Plasma ADAMTS13 activity inhibition in <i>P. falciparum</i> malaria.

<p>(A&B) To investigate further the mechanisms responsible for the marked and discrepant increase in plasma VWF∶CB, and the significant reduction in ADAMTS13 activity, we investigated the effects of mixing malaria plasma with normal plasma. Plasma from four different children (□, ●, △, ◆) with SM (each with baseline ADAMTS13 activities of ∼0.4 U/dl) were mixed in various proportions with pooled normal plasma, and ADAMTS13 activity determined. No evidence of an immediate ADAMTS13 inhibitor effect was observed (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000349#ppat-1000349-g004" target="_blank">Fig 4A</a>). However following incubation at 37°C for 15 min or 30 min (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000349#ppat-1000349-g004" target="_blank">Fig 4B</a>), significant ADAMTS13 inhibition was observed in malaria plasmas at either 75%∶25% (▲), or 50%∶50% (●), but not in normal control plasma (□). All results represent mean±SEM. (C) To further investigate whether malarial plasma contained an ADAMTS13 inhibitor, individual malaria plasma samples (n = 4) and control plasmas (n = 4) were spiked with recombinant human ADAMTS13. Again, significant inhibition of rADAMTS13 activity (means±SEM) was observed only in malaria plasma (◆) but not in normal plasma (■).</p

Severe <i>Plasmodium falciparum</i> malaria influences plasma VWF antigen level and collagen binding activity.

<p>(A) Plasma VWF∶Ag levels (<i>white bars – left Y axis</i>) were measured by ELISA, and VWF activity by collagen binding activity (VWF∶CB) (<i>grey bars – right Y axis</i>). Each plasma sample was tested in duplicate at three dilutions, and median values for each group are shown. VWF∶Ag and VWF∶CB levels were markedly elevated in patients with cerebral malaria and in children with severe malaria at presentation compared to levels in healthy control children. (B) In a cohort of children with cerebral malaria (CM), the time-course of VWF∶Ag and VWF∶CB levels following admission and commencement of anti-malarial therapy was assessed using follow-up plasma samples collected after 24 and 72 hours respectively. (C) Although both VWF∶Ag and VWF∶CB were increased in all cases of <i>P. falciparum</i> malaria, the relative increase observed in plasma VWF∶CB levels was significantly higher (p<0.05), such that the ratio of CB to Ag was consistently >1 in children with CM (n = 13; ●) or SM (n = 20; ▲) at presentation compared to healthy control subjects (n = 25; □). (<i>Hashed line indicates 1∶1 ratio</i>).</p

Clinical and laboratory characteristics by diagnosis of children with and without <i>Plasmodium falciparum</i> malaria – medians (ranges).

<p>n.t.: not tested.</p>1<p>not recorded for 1 child in NMFI group.</p>2<p>not recorded for 1 child in CM retinopathy positive group.</p>3<p>not recorded for 5 children in CM retinopathy positive group, 1 child in CM retinopathy negative group, 10 children in MM group and 22 children in NMFI group.</p>4<p>not recorded for 10 children in MM group and 22 children in NMFI group.</p>5<p>not recorded for 2 children in CM retinopathy positive group and 1 child in CM retinopathy negative group.</p><p>Normal range for lactate: 0.7–2.1 nM.</p

Plasma concentration of VWF and propeptide in <i>Plasmodium falciparum</i> cerebral malaria patients with retinopathy.

<p>Graphs showing median and scatter of plasma VWF and VWF propeptide levels in retinopathy positive children with cerebral malaria who died (Died) or recovered (Recovered) as measured by ELISA (Mann Whitney U test, p = 0.873, p = 1.000, respectively).</p

Plasma concentration of VWF and propeptide in severe <i>Plasmodium falciparum</i> malaria patients with and without retinopathy.

<p>Graphs showing median and scatter of plasma VWF and VWF propeptide levels in cerebral malaria children with (+) and without (−) retinopathy as measured by ELISA (Mann Whitney U test, p = 0.832 and p = 0.143, respectively).</p

Plasma concentration of von Willebrand factor (VWF) and VWF propeptide in various subsets of children with and without <i>Plasmodium falciparum</i> malaria.

<p>Graphs showing median and scatter of plasma VWF and VWF propeptide levels in children with cerebral malaria (CM) and mild malaria (MM), and in non-malaria febrile illness (NMFI) and non-febrile illness (NFI) controls as measured by ELISA (Kruskal-Wallis, *** P<0.001; **P<0.01 after Bonferroni correction for post-hoc multiple pairwise testing). Dotted lines indicate median levels in local healthy Malawian adults.</p

Plasma concentration of VWF and propeptide in <i>Plasmodium falciparum</i> cerebral malaria patients during and after treatment.

<p>Plasma levels of VWF and VWF propeptide were measured at admission, 2 days and 30 days post-treatment in a cohort of retinopathy positive children with cerebral malaria (Friedman Test, *P<0.05). Dotted lines indicate median levels in local healthy Malawian adults.</p