本研究はラットにおける血栓形成に, アラキドン酸代謝系が関与しているか否かを明らかにするため, 合成のトロンボキサン合成酵素阻害剤DP-1904のヘリウム-ネオンレーザー惹起血栓形成に対する抑制効果を検討した. DP-1904は細動脈における血栓形成を用量依存的に阻害した. しかしこの阻害は一過性で, 経口投与6時間後に消失した. 阻害は細静脈では認められなかった. DP-1904の阻害作用は ex vivo において血小板粘着, トロンビンおよびコラーゲン惹起血小板凝集のような血小板機能測定で有意差を認めなかった. 以上の結果から 1) DP-1904は優れた抗血栓薬であること, 2) アラキドン酸代謝系はラットにおける血栓形成に関与していること, その役割は細動脈と細静脈では異なることが示唆された.

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We have purified 7kDa and 18kDa cationic antibacterial proteins (CAP-7 and CAP-18) with LPS-binding and LPS-neutralizing activities from rabbit granulocytes. CAP-7 was identified as the C-terminal 37 amino acid fragment of CAP-18. Synthetic peptide #197-1 (identical sequence to CAP-7, Gly₁-Tyr₃₇) and #36-1 (a truncation of CAP-7 consisting of 32 amino acid residues, Gly₁-Ala₃₂) showed LPS-binding activity. Both peptides inhibited tissue factor- and Xa-induced plasma clotting. Using synthetic chromogenic substrates, both CAP-7 peptides blocked the coagulation cascade at two sites, activation of factor X to Xa and conversion of Factor II (prothrombin) to factor IIa (thrombin). In vivo treatment of peptide #197-1 prevented acute lethality in mice injected with tissue factor. Two other peptides, #32-1 (Gly₁-Phe₉) and #50-2 (Ile₁₃-Tyr₃₇) failed to demonstrate LPS-binding and anticoagulant activities. The active peptides but not the inactive peptides maintain a putative heparin binding domain at their N-termini. This heparin binding domain is participated in the LPS-binding and anticoagulant activites of CAP-7. These active peptides may have therapeutic potential for treatment for DIC due to sepsis and endotoxin shock.

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Microparticles (MP) are known to be released form the platelet membrane in association with platelet activation. They are enriched in binding sites for factor Va. MP generation paralleled the expression of catalytic surface for the prothrombinase enzyme complex. Disseminated intravascular coagulation (DIC) is an acquired disorder in which intravascular coagulation may lead to the formation of microvascular fibrin and hemorrhagic diathesis. We assayed MP by flow cytometric methods in a rabbit DIC model obtained by continuous injection of endotoxin. We found that DIC occured following MP generation, and that there was a correlation between the number of MP and the severity of DIC. These results suggest that MPs play an important role in the occurrence and exacerbation of DIC and may be a useful parameter for the clinical diagnosis of DIC.

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Plasma tissue factor (TF) antigen level and hemostatic markers were measured in 55 patients with disseminated intravascular coagulation (DIC), 15 patients suspected with DIC and 10 patients without DIC. Plasma TF antigen was detected in normal volunteers and it was significantly higher in patients with DIC than in patients suspected with DIC and without DIC. But in some patients with DIC plasma TF antigen level was within normal range. Plasma TF antigen level in patients with DIC significantly decreased after therapy compared with before. And it was not correlated with organ failure and its prognosis. Plasma TF antigen level in patients with DIC was slightly positively correlated with thrombin-antithrombinIII complex but not correlated with other hemostatic markers. In hematopoietic neoplasma with DIC plasma TF antigen level showed a tendency to increase in nonlymphoid leukemia as compared to lymphoid tumor. It was suggested that TF was implicated in occurrence and progression of DIC.

