蛇毒型神经生长因子通过影响候选可塑性相关基因15和核转录因子κB的表达促进脑缺血再灌注损伤后神经重塑

Objective To investigate the significance and mechanism of intracerebroventricular injection viper venom nerve growth factor (Vngf) in rat neural plasticity after cerebral ischemia reperfusion injury.Methods Ninety Wistar male rats were randomly assigned into Vngf-25 U group (n = 18), Vngf-50 U group (n = 18), Vngf-100 U group (n = 18), ischemia reperfusion group (n = 18) and sham operated group.The expression of candidate plasticity-related gene 15(cpg-15) Mrna and nuclear factor of kappa B ( NF-Κb ) Mrna in rat brain tissues which were collection at 2,7,14 days after surgery were evaluated by the real time PCR.Results The expression of cpg-15 Mrna and NF-Κb Mrna began to increase after surgery( the F value of cpg-15:70.43, 34.11, 31.89, the F value of NF-Κb: 27.47, 34.56, 31.89,P＜0.01).At the same time, expression of cpg-15 Mrna and NF-Κb Mrna in the Vngf groups was significantly different from the I/R group and the sham operated group (the F value of cpg-15:48.18, 55.93, 78.43, the F value of NF-Κb: 45.92, 55.72, 50.49, P ＜0.01).The more Vngf were injected, the more cpg-15 Mrna and NF-Κb Mrna were expressed in Vngf groups.Conclusions The Vngf could accelerate neural plasticity and restore neurofunctional defect through up-regulated the expression of cpg-15 and NF-Κb.     

蛇毒型神经生长因子通过影响候选可塑性相关基因15和核转录因子κB的表达促进脑缺血再灌注损伤后神经重塑

目的 探讨蛇毒型神经生长因子(viper venom nerve growth factor,Vngf)促进脑缺血再灌注损伤后大鼠神经重塑的可能机制.方法 雄性Wistar大鼠90只根据随机数字表法分成5组:Vngf-25 U组、Vngf-50 U组、Vngf-100 U组、缺血再灌注组和假手术组,每组18只.造模后第2、7、14天分批处死大鼠取脑组织提取总RNA,用荧光定量PcR检测候选可塑性相关基因15(cpg-15)Mrna和核转录因子κB(NF-κB)mRNA的表达.结果 随着时间的延长,vNGF各组的cpg-15mRNA和NF-κB mRNA表达逐渐增加(cpg-15和NF-κB的F值分别为:70.43、34.11、31.89和27.47、34.56、31.89,均P＜0.01);术后同一时问,cpg.15 mRNA和NF.xB mRNA在vNGF各组、缺血再灌注组和假手术组的表达值比较差异有统计学意义(cpg-15和NF-κB的F值分别为:48.18、55.93、78.43和45.92、55.72、50.49,均P＜0.01),并且cpg-15 mRNA和NF-κB mRNA的表达随着vNGF浓度的升高而增加.结论 脑缺血再灌注损伤后经侧脑室给予vNGF,可以通过正性调节cpg-15和NF-κB转导通路促进神经再生和神经缺损功能恢复.

Abstract：

Objective To investigate the significance and mechanism of intracerebroventricular injection viper venom nerve growth factor (Vngf) in rat neural plasticity after cerebral ischemia reperfusion injury.Methods Ninety Wistar male rats were randomly assigned into Vngf-25 U group (n = 18), Vngf-50 U group (n = 18), Vngf-100 U group (n = 18), ischemia reperfusion group (n = 18) and sham operated group.The expression of candidate plasticity-related gene 15(cpg-15) Mrna and nuclear factor of kappa B ( NF-Κb ) Mrna in rat brain tissues which were collection at 2,7,14 days after surgery were evaluated by the real time PCR.Results The expression of cpg-15 Mrna and NF-Κb Mrna began to increase after surgery( the F value of cpg-15:70.43, 34.11, 31.89, the F value of NF-Κb: 27.47, 34.56, 31.89,P＜0.01).At the same time, expression of cpg-15 Mrna and NF-Κb Mrna in the Vngf groups was significantly different from the I/R group and the sham operated group (the F value of cpg-15:48.18, 55.93, 78.43, the F value of NF-Κb: 45.92, 55.72, 50.49, P ＜0.01).The more Vngf were injected, the more cpg-15 Mrna and NF-Κb Mrna were expressed in Vngf groups.Conclusions The Vngf could accelerate neural plasticity and restore neurofunctional defect through up-regulated the expression of cpg-15 and NF-Κb.     

