t-PA has been well known to show thrombolytic activity mediated through the activation of plasminogen. The activation is markedly stimulated in the presence of fibrin. Although t-PA tightly binds to fibrin, it has not been clarified how long the activity of the binding t-PA to thrombus is sustained in the physiological condition. Therefore, we examined the correlation between the binding amount of t-PA to thrombus and the duration of the thrombolytic activity induced by only the binding t-PA in vivo. After human plasma clots were immersed in human plasma containing various concentration of t-PA or LUK for 1min, the clots were washed with saline and immersed in the plasma without t-PA or LUK. The clots pretreated with t-PA were lysed for 3 hours continuously and the clot lysis is clearly correlated with the binding amount of t-PA to clot. On the other hand, the clots pretreated with LUK lacking in fibrin affinity were never lysed significantly. When the rabbit with pulmonary embolism was injected with t-PA (300, 000IU/kg i. v.), the circulating t-PA was quickly disappeared and was detected only slight (17±10IU/ml) after 30min. However, the degree of thrombolysis was increased for 120min after the injection of t-PA (17±3.5% at 30min; 31±3.2% at 120min). These results indicate that the binding t-PA not only to plasma clots but also to thrombi is active and the thrombi bound with t-PA continue lysing at least for several hours even in the absence of free form of t-PA in plasma.
We have investigated the difference of the coagulo-fibrinolytic factors between 6 patients of thrombotic thrombocytopenic purpura (TTP) and 27 patients of disseminated intravascular coagulation (DIC) using some hemostatic molecular markers. The changes of coagulo-fibrinolytic factors containg a high level of fibrin-fibrinogen degradation products (FDP) were small in TTP patients. Though we found a significant increase of D-dimer FDP level and thrombin-antithrombin III complex (TAT) level except normal plasmin-α2 plasmin inhibitor complex (PIC) level in TTP patients, these increases were much smaller than those of DIC patients. The tissue-type plasminogen activator (t-PA) level was equally high against normal level of urinary-type plasminogen activator (u-PA) in both patients with TTP and DIC, and there were three patients whose plasminogen activator inhibitor type-1 (PAI-1) was much higher than those of DIC patients. We found that the increase of hemostatic molecular makers (D-dimer FDP, TAT, PIC) in TTP patients was much smaller than in DIC patients.
The effects of endothelin (ET-1) on cultured human umbilical vein endothelial cells (HUVEC) and platelet aggregation were investigated with its mode of action. Platelet aggregation (PRP: 30×104 platelets/μl) induced by ADP (2×10-6M) was measured using aggregometer. PGI2, TXB2 and cAMP concentration ([cAMP]i) were assayed by radioimmunoassay. And cytosolic free Ca++ concentration ([Ca++]i) was measured using Fura-2/AM. The results were as follows: The PGI2 generation was enhanced by ET-1 (2×10-7, 5×10-8, 5×10-9M) without change of [cAMP]i or [Ca++]i, and when HUVEC were pretreated with acetylsalicylic acid (ASA), its enhancement was completely inhibited, although the inhibited platelet aggregation by addition of conditioned media of HUVEC incubated with ET-1 was not completely abolished. The addition of conditioned media of HUVEC incubated with ET-1 decreased platelet aggregation and TXB2 generation, but increased [cAMP]i, however the addition of ET-1 alone had no effect on them. These decreased TXB2 generation and increased [cAMP]i in the platelet were suppressed by the pretreatment of HUVEC with ASA. These results suggested that ET-1 attenuated platelet aggregation through its decreased TXB2 generation and increased [cAMP]i via the increased release of PGI2 and the other mediators from HUVEC.
