF, Montgomery RK, Fedyk CG, Aden JK, Ramasubramanian AK, Cap AP (2014) Hemostatic function of apheresis platelets stored at 4°C and 22°C. Shock 41(Suppl 1):54–61. https://doi.org/10.1097/SHK.0000000000000082
Reddoch-Cardenas KM, Bynum JA, Meledeo MA, Nair PM, Wu X, Darlington DN, Ramasubramanian AK, Cap AP (2018) Cold-stored platelets: a product with function optimized for hemorrhage control. Transfus Apher Sci. https://doi.org/10.1016/j.transci.2018.12.012
Rowe SL, Lee S, Stegemann JP (2007) Influence of thrombin concentration on the mechanical and morphological properties of cell-seeded fibrin hydrogels. Acta Biomater 3:59–67. https://doi.org/10.1016/j.actbio.2006.08.006
Ryan EA, Mockros LF, Weisel JW, Lorand L (1999) Structural origins of fibrin clot rheology. Biophys J 77:2813–2826. https://doi.org/10.1016/S0006-3495(99)77113-4
Schwarz Henriques S, Sandmann R, Strate A, Köster S (2012) Force field evolution during human blood platelet activation. J Cell Sci 125:3914–3920. https://doi.org/10.1242/jcs.108126
Shlens J (2014) A tutorial on principal component analysis. arXiv:1404.1100v1
Sprouse LR 2nd, Peeters P, Bosiers M (2005) The capture of visible debris by distal cerebral protection filters during carotid artery stenting: is it predictable? J Vasc Surg 41:950–955. https://doi.org/10.1016/j.jvs.2005.02.048
Stein AM, Vader DA, Jawerth LM, Weitz DA, Sander LM (2008) An algorithm for extracting the network geometry of three-dimensional collagen gels. J Microsc 232:463–475. https://doi.org/10.1111/j.1365-2818.2008.02141.x
Taylor SE, Cao T, Talauliker PM, Lifshitz J (2013) Objective morphological quantification of microscopic images using a fast Fourier transform (FFT) analysis. Curr Protoc Essent Lab Tech 95:9.5.1–9.5.12. https://doi.org/10.1002/9780470089941.et0905s07
Todo A, Minaeian A, Sahni R, Chao KH (2013) Incidence and outcome of procedural distal emboli using the Penumbra thrombectomy for acute stroke. J Neurointerv Surg 5:135–138. https://doi.org/10.1136/neurintsurg-2011-010216
Undas A, Ariëns RAS (2011) Fibrin clot structure and function. Arterioscler Thromb Vasc Biol. https://doi.org/10.1161/atvbaha.111.230631
Varjú I, Sótonyi P, Machovich R, Szabó L, Tenekedjiev K, Silva MMCG, Longstaff C, Kolev K (2011) Hindered dissolution of fibrin formed under mechanical stress. J Thromb Haemost 9:979–986. https://doi.org/10.1111/j.1538-7836.2011.04203.x
Weisel JW, Litvinov RI (2017) Fibrin formation, structure and properties. In: Parry DAD, Squire J (eds) Fibrous proteins: structures and mechanisms, subcellular biochemistry. Springer, Cham, pp 405–456. https://doi.org/10.1007/978-3-319-49674-0_13
Wen Q, Basu A, Winer JP, Yodh A, Janmey PA (2007) Local and global deformations in a strain-stiffening fibrin gel. New J Phys 9:428–428. https://doi.org/10.1088/1367-2630/9/11/428
Wen Q, Basu A, Janmey PA, Yodh AG (2012) Non-affine deformations in polymer hydrogels. Soft Matter 8:8039–8049. https://doi.org/10.1039/c2sm25364j
Yang M, Yu Y, Walsh WR, Yang J-L, Baker L, Lennox AF, Crowe PJ, Varcoe RL (2016) A microscopic and biomarker evaluation of embolic filter debris collected during carotid artery stenting. J Endovasc Ther 23:275–284. https://doi.org/10.1177/1526602816628284
Žagar G, Onck PR, van der Giessen E (2015) Two fundamental mechanisms govern the stiffening of cross-linked networks. Biophys J 108:1470–1479. https://doi.org/10.1016/j.bpj.2015.02.015
Acknowledgements
This work was supported in part by the AHA Institutional Research Enhancement Award (AIREA) program of the American Heart Association (Award 18AIREA33960524) and in part by the Kordestani Foundation at San José State University.
Author information
Authors and Affiliations
Contributions
SJL and AKR designed and planned the research, analyzed the data, and wrote the majority of the paper. DMN performed image analysis. SJL, HSG, CP, and AKS performed FEA simulations. PMN, APC, and AKR generated SEM images.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Fig. S1
Fast Fourier transform (FFT) analysis of a representative clot network
Fig. S2
PC1 and PC2 vs. platelet storage temperature for 66 images of clots prepared from fresh (FR) platelets, 49 images of clots from platelets stored at room temperature (RT), and 45 images of clots prepared from platelets stored at 4 °C
Fig. S3
Mesh convergence. The geometry was discretized using quadratic triangular elements. Mesh convergence was verified by running the simulation with increasing mesh density. (a) Plots of strain energy density versus number of elements for a representative specimen at maximum stretch ratio of 1.5. A stable value for strain energy density occurs beyond 250,000 elements, and discretization settings were selected accordingly. (b) A 5 µm x 5 µm close-up view of a representative mesh for a specimen having approximately 270,000 elements in the overall 25 µm x 17 µm network
Fig. S4
Scatter plots of stiffness (per unit µm thickness) vs. morphometric parameters. The most pronounced correlation is observed between stiffness and porosity, having coefficient of determination R2 = 0.85 with respect to a least-squares straight-line fit
Fig.
S5 Effect of geometric nonlinearity: (a) undeformed mesh, (b) deformed mesh, and (c) contrast in simulated mechanical response when geometric linearity is taken into consideration or not. The overall dimensions are 10 µm x 10 µm in a plane stress model with 1 µm assigned depth and 1 µm fiber diameter along the diagonals. This simplified model applies a uniaxial extension with stretch ratio of 1.5. Neglecting geometric nonlinearity would assume incorrectly that elements have insignificant rotation in the model reference frame
Fig. S6
Skewness of diameter from 160 clot images. A very small fraction of outliers are present at the upper-tail, but the data are otherwise centered at 1.0 with almost equal distribution higher and lower
Rights and permissions
About this article
Cite this article
Lee, SJ.J., Nguyen, D.M., Grewal, H.S. et al. Image-based analysis and simulation of the effect of platelet storage temperature on clot mechanics under uniaxial strain. Biomech Model Mechanobiol 19, 173–187 (2020). https://doi.org/10.1007/s10237-019-01203-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10237-019-01203-8