Inhibition of Protease-Activated Receptor (PAR1) Reduces Activation of the Endothelium, Coagulation, Fibrinolysis and Inflammation during Human Endotoxemia

Christian Schoergenhofer; Michael Schwameis; Georg Gelbenegger; Nina Buchtele; Barbara Thaler; Marion Mussbacher; Gernot Schabbauer; Johann Wojta; Petra Jilma-Stohlawetz; Bernd Jilma

doi:10.1055/s-0038-1655767

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2018; 118(07): 1176-1184
DOI: 10.1055/s-0038-1655767

Coagulation and Fibrinolysis

Georg Thieme Verlag KG Stuttgart · New York

Inhibition of Protease-Activated Receptor (PAR1) Reduces Activation of the Endothelium, Coagulation, Fibrinolysis and Inflammation during Human Endotoxemia

Christian Schoergenhofer

¹Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria

,

Michael Schwameis

¹Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria

,

Georg Gelbenegger

¹Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria

,

Nina Buchtele

¹Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria

,

Barbara Thaler

²Department of Cardiology, Medical University of Vienna, Vienna, Austria

,

Marion Mussbacher

³Department of Thrombosis Research and Vascular Biology, Medical University of Vienna, Vienna, Austria

,

Gernot Schabbauer

³Department of Thrombosis Research and Vascular Biology, Medical University of Vienna, Vienna, Austria

,

Johann Wojta

²Department of Cardiology, Medical University of Vienna, Vienna, Austria

,

Petra Jilma-Stohlawetz

⁴Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria

,

Bernd Jilma

¹Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria

› Author Affiliations

Abstract

The protease-activated receptor-1 (PAR-1) is critically involved in the co-activation of coagulation and inflammatory responses. Vorapaxar is a reversible, orally active, low molecular weight, competitive antagonist of PAR-1.We investigated the effects of PAR-1 inhibition by vorapaxar on the inflammatory response, the activation of coagulation, fibrinolysis and endothelium during experimental endotoxemia. In this randomized, double blind, crossover trial, 16 healthy volunteers received a bolus infusion of 2 ng/kg lipopolysaccharide (LPS) ± placebo/vorapaxar with a washout period of 8 weeks. Vorapaxar dosing was guided by thrombin receptor-activating peptide-6-induced whole blood aggregometry. Participants received 10 mg vorapaxar or placebo as an initial dose and, depending on the aggregometry, potentially an additional 10 mg. Goal was > 80% inhibition of aggregation compared with baseline. Vorapaxar significantly reduced the LPS-induced increase in pro-thrombin fragments F1 + 2 by a median of 27% (quartiles: 11–49%), thrombin–anti-thrombin concentrations by 22% (–3 to 46%) and plasmin–anti-plasmin levels by 38% (23–53%). PAR-1 inhibition dampened peak concentrations of tumour necrosis factor -α, interleukin-6 and consequently C-reactive protein by 66% (−11–71%), 50% (15–79%) and 23% (16–38%), respectively. Vorapaxar decreased maximum von Willebrand factor levels by 29% (26–51%) and soluble E-selectin concentrations by 30% (25–38%) after LPS infusion. PAR-1 inhibition did not affect thrombomodulin, soluble P-selectin and platelet factor-4 concentrations.

PAR-1 inhibition significantly reduced the activation of coagulation, fibrinolysis, the inflammatory response and endothelial activation during experimental human endotoxemia.

