CC BY-NC-ND 4.0 · Thromb Haemost 2018; 118(09): 1600-1611
DOI: 10.1055/s-0038-1668151
New Technologies, Diagnostic Tools and Drugs
Georg Thieme Verlag KG Stuttgart · New York

Quantification of Platelet Contractile Movements during Thrombus Formation

Kjersti Tunströmer
1   Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
,
Lars Faxälv
1   Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
,
Niklas Boknäs
1   Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
2   Department of Haematology, Linköping University, Linköping, Sweden
,
Tomas L. Lindahl
1   Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
3   Department of Clinical Chemistry, Linköping University, Linköping, Sweden
› Author Affiliations
Funding This study was supported by a grant from the Swedish Research Council VR3R, Project No K2015-79X-22644-01-3 and DNr 2017-01177, the Swedish Heart-Lung foundation No 2017-0440 and by Linköping University.
Further Information

Publication History

05 March 2018

19 June 2018

Publication Date:
15 August 2018 (online)

Abstract

Imaging methods based on time-lapse microscopy are important tools for studying the dynamic events that shape thrombus formation upon vascular injury. However, there is a lack of methods to translate the vast amount of visual data generated in such experiments into quantitative variables describing platelet movements that can be subjected to systematic analysis. In this study, we developed experimental and computational protocols allowing for a detailed mathematical analysis of platelet movements within a developing thrombus. We used a flow chamber-based model of thrombosis wherein a collagen strip was used to initiate platelet adhesion and activation. Combining the use of a platelet staining protocol, designed to enable identification of individual platelets, and image processing, we tracked the movements of a large number of individual platelets during thrombus formation and consolidation. These data were then processed to generate aggregate measures describing the heterogeneous movements of platelets in different areas of the thrombus and at different time points. Applying this model and its potential, to a comparative analysis on a panel of platelet inhibitors, we found that total platelet intra-thrombus movements are only slightly reduced by blocking the interactions between glycoproteins IIb/IIIa and Ib and their ligands or by inhibiting thromboxane synthesis or P2Y12 signalling. In contrast, whereas 30 to 40% of the platelets movements (for the CD42a-labelled platelets) and 20% (for the pro-coagulant platelets), within a thrombus, are contractile, i.e., towards the centre of the thrombus, this contractile component is almost totally abolished in the presence of agents inhibiting these pathways.

Authors' Contributions

K. Tunströmer performed the experiments and wrote the first draft of the manuscript. K. Tunströmer and L. Faxälv performed data analysis. All authors contributed to the design of the study, interpretation of results, writing and final approval of the manuscript.


