A1-A2 Junction Research Articles

Tissue Factor-Dependent Activation of Factor VIII by Factor VIIa in the Early Phase of Blood Coagulation

Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation through activation of factor X in the initiation phase. FVIIa can catalyze intrinsic clotting factors such as not only factor IX, but also factor VIII (FVIII). However the role and the mechanisms of the FVIIa-catalyzed FVIII are poorly understood. We first examined FVIIa-catalyzed FVIII activation in the presence of phospholipid (PL) using a one-stage clotting assay. The levels of FVIII activity elevated rapidly by ~4-fold within 30 sec after the addition of FVIIa (1 nM)-TF (1 nM)complex, and subsequently decreased to the initial level within 20 min. This time-dependent reaction was enhanced by the presence of TF and PL in dose-dependent manners, but was moderately inhibited (~50%) in the presence of von Willebrand factor at physiological concentration of 10 μg/mL. FVIII cleavage was evaluated using western blotting immediately after the addition of FVIIa-TF complex. The heavy chain of FVIII was proteolyzed more rapidly (at 15 sec) by cleavages at Arg740 (A2-B junction) and Arg372 (A1-A2 junction) by FVIIa-TF complex, whilst the cleavage at Arg336 in the A1 domain was appeared at ~2.5 min. However little cleavage of the light chain of FVIII was observed, supporting that cleavages at Arg740/Arg372 and Arg336 by FVIIa-TF complex contribute to the up- and down-regulation of FVIII(a) activity, respectively. Of interest, no proteolysis of isolated intact heavy chain was observed, indicating that the proteolysis of the heavy chain was governed by the presence of the light chain. Compared to FVIII activation by thrombin (0.1–1 nM), the activation by FVIIa (0.1–1 nM)-TF (1 nM) complex was observed more rapidly. The activation rate observed by the addition of FVIIa-TF complex was ~50-fold greater than that by thrombin. Kinetics by the chromogenic Xa generation assay showed the catalytic efficiency (kcat/Km; 8.9 min−1/32.8 nM) on FVIIa-TF complex-catalyzed FVIII activation showed ~4-fold greater than that on thrombin-catalyzed activation (kcat/Km; 7.5 min−1/86.4 nM). Furthermore, the catalytic efficiencies on cleavages at Arg740 and Arg372 of FVIII by FVIIa-TF complex were ~3- and ~20-fold greater compared to those by thrombin, respectively. These findings suggested that FVIIa-TF complex was a greater FVIII activator than thrombin in very early phase. In order to localize the binding region for FVIIa, we evaluated the interactions between FVIII subunit and Glu-Gly-Arg-active site modified FVIIa, lacking enzymatic activity, in a surface plasmon resonance-based assay. The heavy chain of FVIII bound to EGR-FVIIa with higher affinity than the light chain (Kd; 2.1 and 45 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains (Kd; 34, 37, and 44 nM, respectively), whilst the A1 domain failed to bind. In conclusion, we demonstrated that FVIIa-TF complex rapidly activated FVIII by proteolysis of the heavy chain and the activation was governed by the presence of the light chain. Furthermore, present results suggested the role of TF-dependent FVIII activation by FVIIa which is responsible for the initiation phase of blood coagulation.

Mechanisms of Factor VIII Activation by Recombinant Factor VIIa Analog through Tissue Factor-Independent Manner

Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation. Analog of recombinant FVIIa (rFVIIa), NN1731 (V158D/E296V/M298Q) possesses a greater hemostatic effect than rFVIIa and has been expected in clinical application. Factor X activation rate of NN1731 compared to rFVIIa was 1.2-fold in the presence of TF (TF(+)), and was 30-fold on activated platelets in its absence (TF(−))(Allen, Arterioscler Thromb Vasc Biol. 2007; 27: 683). This TF-independent mechanism likely attributes to excellent effects by NN1731. More recently, we reported the physiological role of FVIIa/TF-dependent FVIII activation in the early phase of blood coagulation. Therefore, we were tempted to investigate the action of NN1731 in FVIII activation. Time-dependent change in FVIII activity after the addition of rFVIIa/NN1731 was examined by one-stage clotting assay under the presence of phospholipids (PS:PC:PE=1:6:3), CaCl2 and TF(+)/TF(−). NN1731 raised FVIII activity up to peak level rapidly within 30 sec (TF(+)), following by inactivation. Peak level of FVIII activity by NN1731 in TF(−) reached to the same peak level of that in TF(+) within 5 min, and this peak level persisted for ~30 min. Whilst, peak FVIII level by rFVIIa in TF(−) showed only ~35% of that in TF(+) even at 30 min. FVIII activating rate of NN1731 was observed to be 1.2-fold (TF(+)) and 3.8-fold (TF(−)) of rFVIIa-catalyzed activation. Kinetics by the Xa generation assay showed the Km values of NN1731 in FVIII activation were ~1.5-fold lower than those of rFVIIa (NN1731/rFVIIa; TF(+) 27.3/49.2 nM and TF(−) 50.5/68.1 nM). Vmax values of NN1731 in FVIII activation, however, showed the obvious difference between TF(+) (2.3-fold; NN1731/rFVIIa 70.0/30.4 nM•min−1) and TF(−) (7.9-fold; 92.5/11.7 nM•min−1), compared to rFVIIa. Inactivation of FVIIIa by NN1731 was somewhat faster than that by rFVIIa. FVIII cleavages by NN1731 were analyzed using SDS-PAGE/Western blotting. The heavy chain of FVIII was proteolyzed at Arg740 (A2-B junction), Arg372 (A1-A2 junction) and Arg336 (within the A1), faster by NN1731 than by rFVIIa. These predominant cleavages by NN1731 were more evident in TF(−). However, little cleavage of the light chain of FVIII was observed by both proteases. FVIII cleavages were correlated with the observations of FVIII activation and/or inactivation. To further localize the binding region for NN1731, we evaluated the interactions between FVIII and Glu-Gly-Arg-active site modified (EGR-) NN1731, lacking enzymatic activity, in a surface plasmon resonance-based assay. The Kd value of EGR-NN1731 with FVIII was similar to that of EGR-rFVIIa (6.3 and 7.8 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains, whilst the A1 domain failed to bind, similar to the results obtained by rFVIIa. We demonstrated that NN1731 possesses higher potential as an activator for up-regulation of FVIII activity than rFVIIa. Furthermore, catalytic activity of NN1731 in TF(−), rather than binding affinity, likely attributes to this potential of its analog. We concluded that the analog has another novel mechanism in its potent hemostatic effect through FVIII activation in TF-independent manner.

Thrombin Cleavage at Arg1689 Influences Subsequent Cleavage at Arg372 during Activation of Factor VIII

Hemophilia A is the most common of the severe inherited bleeding disorders and is caused by defects or deficiencies in the blood coagulation protein, factor VIII. Factor VIII circulates as an inactive procofactor and is converted to factor VIIIa by thrombin or factor Xa-catalyzed cleavage at Arg740 (A2-B junction), Arg372 (A1-A2 junction), and Arg1689 (a3-A3 junction). Factor VIII cleavage by thrombin is suggested to be an ordered mechanism, with initial cleavage at Arg740 facilitating cleavages at Arg372 and Arg1689 (Newell and Fay, J. Biol. Chem. 282: 25376, 2007). The catalytic efficiency for thrombin cleavage at Arg740 is greater than at either Arg1689 or Arg372, while cleavage at Arg372 appears rate-limiting. For this reason, we investigated whether cleavage at Arg1689 influenced catalysis at the Arg372 site. We constructed, stably transfected, and expressed two recombinant factor VIII mutants, Arg1689His and Arg1689Gln, to slow and eliminate, respectively, cleavage at this site. The specific activity of the Arg1689His and Arg1689Gln factor VIII variants were reduced to 47% and 9% the WT factor VIII value, respectively. This result was consistent with impaired cleavage rates of the light chain impacting this parameter. SDS-PAGE and Western blotting analysis of the Arg1689His variant showed a ~150-fold reduction in the rate of cleavage at Arg1689, while the Arg1689Gln variant was resistant to cleavage at this site. Furthermore, the Arg1689His and Arg1689Gln variants showed 2-fold and 7-fold rate reductions, respectively, in the generation of the A2 subunit following thrombin-catalyzed cleavage at Arg372 as compared to WT factor VIII. This result indicated a linkage between cleavage of the light chain and the primary activating cleavage in the factor VIII heavy chain. In monitoring thrombin activation time courses, we observed a 2-fold reduction in the peak activation for the Arg1689His variant and a 12-fold reduction for Arg1689Gln variant compared to wild type factor VIII. To investigate thrombin binding to Arg1689Gln factor VIII, this variant was used as a competitor of thrombin activation of WT factor VIII. The apparent Ki for Arg1689Gln factor VIII (59 nM) was similar to the Km (apparent) of WT factor VIII (55 nM), suggesting that the Arg1689Gln mutation does not impair binding of thrombin to factor VIII. Taken together, these results suggest thrombin cleavage of factor VIII Arg1689 is preferred before cleavage at Arg372 but not required. Since factor Xa also cleaves factor VIII at the same sites as thrombin, we evaluated factor Xa cleavage of factor VIII using SDS-PAGE and Western blot analysis. These results showed that factor Xa cleavage at Arg372, as judged by A2 subunit generation, for both Arg1689 variants was ~2-fold slower than that for WT factor VIII. Moreover, factor Xa cleavage of light chain at the Arg1721 site was reduced by ~2–3 fold for the variants suggesting that factor Xa cleavage at the Arg1689 site is preferred over the Arg1721 site. Together, these results suggest a less selective mechanism for cleavage order using factor Xa as compared with thrombin.

