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Targeted inhibition of intrinsic coagulation limits cerebral injury in stroke without increasing intracerebral hemorrhage.

Choudhri TF, Hoh BL, Prestigiacomo CJ, Huang J, Kim LJ, Schmidt AM, Kisiel W, Connolly ES, Pinsky DJ - J. Exp. Med. (1999)

Bottom Line: A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site.Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections.At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurological Surgery, University College of Physicians and Surgeons, New York 10032, USA.

ABSTRACT
Agents that restore vascular patency in stroke also increase the risk of intracerebral hemorrhage (ICH). As Factor IXa is a key intermediary in the intrinsic pathway of coagulation, targeted inhibition of Factor IXa-dependent coagulation might inhibit microvascular thrombosis in stroke without impairing extrinsic hemostatic mechanisms that limit ICH. A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site. In a modified cephalin clotting time assay, in vivo administration of Factor IXai caused a dose-dependent increase in time to clot formation (3.6-fold increase at the 300 micrograms/kg dose compared with vehicle-treated control animals, P < 0.05). Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections. At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH. Factor IXai was cerebroprotective even when given after the onset of stroke, indicating that microvascular thrombosis continues to evolve (and may be inhibited) even after primary occlusion of a major cerebrovascular tributary.

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(A) The effect of stroke on the accumulation of radiolabeled platelets, and the inhibitory effects of Factor IXai on platelet accumulation. 111In-labeled platelets were administered either to control animals not subjected to stroke  or to animals immediately before stroke treated preoperatively with vehicle  or with Factor IXai . Platelet accumulation at 24 h is expressed as the ipsilateral cpm/contralateral cpm. Means ± SEM are shown. *P < 0.05 vs. No Stroke and vs. Stroke + IXai. (B) Accumulation of fibrin in infarcted cerebral tissue. After 45 min of right MCAO and 24 h of reperfusion, brains were harvested from representative mice which had been treated before surgery with either vehicle (left lanes) or Factor IXai (300 μg/kg, right lanes). The brains were divided into ipsilateral (R) and contralateral (L) hemispheres, and plasmin digestion was performed to solubilize accumulated fibrin. Immunoblotting was performed using a primary antibody directed against a neoepitope expressed on the gamma-gamma chain dimer of cross-linked fibrin. (C) Immunohistochemical identification of sites of fibrin formation in stroke. Using the same procedures described in B, brains were harvested at 24 h, formalin fixed/paraffin embedded, and fibrin was detected immunohistochemically using the antifibrin antibody. Cerebral microvessels, shown in the center of each field, stained prominently for fibrin (sepia) in the ipsilateral hemisphere of vehicle-treated animals (top right). In contrast, microvessels from the ipsilateral hemisphere of Factor IXai–treated mice rarely demonstrated intravascular fibrin (bottom right). (D) Effect of Factor IXai on CBF in a murine stroke model. Serial measurements of relative CBF were made using a laser doppler over precisely defined neuroanatomic landmarks (reference 21), expressed as ipsilateral/contralateral CBF. Experiments were performed as described in B; , . Means ± SEM are shown. *P < 0.05.
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Figure 1: (A) The effect of stroke on the accumulation of radiolabeled platelets, and the inhibitory effects of Factor IXai on platelet accumulation. 111In-labeled platelets were administered either to control animals not subjected to stroke or to animals immediately before stroke treated preoperatively with vehicle or with Factor IXai . Platelet accumulation at 24 h is expressed as the ipsilateral cpm/contralateral cpm. Means ± SEM are shown. *P < 0.05 vs. No Stroke and vs. Stroke + IXai. (B) Accumulation of fibrin in infarcted cerebral tissue. After 45 min of right MCAO and 24 h of reperfusion, brains were harvested from representative mice which had been treated before surgery with either vehicle (left lanes) or Factor IXai (300 μg/kg, right lanes). The brains were divided into ipsilateral (R) and contralateral (L) hemispheres, and plasmin digestion was performed to solubilize accumulated fibrin. Immunoblotting was performed using a primary antibody directed against a neoepitope expressed on the gamma-gamma chain dimer of cross-linked fibrin. (C) Immunohistochemical identification of sites of fibrin formation in stroke. Using the same procedures described in B, brains were harvested at 24 h, formalin fixed/paraffin embedded, and fibrin was detected immunohistochemically using the antifibrin antibody. Cerebral microvessels, shown in the center of each field, stained prominently for fibrin (sepia) in the ipsilateral hemisphere of vehicle-treated animals (top right). In contrast, microvessels from the ipsilateral hemisphere of Factor IXai–treated mice rarely demonstrated intravascular fibrin (bottom right). (D) Effect of Factor IXai on CBF in a murine stroke model. Serial measurements of relative CBF were made using a laser doppler over precisely defined neuroanatomic landmarks (reference 21), expressed as ipsilateral/contralateral CBF. Experiments were performed as described in B; , . Means ± SEM are shown. *P < 0.05.

