The research referring to leukocyte cathepsin G (chymotrypsin like proteinase, CLP) is not sufficient compared with that of leukocyte elastase (ELP), and consequently the physiological role is not evident. In order to investigate the physiological role of CLP we devised a simple purification method which is able to separate CLP from ELP. CLP was purified with an affinity column, Suc-L-Tyr-D-Leu-D-Val-pNA-sepharose. CLP in leukocyte extract adsorbed to the column at low concentration of NaCl (0.2M), and was eluted with tris-HCl buffer (0.1M, pH 7.5) containing 2M NaCl. Purified CLP preparation contained no ELP activity. Although the proteolytic activity of CLP against fibrinogen and fibrin was very weak compared with those of ELP, CLP acted synergistically with ELP in the fibrinogenolysis. Furthermore the effect was dependent on the amount of CLP. Inhibitory effect of each eglin c fragments for CLP and ELP was different. Ki values of H-(41-49)-OMe, fragment containing active center of eglin c, were 4×10-5M for CLP and >2×10-3M for ELP. On the other hand, eglin c and H-(8-70)-OMe inhibited CLP and ELP at low concentration. Although the physiological role of CLP is unclear, it was suggested that the proteolysis of CLP might be shown under coexistence of ELP.
Properties of factor VIII (FVIII) in recombinant FVIII concentrates (rFVIII, BAY w 6240) were investigated using 6 kinds of monoclonal antibodies to FVIII. The FVIII: C levels obtained by the one-stage method using severe hemophilia A plasma as a substrate were identical with those indicated on the label (100u/ml). The FVIII: C levels obtained by the chromogenic method were relatively higher than those by the one-stage method. The FVIII: Ag levels assayed by polyclonal ELISA agreed with those by NMC-VIII/1 (recognizes the 80KDa fragment of L-chain) or NMC-VIII/5 (the 70KDa of L-chain) monoclonal ELISA, whereas the FVIII: Ag levels by C 5 (the 54KDa of H-chain) monoclonal ELISA were 2.5-3.0 times higher than those by other monoclonal ELISAs. The ratio between FVIII: Ag and FVIII: C was 1.4. It was considered that FVIII in the recombinant concentrates lost its activity less than FVIII in the heat-treated concentrates commercially available. No von Willebrand factor was detectable by the sandwich ELISA system. SDS-PAGE pattern showed almost no contamination except albumin as a stabilizer. Immunoblot analysis revealed that FVIII in recombinant concentrates could react not only with monoclonal antibodies to L-chain and H-chain, but also with monoclonal antibodies to middle portion of FVIII molecule. These results suggest that the fundamental structure of FVIII molecule for coagulant activity is preserved in the rFVIII concentrates, and that it is expected to be useful for the treatment of hemophiliacs.
We evaluated a method for assessing fibrinogenolysis in which Bβ 1-42 peptide (Bβ 1-42) was measured by enzyme immunoassay using a monoclonal antibody against Bβ 1-42. Bβ 1-42 obtained from plasmic-digestion of fibriongen and des A fibrinmonomer in vitro specifically reacted in this assay. Smaller peptides from Bβ 1-42 and subjected to further digestion lost their reactivities. However all peptide fractions obtained by serial plasmicdigestion of crosslinked fibrin showed little crossreactivities. Also no reaction was observed with purified Bβ 15-42 peptide in this assay. Plasma Bβ 1-42 levels in 14 normal subjects were below 6pmol/ml. In randomly selected samples from 58 patients with accelerated fibrinolysis, the Bβ 1-42 levels widely varied from 2 to 234pmol/ml, but 27 of them showed considerably high levels. In these samples, Bβ 1-42 levels showed no correlations with fibrinogen, fibrinopeptide A levels, antithrombin III, plasminogen or α2PI activity. However Bβ 1-42 levels correlated with FDP-E (r=0.33, p<0.025), plasmin-α2PI complex (r=0.53, p<0.025) and Bβ 1-42 by radioimmunoassy (r=0.55, p<0.025). Bβ 1-42 did not correlate with D dimer levels (r=0.30). In conclusion, Bβ 1-42 assay reflected only in vitro fibrinogenolysis.
To study the coagulation and fibrinolytic state in lung cancer, molecular markers for coagulation and fibrinolysis, i. e. plasma fibrinopeptide A (FPA), fibrinopeptide Bβ1-42 (Bβ1-42), fibrinopeptide Bβ15-42 (Bβ15-42), tissue-type plasminogen activator antigen (t-PA antigen) and activity (t-PA activity), plasminogen activator inhibitor (PAI), D-dimer, plasminogen, α2-plasmin inhivitor (α2PI) α2PI-plasmin-complex (PIC), thrombin-antithronbinIII-complex (TAT), ATIII and serum FDP-E levels were assayed in 31 patients with lung cancer. And we also discussed the pathophysiology of coagulation and fibrinolysis according to the histological classification and the clinical staging of lung cancer. All these molecular markers except PAI changed significantly compared with the value in normal subjects. As for coagulation, hypercoagulable state was observed because both FPA and TAT increased and showed positive correlation each other. As for fibrinolytic state, increase of fibrinolysis was observed because of increment of PIC, D-dimer, FDP-E, Bβ1-42, Bβ15-42, t-PA antigen, and activity levels and decrease in plasminogen and α2PI levels. In addition, these changes might be induced through secondary fibrinolysis because the levels of D-dimer and FDP-E were well correlated with the levels of TAT. But compared with the changes in lung cancers with DIC, the grade of these changes were slight. There was no difference of molecular marker levels among the histological classifications and the clinical stagings. These results indicated that these patients with lung cancer might be at the risk of thromboembolic complication because of dynamic changes of coagulation and fibrinolysis.
