PDF herunterladen
Thromb Haemost 2019; 119(06): 916-929
DOI: 10.1055/s-0039-1685139
Cellular Haemostasis and Platelets
Georg Thieme Verlag KG Stuttgart · New York

The Direct Thrombin Inhibitors Dabigatran and Lepirudin Inhibit GPIbα-Mediated Platelet Aggregation

Katharina Trabold   *
1   Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany
,
Stephanie Makhoul   *
1   Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany
,
Stepan Gambaryan
1   Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany
2   Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia
3   Department of Cytology and Histology, St. Petersburg State University, St. Petersburg, Russia
,
Joanne van Ryn
4   Department of Cardiometabolic Disease Research, Boehringer Ingelheim Pharma GmbH, Biberach, Germany
,
Ulrich Walter
1   Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany
,
Kerstin Jurk
1   Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany
› Institutsangaben Funding This study was supported by a research grant from Boehringer Ingelheim (to K.J. and U.W.) and by the German Federal Ministry of Education and Research (BMBF 01EO1003 and 01EO1503 to K.J. and U.W.). S.G. was supported by a grant from RFBR N° 17–00–00141 (17–00–00139).
Weitere Informationen

Publikationsverlauf

17. September 2018

20. Februar 2019

Publikationsdatum:
20. April 2019 (online)

Auch verfügbar auf
    Lizenzen und Reprints

Abstract

The direct thrombin inhibitor (DTI) dabigatran is a non-vitamin K antagonist oral anticoagulant for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation. In addition to its anti-thrombotic efficacy, dabigatran has been suggested to exert some pro-thrombotic effect due to fostering the ligation of thrombin to its high affinity platelet receptor glycoprotein (GP) Ibα in patients with atrial fibrillation. On the other hand, we provided evidence that a member of another class of DTIs, lepirudin, stimulates the inhibitory cyclic guanosine monophosphate (cGMP)/soluble guanylate cyclase pathway in human platelets. Here, we investigated the effect of lepirudin and dabigatran spiked to platelets from healthy volunteers on GPIbα-mediated platelet aggregation and agglutination. Ristocetin/von Willebrand factor (vWF)-induced aggregation of platelets in the presence or absence of plasma was significantly inhibited by lepirudin, dabigatran and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK). However, ristocetin/vWF-mediated platelet agglutination and binding of vWF to platelets were not affected by the DTIs. The anti-aggregatory effect was confirmed by using the GPIbα-specific agonist echicetin beads for human and murine platelets. DTIs diminished echicetin beads-induced Syk Y352 phosphorylation (used here as readout for an early signal occurring during echicetin-induced platelet aggregation), but did not inhibit adenosine diphosphate- or thromboxane A2-induced platelet aggregation. Thrombin was not generated in response to ristocetin/vWF or echicetin beads and therefore did not explain the inhibitory effect of the DTIs. Therapeutic concentration of lepirudin and dabigatran did not affect significantly platelet vasodilator-stimulated phosphoprotein S239 phosphorylation or cGMP and cyclic adenosine monophosphate levels. These data suggest that the DTIs, lepirudin and dabigatran, impair platelet activation measured during platelet aggregation induced by ristocetin/vWF or echicetin beads.

Keywords

platelets - aggregation - direct thrombin inhibitors - von Willebrand factor - GPIb/V/IX

* Both the authors contributed equally and share the first authorship.