Anterior Communicating Artery Aneurysms: An Overview

AComA aneurysms are most commonly found at the A1-A2 junction on the dominant side. The angle of the arteries at the bifurcation and the direction of blood flow are factors of hemodynamic stress in the apical region where these aneurysms often develop. They exist at the bifurcation of dominant A1, A2 and AComA and usually point in the direction away from the dominant A1. They are more prone to rupture and demonstrate the highest incidence of post-operative morbidity among anterior circulation aneurysms. Consideration of aneurysm morphology may be used to guide approaches in AComA aneurysms. Resection of the gyrus rectus in combination with a pterional approach was popularized by Yasargil and it became the standard for treatment or exposure of AComA aneurysms, although other skull base approaches are also widely used. Clip selection is of extreme importance and the preservation of blood flow to the perforators should be emphasized. Adequate dissection and exposure of the entire "H" complex prior to clipping is the key to a successful outcome. Separating the perforators from the neck or dome of the artery and preserving the parent vessel presents a substantial challenge to the surgeon when the aneurysm is behind the parent artery, making it difficult to achieve a good outcome.

Dissecting Aneurysms of Bilateral Anterior Cerebral Artery Complicated by Subarachnoid Hemorrhage after Cerebral Infarction: A Case Report

Intracranial dissecting aneurysms have been increased due to recent advancements in diagnostic imaging. However there have been little article with subarachnoid hemorrhage and cerebral infarction occurring almost at the same time. We performed the surgical treatment and obtained good result. A 47-year-old male presented to our hospital with chief complaints of sudden headache and mild paralysis of the left lower extremity. Brain imaging at admission revealed cerebral infarction in the right frontal lobe and subarachnoid hemorrhage in the frontal convexy and anterior interhemispheric fissure. The left and right internal carotid angiography showed a bulging cerebral aneurysm at the left A1-A2 junction and stenosis and arterial dissections in the peripheral of the bilateral anterior cerebral artery. Wrapping was performed for the dissecting aneurysm of the left anterior cerebral artery. For the right anterior cerebral artery, trapping was performed at the A2 segment without vascular anastomosis. The patient's postoperative course was uneventful. A consensus has not been reached on the treatment for intracranial dissecting aneurysms. Proximal trapping without vascular reconstruction was performed for the right anterior cerebral artery without vascular anastomosis to prevent rebleeding. However no symptoms of neurological deficiency were observed. Proximal trapping of dissecting aneurysm seems to be a good option when patient's functional and life prognosis are taken into account in case that vascular reconstruction will be anticipated difficulty.

Thrombin-Catalyzed Cleavages of Factor VIII at Arg372 and Arg1689 Are Facilitated by the Acidic Residues Glu720-Asp725.

Factor VIIIa acts as an essential cofactor for the serine protease factor IXa, together forming the Xase complex which catalyzes the conversion of factor X to factor Xa. The procofactor, factor VIII circulates as a heterodimeric protein comprised of a heavy chain (A1–A2-B domains) and a light chain (A3-C1-C2 domains) and is activated by proteolytic cleavage by thrombin at Arg372 (A1–A2 junction), Arg740 (A2-B junction), and Arg1689 (near the N-terminus of A3). The regions adjacent to the A1, A2, and A3 domains contain high concentrations of acidic residues and are designated a1 (residues 337–372), a2 (residues 711–740), and a3 (residues 1649–1689). In addition, the N-terminus of the A2 domain (residues 373–395) is rich in acidic residues, and results from a previous study revealed that this region contributes to the rate of thrombin-catalyzed cleavage at Arg740 (Nogami et. al., J. Biol. Chem . 280:18476, 2005). In this study we reveal a role for the acidic region following the A2 domain (a2, residues 717–725) in thrombin-catalyzed cleavage at both Arg372 and Arg1689. The factor VIII mutations Asp717Ala, Glu720Ala, Asp721Ala, Glu724Ala, Asp725Ala, and the double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala were constructed, expressed, and purified from stably-transfected BHK cells as B-domainless protein. Specific activity values for the variants, relative to the wild type value were reduced to 70% for Asp717Ala; ∼50% for Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala; and ∼30% for Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala. SDS-PAGE and western blotting of reactions containing the factor VIII variants and thrombin showed reductions in the rates of thrombin cleavage at both Arg372 and Arg1689 as compared to wild-type factor VIII. The cleavage rates for the single mutations comprising acidic residues 720–724 of factor VIII were reduced from ∼3-5-fold at Arg372, whereas this rate for the Asp717Ala mutant was similar to the wild-type value. The double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala showed rate reductions of ∼7- and ∼27-fold, respectively at Arg372. While the rate for thrombin-catalyzed cleavage at Arg1689 in the Glu720Ala variant was similar to wild-type, rates for cleavage at this site were reduced ∼30-fold compared to wild-type factor VIII for the Asp721Ala, Glu724Ala, Asp725Ala, and Glu720Ala/Asp721Ala mutants, and ∼50-fold for the Glu724Ala/Asp725Ala variant. Furthermore, the generation of factor VIIIa activity following reaction with thrombin as assayed by factor Xa generation showed that all the mutants possessed peak activity values that were ∼2-3-fold reduced compared to wild type factor VIIIa. Moreover, in all the mutants the characteristic peak of activation was replaced with a slower forming, broad plateau of activity, with the double mutants showing the broadest activation profiles. These results suggest that residues Glu720, Asp721, Glu724, and Asp725 following the A2 domain modulate thrombin interactions with factor VIII facilitating cleavage at Arg372 and Arg1689 during procofactor activation.

