Exploring a new mechanism for thrombocytopenia in experimental malaria

La Jolla Infectious Disease Institute, San Diego CA

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Abstract

The mechanisms underlying thrombocytopenia (TP), a hallmark of malaria, are ill defined. TP is likely important in severe malarial (SM) pathogenesis because (i) TP in a febrile patient is an indicator for malaria, (ii) TP is a marker for SM severity and (iii) for a poor prognosis. TP, therefore, occurs during malaria and is likely important for SM pathogenesis. TP has not been used clinically or targeted for adjunctive therapy because the mechanism(s) underlying TP and the clinical significance of TP in malaria remain to be determined. We propose to explore (i) a new mechanism for TP in malaria and (ii) determine whether this mechanism functions in SM pathogenesis. We hypothesize that superoxide (SO) produced during malaria leads to translocation of platelet neuraminidase 1 (Neu1) from platelet granules to the platelet surface. Neu1 desialylates (removes sialic acid) platelet surface proteins that results in adhesion of desialylated platelets to hepatocytes via the Ashwell-Morell Receptor (AMR). The removal of circulating platelets then leads to TP. The 2 major complications of SM are cerebral malaria (CM) and severe malarial anemia (SMA). We propose that this TP mechanism (abbreviated SO?TP) is increased in CM compared with SMA and uncomplicated malaria (UM) and/or an alternate mechanism for TP predominates in SMA and UM. We will use animal models of malaria so we can test the importance of this SO?TP mechanism to the development of experimental CM (eCM) and experimental SMA (eSMA). Our preliminary data provide support that each of the steps in the SO?TP mechanism occurs in eCM. The GSSG:GSH ratio is significantly (p<0.05) decreased in eCM, indicating oxidant stress. Platelet desialylation occurs on day 3 of eCM, just prior to the onset of TP on day 4; elevated free sialic acid is also detected on day 4 of eCM. Platelets are detected on AMR+ hepatocytes during eCM. In Aim 1, we will test whether the extent of oxidative stress, platelet desialylation, platelet binding to hepatocyte AMR, and tissue localization in 1: eCM, 2: eSMA, and 3: experimental uncomplicated malaria (eUM). If SO?TP mechanism is marked only in eCM, then this mechanism may be specific to eCM. If SO?TP mechanism is also marked in eSMA but not eUM, then it is common to experimental severe malaria. If it is similar, then the SO?TP mechanism is required for pathogenesis, but other independent processes (e.g., inflammation or coagulopathy) determine whether eCM or eSMA occur. In Aim 2, we test the importance of each step in the pathway (SO ? platelet surface Neu1 ? platelet desialylation ? binding to hepatocyte AMR) to the genesis of 1: eCM, 2: eSMA. Scavenging of SO significantly (p<0.05) protects against eCM and TP, indicating the importance of this aim. We anticipate that blocking each step of pathway will similarly protect against eCM because it is blocking the same SO?TP pathway. Whether this pathway functions in eSMA remains to be determined. This proposal meets R21 exploratory grant guidelines because it is focused on testing a new potential mechanism for TP in experimental malaria and determines whether the SO?TP mechanism functions in eCM and/or eSMA. Because this is an exploratory grant, we cannot address all aspects of our hypothesis, such as whether this mechanism occurs in human CM and SMA; and how platelet binding to hepatocytes leads to cerebral dysfunction in eCM. However, distant organ damage is a phenomenon known to affect the brain. Moreover, hepatocytes are central in arginine metabolism (urea cycle) and hypoargininemia and low nitric oxide bioavailability are critical for CM pathogenesis. Future RO1 studies will test these and other potential mechanisms as well as translate our findings into adjunctive therapy. Potential adjunctive therapies include: SO scavenger, neuraminidase inhibitor, and AMR blocking mAb. Our proposed studies test whether a proposed novel mechanism for TP occurs in experimental malaria, correlates with eCM and/or eSMA, and whether it functions in eCM and/or eSMA pathogenesis.