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遺伝子組み換えにより調製した一本鎖組織プラスミノゲン・アクチベーター (sct-PA) および二本鎖t-PA (tct-PA) の乏血小板血漿 (PPP), 多血小板血漿 (PRP) および全血よりクロットを調製する際のクロットに対する結合ならびに予め調製したPPPクロットに対する結合を¹²⁵Iで標識したt-PAを用いて検討した. 50ng/ml以下の酵素濃度では, sct-PAのクロット形成時における結合率はtct-PAにくらべて高かったが, 治療濃度 (1μg/ml) では, 両者は同等の結合率を示した. 予め調製したPPPクロットに対し, 25～1,000ng/mlにおいて, sct-PAはtct-PAより高い結合率を示した. 両t-PAのフィブリン分解およびフィブリノゲン分解活性は, 精製系およびPPP中において検討した. 精製系において, 生理的濃度のフィブリン存在下でのsct-PAによるプラスミノゲン活性化は, tct-PAにくらべて少なかった. PPP中においては, sct-PAのフィブリン分解活性はtct-PAにくらべて高かったが, PPP中のα₂-アンチプラスミン, プラスミノゲン及びフィブリノゲンの減少は, tct-PAにくらべsct-PAで軽度であった. 以上の結果より, sct-PAはtct-PAにくらべクロットに選択的であることが示唆された.

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The role of cAMP/cAMP dependent protein kinase (A-kinase) system in the regulation of hepatic tissue-type plasminogen activator (h-tPA) production was studied by use of primary cultured rat hepatocytes. Cholera toxin (CT) which is known to elevate intracellular cAMP concentration, augmented h-tPA production in a dosage-dependent manner. Hepatic-tPA mRNA expression was also augmented to 220% of the control by 10^-7M CT. A protein kinase inhibitor H-8, which preferably inhibits A-kinase, lowered the level augmented by CT to the control level, and actinomycin D was completely inhibited h-tPA expression. Cycloheximide, a protein synthesis inhibitor, brought marked accumulation of h-tPA mRNA rather than inhibition of the h-tPA expression. These data showed that the de novo protein synthesis is not necessary for the augmentation mechanism, and suggest that the existence of a repressor protein for h-tPA gene.

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In our previous study, we have reported the changes of plasma and urinary coagulation-fibrinolysis and kallikrein-kinin systems during normal pregnancy, labor and puerperium, and those in preeclampsia suggesting that preeclampsia is a state of chronic DIC pattern.
The purpose of this study is to investigate further clinical significance of coagulation-fibrinolysis in plasma during normal pregnancy and in the utero-placental circulation in severe cases of preeclampsia especially on tPA, inhibitior of tPA (active PAI-1) and the formation of complex of tPA with the active PAI-1 (tPA/PAI/C), complex of antiplasmin with plasmin (PIC) and a specific peptide of fragment resulting from the degradation of cross-linked fibrin (D-dimer).
In the peripheral venous blood of patients with severe preeclampsia, the levels of all parameters such as plasma thrombin/antithrombin-III complex (TAT) 10.9±1.69ng/ml M±SE n=24 P<0.02, fibrinopeptid A (FPA) 4.7±0.57ng/ml M±SE n=22 P<0.01, tPA 6.1±1.01ng/ml n=10 P<0.01, Bβ15-42 23.0±1.78ng/ml n=25 P<0.001, tPA/PAI/C80.1±10.21ng/ml n=24 P<0.05, active PAI 407.4±33.9ng/ml n=24 P<0.05, PIC 0.49±0.085μg/ml n=24 P<0.001 and D-dimer 435.1±51.3ng/ml n=8 P<0.001 were significantly increased in severe preeclampsia as compared with those of 20 normal control gravidas of 28 to 42 weeks of gestation (TAT 6.1±0.44ng/ml, FPA 2.6±0.24ng/ml, tPA 3.6±0.34ng/ml, Bβ 9.3±0.82ng/ml, tPA/PAI/C 57.94±6.88ng/ml, active PAI 304.2±33.23ng/ml, PIC 0.49±0.053μg/ml, D-dimer 282.9±16.83ng/ml).
These changes of plasma coagulation-fibrinolysis system show that patients with severe preeclampsia are in a state of chronic DIC pattern with marked changes of fibrinolytic system.
In the uterine venous blood (UVB) of severely preeclamptic patients, levels of TAT 43.0±15.78ng/ml n=6M±SE P<0.001, tPA/PAI/C 199.75±30.21ng/ml P<0.001 and PIC 2.12±0.33μg/ml P<0.001 were markedly increased, but active PAI 203.98±56.57ng/ml P<0.01 was markedly decreased as compared with those in peripheral venous blood (PVB) TAT 10.9±1.69ng/ml, tPA/PAI/C 85.07±10.28ng/ml, PIC 0.84±0.09μg/ml, active PAI 407.41±33.89ng/ml n=20.
These findings suggest that fibrin deposition and secondary fibrinolysis in the uteroplacental circulation are locally dominant.

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