CN1211280A - 凝血酶突变蛋白质作为凝血酶抑制剂的解毒剂 - Google Patents
凝血酶突变蛋白质作为凝血酶抑制剂的解毒剂 Download PDFInfo
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Abstract
与天然凝血酶相比在序列上有如下不同的凝血酶突变蛋白质(a)Ala取代Gly,Gly位于序列环境Tyr-Gly-Phe并占据天然人类凝血酶原第558位(序列号14);(b)下列残基中至少一个取代或缺失(b1)位于序列环境Ala-His-Cys并占据天然人类凝血酶原第363位(序列号14)的His;(b2)位于序列环境Arg-Asp-Ile并占据天然人类凝血酶原第419位(序列号14)的Asp;(b3)位于序列环境Asp-Ser-Gly并占据天然人类凝血酶原第525位(序列号14)的Ser;用作解毒剂。
Description
本发明涉及新的凝血酶突变蛋白质，涉及其制备及其用作凝血酶抑制剂的解毒剂。
依据对凝血酶直接抑制的原理而起作用的抗凝药对于抗凝血治疗逐渐变得重要。广泛应用抗凝药物的必须前提是在过量情况下，中和其效果的可能性，其中使衰竭减轻或分泌降低，这样抑制出血的副作用，这些副作用随后会很危险，如腹膜，心包膜，肺膜区的出血。而已有的肝素高效解毒剂为硫酸鱼精蛋白，聚赖氨酸和血小板因子4，迄今还没有用于人类的中和凝血酶抑制剂的合适的解毒剂。
文献中描述了不同的解毒剂原理。Fareed提示活化的血浆部分，如自身复合产物或FEIBA，作为中和水蛭素的成分：然而，由于它们的活性类似于凝血酶原激酶而不适合用于中和。(Fareed,Fed Proc 3(1989)A 328；Welenga Sem.凝血.止血.15(1989)316-333)。
Markwardt(血栓形成研究74(1994)1-23)描述了凝血酶和凝血酶衍生物的应用。例如凝血酶)，凝血酶/2-巨球蛋白复合物和meizothrombin。化学失活的凝血酶如DFP-凝血酶或苯甲酰-凝血酶中和水蛭素的应用也有描述(Markwardt，药学，44(1989)648-649)。
然而，这些产物均不适于用作人类解毒剂，由于其凝血活性及其对水蛭素低至1000倍的亲和活性(F.Doyle酶学方法；222卷，299页，Moriat酶学方法；80卷，303页，Stone等生物化学26(1987)4617-4624,Markwardt，药学，44(1989)648-649)。
人类的凝血酶原已由Friezner等了解并描述(生物化学，22(1983)2087-2097)。人类凝血酶原的氨基酸序列也在序列号NO:14中表示。在文献中可发现多种人类凝血酶序列编号方法(zahlweisen)，这就是除非另外指出下文皆用序列号NO:14编号方法的原因。
一种基因工程的酶学失活的凝血酶由Lentz等描述(生物化学杂志266,15,9598-9604)。这其中，催化必需的位于205(序列号NO:14中525)位置的丝氨酸残基被丙氨酸取代，导致酶学失活的凝血酶变异物，它对合成底物和天然底物纤维蛋白原无剪切活性(Lent等，生物化学杂志266,15,9598-9694)但对凝血酶抑制剂苄基精氨酸-N-(3-乙基-1,5-二戊烷基)酰胺有弱的结合活性。
Stone等的研究(生物化学30,6392-6397)发现了天然的凝血酶变异体QickⅡ.QuickⅡ凝血酶与天然凝血酶只在位置238有区别(序列号NO:14中位置558)。此时天然凝血酶238位的甘氨酸残基突变为疏水残基缬氨酸。酶的P1口袋中这一小突变导致纤维蛋白原的剪切速率的显著下降(原始活性的2％)，同时，对低分子量底物和水蛭素的结合都急剧下降。Stone指出突变蛋白质对水蛭素的结合常数退化了1000倍，且通过结合空腔的空间阻碍和活性中心的环境构象识别解释此结果。
迄今发现凝血酶突变蛋白质不适用作为凝血酶抑制剂的解毒剂，由于它们对凝血酶抑制剂的亲和力太弱或其对纤维蛋白原的结合和酶促活性太高。
本发明的一个目标是提供新的凝血酶突变蛋白质，它们是基平地酶活性失活的，与凝血酶抑制剂结合良好的，同时，与未修饰的凝血酶相比，其结合天然底物纤维蛋白原亲和力低。
我们已发现通过与天然凝血酶相比具有如下序列差异的凝血酶突变蛋白质达到此目的。
(a)Gly被Ala取代，其中Gly位于序列环境Tyr-Gly-Phe并在天然凝血酶中占据位置558(序列号NO:14)；
(b)至少如下残基中的一处被取代或缺失
(b1)His，位于序列环境Ala-His-Cys中，在天然人类凝血酶原中占据位置363(序列号NO:14)；
(b2)Ser，位于序列环境Arg-Asp-Ile中，在天然人类凝血酶原中占据位置419(序列号NO:14)；
(b3)Ser，位于序列环境Asp-Ser-Gly中，在天然人类凝血酶中占据位置241(序列号NO:14)；
新的凝血酶突变蛋白质可从来自人类或其它哺乳动物的已知天然凝血酶开始制得，如灵长类，牛，猪，狗，猫，大鼠，小鼠。天然凝血酶是指具有凝血酶活性在生物体内天然产生的多肽序列。
凝血酶突变蛋白质不仅指二硫键连接的两条链分子(A和B链)还指单链形式的分子如凝血酶原或只含B链或部分B链，优选N-和/或C-端截短B链，的蛋白质。根据本发明就凝血酶突变蛋白质而言，具有结合凝血酶抑制剂的活性是非常重要的。
根据本发明凝血酶突变蛋白质还可从已知的凝血酶蛋白水解产物，如β-凝血酶，γ-凝血酶，ω-凝血酶或其它凝血酶前体如凝血酶原，凝血酶中间体，或其它meizothrombins起始制得，然后它们可通过合适的剪切方法，例如利用来自Ecthis carinatus或Oxyranusscutellatus蛇毒级分因子Xa转变为有活性的凝血酶分子。
本发明的凝血酶突变蛋白质适用于中和水蛭素，水蛭素衍生物或其它凝血酶抑制剂。
优选基因工程制备，其中制备合适的凝血酶突变蛋白质基因并将此基因在适合宿主生物中表达。
新的凝血酶突变蛋白质与天然凝血酶至少有两处序列差别，第一个序列差别是丙氨酸残基取代甘氨酸残基。取代的位置取决于起始天然凝血酶的来源。而天然人类凝血酶原中甘氨酸位置位于序列环境Tyr-Gly-Phe中且占据558位(序列号NO:14)。