Supplementary Material

 

  • References

  • 1 Bergmeier W, Chauhan AK, Wagner DD. Glycoprotein Ibalpha and von Willebrand factor in primary platelet adhesion and thrombus formation: lessons from mutant mice. Thromb Haemost 2008; 99 (02) 264-270
    Artikel in Thieme ConnectPubMedGoogle Scholar
  • 2 Stel HV, Sakariassen KS, de Groot PG, van Mourik JA, Sixma JJ. Von Willebrand factor in the vessel wall mediates platelet adherence. Blood 1985; 65 (01) 85-90
    CrossrefPubMedGoogle Scholar
  • 3 Ruggeri ZM, Dent JA, Saldívar E. Contribution of distinct adhesive interactions to platelet aggregation in flowing blood. Blood 1999; 94 (01) 172-178
    CrossrefPubMedGoogle Scholar
  • 4 Savage B, Saldívar E, Ruggeri ZM. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell 1996; 84 (02) 289-297
    CrossrefPubMedGoogle Scholar
  • 5 Bryckaert M, Rosa JP, Denis CV, Lenting PJ. Of von Willebrand factor and platelets. Cell Mol Life Sci 2015; 72 (02) 307-326
    CrossrefPubMedGoogle Scholar
  • 6 Harmon JT, Jamieson GA. The glycocalicin portion of platelet glycoprotein Ib expresses both high and moderate affinity receptor sites for thrombin. A soluble radioreceptor assay for the interaction of thrombin with platelets. J Biol Chem 1986; 261 (28) 13224-13229
    CrossrefPubMedGoogle Scholar
  • 7 Ruggeri ZM, Zarpellon A, Roberts JR, Mc Clintock RA, Jing H, Mendolicchio GL. Unravelling the mechanism and significance of thrombin binding to platelet glycoprotein Ib. Thromb Haemost 2010; 104 (05) 894-902
    Artikel in Thieme ConnectPubMedGoogle Scholar
  • 8 Estevez B, Kim K, Delaney MK. , et al. Signaling-mediated cooperativity between glycoprotein Ib-IX and protease-activated receptors in thrombin-induced platelet activation. Blood 2016; 127 (05) 626-636
    CrossrefPubMedGoogle Scholar
  • 9 Kasirer-Friede A, Cozzi MR, Mazzucato M, De Marco L, Ruggeri ZM, Shattil SJ. Signaling through GP Ib-IX-V activates alpha IIb beta 3 independently of other receptors. Blood 2004; 103 (09) 3403-3411
    CrossrefPubMedGoogle Scholar
  • 10 Gardiner EE, Arthur JF, Shen Y. , et al. GPIbalpha-selective activation of platelets induces platelet signaling events comparable to GPVI activation events. Platelets 2010; 21 (04) 244-252
    CrossrefPubMedGoogle Scholar
  • 11 Navdaev A, Subramanian H, Petunin A, Clemetson KJ, Gambaryan S, Walter U. Echicetin coated polystyrene beads: a novel tool to investigate GPIb-specific platelet activation and aggregation. PLoS One 2014; 9 (04) e93569
    CrossrefPubMedGoogle Scholar
  • 12 Peng M, Lu W, Beviglia L, Niewiarowski S, Kirby EP. Echicetin: a snake venom protein that inhibits binding of von Willebrand factor and alboaggregins to platelet glycoprotein Ib. Blood 1993; 81 (09) 2321-2328
    CrossrefPubMedGoogle Scholar
  • 13 Peng M, Emig FA, Mao A. , et al. Interaction of echicetin with a high affinity thrombin binding site on platelet glycoprotein GPIb. Thromb Haemost 1995; 74 (03) 954-957
    Artikel in Thieme ConnectPubMedGoogle Scholar
  • 14 Navdaev A, Dörmann D, Clemetson JM, Clemetson KJ. Echicetin, a GPIb-binding snake C-type lectin from Echis carinatus, also contains a binding site for IgMkappa responsible for platelet agglutination in plasma and inducing signal transduction. Blood 2001; 97 (08) 2333-2341
    CrossrefPubMedGoogle Scholar
