CCL5 derived from platelets increases megakaryocyte proplatelet formation

Circulating blood platelets are specialized cells that function to minimize bleeding and blood vessel injury. As such, platelets play a critical role in both normal and disease physiology. Large progenitor cells in the bone marrow called megakaryocytes (MKs) release platelets by extending long processes, designated proplatelets, into sinusoidal blood vessels.¹  Despite the importance of platelets in thrombosis and hemostasis, the mechanism by which MKs complete differentiation and release platelets is poorly understood. Specifically, very little is known about what triggers mature, resting MKs to form proplatelets. Platelet counts rise transiently in the setting of physiological stress, such as myocardial infarction, infection, inflammation, and malignancy.^2-4  What initiates this upregulation is not well understood and has largely been attributed to an inflammatory response and increased cytokine release.^5-7  One cytokine that is highly expressed in inflammatory states is CCL5 (RANTES).⁸  CCL5, which is abundant in human platelets, signals predominantly through CCR5, a 7-transmembrane G-protein–coupled receptor that mediates diverse signaling cascades.⁹ 

Blood collection was performed with institutional review board/institutional animal care and use committee approval and in accordance with the Declaration of Helsinki. Platelets were isolated from healthy volunteers or mice as described previously.¹⁰  Platelets were activated for 10 minutes at 37°C and CCL5 measured by enzyme-linked immunosorbent assay (R&D Systems).

Murine fetal livers were collected from CD1 mice (Charles River Laboratories) on embryonic day 13.5 and cultured in the presence of 70 ng/mL recombinant mouse thrombopoietin (rTPO). Mature MKs were isolated as described elsewhere.^11,12 

MKs were purified and probed as previously described.^11,12 

MK and platelet CCR5 expression and MK number were determined by flow cytometry (BD FACSCanto II) using anti-CCR5 (R&D Systems) and anti-CD41/61 (Emfret), respectively. Ploidy was determined by DNA binding via propidium iodide. Data were analyzed with BD FACSDiva 6.1.3 software.

Dextran sulfate sodium (5% wt/vol in drinking water) was used to induce acute colitis in C57/BL/6 mice. Maraviroc (100 mg/kg) or saline vehicle was injected intraperitoneally daily. After 7 days, mice were euthanized and blood was collected.

Platelets contain proteins such as platelet factor 4 that act on MKs to negatively regulate platelet production.¹³  We hypothesized that platelets also contain positive regulators of megakaryopoiesis. We therefore tested the effect of total platelet releasate on MK proplatelet production. Releasate derived from TRAP (thrombin receptor activator peptide)-activated platelets was added to MK cultures (Figure 1A). Intriguingly, platelet releasate increased MK proplatelet production 47% (Figure 1B-C).^14-16  This novel and unexpected finding prompted further exploration. Previously, we observed that platelets release agonist-dependent factors and cytokines including abundant amounts of CCL5.¹⁰  We hypothesized that CCL5 may be the component of platelet releasate causing increased proplatelet formation. We pretreated MKs with maraviroc, an antagonist specific for the CCL5 receptor CCR5, prior to addition of platelet releasate or immunodepleted CCL5 from the platelet releasate using a neutralizing antibody. Maraviroc and CCL5 neutralization diminished the effect of platelet releasate on proplatelet production by 95% and 70%, respectively, suggesting that platelet-derived CCL5 significantly increased MK proplatelet production through CCR5.

We measured the amount of CCL5 released from platelets after stimulation with various agonists including TRAP, ADP, thromboxane A₂, or MCF-7 tumor cells (Figure 1D). Consistent with previous work, CCL5 release was not agonist dependent.¹⁷ 

Using immunofluorescence, we confirmed that platelets and MKs contain CCL5 (Figure 1E).¹⁸  We measured CCR5 surface expression on human and mouse platelets and MKs utilizing flow cytometry (Figure 1F-G), finding that MKs expressed CCR5 on their surface whereas platelets did not, suggesting that platelet-released CCL5 can signal through MK-localized CCR5. Although lack of CCR5 on platelets has been demonstrated,¹⁹  it is rare for a receptor to be present on MKs but not platelets, indicating that MK CCR5 is specifically excluded or degraded from the platelet surface.

We next tested whether recombinant CCL5 could recapitulate the increased proplatelet production seen with platelet releasate. Indeed, CCL5 significantly increased proplatelet production by 50% (Figure 2A), and Maraviroc significantly and dose-dependently reversed the effect of CCL5 up to 97% (Figure 2B-C).

