Thrombin inhibitors. 2. Amide derivatives of N alpha-substituted L-arginine.

A series of N alpha-(arylsulfonyl)-L-arginine amide derivatives with substituted or unsubstituted naphthalene and heterocyclic compounds as the N alpha-substituent was prepared and tested as inhibitors of the clotting activity of thrombin. N-n-Butyl and N-n-butyl-N-methyl derivatives of N alpha-dansyl-L-arginine amide were the most inhibitory of N-alkyl and N,N-dialkyl derivatives of N alpha-dansyl-L-arginine amide. Their inhibitory effect was as potent as that of N alpha-dansyl-L-arginine-n-butyl ester with an I50 of 2 X 10(-6) M. N alpha-Substituted naphtalenesulfonyl-L-arginine amide derivatives of 4-methyl- and 4-ethylpiperidine also showed a potent inhibition with an I50 of 10(-7) to 10(-6) M. The most potent inhibitior in this study was 1-[N alpha-(4,6-dimethoxynaphthalene-2-sulfonyl)-arginyl]-4-methylpiperidine, with an I50 of 7.5 X 10(-8) M. Arginine amide derivatives of 4-methyl- or 4-ethylpiperidine with tetralin or an oxygen-containing heterocyclic compound as a N alpha-substituent showed an inhibition with an I50 less than 10(-5) M. N-Monosubstituted derivatives of N alpha-dansyl-L-arginine amide were not hydrolyzed at all by thrombin and were hydrolyzed very slowly by trypsin, and N,N-disubstituted derivatives were not hydrolyzed at all by both enzymes.

Alterations in vascular endothelial cell-related plasma proteins in thalassaemic patients and their correlation with clinical symptoms.

An increased level of plasma thrombomodulin (TM) in alpha- and beta-thalassaemia was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Nonsplenectomized patients with beta-thalassaemia/haemoglobin E (BE) had higher levels of TM than splenectomized cases (BE-S). Patients with leg ulcers (BE-LU) were found to have the highest increase in TM level. Appearance of larger platelets in all types of thalassaemic blood was observed indicating an increase in the number of younger platelets. These data indicate that injury of vascular endothelial cells is present in thalassaemic patients.

Enhancement of collagen-induced aggregation of platelets in whole blood.

In order to elucidate the features of platelet aggregation in whole blood, studies were carried out on human blood. The platelet aggregation reaction was monitored by counting the residual free platelet number with an electronic particle counter (Coulter). Platelets in citrated whole blood were aggregated by a very small amount of collagen which did not aggregate platelets in citrated plasma. Such enhancement was not observed if ADP or epinephrine was used. By addition of isolated erythrocytes to platelet rich plasma, enhancement of the platelet response to collagen was obtained. The erythrocyte membrane stabilizer, Dilazep, abolished the enhancement effect of erythrocytes. Those results indicated that erythrocytes enhanced the platelet response to collagen. Although participation of ADP from the erythrocytes in the enhancement was suggested, the results of ADP determinations on suspensions of erythrocytes indicated that other factors of the erythrocytes might be involved in the enhancement.

An evaluation of prothrombin assay method using MD805 by means of warfarin-treated plasma.

The prothrombin assay method using the synthetic thrombin-inhibitor MD805 was standardized by fixing the concentrations of MD805 and S-2238 through their extinction coefficients (epsilon 333 for MD805 and epsilon 316 for S-2238). The prothrombin assay was directly proportional to the concentration of plasma up to 200% of the normal level and was not significantly influenced by the variety of three kinds of commercially available tissue thromboplastin preparations. Using plasma from Warfarin-treated patients and healthy volunteers, the correlation was studied between the prothrombin assay and the conventional coagulation tests such as Prothrombin time (INR), Thrombotest and Hepaplastintest (Normotest), and the correlation coefficients of -0.85, 0.81 and 0.94 were obtained respectively. FUT-175 and MD805 in the test plasma hardly affected the prothrombin assay in the concentration ranges which affected remarkably the conventional coagulation tests. These results indicated that the prothrombin assay was useful for monitoring the hyper- or hypoprothrombin state even on anticoagulant therapy. Eighteen healthy volunteers at 18 to 20 years old showed the mean and standard deviation of 0.96 +/- 0.097.