Cleavage at Arg740 Appears Essential for Thrombin-Catalyzed Cleavage at Arg372 during the Activation of Factor VIII.

Factor VIIIa serves as an essential cofactor for the factor IXa-catalyzed activation of factor X during the propagation phase of coagulation. The factor VIII procofactor is converted to factor VIIIa by thrombin-catalyzed proteolysis of three P1 positions at Arg372 (A1–A2 junction), Arg740 (A2–B junction), and Arg1689 (a3–A3 junction). Cleavage at Arg372 exposes a cryptic functional factor IXa-interactive site, while cleavage at Arg1689 liberates factor VIII from von Willebrand factor and contributes to factor VIIIa specific activity, thus making both sites essential for procofactor activation. However, cleavage at Arg740, separating the A2–B domainal junction, has not been rigorously studied. To evaluate thrombin cleavage at Arg740, we prepared and stably expressed two recombinant factor VIII mutants, Arg740His and Arg740Gln. Results from a previous study examining proteolysis at Arg372 revealed substantially reduced cleavage rates following substitution of that P1 Arg with His, whereas replacing Arg with Gln at residue 372 yielded an uncleavable bond at that site (Nogami et al., Blood, 2005). Specific activity values for the factor VIII Arg740His and Arg740Gln variants as measured using a one-stage clotting assay were approximately 50% and 18%, respectively, that of the wild type protein. SDS-PAGE and western blotting following a reaction of factor VIII Arg740His with thrombin showed reduced rates of cleavage at His740 as well as at Arg372 relative to the wild type. Alternatively, factor VIII Arg740Gln was resistant to thrombin cleavage at Gln740 and showed little, if any, cleavage at Arg372 over an extended time course. The mutant proteins assayed in a purified system by factor Xa generation showed a slight increase in activity for the Arg740His variant compared with the Arg740Gln variant in both the absence and presence of thrombin, and the activities for both variants were reduced compared with wild type factor VIII. These results suggest that cleavage at residue 740 affects subsequent cleavage at Arg372 and generation of the active cofactor factor VIIIa. Preliminary results obtained evaluating proteolysis of these mutants by factor Xa, which cleaves the same sites in factor VIII as thrombin, also revealed slow proteolysis at the P1 His and no cleavage at the P1 Gln. However, subsequent cleavage at Arg372 exhibited less dependence on initial cleavage at residue 740. These observations may explain the higher than predicted specific activity values obtained for the two variants and suggest a different mechanism of action for the two activating proteinases. Overall, these results support a model whereby cleavage of factor VIII heavy chain by thrombin is an ordered pathway with initial cleavage at Arg740 required to facilitate cleavage at the critical Arg372 site to yield the active cofactor.

Characterization of an Anti-Factor VIII Monoclonal Antibody with A1 and A3 Epitopes Which Increases Factor VIII Activity.

