Polyphosphate delays fibrin polymerisation and alters the mechanical properties of the fibrin network

Summary

Polyphosphate (polyP) binds to fibrin(ogen) and alters fibrin structure, generating a heterogeneous network composed of ‘knots’ interspersed by large pores. Here we show platelet-derived polyP elicits similar structural changes in fibrin and examine the mechanism by which polyP alters fibrin structure. Polymerisation of fibrinogen with thrombin and CaCl₂ was studied using spinning disk confocal (SDC) microscopy. PolyP delayed fibrin polymerisation generating shorter protofibrils emanating from a nucleus-type structure. Consistent with this, cascade blue-polyP accumulated in fibrin ‘knots’. Protofibril formation was visualized by atomic force microscopy (AFM) ± polyP. In the presence of polyP abundant monomers of longer length were visualised by AFM, suggesting that polyP binds to monomeric fibrin. Shorter oligomers form in the presence of polyP, consistent with the stunted protofibrils visualised by SDC microscopy. We examined whether these structural changes induced by polyP alter fibrin’s viscoelastic properties by rheometry. PolyP reduced the stiffness (G’) and ability of the fibrin network to deform plastically G”, but to different extents. Consequently, the relative plastic component (loss tangent (G”/G’)) was 61 % higher implying that networks containing polyP are less stiff and more plastic. Local rheological measurements, performed using magnetic tweezers, indicate that the fibrin dense knots are stiffer and more plastic, reflecting the heterogeneity of the network. Our data show that polyP impedes fibrin polymerisation, stunting protofibril growth producing ‘knotted’ regions, which are rich in fibrin and polyP. Consequently, the mechanical properties of the fibrin network are altered resulting in clots with overall reduced stiffness and increased ability to deform plastically.

Supplementary Material to this article is available online at www.thrombosis-online.com.

 

MissingLinkText

Keywords

Fibrin(ogen) - polyphosphate - polymerisation

Video 1 Video 1

Video 2 Video 2

 

MissingLinkText

Conflicts of interest

None declared.

