ニワトリの線維素溶解現象に関する研究 Studies on fibrinolysis in the domestic chicken

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著者

    • 山田, 學 ヤマダ, マナブ

書誌事項

タイトル

ニワトリの線維素溶解現象に関する研究

タイトル別名

Studies on fibrinolysis in the domestic chicken

著者名

山田, 學

著者別名

ヤマダ, マナブ

学位授与大学

麻布大学

取得学位

獣医学博士

学位授与番号

乙第259号

学位授与年月日

1987-09-09

注記・抄録

博士論文

線維素溶解現象(線溶)の生理学的な役割とは、血液凝固系、キニン系、補体系などと強い相互作用を持ちながら、生体内での凝血形成の予防と排除とを主な機能とする現象である。病理学あるいは臨床においても出血、血行障害、組織の修復などと大きな関連性を有することから、本現象の解明は診断や治療を含めた獣医学全体としても意義あるものであると考える。線溶の機能的発達は、概略では動物の進化と並行していると考えられている。従って、線溶機構を考察する上では、できるかぎり多くの動物についての情報が要求される。 しかしながら、ヒトあるいはヒトのモデルとしての実験動物以外の脊椎動物を対象とした研究は遅れており、中でも鳥類の報告は非常に少ない。このことは、生理学的な面から鳥類の線溶機構そのものの未解決が問題であるばかりでなく、動物全体として線溶を考察するとか、臨床的にも大いに注目されよう。 本研究は飼育羽数の点から家禽の代表的なものとしてニワトリを研究対象に選び、未だ確立されていない線溶の測定に有効な方法を模索しつつ、成鶏の線維素溶解現象の作用機構について計測と結果の検討を重ね、以下の結論を得た。1. in vivoにおけるニワトリの血液塊除去機能について 全血1mlから形成した血液凝塊をニワトリ皮下に埋没したところ、形成から1週間以内では主に血餅退縮と線溶、2週目以降は線溶と器質化の機能によって縮小および分解過程が進行し、3週間でほとんどの血液凝塊は除去された。 尺骨皮静脈の長さ3cmに渡る両端を結紮した内部に形成したトロンビン血栓は、血栓の形成後に結紮を完全に除去し、形成部位に血流が存在するものでは、1日以内に障害部位から血栓は消失した。緩やかな血栓流失防止のための結紮を行ない、血流阻害を受けた場合には半数の血栓がなくなるのに形成から4日を要し、さらに長期残存例では血管内膜との強い癒着が形成された。 以上のことから、ニワトリには哺乳動物と機能的に類似し、能力としても充分な血液塊除去機能が生体内に存在することが明らかとなった。2. 内因系線維素溶解現象について 白色レグホン種の雌成鶏60羽の正常なユーグロブリン溶解時間は、Allenの方法によると5.1±1.6時間(平均値±標準偏差)である。このときのユーグロブリンに線溶活性化物質であるデキストランサルフェート、ストレプトキナーゼ、ウロキナーゼを加えると、これらの添加量にユーグロブリン溶解時間は反比例した。この傾向は-20℃に保存した血漿を用いたときに特に顕著であった。しかし、高濃度のデキストランサルフェートはこの反応を抑制した。 ニワトリフィブリン平板では50-100mg/mlのデキストランサルフェートに同量の血漿あるいはユーグロブリンを加えたものを検体としたとき溶解した。リジンセファロースのカラムクロマトグラフィを使って得た画分は、デキストランサルフェートおよびウロキナーゼで活性化され、このうちデキストランサルフェートによる活性化は同種の平板上でのみ観察された。ストレプトキナーゼによる活性化はいずれの測定においても認められなかった。血漿のユーグロブリン処理は抗線溶物質除去の機能を果たしていた。また、プラスミノーゲン活性化において、カルシウムイオンは活性増強の傾向を示した。 以上のことから、ニワトリには弱い内因系線溶活性系が存在し、抗線溶物質の除去と反応性の高い測定法とを選択することによって、測定できる可能性が示唆された。