Thrombin activation of PAR-1 contributes to microvascular stasis in mouse models of sickle cell disease

Sickle cell disease (SCD) results in an altered erythrocyte physiology that causes vascular complications, such as hemolytic anemia, vaso-occlusion, chronic inflammation, and activation of coagulation.¹  A hypercoagulable state and an increased risk for venous thromboembolism have been well documented in sickle patients.^1-3  Several recent studies have identified coagulation activation as a mediator of disease pathology.^4-6  Furthermore, it has been shown that activation of endothelial protease activated receptor-1 (PAR-1) with thrombin or agonist peptide enhances the interaction between sickle erythrocytes and endothelial cells, dependent on exocytosis of P-selectin (pSel) and von Willebrand factor (VWF) from endothelial Weibel-Palade bodies.^7,8  Therefore, we hypothesized that tissue factor (TF)-mediated activation of coagulation might promote vaso-occlusion in SCD via thrombin-dependent PAR-1 activation on endothelial cells. We tested this hypothesis in 2 mouse models of vaso-occlusion: heme-induced stasis in skin microvasculature⁹  and lipopolysaccharide (LPS)-induced pulmonary vaso-occlusion mediated by arteriolar neutrophil-platelet microemboli.¹⁰ 

All animal experiments were approved by the University of North Carolina, University of Pittsburgh, and University of Minnesota Institutional Animal Care and Use Committees. We used male and female NY1DD¹¹  and HbSS-Townes¹²  transgenic sickle mice. Generation of PAR-1^+/+ and PAR-1^−/− mice has been described previously.¹³  Detailed experimental methods are included in supplemental Materials and methods (available on the Blood Web site).

To determine whether coagulation contributes to microvascular stasis in sickle mice, we used a dorsal skinfold chamber model of vaso-occlusive crisis (VOC).⁹  In NY1DD mice pretreated with control immunoglobulin G, hemoglobin induced stasis in 28 ± 2.9% (mean ± standard deviation) of preselected vessels 1 hour after infusion. This was reduced significantly in NY1DD mice pretreated with an inhibitory anti-TF antibody 1H1 (Figure 1A). To determine whether TF contributes to stasis via generation of the downstream protease factor Xa or thrombin, NY1DD mice were treated with rivaroxaban or dabigatran, respectively. Similar to TF inhibition, factor Xa or thrombin inhibition also significantly reduced microvascular stasis 1 hour after hemoglobin infusion (Figure 1B). Previously, we demonstrated that the same doses of rivaroxaban and dabigatran efficiently anticoagulated sickle mice and reduced plasma levels of thrombin antithrombin complexes to the baseline levels observed in nonsickle mice.⁵  No spontaneous bleeding was observed; however, in a femoral vein bleeding model, hemostatic responses were significantly delayed by both anticoagulants.⁵  It is difficult to speculate what doses of these oral anticoagulants should be used to investigate their effect on the incidence of VOC in sickle cell patients. Importantly, 2 recent studies indicated that the approved doses of rivaroxaban were safely tolerated in sickle patients treated for venous thromboembolism, with noninferiority being demonstrated compared with warfarin.^14,15 

To determine whether thrombin contributes to stasis through activation of PAR-1, we used the PAR-1 inhibitor vorapaxar.¹⁶  Vorapaxar was developed as an inhibitor of human PAR-1; however, it also inhibits mouse PAR-1.^17,18  In NY1DD mice treated with vehicle, microvascular stasis was observed in 34 ± 1.7% of preselected vessels. Importantly, vorapaxar treatment significantly attenuated microvascular stasis 1 hour after hemoglobin infusion (Figure 1C). In contrast to human platelets, mouse platelets do not express PAR-1, and thrombin-mediated platelet activation occurs via cleavage of PAR-3, with subsequent transactivation of PAR-4.¹⁹  Therefore, the protective effect of PAR-1 inhibition in sickle mice is independent of platelets and likely mediated by attenuation of PAR-1 signaling in endothelial cells. To confirm the role of endothelial PAR-1 in hemoglobin-induced stasis, we generated sickle mice that lack PAR-1 expression on all nonhematopoietic cells using bone marrow transplantation.⁵  Efficient bone marrow reconstitution was confirmed by hemoglobin electrophoresis (data not shown). Compared with sickle mice with normal expression of PAR-1, stasis was significantly reduced in sickle mice lacking PAR-1 on all nonhematopoietic cells (Figure 1D). Indeed, we found that this protection was associated with significantly reduced intensity of VWF staining, as well as a nonsignificant trend toward reduced pSel staining, in the lungs of sickle mice lacking PAR-1 (Figure 1E-G). We showed previously that inhibition of pSel or VWF markedly inhibits stasis in sickle mice.⁹  Together, these experiments indicate that thrombin contributes to stasis, in part, via activation of endothelial cell PAR-1. Interestingly, these results differ from our previous observation demonstrating that thrombin-dependent vascular inflammation in sickle cell mice was not attenuated by PAR-1 deficiency on all nonhematopoietic cells.⁵ 