序列的第二个序列差别是形成蛋白酶凝血酶催化三分体(diekatalytisch Triade)的三个位置(His,Asp,Ser)之一的氨基酸残基的取代或缺失。天然人类凝血酶原中这些位置是His336位，Asp419位，Ser525位。在其它的非人类的凝血酶中，这些位置可依据分子的总长度而挪动。序列环境，即在相应氨基酸之前或之后的氨基酸残基，在凝血酶中是保守的，因此，在序列环境的基础上可容易地确定相应氨基酸残基的精确位置。
根据本发明凝血酶突变蛋白质可在催化三分体有若干个改变(删除或取代)。优选在催化三分体上只有一个改变的凝血酶突变蛋白质。
优选改变包括催化三分体中氨基酸的取代。优选组氨酸残基被极性或疏水性氨基酸取代，特别是由Phe,Tyr,Ala或Val。天冬氨酸残基优选由其它极性氨基酸如Gln或Asn取代。
特别优选凝血酶突变蛋白质，其中丝氨酸残基改变，特别是丝氨酸由无羟基功能基团的氨基酸取代，特别是与丝氨酸有相似体积的氨基酸。特别优选由Ala取代Ser。
这些凝血酶突变蛋白质的活性可以通过分子内引入其它改变进一步提高。这样，例如，可以想象进一步改善蛋白水解稳定性以便，例如，使体内作用期(die Wirkdauer)变长成为可能。这是可能的，例如，用另外的非碱性氨基酸取代碱性残基Arg393,Lys465,Arg390或Arg382(序列号NO:14)。
本发明还涉及核苷酸序列，特别是DNA序列，它编码所述凝血酶突变蛋白质。通过按照遗传密码将多肽序列回译成相应的DNA序列这些可很容易确定。优选使用密码子为在所需宿主生物中导致良好表达的。
核苷酸序列可通过点特异突变从天然凝血酶基因序列开始或完全通过DNA合成来制备。
根据本发明的凝血酶突变蛋白质特别适合用作凝血酶抑制剂的解毒剂，如水蛭素，水蛭素衍生物和低分子量凝血酶抑制剂。
水蛭素是最初分离自水蛭的蛋白质，含65个氨基酸且有极高的对凝血酶的的亲和力(ki=10-12mol/l)和极好选择性。水蛭素已在动物试验和人类中显示有活性。除最初分离的水蛭素还有几种水蛭素突变蛋白质和其序列与天然水蛭素相比有序列改变的异水蛭素。因此水蛭素不仅指天然水蛭素还包括相对来自医用水蛭序列中一个或多个修饰且至少有对凝血酶同样高的亲和力的水蛭素。
水蛭素易于用基因工程的方法得到，其半衰期在人类中约45分钟。
也已知其中化学上的修饰改变了水蛭素的药物动力学的水蛭素衍生物。Markwardt描述了作用时间延长的水蛭素与右旋糖苷的缀合物。EP345616,EP502962和EP557199揭示了作用时间延长的聚合物缀合体。WO9515183描述了作用时间延长的疏水水蛭素衍生物。储存形式和作用时间延长的水蛭素二聚体(WO9504823)类似已知。进一步的凝血酶抑制剂是所揭示的低分子量凝血酶抑制剂，例如，在EP236164,DE2801478,EP293881或EP185390中。
进一步凝血酶抑制剂是重组二硫水蛭素，水蛭素突变蛋白质，多聚物修饰的水蛭素如右旋糖苷-水蛭素，PEG-水蛭素，疏水水蛭素衍生物，和具有延长作用期的水蛭素突变蛋白质。新的凝血酶能进一步用于拮抗Hirudisin，截断的水蛭素和来自水蛭素类似物的部分序列，如hirulog或C端水蛭素多肽。中和其它的凝血酶抑制多肽也是可能的，例如所描述的来自吸血生物且基因工程制备用于治疗的，如rhodniin；infestin。
能够被中和的凝血酶抑制剂的例子也是合成的，优选低分子量，凝血酶抑制剂如三肽D-Phe-Pro-Arg的衍生物，NAPAP的硼酸衍生物，Arginals或苄脒衍生物。本发明用作上述所有凝血酶抑制剂的解毒剂均可行。
根据本发明凝血酶突变蛋白质对凝血酶抑制剂的中和作用优于迄今为止所述的用于中和凝血酶抑制剂的方法。根据本发明凝血酶应用是安全的，甚至在过量情况下，并且因此使在紧急情况下迅速而有效的中和凝血酶抑制剂成为可能。
根据本发明凝血酶突变蛋白质以适合非肠道给药的形式提供，即皮下，肌内，或静脉内给药。优选静脉内给药。在必要的情况下根据本发明的凝血酶突变蛋白质也能持续浸渍(Dauerinfusion)给药。将要给予的失活的凝血酶的数量取决于，例如过量程度，体重及所选给药形式。过量程度可由熟练人员在给凝血酶之前或者过程中通过适当的试验测定。
根据本发明凝血酶突变蛋白质可通过基因工程方法制备。优选以基因工程制备凝血酶突变蛋白质的前体形式(与凝血酶原类似)，凝血酶突变蛋白质可通过蛋白水解活化从该前体形成获得。
另一个优选的制备形式，尤其对于只包含B链的凝血酶突变蛋白质，是根据本发明的修饰的B链基因在微生物中表达，优选细菌如大肠杆菌。
必要的所得的表达产物必须通过熟练人员熟悉的方法复性以获得有活性的凝血酶突变蛋白质分子。在细菌中表达B链时，已发现用另一个氨基酸残基，例如Ser或Ala取代Cys残基是有利的，Cys残基在天然人类凝血酶中，用于形成二硫键。
以下实施例用于进一步阐明本发明，但并不限制之。
凝血酶原按照Degen等发表的方法，克隆自人类cDNA库(生物化学22(1983)2087-97)。
根据本发明突变蛋白质通过在凝血酶部分中定靶基因突变制备。
实施例1
制备Ala 558-凝血酶原
通过定靶基因突变人类凝血酶原558位的编码氨基酸甘氨酸GGC(序列号No:14)突变成编码Ala的密码子GCC。
序列号No.5的寡核苷酸以基因的有义方向合成，互补的序列号No:6的寡核苷酸以反义方向，具有适当的核苷酸改变。序列号1的寡核苷酸按照凝血酶原基因的5’末端以有义方向合成。Eco R1限制性内切酶切点序列另外附加于其5’末端。序列号2的寡核苷酸依照凝血酶原基因3’末端以反义方向合成，Rco R1限制性内切酶切点序列加于其5’末端。