Epitopes of anti-factor VIII (FVIII) neutralizing antibodies distribute all of the FVIII domains. They inhibit FVIII for instance by blocking the FVIII interaction with factor IXa, factor X/factor Xa (FX/FXa), phospholipid, von Willebrand factor, or thrombin. Therefore, localization of such FVIII inhibitory effect gives us useful information for understanding of FVIII structure and function. We expect FVIII enhancing antibody also may be a useful tool. In this study, we found an anti-FVIII monoclonal antibody (named by moAb216) that increased the FVIII cofactor activity. The addition of moAb216 increased FVIII activity by ~1.6-fold dose-dependently in one-stage clotting assay. The increase of FVIII activity in the presence of moAb216 correlated with that of generated thrombin or factor Xa in thrombin or FXa generation based-assay. Blotting analysis revealed that this antibody reacted with only intact FVIII molecule, whilst failed to react with either SDS-treated FVIII, FVIIIa (active-form), or isolated each A1, A2, and A3-C1-C2 subunit. Individual monoclonal antibody, with an epitope of the A1 or A3 acidic region in FVIII, competitively inhibited FVIII binding to moAb216 as well as the increase of FVIII activity by its antibody. However, anti-A2 or anti-C2 monoclonal antibody did not affect. These results supported that this unique antibody, with a discontinuous epitope spanning both acidic regions in the A1 and A3 domains, recognized the native conformation of FVIII. To examine the mechanism(s) of increasing effect by moAb216, we focused on the activation and/or inactivation of FVIII. Rate constants on thrombin- and FXa-catalyzed activation of FVIII in the presence of moAb216 were ~2 and 3-fold greater, respectively, in dose-dependent manners compared with that of FVIII in its absence. On the other hand, the antibody inhibited the activated protein C (APC)-catalyzed FVIII inactivation with ~10-fold lower of inactivation rate constant. SDS-PAGE analysis revealed that moAb216 accelerated the cleavage at Arg372 in the A1-A2 junction by thrombin and FXa, whilst decelerated the cleavage at Arg336 within the A1 domain by APC. In addition, FVIII activity in the presence of moAb216 was more stable following heat denaturation analysis than that in its absence. We demonstrated that the increasing effect of FVIII activity by moAb216 was attributed to the change of cleavage at Arg372 and/or Arg336 in the heavy chain caused by interaction with the A1/A3 domains. Furthermore, moAb216 would be useful as a new replacement therapy for hemophilia A patients, since this increases and stabilizes FVIII activity, and can function even in the presence of anti-FVIII (A2 and C2) inhibitors.

3T MRI — 3D DSA Fusion Technique on Posterior Cerebral Artery Dissecting Aneurysm: Understanding a Potential Pathophysiologic Mechanism

We aimed to show the anatomical relationship between a dissecting aneurysm of the posterior cerebral artery (PCA) and tentorial free edge to understand the pathophysiologic mechanism. A 52-year-old woman with a history of head trauma presented with dizziness and numbness in her left fingers. 3D DSA showed a dissecting aneurysm of the right P2-P3 segment of PCA. The fusion of 3D DSA and 3T MRI was performed at the dedicated workstation using three pairs of landmarks including the ICA termination, MCA bifurcation and A1-A2 junction of the right ACA. Fusion of 3D DSA and 3T MRI clearly demonstrated the dissected segment of PCA crossed the tentorial free edge twice. The fusion images support the direct trauma hypothesis of dissecting aneurysm of the P2-P3 segment of PCA. This novel imaging technique shows future potential to be used to understand the anatomical relationships between various vascular lesions and surrounding structures.

Factor VIII A2 domain residue Asp721 facilitates thrombin–catalyzed cleavage at Arg372

Blood coagulation factor VIIIa serves as an essential cofactor for the factor IXa-catalyzed activation of factor X. Factor VIIIa is generated from the procofactor, factor VIII, a heterodimeric protein comprised of a heavy chain (A1-A2-B domains) and a light chain (a3-A3-C1-C2 domains) following cleavage by thrombin at Arg372 (A1-A2 junction), Arg740 (A2-B junction), and Arg1689 (a3-A3 junction). Both N- and C-termini of the A2 domain (residues 373–740) are rich in acidic residues. Results from a previous study revealed that the N-terminal acidic region modulated cleavage at the A2-B domain junction (Nogami et. al., J. Biol. Chem. 280:18476, 2005). We now show a role for the A2 C-terminal acidic region (residues 717–725) in thrombin-catalyzed cleavage at the A1-A2 junction. In this study, Asp721Ala Factor VIII was constructed, expressed, and purified from stably-transfected BHK cells as a B-domainless protein. The cleavage rate of Asp721Ala factor VIII by thrombin was reduced ~5-fold at Arg372 compared with wild type (WT) factor VIII as judged by SDS-PAGE and western blotting, whereas cleavage rates for the mutant at Arg740 and Arg1649 were equivalent to the WT values. Furthermore, the mutant protein possessed an ~2-fold reduced activity in generating factor Xa, consistent with its reduced activation rate. These results suggest that Asp721, and possibly other residues within this C-terminal acidic cluster, selectively modulate thrombin interaction with factor VIII facilitating cleavage at Arg372 during procofactor activation. This work is supported by NIH grants HL38199 and HL76213.

“Kissing Aneurysms” of the Anterior Communicating Artery Treated With Coil Embolization

To present a case of kissing aneurysms of the anterior communicating artery treated with endovascular coil embolization and discuss the advantages and disadvantages of this technique compared with neck clipping. A 48-year-old man became drowsy and was admitted to the hospital; he had right hemiparesis and aphasia. Computed tomography revealed diffuse subarachnoid hemorrhage; diagnostic angiography identified an aneurysm at the left A1-A2 junction of the anterior communicating artery and another in the distal anterior cerebral artery (ACA). Endovascular coil embolization was performed on the same day. During the procedure, the 3-mm-diameter junctional aneurysm was successfully packed with coils, but an additional aneurysm was suspected; right carotid angiography following embolization of the left aneurysm clearly showed a mirror image aneurysm of the right A1-A2 junction. The right aneurysm was treated using the same technique. The broad-necked ACA aneurysm was unsuitable for embolization, so neck clipping was performed 5 weeks later. The patient was discharged to his home following complete recovery 7 weeks after the coil embolization. Kissing aneurysms are a rare and specific type of multiple aneurysms that require caution in diagnosis and surgical management. Endovascular treatment may be suitable because it does not involve dissection around the aneurysms.

