STUDIES ON THE PATHOPHYSIOLOGY OF THROMBOEMBOLIC DISEASES WITH SPECIAL REFERENCE TO THE UNDERLYING IMPAIRED BLOOD COAGULATION AND ITS REGULATION

血栓性疾病的病理生理学研究，特别是潜在的凝血受损及其调节

• 批准号: 11470250
• 负责人: MATSUDA Michio
• 金额: $ 7.81万
• 依托单位: Jichi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470250/
• 关键词: fibrinogen; hereditary dysfibrinogen; structural alteration; thrombosis; bleeding tendency; fibrin ultra-structure; monoclonal antibody; anti-angiogenesis; 点変異; 余剰糖鎖; 血栓性素因; 電顕学的分析; フィブリンの超微細構造; 抗フィブリノゲン・モノクロナル抗体

1. Studies on the hereditary dysfibrinogens associated with thrombosis and/or bleeding In fibrinogen (Fbg) Osaka VI, a hereditary dysfibrinogen derived from a 36-year-old woman with a history of severe bleeding on two childbirths, we have identified an exchange of T to A in the stop codon (TAG) of the fibrinogen Bβ-chain gene leading to formation of a codon for Lys (AAG). Thus, 12 amino acid residues are translated to elongate the aberrant Bβ-chain and indeed, this extension has been confirmed by sequence analysis. There is a Cys residue next to the new carboxyl-terminus, which forms a disulfide bridge with its counterpart in another dysfibrinogen molecule. Thus, two types of end-linked Fbg-dimers exist in the patient's Fbg, i.e., a bilayer dimer end-linked at both ends and a longitudinally aligned dimer end-linked at either one of the two carboxyl-terminal ends. These dimers had been predicted by SDS-PAGE run under non-reducing conditions, where two molecular species, i.e., a normal s … More ized and a double-sized Fbg species had been visualized.By transmission electron microscopy, the fibrin fibers appear extremely thin but the fibrin networks are highly branched and compact as compared with those of normal fibrin clots. We thus hypothesized that the abnormal branching may occur at the points in the double-stranded fibrin proitofibrils, where the abnormal dimeric molecules are incorporated. Furthermore, the Osaka VI fibrin clots are mechanically readily compressible leading to form very fragile fibrin clots. These fibrin clots may fail to exert their hemostatic function and thereby lead to easy bleeding (Blood 96(12) : 3779-3785, 2000). These findings seem to be comparable to those of Fbg Marburg associated with thrombosis besides bleeding after surgery. Namely, this dysfibringen also forms fine fibrin fibers and compact fibrin networks, but the fibrin fibers are highly resistant against plasmin, accounting for thromboembolic complications (Blood 91(9) : 3282-3288, 1999).We have also studied the role of oligosacharides linked to Asn residue due to formation of a new glycosylation sequence of Asn-X-Thr/Ser in Fbgs Asahi and Lima (Ann NY Acad Sci, in press). Matsuda, the chief researcher, had chances to review these data at several international congresses and symposia (see the attached publication list).2. Other studies1) We have produced a monoclonal antibody, IF-123, that specifically recognizes elastase-digests of human fibrinogen/fibrin, and its epitope determination and clinical application have been successfully attempted (Blood 96(5) : 1721-1728, 2000).2) A novel strategy for the tumor angiogenesis-targeted gene therapy has been established by generation of angiostatin from endogenous plasminogen by protease gene transsfer in mice (Cancer Gene Therapy 7(5) : 589-596, 2000). Less