Plasminogen activator activity of cultured endothelial cells derived from canine coronary vessel and human umbilical artery and vein.

The present study was undertaken to determine which cells participate in plasminogen activator (PA) release from the vascular wall. Canine coronary vessel was confirmed to release PA when various agents were administered in perfusion experiments. Endothelial cells from the coronary vessel were then isolated and cultured for 2 days. PA activity was observed in lysates of the cultured cells, and the medium used for the cultivation was found to contain little PA activity. This suggests that the endothelial cells participate in PA release from the vascular wall. In addition, the PA synthesis was also studied in cultured endothelial cells from human umbilical artery and vein. Both contained little PA activity, indicating that vascular endothelial cells may vary in PA synthesis and release according to the vessel.

Prostacyclin release from the coronary vascular wall by vasoactive substances.

Which vasoactive substances that are synthesized in vivo could induce the release of a sufficient amount of prostacyclin (PGI2) to inhibit platelet aggregation from the vascular wall was investigated in the isolated dog heart perfused by a modified method of Langendorff. Infusion of 5 microM bradykinin or 25 u/ml crude thrombin into the heart for 30 sec resulted in the transient appearance of inhibitory activity of platelet aggregation. The inhibitory activity was stable at alkaline pH but unstable at acidic pH and thermolabile. The appearance of the inhibitory activity was prevented by treatment of the coronary vessel with 30 microM indomethacin or 1 mM tranylcypromine. These results indicated that the inhibitory activity was caused by PGI2. When 25 microM acetylcholine, 25 microM noradrenaline, 25 microM isoproterenol, 10 microM adenosine triphosphate (ATP), 5 microM adenosine, 1 microM angiotensin II, 25 microM histamine or 1 microM serotonin was infused for 30 sec, no inhibitory activity of platelet aggregation was observed. Bradykinin (5 X 10(-9) approximately 5 X 10(-6) M) and purified thrombin (1 X 10(-9) approximately 1 X 10(-7) M) induced a dose-dependent release of PGI2 which was assayed using a radioimmunoassay for 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha).

Intravenous injection of sonicated blood induces pulmonary microthromboembolism in rabbits with ligation of the splenic artery.

Pulmonary thromboembolism (PTE) is found in long hospitalized patients. Chronic PTE has been reported to play an important role in cardiac failure in thalassemic patients after splenectomy. However, the mechanism of PTE in these patients remains unclear. In this study, we attempted to establish an animal model of PTE. We divided New Zealand white rabbits into three groups: Group I was injected sonicated blood, II was injected non-sonicated blood after ligation of the splenic artery, and III was injected sonicated blood after ligation of the splenic artery. After injection of the sonicated blood, we examined the platelet counts every 10 minutes until 1 hour and the rabbits were sacrificed for histological examination. Platelets significantly decreased in number immediately after the injection of sonicated blood in Groups I and III. Many pulmonary thromboemboli composed mainly of platelets were found in Group III but not in other groups. These pathological changes seem to be partly similar to those of thalassemic patients after splenectomy. This animal model is thought to be useful to study the pathogenesis of pulmonary thromboembolism, especially in thalassemic patients after splenectomy.

Increase in factor VIII clotting activity in the perfusate of isolated dog hind leg and heart by components of kallikrein-kinin system.

Transient increase in Factor VIII clotting (F.VIIIc) activity was observed by infusion of kallikrein or bradykinin (BK) to the isolated dog leg or heart preparation which was perfused with physiological solution, but the increase in Factor IX activity was not observed, suggesting that the increase in F.VIIIc activity was not due to contaminated plasma components of the perfusate. The increase coincided with the increase in prostacyclin (PGI2) which was determined by immunoassay of 6-keto-PGF1 alpha and by platelet aggregation inhibiting activity. Although increase in PGI2 by BK was inhibited by the pretreatment of the preparations with indomethacin or tranylcypromine, the increase in F.VIIIc activity was not inhibited, suggesting that the increase in F.VIIIc activity was not mediated by PGI2.

