Immune-mediated thrombotic thrombocytopenic purpura plasma induces calcium- and IgG-dependent endothelial activation: correlations with disease severity

Abstract

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by a severe ADAMTS13 deficiency due to the presence of anti-ADAMTS13 autoantibodies, with subsequent accumulation of circulating ultra-large von Willebrand Factor (VWF) multimers. The role of endothelial cell activation as a trigger of the disease has been suggested in animal models but remains to be demonstrated in humans. We prospectively obtained plasma from the first plasma exchange of 25 patients during iTTP acute phase. iTTP but not control plasma, induced a rapid VWF release and P-selectin exposure on dermal human micro-vascular endothelial cell (HMVEC-d) surface, associated with angiopoietin-2 and endothelin-1 secretion, consistent with Weibel-Palade bodies exocytosis. Calcium (Ca2+) blockade significantly decreased VWF release, whereas iTTP plasma induced a rapid and sustained Ca2+ flux in HMVEC-d which correlated in retrospect, with disease severity and survival in 62 iTTP patients. F(ab)’2 fragments purified from the immunoglobulin G (IgG) fraction of iTTP plasma mainly induced endothelial cell (EC) activation with additional minor roles for circulating free heme and nucleosomes, but not for complement. Furthermore, two anti-ADAMTS13 monoclonal antibodies purified from iTTP patient B cells, but not serum from hereditary TTP, induced endothelial Ca2+ flux associated with Weibel-Palade bodies exocytosis in vitro, whereas inhibition of endothelial ADAMTS13 expression using small interference RNA, significantly decreased the stimulating effects of iTTP IgG. In conclusion, Ca2+-mediated endothelial cell activation constitutes a second “hit” of iTTP, is correlated with the severity of the disease and may constitute a possible therapeutic target.

Is Endothelial Activation a Critical Event in Thrombotic Thrombocytopenic Purpura?

“…Moreover, Ren et al showed that administration of xenogeneic AECA to rats led to dose-dependent and complement-mediated TMA features [110]. We also demonstrated a potential role of ADAMTS13 antibodies: anti-ADAMTS13 monoclonal antibodies purified from iTTP patients B cells induced an ECs activation calcium dependent leading to a degranulation of WPBs [68].…”

“…We have recently shown high levels of plasmatic free heme in TTP patients. Furthermore, ECs treatment with hemopexin mildly decreased calcium influx and WPB degranulation in vitro, which supports a modest amplification role of free heme [68].…”

“…Discordances between these studies may be at least partly explained by the use of different experimental methods [62,67]. Finally, our team recently showed that plasmas from acute TTP patients induced a calcium and IgG WPB degranulation in vitro with a strong correlation with the pathology severity [68].…”

Reversible skin microvascular hyporeactivity in patients with immune-mediated thrombocytopenic thrombotic purpura

“…In iTTP patients, circulating EC number and plasma biomarkers reflecting endothelial activation are increased and correlated with the outcome supporting a role of the endothelium in the pathophysiology of iTTP [ 24 ]. Recently, Tellier et al, reported that several plasmatic components, including anti-ADAMTS13 IgG, free heme and possibly others converge to induce EC activation ex vivo [ 25 ]. Interestingly the intensity of the ex vivo EC activation induced by iTTP patients' plasmas correlated with the disease severity [ 25 ].…”

“…Recently, Tellier et al, reported that several plasmatic components, including anti-ADAMTS13 IgG, free heme and possibly others converge to induce EC activation ex vivo [ 25 ]. Interestingly the intensity of the ex vivo EC activation induced by iTTP patients' plasmas correlated with the disease severity [ 25 ]. Among soluble factors released in iTTP, hemolysis-derived products (including cell-free heme, free-hemoglobin and bilirubin at high concentration [ 26 , 27 ]) are well known to be highly toxic for the endothelium [ 28 , 29 ].…”