Supplementary Material

 
  • References

  • 1 de Witt SM, Swieringa F, Cavill R. , et al. Identification of platelet function defects by multi-parameter assessment of thrombus formation. Nat Commun 2014; 5 (May): 4257
  • 2 Nieswandt B, Brakebusch C, Bergmeier W. , et al. Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen. EMBO J 2001; 20 (09) 2120-2130
  • 3 Maloney SF, Brass LF, Diamond SL. P2Y12 or P2Y1 inhibitors reduce platelet deposition in a microfluidic model of thrombosis while apyrase lacks efficacy under flow conditions. Integr Biol 2010; 2 (04) 183-192
  • 4 Li R, Diamond SL. Detection of platelet sensitivity to inhibitors of COX-1, P2Y1, and P2Y12 using a whole blood microfluidic flow assay. Thromb Res 2014; 133 (02) 203-210
  • 5 Lee H, Sturgeon SA, Jackson SP, Hamilton JR. The contribution of thrombin-induced platelet activation to thrombus growth is diminished under pathological blood shear conditions. Thromb Haemost 2012; 107 (02) 328-337
  • 6 Tovar-Lopez FJ, Rosengarten G, Westein E. , et al. A microfluidics device to monitor platelet aggregation dynamics in response to strain rate micro-gradients in flowing blood. Lab Chip 2010; 10 (03) 291-302
  • 7 Stalker TJ, Traxler EA, Wu J. , et al. Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network. Blood 2013; 121 (10) 1875-1885
  • 8 Munnix ICA, Kuijpers MJE, Auger J. , et al. Segregation of platelet aggregatory and procoagulant microdomains in thrombus formation: regulation by transient integrin activation. Arterioscler Thromb Vasc Biol 2007; 27 (11) 2484-2490
  • 9 Heemskerk JWM, Mattheij NJA, Cosemans JMEM. Platelet-based coagulation: different populations, different functions. J Thromb Haemost 2013; 11 (01) 2-16
  • 10 Tutwiler V, Litvinov RI, Lozhkin AP. , et al. Kinetics and mechanics of clot contraction are governed by the molecular and cellular composition of the blood. Blood 2016; 127 (01) 149-159
  • 11 Ono A, Westein E, Hsiao S. , et al. Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth. Blood 2008; 112 (01) 90-99
  • 12 Léon C, Eckly A, Hechler B. , et al. Megakaryocyte-restricted MYH9 inactivation dramatically affects hemostasis while preserving platelet aggregation and secretion. Blood 2007; 110 (09) 3183-3191
  • 13 Lam WA, Chaudhuri O, Crow A. , et al. Mechanics and contraction dynamics of single platelets and implications for clot stiffening. Nat Mater 2011; 10 (01) 61-66
  • 14 Aoki N. Clot retraction increases clot resistance to fibrinolysis by condensing alpha 2-plasmin inhibitor crosslinked to fibrin. Thromb Haemost 1993; 70 (02) 376
  • 15 Sutton JT, Ivancevich NM, Perrin Jr SRJ, Vela DC, Holland CK. Clot retraction affects the extent of ultrasound-enhanced thrombolysis in an ex vivo porcine thrombosis model. Ultrasound Med Biol 2013; 39 (05) 813-824
  • 16 Munnix ICA, Cosemans JMEM, Auger JM, Heemskerk JWM. Platelet response heterogeneity in thrombus formation. Thromb Haemost 2009; 102 (06) 1149-1156
  • 17 Welsh JD, Stalker TJ, Voronov R. , et al. A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets. Blood 2014; 124 (11) 1808-1815
  • 18 Claesson K, Lindahl TL, Faxälv L. Counting the platelets: a robust and sensitive quantification method for thrombus formation. Thromb Haemost 2016; 115 (06) 1178-1190
  • 19 Fontayne A, Vanhoorelbeke K, Pareyn I. , et al. Rational humanization of the powerful antithrombotic anti-GPIbalpha antibody: 6B4. Thromb Haemost 2006; 96 (05) 671-684
  • 20 Roest M, Reininger A, Zwaginga JJ, King MR, Heemskerk JWM. ; Biorheology Subcommittee of the SSC of the ISTH. Flow chamber-based assays to measure thrombus formation in vitro: requirements for standardization. J Thromb Haemost 2011; 9 (11) 2322-2324
  • 21 Van Kruchten R, Cosemans JMEM, Heemskerk JWM. Measurement of whole blood thrombus formation using parallel-plate flow chambers - a practical guide. Platelets 2012; 23 (03) 229-242
  • 22 Heemskerk JW, Vuist WM, Feijge MA, Reutelingsperger CP, Lindhout T. Collagen but not fibrinogen surfaces induce bleb formation, exposure of phosphatidylserine, and procoagulant activity of adherent platelets: evidence for regulation by protein tyrosine kinase-dependent Ca2+ responses. Blood 1997; 90 (07) 2615-2625
  • 23 Crocker J, Grier D. Methods of digital video microscopy for colloidal studies. J Colloid Interface Sci 1996; 179 (01) 298-310
  • 24 Stalker TJ, Welsh JD, Tomaiuolo M. , et al. A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity. Blood 2014; 124 (11) 1824-1831
  • 25 Neeves KB, Onasoga AA, Hansen RR. , et al. Sources of variability in platelet accumulation on type 1 fibrillar collagen in microfluidic flow assays. PLoS One 2013; 8 (01) e54680
  • 26 Vaiyapuri S, Jones CI, Sasikumar P. , et al. Gap junctions and connexin hemichannels underpin hemostasis and thrombosis. Circulation 2012; 125 (20) 2479-2491 abstract
  • 27 Vaiyapuri S, Moraes LA, Sage T. , et al. Connexin40 regulates platelet function. Nat Commun 2013; 4: 2564