Mentions: Using a murine model of middle cerebral artery occlusion (MCAO) with an intraluminal vascular suture, which is removed after 45 min to initiate reperfusion, the occurrence of microvascular thrombosis distal to the site of primary occlusion was examined. Platelet-rich thrombotic foci occur within the ischemic cerebral hemisphere, as shown by experiments in which 111In-labeled platelets were administered to mice immediately before ischemia and their accumulation in the ipsilateral hemisphere measured at 24 h. In animals not subjected to the surgical procedure to create stroke, the presence of platelets was approximately equal between the right and left hemispheres, as would be expected (Fig. 1 A, left bar). However, when animals were subjected to stroke (and received only saline vehicle for control), radiolabeled platelets preferentially accumulated in the ischemic (ipsilateral) hemisphere, compared with significantly less deposition in the contralateral (nonischemic) hemisphere (Fig. 1 A, middle bar). These data support the occurrence of platelet-rich thrombi in the ischemic territory. Another line of evidence also supports the occurrence of microvascular thrombosis in stroke. These data come from the immunodetection of fibrin, using an antibody directed against a neoepitope on the gamma-gamma chain dimer of cross-linked fibrin. Immunoblots demonstrate a band of increased intensity in the ipsilateral (right) hemisphere of vehicle-treated animals subjected to focal cerebral ischemia and reperfusion (Fig. 1 B, Vehicle). To demonstrate that fibrin accumulation was due to the deposition of intravascular fibrin (rather than due to nonspecific permeability changes and exposure to subendothelial matrix), fibrin immunostaining clearly localized the increased fibrin to the lumina of ipsilateral intracerebral microvessels (Fig. 1 C, top panels). As an in vivo physiological correlate of microvascular thrombosis, relative CBF was measured by laser doppler during the occlusive period as well as after stroke. These data (Fig. 1 D, bars labeled Vehicle) show that the intraluminal suture technique significantly reduces ipsilateral CBF during the occlusive period (Fig. 1 D, middle). Blood flow remains depressed even 24 h after removing the intraluminal occluding suture (Fig. 1 D, right), corresponding to the platelet, fibrin immunoblot, and fibrin immunostaining data indicating the presence of postischemic microvascular thrombosis.


Targeted inhibition of intrinsic coagulation limits cerebral injury in stroke without increasing intracerebral hemorrhage.

Choudhri TF, Hoh BL, Prestigiacomo CJ, Huang J, Kim LJ, Schmidt AM, Kisiel W, Connolly ES, Pinsky DJ - J. Exp. Med. (1999)

(A) The effect of stroke on the accumulation of radiolabeled platelets, and the inhibitory effects of Factor IXai on platelet accumulation. 111In-labeled platelets were administered either to control animals not subjected to stroke  or to animals immediately before stroke treated preoperatively with vehicle  or with Factor IXai . Platelet accumulation at 24 h is expressed as the ipsilateral cpm/contralateral cpm. Means ± SEM are shown. *P < 0.05 vs. No Stroke and vs. Stroke + IXai. (B) Accumulation of fibrin in infarcted cerebral tissue. After 45 min of right MCAO and 24 h of reperfusion, brains were harvested from representative mice which had been treated before surgery with either vehicle (left lanes) or Factor IXai (300 μg/kg, right lanes). The brains were divided into ipsilateral (R) and contralateral (L) hemispheres, and plasmin digestion was performed to solubilize accumulated fibrin. Immunoblotting was performed using a primary antibody directed against a neoepitope expressed on the gamma-gamma chain dimer of cross-linked fibrin. (C) Immunohistochemical identification of sites of fibrin formation in stroke. Using the same procedures described in B, brains were harvested at 24 h, formalin fixed/paraffin embedded, and fibrin was detected immunohistochemically using the antifibrin antibody. Cerebral microvessels, shown in the center of each field, stained prominently for fibrin (sepia) in the ipsilateral hemisphere of vehicle-treated animals (top right). In contrast, microvessels from the ipsilateral hemisphere of Factor IXai–treated mice rarely demonstrated intravascular fibrin (bottom right). (D) Effect of Factor IXai on CBF in a murine stroke model. Serial measurements of relative CBF were made using a laser doppler over precisely defined neuroanatomic landmarks (reference 21), expressed as ipsilateral/contralateral CBF. Experiments were performed as described in B; , . Means ± SEM are shown. *P < 0.05.
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Related In: Results  -  Collection