Hematology laboratory standardization: a plan for harmonization in Asia.

Hematology laboratory is generally required in the hospital. At the macroscale, hematology laboratories have served a large number of population. In Asia, more than 3,000 million people are potentially to use the hematology laboratory service, particularly the complete blood count. Since 1970s, automated technology has been introduced to Asia and as years passed by, technology diversity is increasing. However, there are considerable number of hematology laboratories that have no automated machine. They are still relied on manual technology which is still variable in spectrophotometer for hemoglobin determination, centrifuge for hematocrit and diluting pipet for cell counting. In particular, blood smear preparation and interpretation are very difficult to control for standardization from person to person and laboratory to laboratory. Different methodology and a large population in the huge geographical area in Asia, the agreement of standard criteria is greatly important. This report has shown strategy and action plan to reach the goal of hematology laboratory standardization in Asia.

A simple and quantitative method for the determination of tissue factor.

Tissue factor (TF), a potent initiator of the extrinsic coagulation pathway, is believed to have a critical role in thrombogenesis and haemostasis. To elucidate the role of TF in the development of various syndrome, we developed a quantitative assay method for the determination of TF using FIX complex (Profilnine) and the synthetic chromogenic substrate S-2238, all of which are commercially available. The method is simple, very sensitive, good linearity and applicable to the tissue culture plate, indicating its promising usage for the quantitation of TF activity of cells.

Red blood cell and platelet induced differentiation of endothelial cells into capillary-like structure.

We cultivated endothelial cells of human umbilical vein origin in the presence of red blood cells and platelet-rich plasma, and observed the phenomena which occurred in the petri dish under phase contrast microscopy. Many small particles were observed after an overnight incubation. We washed the dish two times, then added thrombin to the dish. The network of thread-like strands appeared within 10 to 20 minutes of the addition, and at the same time the small particles adhered on the surface of the strands, swelled and fused gradually to cover the surface of the strands completely. Within 30 to 60 minutes the network of the strands changed into a capillary-like structure. These phenomena were not observed if we omitted red blood cells or platelet-rich plasma. Studies by transmission electron microscopy revealed that the inner surface of the lumen of the structure was covered with cells. The cells isolated from the lumen by trypsin grew to confluence in the conventional culture medium, and showed vWF antigen on their surface. These observations indicated that the method described is useful for in vitro study of angiogenesis.

Increase in spontaneous platelet aggregation in beta-thalassemia/hemoglobin E disease: a consequence of splenectomy.

Clinical symptoms related with disturbances of the circulatory system are often observed in beta-thalassemia/hemoglobin E (beta-thal/HbE) patients after splenectomy. Pulmonary thrombosis is one of the important contributing factors. However, the pathogenesis of this phenomenon was not known. Previous studies on platelet functions were controversial as platelet-rich plasma (PRP) was employed for all of the studies. By centrifugation, most of the hyperactive platelets were excluded before platelet aggregation tests were performed. Besides, the role of red cells related to platelet aggregation was not investigated. In this study, a platelet function test was designed to avoid these two handicaps of previous work as mentioned, by using whole blood from 15 normal and 40 beta-thal/HbE patients (15 nonsplenectomized and 25 splenectomized) to study spontaneous platelet aggregation. The principle of the test was to evaluate platelet number in whole blood by electronic platelet counter at time 0 (45 minutes after blood collection) and this number was used as 100% of free unaggregated platelets. Then the same specimen of whole blood was incubated at 37 degrees C with continuous stirring by magnetic stirrer in an aggregometer for 8 minutes; at 1 minute intervals free unaggregated platelets were evaluated and calculated as a percentage of the initial control value. The results indicated increased spontaneous platelet aggregation in whole blood of post-splenectomized beta-thal/HbE patients. The residual free platelet number were 24% at 8 minutes after incubation. Effects of red blood cells on spontaneous platelet aggregation were studied by mixing autologous beta-thal/HbE red cells obtained from splenectomized and non-splenectomized patients with platelet rich plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Three possible triggers to induce thrombocytopenia in dengue virus infection.