  • 15 Eisert WG, Hauel N, Stangier J, Wienen W, Clemens A, van Ryn J. Dabigatran: an oral novel potent reversible nonpeptide inhibitor of thrombin. Arterioscler Thromb Vasc Biol 2010; 30 (10) 1885-1889
    CrossrefPubMedGoogle Scholar
  • 16 Petros S. Lepirudin in the management of patients with heparin-induced thrombocytopenia. Biologics 2008; 2 (03) 481-490
    PubMedGoogle Scholar
  • 17 Kobsar A, Koessler J, Kehrer L, Gambaryan S, Walter U. The thrombin inhibitors hirudin and Refludan(®) activate the soluble guanylyl cyclase and the cGMP pathway in washed human platelets. Thromb Haemost 2012; 107 (03) 521-529
    Artikel in Thieme ConnectPubMedGoogle Scholar
  • 18 Bye AP, Unsworth AJ, Gibbins JM. Platelet signaling: a complex interplay between inhibitory and activatory networks. J Thromb Haemost 2016; 14 (05) 918-930
    CrossrefPubMedGoogle Scholar
  • 19 Makhoul S, Walter E, Pagel O. , et al. Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets. Nitric Oxide 2018; 76: 71-80
    CrossrefPubMedGoogle Scholar
  • 20 Kanaji T, Russell S, Ware J. Amelioration of the macrothrombocytopenia associated with the murine Bernard-Soulier syndrome. Blood 2002; 100 (06) 2102-2107
    CrossrefPubMedGoogle Scholar
  • 21 Gambaryan S, Kobsar A, Rukoyatkina N. , et al. Thrombin and collagen induce a feedback inhibitory signaling pathway in platelets involving dissociation of the catalytic subunit of protein kinase A from an NFkappaB-IkappaB complex. J Biol Chem 2010; 285 (24) 18352-18363
    CrossrefPubMedGoogle Scholar
  • 22 Beck F, Geiger J, Gambaryan S. , et al. Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition. Blood 2017; 129 (02) e1-e12
    CrossrefPubMedGoogle Scholar
  • 23 Grüner S, Prostredna M, Aktas B. , et al. Anti-glycoprotein VI treatment severely compromises hemostasis in mice with reduced alpha2beta1 levels or concomitant aspirin therapy. Circulation 2004; 110 (18) 2946-2951
    CrossrefPubMedGoogle Scholar
  • 24 Loroch S, Trabold K, Gambaryan S. , et al. Alterations of the platelet proteome in type I Glanzmann thrombasthenia caused by different homozygous delG frameshift mutations in ITGA2B. Thromb Haemost 2017; 117 (03) 556-569
    Artikel in Thieme ConnectPubMedGoogle Scholar
  • 25 Jurk K, Schulz AS, Kehrel BE. , et al. Novel integrin-dependent platelet malfunction in siblings with leukocyte adhesion deficiency-III (LAD-III) caused by a point mutation in FERMT3. Thromb Haemost 2010; 103 (05) 1053-1064
    Artikel in Thieme ConnectPubMedGoogle Scholar
  • 26 Hemker HC, Giesen PL, Ramjee M, Wagenvoord R, Béguin S. The thrombogram: monitoring thrombin generation in platelet-rich plasma. Thromb Haemost 2000; 83 (04) 589-591
    Artikel in Thieme ConnectPubMedGoogle Scholar
  • 27 Jurk K, Lahav J, Van Aken H, Brodde MF, Nofer JR, Kehrel BE. Extracellular protein disulfide isomerase regulates feedback activation of platelet thrombin generation via modulation of coagulation factor binding. J Thromb Haemost 2011; 9 (11) 2278-2290
    CrossrefPubMedGoogle Scholar
  • 28 Wienen W, Stassen JM, Priepke H, Ries UJ, Hauel N. In-vitro profile and ex-vivo anticoagulant activity of the direct thrombin inhibitor dabigatran and its orally active prodrug, dabigatran etexilate. Thromb Haemost 2007; 98 (01) 155-162