3. 外因系線維素溶解現象について フィブリンスライド法による観察では、二ワトリ新鮮心臓の心外膜からブラスミノーゲンアクチベーター作用を有する物質の溶出があり、プラスミノーゲンを含むウシフィブリン膜を溶解した。 プラスミノーゲンアクチベーター作用は2Mロダンカリ、0.3M酢酸カリウム、5M尿素および硫酸マグネシウム溶液3.9-62.5mM、0.5-2Mを溶媒とした新鮮心臓ホモジネート中に存在した。MgSO_4溶液でニワトリ心臓をホモジネートしたときの上清とニワトリプラスミノーゲンとの混合液を、37℃、4時間加温したものにCaCl_2を加えるとき、フィブリン平板法において最高活性を認めた。これに対し.ウロキナーゼ活性化では37℃、1時間の加温でピークに達した。これらの添加順序を変えた場合の活牲は弱くなった。ニワトリプラスミノーゲン、MgSO_4抽出ニワトリ心臓ホモジネート上清、CaCl_2の容量比が2:1:1であるとき、CaCl_2の至適濃度は33mMであった。 ニワトリ心臓抽出プラスミノーゲンアクチベーターとウロキナーゼの活性はいずれも片対数グラフ上で直線性を示す量的比例関係のある反応であり、∈-アミノカプロン酸は主にニワトリ心臓由来プラスミノーゲンアクチベーターによるプラスミノーゲンの活性化反応系を強く抑制した。 この結果から、ニワトリには組織プラスミノーゲンアクチベーターがプラスミノーゲンを活性化する機構があり、できたプラスミンの酵素作用はCaイオンで増強されることが示唆された。4. ニワトリプラスミノーゲンの分離とカルシウムの線溶増強作用について ニワトリ血漿からのプラスミノーゲンの分離と、プラスミノーゲン活性化機構においてカルシウムの果たす役割について検討した。ニワトリ血漿から硫安分画、リジン-セファロースによるアフィニティクロマトグラフィと限外濾過法等によってプラスミノーゲンを分離、精製した。SDS-PAGEでは、ニワトリプラスミノーゲンの分子量はおよそ77,000の単一バンドで示された。プラスミノーゲンは高分子のデキストランサルフェートやストレプトキナーゼでは活性化を受けなかったが、ヒトウロキナーゼにより活性化された。 ウロキナーゼ活性化プラスミンにカルシウムを加えると酵素活性の増強効果が見られ、基質側に加えても同様の効果が観察された。∈-アミノカプロン酸やトラネキサム酸はこの酵素作用を抑制したけれども、効果の比較では後者が約1,000倍の強さを示した。 これらの結果から、ニワトリプラスミノーゲンの分子量は約77k daltonで、カルシウムはプラスミンの作用を増強する効果を持っていることが明らかとなった。5. 血漿中の抗線溶因子について ニワトリ血漿中の線溶抑制因子の活性能を、ヒトで応用されている方法を用いて測定した。単純一元免疫拡散法による測定では、アンチトロンビンⅢとα2-マクログロブリンは反応性があってヒト由来の因子との共通抗原性を認めたけれども、ヒトの正常値に比べるとかなり低値であった。C1インアクチベーターは全く反応がなく、因子の定性的と定量的な確認が不可能であった。合成発色基質を利用した方法から得られた結果では、ヒトプラスミンの活性を抑制する機能からみた比較という意味で、ニワトリのα2-プラスミンインヒビターとアンチトロンビンIIIの測定に利用できる可能性が示唆された。得られたニワトリ正常血漿の結果から、血漿中のα2-プラスミンインヒビターは平均値でヒトの25%程度と低く、アンチトロンビンIIIは125%程度でやや高い傾向を示した。 以上のことから、ニワトリ血漿が量的な差はあるけれども、哺乳勤物と同程度の線溶抑制効果を示す因子を保有していることが示された。 上記の結論を集約すると、ニワトリ線溶が哺乳動物の線溶に比べると、相対的な活性力の弱さはあるものの、基本的な線溶機構には何ら変りのないことが明らかとなった。その結果、本論の初めに提示した哺乳動物の線溶機描の概要に対して、以下のようなニワトリ線溶機構のschemaが想定できた。(以下図)

