Prognostic Value of Circulating Microvesicle Subpopulations in Ischemic Stroke and TIA

Abstract

Platelet microvesicles (PMV) have previously been found elevated in acute ischemic stroke (IS) and could be biomarkers for risk of recurrence. PMV surface antigens such as P-selectin and phosphatidylserine (PS) reflect platelet activation and procoagulance. Tissue factor-positive microvesicles (TF⁺MV) are considered procoagulant, in particular if co-expressing PS. We enumerated MV subpopulations with these surface antigens in a cohort of 211 patients with primarily non-cardioembolic IS or transient ischemic attack (TIA) and investigated their association with long-term outcome. MV concentrations were determined by flow cytometry in the acute and convalescent phase. Primary outcome was a composite of fatal and non-fatal recurrent IS or myocardial infarction. Secondary outcomes were recurrent IS and all-cause mortality. Outcome events were obtained from Swedish registers during a follow-up of 1100 patient years. Concentrations of PS-positive and PS-negative MV populations were elevated in patients compared with healthy controls in both the acute and convalescent phase. PS⁺TF⁺PMV displayed pronounced elevations, median fold change 77 in the acute phase (p < 0.0001) but were not associated with outcome, neither were PS⁺P-selectin⁺PMV. The only subpopulation positively associated with primary outcome was PS^-TF⁺PMV, with adjusted hazard ratio of 1.86 (1.04-3.31, p = 0.036) by Cox regression. Unexpectedly, several MV subpopulations tended to be associated with reduced risk of poor long-term outcome. Our results suggest that PS⁺TF⁺PMV may be a promising marker for cerebral ischemia, and that the in vivo generation of PS^-MV after IS/TIA warrants further study. Future MV studies should ideally enumerate PS⁺ and PS^-MV subpopulations separately.

Keywords: Ischemic stroke; Microvesicles; Phosphatidylserine; Platelet; TIA.

Fig. 1

Schematic view of analyzed platelet microvesicles (PMV) and tissue factor (TF)-positive microvesicles (TF⁺MV) with their presumed cell of origin. Platelets express surface integrin GPIIbIIIa (dark purple) abundantly. Platelet activation causes exposure of phosphatidylserine (PS, red). Activated platelets release different PMV (light blue), positive or negative for PS. P-selectin-positive (dark blue) PMV (P-selectin⁺PMV) signal platelet activation including degranulation of α-granules. Primarily monocytes are thought to express TF (dark green). Monocyte TF may be transferred to platelets via uptake of monocyte MV (light green) or cell interactions (broad light green arrow). Subsequent platelet activation may lead to release of TF⁺PMV. Non-platelet TF⁺MV (npTF⁺MV, green) primarily from monocytes can also be released directly into the circulation. PMV lacking activation markers P-selectin and TF form a subset of PMV and were not enumerated separately. Sizes of cells, MV, and surface markers are non-proportional for clarity. The figure was created with images from Servier Medical Art, licensed under Creative Common Attribution 3.0 Unported License

Fig. 2

a Concentrations of PS⁺PMV and PS⁺P-selectin⁺PMV and b PS⁻PMV and PS⁻P-selectin⁺PMV. Red, patients in the acute phase; gray, patients in the convalescent phase; and white, healthy controls. ***p < 0.0001. Box, 25–75%; whiskers, 5–95%

Fig. 3

Concentrations of PS⁺TF⁺PMV and PS⁻TF⁺PMV. Red, patients in the acute phase; gray, patients in the convalescent phase; and white, healthy controls. ***p < 0.0001. Box, 25–75%; whiskers, 5–95%

Fig. 4

Concentrations of non-platelet (np) PS⁺TF⁺MV and PS⁻TF⁺MV. Red, patients in the acute phase; gray, patients in the convalescent phase; white, healthy controls. ***p < 0.0001; NS, non-significant. Box, 25–75%; whiskers, 5–95%

Fig. 5

Univariate Kaplan-Meier curves for recurrent ischemic events versus respective MV concentrations above (red curves) and below median (black curves). a Primary outcome for patients with PS⁻TF⁺PMV above and below median in the acute phase. b Primary outcome for PS⁺PMV above and below median in the acute phase. c Primary outcome for patients with PS⁻P-sel⁺PMV above and below median in the convalescent phase. d Secondary outcome recurrent IS for patients with non-platelet PS⁺TF⁺MV above and below median in the convalescent phase