50纳克人类凝血酶原cDNA作为模板，序列号5和2的寡核苷酸每种50皮摩尔，加入2纳摩尔dNTPs，聚合酶缓冲液和Pfu聚合酶(Stratagene)来进行PCR反应，35轮，每轮94℃1分钟，55℃2分钟和72℃3分钟。相应的，用同样的模板和序列号6和序列号1的寡核苷酸进行第二个PCR反应。此法产生的两种DNA片段用标准方法纯化(PCR纯化试剂盒，Quiagen,Hilden)。两种片段各取0.1皮摩尔混合，95℃加热5分钟变性，并室温维持1小时复性，以获得完全的DNA双链，加入2纳摩尔dNTPs，聚合酶缓冲液和Pfu聚合酶进行10轮反应，每轮94℃1分钟，72℃5分钟。为扩增全长片段，加入50皮摩尔外边的寡核苷酸序列号1和序列号2，再次加入Pfu聚合酶后，进行35轮另一个PCR反应，每轮94℃1分钟，55℃2分钟和72℃3分钟。此法制备的558位Ala取代Gly的突变凝血酶原cDNA用标准方法纯化，然后用限制性内切酶Eco R1酶切并用标准方法以T4连接酶克隆进也用Eco R1酶切的pUC18载体(Pharmacia Biotech Europe GmbH,Friburg)。测序验证DNA。
实施例2
制备Ala 525,Ala 558凝血酶原
进行基因定靶突变使人类凝血酶原525位(序列号14)的编码Ser的密码子AGT突变为编码Ala的CGT，且558位(序列号14)的编码GLY的密码子GGC突变为编码Ala的密码子GCC。
为此，在实施例1凝血酶原突变蛋白质DNA基础上进行PCR反应，利用以有义方向的具有合适的525位ALA核苷酸取代的序列号3寡核苷酸及序列号2寡核苷酸(凝血酶原3’末端)。试验条件依照实施例1。利用与序列号3互补的序列号4寡核苷酸，和序列号1寡核苷酸(凝血酶原5’末端)进行第二个PCR反应。此法获得的DNA片段变性，杂交，填入以提供双链，并依照实施例1用外边的寡核苷酸序列号1和序列号2进行另一个PCR反应。
纯化用此法制备的凝血酶原突变蛋白质cDNA，然后用Eco R1酶切，克隆进pUC18载体，并测序验证。
实施例3
制备Ala 363,Ala 558凝血酶原
进行基因定靶突变使人类凝血酶原363位(序列号14)的编码His的密码子AGT突变为编码Ala的GCA，且558位(序列号14)的编码GLY的密码子GGC突变为Ala的密码子GCC。
为此，在实施例1凝血酶原突变蛋白质DNA基础上进行PCR反应，利用以有义方向的具有合适的363位ALA核苷酸取代的序列号10寡核苷酸及序列号2寡核苷酸(3’末端凝血酶原)。试验条件依照实施例1。利用与序列号10互补的序列号11寡核苷酸，和序列号1寡核苷酸(凝血酶原5’末端)进行第二个PCR反应。此法获得的DNA片段变性，杂交，填入以提供双链，并依照实施例1用外边的寡核苷酸序列号1和序列号2进行另一个PCR反应。
纯化用此法制备的凝血酶原突变蛋白质cDNA，然后用Eco R1酶切，克隆进pUC18载体，并测序验证。
实施例4
制备Asn 419,Ala 558凝血酶原
进行基因定靶突变使人类凝血酶原419位(序列号14)的编码Asp的密码子GAC突变为编码Asn的CGA，且558位(序列号14)的编码GLY的密码子GGC突变为ALA的密码子GCC。
为此，在实施例1凝血酶原突变蛋白质DNA基础上进行PCR反应，利用以有义方向的具有合适的419位ASN核苷酸取代的序列号12寡核苷酸及序列号2寡核苷酸(凝血酶原3’末端)。试验条件依照实施例1。利用与序列号12互补的序列号13寡核苷酸，和序列号1寡核苷酸(凝血酶原5’末端)进行第二个PCR反应。此法获得的DNA片段变性，杂交，填入以提供双链，并依照实施例1用外边的寡核苷酸序列号1和序列号2进行另一个PCR反应。
纯化用此法制备的凝血酶原突变蛋白质cDNA，然后用Eco R1酶切，克隆进pUC18载体，并测序验证。
实施例5
制备Ala 525-凝血酶原
进行基因定靶突变使人类凝血酶原525位(序列号14)的编码Ser的密码子AGT突变为编码Ala的GCT。
为此，在凝血酶原突变蛋白质DNA基础上进行PCR反应，利用以有义方向的具有合适的525位ALA核苷酸取代的序列号3寡核苷酸及序列号2寡核苷酸(凝血酶原3’末端)。利用与序列号3互补的序列号4寡核苷酸，和序列号1寡核苷酸(凝血酶原5’末端)进行第二个PCR反应。此法获得的DNA片段变性，杂交，填平以提供双链，并依照实施例1用外边的寡核苷酸序列号1和序列号2进行另一个PCR反应。
纯化用此法制备的凝血酶原突变蛋白质cDNA，然后用Eeo R1酶切，克隆进pUC18载体，并测序验证。
实施例6
人类凝血酶原突变蛋白质表达载体的构建
为在CHO哺乳动物细胞中分泌重组表达，人类tPA基因的分泌引导序列被连到突变凝血酶原cDNA的5’末端(参见实施例1到5)(Pennica等，自然301(1983)214-221)。从tPA cDNA开始，利用合成寡核苷酸序列号NO:9(tPA引导区的5’末端和Eco R1切点)和序列号NO:8(tPA引导区的3’末端，凝血酶原的5’末端)进行PCR基础上的tPA分泌引导cDNA扩增。每条突变的凝血酶原突变蛋白质片段用和序列号NO:8互补的寡核苷酸序列号NO:7，和序列号NO:2进行扩增。tPA引导区片段加入相关的凝血酶原突变蛋白质片段。混合物变性，杂交，填平以产生双链。，相关tPA引导区凝血酶原突变蛋白质DNA使用寡核苷酸序列号NO:9和NO:2通过另一PCR反应制备。这些用Eco R1酶切，克隆入pUC18并参见验证。
为在CHO细胞中表达，这些DNA从pUC18载体中用Eco R1切出，分离并插入载体pc DNA3 neo(Invitrogen,San Diego,USA)的Eco R1剪切位点，凝血酶原cDNA的翻译由载体中强的巨细胞病毒启动子控制。转染细胞的筛选通过位于质粒上只允许G418-抗性克隆生长的新霉素抗性基因的方法进行。
转染前，用于在CHO细胞中表达的适合DNA用限制性内切酶PvuI线性化，沉淀并在无菌条件下溶于10毫摩尔Tris缓冲液(pH8.5)。
实施例7
哺乳动物细胞中凝血酶原突变蛋白质的表达
表达载体的DNA用lipofectamine(从Gibco；Life TechnologiesGmbH；Dieselstraβe 5,76334 Eggenstein，德国；NO.530-8324SA)转入哺乳动物细胞。细胞用CHO-Kl(ATCC CCL 61)；293(ATCC CRL1573)；
2×105细胞/毫升引入6-孔培养板的每一孔中的3毫升培养基。第二天进行转染。为此，细胞用无血清培养基洗一次。按照制造商Gibco(Focus 15(1993)73-78)的说明书利用Lipofectamine进行转染。每孔含1微克DNA和6微升Lipofectamine，它们被加入总量1000微升的无血清细胞培养基。37℃孵育6小时后，蒸发掉转染培养基，将细胞在正常生长培养基中(含胎牛血清，FCS)过夜。