Morgagni (1761) was the first to describe that human blood does not coagulate after sudden death. Dastre (1893) also found that canine blood clots once formed, were hydrolyzed by an enzyme and that the blood fluidity is restored. This phenomenon was later termed "fibrinolysis". The fibrinolytic system consists of three components, the proenzyme plasminogen (Plg) which is activated by a limited proteolysis to produce the proteolytic enzyme plasmin (Pln), the plasminogen activators (Act) and the inhibitors which rapidly neutralize Pln or interfere with activation of Plg. The physiological role of fibrinolysis is not only to prevent fibrinformation and to remove blood clots but also related with several other biological phenomena such as tissue repair, malignant transformation, macrophage function, ovulation, embryo implantation and the metabolism of kinins and complements. The blood fibrinolytic mechanism is highly complicated implying that it has phylogenetically developed with animal evolution. Generally in mammals, the fibrinolytic mechanism involves activation and inhibition processes. These processes are separately detectable and the activities are balanced in normal condition. Avian intrinsic fibrinolysis exists in chickens, geese, turkeys, Japanese quails, Plgeons, parrots and some species of wild birds. In all these cases except in wild birds that fly around tree tops, however, the reaction is not detected, or very weak if any, when the activity is measured by the same method as that used for mammals. This is due to the lack of a strong antiplasmin activity. Thus, an improvement of the measuring method may make possible to detect the fibrinolytic reaction in birds. With regard to avian extrinsic fibrinolysis, any reports are not available except for those on urokinase, a particular case of tissue plasminogen activator (t-PA) in the Raus sarcoma virus-transformed chick embryo fibroblast or in the vampire bat saliva. On the other hand, fibrinolytic activity has been found to increase over the normal range with stress caused by various deseases. Accordingly, studies of fibrinolysis in animals other than man or common laboratory mammals seem necessary not only for physiological interest but also for clinical investigations. Broad spectrum studies are the only way in which any physiological mechanism can be fully understood and the basic mode of action of the fibrinolytic system is established. The purpose of the present study is to. elucidate the nature of chicken fibrinolysis in comparison with the mammalian mechanism and to establish a reliable method of measuring its activity. Adult White Leghorn hens of Shaver strain kept at Hiroshima Agricultural College Farm were used in this study.1. Dissolution of thrombus in vivo This study was carried out in order to clarify the mechanism of thrombolysis in vivo. Five milliliters of venous blood was collected from 5 chickens and pooled. One milliliter of the blood was placed in a 10-mm-diameter glass tube and let to coagulate spontaneously by incubation at 37℃ for 1 hour. The skin of upper area of the M. brachialis under the wing was incised about 2 cm in length. The thrombus previously prepared was embedded in this part and the wound was sutured. Observation of the clot at this site was carried out once a week and its weight was determined. The V. cutanea ulnaris was ligated for a length of 3 cm with suture thread under anesthesia with pentobarbital IP injection. A thrombus was produced in the ligated vessel by an IV injection of 10 μl of bovine thrombin (50 NIH units/ml physiological saline). The needle was left in place for 5 min. The ligatures in one group were completely removed 1 hour after thrombin injection and those in another group were left loose. The thrombi in vessels were taken out under anesthesia and weighed. After incubation of 1 ml of chicken blood at 37℃ for 1 hour in a glass tube, the wet weight of the clot was 0.79 ± 0.10 g (mean ± standard deviation) after losing serum. These clots were embedded under the skin of wing. The clots were observed and weighed once a week. The weight of thrombi decreased by 1/10 in the first week, though they were still separable from surrounding tissues at this time. After the end of the second week, the color changed from red-brown to dark green, the clot was covered by a connective tissue producing a formation connected with surrounding tissues. After the third week, the weight decreased to less than 1/10 of the previous week, and the clot sometimes splited into several pieces. The thrombi in the V.cutanea ulnaris weighed 14.3± 5.8 mg ( mean ± standard deviation) 