Keywords

PAR - endothelium - endotoxemia - inflammation - coagulation

Full Text

References

References
1 Huntington JA. Natural inhibitors of thrombin. Thromb Haemost 2014; 111 (04) 583-589
2 Siller-Matula JM, Schwameis M, Blann A, Mannhalter C, Jilma B. Thrombin as a multi-functional enzyme. Focus on in vitro and in vivo effects. Thromb Haemost 2011; 106 (06) 1020-1033
3 O'Brien PJ, Molino M, Kahn M, Brass LF. Protease activated receptors: theme and variations. Oncogene 2001; 20 (13) 1570-1581
4 Kahn ML, Nakanishi-Matsui M, Shapiro MJ, Ishihara H, Coughlin SR. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 1999; 103 (06) 879-887
5 Schmidt VA, Nierman WC, Maglott DR. , et al. The human proteinase-activated receptor-3 (PAR-3) gene. Identification within a Par gene cluster and characterization in vascular endothelial cells and platelets. J Biol Chem 1998; 273 (24) 15061-15068
6 López ML, Bruges G, Crespo G. , et al. Thrombin selectively induces transcription of genes in human monocytes involved in inflammation and wound healing. Thromb Haemost 2014; 112 (05) 992-1001
7 Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R. Proteinase-activated receptors. Pharmacol Rev 2001; 53 (02) 245-282
8 Tressel SL, Kaneider NC, Kasuda S. , et al. A matrix metalloprotease-PAR1 system regulates vascular integrity, systemic inflammation and death in sepsis. EMBO Mol Med 2011; 3 (07) 370-384
9 Pawlinski R, Pedersen B, Schabbauer G. , et al. Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia. Blood 2004; 103 (04) 1342-1347
10 French SL, Paramitha AC, Moon MJ, Dickins RA, Hamilton JR. Humanizing the protease-activated receptor (PAR) expression profile in mouse platelets by knocking PAR1 into the Par3 locus reveals PAR1 expression is not tolerated in mouse platelets. PLoS One 2016; 11 (10) e0165565
11 Becker RC, Moliterno DJ, Jennings LK. , et al; TRA-PCI Investigators. Safety and tolerability of SCH 530348 in patients undergoing non-urgent percutaneous coronary intervention: a randomised, double-blind, placebo-controlled phase II study. Lancet 2009; 373 (9667): 919-928
12 Tricoci P, Huang Z, Held C. , et al; TRACER Investigators. Thrombin-receptor antagonist vorapaxar in acute coronary syndromes. N Engl J Med 2012; 366 (01) 20-33
13 Morrow DA, Braunwald E, Bonaca MP. , et al; TRA 2P–TIMI 50 Steering Committee and Investigators. Vorapaxar in the secondary prevention of atherothrombotic events. N Engl J Med 2012; 366 (15) 1404-1413
14 Schoergenhofer C, Schwameis M, Hobl EL. , et al. Potent irreversible P2Y12 inhibition does not reduce LPS-induced coagulation activation in a randomized, double-blind, placebo-controlled trial. Clin Sci (Lond) 2016; 130 (06) 433-440
15 Derhaschnig U, Schweeger-Exeli I, Marsik C, Cardona F, Minuz P, Jilma B. Effects of aspirin and NO-aspirin (NCX 4016) on platelet function and coagulation in human endotoxemia. Platelets 2010; 21 (05) 320-328
16 Mayr FB, Jilma B. Coagulation interventions in experimental human endotoxemia. Transl Res 2006; 148 (05) 263-271
17 Pernerstorfer T, Schmid R, Bieglmayer C, Eichler HG, Kapiotis S, Jilma B. Acetaminophen has greater antipyretic efficacy than aspirin in endotoxemia: a randomized, double-blind, placebo-controlled trial. Clin Pharmacol Ther 1999; 66 (01) 51-57
18 Pernerstorfer T, Stohlawetz P, Hollenstein U. , et al. Endotoxin-induced activation of the coagulation cascade in humans: effect of acetylsalicylic acid and acetaminophen. Arterioscler Thromb Vasc Biol 1999; 19 (10) 2517-2523
19 Thomas MR, Outteridge SN, Ajjan RA. , et al. Platelet P2Y12 inhibitors reduce systemic inflammation and its prothrombotic effects in an experimental human model. Arterioscler Thromb Vasc Biol 2015; 35 (12) 2562-2570
20 Kiers D, van der Heijden WA, van Ede L. , et al. A randomised trial on the effect of anti-platelet therapy on the systemic inflammatory response in human endotoxaemia. Thromb Haemost 2017; 117 (09) 1798-1807
21 Jilma-Stohlawetz P, Gilbert JC, Gorczyca ME, Knöbl P, Jilma B. A dose ranging phase I/II trial of the von Willebrand factor inhibiting aptamer ARC1779 in patients with congenital thrombotic thrombocytopenic purpura. Thromb Haemost 2011; 106 (03) 539-547
22 Sibbing D, Braun S, Jawansky S. , et al. Assessment of ADP-induced platelet aggregation with light transmission aggregometry and multiple electrode platelet aggregometry before and after clopidogrel treatment. Thromb Haemost 2008; 99 (01) 121-126
