is a preeminent human pathogen and one of the leading causes of death worldwide, especially in infants and young children39,40
. Sepsis caused by S. pneumoniae
infection can trigger disseminated intravascular coagulation (DIC), a life-threatening complication involving the consumption of procoagulants and platelets with the deposition of intravascular fibrin throughout the body, resulting in multi-organ failure35,36,40
. This thrombohemorrhagic pathology is lethal among a significant fraction of patients with severe septicemia and in intensive care. Our findings indicate that the marked thrombocytopenia closely associated with S. pneumoniae
sepsis is neither mediated by the pathogen per se
nor due to platelet consumption in DIC. Instead, thrombocytopenia is the result of Ashwell receptor–dependent clearance of platelets that are first desialylated by the NanA sialidase of the pathogen. Host glycoprotein remodeling by S. pneumoniae
NanA retards the onset of severe hematologic changes that are indicative of acute DIC. Consequently, a subset of normal mice can survive challenge with limiting doses of S. pneumoniae
that are lethal to littermates deficient in Ashwell receptor function.
Both the Asgr-1 and Asgr-2 chains of the Ashwell receptor are required for asialo-platelet clearance, and a deficiency of either leads to a shortened survival time in S. pneumoniae sepsis. The time to death upon challenge with a lethal dose of S. pneumoniae WT was prolonged to a lesser extent by Asgr-2 deficiency as compared to Asgr-1 deficiency. This suggests that asialo-vWF clearance, mediated by Asgr-1 but not Asgr-2, may be important for the relative efficacy of Asgr-1 deficiency in prolonging survival. However, an increased protective effect of Asgr-1 deficiency as compared to Asgr-2 deficiency against severe DIC and morbidity is not yet clearly established, and additional studies are needed to determine the precise lethal dose range of S. pneumoniae in mice deficient in either Asgr-1 or Asgr-2.
The Asgr-1 chain of the Ashwell receptor normally participates in plasma vWF homeostasis independently of sialylation and sepsis. Although we found that asialo-vWF is rapidly removed from circulation by Asgr-1, asialo-vWF accumulation did not occur in Asgr-1–deficient mice. This may be explained by the previously documented ability of the Ashwell receptor to bind sialo- as well as asialo-glycoproteins as potential endogenous ligands9
. We observed increased colocalization of vWF with Asgr-2 in Asgr-1–deficient mice, even after adjustment for reduced hepatocyte vWF abundance in these mice, consistent with the possibility that there may be some compensation by Asgr-2. Nevertheless, plasma vWF abundance is not altered by Asgr-2 deficiency. There seems to be less interdependence and overlap between Asgr-1 and Asgr-2 function than might be expected, and we observed that either chain of the Ashwell receptor can be expressed in the absence of the other in vivo
. Our findings reveal that vWF and platelets are endogenous ligands of the Ashwell receptor, implying that hepatocytes are involved in the clearance of these coagulation factors from the circulation, which is an unexpected biological activity for this cell type.
Respiratory pathogens other than S. pneumoniae
, including Haemophilus influenzae, Pseudomonas aeruginosa
and influenza virus, also express one or more sialidases that target host glycoconjugates to reveal cellular receptors for mucosal adherence or cell penetration41–44
. Not all pathogens that evoke DIC express sialidase activity, however; thus, for these pathogens, the Ashwell receptor would not be expected to modulate pathogenicity by asialoglycoprotein clearance. All S. pneumoniae
strains isolated in nature express the NanA sialidase as a virulence factor that promotes mucosal colonization45,46
. However, we find that NanA also reduces the severity of DIC, revealing an antivirulence activity of NanA that may serve the S. pneumoniae
pathogen by lessening the likelihood of host mortality. NanA expressed by S. pneumoniae
during sepsis removes a subset of α2,3-linked sialic acids but does not appear to remove this sialic acid linkage from the core 1 O
-glycan structure and thus does not expose the Thomsen-Friedenreich antigen, as indicated by unaltered peanut agglutinin lectin binding on either platelets or red blood cells. Although different sialic acid linkages often have unique functions47
, loss of sialic acids in general can reduce the negative charge at the platelet surface and may in some cases alter their aggregation properties and contribute to disease48
. We hypothesize that the effect of such changes may be exaggerated by aspects of the acute cellular pathophysiology of sepsis, including endothelial dysfunction and leukocyte activation. Of note, the asialo-platelets that accumulate in uninfected ST3Gal-IV deficient mice did not show altered activation profiles or responses (P.K.G., S.U., D.D., N.V., D.T.L. et al.
, unpublished data), and the substantial elevation of circulating asialo-platelet concentration in mice lacking ST3Gal-IV and either Asgr-1 or Asgr-2 was not associated with obvious pathologic changes.
Ashwell receptor expression in hepatocytes is induced rapidly upon birth; the fetus lacks this mechanism of removing circulating glycoproteins49
. As a post-partum glycoprotein and platelet clearance system, the Ashwell receptor may have had a major evolutionary role in the outcome of infection by S. pneumoniae
and perhaps other systemic pathogens. Moreover, the changing ligand specificity of Ashwell receptors in phylogeny50,51
reflects increasing glycan complexity in the host and perhaps mirrors the evolving infectious nature of various pathogens. It is unclear whether the Ashwell receptor provides a selective advantage among vertebrates in the absence of infection. ST3Gal-IV and perhaps other sialyltransferases may be regulated in some circumstances to desialylate endogenous glycoproteins and thereby engage Ashwell receptor clearance activity. Such regulation has yet to be documented, however, and the normal turnover of endogenous circulating glycoproteins has not been linked to ASGPR activity10
. Nevertheless, endogenous sialidases in vertebrates may be involved in some clinical and pathogenic contexts. For example, loss of both sialic acid and galactose from the surface of chilled platelets prepared for transfusion may reflect extracellular glycosidase activities that thereby provoke rapid platelet clearance via binding of exposed N
-acetylglucosamine to αM
integrin on liver-resident Kupffer cells31
The Ashwell receptor provides an adaptive response to S. pneumoniae
infection by detecting the byproducts of NanA sialidase activity on host blood factors and removing these prothrombotic components from circulation, preventing them from promoting the lethal complications of DIC. These results reveal a critical hemostatic role for hepatocytes in the acute pathology of sepsis and show that the Ashwell receptor is a key factor in determining host survival. Similar hemostatic modulation may be induced by treatments that likewise deplete platelet and prothrombotic coagulation factor abundance and function. Exogenously administered sialidase is, for example, capable of desialylating platelets and inducing thrombocytopenia52
. Methods to engage and enhance the Ashwell receptor system may provide a new therapeutic opportunity for impeding the development of DIC in life-threatening systemic infections.