1 hour after thrombin injection. In none of the ligated 12 veins, any vessels completety free from thrombus existed after 1 day. In the loosely ligated venous, the thrombi did not disappear and the thrombus weight decreased by 57% in the first day, and gradually diminished during the following 3 days. After 7 days, only 3 vessels out of 12 held a small thrombus loosely adhering to the inner surface of the vein. Thus, it was indicated that chickens possessed an ability to remove blood clot at outside of vessels, by dissolving and absorbing it within 3 weeks. Venous thrombi produced by thrombin injection were entirely dissipated within one day in vessels without ligation. The thrombi in vessels with loose ligation took about 3 days for dissolving by thrombolysis, and further surviving thrombi were absorbed by organization.2. Intrinsic fibrinolysis in the chicken blood The intrinsic activity of avian fibrinolysis has not been accurately measured by methods for the human blood because avian fibrinolysis has a weak or small contact phase and a anti-fibrinolytic activity, with a few exceptions. Separation and purification of fibrinolytic substances from the blood of vertebrates have been facilitated by an application of affinity chromatography with lysine sepharose. Then, a study was carried out to determine the authentic activity of chicken intrinsic fibrinolysis by using partially purified fibrinolytic substances in the absence of an interference by avian plasma in the assay procedure. The activity was measured by the euglobulin clot-lysis time (ELT) and a fibrin plate method. Normal chicken ELT determined by the method of Allen (1972) was 5.1± 1.6 hours (mean ± standard deviation). The euglobulin fraction from fresh chicken plasma was not activated by dextran sulfate (DS), streptokinase (SK), or urokinase (UK) as fibrinolytic activators. ELT was short when plasma samples kept at -20℃ for 3 months were used. DS at the concentrations higher than 13.7 μg/ml and than 41.1 μg /ml inhibited the actions of Pln when fresh plasma and stored plasma was used, respectively. It was considered that the difference between the fresh and stored plasmawas attributable to the difference in the amount of proactivator (Proact) or Act in samples. The Act might be produced actively when a sufficient amount of Proact was present in the fresh plasma, irrespectively of the presence of Act SK or UK. Conversely, fibrinolytic effects of SK or UK were exhibited following a destruction of native activators in plasma under an influence of storage. Four kinds of fibrin plates were made from bovine plasminogen-rich fibrinogen, bovine plasminogen-free fibrinogen or Cohn I fraction of chicken thrombocyte-poor plasma. Affinity chromatography using lysine-sepharose was conducted for removing Plg from the Cohn I fraction. Activation by DS, SK or UK of chicken plasma was not detected in the fibrin plate method. Chicken euglobulin activated by UK solubilized the bovine standard fibrin plate (BSP). DS-activated euglobulin slightly dissolved the chicken fibrin plate, irrespectively of the presence of Plg. Three fractions were obtained employing the method of Igarashi et al.(1973) with lysine-sepharose affinity chromatography. The fraction I was eluted with 0.005 M phosphate buffer (pH 7.5), the fraction II was eluted with 1 M NaCl-0.05 M phosphate buffer (pH 7.5), while the fraction III was eluted with 0.1 M acetic acid. BSP were solubilized to a small extent by these fractions which were activated with UK. Bovine Plg free fibrin plate (BfP) was not dissolved by any preparations. Chicken fibrin plates were dissolved by the DS-activated fractions as well as by fractions II and III activated with UK. Fraction III showed activity in the presence of CaCl_2 on avian Plg-free fibrin plates. These results indicated that the chicken blood possesses fibrinolytic activity with a weak SK sensitivity, a considerable reactivity to DS, UK or CaCl_2. Therefore, it was concluded that intrinsic fibrinolytic activity in chicken blood can be determined by eliminating the anti-fibrinolytic substances from samples and by using a more sensitive assay method.3. Extrinsic fibrinolytis in chickens The main system of avian blood coagulation works through an extrinsic pathway. Therefore, the existence of unidentified thrombolytic activity in the extrinsic phase, as in mammals, is assumed in the avian blood coagulation. Astrup (1981) have studied extensively the various t-PA of the body. The distribution of t-PA varies with organs and species of animals. In human, uterus, adrenals, lymph nodes, prostrate, thyroid, lung, ovary, pituitary, kidney, and