The effect of daunorubicin (DNR) on von Willebrand factor (vWF) in plasma was studied. Ristocetin induced-platelet aggregation (RIPA) in five acute myeloid leukemia (AML) patients was decreased after administration of DNR (p<0.05). And then plasma vWF antigen (vWF: Ag) and ristocetin cofactor (RCof) in seven AML patients were measured. After administration of DNR, vWF: Ag was increased (p<0.05), whereas RCof was decreased (p<0.01). RCof was decreased significantly (p<0.005) in the DNR-treated plasma (DTP) of normal subjects, in which DNR was added and incubated at 37°C for 30min, in comparison with the control in which DNR was not added. Further more, RCof was decreased significantly (p<0.01) even in the DTP from which DNR was removed by gel filtration. Change of the multimeric composition of vWF were investigated by crossed immunoelectrophoresis (CIE) and SDS-agarose gel electrophoresis. In the plasma of AML patients after administration of DNR and DTP, the components corresponding to the small multimer increased. These results suggested that DNR could induce the molecular abnormality of vWF.
Among monoclonal antibodies reacting to platelets, some GPIIb/IIIa antibodies have been reported to react to the platelets of different animals. In this study, we searched for common antigens for platelets of dogs and humans, using 11 monoclonal anti-human-platelet antibodies. By flow cytometry, we examined the reactivity of various monoclonal antibodies to dog platelets. Anti-GPIIb/IIIa antibody NNKY2-11 reacted with about 80%, NNKY2-18 about 50%, CD9 antibody NNKY1-19 about 60%, and MALL13 about 95% of dog platelets, indicating that these monoclonal antibodies recongnize a common antigen in the platelet membranes of dog and human. These anti-GPIIb/IIIa antibodies also inhibited the ADP and collagen aggregation of dog platelets but significantly. When dog platelets were treated with EDTA, the reactivity of the anti-GPIIb/IIIa antibodies decreased significantly. It suggested that the structure of the epitope recognized is dipendent upon Ca++, similar to the human platelet GPIIb/IIIa. However, dog platelets did not show an increase in antibody reactivity upon activation. Autoradiography showed that the antigen recognized by NNKY2-11 on the dog platelet membrane is not exactly the same as GPIIb/IIIa of human platelets. The CD9 antibody MALL13 aggregated dog platelets. We suggest that these monoclonal antibodies may be useful studying platelet function in dogs.
The blood level of interleukin-1β (IL-1β) was determined in 100 normal individuals, 10 patients with thrombotic thrombocytopenic purpura (TTP), 41 patients with established disseminated intravascular coagulation (DIC), 15 in pre-DIC period (within 7 days before the onset of DIC), and 23 non-DIC patients. In TTP, the blood IL-1β concentration was increased at onset but decreased at remission. The blood IL-1β concentration was significantly higher (p<0.01) in the DIC group (0.19±0.19ng/ml) than in the pre-DIC group (0.05±0.08ng/ml) or the non-DIC group (0.09±0.01ng/ml). According to underlying diseases, the blood IL-1β level was not markedly elevated in leukemia patients even in the DIC group, but it was significantly increased in the DIC group of solid cancer patients. It was generally elevated in sepsis patients. It was lower in DIC patients with no organ failure than in the entire DIC group, and was markedly elevated in those with organ failure at 0.29±0.26ng/ml. From those results, IL-1 is considered to be closely related to the pathogenesis of DIC and TTP, and it was associated organ failure.
