Two types of congenital dysfibrinogenemias, designated as fbrinogens Hamasaka and Tokyo III, with impaired release of fibrinopeptide A (FPA) were found in a 3-year-old girl and a 63-year-old man, respectively. They were both heterozygotes for abnormality and had been asymptomatic. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fibrinogen Hamasaka displayed delayed release of FPA by thrombin and defective release by Ancrod, a thrombin-like snake venom enzyme derived from Agkistrodon rhodostoma (Fig. 1). There were two species of FPA, normal and abnormal, and the abnormal FPA was eluted earlier than its normal counterpart in only half the normal amount on reverse phase high-performance liquid chromatography (Fig. 2). From fibrinogen Tokyo III, no aberrant FPA was released by thrombin or Ancrod (Fig. 3). In these abnormal fibrinogens, polymerization of fibrin monomers was not affected (Fig. 4). Reduced and S-carboxymethylated fibrinogen Tokyo III showed the broad Aα chain on SDS-PAGE, suggesting the presence of an excess cysteine residue in the aberrant Aα chain (Fig. 5). Direct amino acid sequencing of the abnormal FPA or Aα chain revealed substitutions of Aα arginine-16 to histidine in fibrinogen Hamasaka and to cysteine in fibrinogen Tokyo III. These rather common mutations are accounted for by a possible single base exchange compatible with the C to T hot spot mutation theory, either in the antisense strand for the former or in the sense strand for the latter.

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Effects of X-ray irradiation to the liver were studied in rabbits with special reference to the free radicals induced by the irradiation at various doses of 5, 10, 20 and 40Gy. Results were summarized as follows:
1) APTT and PT significantly prolonged immediately after irradiation and on day 1, and recovered thereafter to the control levels.
2) Levels of blood coagulation factors I, II and plasminogen, t-PA and plasma prekallikrein showed similar trends, namely, an initial rapid decrease immediately after irradiation, followed by a transient increase on day 3 and a subsequent recovery to the control level on days 7 and 10.
These changes coincided with the prolongation of APTT and PT.
3) The SOD activity and the plasma albumin level also decreased immediately after irradiation, recovered once from 3 to 7 days and decreased again on days 10.
The decreased SOD activity noted immediately after irradiation indicates production of free radiacals induced by the irradiation. This finding may suggest that the free radicals thus produced may have perturbed blood coagulation and fibrinolysis, at least in part, by blocking the secretion of relevant proteins from the hepatocytes.

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In order to reduce risk factors in the treatment of hemophilia B with prothrombin complex concentrates, a new coagulation factor IX concentrate (GA-1013) was developed by Green Cross Corporation, Osaka, Japan. This product is prepared by immunoaffinity chromatography using a monoclonal antibody (3A6) and virally inactivated by solvent/detergent treatment. GA-1013 has a specific activity of 173 factor IX units/mg of protein, representing a 13, 700 fold purification from cryosupernatant.
A multicenter clinical study was conducted in 24 previously treated and 1 previously untreated hemophilia B patients with no inhibitor to evaluate the safety and efficacy of GA-1013. We determined the half life and in vivo recovery of this FIX concentrate.
The mean age of the patients was 29.0 years with a range of 9 months 62 years. The mean T1/2 value (n=22) was 20.3±8.93hr and the mean in vivo recovery rate was 69.0±20.12% calculated from the labelled FIX: C content (47.6±14.46% calculated from the actual FIX: C content).
A total of one hundred and seventy-six bleeding episodes were assessed during the 6 months treatment, chiefly on a demand basis. Hemostatic effectiveness was confirmed with an efficacy rate of 93.7% (165/176). No adverse reactions attributed to GA-1013 were obserbed in any infusion. Neither FIX inhibitors nor antibodies to mouse or goat protein were detected. No significant changes attributed to GA-1013 were seen in vital signs or in laboratory findings including CBC, blood chemistries, virological parameters and immunological data (CD4 and CD8 counts, lymphocyte blastogenesis index and serum immunoglobulins at 1, 3 and 6 months).
These results suggest that monoclonal antibody-purified coagulation FIX concentrate (GA-1013) is safe and efficacious as replacement therapy for hemophilia B.