skeletal muscle possess t-PA activity, while little or no activity is shown in the testes, spleen, and liver. The t-PA activity was found to have a species-specificity in mammals. A study was therefore carried out to show the presence of t-PA and its mode of action. A chicken heart was minced and homogenized with a 10-fold volume of a solution using a Bio-mixer at 4℃ for 10 min. Freezing and thawing of the homogenates were repeated 3 times. The supernatant, separated by centrifugation at 12,000×g for 30 min, was used for a t-PA solution. The Act activities of the supernatants were determined by the fibrin plate method when the heart was homogenized with KSCN, CH_3COOK, (NH_2)_2C0 , or MgSO_4 solution. When MgSO_4 solution was used for extracting t-PA from fresh chicken hearts, the activity showed a tendency to decrease with reducing MgSO_4 concentration at the range of 0.5-2 M. The highest activity was shown at 62.5 mM and the activity decreased at higher concentrations. t-PA activity could be observed pH between 3.9-4.5, with an optimum pH of 4.2. Therefore, the supernatant of chicken heart homogenate extracted with 62.5 mM MgSO_4 solution after adjusting pH to 4.2 with 1% acetic acid was used in the following studies as a t-PA solution. The effect of CaCl_2 on t-PA was examined where the Plg:t-PA:CaCl_2 volume ratio was 2:1:1, and the CaCl_2 concentration was changed in the range of 3.9 mM-1 M. At the range of 0.25-1 M of CaCl_2, the activity was completely inhibited, while the activity could he detected at the presence of CaCl_2 less than 125 mM. As the maximum activity was shown at 33 mM, this concentration was used in the following studies. The effect of UK or t-PA concentration in activation was studied where the volume ratio of Plg : CaCl_2 : t-PA or UK was 2:1:1. Both activators showed linear curves on a semi-log graph. Furthermore, the slopes of two curves closely resembled with each other. This suggested that both activators shared a similar action in the chicken Plg activation. After incubation of Plg and t-PA mixture at 37℃ for 0.5, 1, 2, 4, 6, 8 or 24 hours, a constant amount of CaCl_2 was added, and 200 IU/ ml UK was used as a control of Act. The action of t-PA was enhanced by incubation for 4 hours, but further incubation rather decreased the activity. UK-activation reached a maximum level after 1 hour, but by during subsequent incubation for 8 hours the activity was maintained at the level which was half that of the maximum, and thereafter it gradually decreased. Incubation for 24 hours caused a sharp drop in activity and both activators showed approximately the same effect. When CaCl_2 was added after incubation of Plg and t-PA mixture for 4 hours, the maximum activity was produced. The activation was slightly low when a mixture of Plg, t-PA and CaCl_2 was incubated. This was observed when UK was employed instead of t-PA. After incubation of Plg alone, or t-PA and CaCl_2 mixture, only a weak activation was observed. However, after incubation of t-PA or CaCl_2 alone, the mixture brought about the least activation. Thus, it was demonstrated that CaCl_2 had a stimulating effect on t-PA-activated Pln action. The inhibitory effect of EACA against fibrinolysis by Plg and t-PA before and after incubation at 37℃ for 4 hours was measured and the degree of inhibition was expressed as a percentage of the control. When 2 M EACA was added before incubation, the inhibitory effect against Plg activation was completely abolished. The inhibition was biphasic with the EACA concentration range. An inhibition of t-PA-activated Pln activity was observed by an addition of EACA after incubation. The maximum forty percent inhibition was observed at the concentration of 2 M, but no such inhibition was observed below 250 mM.4. Separation of plasminogen from chicken plasma Fibrinolysis is specifically distinct from proteolysis, and involves the enzymatic breakdown of fibrin and fibrinogen. The active blood fibrinolytic agent responsible is known as Pln. Pln is derived from its inactive precursor, Plg, which is always present in circulating blood and is firmly bound to fibrinogen. The primary site of synthesis of Plg has not been established in any species. However, several studies have provided the indirect evidence which suggests both considerable species variation as well as possible multiple sites of synthesis and/or storage. Pln itself is not found in the circulation except in pathological states, since it is rapidly neutralized by antiplasmins. It only appears when it is produced in such excess that the antiplasmins are insufficient to inactivate