23 Spiel AO, Bartko J, Schwameis M. , et al. Increased platelet aggregation and in vivo platelet activation after granulocyte colony-stimulating factor administration. A randomised controlled trial. Thromb Haemost 2011; 105 (04) 655-662
24 Tóth O, Calatzis A, Penz S, Losonczy H, Siess W. Multiple electrode aggregometry: a new device to measure platelet aggregation in whole blood. Thromb Haemost 2006; 96 (06) 781-788
25 Reiter R, Derhaschnig U, Spiel A. , et al. Regulation of protease-activated receptor 1 (PAR1) on platelets and responsiveness to thrombin receptor activating peptide (TRAP) during systemic inflammation in humans. Thromb Haemost 2003; 90 (05) 898-903
26 Derhaschnig U, Pernerstorfer T, Knechtelsdorfer M, Hollenstein U, Panzer S, Jilma B. Evaluation of antiinflammatory and antiadhesive effects of heparins in human endotoxemia. Crit Care Med 2003; 31 (04) 1108-1112
27 Spiel AO, Derhaschnig U, Schwameis M, Bartko J, Siller-Matula JM, Jilma B. Effects of prasugrel on platelet inhibition during systemic endotoxaemia: a randomized controlled trial. Clin Sci (Lond) 2012; 123 (10) 591-600
28 Mayr FB, Spiel AO, Leitner JM. , et al. Racial differences in endotoxin-induced tissue factor-triggered coagulation. J Thromb Haemost 2009; 7 (04) 634-640
29 Storey RF, Kotha J, Smyth SS. , et al. Effects of vorapaxar on platelet reactivity and biomarker expression in non-ST-elevation acute coronary syndromes. The TRACER Pharmacodynamic Substudy. Thromb Haemost 2014; 111 (05) 883-891
30 Nieuwenhuizen L, Falkenburg WJ, Schutgens RE. , et al. Stimulation of naïve monocytes and PBMCs with coagulation proteases results in thrombin-mediated and PAR-1-dependent cytokine release and cell proliferation in PBMCs only. Scand J Immunol 2013; 77 (05) 339-349
31 Pernerstorfer T, Hollenstein U, Hansen J. , et al. Heparin blunts endotoxin-induced coagulation activation. Circulation 1999; 100 (25) 2485-2490
32 Derhaschnig U, Bergmair D, Marsik C, Schlifke I, Wijdenes J, Jilma B. Effect of interleukin-6 blockade on tissue factor-induced coagulation in human endotoxemia. Crit Care Med 2004; 32 (05) 1136-1140
33 DeLa Cadena RA, Majluf-Cruz A, Stadnicki A. , et al. Recombinant tumor necrosis factor receptor p75 fusion protein (TNFR:Fc) alters endotoxin-induced activation of the kinin, fibrinolytic, and coagulation systems in normal humans. Thromb Haemost 1998; 80 (01) 114-118
34 van der Poll T, Coyle SM, Levi M. , et al. Effect of a recombinant dimeric tumor necrosis factor receptor on inflammatory responses to intravenous endotoxin in normal humans. Blood 1997; 89 (10) 3727-3734
35 Aisiku O, Peters CG, De Ceunynck K. , et al. Parmodulins inhibit thrombus formation without inducing endothelial injury caused by vorapaxar. Blood 2015; 125 (12) 1976-1985
36 Mosnier LO, Sinha RK, Burnier L, Bouwens EA, Griffin JH. Biased agonism of protease-activated receptor 1 by activated protein C caused by noncanonical cleavage at Arg46. Blood 2012; 120 (26) 5237-5246
37 Austin KM, Covic L, Kuliopulos A. Matrix metalloproteases and PAR1 activation. Blood 2013; 121 (03) 431-439
38 Judge HM, Jennings LK, Moliterno DJ. , et al. PAR1 antagonists inhibit thrombin-induced platelet activation whilst leaving the PAR4-mediated response intact. Platelets 2015; 26 (03) 236-242
39 Zhang G, Han J, Welch EJ. , et al. Lipopolysaccharide stimulates platelet secretion and potentiates platelet aggregation via TLR4/MyD88 and the cGMP-dependent protein kinase pathway. J Immunol 2009; 182 (12) 7997-8004
40 Huang Z, Liu P, Zhu L, Li N, Hu H. P2 × 1-initiated p38 signalling enhances thromboxane A2-induced platelet secretion and aggregation. Thromb Haemost 2014; 112 (01) 142-150
41 Joshi S, Whiteheart SW. The nuts and bolts of the platelet release reaction. Platelets 2017; 28 (02) 129-137
42 Heijnen H, van der Sluijs P. Platelet secretory behaviour: as diverse as the granules … or not?. J Thromb Haemost 2015; 13 (12) 2141-2151
43 Totani L, Dell'Elba G, Martelli N. , et al. Prasugrel inhibits platelet-leukocyte interaction and reduces inflammatory markers in a model of endotoxic shock in the mouse. Thromb Haemost 2012; 107 (06) 1130-1140

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