Mechanism of Interaction between the Heavy Chain of Factor VIII and Thrombin.

Factor VIII is activated by proteolytic cleavages catalyzed by thrombin or factor Xa. An earlier study indicated that thrombin binding within the C2 domain facilitated cleavage at Arg1689 of factor VIII light chain (Nogami et al. (2000) J. Biol. Chem. 275, 25774–25780). However, thrombin-interactive region(s) within the heavy chain involved with cleaving the A1-A2 and A2-B domainal junctions remain to be determined. Several approaches were employed to examine the interactions between factor VIII heavy chain and thrombin. Fluorescence energy transfer using acrylodan-labeled A1 or A2 subunits (fluorescence donors) and a fluorescein-labeled, Phe-Pro-Arg-chloromethyl ketone active site-modified thrombin (Fl-FPR-thrombin; fluorescence acceptor) showed that FPR-thrombin bound to the A2 subunit with an ~6-fold higher affinity (Kd =36.6 nM) compared with the A1 subunit (Kd=234 nM). Solid phase binding assays using immobilized thrombin S205A, where the active-site Ser205 was converted to Ala by site directed mutagenesis, showed that the binding affinity of A2 subunit was ~3-fold greater than that of A1 subunit. Similar solid phase assays indicated that hirudin, a ligand for anion-binding exosite I of thrombin (ABE-I), effectively blocked thrombin interaction with A1 subunit while having little if any effect on its interaction with A2 subunit. Conversely, heparin, which binds ABE-II, blocked thrombin interaction with A2 subunit while showing only a marginal effect on A1 subunit binding. To identify an interactive site for thrombin in the A2 domain, we focused on two regions containing clustered acidic residues (389GluGluGluAspTrpAsp394 and 720GluAspSerTyrGluAsp725), which are localized near the N- and C-termini of the A2 domain, respectively. SDS-PAGE analyses using isolated factor VIII heavy chain as substrate showed peptides with the sequences 373–395 and 719–740 encompassing these acidic regions, blocked thrombin cleavage at both Arg372 (A1–A2 junction) and Arg740 (A2–B junction) while a 373–385 peptide did not block either cleavage. The 373–395 and 719–740 peptides competitively inhibited A2 binding to S205A thrombin in a solid phase assay (Ki=11.5 and 12.4 μM, respectively), and quenched the fluorescence of Fl-FPR-thrombin. These data demonstrate that both A2 terminal regions support interaction with thrombin. Furthermore, a B-domainless, factor VIII double mutant D392A/D394A was constructed and possessed specific activity equivalent to a severe hemophilia phenotype (<1% compared with wild type). This mutant was resistant to cleavage at Arg740 whereas cleavage at Arg372 was not appreciably affected. Thus the low specific activity of this mutant was attributed to small C-terminal extensions on the A2 subunit that were not removed following cleavage at Arg740. However, factor Xa cleavage of the mutant at Arg740 was not affected. These data suggest the acidic region comprising residues 389–394 in factor VIII A2 domain interacts with thrombin via ABE-II of the proteinase facilitating cleavage at Arg740.

Identification of a Factor Xa-interactive Site within Residues 337–372 of the Factor VIII Heavy Chain

We recently demonstrated that the residues 337-372, comprising the acidic C-terminal region in A1 subunit, interact with factor Xa during the proteolytic inactivation of factor VIIIa (Nogami, K., Wakabayashi, H., and Fay, P. J. (2003) J. Biol. Chem. 278, 16502-16509). We now show this sequence is important for factor Xa-catalyzed activation of factor VIII. Peptide 337-372 markedly inhibited cofactor activation, consistent with a delay in the rate of cleavage at the A1-A2 junction. Studies using the isolated factor VIII heavy chain indicated that the peptide completely blocked cleavage at the A1-A2 junction (IC50 = 11 microm) and partially blocked cleavage at the A2-B junction (IC50 = 100 microm). Covalent cross-linking was observed between the 337-372 peptide and factor Xa following reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the peptide quenched the fluorescence of dansyl-Glu-Gly-Arg active site-modified factor Xa, suggesting that residues 337-372 directly interact with factor Xa. Studies using a monoclonal antibody recognizing residues 351-365 as well as the peptide to this sequence further restricted the interactive region. Mutant factor VIII molecules in which clustered acidic residues in the 337-372 segment were converted to alanine were evaluated for activation by factor Xa. Of the mutants tested, only factor Xa-catalyzed activation of the D361A/D362A/D363A mutant was inhibited with peak activity of approximately 50% and an activation rate constant of approximately 30% of the wild type values. These results indicate that the 337-372 acidic region separating A1 and A2 domains and, in particular, a cluster of acidic residues at position 361-363 contribute to a unique factor Xa-interactive site within the factor VIII heavy chain that promotes factor Xa docking during cofactor activation.