it completely. Since 1970, mostly used application of the antifibrinolytic amino acids to the purification of Plg is the affinity chromatographic technique of Deutsch and Mertz in which lysine coupled to sepharose is used as absorbant of Plg. Several modifications of this technique have been developed for the purification of Plg from plasma and different crude plasma fractions. Purification of chicken Plg is required in order to study its real properties. There are few reports concerning the purification and the enzymological properties of chicken Plg. Accordingly, an attempt was made in this study to separate the Plg from chicken plasma using the same method as for mammals, and to ascertain some of the properties of Plg fraction obtained. Saturated ammonium sulfate was slowly added to a concentration of 50% in chicken plasma, and stirred for 2 hours. After being allowed to settle for 2 hours, this mixture was centrifuged at 2,000 rpm for 10 min, and the precipitates was dissolved in 0.005 M phosphate buffer (pH 7.5) and dialyzed overnight against an approximate ten-fold quantity of the same buffer. The Plg fraction was isolated by affinity chromatography on a column packed with lysine-sapharose 4B using a procedure similar to that of Hishikawa, et al. (1973). This fraction was eluted with 0.2 M EACA and subjected to ultrafiltration with a 10-fold volume of phosphate buffer. A volume equivalent to that of the starting material was obtained and stored at -80℃ until examination. The protein purified by this procedure showed a single band by SDS-PAGE and its molecular weight was estimated to be 77,000 daltons, which was different from that of Glu-plasminagen or Lys-plasminogen in man. However, this fraction was activated with UK and CaCl_2 mixture, and dissolved bovine fibrin. Tranexamic acid and EACA completely inhibited this activity. From the properties mentioned above, the fraction was considered to be chicken Plg. This Plg was not activated by DS or SK, and activation by UK was relatively weak when compared with that in mammals. Therefore, chicken Plg activation by SK and DS was considered to be so slight as to be unmeasurable by the fibrin plate method, or to be completely absent.5. Stimulating effect of CaCl_2 on chicken plasmin activity Allen (1872) reinvestigated the kind of buffer solution, ionic strength and calcium ion in the solvent of euglobulin when chicken ELT was measured. And it became clear that the selection of certain buffers as a solvent and a suitable density of calcium ion in the euglobulin permitted measurement of chicken ELT. Also, CaCl_2 was used in fibrin plate method for accleration of the activity. As a series of studies concerning with the effect of calcium in chicken fibrinolysis, the present section describes the activation of purified Plg by CaCl_2 in Vitro. At various concentrations CaCl_2 was added to a mixture containing a fixed amount of Plg and UK. The lysis area showed the maximum at 7.8 mM, and reduction from this concentration resulted in a decrease of activity. Accordingly, 7.8 mM CaCl_2 solution with UK, DS or SK was added to fractions obtained at purification steps. Fibrinolytic activity in BSP was shown in the ammonium sulfate fraction and Plg solution, and only the latter dissolved BfP. Combined mixture of Plg, UK and CaCl_2 solutions were preincubated at 4℃, 20℃, and 37℃ for 4 hours. Plg and CaCl_2 mixture with or without UK showed the highest activity upon incubation at 4℃ for 4 hours. Plg with UK incubated at 37℃ for 1 hour and UK with CaCl_2 incubated at 4℃ for 2 hours also showed a higher activity than under others. When a fixed amount of CaCl_2 (7.8 mM) was added to the samples, a relation of what was observed between CaCl_2 present in substrates and lysis area. Within a concentration range from 31.3 mM - 1 M, the lysis area was not dependent upon the presence of CaCl_2 and activity increased gradually in inverse proportion. The presence of CaCl_2 in samples inhibited the activity when the concentration of CaCl_2 in the substrate was below 7.8 mM. The inhibitory effect of tranexamic acid and EACA in 0.85% saline on chicken Plg activation with UK in the presence of CaCl_2 was observed using the fibrin plate method and expressed as an inhibitory percentage in comparison with the control. Both inhibitors controlled the activation completely, and its relation between the natural logarithm of concentration and lysis area showed a negative correlation (p <0.01). The action of tranexamic acid was approximately 1,000-times stronger than the action of EACA when the activities were calculated using the experimental results