The role of Ca++ mobilization from storage sites on the prostacyclin (PGI2) generation was investigated utilizing saponized human umbilical vein endothelial cells (HUVEC). Bradykinin and thrombin increased PGI2 generation, Inositol 1, 4, 5-trisphosphate (IP3) formation and cytosolic free Ca++ concentration ([Ca++]i). And these increases induced by bradykinin or thrombin were not observed in Ca++ free solution, while their basal levels were maintained (Fig. 4, 6, 7). IP3 increased 45Ca release from the storage sites in saponized HUVEC, and it was 35.7% of Ca ionophore releasable 45Ca. Its increase was not influenced by the pretreatment of antimycin and oligomycin (Fig. 8). Through these results it was concluded that; 1) The extracellular Ca++ was not required for the maintenance of basal PGI2 generation. 2) The enhancement of PGI2 generation induced by bradykinin or thrombin was brought about from the increase of [Ca++]i, which was induced by the enhanced Ca++ influx from the extracellular space and by the Ca++ release from the storage sites triggered by IP3 generation. 3) The first direct evidence was shown that IP3 enhanced Ca++ release from the non-mitochondrial storage sites in HUVEC, and the amount was 35.7% of Ca ionophore releasable Ca++.
Protein S is a vitamin K-dependent plasma protein which acts as a cofactor for an anticoagulant, activated protein C. We observed a patient with recurrent deep vein thrombosis complicated by fever, central nervus system involvement and acute abdomen. This case, a 17-year-old Japanese male, was hospitalized on 13-year-old at the first time. He complained fever, chest pain, and convulsion of unknown origin. Laboratory studies showed an elevated erythrocyte sedimentation rate and an elevated C-reactive protein. He had a meningismus and a severe abdominal pain (acute abdomen), but no inflammatory change. A phleborreogram showed evidence of left femoral deep venous thrombosis. He had recurrent thrombosis and familial thrombosis. The family analysis revealed that the reduced levels of protein S activity, total and free forms of protein S antigen and plasminogen activity in his father and himself. This case suggests that patients with a protein S deficiency associated with dysplasminogenemia may have various clinical features.
Since the fisrt report by Oeri et al. in 1954, approximately 30 cases including 4 our cases of congenital combined deficiency of factor V and factor VIII have been reported in literarures. This combined deficiency is extremely rare and its pathogenesis is still unclear. We report here the changes of coagulation parameters during pregnancy in a patient from first trimester until successful vaginal parturition. The case was 26-year-old woman. Factor VIII, facotr VIII: Ag and vWF: Ag were increased during pregnancy. The level of factor V in non-pregnant period was 6% of normal. Although the level of factor V did not rise until 42 weeks of pregnancy, during 2 days before parturition, the patient showed marked elevation of factor V from 6% to 20% that lasted until 2 days after parturition. This is the first observation that the level of factor V increased before parturition not only in patients with combined deficiency of factor V and factor VIII or deficiency of factor V, but in normal women.
A forty-six year old female with severe mitral valve stenosis complicated by thrombocytopenia and a positive test for lupus anticoagulant was reported. Ten years ago, she was diagnosed to have thrombocytopenia when she received a mastectomy. She had a history of total of 6 spontaneous abortions, but no history of thromboembolic episodes. She received a steroid hormone therapy for thrombocytopenia for 5 years, but thrombocyte count did't show an increase. A year ago, she suffered from a congestive heart failure. At a local hospital, digitalis and a diuretics were given to her, but her condition remained unimproved. She was referred to our hospital. On phisical examination, a diastolic rumble and an opening snap were audible at the apex of the heart. A hepatosplenomegaly was noted. Laboratory tests revealed a thrombocytopenia with the count of 58, 000/mm3, and the prolongation of APTT as well as bleeding time. Radioisotopic examination employing111 In revealed a platelet life span of 4.6 days. A serum β-TG and a serum PF-4 levels were markedly increased. The serum anticardiolipin antibody titre level was significantly high. The serum mixing test between the sera from this patient and the sera from healthy persons showed a marked prolongation of APTT. A cardiac catheterization revealed severe mitral valve stenosis with a transvalvular pressure gradient of 25.2mmHg, and a pulmonary arterial pressure of 68/32mmHg. Since the risk for an open-heart surgery was too high in a patient with coagulopathy and thrombocytopenia, the treatment of choice seemed to be a percutaneous transvenous mitral commissurotomy (PTMC). In our case, the transmitral pressure gradient dropped from 25.2 to 12.9mmHg after PTMC. She was able to go back to her daily life.