1. 纤维蛋白原（Fbg）大阪六世（Osaka VI）是一种遗传性纤维蛋白异常原，来源于一名36岁、两次分娩有严重出血史的女性，我们发现纤维蛋白原b β-链基因的终止密码子（TAG）中T到a的交换导致了Lys （AAG）密码子的形成。因此，12个氨基酸残基被翻译成延长了异常的b β链，这种延长已经被序列分析证实。在新的羧基末端旁边有一个Cys残基，它与另一个异常纤维蛋白原分子中的对应物形成一个二硫桥。因此，在患者的Fbg中存在两种末端连接的Fbg-二聚体，即在两端连接的双层二聚体和在两个羧基末端连接的纵向排列的二聚体。这些二聚体在非还原条件下通过SDS-PAGE进行了预测，其中两种分子物种，即正常的s . More化和两倍大小的Fbg物种已被可视化。在透射电镜下，与正常纤维蛋白凝块相比，纤维蛋白纤维看起来非常薄，但纤维蛋白网络具有高度分支和致密性。因此，我们假设异常分支可能发生在双链纤维蛋白原纤维的点上，在那里异常的二聚体分子被合并。此外，大阪VI纤维蛋白凝块是机械容易压缩导致形成非常脆弱的纤维蛋白凝块。这些纤维蛋白凝块可能无法发挥其止血功能，从而导致容易出血（Blood 96(12): 3779- 3785,2000）。这些发现似乎与手术后出血外伴有血栓形成的马尔堡出血热相似。也就是说，这种异常纤维原也会形成细小的纤维蛋白纤维和致密的纤维蛋白网络，但纤维蛋白纤维对纤溶酶具有高度抗性，这是导致血栓栓塞并发症的原因（Blood 91(9): 3282-3288, 1999）。我们还研究了由于在Fbgs Asahi和Lima中形成新的Asn- x - thr /Ser糖基化序列而与Asn残基连接的低聚糖的作用（Ann NY Acad Sci, in press）。首席研究员Matsuda有机会在几个国际会议和专题讨论会上审查这些数据（见所附出版物清单）。1)我们已经生产了一种单克隆抗体IF-123，它可以特异性识别人纤维蛋白原/纤维蛋白的弹性酶消化，其表位的确定和临床应用已经成功尝试（Blood 96(5): 1721- 1728,2000）。2)内源性纤溶酶原通过蛋白酶基因转移生成血管抑制素，为肿瘤血管生成靶向基因治疗提供了一种新的策略（癌症基因治疗7(5):589-596,2000）。少

项目成果

Michio Matsuda: "Structure and function of fibrinogen inferred from hereditary dysfibrinogens"Intern J Haematol. (in press).

Michio Matsuda：“从遗传性纤维蛋白原异常推断的纤维蛋白原的结构和功能”Intern J Haematol。

Kikuchi J: "Induction of ubiquitin-conjugating enzyme by aggregated low density lipoprotein in human macrophages and its implications for"Arterioscler Thromb Basc Biol. 20. 128-134 (2000)

Kikuchi J：“人巨噬细胞中聚集的低密度脂蛋白诱导泛素结合酶及其对动脉硬化血栓基础生物学的影响”。

Seiji Madoiwa: "Developmental expression of plasminogen activator inhibitor-1 associated with thrombopoietin-dependent megakaryocytic differentiation"Blood. 94(2). 475-482 (1999)

Seiji Madoiwa：“与血小板生成素依赖性巨核细胞分化相关的纤溶酶原激活剂抑制剂-1 的发育表达”血液。

Jiro Kikuchi: "Induction of ubiquitin-conjugating ing enzyme by aggregated low density lipoprotein in human macrophages and its implications for atherosclerosis"Arterioscler Thromb Basc Biol. 20. 128-134 (2000)

Jiro Kikuchi：“通过人巨噬细胞中聚集的低密度脂蛋白诱导泛素结合酶及其对动脉粥样硬化的影响”Arterioscler Thromb Basc Biol。

Kohno I: "A monoclonal antibody specific to the granulocyte-derived elastase-fragment D species of human fibrinogen and fibrin : Its application to the measurement of granulocyte-derived elastase-digests in plasma."Blood. 95. 1721-1728 (2000)

Kohno I：“一种对人纤维蛋白原和纤维蛋白的粒细胞来源的弹性蛋白酶片段 D 种具有特异性的单克隆抗体：其在测量血浆中粒细胞来源的弹性蛋白酶消化物中的应用。”血液。