of completely inhibiting concentration. In human fibrinolysis, the accelerating effect of calcium ion on ELT has been described by Ratnoff (1952). According to this report, calcium showed an indirect mediation, but did not appear to potentiate fibrinolysis by bovine or human active plasma proteolytic enzyme and it didnot accelerate the activation of precursors by chloroform or SK. It was also confirmed by Kowalski, et al. (1959) that calcium activation of euglobulin clot lysis differed from SK activation in that the calcium activation released more trichloroacetic acid-soluble tyrosine compounds. Hindersin, et al. (1974) observed microfluorometrically that an increase of calcium in euglobulin coagulated with thrombin accelerated lysis, while Helle (1968) reported that ELT was dependent upon the calcium concentration in samples. On the other hand, an inhibitory effect of calcium was reported by Sherry, et al. (1959). Bruce (1964) showed that fibrinolytic activity was accelerated by calcium in the absence of inhibitors, although anti-plasmin activity was enhanced with calcium. The results of this study indicated that a certain amount of calcium ion is necessary for activation of chicken Plg, through its reaction mechanism is unknown. Therefore, incubation of the Plg and UK mixture at 37℃ for more than 4 hours followed by an addition of calcium seems the best way to induce chicken Plg activation.6. Antifibrinolytic factors in chicken plasma The presence of antifibrinolytic substances has been known since at least the beginning of this century. There are two main types of inhibitors which inhibit the action of Pln and the activation of Plg. Many substances derived from biological fluids, tissues, plants, or microorganisms inhibit fibrinolysis. Some active-site titrants of serine proteases are potent inhibitors of fibrinolytic enzymes. Certain amino acids, such as lysine, 6-aminohexanoic acid, or tranexamic acid, inhibit the digestion of fibrin. Mammalian blood possesses large amounts of protease inhibitors which take place over 10% of whole plasma protein. AntithrombinIII (ATIII), α_2-plasmin inhibitor (α_2PI), α_1-antitrypsin (α_1AT), C1-inactivator (C1INA), α_2-macroglabulin (α_2MG) and antiactivator are known to be present and to show antifibrinolytic properties in mammalian plasma as physiological inhibitors. Plasma from amphibians, reptiles, birds and mammals have anti-fibrinolytic activity which shows a species specificity. The inhibition by chicken plasma of 1 IU/ml swine plasmin in a bovine fibrin plate was 80.2 ±14.2% (mean ± standard deviation). This value was somewhat low in comparison with the values in plasma of some domestic animals except the cat. A study was conducted to quantify the inhibitors present in normal chicken plasma using a simple immunodiffusion method and spectrophotometric measurement using synthetic chromogenic sustrates. ATIII, C1INA and α_2MG in 9 normal plasma samples were measured using the single radial immunodiffusion plate method. Lyophilized human standard plasma diluted with distilled water was used for the control. ATIII was present at 10.2± 7.3 mg/dl, and α_2MG at 50.3 ± 13.7 mg/dl (mean ± standard deviation) but C1INA was not detected. It was indicated that the chicken AT III had species-specific structure, immunological, functional properties as was shown in those in plasma of most all mammals. α_2MG was proved to have common antigenicity among many mammals because of the presence of cross-reactivity to anti-human α_2MG antibody in rabbit serum. Among three antifibrinolytic factors in the chicken plasma, ATIII and α_2MG were found to have some common immunological features with human factors. The presence and the antigenic specificity of the avian C1INA remained unexplained. α_2PI and ATIII in 26 normal chicken plasma samples were measured by an end-point method using chromogenic substrates, S-2251 and S-2238. The level of α_2PI was found to be 26.7 ±27.3% while that of human α_2PI was 100 ± 17%. Moreover, in 9 out of 26 samples was not it detected. Chicken AT III level was 125 ± 28%, in comparison with the human standard range of 103 ±10%. This method for assaying anti-fibrinolytic factors using chromogenic substrates measures the relative inhibitory effect against human Pln action or Plg activation. It is impossible to quantify the absolute quantity. It was thus demonstrated that chicken plasma contains a synthetic anti-fibrinolytic function approximately equal to that of mammals although inhibitors differed from those of human quantitatively and/or qualitatively.

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