Circulating CD62E Occurs in Soluble Form as Well as Bound to Endothelial Microparticles (EMP): Functional Differences in Platelet Aggregation in Thrombotic Thrombocytopenic Purpura (TTP).

Abstract

Injured endothelial cells (EC) are believed to play a critical role in the pathophysiology of TTP. Soluble markers of endothelial disturbance measured by enzyme-linked immunoassay (ELISA) have been found elevated in TTP. We have recently demonstrated an increase in the release of CD31/42b- EMP, and CD62E+ EMP. Moreover, we have observed that CD62E+ EMP also express vWF. The aim of this study was to quantitate soluble (s) vs. EMP-bound CD62E (bCD62E) in vitro and in vivo, in relation to the functional activity of vWF+ EMP.METHODS: Brain and renal microvascular endothelial cells (MVEC) were cultured and treated with 10ng/mL TNF-α to induce activation, or deprived of serum and growth factors (GFD) to induce apoptosis. Culture supernatants were collected and evaluated in a time-dependent manner. For in vivo studies, platelet-poor plasma was obtained from 4 TTP patients during the acute phase and upon remission. Filtration through 0.1μm, which retains most EMP, was employed to discriminate between (s) and bCD62E. sCD62E was measured by ELISA post-filtration and bCD62E by ELISA pre-filtration. Additionally, CD62E+ and CD62E+/vWF+ EMP were measured by flow cytometry. To assess pro-aggregatory function, EMP were added to washed platelets in the presence of 1 mg/mL ristocetin and aggregates were measured by flow cytometry.RESULTS:In vitro: Activation did not induce release of sCD62E at 3 hours, although bCD62E was present (1.5±0.5X106 EMP/mL). At 6 hours, some sCD62E was detected in the filtrate (0.09±0.02 ng/mL), but most was present in the unfiltered medium (3.5±0.85 ng/mL), signifying that the majority was bCD62E, confirmed by a doubling of CD62E+ EMP (3.0±0.6X106/mL). Subsequently, sCD62E levels were 1.0±0.2 ng/mL at 12 hr, 3.5±0.7 ng/mL at 18 hr, and 5±0.9 ng/mL at 24 hr. In contrast, EMP counts at 12, 18 and 24 hours were 4.6±1, 7±1.3 and 9±1.8 X106/mL (p=0.01, p=0.01, p=0.02, respectively). For all time periods, 40-60% of CD62E were positive for vWF. In control or GFD cultures, there was not a significant increase in sCD62E or CD62E+ EMP at any time period. MVEC from renal gave similar results. In acute TTP plasma samples, CD62E measured by ELISA was significantly increased (65±22 ng/mL) vs. remission (30±6 ng/mL). bCD62E accounted for 50% in acute and 15% in remission. CD62E+/vWF+ EMP were significantly elevated in plasma from acute TTP patients vs. remission (15±4.5 vs. 3±0.5, p=0.01). Sample filtration resulted in a decrease of >95% EMP in both acute and remission TTP plasma. MVEC-derived CD62E+/vWF+ EMP resulted in a dose-dependent increase in platelet aggregation. Additionally, plasma from 4 TTP patients with elevated CD62E+/vWF+ EMP obtained during the acute phase enhanced the formation of platelet aggregates by 48±12% (p=0.02) above remission plasma with low EMP counts.CONCLUSIONS: The results demonstrate that CD62E heretofore regarded as a soluble marker of endothelial dysfunction, in reality exists in both a soluble and EMP-bound form. Indeed, this distinction is highly relevant because CD62E+ EMP also express vWF and are pro-aggregatory to platelets. These EMP have been shown to be elevated during the acute phase of TTP and decrease upon remission. Thus, CD62E+/vWF+ EMP may be active participants in the formation of platelet-rich thrombi in TTP.