Vorapaxar is approved by the US Food and Drug Administration as an antiplatelet agent for the treatment of myocardial infarction and peripheral arterial disease; however, it is contraindicated in patients with a history of stroke because of increased bleeding risk.¹⁶  Thus, it is unlikely to be used in sickle cell patients, who are at high risk to develop stroke. Importantly, however, this proof-of-concept study demonstrates that targeting PAR-1 has the potential to attenuate VOC in sickle patients. Future studies should explore alternative approaches to interrupt thrombin–PAR-1 signaling on endothelial cells (eg, by inducing biased PAR-1 signaling).²⁰ 

A recent finding suggests that gut microbiota–derived Toll-like receptor ligands can promote vaso-occlusion in SCD.²¹  Using quantitative fluorescent intravital lung microscopy (qFILM), we previously demonstrated pulmonary vaso-occlusion in SCD mice following IV challenge with a low-nanogram dose of LPS.¹⁰  Interestingly, unlike systemic vaso-occlusion, which never occurs in arterioles,²²  pulmonary vaso-occlusion in sickle mice involved microembolism of precapillary pulmonary arterioles by large neutrophil-platelet aggregates.¹⁰  Importantly, inhibition of TF with 1H1 antibody attenuated pulmonary arteriole microembolism in sickle mice, as demonstrated by a reduced number of neutrophil-platelet aggregates and total aggregate area (Figure 2; supplemental Videos 1-4). These results highlight the therapeutic potential of targeting TF-initiated activation of coagulation to prevent systemic vaso-occlusion, as well as lung microembolism that might contribute to acute chest syndrome.¹⁰ 

Despite the well-described hypercoagulable state, only 1 adequately powered placebo-controlled study has investigated the effect of anticoagulation in SCD, using the low molecular weight heparin tinzaparin. Once-daily administration of tinzaparin (175 IU/kg) upon admission for VOC significantly reduced the duration of painful crisis and hospital stay.²³  It has been demonstrated that the concentrations of low molecular weight heparins used clinically to achieve anticoagulation are not sufficient to effectively block pSel.²⁴  Therefore, it is likely that protection provided by tinzaparin was mediated by its anticoagulant properties rather than the direct inhibition of pSel‐mediated cellular interactions.^25,26  This notion is further supported by 2 recent clinical trials. The first investigated the effects of sevuparin, a heparin derivative that retains antiadhesive (anti-pSel) properties but has very low anticoagulant activity (NCT02515838).²⁷  In preclinical models, sevuparin reduced sheep red blood cell adhesion to endothelium and reduced microvascular stasis in animal models²⁷ ; however, daily administration to sickle patients upon admission for painful crisis did not improve resolution of VOC.²⁸  Similar negative results were reported for rivipansel, a pan-selectin inhibitor.²⁹  This is in contrast to the pSel-neutralizing antibody crizanlizumab, which reduced the incidence of VOC in sickle patients when given prophylactically.³⁰  Together with our results, the outcomes of these clinical trials suggest that anticoagulants may reduce the incidence and, possibly, duration of VOC,²³  whereas targeting pSel is only effective in preventing VOCs.

As with all preclinical studies using animal models, one should be careful about overinterpreting results until appropriate clinical trials confirm basic research observations. The single time point used to assess VOC and treatments prior to VOC induction are the main limitations of our study; however, the robust protection reported in our study, together with the limited range of therapeutic options for SCD patients in VOC, highlights the need to investigate anticoagulants as a possible treatment strategy.

This work was supported by National Institutes of Health, National Heart, Lung, and Blood Institute grants R01 HL142604, R01 HL114567, R01 HL128297, R01 HL141080, and T32 HL007149.

Contribution: E.M.S. designed research, performed research, analyzed data, and wrote the manuscript; C.C., T.B., J.N., and S.W. performed research; P.S. and G.M.V. provided analytical tools; N.S.K. designed research and edited the manuscript; J.D.B. designed research and provided analytical tools; and R.P. designed research, analyzed data, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Rafal Pawlinski, UNC Blood Research Center, Division of Hematology/Oncology, Department of Medicine, University of North Carolina at Chapel Hill, 116 Manning Dr, 8008C Mary Ellen Jones Building, Chapel Hill, NC 27599; e-mail: rafal_pawlinski@med.unc.edu; and John D. Belcher, Division of Hematology, Oncology and Transplantation, Department of Medicine, University of Minnesota, 420 Delaware St SE, MMC 480, Minneapolis, MN 55455; e-mail: belcher@umn.edu.