实施例8
凝血酶原突变蛋白质在293细胞中的暂时表达
293细胞保存在DMEM(Gibco No.41965)+10％FCS(BiowhittakerNo.14601 B)中。
转染按上述描述进行。这种情况下，为增加表达，以质粒pAdVantange(来自Promega E1711号)进行共转染。0.2微克pAdVantange DNA和0.8微克在表达载体pcDNA3中的特殊凝血酶原突变蛋白质的DNA按上述转染。转染后第一天，蒸发掉含血清的培养基，并用无血清培养基取代(无酚红)。转染后第3天，移去细胞培养上清并存于-20℃直至测定。
实施例9
凝血酶原突变蛋白质在CHO-K1细胞中的稳定表达
按以上描述进行转染。CHO细胞在DMEM-F12=1∶1(GibcoNo.21331-020)+10％FCS中保存和培养。转染后第一天，将细胞分开以使从六孔板中的一孔分布到20-100厘米的皮氏培养皿上。同时，开始用G418(来自Gibco 1200微克/毫升培养基中)处理筛选重组细胞。所得抗性克隆用克隆圆柱法分离，并进一步培养至足够细胞数目后检查适当凝血酶突变蛋白质的表达。通过从汇合的细胞中蒸去培养基并用新鲜的，无血清培养基取代进行。经过一定的时间，例如24小时，48小时，移去孵育细胞的培养上清并用ELISA检测凝血酶原的存在。
某些情况下，通过微滴定板中分离克隆所得重组细胞以增加表达。
为了提供足量的无血清细胞培养上清，显示良好表达的CHO细胞在含10％FCS的培养基中在不同的容器中培养至汇合。细胞然后用PBS清洗，用无血清DMEM-12孵育(DMEM:Gibco No.11880-028；F12:GibcoNo.21765-029:混合物包括等体积比)。用于此目的的DMEM不含酚红。两至三天后更换培养基以便收获含凝血酶原突变蛋白质的细胞培养上清。用此法可能从一个培养容器中收获达十次。结合来自不同培养容器中的不同收获物以保持纯化所需的合适体积。
实施例10
重组凝血酶原突变蛋白质表达的检测
通过用ELISA检查来自汇合细胞的细胞培养上清中凝血酶原的存在，或凝血酶原剪切后凝血酶活性或凝血酶抗原，来检测表达凝血酶原并将其分泌到细胞培养基中的细胞中重组凝血酶原的存在。为此，培养不同时间后移去无血清细胞培养上清。细胞培养上清中存在的凝血酶原剪切用蛇毒(Sigma；cat No.V3129)来获得。每一混合物用以下方法进行：
388微升样品(无血清细胞培养上清或凝血酶原)
63微升蛇毒(0.2毫克/毫升水中)
50微升缓冲液(1摩尔NaCl；100毫摩尔CaCl2；200毫摩尔Tris PH7.4)
100微升水
剪切混合物在室温孵育45分钟，所得凝血酶接着在不同试验中表征。
实施例11
用ELISA的方法检测凝血酶原突变蛋白质和凝血酶突变蛋白质
测定凝血酶原和凝血酶的ELISA，按以下方案所示进行：
-用0.1毫升/孔抗-凝血酶抗体包被微滴定板；5微克/毫升0.05 M NaHCO3,pH9.2；16小时/4℃。
-用1％BSA/PBS饱和；0.3毫升/孔；0.5-1小时/室温
-用0.05％吐温20/PBS洗3遍
-11标准2倍稀释人类凝血酶(Calbiochem 605195；3.159 NIHU/毫克)，
以10纳克/毫升0.1％BSA/0.05％吐温20/PBS开始；待测定样品平行稀释；0.1毫升/孔；2-4小时/室温
-如上清洗
-0.1毫升/孔生物素化的抗凝血酶抗体1∶200；稀释于0.1％BSA/0.05％吐温20/PBS；2-4小时/室温
-如上清洗
-0.1毫升/孔抗生蛋白链菌素-过氧化酶复合物(B.M.1089153)；1∶10000稀释于0.1％BSA/0.05％吐温20/PBS；30分钟/室温
-如上清洗
-0.1毫升/孔过氧化酶底物
-以0.1毫升/孔2M H2SO4停止反应
-450纳米处测量吸收值
过氧化酶底物：将0.1毫升TMB溶液(42mM TMB的DMSO溶液)与10毫升底物缓冲液混合(0.1M乙酸钠PH 4.9)；然后加入14.7微升3％H2O2
实施例12
抗人类凝血酶抗体的制备
为诱导多克隆抗体，按如下方法免疫兔子：
第1天200微克人类凝血酶于0.5毫升PBS中/完全Freund氏佐剂(Sigma F5881)
第14天200微克人类凝血酶于0.5毫升PBS中/不完全Freund氏佐剂(Sigma F5506)
第28天200微克人类凝血酶于0.5毫升PBS中/不完全Freund氏佐剂
第42天200微克人类凝血酶于0.5毫升PBS中
最后一次免疫7天后，从兔耳静脉中取血。
兔的多克隆抗体在蛋白A Sepharose上从所收集的血清中纯化(如Pharmacia制备手册规定)，产率为65毫克IgG/10毫升血清。生物素化前，抗体用0.05MNaHCO3 PH9.0(2×21)透析，然后每600微升抗体中加入200微升生物素×NHS酯(Calbiochem 203189；1毫克/毫升水)，室温振荡2小时。
最后混合物用PBS透析3次。
用所得抗体构建一个多层ELISA。
-用0.05M NaCO3，PH9.2中的5微克/毫升抗-凝血酶抗体0.1毫升/孔包被微滴定板；16小时/4℃
-用1％BSA饱和；0.3毫升/孔；0.5小时/室温
-用0.05％吐温20/PBS洗3遍
-以10纳克/毫升0.1％BSA/0.05％吐温20/PBS开始11标准2倍稀释人类凝血酶(Calbiochem 605195；3.159 NIH U/毫克)，待测定样品平行稀释；0.1毫升/孔；2小时/室温
-如上清洗
-0.1毫升/孔生物素化的抗凝血酶抗体1∶200；稀释于0.1％BSA/0.05％吐温20/PBS；2小时/室温
-如上清洗
-0.1毫升/孔抗生蛋白链菌素-过氧化酶复合物(B.M.1089153)；1∶10000稀释于0.1％BSA/0.05％吐温20/PBS；30分钟/室温
-如上清洗
-0.1毫升/孔过氧化酶底物
-以0.1毫升/孔2M H2SO4停止反应
-450纳米处测量吸收值(OD)
吸收值(OD)对凝血酶浓度作图见图1.