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Effects of calpain-I purified from human erythrocytes on phosphatidylinositol hydrolysis activity of phospholipase C (PI-PLC) were investigated by using human platelet lysate and the partial purified enzymes. PI-PLC activity in human platelet lysate prepared in the absence of thiolprotease inhibitors (leupeptin and EST) was higher than that prepared in the presence of inhibitors. This result implied that PI-PLC was activated by thiolprotease in platelets during preparation of platelet lysate. When the thiolprotease inhibitors were removed by gel filtration from platelet lysate prepared in the presence of the inhibitors and calpain-I was added into the preparation, the PI-PLC activity was enhanced in correlation with the amount of calpain-I. PI-PLC in platelets lysate was partially purified by phenyl-Sepharose and Ultrogel AcA22 column chromatography. PI-PLC was separated to two isozymes by phenyl-Sepharose chromatography. The apparent molecular weight of these isozymes was about 200kDa in AcA22 gel filtrations. One of the PI-PLC, which was adsorbed with phenyl-Sepharose column and eluted with the buffer containing 50% ethyleneglycol, was not influenced by the preincubation with calpain-I, but the activity of the other one, which was not adsorbed with phenyl-Sepharose column, was enhanced three times by the preincubation with calpain-I. These results indicate that two isozymes of PI-PLC are located in platelets and one of the enzymes is activated by calpain-I.

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Effect of endothelin on platelets was studied. Affinity labeling of bovine platelet membrane with 125I-endothelin-1 by a cross-linking reagent followed by SDS-polyacrylamide gel electrophoresis and autoradiography revealed the existence of a speciffic binding site with apparent molecular mass of 37kDa on platelet membrane. When added to human platelet-rich plasma, endothelin showed a potentiating effect on the second wave of ADP-induced platelet aggregation, though it alone had no effect. This effect on ADP-induced aggregation was abolished by the treatment of platelets with aspirin, suggesting the involvement of arachidonic acid metabolism in the signal transduction elicited by endothelin.

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We have established a solid phase binding assay using biotinylated fibrinogen as a ligand. Biotinylation did not alter its binding capacity and affinity to solid phase GPIIb/IIIa. Biotinylated fibrinogen binding, as measured by color development of enzymatic reaction, was specific, time-dependent, reversible, saturable and divalent cation-dependent. Unlabeled fibrinogen and three peptide fragments of fibrinogen, RGDV, RGDS and HHLGGAKQAGDV (gamma-chain 400-411) competitively inhibited the binding with IC₅₀ values of 3.7, 49, 155 and 715nM, respectively. The apparent dissociation constant (Kd) of biotinylated fibrinogen was determined to be 0.33nM.
This simple and high-flux solid phase binding assay can be useful to directly detect the binding between fibrinogen and GPIIb/IIIa.

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Fifty six year old male presented with a sudden, tender swelling of his right leg. A diagnosis of femoral vein thrombosis was made because of typical purple skin color and palpable thrombosed femoral vein with intact arterial flow and then anticoagulant and fibrinolytic therapy was initiated. Subsequently, he suffered from disorientation, spiking fever, thrombocytopenia, hemolytic anemia and renal failure, all of which were compatible with thrombotic thrombocytopenic purpura (TTP). As the swelling of the leg subsided, the entire skin of the leg became gangrenous in spite of the satistactory arterial circulation, which necessitated above knee amputation. Medical treatment consisted of corticosteroid, protease inhibitor and gamma globlin resulted in rapid improvement in all the clinical manifestations of TTP. Histological examination of the amputated of leg revealed the presence of multiple thrombotic occlusion of arterioles in calf muscles, which is compatible with changes in TTP. In this case, TTP was more likely triggered by the deep vein thrombosis, which has not been reported before as the underlying disease of TTP. Arteriole thrombosis was formed because of the stagnant venous drainage, which is also rare sequelae of TTP.

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