Oxidative phosphorylated neurofilament protein M protects spinal cord against ischemia/reperfusion injury

Previous studies have shown that neurofilament protein M expression is upregulated in the early stage of spinal cord ischemia/reperfusion injury, indicating that this protein may play a role in the injury process. In the present study, we compared protein expression in spinal cord tissue of rabbits after 25 minutes of ischemia followed by 0, 12, 24, or 48 hours of reperfusion with that of sham operated rabbits, using proteomic two-dimensional gel electrophoresis and mass spec- trometry. In addition, the nerve repair-related neurofilament protein M with the unregulated expression was detected with immunohistochemistry and western blot analysis. Two-dimen- sional gel electrophoresis and mass spectrometry showed that, compared with the sham group, upregulation of protein expression was most significant in the spinal cords of rabbits that had undergone ischemia and 24 hours of reperfusion. Immunohistochemical analysis revealed that neurofilament protein M was located in the membrane and cytoplasm of neuronal soma and axons at each time point after injury. Western blot analysis showed that neurofilament protein M expression increased with reperfusion time until it peaked at 24 hours and returned to baseline level after 48 hours. Furthermore, neurofilament protein M is phosphorylated under oxidative stress, and expression changes were parallel for the phosphorylated and non-phosphorylated forms. Neurofilament protein M plays an important role in spinal cord ischemia/reperfusion injury, and its functions are achieved through oxidative phosphorylation.     

大鼠局灶性脑缺血再灌注后凋亡诱导因子的表达与神经元凋亡的关系

Objective To study the relationship between the expression of apoptosis inducing factor (AIF) and neuronal apoptosis in rats with focal ischemia reperfusion.Methods Wistar rats were divided into sham-operated (n=6) and model (n=30) groups.Middle cerebral artery occlusion (MCAO)models were established by thread ligation.The expression of AIF in the ischemic penumbra was observed in the sham-operated and model groups 6 h and 1,2,3 and 7 d after the reperfusion; the changes of neuronal apoptosis in corresponding region were observed by TUNEL staining at the same time.Results AIF-positive cells in the ischemic penumbra were significantly increased in the model group 6 h after the ischemic reperfusion,reaching its peak level at 48 h of reperfusion (130.47±11.32 cells).Cell apoptosis occurred at each time point of ischemic reperfusion and the percentage of apoptosis cells at 48 h ofreperfusion (118.53±11.67 ceils) was the highest among all the time points.Significant differences between the 2 groups were found between each 2 time points (P<0.05).Conclusion Focal ischemia reperfusion can induce the increased expression of AIF positive cells and neuronal apoptosis.     

大鼠局灶性脑缺血再灌注后凋亡诱导因子的表达与神经元凋亡的关系

Objective To study the relationship between the expression of apoptosis inducing factor (AIF) and neuronal apoptosis in rats with focal ischemia reperfusion.Methods Wistar rats were divided into sham-operated (n=6) and model (n=30) groups.Middle cerebral artery occlusion (MCAO)models were established by thread ligation.The expression of AIF in the ischemic penumbra was observed in the sham-operated and model groups 6 h and 1,2,3 and 7 d after the reperfusion; the changes of neuronal apoptosis in corresponding region were observed by TUNEL staining at the same time.Results AIF-positive cells in the ischemic penumbra were significantly increased in the model group 6 h after the ischemic reperfusion,reaching its peak level at 48 h of reperfusion (130.47±11.32 cells).Cell apoptosis occurred at each time point of ischemic reperfusion and the percentage of apoptosis cells at 48 h ofreperfusion (118.53±11.67 ceils) was the highest among all the time points.Significant differences between the 2 groups were found between each 2 time points (P<0.05).Conclusion Focal ischemia reperfusion can induce the increased expression of AIF positive cells and neuronal apoptosis.     