过氧化酶底物：将0.1毫升TMB溶液(42mM TMB的DMSO溶液)与10毫升底物缓冲液混合(0.1M乙酸钠PH 4.9)；然后加入14.7微升3％H2O2.
通过ELISA检测凝血酶原突变蛋白和凝血酶突变蛋白的存在。
检测凝血酶原突变蛋白和凝血酶突变蛋白的的ELISA按以下所示方案进行：
-用0.1毫升/孔抗-凝血酶抗体包被微滴定板；5微克/毫升0.05 M NaHCO3，pH9.2；16小时/4℃.
-用1％BSA/PBS饱和；0.3毫升/孔；0.5-1小时/室温
-用0.05％吐温20/PBS洗3遍
-11标准2倍稀释人类凝血酶(Calbiochem 605195；3.159 NIHU/毫克)，
以10纳克/毫升0.1％BSA/0.05％吐温20/PBS开始；待测定样品平行稀释；0.1毫升/孔；2-4小时/室温
-如上清洗
-0.1毫升/孔生物素化的抗凝血酶抗体1∶200；稀释于0.1％BSA/0.05％吐温20/PBS；2-4小时/室温
-如上清洗
-0.1毫升/孔抗生蛋白链菌素-过氧化酶复合物(B.M.1089153)；1∶10000稀释于0.1％BSA/0.05％吐温20/PBS；
30分钟/室温
-如上清洗
-0.1毫升/孔过氧化酶底物
-以0.1毫升/孔2M H2SO4停止反应
-450纳米处测量吸收值
过氧化酶底物：将0.1毫升TMB溶液(42mM TMB的DMSO溶液)与10毫升底物缓冲液混合(0.1M乙酸钠PH 4.9)；然后加入14.7微升3％H2O2
实施例13a
从血浆中纯化人类凝血酶
人类凝血酶原按照Henriskson所述(酶学方法，第222卷，312-3279页)从加入了柠檬酸盐的人类血浆中纯化。凝血酶原剪切和用肝素色谱纯化凝血酶，依照实施例14和15进行。含有凝血酶的部分用S2238通过产色凝血酶试验确认并通过超滤浓缩至1毫克/毫升，用PBS再次过滤。
用产色底物S2238测定凝血酶活性
凝血酶突变蛋白的活性通过三肽的断裂来测定。用于此目的的移液方案如下：
50微升样品(0.5U/毫升凝血酶；或不同溶液中的剪切的凝血酶原)
100微升H2O
50微升S2238(1毫克/毫升水溶液；来自Chromogenix，Molndal；Sweden)。
混合物37℃孵育10-15分钟。然后在波长405纳米处测定吸收，凝血酶的量从0.01至1U/毫升的校准曲线上得到。
实施例13b
通过阴离子交换色谱纯化凝血酶原突变蛋白
来自CHO细胞培养液的35升含凝血酶原的细胞培养上清通过4C(SPS 400 Memberane,Fresenius,St.Wendel)超滤首先浓缩至400毫升，然后用10mM Tris PH 7.5再滤至导电率为1.1mS/厘米。脱盐滤液上Q-Sepharose FF柱(直径5厘米，体积530毫升)，流速6毫升/分钟，柱子以起始缓冲液冲洗(20mM Tris PH 7.5)，然后逐渐以15个柱体积梯度洗至400mMNaCl。含凝血酶原的部分通过ELISA确认。凝血酶原突变蛋白在280mM NaCl时洗脱出。从该柱中分离到约150-200毫克蛋白。
实施例14
凝血酶突变蛋白从凝血酶原中释放
在Amicon YM 30(终体积20毫升，175毫克总蛋白)中浓缩的Q-Sepharose色谱部分以2升20mMTris/HCl PH7.5,100mMNaCl透析，通过加入用于剪切的1mMCaCl2,20mMTris/HCl PH7.5调节终浓度至10mM。加入30毫克来自Oxyranus scutellatus蛇的毒液，然后4℃搅拌下剪切，12小时后加入2毫升20mMEGTA溶液，PH7.5终止反应。
实施例15
通过肝素色谱纯化凝血酶突变蛋白
将实施例14所得的含凝血酶的剪切产物上肝素-Sepharose柱(14厘米×1厘米，Pharmacia)流速38厘米/小时。以20毫升20mm磷酸钠，0.1M氯化钠PH7.5,0.01％Pluriol F68清洗柱子后，用150毫升线性梯度洗至600mm磷酸钠，20mM氯化钠PH7.5,0.01％Pluriol F68.然后通过ELISA检测凝血酶突变蛋白。突变蛋白在约400mM NaCl时洗脱出。得到30-35毫克突变蛋白，加入BSA(1毫克/毫升)后，用PBS,0.01％Pluriol F68透析，存于-20℃。
实施例16
体外系统中水蛭素的中和
用凝血酶活性试验检测失活重组凝血酶对水蛭素的解毒效果，通过一起孵育水蛭素，待试验凝血酶突变蛋白质，激活凝血酶和S-2238来进行。此试验所依据的原理是失活待测凝血酶中和(或竞争)水蛭素，和激活凝血酶的所得高活性。
移液方案如下：
100微升水蛭素HL20(1.5U/毫升)或TBS PH 7.5中的0.1％BSA和100微升凝血酶突变蛋白质(实施例2制备)在室温前孵育5分钟。加入50微升活化凝血酶(0.5U/毫升)后，通过加入50微升S-2238(1毫克/毫升水中；来自Chromogenix,Molndal，瑞典)开始反应。混合物37℃孵育15分钟。接着，在波长410纳米处测定吸收，失活凝血酶突变蛋白质中和水蛭素导致吸收增加(图2)。
实施例17
在狗中中和PEG-水蛭素
Beagle狗(9.8-13.5公斤)以10毫克/千克的剂量接受PEG-水蛭素皮下给药。注射PEG-水蛭素180分钟后，实施例2中制备的凝血酶突变蛋白(Thrombinmut#3)在50分钟内以20克/千克的剂量静脉输注。血样通过穿刺肱静脉在试验开始时从狗采得，在150长达6小时的时间后再次采样。通过2000g离心20分钟从枸掾酸化的血中获得血浆，并储存于-20℃至分析。
血浆样品中的抗凝血酶活性按Spannagel等所述测定，血凝纤溶2(1991)121-127。抗凝血活性(aPTT)用Pathromtin系统(Behring)测定。
结果总结于图3。鉴于对照动物给予PEG-水蛭素后抗凝血酶活性迅速升高，在用解毒剂处理的动物中阻止抗凝血酶活性(图3B)的升高是可能的。解毒剂注入完成后，PEG-水蛭素血浆水平以与对照动物可比的速率进一步上升。定量地在aPTT中也观察到了类似情况(图3A)。
序列列表(1)一般信息：
(ⅰ)申请者
(A)姓名：BASF Aktiengesellschaft
(B)街：Carl-Boseh-Strasse 38
(C)城市：Ludwigshafen
(E)国家：Federal Republic of Germany
(F)邮编：67056
(G)电话：0621/6048526
(H)电传：0621/6043123
(I)TELEX：1762175170
(ⅱ)申请题：新的凝白酶突变蛋白质和其解毒剂应用
(ⅲ)序列数量：14
(ⅳ)计算机可清形式
(A)介质：软盘
(B)计算机：IBM PC兼容
(C)操作系统：PC-DOS/MS-DOS
(D)软件：PatentIn Release#1.0,Version#1.25
(EPA)(2)序列号NO:1信息
(ⅰ)序列特点：
(A)长度：33bp
(B)类型：核酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：mRNA的cDNA
(ⅲ) 假说：非
(ⅹⅰ)序列描述：序列号NO:1:
GGGGGGGAAT TCGCCAACAC CTTCTTGGAG GAG(2)序列号NO:2信息
(ⅰ)序列特点
(A)长度：33bp
(B)类型：核酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：mRNA的cDNA
(ⅲ) 假说：非
(ⅳ)反义：非
(ⅹⅰ) 序列描述：序列号NO:2
GGGGGGGAAT TCCTACTCTC CAAACTGATC AAT(2)序列号NO:3信息
(ⅰ)序列特点
(A)长度：40bp
(B)类型：核酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：mRNA的cDNA
(ⅲ)假说：非
(ⅲ)反义：非
(ⅹⅰ) 序列描述：序列号NO:3GGATGCCTGT GAAGGTGACG CTGGGGGACC CTTTGTCATG(2)序列号NO:4信息
(ⅰ)序列特点
(A)长度：40bp
