Masaharu Hanano

Primary Thrombocythemia in Japan: A Survey of 225 Patients

Data on 225 Japanese patients with primary or essential thrombocythemia (ET) were analyzed in an attempt to characterize the clinical and laboratory features in subgroups with thrombosis (T), hemorrhages (H), thrombohemorrhagic events (TH) or a non-thrombohemorrhagic (O) group, and in order to examine survival and the incidence of blastic transformation in the entire group and in the different subgroups. Higher platelet and leukocyte counts were related to hemorrhage (H and TH), prolonged activated partial thromboplastin times and high LDH levels to H while elevated FDP levels were more frequently linked to T. Increased spontaneous platelet aggregation (SPA) was noted in 80.3% of the entire group, independent of whether there was a tendency for thrombohemorrhagic events or not. Bleeding time, as measured by the Duke method, and hemoglobin levels were not different in the various subgroups. Transformation occurred in 11 patients (1.9% per year); seven developed acute leukemia (myeloblastic 4, lymphoblastic 2, megakaryoblastic 1) at a rate of 1.2% per year; and 4 developed other types of chronic myeloproliferative disorders. Nineteen patients died (3.3% per year), six from leukemia (32%), 4 from bleeding (21%) and 9 from unrelated diseases (47%). Survival was estimated to be 65% at ten years, and was significantly longer in females, younger individuals, and the groups with lower leukocyte counts, but did not differ between the subgroups when platelet count and hemoglobin level were considered. Survival was similar in patients with platelet counts between 700-1000 × 10(9)/L and in those with an even higher platelet count. These findings suggest that (1) young female patients with low leukocyte counts may survive longer, (2) SPA is not indicative of either a thrombotic or an hemorrhagic tendency and (3) the limit of the platelet count for establishing the diagnosis of this disorder could perhaps be lowered to 700 × 10(9)/L.

Heat-Treated Factor VIII/von Willebrand Factor Concentrate in Platelet-Type von Willebrand’s Disease

Cryoprecipitate has proved to correct the hemostatic defects in von Willebrands disease (vWD) and platelet-type vWD. However, recent studies have revealed that transmission of the AIDS retrovirus (HIV) occurs through exposure to blood products including cryoprecipitate. Treatment with heat-treated factor VIII/von Willebrand factor (vWf) concentrates may have certain advantages over treatment with nonheated products, if these preparations are efficacious in these disorders. We found that a commercially available factor VIII/vWf concentrate, Haemate P, contained the high-molecular-weight multimers of vWf and had a ratio of ristocetin cofactor (RCof) to vWf antigen (vWf:Ag) close to unity. In addition, its capacity to directly induce aggregation of platelet-type vWD platelets in vitro was similar to that for cryoprecipitate. When infused into a patient with platelet-type vWD, Haemate P shortened the prolonged bleeding time and caused spontaneous platelet aggregation in vitro with a mild diminution of platelet count. These results indicate that some of the heat-treated factor VIII/vWf concentrates may provide a safer, yet still effective, treatment for platelet-type vWD.

Behavior of protein S during long-term oral anticoagulant therapy.

It has recently been reported that a natural anticoagulant, protein S (PS), is depressed during oral anticoagulation. Since more detailed information is required from the clinical standpoint, we measured plasma levels of PS [both total and free (not complexed) PS antigen], C4b-binding protein (C4bp) and other vitamin K-dependent proteins (factors II, VII, IX, X and protein C) in 60 plasma samples from patients on long-term oral anticoagulant therapy with warfarin. Together with the reduction of other vitamin K-dependent plasma proteins, PS decreased during warfarin treatment, being dependent on the intensity of the therapy. A considerable variation in plasma PS levels was also observed among individuals with a similar intensity of anticoagulation. Plasma concentration of C4bp was closely correlated with total PS level, and free PS/total PS ratio was independent of thrombotest values. These findings indicate that long-term oral anticoagulant therapy results in the suppression of the synthesis of PS, and that its reduction is on the whole balanced with C4bp and vitamin K-dependent coagulation factors. It was suggested that the metabolism of C4bp might be regulated by the plasma PS level, although this hypothesis needs further exploration.

Von Willebrand Factor Fragment in Type IIA von Willebrand’s Disease: Demonstration of Two Different Forms of Fragments

A fast-migrating precipitin peak which showed a reaction of partial immunochemical identity with the major von Willebrand factor (vWf) component was detected by crossed immunoelectrophoresis in agarose gel in plasma but not in platelets from a patient with type IIA von Willebrands disease (vWD). Another patient undergoing urokinase therapy had a vWf fragment in plasma which was antigenically cross-reactive with the major vWf component. When plasma from both patients was mixed and electrophoresed together in the first dimension, two fast-moving precipitin arcs were demonstrated in the second dimension. These data indicate that two different kinds of vWf fragments can be generated in vivo.