Plasma levels of thrombin-antithrombin III complex (TAT) and plasmin-α2-plasmin inhibitor complex (PAP) were measured by enzyme-linked immunosorbent assays (ELISA) together with other coagulation parameters in patients with hematological diseases, liver diseases, diabetes mellitus, systemic lupus erythematosus, thrombotic diseases and disseminated intravascular coagulation (DIC). The mean plasma levels of TAT and PAP in 28 healthy subjects were 1.26±SD 0.71ng/ml and 0.22±0.14μg/ml, respectively. Both TAT and PAP were elevated in a variety of diseases, especially in DIC (22.97±19.66ng/ml and 4.33±3.08μg/ml, respectively), followed by liver diseases, thrombotic diseases and hematological malignancies. On the whole, the PAP value was correlated with TAT (r=0.533, p<0.00001). Plasma concentrations of TAT and PAP were correlated positively with concurrently assayed prothrombin time, FDP and von Willebrand factor antigen/factor VIII activity ratio, and negatively with fibrinogen, α2-plasmin inhibitor, plasminogen and ristocetin cofactor/von Willebrand factor antigen ratio. No correlation was found between TAT and antithrombin III. These findings indicate that excessive amounts of thrombin and plasmin are actually generated not only in DIC patients but also in patients with a variety of diseases. In addition, measurements of TAT and PAP in plasma would be sensitive parameters for specific detection of activation of blood coagulation and fibrinolysis in selected disease states.
DNA samples from white blood cells of 9 patients and 10 healthy members in 3 Japanese kindreds (Family Mo, Mi and Tu) with congenital antithrombin III (AT III) deficiency, and of 48 normal Japanese individuals were analysed by Southern blotting method using AT III cDNA probe PA62. Restriction fragment length polymorphism (RFLP) by uses of Pst I and PA62 demonstrated + allele in 50%, - allele in 50%, F allele in 59% and S allele in 41% of 96 alleles from 48 normal Japanese individuals, indicating that the frequencies of RFLP in Japanese are almost the same as those in other races reported before. This suggests that DNA analysis on Japanese families with congenital AT III deficiency was possible using RFLP of AT III gene because of high frequencies of RFLP in normal Japanese. All patients in 3 kindreds with congenital AT III deficiency had a -/+ genotype, indicating that complete deletion of one allele of AT III gene can be neglected. No abnormal DNA fragments were observed by Southern blot analysis of genomic DNAs from the patients in the 3 kindreds digested with various restriction enzymes (Bam HI, Eco RI, Hind III, Bgl II, Bcl I, Kpn I, Pvu II, Sac I, Taq I and Xba I). This suggests that AT III deficiency in our 3 kindreds is not caused by major structural alterations such as partial deletion, rearrangement and duplication, small deletion, insertion or limited nucleotide substitution in the AT III gene. In addition, it was suggested by analysis of polymorphism of AT III gene in the members of Family Mo and Mi that abnormal AT III gene existed on -, F allele in these families.
The blood level of tumor necrosis factor (TNF) was determined in 20 normal individuals, 52 patients with disseminated intravascular coagulation (DIC), 22 pre-DIC patients, and 39 non-DIC patients and 10 thrombotic thrombocytopenic purpura (TTP) patients. TNF was not detected in the normal subjects and the level was very low in non-DIC patients. However, the level was significantly elevated in DIC patients. TNF was also increased significantly in pre-DIC patients shortly before the onset of DIC as compared with non-DIC patients. This increase in circulating TNF may be associated with DIC. In TTP, the blood level of TNF was increased at onset but decreased at remission. According to underlying diseases, TNF was higher in DIC associated with solid cancer than in DIC associated with leukemia or sepsis. In solid cancer, a large amount of tissue factor may be produced as the immune system is activated, cytokines such as TNF are released into the circulation, and endothelial cells and monocytes are activated. The blood TNF level was not significantly different between DIC patients with organ failure and DIC those without organ failure. From these findings, the increase in circulating TNF is considered to be a direct pathogenic factor in DIC or TTP rather than a consequence of organ failure due to DIC.
To assess the state of thrombosis and fibrinolysis, we measured the plasma concentrations of fibrinopeptide A (FPA), fibrinopeptide Bβ15-42 (FPBβ15-42), D-dimer, protein C (PC) activity and antigen, and antithrombin III (AT III) in 17 patients of cardioembolic stroke. Patient group had significantly high concentrations of FPA and FPBβ15-42, and significantly low concentrations of PC and AT III compared with healthy control group (p<0.005-0.05). D-dimer was increased in 5 of 7 patients, and both FPA and FPBβ15-42 were increased in 15 of 16 patients. In contrast, PC activity, PC antigen and AT III were reduced only in 6 of 12, 4 of 12 and 5 of 15 patients, respectively. These findings suggest that FPA, FPBβ15-42 and D-dimer may be useful to detect the activities of thrombosis and fibrinolysis in patients with cardioembolic stroke.
