Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis

Abstract

Formation of fibrin is critical for limiting blood loss at a site of blood vessel injury (hemostasis), but may also contribute to vascular thrombosis. Hereditary deficiency of factor XII (FXII), the protease that triggers the intrinsic pathway of coagulation in vitro, is not associated with spontaneous or excessive injury-related bleeding, indicating FXII is not required for hemostasis. We demonstrate that deficiency or inhibition of FXII protects mice from ischemic brain injury. After transient middle cerebral artery occlusion, the volume of infarcted brain in FXII-deficient and FXII inhibitor-treated mice was substantially less than in wild-type controls, without an increase in infarct-associated hemorrhage. Targeting FXII reduced fibrin formation in ischemic vessels, and reconstitution of FXII-deficient mice with human FXII restored fibrin deposition. Mice deficient in the FXII substrate factor XI were similarly protected from vessel-occluding fibrin formation, suggesting that FXII contributes to pathologic clotting through the intrinsic pathway. These data demonstrate that some processes involved in pathologic thrombus formation are distinct from those required for normal hemostasis. As FXII appears to be instrumental in pathologic fibrin formation but dispensable for hemostasis, FXII inhibition may offer a selective and safe strategy for preventing stroke and other thromboembolic diseases.

Figure 1.

Infarct volumes and functional outcomes 24 h after focal cerebral ischemia in WT and FXII^−/− mice, and in FXII^−/− mice infused with human FXII. (A) Representative images of three corresponding coronal sections of WT (left), FXII^−/− (middle), and FXII^−/− mice reconstituted with human FXII (huFXII, 2 μg/g body weight i.v. 10 min before the MCAO; right) stained with TCC. (B) Brain infarct volumes in WT (n = 18), FXII^−/− (n = 18), and FXII^−/− mice reconstituted with huFXII (n = 8); **P < 0.01. (C) Neurological Bederson score assessed at day 1 after tMACO for WT (n = 18), FXII^−/− (n = 18), and huFXII-treated FXII^−/− animals (n = 8); **P < 0.01. n.s., not significant.

Figure 2.

Comparison of FXII^−/− and FXI^−/− mice in the tMCAO model. (A) Representative TTC-stained coronal sections of FXI^−/− and WT mice. Infarct volumes were assessed 24 h after tMCAO (WT, n = 18 and FXI^−/−, n = 11; *P < 0.05). (B) Accumulation of fibrin in the infarcted (+) and contralateral (−) hemispheres of WT, FXI^−/−, and FXII^−/− mice. Fibrin formation 24 h after ischemia was analyzed by immunoblotting using the fibrin-specific antibody 59D8. (C) Immunohistochemical colocalization of fibrin (59D8 antibody) and platelets (anti-GP Ib antibody) in the lumens of microvessels after 24 h in the infarcted hemisphere of WT mice. Bar, 100 μm. (D) Hematoxylin and eosin–stained sections of corresponding territories in the ischemic hemispheres of WT, FXI^−/−, and FXII^−/− mice. The dark arrows indicate blood vessels that are shown magnified in the inserts. Note the reduced microvascular patency in the ischemic areas of WT animals compared with FXI^−/− and FXII^−/− mice. Bar, 25 μm. The asterisk denotes the ischemic lesion.

Figure 3.

Inhibition of FXII activity inhibits clotting in vitro and thrombus formation in vivo. (A) Normal human plasma (open symbols) and WT mouse plasma (filled symbols) were incubated with increasing concentrations of PCK (1–200 μg/ml final concentration), an inhibitor that blocks FXIIa activity and activation. Clotting was initiated by adding kaolin and CaCl₂ (triangles) or TF (squares) to determine the aPTT and the PT, respectively. (B and C) PCK (8 μg/g of body weight) was infused intravenously into WT mice (n = 8) before tMCAO. 24 h after stroke, treated animals were analyzed and compared with untreated controls (ctrl, n = 18 per group). (B) Infarct volumes determined from TTC-stained sequential coronal sections (*P < 0.05) and (C) the neurological function assessed by the Bederson Score for PCK-treated and untreated mice (***P < 0.0001).

Figure 4.

Inhibition of FXII activity does not affect normal hemostasis. (A) Tail bleeding times for PCK-treated (8 μg/g of body weight) and untreated control mice (ctrl; n = 12 per group; ***P < 0.0001). Heparin-infused (hep) mice are shown for comparison. (B) Serial coronal T2-weighted MRI brain images from untreated (ctrl), PCK-treated (8 μg/g of body weight), and FXII^−/− mice at days 1, 3, and 7 after tMCAO (n = 5 per group). The asterisk indicates hydrocephalus of the left lateral ventricle as an indicator of infarct-related swelling.