ACoA angle measured by computed tomographic angiography and its relevance in the pterional approach for ACoA aneurysms

The purpose of this study was to analyze the spatial disposition of the anterior communicating artery (ACoA) complex and the orientation of the ACoA plane by computed tomographic angiography with threedimensional reconstruction (3D-CTA) and to evaluate the relevance of the orientation of the ACoA plane in the planification of the surgical approach to the ACoA complex aneurysms. The dominance of the right or left A1 segment of the anterior cerebral artery, the position of the A1-A2 junction in relation to the transverse plane and the angulation of plane of the ACoA traject were studied using 3D-CTA in 30 patients with subarachnoid hemorrhage. Twelve patients harbored an aneurysm on the ACoA complex and the most relevant anatomic characteristics of these lesions were recorded. In 56.7% of cases both A1 segments were similar in size, whereas in 33.3% of cases the left A1 segment was dominant and in the remaining 10% the right A1 segment was dominant. The position of the left A1-A2 junction was anterior to the transverse plane in 30% of cases with an angulation of the ACoA plane of 42 ± 6 degrees. All patients with this anatomical arrangement and left A1 dominance (seven cases) harbored an aneurysm of the ACoA complex located on the left A1-A2 junction. The right A1-A2 junction was anterior to the transverse plane in 13.3% of cases with an angulation of the ACoA plane of 37 ± 9 degrees. All patients in this situation and right A1 dominance (three cases) had an aneurysm on the ACoA complex. Finally, in 50% of cases the ACoA plane was on the transverse plane. In this group of cases only two patients harbored an aneurysm located on the ACoA segment. The dominance of an A1 segment of the anterior cerebral artery may develop anatomical changes in the ACoA complex due to the hemodynamic stress. The main result is the advance of the A1-A2 junction point in the dominant side and the angulation of the ACoA plane in relation to the transverse plane as well as the development of aneurismatic lesions. The angulation of the ACoA plane, in combination with the orientation of the aneurysm, must be included among the criteria for the selection of the route of the surgical approach. This information is given by the 3D-CTA. All of these data allows a pre-operative simulation of the surgical approach to the lesion for a safer clipping of the neck of the aneurysm. [Neurol Res 2002; 24: 291-295]

内頚動脈閉塞に合併した脳動脈りゅう

We investigated the prevalence and clinical significance of cerebral aneurysm cases that were associated with occlusion of the internal carotid artery (IC). We found 10 such patients (0.9%) among serial patients who were operated on by a senior neurosurgeon. All 3 cases with bilateral occlusion had aneurysms in the P1P2 junction. Cases with unilateral occlusion had a total of 10 aneurysms; 4 in the contralateral A1A2 junction, 3 in the basilar artery or P1P2 junction, 2 in the contralateral IC and 1 in the ipsilateral IC. Four of 11 patients (36%) with P1P2 junction aneurysm were combined with carotid artery occlusion. These findings strongly suggest that hemodynamic stress plays an important role in formation of those aneurysms. Though surgical outcome of those patients did not statistically differ from other patients without IC occlusion, hemodynamic compromise should be considered in treatment of such patients. Furthermore, in patients with occlusion of the major cerebral artery, incidental aneurysm should be taken into account.

A stump of occluded posterior cerebral artery mimicking a ruptured aneurysm: Case report

We report on a 63-year-old female with subarachnoid hemorrhage who had a stump of occluded posterior cerebral artery (PCA) mimicking a ruptured aneurysm of the basilar bifurcation. Intraoperatively, the aneurysmal opacification on preoperative angiograms proved to be the residual lumen of the occluded right P1 segment. Because of the nodular appearance and upward direction of the stump of the right P1 segment, it was misinterpreted as an aneurysm. During operation, a tiny ruptured aneurysm missed on preoperative angiograms was found in the left A1-A2 junction and was clipped safely.