Although the relationship between ploidy and proplatelet formation is complex, elevated ploidy correlates with increased proplatelet formation.²⁰  Therefore, we probed CCL5’s impact on MK endomitosis (repeated DNA replication without cell division).²¹  Interestingly, CCL5 treatment caused significantly higher ploidy; 65% fewer MKs were 2N, whereas 76% more were 16N (Figure 2D). Maraviroc pretreatment inhibited the CCL5 augmented ploidy, substantiating the idea that the increased ploidy was mediated though CCR5.²⁰ 

We next examined the mechanism by which the CCL5/CCR5 axis enhanced MK maturation and proplatelet formation. We examined the Akt pathway because it is downstream of CCR5 and has a role in megakaryocyte maturation.²²  We performed an antibody-based bioarray probing AKT pathway signaling (Cell Signaling Technology). The results from the array revealed a >1.5-fold increase in BAD, 4E-BP1, and PDK1 phosphorylation, proteins crucial for apoptosis suppression (Figure 2E).^23-25  Maraviroc alone had no affect on BAD, 4E-BP1, and PDK1 phosphorylation, indicating that maraviroc does not have off-target effects. Additionally, the increase observed with CCL5 administration was reversed when megakaryocytes were pretreated with maraviroc, confirming the specificity of CCL5 for CCR5 (Figure 2E). In megakaryocytes, the role of apoptosis in proplatelet formation is an area of active investigation, and recent studies have revealed that the intrinsic apoptosis pathway must be restrained in order for cells to undergo proplatelet formation.^26,27  Therefore, these results suggest that CCL5/CCR5 may support platelet production by facilitating apoptosis inhibition.

Finally, we employed an in vivo murine colitis model in which CCL5 has been linked with inflammation. Mice were given dextran sodium sulfate (DSS) with or without maraviroc to examine the role of the CCL5/CCR5 axis in platelet production during acute inflammation. The platelet counts of DSS-treated mice positively correlated with the white blood cell (WBC) count (Figure 2F; Pearson’s correlation, P = .018). This correlation was not present in sham-treated mice (water or maraviroc alone). Importantly, maraviroc eliminated the correlation between platelet and WBC counts (Figure 2F), suggesting that the component of the inflammatory response that regulates platelet count is mediated through CCR5.

Our data demonstrate that in vitro, CCL5 increased MK ploidy and subsequent proplatelet formation in a CCR5-dependent manner. We propose that CCL5 may increase platelet counts during acute inflammation (Figure 2G, schematic); this is supported by our in vivo data. CCR5 knockout mice do not demonstrate thrombocytopenia, suggesting that CCL5/CCR5 functions in times of stress and not hemostatic platelet production. Although a prior mechanistic link between platelet production and CCL5 has not been identified, clinical data support this association; patients with thrombocytopenia due to idiopathic thrombocytopenic purpura or aplastic anemia have a direct correlation between CCL5 level and platelet count.²⁸  Our data (1) provide a possible explanation for the elevated platelet counts observed in reactive thrombocytosis and (2) suggest that CCR5 may be a therapeutic target for treating thrombocytopenia. Although CCL5 itself may not be a good therapeutic candidate owing to its role in the body’s immunogenic response, small molecules could work to stimulate CCR5 directly. This would provide a therapeutic intervention to stimulate platelet release from existing bone marrow MKs, resulting in an immediate increase in platelet count, representing a significant advantage over current TPO-based therapies, which take 5 to 12 days to be effective.

The authors thank Dr Nancy Berliner, Dr Robert Flaumenhaft, and Dr Marsha Moses for their insightful feedback and unyielding support.

This work was supported by National Institutes of Health, National Heart, Lung, and Blood Institute grants F32HL118865 (K.R.M.), T32HL116324 (K.E.J.), and R01HL68130 (J.E.I.).

Contribution: K.R.M. and K.E.J. designed and carried out experiments, analyzed data, and wrote the manuscript; S.W., J.A.F., M.D.T., T.S.S., R.K., S.H.E.-H., and S.K.W. carried out experiments and analyzed data; R.S.W. and J.E.I. contributed to the manuscript; and E.M.B. supervised all research, contributed to design of experiments, analyzed data, and wrote manuscript.

Conflict-of-interest disclosure: J.E.I. has financial interest in and is a founder of Platelet BioGenesis, a company that aims to produce donor-independent human platelets from human-induced pluripotent stem cells at scale. J.E.I. is an inventor on this patent. The interests of J.E.I. were reviewed and are managed by the Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict-of-interest policies. The remaining authors declare no competing financial interests.

Correspondence: Elisabeth M. Battinelli, Brigham and Women’s Hospital, 1 Blackfan Circle, Karp 5, Boston, MA 02115; e-mail: ebattinelli@partners.org.