Show All Figures
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Figure 1: (A) The effect of stroke on the accumulation of radiolabeled platelets, and the inhibitory effects of Factor IXai on platelet accumulation. 111In-labeled platelets were administered either to control animals not subjected to stroke or to animals immediately before stroke treated preoperatively with vehicle or with Factor IXai . Platelet accumulation at 24 h is expressed as the ipsilateral cpm/contralateral cpm. Means ± SEM are shown. *P < 0.05 vs. No Stroke and vs. Stroke + IXai. (B) Accumulation of fibrin in infarcted cerebral tissue. After 45 min of right MCAO and 24 h of reperfusion, brains were harvested from representative mice which had been treated before surgery with either vehicle (left lanes) or Factor IXai (300 μg/kg, right lanes). The brains were divided into ipsilateral (R) and contralateral (L) hemispheres, and plasmin digestion was performed to solubilize accumulated fibrin. Immunoblotting was performed using a primary antibody directed against a neoepitope expressed on the gamma-gamma chain dimer of cross-linked fibrin. (C) Immunohistochemical identification of sites of fibrin formation in stroke. Using the same procedures described in B, brains were harvested at 24 h, formalin fixed/paraffin embedded, and fibrin was detected immunohistochemically using the antifibrin antibody. Cerebral microvessels, shown in the center of each field, stained prominently for fibrin (sepia) in the ipsilateral hemisphere of vehicle-treated animals (top right). In contrast, microvessels from the ipsilateral hemisphere of Factor IXai–treated mice rarely demonstrated intravascular fibrin (bottom right). (D) Effect of Factor IXai on CBF in a murine stroke model. Serial measurements of relative CBF were made using a laser doppler over precisely defined neuroanatomic landmarks (reference 21), expressed as ipsilateral/contralateral CBF. Experiments were performed as described in B; , . Means ± SEM are shown. *P < 0.05.
Mentions: Using a murine model of middle cerebral artery occlusion (MCAO) with an intraluminal vascular suture, which is removed after 45 min to initiate reperfusion, the occurrence of microvascular thrombosis distal to the site of primary occlusion was examined. Platelet-rich thrombotic foci occur within the ischemic cerebral hemisphere, as shown by experiments in which 111In-labeled platelets were administered to mice immediately before ischemia and their accumulation in the ipsilateral hemisphere measured at 24 h. In animals not subjected to the surgical procedure to create stroke, the presence of platelets was approximately equal between the right and left hemispheres, as would be expected (Fig. 1 A, left bar). However, when animals were subjected to stroke (and received only saline vehicle for control), radiolabeled platelets preferentially accumulated in the ischemic (ipsilateral) hemisphere, compared with significantly less deposition in the contralateral (nonischemic) hemisphere (Fig. 1 A, middle bar). These data support the occurrence of platelet-rich thrombi in the ischemic territory. Another line of evidence also supports the occurrence of microvascular thrombosis in stroke. These data come from the immunodetection of fibrin, using an antibody directed against a neoepitope on the gamma-gamma chain dimer of cross-linked fibrin. Immunoblots demonstrate a band of increased intensity in the ipsilateral (right) hemisphere of vehicle-treated animals subjected to focal cerebral ischemia and reperfusion (Fig. 1 B, Vehicle). To demonstrate that fibrin accumulation was due to the deposition of intravascular fibrin (rather than due to nonspecific permeability changes and exposure to subendothelial matrix), fibrin immunostaining clearly localized the increased fibrin to the lumina of ipsilateral intracerebral microvessels (Fig. 1 C, top panels). As an in vivo physiological correlate of microvascular thrombosis, relative CBF was measured by laser doppler during the occlusive period as well as after stroke. These data (Fig. 1 D, bars labeled Vehicle) show that the intraluminal suture technique significantly reduces ipsilateral CBF during the occlusive period (Fig. 1 D, middle). Blood flow remains depressed even 24 h after removing the intraluminal occluding suture (Fig. 1 D, right), corresponding to the platelet, fibrin immunoblot, and fibrin immunostaining data indicating the presence of postischemic microvascular thrombosis.

Bottom Line: A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site.Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections.At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurological Surgery, University College of Physicians and Surgeons, New York 10032, USA.

ABSTRACT
Agents that restore vascular patency in stroke also increase the risk of intracerebral hemorrhage (ICH). As Factor IXa is a key intermediary in the intrinsic pathway of coagulation, targeted inhibition of Factor IXa-dependent coagulation might inhibit microvascular thrombosis in stroke without impairing extrinsic hemostatic mechanisms that limit ICH. A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site. In a modified cephalin clotting time assay, in vivo administration of Factor IXai caused a dose-dependent increase in time to clot formation (3.6-fold increase at the 300 micrograms/kg dose compared with vehicle-treated control animals, P < 0.05). Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections. At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH. Factor IXai was cerebroprotective even when given after the onset of stroke, indicating that microvascular thrombosis continues to evolve (and may be inhibited) even after primary occlusion of a major cerebrovascular tributary.

Show MeSH
Related in: MedlinePlus