Wataru Tatewaki

Activation of blood coagulation and fibrinolysis in diabetes mellitus: Evaluation by plasma levels of thrombin-antithrombin III complex and plasmin-alpha 2-plasmin inhibitor complex

Diabetes mellitus (DM) is associated with an increased incidence of vascular complications. Abnormalities in the hemostatic system contribute at least in part to the development of vascular disease or atherosclerosis. In order to assess the actual degree of activation of the coagulation and fibrinolytic systems in diabetics, plasma levels of thrombin-antithrombin III complex (TAT) and plasmin-alpha 2-plasmin inhibitor complex (PAP) were measured together with tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) in 18 patients with DM (three patients with type I DM and 15 with type II DM). Mean plasma levels of TAT (2.5 +/- SD 1.2 ng/mL) and PAP (0.9 +/- 1.2 micrograms/mL) were significantly elevated in diabetics as compared with healthy subjects (1.7 +/- 0.3 ng TAT and 0.2 +/- 0.1 micrograms PAP per mL of plasma; p = 0.009 and 0.02, respectively). Plasma antigen concentration of t-PA but not of PAI-1 was also elevated. No difference was found in the levels of these variables between type I and type II diabetics or between patients with and without retinopathy or nephropathy. These findings indicate that continuous activation of coagulation and fibrinolysis actually occurs in the majority of the patients with DM.

Plasma protein S in disseminated intravascular coagulation, liver disease, collagen disease, diabetes mellitus, and under oral anticoagulant therapy

Plasma levels of protein S (PS) antigen, both total and free fractions, were measured together with C4b-binding protein (C4bp) and protein C (PC) antigen in 39 patients with disseminated intravascular coagulation (DIC), 34 with liver disease, 17 with collagen disease, 17 with diabetes mellitus, and 51 under stabilized warfarin treatment. In patients with DIC, mean concentrations of total PS and free PS were normal, while PC was reduced and C4bp were elevated. Total PS, free PS, C4bp and PC were all decreased in liver disease, elevated in diabetes mellitus, and normal in collagen disease. In warfarin-treated patients, total PS, free PS and PC were moderately decreased, but the decrease in C4bp was minimal. The concentration of PS correlated positively with PC in liver disease, diabetes mellitus, and during oral anticoagulation, but did not in DIC. These results indicate that PS and PC behave similarly when liver synthetic function is principally affected, but in contrast to PC, PS is hardly consumed during intravascular coagulation.

Paroxysmal Nocturnal Hemoglobinuria with Myelofibrosis: Progression to Acute Myeloblastic Leukemia

A 58-year-old male was diagnosed as having paroxysmal nocturnal hemoglobinuria (PNH) with myelofibrosis in 1984. The administration of hydroxyurea and low dose splenic irradiation were initiated for abdominal distention due to splenomegaly in 1987. In May 1990 the patient developed smouldering acute myeloblastic leukemia (AML); and the blasts proliferated in response to G-CSF administered for refractory pneumonia. The patient died of pneumonia and pleural involvement of leukemia in September 1990. FACS analysis of the blasts using anti-decay accelerating factor (DAF) (CD55) and CD59 (membrane attack complex inhibition factor: MACIF) monoclonal antibodies demonstrated that 25.5% and/or 87.3% of the blasts were negative for DAF or CD59 respectively. There is the earlier evidence that about 90% leukemic myeloblasts from non-PNH AML patients are positive for DAF, and nearly 100% of non-PNH neutrophils have been shown to be positive for both DAF and CD59. Our data suggest that the leukemic blasts from this patient may have derived from the PNH clone.

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.

Plasmin-a2-plasmin inhibitor complex in plasma of patients with thromboembolic diseases

Plasmin generation in vivo was assessed by measuring plasma levels of plasmin-a2-plasmin inhibitor complex (PAP) with an ELISA in 42 patients with arterial or venous thromboembolic diseases. Plasma concentration of PAP was markedly elevated in patients with venous thromboembolic diseases during acute illness (3.32 +/- 3.71 ug/ml, mean +/- SD) as compared to healthy subjects (0.24 +/- 0.13 ug/ml, n = 14), while it was nearly normal (0.30 +/- 0.13 ug/ml) in patients with venous thromboembolic diseases in chronic stage. Patients with arterial thromboembolism had modestly elevated PAP; 1.05 +/- 0.77 ug/ml during acute episode, and 0.84 +/- 0.40 ug/ml in chronic stage. These results indicate that excessive activation of fibrinolytic system (plasmin generation in vivo) occurs actually in many patients with thrombotic diseases, especially in venous thromboembolic diseases during acute illness.

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.

Plasma von Willebrand factor proteolysis in patients with chronic myeloproliferative disorders: No possibility of ex vivo degradation by calcium-dependent proteases

Patients with chronic myeloproliferative disorders (CMPD) frequently have abnormalities of plasma von Willebrand factor (vWf) multimers. The pathogenesis of this phenomenon is still unknown. In order to evaluate the possibility of ex vivo degradation of vWf during blood processing, we compared vWf antigen, ristocetin cofactor and the multimeric composition of vWf in plasmas obtained in the presence of trisodium citrate with or without calcium-dependent protease inhibitors (leupeptin, N-ethylmaleimide and Na2EDTA). The subjects included 20 patients with CMPD, 11 with other diseases and 8 normal subjects. In patients with CMPD and normal subjects, the values of vWf antigen, ristocetin cofactor, ristocetin cofactor/vWf antigen ratio and the relative amount of large multimers of vWf did not significantly differ from each other in plasma samples with and without protease inhibitors. In other diseases, especially in a patient with disseminated intravascular coagulation, a somewhat higher amount of large multimers were found in plasma with protease inhibitors than without inhibitors. These findings indicate that the ex vivo proteolysis during blood processing is negligible in patients with CMPD, and that the observed abnormalities in vWf is an in vivo phenomenon.

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.