Effects of basic fibroblast growth factor on hippocampal and parietal cortical neuronal cAMP-response element-binding protein expression in a rat model of focal cerebral ischemia/reperfusion

BACKGROUND： cAMP-response element binding protein （CREB） is a key modulator of various signaling pathways. CREB activation initiates a series of intracellular signaling pathways that promote neuronal survival.
OBJECTIVE： To investigate the regulatory effects of basic fibroblast growth factor （bFGF） on cerebral neuronal CREB expression following ischemia/reperfusion injury.
DESIGN, TIME AND SETTING： An immunohistochemical detection experiment was performed at the Department of Anatomy, Shenyang Medical College, between October 2006 and April 2008.
MATERIALS： A total of 60 healthy, adult, Wistar rats were randomly divided into three groups： sham-operated （n =12）, ischemia/reperfusion （n = 24）, and bFGF-treated （n = 24）. Rabbit anti-rat CREB （1： 100） and biotin labeled goat anti-rabbit IgG were purchased from the Wuhan Boster Company, China. MetaMorph-evolution MP5.0-BX51 microscopy imaging system was provided by China Medical University, China. METHODS： Rat models of cerebral ischemia/reperfusion injury were developed using the suture method for right middle cerebral artery occlusion. Two-hour ischemia was followed by reperfusion. Rats from the bFGF-treated and ischemia/reperfusion groups were intraperitoneally administered endogenous bFGF （500 IU/mL, 2 000 IU/kg） or an equal amount of physiological saline. Rats from the sham-operated group underwent a similar surgical procedure, without induction of ischemia/reperfusion injury and drug administration.
MAIN OUTCOME MEASURES： After 48-hour reperfusion, hippocampal and parietal cortical neuronal CREB expression was detected by immunohistochemistry, and the absorbance of hippocampal CREB-positive products was determined using MetaMorph-evolutionMP5.0-BX51 microscopy imaging system.
RESULTS： The sham-operated group exhibited noticeable CREB expression in hippocampal and parietal cortical neurons. In the ischemia/reperfusion group, the CREB expression was discrete and neurons were poorly arranged. The bFGF-treated group exhibited increased CREB expression and better neuronal arrangement compared with the ischemia/reperfusion group. The mean absorbance of CREB-immunoreactive products in the hippocampus and parietal cortex was significantly higher in the ischemia/reperfusion group than in the sham-operated group （P 〈 0.05）, and significantly higher in the bFGF-treated group than in the ischemia/reperfusion group （P 〈 0.05）. CONCLUSION： bFGF significantly upregulates CREB expression in hippocampal and parietal cortical neurons following ischemia/reperfusion injury.     

电针治疗脑缺血：促胰岛素样生长因子1表达的作用

Background Growth factors such as basic fibroblast growth factor (bFGF) have shown neuroprotective effects on cerebral ischemia. Electroacupuncture (EA) could be a potential treatment for cerebral ischemia because it increases local cerebral blood flow and up-regulates the expression levels of bFGF. This study aimed to investigate the neuroprotective effects of EA against cerebral ischemia as well its effect on insulin-like growth factor-1 (IGF-1) expression following middle cerebral artery occlusion (MCAO) in rats. Methods This study compared EA with sham in a sample of 41 male Sprague Dawley rats, weighting between 280-300 g. The animals were randomized into two groups: 12 in the cDNA microarray study and 29 in the immunohistochemical and in situ hybrization study. In the histochemical and in situ hybrization study: normal control (n=1), sham (n=4), Ischemia Ⅰ (9h reperfusion, n=6), Ischemia Ⅰ+EA (9h reperfusion, n=6), Ischemia Ⅱ (24h reperfusion, n=6), Ischemia Ⅱ+EA (24h reperfusion, n=6). Overall changes of gene expression were analyzed with cDNA microarray, and IGF-1mRNA expression was confirmed by in situ hybridization, then the IGF-1 protein expression was detected by immunohistochemistry. EA was given 15 minutes after MCA occlusion and lasted for 1 hour between the Baihui acupoint (GV. 20) and the Renzhong acupoint (GV. 26) using the dense-sparse waveforms, which can be transformed into each other when a dense or sparse wave is terminated. Results After MCA occlusion, IGF-1mRNA and protein expression levels decreased. EA attenuated brain edema, decreased the infarct volume, and led to the upregulation of the decreased IGF-1mRNA and protein expression in the striatum. Conclusions EA is effective to extenuate cerebral ischemia and up-regulate endogenous IGF-1 expression following MCA occlusion, which might be an important mechanism for the neuroprotective effects of EA against cerebral ischemia.     

