Severe Plasmodium Falciparum Malaria Is Associated with Circulating Ultra-Large Von Willebrand Multimers and ADAMTS13 Inhibition

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IE) to activated endothelial cell (EC) surfaces plays a key role in the pathophysiology of cerebral malaria. Moreover, recent evidence suggests that platelet adhesion and aggregation are critical in facilitating this cytoadhesion process. We have recently reported marked increased plasma VWF and VWF propeptide levels in severe P. falciparum infection, indicative of acute EC perturbation. Furthermore, plasma VWF:Ag levels in patients with malaria inversely correlated with platelet count, and plasma VWF propeptide levels correlated with other established biochemical markers of malaria severity, including plasma lactate. Nevertheless, it remains unclear whether VWF plays any direct role in mediating IE cytoadhesion, or whether the increased plasma levels of both VWF and propeptide merely serve as a marker of acute EC activation.

To address this question, we collected plasma samples from a cohort of African children presenting with cerebral malaria (CM), or non-cerebral severe malaria (SM). In children with CM and SM, plasma VWF:Ag levels were significantly elevated (medians 3.1 and 3.4 IU/ml) as before. VWF collagen binding (VWF:CB) levels were also markedly increased (medians 7.6 and 7.0 IU/ml). Furthermore, the relative rise in VWF:CB was much greater, so that the ratio of CB:Ag was consistently increased. VWF multimer analysis demonstrated abnormal circulating ULVWF multimers in children with CM and SM, consistent with acute, regulated VWF secretion. To characterize mechanisms responsible for the markedly increased VWF:CB and circulating ULVWF in malarial plasma, we investigated ADAMTS13 antigen and activity levels in CM and SM plasma. Plasma ADAMTS13 activity levels (FRETS-VWF73 assay) and antigen levels were both significantly reduced (medians = 0.63 U/ml and 0.56 U/ml; p< 0.001) in children with CM and SM compared to controls. Classical mixing studies of malaria and normal plasma demonstrated no evidence of immediate ADAMTS13 inhibition. However, ADAMTS13 activity in normal plasma was significantly reduced (~60%) following 30 min incubation with malarial plasma (75%:25% mix). This inhibitory ability of malaria plasma was further confirmed by spiking malarial plasmas with recombinant human ADAMTS13. Significant time-dependent inhibition of FRETS-VWF73 activity was again observed in the plasmas of children with severe P. falciparum, but not in normal control plasmas. Potential inhibitors of ADAMTS13 in vivo include interleukin-6 (IL-6), thrombospondin-1 (TSP-1), thrombin and plasmin, free plasma haemoglobin, and reduced FVIII levels. Although plasma IL-6 levels were significantly elevated in children with either CM (mean 240 pg/ml; p<0.001) or SM (mean 217 pg/ml; p=0.01) compared to normal controls, levels did not approach those previously reported necessary to inhibit ADAMTS13 activity. Disseminated intravascular coagulation (DIC) is associated with enhanced thrombin generation, and consumption of FVIII, both of which can inhibit VWF proteolysis by ADAMTS13. However, in children with CM or SM respectively, we observed significantly increased plasma FVIII:C levels. Although intravascular haemolysis is also a recognised complication of malarial infection, we observed only minor increased plasma haemoglobin concentrations, again well below that previously described to significantly inhibit ADAMTS13 activity. Finally, in contrast to the increased plasma levels of IL-6 and FVIII:C, we found that plasma TSP-1 levels were not significantly elevated in children with either CM or SM compared to pooled normal plasma.

In conclusion, we demonstrate that the presence of ULVWF multimers in the plasma of children with severe P. falciparum malaria is the result of

• acute EC activation and release of ULVWF from WP bodies;

• significantly reduced plasma ADAMTS13 antigen levels

• a circulating but unidentified inhibitor of human ADAMTS13 activity.

In view of the critical role played by VWF in mediating platelet adhesion/aggregation, and the accumulating evidence suggesting that platelet adhesion/aggregation also facilitate cytoadhesion of IE, we propose a novel role for ULVWF multimers in the pathophysiology of severe P. falciparum malaria.