(B)类型：核酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：mRNA的cDNA
(ⅲ)假说：非
(ⅲ)反义：非
(ⅹⅰ) 序列描述：序列个NO:4CATGACAAAG GGTCCCCCAG CGTCACCTTC ACAGGCATCC(2)序列号NO:5信息
(ⅰ)序列特点
(A)长度：40bp
(B)类型：核酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：mRNA的cDNA
(ⅲ)假说：非
(ⅲ)反义：非
(ⅹⅰ) 序列描述：序列个NO:5GACCGGGATG GGAAATATGC CTTCTACACA CATGTGTTCC(2)序列号NO:6信息
(ⅰ)序列特点
(A)长度：40bp
(B)类型：核酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：mRNA的cDNA
(ⅲ)假说：非
(ⅲ)反义：非
(ⅹⅰ)序列描述：序列个NO:6GGAACACATG TGTGTAGAAG GCATATTTCC CATCCCGGTC(2)序列号NO:7信息
(ⅰ)序列特点
(A)长度：40bp
(B)类型：核酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：mRNA的cDNA
(ⅲ)假说：非
(ⅲ)反义：非
(ⅹⅰ)序列描述：序列个NO:7TCCATGCCCG ATTCAGACGC GCCAACACCT TCTTGGAGGA(2)序列号NO:8信息
(ⅰ)序列特点
(A)长度：40bp
(B)类型：核酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：mRNA的cDNA
(ⅲ)假说：非
(ⅲ)反义：非
(ⅹⅰ)序列描述：序列个NO:8
TCCTCCAAGA AGGTGTTGGC GCGTCTGAAT CGGGCATGGA(2)序列号NO:9信息
(ⅰ)序列特点
(A)长度：27bp
(B)类型：核酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：mRNA的cDNA
(ⅲ)假说：非
(ⅲ)反义：非(ⅹⅰ)序列描述：序列个NO:9
GGGGGGGAAT TCGTGAAGCA ATCATGG(2)序列号NO:10信息
(ⅰ)序列特点
(A)长度：30bp
(B)类型：核酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：mRNA的cDNA
(ⅲ)假说：非
(ⅲ)反义：非
(ⅹⅰ)序列描述：序列个NO:10
CTCACCGCCG CCGCATGCCT CCTGTACCCG(2)序列号NO:11信息
(ⅰ)序列特点
(A)长度：30bp
(B)类型：核酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：mRNA的cDNA
(ⅲ)假说：非
(ⅲ)反义：非
(ⅹⅰ)序列描述：序列个NO:11
CGGGTACAGG AGGCACACGG CGGCGGTGAG(2)序列号NO:12信息
(ⅰ)序列特点
(A)长度：27bp
(B)类型：核酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：mRNA的cDNA
(ⅲ)假说：非
(ⅲ)反义：非
(ⅹⅰ)序列描述：序列个NO:12
GAACCTGGAC CGGAACATTG CCCTGAT(2)序列号NO:13信息
(ⅰ)序列特点
(A)长度：27bp
(B)类型：核酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：mRNA的cDNA
(ⅲ)假说：非
(ⅲ)反义：非
(ⅹⅰ)序列描述：序列个NO:13
ATCAGGGCAA TGTTCCGGTC CAGGTTC(2)序列号NO:14信息
(ⅰ)序列特点
(A)长度：579aa
(B)类型：氨基酸
(C)链型：单
(D)拓扑型：线性
(ⅱ)分子类型：蛋白
(ⅵ)来源：
生物：人类
(ⅹⅰ)序列描述：序列NO:14Cys Val Glu Glu Thr Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu
20 25 30Ser Ser Thr Ala Thr Asp Val Phe Trp Ala Lys Tyr Thr Ala Cys Glu
35 40 45Thr Ala Arg Thr Pro Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn
50 55 60Cys Ala Glu Gly Leu Gly Thr Asn Tyr Arg Gly His Val Asn Ile Thr65 70 75 80Arg Ser Gly Ile Glu Cys Gln Leu Trp Arg Ser Arg Tyr Pro His Lys
85 90 95Pro Glu Ile Asn Ser Thr Thr His Pro Gly Ala Asp Leu Gln Glu Asn
100 105 110Phe Cys Arg Asn Pro Asp Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr
115 120 125Thr Asp Pro Thr Val Arg Arg Gln Glu Cys Ser Ile Pro Val Cys Gly
130 135 140Gln Asp Gln Val Thr Val Ala Met Thr Pro Arg Ser Glu Gly Ser Ser145 150 155 160Val Asn Leu Ser Pro Pro Leu Glu Gln Cys Val Pro Asp Arg Gly Gln
165 170 175Gln Tyr Gln Gly Arg Leu Ala Val Thr Thr His Gly Leu Pro Cys Leu
180 185 190Ala Trp Ala Ser Ala Gln Ala Lys Ala Leu Ser Lys His Gln Asp Phe
195 200 205Asn Ser Ala Val Gln Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly
210 215 220Asp Glu Glu Gly Val Trp Cys Tyr Val Ala Gly Lys Pro Gly Asp Phe225 230 235 240Gly Tyr Cys Asp Leu Asn Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr
245 250 255Gly Asp Gly Leu Asp Glu Asp Ser Asp Arg Ala Ile Glu Gly Arg Thr
260 265 270Ala Thr Ser Glu Gln Gln Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser
275 280 285Gly Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu
290 295 300Glu Asp Lys Thr Glu Arg Glu Leu Leu Glu Ser Tyr Ile Asp Gly Arg305 310 315 320Ile Val Glu Gly Ser Asp Ala Glu Ile Gly Met Ser Pro Trp Gln Val
325 330 335Met Leu Phe Arg Lys Ser Pro Gln Glu Leu Leu Cys Gly Ala Ser Leu
340 345 350Ile Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro
