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Thromb Haemost 2021; 121(01): 046-057
DOI: 10.1055/s-0040-1715648
Coagulation and Fibrinolysis

Intrathrombus Fibrin Attenuates Spatial Sorting of Phosphatidylserine Exposing Platelets during Clotting Under Flow

Kevin T. Trigani
1   Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States
,
Scott L. Diamond
1   Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States
› Author Affiliations Funding This study was supported by National Institutes of Health grants U01-HL-131053 and R01-HL-103419 to S.L.D.
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Abstract

Background As thrombosis proceeds, certain platelets in a clot expose phosphatidylserine (PS) on their outer membrane. These PS+ platelets subsequently sort to the perimeter of the mass via platelet contraction. It remains unclear how thrombin and fibrin may alter PS+ platelet sorting within a clot.

Objective We investigated the role of fibrin in PS+ platelet sorting.

Methods We used an 8-channel microfluidic assay of clotting over collagen (±tissue factor) at 100 s−1 initial wall shear rate. Temporal PS+ platelet sorting was measured using a Pearson's correlation coefficient between the annexin V distribution in a clot at 9 versus 15 minutes. Spatial PS+ platelet sorting was measured using an autocorrelation metric of the final annexin V distribution.

Results By 6 minutes, PS+ platelets were distributed throughout the platelet deposits and became highly spatially sorted by 15 minutes when thrombin and fibrin were blocked with Phe-Pro-Arg-chloromethylketone (PPACK). Fibrin polymerization (no PPACK) attenuated temporal and spatial PS sorting and clot contraction. With Gly-Pro-Arg-Pro (GPRP) added to block fibrin polymerization, PS sorting was prominent as was clot contraction. Exogenously added tissue plasminogen activator drove fibrinolysis that in turn promoted clot contraction and PS sorting, albeit to a lesser degree than the PPACK or GPRP conditions. Clots lacking fibrin displayed 3.6 times greater contraction than clots with fibrin.

Conclusion PS sorting correlated with clot contraction, as previously reported. However, fibrin inversely correlated with both percent contraction and PS sorting. Fibrin attenuated clot contraction and PS sorting relative to clots without fibrin.

Keywords

contraction - fibrin - phosphatidylserine - platelet - thrombin

Authors' Contributions

K.T.T. and S.L.D. designed experiments, analyzed and interpreted data, and wrote the manuscript. K.T.T. conducted experiments.