Multimeric composition of plasma von Willebrand factor in chronic myelocytic leukaemia

We studied the multimeric composition of plasma von Willebrand factor (vWf) in 26 patients with chronic myelocytic leukaemia (CML); 13 in chronic phase, 8 in blast crisis, 5 in both chronic phase and blast crisis. The relative amount of large (multimer band greater than or equal to 11) multimers calculated by densitometer scan following SDS-agarose gel electrophoresis was 14.6 +/- 2.9% (mean +/- SD) in normal controls, 8.7 +/- 4.7% in chronic phase and 15.0 +/- 5.2% in blast crisis. CML patients in chronic phase (but not in blast crisis) had a significantly lower percentage of large multimers than normal controls (p less than 0.001). The relative amount of large multimers was positively correlated with a ratio of ristocetin cofactor/vWf antigen (p less than 0.005), and was negatively correlated with platelet count (p less than 0.001), WBC count (p less than 0.05) and granulocyte count (p less than 0.05). We conclude that some patients with CML, especially in chronic phase, lack large multimers. The negative correlation of the relative amount of large multimers with platelet and granulocyte count suggests that large multimers may be consumed in high platelet count states or degraded by protease(s) from increased blood cells.

Fast functional assay of protein C in whole plasma using a snake venom activator: evaluation in patients with congenital and acquired protein C deficiencies.

Both anticoagulant and amidolytic activities of protein C (PC) were measured using a snake venom activator in 4 patients with hereditary PC deficiency, 37 with disseminated intravascular coagulation (DIC), and 30 under stabilized warfarin therapy. The results were compared with those obtained by an immunological assay (ELISA). PC levels measured by different functional and immunological assays were very close in patients with hereditary PC deficiency and DIC. In patients under stable oral anticoagulant therapy, there was no detectable difference between amidolytic activity and antigen levels of PC in each patient plasma, whereas a decrease in anticoagulant activity was much more pronounced. These results indicate that the present activity assays measure PC specifically, and that the snake venom activator is capable of activating both carboxylated and hypocarboxylated forms of PC, but only anticoagulant assay can evaluate the physiological PC function in vitamin K-deficient states.

Plasma Urokinase-Type Plasminogen Activator in Patients with Leukemias

Plasma levels of urokinase-type plasminogen activator (u-PA) were measured with an enzyme-linked immunosorbent assay in patients with leukemias. As compared with healthy subjects (0.73 +/- SD 0.17 ng/ml), plasma u-PA antigen level was markedly elevated in patients with acute promyelocytic leukemia (APL) (1.76 +/- 0.89 ng/ml) at disease onset. Mean u-PA concentrations in patients with other acute nonlymphoblastic leukemia (0.57 +/- 0.51 ng/ml), acute lymphoblastic leukemia (0.77 +/- 0.82 ng/ml) and chronic myelocytic leukemia in blastic crisis (1.30 +/- 1.35 ng/ml) were not significantly elevated, but some of them showed an elevation of plasma u-PA. Plasma u-PA values were correlated with some of the fibrinolytic parameters such as FDP and D-dimer. Plasma u-PA antigen was decreased after the administration of antileukemic drugs in patients with APL. These results suggest that the coagulopathy in patients with various leukemias may in part be associated with u-PA release from the leukemic cells, especially in patients with APL.

Platelet-type von willebrand disease platelet aggregating factor: A novel functional assay of von willebrand factor

Blood platelets from patients with platelet-type von Willebrand disease (vWD) aggregate upon the addition of human von Willebrand factor (vWf) in the absence of ristocetin or other stimulating factors. We measured quantitatively the ability of vWf to induce directly aggregation of platelet-type vWD platelets (platelet-type vWD platelet aggregating factor [PT-PAF]). Cryoprecipitate and factor VIII concentrates were used as a source of vWf of various multimeric composition. The PT-PAF activity was dependent on the multimer size of vWf, like in the case of ristocetin cofactor (RCof) activity. However, PT-PAF activity was not equivalent to RCof activity and the relative PT-PAF/RCof ratio ranged from 1.00 to 0.18 in the materials studied. The preparations containing the higher-molecular-weight multimers had higher PT-PAF/RCof ratio. These findings suggest that PT-PAF activity is a functional expression of more highly polymerized multimers of vWf as compared with RCof activity. Measurement of PT-PAF would serve as a novel functional assay of vWf.

Factor VIII lability, protein C and other vitamin K-dependent proteins.

The stability of factor VIII was studied in plasmas from patients on long-term warfarin therapy. The percent residual factor VIII activity (F.VIII:C) after incubation at 37 degrees C for 4 hr was higher in warfarinized patients than in normal subjects; 76.9 +/- 10.8% (mean +/- SD) of the initial F.VIII:C in the patients versus 61.6 +/- 5.8% in normal subjects (p less than 0.001). On the whole, neither protein C nor vitamin K-dependent coagulation factors except factor VII activity (F.VII:C) correlated with the residual F.VIII:C. There was a negative and weak correlation between the residual F.VIII:C at 4 hr and either the initial F.VIII:C or F.VII:C. Another experiment using protein C depleted plasma showed a relatively enhanced stability of F.VIII:C in the protein C deficiency. These results indicate that factor VIII is more stable in warfarinized plasma, and that protein C and vitamin K-dependent coagulation factors are not the sole, main factor responsible for such a phenomenon.