    Artikel in Thieme ConnectPubMedGoogle Scholar
  • 29 Suzuki-Inoue K, Wilde JI, Andrews RK. , et al. Glycoproteins VI and Ib-IX-V stimulate tyrosine phosphorylation of tyrosine kinase Syk and phospholipase Cgamma2 at distinct sites. Biochem J 2004; 378 (Pt 3): 1023-1029
    CrossrefPubMedGoogle Scholar
  • 30 Bradshaw JM. The Src, Syk, and Tec family kinases: distinct types of molecular switches. Cell Signal 2010; 22 (08) 1175-1184
    CrossrefPubMedGoogle Scholar
  • 31 Liu J, Pestina TI, Berndt MC, Steward SA, Jackson CW, Gartner TK. The roles of ADP and TXA in botrocetin/VWF-induced aggregation of washed platelets. J Thromb Haemost 2004; 2 (12) 2213-2222
    CrossrefPubMedGoogle Scholar
  • 32 Canobbio I, Bertoni A, Lova P. , et al. Platelet activation by von Willebrand factor requires coordinated signaling through thromboxane A2 and Fc gamma IIA receptor. J Biol Chem 2001; 276 (28) 26022-26029
    CrossrefPubMedGoogle Scholar
  • 33 Connolly SJ, Ezekowitz MD, Yusuf S. , et al; RE-LY Steering Committee and Investigators. Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med 2009; 361 (12) 1139-1151
    CrossrefPubMedGoogle Scholar
  • 34 Connolly SJ, Ezekowitz MD, Yusuf S, Reilly PA, Wallentin L. ; Randomized Evaluation of Long-Term Anticoagulation Therapy Investigators. Newly identified events in the RE-LY trial. N Engl J Med 2010; 363 (19) 1875-1876
    CrossrefPubMedGoogle Scholar
  • 35 Hoffman M, Monroe DM. Impact of non-vitamin K antagonist oral anticoagulants from a basic science perspective. Arterioscler Thromb Vasc Biol 2017; 37 (10) 1812-1818
    CrossrefPubMedGoogle Scholar
  • 36 Petzold T, Thienel M, Konrad I. , et al. Oral thrombin inhibitor aggravates platelet adhesion and aggregation during arterial thrombosis. Sci Transl Med 2016; 8 (367) 367ra168
    CrossrefPubMedGoogle Scholar
  • 37 Olivier CB, Weik P, Meyer M. , et al. TRAP-induced platelet aggregation is enhanced in cardiovascular patients receiving dabigatran. Thromb Res 2016; 138: 63-68
    CrossrefPubMedGoogle Scholar
  • 38 Achilles A, Mohring A, Zeus T, Kelm M, Polzin A. Dabigatran enhances platelet reactivity and platelet thrombin receptor expression in patients with atrial fibrillation: reply. J Thromb Haemost 2017; 15 (07) 1524-1525
    CrossrefPubMedGoogle Scholar
  • 39 Eller T, Busse J, Dittrich M. , et al. Dabigatran, rivaroxaban, apixaban, argatroban and fondaparinux and their effects on coagulation POC and platelet function tests. Clin Chem Lab Med 2014; 52 (06) 835-844
    PubMedGoogle Scholar
  • 40 Olivier CB, Weik P, Meyer M. , et al. Dabigatran and rivaroxaban do not affect AA- and ADP-induced platelet aggregation in patients receiving concomitant platelet inhibitors. J Thromb Thrombolysis 2016; 42 (02) 161-166
    CrossrefPubMedGoogle Scholar
  • 41 Hauel NH, Nar H, Priepke H, Ries U, Stassen JM, Wienen W. Structure-based design of novel potent nonpeptide thrombin inhibitors. J Med Chem 2002; 45 (09) 1757-1766
    CrossrefPubMedGoogle Scholar
  • 42 López JA. The platelet glycoprotein Ib-IX-V complex. In: Gresele P, Kleiman NS, Lopez JA, Page CP. , eds. Platelets in Thrombotic and Non-Thrombotic Disorders: Pathophysiology, Pharmacology and Therapeutics: An Update. Cham, Switzerland: Springer International Publishing; 2017: 85-97
    CrossrefGoogle Scholar