Sites in the A2 Subunit Involved in the Interfactor VIIIa Interaction

Sites in the A2 Subunit Involved in the Interfactor VIIIa Interaction

A case of vessel perforation during interventional neuroradiological procedure operative findings of the perforated vessel

A case of vessel perforation by a guide wire during an interventional neuroradiological procedure is reported. The patient was a 59-year-old woman with a left frontal basal arteriovenous malformation (AVM) fed by the left anterior cerebral artery. Transarterial embolization of the AVM was attempted. During the procedure, vessel perforation by the guide wire occurred at the left A1-A2 junction and resulted in subarachnoid hemorrhage, which stopped spontaneously. The patient developed progressive obstructive hydrocephalus, and surgical treatment was performed. The AVM was totally removed after ventricular drainage, and the arterial perforation site was explored. When clot around the left A1-A2 junction was removed, hemorrhage recurred. This hemorrhage was similar to what has been observed when a small perforating artery was avulsed. The hemorrhage site was coagulated under temporary occlusion of both A1 segments. Surgical intervention was probably not necessary for this type of bleeding if it had stopped spontaneously, because the rebleeding from the small pinhole would be unlikely, and the operation was more hazardous than the usual aneurysmal surgery.

破裂前交通動脈瘤に対する手術法の検討II: 血管写上の特徴と手術法の選択について

In this paper, operative indications, especially as regards the selection of surgical approach for anterior communicating artery aneurysms (ACAA) are discussed with precise and detailed analyses of angiographical anatomy of the aneurysm and the surrounding arteries in 120 surgical cases.Aneurysms originating in the anterior communicating artery itself were much less frequently observed (18.3%), and the majority have their origins on the A1-A2 junction (81.7%). Fifty percent of the A1 segments of the anterior cerebral arteries were dominant on the left side. The right-dominant and the equi-A, type comprised 25% each. In 60% of the cases, the left A2 was anterior to the right one, in 25% vice versa and only in 15% both A, were running in parallel. These inequalities of the arteries surrounding the ACAA seemingly affect the incidence of aneurysmal type. That is, about one third (31.7%) of the ACAA projected laterally from the origin on the A1-A2 junction, and for this lateral-type aneurysm the most suitable surgical approach is the interhemispheric (IH), among numbers of access methods.The location of the neck of the aneurysm, the direction of its dome and the side of the dominant A1 segment of the anterior cerebral artery are important factors in deciding what type of surgical approach is to be done. Though the anteriority of the A, segment is a factor that cannot be disregarded in the pterional (PT) approach, the authors believe that it is not essential in considering the IH approach because this approach can be used for any type of ACAA. At the same time, surgeons should not stick to a single method, especially in the acute stage of subarachnoid hemorrhage because, in this situation, they must try to accomplish the operation as safely, quickly and, of course, completely as possible. In the authors' series, the pterional approach was used in 66 cases (55.0%), the interhemispheric approach in 47 (39.2%), and the bilateral subfrontal and trans-hematoma access were chosen for 4 and 3 cases respectively. And there were 11 cases in which the IH approach with an additional PT approach was used for securing the Al segment which was hidden behind the dome of the aneurysm from the IH surgical view and/or for removal of diffuse and thick subarachnoid clots.

Pterional approach による前交通動脈瘤の手術

The essentials for safe, reliable and prompt ruptured anterior communicating aneurysm surgery through the pterional approach were retrospectively studied by the analysis of preoperative cerebral angiogram (AG) and operative procedure, including topographical microanatomy.The subjects of the study were 28 patients who had undergone acute neck clipping of aneurysms performed by the same surgeon over the last three years.As for preoperative AG, left A1 dominancy was observed in 12 cases (43%), right A1 dominancy in 7 cases (25%) and no dominancy in 9 cases (32%). The number of cases classified according to the projection of the aneurysm were 19 (68%) for anterior, 7 (25%) for inferior and 2 (7%) for dorsalposterior.The size of the aneurysm was 4~14mm (mean 7mm), though the height was 2~14mm (mean 7mm). Concerning the antero-posterior relationship of bilateral A2, segment, there were nine cases (32%) of right A2 posterior to left and 11 cases (39%) of left A2 posterior to right. In eight cases (29%) there was no difference.As regards the essentials of the operative procedure, 15 patients (54%) underwent right craniotomy, while 13 patients (46%) underwent left craniotomy. The area of the craniotomy was extended further to the anterior and the midline in left craniotomy than in right.Premature rupture happened in 2 cases (7%) with inferior projection. A temporary clip on the dominant A1 was applied in 7 cases (25%), two of which were cases of premature rupture and five of which were cases of intentional clipping on dissection of the aneurysmal complex. The mean occlusion time was 9 min.. Rectal gyrus was partially removed in only 2 cases (7%).From these observations, the following conclusions were made. The dominant side of the A1 segment was basically used as the side for the craniotomy. When no dominancy was seen, the posterior side of the A2 segment was chosen.The further extended craniotomy on the left side should be used by right handed surgeons. The advantage of the approach through the same side as the dominant A1 segment was that surgical procedure was extremely safe because of premature rupture and dissection of the aneurysmal complex. On the other hand, it is a disadvantage when the A2 segment is situated anteriory to the oposite side. In these cases, oposite A1 and A2 could be dissected through the“dorsal A1-A2 junction related trangle”. Furthermore, the use of the fenestrated clip was effective.