Changes in platelet count by dengue virus-platelet interaction and the participation of anti-DV antibody to such changes were studied in vitro with the aim to investigate a mechanism of how the DV causes prominent thrombocytopenia characteristically seen in DHF. The results obtained showed that: (1) DV antigen attached to human platelets without immune-mediated reaction, (2) a decrease in platelet count was more markedly demonstrated by the binding of anti-DV antibody on the DV antigen associated with platelets than by the binding of the antigen-antibody complex on platelets, (3) a modulation of endothelial cell by the infection of DV to the cell was suggested as one of the causes of the thrombocytopenia.

DHF characterized by acute type DIC with increased vascular permeability.

Decreases in platelet count, fibrinogen concentration, factor VIII, antithrombin III and alpha 2-antiplasmin activities, increase in FDP-D fraction, and pleural effusion were observed transiently at early fever stage of DHF at grade II, indicating DHF patients had manifestations of the acute type of DIC with increased permeability of vascular wall.

Automatic measurement of hemoglobin F in blood obtained from patients with hemoglobin E/E and beta-thalassemia/hemoglobin/E.

This paper presents an automated determination of hemoglobin (Hb) F in Hb E/E disease using Hi-Auto A1c. Blood specimens collected in Bangkok were frozen, and sent to Japan by air mail for the determination. The automatically determined values showed a high correlation with the values obtained by the classical alkali denaturation method. Hb E/E cases showed 4.24 +/- 1.75% of Hb F. On the other hand, Hb, Hct, MCV and MCH in the disease samples were lower than in the controls, but higher than those of beta-thalassemia/HbE disease. From the results it was concluded that Hb E/E could be differentiated from beta-thalassemia/HbE by combination of Hb F value and MCH or Hb in CBC.

Interaction between endothelial cells and thalassemic red cells in vitro.

Erythrocytes from 45 patients with thalassemia and/or hemoglobinopathies were studied for their cytoadherence property to the vascular endothelial cells in vitro. In plasma free medium, erythrocytes from patients with beta-thal/Hb E both splenectomized and nonsplenectomized, HbH diseases (alpha-thal 1/alpha-thal 2 and alpha-thal 1/Hb Constant Spring genotypes) and homozygous Hb E subjects bind to endothelial cells at a greater number as compared to the binding cell number of normal erythrocytes (p-value < 0.05 in all types). Addition of autologous platelet-rich plasma or whole blood to the culture system causes further increase in the number of adhering beta-thalassemia red cells. Platelet-rich plasma had more enhancement effect than the whole blood. However, no such enhancement of both platelet-rich plasma and whole blood was demonstrated in the culture of normal or alpha-thalassemia erythrocytes. Increased binding between red cells and endothelial cells may contribute to the greater risk of vascular occlusion in thalassemic patients.

Thalassemic serum impairs endothelial cell growth in vitro.

Endothelial cells cultured for 3 days in the presence of hemoglobin H pooled sera had significantly decreased cell proliferation compared to those in normal serum. Inhibition was demonstrated at a concentration of 20% pooled serum in the cultured medium. Further decrease was shown in the presence of 30% pooled hemoglobin H sera. Sera from two genotypes of Hb H disease (alpha-thal 1/alpha-thal 2 and alpha-thal 1/Hb Constant Spring) had the same degree of inhibitory effect. Pooled sera from beta-thal/Hb E patients (both splenectomized or nonsplenectomized cases) had no such inhibitory effect. However, at day 4 and 5, the growth pattern relatively declined. Bilirubin at a concentration greater than 4.0 mg% in the medium 199 also caused significant decrease in cell proliferation. Since the diluted Hb H serum had bilirubin less than 4.0 mg%, the inhibitory effect of the pooled HbH serum is thus not due to effect of bilirubin. The difference between HbH and beta-thal/HbE sera in terms of inhibition of endothelial cell proliferation is the new finding that needs further investigation to explain vascularization and hemostasis in the patients of these two genotypes.