Time-dependent effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in focal cerebral ischemia rats**☆

BACKGROUND： The onset of focal cerebral ischemia activates extracellular signal-regulated kinases 1 and 2, regulates cell cycle, promotes cell proliferation and differentiation, and affects the normal stage and function of brain cells. OBJECTIVE： To observe the effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in the lateral cerebral ventricle wall of rats with focal cerebral ischemia. The effects were analyzed at different time points after intervention. DESIGN： Randomized controlled study. SETTING： Department of Anatomy, Sun Yat-Sen University. MATERIALS： A total of 60 healthy adult male Wistar rats weighing （250±10） g were provided by the Experimental Animal Center, Medical College of Sun Yat-Sen University. The animal experiment was conducted with confirmed consent by the local ethics committee. The GB6805-Ⅱ electric acupuncture apparatus was provided by Shanghai Medical Equipment High-techno Company. METHODS： The experiment was performed at the Laboratory of Anatomy, Sun Yat-Sen University, from February to July 2007. All experimental animals were randomly divided into the following groups： normal group （n = 6）, sham operation group （n = 18）, model group （n = 18）, and electroacupuncture group （n = 18）. Middle cerebral artery occlusion （MCAO） was performed in the model group and electroacupuncture group. Zea Longa＇s grading standard was used to assess neurological impairment after reperfusion; animals whose grades were between l and 4 were included in this study. The normal control group was not exposed to MCAO. In sham operation animals, the right common carotid artery （CCA） was isolated, and the external carotid artery （ECA） was damaged, but no embolism was induced. The electroacupuncture group was given acupuncture on the second day after surgery. The acupoint locations were chosen according to Experimental Acupuncture （People＇s Publishing House; 1997; First Edition）. The Chengjiang, Qihai, and     

Key genes expressed in different stages of spinal cord ischemia/reperfusion injury

The temporal expression of microRNA atfer spinal cord ischemia/reperfusion injury is not yet fully understood. In the present study, we established a model of spinal cord ischemia in Sprague-Dawley rats by clamping the abdominal aorta for 90 minutes, before allowing reperfusion for 24 or 48 hours. A sham-operated group underwent surgery but the aorta was not clamped. The damaged spinal cord was removed for hematoxylin-eosin staining and RNA extraction. Neuronal degeneration and tissue edema were the most severe in the 24-hour reperfusion group, and milder in the 48-hour reperfusion group. RNA ampliifcation, labeling, and hybridization were used to obtain the microRNA expression proifles of each group. Bioinformatics analysis conifrmed four differentially expressed microRNAs (miR-22-3p, miR-743b-3p, miR-201-5p and miR-144-5p) and their common target genes (Tmem69 and Cxcl10). Compared with the sham group, miR-22-3p was continuously upregulated in all three ischemia groups but was highest in the group with no reperfusion, whereas miR-743b-3p, miR-201-5p and miR-144-5p were downregulated in the three ischemia groups. We have successfully identiifed the key genes expressed at different stages of spinal cord ischemia/reperfusion injury, which provide a reference for future investigations into the mechanism of spinal cord injury.     

Effect of cyclovirobuxine D on human growth-associated protein 43 and neurocan expression in ischemic brain tissue of stroke-prone renovascular hypertensive rats*★

BACKGROUND： Experimental data indicate that human growth-associated protein 43 mRNA expression coincides with axonal growth during nerve ganglion development; while neurocan, secreted from astrocytes, can inhibit sprouting and elongation of the axonal growth cone. OBJECTIVE： To verify regulatory effects of cyclovirobuxine D （CVB-D） extracted from Chinese box branchlet on human growth-associated protein 43 （GAP-43）, and neurocan expression in brain tissue of stroke-prone renovascular hypertensive （RHRSP） rats, at different time points after cerebral ischemia/reperfusion. DESIGN： Randomized grouping design and controlled animal study. SETTING： This study was performed at the Center of Guangdong Hospital of Traditional Chinese Medicine （a national key laboratory） from March 2003 to September 2006. MATERIALS： 100 healthy male Sprague-Dawley rats, aged 2 3 months and weighing 90-120 g, were selected for this study. CVB-D was provided by Nanjing Xiaoying Pharmaceutical Factory （Batch number： 307701）. METHODS： The initial tip of renal arteries was clamped bilaterally for 10 weeks to establish the RHRSP model. 100 RHRSP rats were randomly divided into 4 groups： naive group （n = 10）, sham surgery group （n = 10）, CVB-D group （n = 40）, and lesion group （n = 40）. Rats in the naive group did not undergo any treatment, and cervical vessels of rats in the sham surgery group were exposed, but not blocked. The right middle cerebral artery of rats in the CVB-D group and lesion group were occluded to establish cerebral ischemia. Rats in the CVB-D group were intraperitoneally injected with CVB-D （6.48 mg/kg） every day and with saline （1.5 mL/injection） twice a day. Rats in the lesion group were intraperitoneally injected with saline （2 mL/injection）. MAIN OUTCOME MEASURES： Immunohistochemistry was applied to detect GAP-43 and neurocan expression in the ischemic penumbra region of CVB-D and lesion brains at 2 hours post-cerebral ischemia and at 1, 7, 14, and 30 days po     