355 360 365Pro Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu Val Arg Ile Gly
370 375 380Lys His Ser Arg Thr Arg Tyr Glu Arg Asn Ile Glu Lys Ile Ser Met385 390 395 400Leu Glu Lys Ile Tyr Ile His Pro Arg Tyr Asn Trp Arg Glu Asn Leu
405 410 415Asp Arg Asp Ile Ala Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser
420 425 430Asp Tyr Ile His Pro Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser
435 440 445Leu Leu Gln Ala Gly Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu
450 455 460Lys Glu Thr Trp Thr Ala Asn Val Gly Lys Gly Gln Pro Ser Val Leu465 470 475 480Gln Val Val Asn Leu Pro Ile Val Glu Arg Pro Val Cys Lys Asp Ser
485 490 495Thr Arg Ile Arg Ile Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro
500 505 510Asp Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro
515 520 525Phe Val Met Lys Ser Pro Phe Asn Asn Arg Trp Tyr Gln Met Gly Ile
530 535 540Val Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr545 550 555 550Thr His Val Phe Arg Leu Lys Lys Trp Ile Gln Lys Val Ile Asp Gln
565 570 575Phe Gly Glu
Claims (10)
1．与天然凝血酶序列有以下序列区别的一种凝血酶突变蛋白质
(a)Ala取代Gly,Gly位于序列环境Tyr-Gly-Phe中，并占据天然人类凝血酶原第558位(序列号14)；
(b)下列残基中至少一个取代或缺失
(b1)位于序列环境Ala-His-Cys中，并占据天然人类凝血酶原第363位(序列号14)的His；
(b2)位于序列环境Arg-Asp-Ile中，并占据天然人类凝血酶原第419位(序列号14)的Asp；
(b3)位于序列环境Asp-Ser-Gly中，并占据天然人类凝血酶原第525位(序列号14)的Ser；
2．权利要求1所要求的凝血酶突变蛋白质，它与天然人类凝血酶原(序列号14)相比有下列序列区别(序列号14)
(a)558位Gly被Ala取代并
(b)如下残基中至少一处取代或缺失
(b1)363位的His；
(b2)419位的Asp；
(b3)525为的Ser。
3．权利要求2所要求的凝血酶突变蛋白质，其中序列区别(b)为525位的取代。
4．权利要求3所要求的凝血酶突变蛋白质，其中序列区别(b)为Ser被Ala取代。
5．编码权利要求1到4中任一所要求的凝血酶突变蛋白质的核酸序列。
6．权利要求1到4中任一所要求的凝血酶突变蛋白质治疗凝血障碍的应用。
7．权利要求6所要求的作为凝血酶抑制剂的解毒剂的应用。
8．权利要求7所要求的作为水蛭素，水蛭素突变蛋白质或水蛭素衍生物的解毒剂的应用。
9．权利要求8所要求的作为聚乙二醇修饰的水蛭素衍生物的解毒剂的应用。
10．包含权利要求1到4中任一所要求的凝血酶突变蛋白质和药用助剂的凝血酶抑制剂的解毒剂。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19605126A DE19605126A1 (de) | 1996-02-12 | 1996-02-12 | Thrombinmuteine als Antidot für Thrombininhibitoren |
| DE19605126.6 | 1996-02-12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1211280A true CN1211280A (zh) | 1999-03-17 |
Family
ID=7785198
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN97192210A Pending CN1211280A (zh) | 1996-02-12 | 1997-02-11 | 凝血酶突变蛋白质作为凝血酶抑制剂的解毒剂 |
Country Status (18)
| Country | Link |
|---|---|
| US (1) | US6060300A (zh) |
| EP (1) | EP0880591A1 (zh) |
| JP (1) | JPH11512621A (zh) |
| KR (1) | KR19990082479A (zh) |
| CN (1) | CN1211280A (zh) |
| AR (1) | AR005797A1 (zh) |
| AU (1) | AU1768497A (zh) |
| BR (1) | BR9707280A (zh) |
| CA (1) | CA2245546A1 (zh) |
| CO (1) | CO4600682A1 (zh) |
| CZ (1) | CZ247798A3 (zh) |
| DE (1) | DE19605126A1 (zh) |
| HR (1) | HRP970077A2 (zh) |
| HU (1) | HUP9900574A2 (zh) |
| IL (1) | IL125077A0 (zh) |
| NO (1) | NO983677L (zh) |
| WO (1) | WO1997029198A1 (zh) |
| ZA (1) | ZA971107B (zh) |
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| KR20000018933A (ko) * | 1998-09-07 | 2000-04-06 | 김승수 | 내피세포 성장억제 활성을 가지는 사람 프로트롬빈 크링글단백질 및 이를 암호하는 유전자 |
| GB2355161B (en) * | 1999-10-05 | 2001-09-12 | 3Com Corp | Network device incorporating selective compression of stored data packets |
| KR20020008471A (ko) * | 2000-07-20 | 2002-01-31 | 김승수 | 내피세포 성장 억제활성을 가지는 인간 프로트롬빈크링글, 그것의 재조합벡터 및 이를 발현하는 재조합 숙주 |
| CA2644926A1 (en) * | 2006-03-06 | 2007-09-13 | Humagene, Inc. | A method for the preparation of recombinant human thrombin and fibrinogen |
| BRPI0816837B1 (pt) | 2007-09-28 | 2022-10-18 | Portola Pharmaceuticals, Inc | Composições farmacêuticas, polipeptídeo isolado de duas cadeias e uso de uma composição farmacêutica |