In order to evaluate the antithrombotic effect of Danazol, we investigated serial changes in plasma levels of the blood coagulation, fibrinolysis and platelet factors in 4 patients with endometriosis, and we tried a long-term Danazol treatment in a patient with chronic consumption coagulopathy associated with dissecting aortic aneurysm. Clear increases in plasma levels of the regulatory factors for blood coagulation system including antithrombin III (AT III), heparin cofactor II (HC II), protein C (PC) and free protein S (PS) were observed in response to Danazol. Of the promoting factors for blood coagulation system, both factor II and X levels were significantly increased, while fibrinogen decreased significantly. Only plasminogen levels were significantly increased in the fibrinolytic factors including tissue-type plasminogen activator, α2-plasmin inhibitor and plasminogen activator inhibitor 1. No consistent alterations were observed in the platelet factors including platelet counts, ADP-induced platelet aggregability, von Willebrand factor and thromboxane B2 production. Plasma levels of FDP, fibrinopeptide A, fibrinopeptide Bβ15-42 and β-thromboglobulin were gradually decreased, and gradual increases of platelet counts were seen without any thrombo-hemorrhagic phenomena in a patient with chronic consumption coagulopathy during Danazol administration. These results imply that Danazol has a distinct anticoagulant effect chiefly by increasing plasma levels of the potent regulatory factors for blood coagulation system such as AT III, HC II, PC and PS, and would provide a useful adjunct to anticoagulant treatment in congenital deficiency of each regulatory factor for blood coagulation system, plasminogen deficiency and chronic consumption coagulopathy.
It has been reported that many parameters relating to hemostasi, coagulation and fibrinolysis vary by amounts of exercise given. We studied effects on platelet aggregation of abrupt physical exercises given to healthy individuals. A total of twenty healthy adults, 11 males and 9 females (ages 19-33, average 24.7) were subjected 2.6 kilometers of exercise jog and walk, and we determined their platelet counts in peripheral blood, amounts of plasma cetecholamines, apolipoproteins, β-thromboglobulin (β-TG) and thromboxane B2 (TxB2). The values before and after the exercise were compared, where platelet counts showed an increase (p<0.01), and cate-cholomines (p<0.01), apolipoproteines (p<0.05) and β-TG (p<0.01) revealed a rise in their blood concertrations. On the contrary, TxB2 tend to decrease after the exercise. Exercise-induced change in platelet aggregability was variable. Furthermore, concertrations of β-TG and TxB2 in platelet rich plasma (PRP) stimulated with a low concentration of collagen (0.5μg/ml) increased after the exercise. Accordingly, abrupt exercises given to healthy individuals cause no marked platelet activation in vivo, but they make platelets more susceptible to activating agents in vitro.
Three cases in a family of hereditary thrombocytopenic purpura are reported. Each case presented as an idiopathic thrombocytopenic purpura (ITP)-like syndrome. Careful family history revealed that some other members had similar symptom, and the mode of inheritance was autosomal dominant (Fig. 1). The platelet counts of the three family members ranged from 0.8×104/μl to 8.0×104/μl. The major platelet membrane glycoprotein was normally represented in case 3 (Fig. 2). All patients revealed increased PAIgG and PBIgG and slightly increased platelet volume (Table 1). The autoantibodies of the three patients did not recognize same antigen (Fig. 3). Aggregation by ADP, collagen and ristocetin was all normal in case 3 (Fig. 4). Cases 1 and 3 exhibited normal vWF: Ag (Fig. 5). These results suggest that the thrombocytopenia in this family is related to an immunological mechanism.
Bernard-Soulier syndrome (BSS), a function disorder of platelet with autosomal recessive inheritance, is characterized by relatively severe bleeding tendency, giant platelets accompanied by the deficiency of membrane glycoprotein Ib (GPIb) and thrombocytopenia. GPIb is thougt to play an important role in the process of thrombus formation through its adhesive activity to subendothelial tissue, interacting with von Willebrand factor (vWF). BSS platelets fail to aggregate in response to ristocetin despite normal content of factor VIII/vWF in plasma. In this paper, we report clinical and hematological data of a 5 year old homozygous female BSS case and her heterozygous parents. Laboratory examinations revealed moderately decreased amount of vWF antigen and molecular abnormality examined by the crossed immunoelectrophoresis and the SDS agarose gel electrophoresis in proband and her mother. The results of SDS polyacrylamide gel electrophoresis/PAS stain and immunomagnetic platelet separation with monoclonal antibody to GPIb/GPIIbIIIa showed a lack of GPIb in proband and complicated abnormalities of GPIIb/IIIa complex in her parents. Intracellular Ca2+ mobilization under the stimulation of thrombin showed markedly decreased levels in proband and about half amount of normal in her parents.