Dispersity of human factor VIII--Von Willebrand factor.

Human factor VIII — Von Willebrand factor purified by gel chromatography in the presence of dextran has been studied with large pore polyacrylamide gel electrophoresis designed for analysis of highmolecular-weight proteins. It was demonstrated that purified factor VIII-Von Willebrand factor is not a single homogeneous entity, but consists of a series of immunologically related polymers. The polydispersity of factor VIII-Von Willebrand factor was confirmed by an immunochemical variant of the reversal-electrophoresis spreading test. With this technique it could be visualized that also factor VIII in plasma and cryoprecipitate is polydisperse in nature. As a consequence, changes in the polymer distribution of factor VIII-Von Willebrand factor can be an alternative explanation for results of dissociation experiments and for abnormalities in various diseases as demonstrated by cross-immunoelectrophoresis.     

Urinary procoagulant behaves as tissue factor by promoting factor VIIa-catalyzed activation of factor X

We purified the urinary procoagulant from frozen human urine by introducing phenyl-Sepharose hydrophobic chromatography. By this method, the apoprotein of the procoagulant and the lipid-like substance were separately recovered. Upon reassociation with the lipid-like substance or exogenous crude phospholipids, the apoprotein accelerated factor VIIa-catalyzed activation of factor X, probably by forming a stoichiometric complex with the catalytic enzyme. Thus the procoagulant was confirmed to be a tissue factor by its mode of participation in the blood coagulation mechanism.     

Current status on tissue factor activation of factor VIIa

Free factor VIIa displays a zymogen-like behavior with low intrinsic activity. Formation of a complex between factor VIIa and tissue factor is necessary to enhance the procoagulant activity of factor VIIa, not only by providing membrane localization, substrate exosites and positioning the active site at an appropriate distance above the surface but also by allosteric enhancement of the enzymatic activity, and this event signals initiation of blood coagulation. The interaction is of high affinity and all the domains are engaged at the interface. The crosstalk between the protease domain of factor VIIa, in particular residue Met-306, and the N-terminal domain of tissue factor provides the starting point for the allosteric activation of factor VIIa. The pathway(s) of conformational transitions in factor VIIa ensuing tissue factor binding has not been entirely mapped. The present paper is a brief compilation of our current knowledge of the allosteric mechanism by which tissue factor induces and stabilizes the active conformation of factor VIIa.     

Heterogeneity of factor IX in therapeutic factor IX concentrates

Rabbit antibody to human factor IX was used to investigate the factor IX antigenic content and electrophoretic mobility of commercial products as well as experimental “activated products”. Rocket immunoelectrophoresis of all concentrates showed a 1.2–3 fold increased antigenic content/unit factor IX clotting activity when compared to plasma. Two dimensional crossed immunoelectrophoresis of standard Factor IX concentrates produced a single sharp peak whether electrophoresed in Ca²⁺ or EDTA containing buffer. “Activated” concentrates produced a dome shaped precipitin arc. The addition of plasma to standard factor IX concentrates yielded a marked shoulder only when the electrophoresis was run in EDTA. This effect could not be reproduced by the addition of antithrombin III (AT-III). The addition of plasma to some “activated” products revealed an even more pronounced heterogeneity whether in Ca²⁺ or EDTA and the addition of AT-III in the same products produced a second precipitin peak. These results indicate that at least three forms of factor IX exist in Factor IX concentrates. The absence of detectable AT-III reacting material in the standard concentrates is $\text{a priori}$ evidence of the absence of major amounts of IXa, whereas the presence of AT-III reacting material in the “activated” concentrates is evidence of biologically active material.     