To characterize human platelet membrane glycoprotein (GP), we developed an immunomagnetic cell isolation method using monoclonal antibodies (MoAbs) that specifically bind to Ib and IIb-IIIa complexes of GP. When added to plateletrich plasma (PRP), anti-GPIb MoAb inhibited ristocetin-induced platelet aggregation, and anti-GPIIb-IIIa MoAb inhibited ADP- and collagen-induced platelet aggregation. Each MoAb was incubated with monosized polystyrene beads (4.5μm in diameter) coated with secondary antibody and then with normal PRP for 30 minutes. More than 90% of the platelets bound to the beads and were eliminated from PRP. However, the binding between the platelets and the beads coated with each MoAb was inhibited by pretreatment of PRP with the same type of MoAb. Platelets from patients with Bernard Soulier syndrome (BSS) and Glanzmann's thrombasthenia (GT) were added to the beads coated with each MoAb. BSS platelets with defective GPIb and GT platelets with defective GPIIb-IIIa did not bind to the beads coated with anti-GPIb MoAb and those coated with anti-GPIIb-IIIa MoAb, respectively. Our immunomagnetic isolation method using anti-GP MoAbs is very simple and useful for detecting abnormalities such as GP deficiency and especially diagnosing BSS and GT.
Changes in plasma and liver descarboxyprothrombin (PIVKA-II), an abnormal prothrombin without biological activity, were examined in rats after a single injection of warfarin (2.5mg/kg, s. c.) and compared with changes in plasma clotting factor levels. Furthermore, the changes in plasma and liver PIVKA-II levels after a single injection of vitamin K1 (VK1) (200μg/kg, s. c.) were examined in rats kept on a VK-deficient diet for 3 days. The plasma prothrombin and factor VII levels decreased soon after warfarin injection, bottomed out at 24hrs and then began to increase, returning almost to the initial levels at 72hrs. The plasma PIVKA-II level began to increase 3hrs after warfarin injection, peaked at 24hrs and then declined. The liver PIVKA-II levels, however, began to increase at 1hr after warfarin injection, continued to increase for a while, and then declined. Warfarin caused an abrupt increase in the liver PIVKA-II levels, but the increase in plasma PIVKA-II level was delayed about 3hrs. Liver PIVKA-II increased to about 5 fold that of normal rats and began to be excreted into plasma. The liver PIVKA-II levels were normalized within 2hrs after VK1 injection, but the recovery of the plasma PIVKA-II levels required 12-24hrs. The disappearance half-time of liver PIVKA-II was calculated to be 0.86hr while that of plasma PIVKA-II was 11.6hrs. These results suggest that warfarin increases PIVKA-II levels in plasma and liver, but the rise in plasma level is delayed for about 3hrs. During the time lag the liver PIVKA-II level increased about 5 fold that of the normal rat liver, indicating that this value corresponds to the maximal capacity of liver microsomes to restore PIVKA-II. The shorter disappearance time of liver PIVKA-II after VK1 injection than that of plasma suggests that the carboxylation reaction in the liver proceeds quickly.
It has suggested that hypercoaglability was induced activated factor VII by tissue factor in the estrinsic pathway from malignant tumor. Procoaglant activity may relate with tumor gorwth and development of metastasis. The procoaglant activity of four human cancer cells and of culture supernatant was measured by a one-stage plasma recalcifiation assay. The cell suspension of LK-2, LK-17, LK-52 and HLC-1 was shorter markedly the recalcifiation time of normal human plasma, not of factor VII deficient plasma. The cultre suspension of LK-2 was longer markedly the recalcifiation time of normal human plasma. The culture suspension of LK-17 was shoten markedly the recalcifiation time of normal human plasma, not of factor VII deficient plasma. The culture suspension of LK-52 was shoter the recalficication time of both normal human plasma and factor X, of factor VII deficient plasma. These Beta incident that LK-52 cancer cell line products a directer activater of coagulation facter X in cluture medium.