Cytoprotective effect of dilazep on hydrogen peroxide-perturbed vascular endothelial cells.

The effect of dilazep and dimethyl thiourea (DMTU) on the hydrogen peroxide-derived injury of culture pulmonary artery epithelial cells (CPAEC) was assessed by colorimetric assay of MTT formazan (MTT formazan assay). When CPAEC were treated with hydrogen peroxide, neither cell lysis nor detachment of the cells from surface of the well was observed. However, the MTT formazan formation was decreased in a time and dose dependent manner. The decrease in the formation was significantly suppressed in the presence of dilazep (0.1 to 10 microM) or DMTU (0.01 to 0.3 microM). CPAEC treated with hydrogen peroxide in the same way enhanced an activation of prothrombin, and this enhancement was significantly inhibited in the presence of dilazep (1 to 3 microM). These data indicate that dilazep exerts a cytoprotective effect against challenges of intracellular oxidant produced by hydrogen peroxide and suppresses augmented procoagulant activity of injured cells.

Development of a new method for detection of platelet factor 3 like activity.

We asked the question, "Can thalassemic erythrocytes play some role in alteration of the hemostatic system?", because clinical examination of thalassemic patients shows symptoms and signs related to alterations in hemostatic and circulatory systems, and thalassemic erythrocytes are different from normal erythrocytes. We obtained one of the answers to the question: The erythrocytes of postsplenectomized patients of beta-thalassemia/HbE disease could stimulate their own platelets to aggregate spontaneously. To know the role of erythrocytes in platelet aggregation, we wanted to examine the effect of thalassemic erythrocytes on the coagulation system by focusing of PF3-like activity of erythrocytes, because PF3-like activity of the ghosts of erythrocytes had been reported. For the study, we tried to develop a technique that was accurate and sensitive enough to detect PF3-like activity of blood. The system we developed was the following: 1) We activated the intrinsic coagulation pathway of commercial standard plasma by ellagic acid. 2) CaCl2, a fixed amount of PF 3 and synthetic thrombin inhibitor MD 805 were added to the reaction mixture. 3) At a fixed time, thrombin activity in the mixture was measured by using S-2238 as a substrate. At full activation of the contact system by ellagic acid, the amount of thrombin formed in a certain time depended on the amount of PF3-like substances such as cephalin, freeze-thawed platelets or ghosts of erythrocytes added to the test system, indicating that PF3-like activity of those substances can be measured by the activity of thrombin generated in a fixed time.(ABSTRACT TRUNCATED AT 250 WORDS)

PF3 activity in normal subjects and beta-thalassemia trait.

Platelet factor 3 (PF3) is a platelet membrane component that plays an important role in the activation of the coagulation mechanism. Whenever platelet activation occurred, PF3 is released and participates in thrombin formation. Erythrocyte membrane fraction has also some PF3 like activity, and in abnormal erythrocyte membrane disorders, eg thalassemia, some of the membrane fraction accelerates platelet activation by increasing the PF3 activity. Formerly it was difficult to measure the PF3 activity in plasma. Recently a sensitive chromogenic test to determine the PF3 activity, which could detect the changes in PF3 activity with time, was introduced. This study was done to observe the effect of abnormal erythrocyte on platelet activation. The results obtained using the chromogenic method are the following: whole blood taken from normal subjects showed OD 0.11 +/- 0.06 at 0 minutes after blood collection and then increased significantly (p < 0.01) to 0.21 +/- 0.10 after 90 minutes, while the platelet count did not differ significantly (p > 0.05). Those results showed that there were some platelet activation after 90 minutes as seen by the increased PF3 activity, with no significant change in platelet counts. In beta-thalassemic trait subjects the PF3 activity in whole blood at 0 minutes did not differ significantly compared to the normal subjects, but after 90 minutes it was significantly higher (p < 0.01), OD 0.52 +/- 0.35. However the PF3 in platelet rich plasma at 90 minutes did not increase. The platelet count after 90 minutes was significantly decreased (p < 0.01) This result suggest that the increase in PF3 activity was caused by the role of the abnormal erythrocytes.