• References

• 1 Fatah K, Hamsten A, Blomback B. et al. Fibrin gel network characteristics and coronary heart disease: relations to plasma fibrinogen concentration, acute phase protein, serum lipoproteins and coronary atherosclerosis. Thromb Haemost 1992; 68: 130-135.
• 2 Fatah K, Silveira A, Tornvall P. et al. Proneness to formation of tight and rigid fibrin gel structures in men with myocardial infarction at a young age. Thromb Haemost 1996; 76: 535-540.
• 3 Collet JP, Allali Y, Lesty C. et al. Altered fibrin architecture is associated with hy-pofibrinolysis and premature coronary atherothrombosis. Arterioscler Thromb Vasc Biol 2006; 26: 2567-2573.
• 4 Undas A, Nowakowski T, Ciesla-Dul M. et al. Abnormal plasma fibrin clot characteristics are associated with worse clinical outcome in patients with peripheral arterial disease and thromboangiitis obliterans. Atherosclerosis 2011; 215: 481-486.
• 5 Undas A, Zawilska K, Ciesla-Dul M. et al. Altered fibrin clot structure/function in patients with idiopathic venous thromboembolism and in their relatives. Blood 2009; 114: 4272-4278.
• 6 Meade TW, Mellows S, Brozovic M. et al. Haemostatic function and ischaemic heart disease: principal results of the Northwick Park Heart Study. Lancet 1986; 02: 533-537.
• 7 Wolberg AS, Monroe DM, Roberts HR. et al. Elevated prothrombin results in clots with an altered fiber structure: a possible mechanism of the increased thrombotic risk. Blood 2003; 101: 3008-3013.
• 8 Scrutton MC, Ross-Murphy SB, Bennett GM. et al. Changes in clot deformability--a possible explanation for the epidemiological association between plasma fibrinogen concentration and myocardial infarction. Blood Coagul Fibrinolysis 1994; 05: 719-723.
• 9 Undas A, Szuldrzynski K, Stepien E. et al. Reduced clot permeability and susceptibility to lysis in patients with acute coronary syndrome: effects of inflammation and oxidative stress. Atherosclerosis 2008; 196: 551-557.
• 10 Bhasin N, Ariens RA, West RM. et al. Altered fibrin clot structure and function in the healthy first-degree relatives of subjects with intermittent claudication. J Vasc Surg 2008; 48: 1497-1503 503 e1.
• 11 Collet JP, Park D, Lesty C. et al. Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy. Arterioscler Thromb Vasc Biol 2000; 20: 1354-1361.
• 12 Ruiz FA, Lea CR, Oldfield E. et al. Human platelet dense granules contain poly-phosphate and are similar to acidocalcisomes of bacteria and unicellular euka-ryotes. J Biol Chem 2004; 279: 44250-44257.
• 13 Muller F, Mutch NJ, Schenk WA. et al. Platelet polyphosphates are proinflam-matory and procoagulant mediators in vivo. Cell 2009; 139: 1143-1156.
• 14 Smith SA, Mutch NJ, Baskar D. et al. Polyphosphate modulates blood coagulation and fibrinolysis. Proc Natl Acad Sci USA 2006; 103: 903-908.
• 15 Nickel KF, Ronquist G, Langer F. et al. The polyphosphate-factor XII pathway drives coagulation in prostate cancer-associated thrombosis. Blood 2015; 126: 1379-1389.
• 16 Morrissey JH, Choi SH, Smith SA. Polyphosphate: an ancient molecule that links platelets, coagulation, and inflammation. Blood 2012; 119: 5972-5979.
• 17 Smith SA, Morrissey JH. Polyphosphate enhances fibrin clot structure. Blood 2008; 112: 2810-2816.
• 18 Mutch NJ, Engel R, Uitte de Willige S. et al. Polyphosphate modifies the fibrin network and down-regulates fibrinolysis by attenuating binding of tPA and plasminogen to fibrin. Blood 2010; 115: 3980-3988.
• 19 Zhu S, Travers RJ, Morrissey JH. et al. FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow. Blood 2015; 126: 1494-1502.
• 20 Smith SA, Choi SH, Collins JN. et al. Inhibition of polyphosphate as a novel strategy for preventing thrombosis and inflammation. Blood 2012; 120: 5103-5110.
• 21 Travers RJ, Shenoi RA, Kalathottukaren MT. et al. Nontoxic polyphosphate inhibitors reduce thrombosis while sparing hemostasis. Blood 2014; 124: 3183-3190.
• 22 Smith SA, Choi SH, Davis-Harrison R. et al. Polyphosphate exerts differential effects on blood clotting, depending on polymer size. Blood 2010; 116: 4353-4359.
• 23 Chernysh IN, Nagaswami C, Weisel JW. Visualisation and identification of the structures formed during early stages of fibrin polymerisation. Blood 2011; 117: 4609-4614.
• 24 Allan P, Uitte de Willige S, Abou-Saleh RH. et al. Evidence that fibrinogen gamma’ directly interferes with protofibril growth: implications for fibrin structure and clot stiffness. J Thromb Haemost 2012; 10: 1072-1080.
• 25 Evans RM, Tassieri M, Auhl D. et al. Direct conversion of rheological compliance measurements into storage and loss moduli. Phys Rev E Stat Nonlin Soft Matter Phys 2009; 80: 012501.
• 26 Choi SH, Collins JN, Smith SA. et al. Phosphoramidate end labeling of inorganic polyphosphates: facile manipulation of polyphosphate for investigating and modulating its biological activities. Biochemistry 2010; 49: 9935-9941.
• 27 Gray MJ, Wholey WY, Wagner NO. et al. Polyphosphate is a primordial chap-erone. Mol Cell 2014; 53: 689-699.
• 28 Donovan AJ, Kalkowski J, Smith SA. et al. Size-controlled synthesis of granular polyphosphate nanoparticles at physiologic salt concentrations for blood clotting. Biomacromolecules 2014; 15: 3976-3984.
• 29 Blomback B, Carlsson K, Fatah K. et al. Fibrin in human plasma: gel architectures governed by rate and nature of fibrinogen activation. Thromb Res 1994; 75: 521-538.
• 30 Chernysh IN, Weisel JW. Dynamic imaging of fibrin network formation correlated with other measures of polymerisation. Blood 2008; 111: 4854-4861.
• 31 Carr Jr. ME, Hermans J. Size and density of fibrin fibers from turbidity. Macro-molecules 1978; 11: 46-50.
• 32 Janmey PA, Ferry JD. Gel formation by fibrin oligomers without addition of monomers. Biopolymers 1986; 25: 1337-1344.
• 33 Weisel JW. Structure of fibrin: impact on clot stability. J Thromb Haemost 2007; 05 (Suppl. 01) 116-124.
• 34 Weisel JW, Medved L. The structure and function of the alpha C domains of fi-brinogen. Ann NY Acad Sci 2001; 936: 312-327.
• 35 Gorkun OV, Veklich YI, Medved LV. et al. Role of the alpha C domains of fibrin in clot formation. Biochemistry 1994; 33: 6986-6997.
• 36 Medved LV, Gorkun OV, Manyakov VF. et al. The role of fibrinogen alpha C-do-mains in the fibrin assembly process. FEBS Lett 1985; 181: 109-112.
• 37 Collet JP, Moen JL, Veklich YI. et al. The alphaC domains of fibrinogen affect the structure of the fibrin clot, its physical properties, and its susceptibility to fi-brinolysis. Blood 2005; 106: 3824-3830.
• 38 Veklich YI, Gorkun OV, Medved LV. et al. Carboxyl-terminal portions of the alpha chains of fibrinogen and fibrin. Localisation by electron microscopy and the effects of isolated alpha C fragments on polymerisation. J Biol Chem 1993; 268: 13577-13585.
• 39 Lawrence MJ, Sabra A, Mills G. et al. A new biomarker quantifies differences in clot microstructure in patients with venous thromboembolism. Br J Haematol 2015; 168: 571-575.