| WO2010056765A2 (en) | 2008-11-14 | 2010-05-20 | Portola Pharmaceuticals, Inc. | Antidotes for factor xa inhibitors and methods of using the same in combination with blood coagulating agents |
| ES2607935T3 (es) | 2009-03-30 | 2017-04-04 | Portola Pharmaceuticals, Inc. | Antídotos para inhibidores del factor Xa y procedimientos de uso de los mismos |
| CA2767858C (en) * | 2009-07-15 | 2019-02-12 | Portola Pharmaceuticals, Inc. | Unit dose formulation of antidotes for factor xa inhibitors and methods of using the same |
| ES2620421T3 (es) | 2009-10-30 | 2017-06-28 | Senova Gesellschaft für Biowissenschaft und Technik mbH | Peptidasas acopladas a polímero |
| AR079944A1 (es) | 2010-01-20 | 2012-02-29 | Boehringer Ingelheim Int | Anticuerpo neutralizante de la actividad de un anticoagulante |
| EP2691156A1 (en) | 2011-03-30 | 2014-02-05 | Boehringer Ingelheim International GmbH | Anticoagulant antidotes |
| JP6640198B2 (ja) | 2014-05-26 | 2020-02-05 | アカデ−ミッシュ ズィーケンハウス ライデンＡｃａｄｅｍｉｓｃｈ Ｚｉｅｋｅｎｈｕｉｓ Ｌｅｉｄｅｎ | 出血治療のための止血促進タンパク質 |
| EP3971576A1 (en) | 2020-09-21 | 2022-03-23 | Instrumentation Laboratory Company | Detecting and monitoring oral anticoagulants or intravenous direct thrombin inhibitors in a blood sample |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4203965A1 (de) * | 1992-02-11 | 1993-08-12 | Max Planck Gesellschaft | Antidot fuer hirudin und synthetische thrombininhibitoren |
-
1996
- 1996-02-12 DE DE19605126A patent/DE19605126A1/de not_active Withdrawn
-
1997
- 1997-02-11 AU AU17684/97A patent/AU1768497A/en not_active Abandoned
- 1997-02-11 CA CA002245546A patent/CA2245546A1/en not_active Abandoned
- 1997-02-11 CO CO97006960A patent/CO4600682A1/es unknown
- 1997-02-11 ZA ZA971107A patent/ZA971107B/xx unknown
- 1997-02-11 IL IL12507797A patent/IL125077A0/xx unknown
- 1997-02-11 EP EP97903254A patent/EP0880591A1/de not_active Withdrawn
- 1997-02-11 HR HR19605126.6A patent/HRP970077A2/hr not_active Application Discontinuation
- 1997-02-11 JP JP9528173A patent/JPH11512621A/ja active Pending
- 1997-02-11 CZ CZ982477A patent/CZ247798A3/cs unknown
- 1997-02-11 KR KR1019980706212A patent/KR19990082479A/ko not_active Application Discontinuation
- 1997-02-11 BR BR9707280A patent/BR9707280A/pt not_active Application Discontinuation
- 1997-02-11 WO PCT/EP1997/000612 patent/WO1997029198A1/de not_active Application Discontinuation
- 1997-02-11 CN CN97192210A patent/CN1211280A/zh active Pending
- 1997-02-11 HU HU9900574A patent/HUP9900574A2/hu unknown
- 1997-02-11 US US09/117,708 patent/US6060300A/en not_active Expired - Fee Related
- 1997-02-12 AR ARP970100548A patent/AR005797A1/es unknown
-
1998
- 1998-08-11 NO NO983677A patent/NO983677L/no unknown
Also Published As
| Publication number | Publication date |
|---|---|
| HUP9900574A2 (hu) | 1999-06-28 |
| EP0880591A1 (de) | 1998-12-02 |
| NO983677D0 (no) | 1998-08-11 |
| CZ247798A3 (cs) | 1999-03-17 |
| IL125077A0 (en) | 1999-01-26 |
| HRP970077A2 (en) | 1998-04-30 |
| JPH11512621A (ja) | 1999-11-02 |
| ZA971107B (en) | 1998-08-11 |
| NO983677L (no) | 1998-08-11 |
| WO1997029198A1 (de) | 1997-08-14 |
| CO4600682A1 (es) | 1998-05-08 |
| KR19990082479A (ko) | 1999-11-25 |
| AR005797A1 (es) | 1999-07-14 |
| CA2245546A1 (en) | 1997-08-14 |
| DE19605126A1 (de) | 1997-08-14 |
| AU1768497A (en) | 1997-08-28 |
| BR9707280A (pt) | 1999-07-20 |
| US6060300A (en) | 2000-05-09 |
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