Microvascular thrombosis, fibrinolysis, ischemic injury, and death after cerebral thromboembolism are affected by levels of circulating α2-antiplasmin

Abstract

Objective: Ischemic stroke is primarily attributable to thrombotic vascular occlusion. Elevated α2-antiplasmin (a2AP) levels correlate with increased stroke risk, but whether a2AP contributes to the pathogenesis of stroke is unknown. We examined how a2AP affects thrombosis, ischemic brain injury, and survival after experimental cerebral thromboembolism.

Approach and results: We evaluated the effects of a2AP on stroke outcomes in mice with increased, normal, or no circulating a2AP, as well as in mice given an a2AP-inactivating antibody. Higher a2AP levels were correlated with greater ischemic brain injury (rs=0.88, P<0.001), brain swelling (rs=0.82, P<0.001), and reduced middle cerebral artery thrombus dissolution (rs=-0.93, P<0.001). In contrast, a2AP deficiency enhanced thrombus dissolution, increased cerebral blood flow, reduced brain infarction, and decreased brain swelling. By comparison to tissue plasminogen activator (TPA), a2AP inactivation hours after thromboembolism still reduced brain infarction (P<0.001) and hemorrhage (P<0.05). Microvascular thrombosis, a process that enhances brain ischemia, was markedly reduced in a2AP-deficient or a2AP-inactivated mice compared with TPA-treated mice or mice with increased a2AP levels (all P<0.001). Matrix metalloproteinase-9 expression, which contributes to acute brain injury, was profoundly decreased in a2AP-deficient or a2AP-inactivated mice versus TPA-treated mice or mice with increased a2AP levels (all P<0.001). a2AP inactivation markedly reduced stroke mortality versus TPA (P<0.0001).

Conclusions: a2AP has profound, dose-related effects on ischemic brain injury, swelling, hemorrhage, and survival after cerebral thromboembolism. By comparison to TPA, the protective effects of a2AP deficiency or inactivation seem to be mediated through reductions in microvascular thrombosis and matrix metalloproteinase-9 expression.

Keywords: brain ischemia; fibrinolysis; plasminogen activator; thromboembolism; tissue-type plasminogen activator.

© 2014 American Heart Association, Inc.

Figures

Figure 1

The effects of circulating and thrombus-bound a2AP on brain infarction, swelling and dissolution of the culprit MCA thrombus. A) Changes in relative cerebral blood flow (mean ± SE) in the ischemic hemisphere in mice with elevated circulating levels of a2AP (⇑a2AP), normal a2AP levels (Ctl) and absent a2AP (a2AP−/−). B) Representative images of a proximal middle cerebral artery thrombus (arrow), and of TTC-stained brain slices showing viable (red) and infarcted tissue (white) 6 h after thromboembolism. Percent C) infarction, D) dissolution of the MCA thrombus, E) hemorrhage and F) swelling of the ischemic hemisphere 6 h after placement of an 125I-fibrin labeled MCA thrombus (containing normal levels of a2AP). G) Immunoblot analysis of a2AP in thromboemboli formed from a2AP+/+ and a2AP−/− mice. Immuno-stained bands consistent with thrombus-bound a2AP (fibrin-cross linked a2AP, arrows) in a2AP+/+ but not a2AP−/− clots. Top panel, immunostaining with polyclonal anti-a2AP antibody; bottom panel, immunostaining with pooled monoclonal a2AP antibodies. H-K) Effects of thrombus-bound a2AP in vivo on the percent H) MCA thrombus dissolution, I) infarction, J) hemorrhage and K) swelling in brains from a2AP−/− mice with MCA thromboemboli containing normal a2AP (a2AP+/+) or no a2AP (a2AP−/−), n = 7 per group, see Methods for additional details. Data in C-F, H-K represent the means ± SE. **p≤0.01, ***p≤0.001, NS not significant.

Figure 2

Binding specificity and effects of an a2AP inactivating (a2AP-I) monoclonal antibody and TPA on thrombus dissolution, infarction, swelling and hemorrhage after thromboembolism. A) The a2AP-I monoclonal antibody binds specifically to mouse a2AP vs. BSA in an ELISA by comparison to a control (anti-digoxin) monoclonal antibody. See Methods for additional details. B) The a2AP-I (o) accelerates the dissolution of 125I-fibrin labeled mouse plasma clots in vitro by comparison to no monoclonal antibody (●). Clot dissolution was determined as described in Methods. Effect of a2AP-I (9 mg/kg) or TPA (10 mg/kg) treatment given 2.5 hour after thromboembolism on the percent C) brain infarction, D) thrombus dissolution, E) brain swelling and F) hemorrhage. Brains were examined 6 h after cerebral thromboembolism. Data represent the means ± SE. n = 7 mice per group (B-E). *p≤0.05, ***p≤0.001, NS not significant.

Figure 3

Effects of a2AP and TPA on development of microvascular thrombosis after MCA thromboembolism. Representative images (40x) of microvascular thrombus (e.g., green arrows) in the ischemic brain detected by Martius-Scarlet-Blue staining in mice with increased a2AP levels (⇑aAP), normal aAP levels (control), a2AP deficiency (a2AP−/−), a2AP inactivation (a2AP-I) or TPA treatment. Bar graph, quantification of microvascular thrombus by digital imaging. The area of microvascular thrombus per 40x microscopic fields was quantitated by digital imaging in 12-15 random fields using Image-Pro Plus software. Line graph, relation between thrombus area and a2AP blood levels in mice supplemented with a2AP, normal and a2AP-deficient mice. Data represent means ± SE of n = 4-5 mice per group. Bar =20 μm. *p≤0.05, **p≤0.01, ***p≤0.001.

Figure 4

MMP-9 expression after MCA thromboembolism. Representative images of MMP-9 immunofluorescence staining (red) in the ischemic hemispheres of mice with increased a2AP (⇑a2AP), normal a2AP levels (control), a2AP deficiency (a2AP−/−) as well as mice treated with a2AP inactivation (a2AP-I) or TPA. MMP-9 expression was assessed by double immunofluorescence staining for MMP-9 (DyLight549, red) and collagen IV (DyLight488, green). Representative micrographs with DAPI-stained nuclei (blue) were taken using a Zeiss LSM 710 confocal microscope at 40x magnification. Bar graph, quantitative digital analysis of the density of MMP-9 immunofluorescence (immunofluorescence units) of ten 40x fields from each stroke hemisphere. Data represent means ± SE of n = 4-5 mice per group. Line graph, relation between of MMP-9 immunofluorescence staining density (units) and a2AP blood levels in mice supplemented with a2AP, normal and a2AP-deficient mice. *p≤0.05, ** p≤0.001, ***p≤0.001. Bar =50 μm.

Figure 5

Effect of a2AP inactivation (a2AP-I) after cerebral thromboembolism on survival, brain infarction, hemorrhage and swelling. A) Survival in mice treated with no agent (control, Ctl), TPA (10 mg/kg), low dose a2AP-I (9 mg/kg) or high dose a2AP-I (as whole antibody (21.3 mg/kg) or Fab (9.3 mg/kg)) thirty minutes after stroke onset. Mice were observed for 7 days post stroke. Effects of treatment on B) brain infarction, C) brain hemorrhage and D) brain swelling after thromboembolism in surviving mice. Survival groups N= 12-16 in each cohort, data represent means ± SE. *p≤0.05, ***p≤0.001, ****p≤0.0001 vs. control or TPA.