Activation of factor IX by factor XIa--a spectrophotometric assay for factor IX in human plasma

The activation of Factor IX by partially purified Factor XIa was followed by active site titration, gelelectrophoresis and by a spectrophotometric assay. The assay is based on the finding that the rate of Factor X activation in the presence of phospholipid and Ca2+ is linear in time and proportional to the amount of Factor IXa present and can be determined with the chromogenic substrate S2222. Conditions were found that allowed complete activation of Factor IX in human plasma by Factor XIa. The amount of Factor IXa present in the plasma sample can be determined with the spectrophotometric assay and is proportional with the amount of plasma present. In plasma from patients receiving vitamin-K antagonists reduced Factor IX activity is found with the spectrophotometric assay and the new assay method may be useful in monitoring oral anticoagulant therapy.     

Cellular activation of factor IX (christmas factor)

We have demonstrated Factor IX activation by sonicated polymorphonuclear leukocytes (PMNs). This activation reaction required the disrupted leukocytes, calcium chloride, and a small amount of normal human plasma. The requirement for normal plasma was met by plasma deficient in all of the known coagulation factors, and thus the substance present in normal plasma which facilitates this reaction was not identified. The fact that factor XI deficient plasma supported the reaction as well as normal plasma implied that factor XIa was not involved in this activation. Strontium ions could not substitute for calcium ions in the activation reaction, also implying that factor XIa was not involved. This factor IX activating principle in leukocytes could provide a mechanism for by-passing the contact factors of blood coagulation, thus providing an explanation for the discrepancy in clinical severity between deficiencies of the contact factors on the one hand and hemophilias A and B on the other.     

The isolation of prothrombin, factor IX and factor X from human factor IX concentrates

A relatively simple and reproducible procedure is described for the isolation of functionally and electrophoretically homogeneous prothrombin, factor IX and factor X from clinical Factor IX Concentrates. The procedure involves ammonium sulphate fractionation; then chromatography on DEAE-Sephadex A50, dextran sulphate-Sepharose and heparin-Sepharose. High recovery of all three procoagulants was obtained: 32% for factor IX; 32% for factor X; 29% for prothrombin.     

Model of a ternary complex between activated factor VII,tissue factor and factor IX

Upon binding to tissue factor, FVIIa triggers coagulation by activating vitamin K-dependent zymogens, factor IX (FIX) and factor X (FX). To understand recognition mechanisms in the initiation step of the coagulation cascade, we present a three-dimensional model of the ternary complex between FVIIa:TF:FIX. This model was built using a full-space search algorithm in combination with computational graphics. With the known crystallographic complex FVIIa:TF kept fixed, the FIX docking was performed first with FIX Gla-EGF1 domains, followed by the FIX protease/EGF2 domains. Because the FIXa crystal structure lacks electron density for the Gla domain, we constructed a chimeric FIX molecule that contains the Gla-EGF1 domains of FVIIa and the EGF2-protease domains of FIXa. The FVIIa:TF:FIX complex has been extensively challenged against experimental data including site-directed mutagenesis, inhibitory peptide data, haemophilia B database mutations, inhibitor antibodies and a novel exosite binding inhibitor peptide. This FVIIa:TF:FIX complex provides a powerful tool to study the regulation of FVIIa production and presents new avenues for developing therapeutic inhibitory compounds of FVIIa:TF:substrate complex.     

Heterogeneity of factor IX BM. Difference of cleavage sites by factor XIa and Ca2+ in factor IX Kashihara, factor IX Nagoya and factor IX Niigata

Abnormal factor IX was isolated from the plasma of a patient with hemophilia B Kashihara and two patients with hemophilia BM. The F.IX was purified to homogeneity by using monoclonal anti-F.IX-Sephrose, heparin-Sepharose and DEAE-Sephadex A-50 affinity chromatography successively. The isolated proteins have the same molecular weight and the same mobility on crossed immunoelectrophoresis as normal F.IX. The limited proteolysis of purified proteins was induced by F.XIa/Ca2+ or by RVV-X/Ca2+. A time course study showed that F.IX Nagoya seemed to be cleaved by neither F.XIa nor RVV-X, F.IX Kashihara was cleaved partially by F.XIa but not by RVV-X, and that F.IX Niigata was cleaved completely at the rate similar to normal F.IX, though the resultant product of F.IX Niigata did not show any F.IXa activity. These results favored the view that hemophilia B+ or BM is a heterogeneous disorder.