Supplementary Material



Publication History

Received: 04 March 2020

Accepted: 14 July 2020

Article published online:
22 September 2020

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 Heemskerk JWM, Bevers EM, Lindhout T. Platelet activation and blood coagulation. Thromb Haemost 2002; 88 (02) 186-193
    Article in Thieme ConnectPubMedGoogle Scholar
  • 2 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
    CrossrefPubMedGoogle Scholar
  • 3 Heemskerk JWM, Mattheij NJA, Cosemans JMEM. Platelet-based coagulation: different populations, different functions. J Thromb Haemost 2013; 11 (01) 2-16
    CrossrefPubMedGoogle Scholar
  • 4 Briedé JJ, Heemskerk JWM, Hemker HC, Lindhout T. Heterogeneity in microparticle formation and exposure of anionic phospholipids at the plasma membrane of single adherent platelets. Biochim Biophys Acta 1999; 1451 (01) 163-172
    CrossrefPubMedGoogle Scholar
  • 5 Pang A, Cui Y, Chen Y. et al. Shear-induced integrin signaling in platelet phosphatidylserine exposure, microvesicle release, and coagulation. Blood 2018; 132 (05) 533-543
    CrossrefPubMedGoogle Scholar
  • 6 Leventis PA, Grinstein S. The distribution and function of phosphatidylserine in cellular membranes. Annu Rev Biophys 2010; 39 (01) 407-427
    CrossrefPubMedGoogle Scholar
  • 7 Agbani EO, van den Bosch MTJ, Brown E. et al. Coordinated membrane ballooning and procoagulant spreading in human platelets. Circulation 2015; 132 (15) 1414-1424
    CrossrefPubMedGoogle Scholar
  • 8 Berny MA, Munnix ICA, Auger JM. et al. Spatial distribution of factor Xa, thrombin, and fibrin(ogen) on thrombi at venous shear. PLoS One 2010; 5 (04) e10415
    Google Scholar
  • 9 Podoplelova NA, Sveshnikova AN, Kotova YN. et al. Coagulation factors bound to procoagulant platelets concentrate in cap structures to promote clotting. Blood 2016; 128 (13) 1745-1755
    CrossrefPubMedGoogle Scholar
  • 10 Suzuki J, Umeda M, Sims PJ, Nagata S. Calcium-dependent phospholipid scrambling by TMEM16F. Nature 2010; 468 (7325): 834-838
    Google Scholar
  • 11 Baig AA, Haining EJ, Geuss E. et al. TMEM16F-mediated platelet membrane phospholipid scrambling is critical for hemostasis and thrombosis but not thromboinflammation in mice-brief report. Arterioscler Thromb Vasc Biol 2016; 36 (11) 2152-2157
    CrossrefPubMedGoogle Scholar
  • 12 Castoldi E, Collins PW, Williamson PL, Bevers EM. To the editor: compound heterozygosity for 2 novel TMEM16F mutations in a patient with Scott syndrome. Blood 2011; 117 (16) 4399-4400
    CrossrefPubMedGoogle Scholar
  • 13 Delaney MK, Liu J, Kim K. et al. Agonist-induced platelet procoagulant activity requires shear and a Rac1-dependent signaling mechanism. Blood 2014; 124 (12) 1957-1967
    CrossrefPubMedGoogle Scholar
  • 14 Munnix ICA, Cosemans JMEM, Auger JM, Heemskerk JWM. Platelet response heterogeneity in thrombus formation. Thromb Haemost 2009; 102 (06) 1149-1156
    Article in Thieme ConnectPubMedGoogle Scholar
  • 15 Cosemans JMEM, Iserbyt BF, Deckmyn H, Heemskerk JWM. Multiple ways to switch platelet integrins on and off. J Thromb Haemost 2008; 6 (08) 1253-1261
    CrossrefPubMedGoogle Scholar
  • 16 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
    CrossrefPubMedGoogle Scholar
  • 17 Nechipurenko DY, Receveur N, Yakimenko AO. et al. Clot contraction drives the translocation of procoagulant platelets to thrombus surface. Arterioscler Thromb Vasc Biol 2019; 39 (01) 37-47
    CrossrefPubMedGoogle Scholar
  • 18 Chen Z, Lu J, Zhang C. et al. Microclot array elastometry for integrated measurement of thrombus formation and clot biomechanics under fluid shear. Nat Commun 2019; 10 (01) 2051
    CrossrefPubMedGoogle Scholar
  • 19 Samson AL, Alwis I, Maclean JAA. et al. Endogenous fibrinolysis facilitates clot retraction in vivo. Blood 2017; 130 (23) 2453-2462
    CrossrefPubMedGoogle Scholar
  • 20 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
    CrossrefPubMedGoogle Scholar
  • 21 Zhu S, Herbig BA, Li R. et al. In microfluidico: recreating in vivo hemodynamics using miniaturized devices. Biorheology 2015; 52 (5-6): 303-318
    CrossrefPubMedGoogle Scholar
  • 22 Petersen NO, Höddelius PL, Wiseman PW, Seger O, Magnusson KE. Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application. Biophys J 1993; 65 (03) 1135-1146
    CrossrefPubMedGoogle Scholar
  • 23 Nohe A, Petersen NO. Image Correlation Spectroscopy. Sci STKE 2007; (417) l7
    PubMedGoogle Scholar
  • 24 Hagen N, Dereniak EL. Gaussian profile estimation in two dimensions. Appl Opt 2008; 47 (36) 6842-6851
    CrossrefPubMedGoogle Scholar
  • 25 Robertson C, George SC. Theory and practical recommendations for autocorrelation-based image correlation spectroscopy. J Biomed Opt 2012; 17 (08) 080801-1
    CrossrefPubMedGoogle Scholar
  • 26 Mohapatra S, Weisshaar JC. Modified Pearson correlation coefficient for two-color imaging in spherocylindrical cells. BMC Bioinformatics 2018; 19 (01) 428
    CrossrefPubMedGoogle Scholar
  • 27 Manders EMM, Stap J, Brakenhoff GJ, van Driel R, Aten JA. Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. J Cell Sci 1992; 103 (Pt 3): 857-862
    CrossrefPubMedGoogle Scholar
  • 28 Vyavahare Scott N, Hanson SR, Kohn JN. In vitro and in vivo evaluation of the site-specific administration of the thrombin inhibitor PPACK. J Control Release 1993; 27: 165-173
    CrossrefPubMedGoogle Scholar
  • 29 Yamazumi K, Doolittle RF. The synthetic peptide Gly-Pro-Arg-Pro-amide limits the plasmic digestion of fibrinogen in the same fashion as calcium ion. Protein Sci 1992; 1 (12) 1719-1720
    CrossrefPubMedGoogle Scholar
  • 30 Whyte CS, Swieringa F, Mastenbroek TG. et al. Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow. Blood 2015; 125 (16) 2568-2578
    CrossrefPubMedGoogle Scholar
  • 31 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
    CrossrefPubMedGoogle Scholar
  • 32 Tutwiler V, Wang H, Litvinov RI, Weisel JW, Shenoy VB. Interplay of platelet contractility and elasticity of fibrin/erythrocytes in blood clot retraction. Biophys J 2017; 112 (04) 714-723
    CrossrefPubMedGoogle Scholar
  • 33 Aleman MM, Byrnes JR, Wang JG. et al. Factor XIII activity mediates red blood cell retention in venous thrombi. J Clin Invest 2014; 124 (08) 3590-3600
    CrossrefPubMedGoogle Scholar
  • 34 Byrnes JR, Duval C, Wang Y. et al. Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin a-chain crosslinking. Blood 2015; 126 (16) 1940-1948
    CrossrefPubMedGoogle Scholar
  • 35 van Geffen JP, Swieringa F, Heemskerk JWM. Platelets and coagulation in thrombus formation: aberrations in the Scott syndrome. Thromb Res 2016; 141 (Suppl. 02) S12-S16
    CrossrefPubMedGoogle Scholar
  • 36 Zhu S, Lu Y, Sinno T, Diamond SL. Dynamics of thrombin generation and flux from clots during whole human blood flow over collagen/tissue factor surfaces. J Biol Chem 2016; 291 (44) 23027-23035
    CrossrefPubMedGoogle Scholar
  • 37 Zhu S, Chen J, Diamond SL. Establishing the transient mass balance of thrombosis: from tissue factor to thrombin to fibrin under venous flow. Arterioscler Thromb Vasc Biol 2018; 38 (07) 1528-1536
    CrossrefPubMedGoogle Scholar
  • 38 Yu X, Diamond SL. Fibrin modulates shear-induced NETosis in sterile occlusive thrombi formed under haemodynamic flow. Thromb Haemost 2019; 119 (04) 586-593
    Article in Thieme ConnectPubMedGoogle Scholar