Prophylactic fresh frozen plasma may prevent development of hepatic VOD after stem cell transplantation via ADAMTS13-mediated restoration of von Willebrand factor plasma levels.

We initially conducted a multicenter, randomized trial (n=43), and subsequently a questionnaire study (n=209) of participating hospitals, to evaluate whether infused fresh frozen plasma (FFP) could prevent the occurrence of hepatic veno-occlusive disease (VOD) after stem cell transplantation (SCT). Forty-three patients were divided into two groups: 23 receiving FFP infusions and 20 not receiving it. VOD developed in three patients not receiving FFP. Plasma von Willebrand factor (VWF) antigen levels were lower at days 0, 7 and 28 after SCT in patients receiving FFP than in those not receiving it, whereas plasma ADAMTS13 activity (ADAMTS13:AC) did not differ between them. Plasma VWF multimer (VWFM) was demonstrated to be defective in the high approximately intermediate VWFM during the early post-SCT phase, but there was a significant increase in high VWFM just before VOD onset. This suggests that a relative enzyme-to-substrate (ADAMTS13/high-VWFM) imbalance is involved in the pathogenesis of VOD. To strengthen this hypothesis, the incidence of VOD was apparently lower in patients receiving FFP infusions than in those not receiving it (0/23 vs 3/20) in the randomized trial. Further, the results combined with the subsequent questionnaire study (0/36 vs 11/173) clearly showed the incidence to be statistically significant (0/59 vs 14/193, P=0.033).

Platelets treated with ticlopidine are less reactive to unusually large von Willebrand factor multimers than are those treated with aspirin under high shear stress.

Much attention has recently been focused on the interaction between unusually large von Willebrand factor multimers (UL-VWFM) and platelets under high shear stress in pathological thrombus formation. The antiplatelet drugs acetylsalicylic acid (aspirin) and a thienopyridine derivative (ticlopidine) are commonly used to treat cerebral ischemia but exert different effects on high-shear-stress-induced platelet aggregation (H-SIPA) in the plasma. To examine the effects of these drugs in the absence of plasma factors, we studied H-SIPA using washed platelets (WPs) and purified UL-VWFM. WPs were prepared from the blood of 9 aspirin-treated and 11 ticlopidine-treated patients with cerebral ischemia, and H-SIPA in the presence of UL-VWFM was measured using a cone plate aggregometer. Plasma levels of VWF antigen with its multimer analysis, ristocetin cofactor and VWF-cleaving protease (ADAMTS13) activity were also measured. Forty-six healthy volunteers from 2 age groups, 20-40 years (n=20) and 41-60 years old (n=26), were also tested as controls. H-SIPA was significantly inhibited for ticlopidine-treated platelets, but it was observed to a lesser extent for aspirin-treated platelets. For both groups, no difference in the plasma levels of VWF antigen, ristocetin cofactor and ADAMTS13 activity was noted. All patients possessed UL-VWFM, and it was detected in healthy volunteers with increasing frequency with increasing age. Under plasma-free conditions, platelets from aspirin-treated patients exhibit marginal but significant inhibition of H-SIPA. Furthermore, the presence of UL-VWFM in the plasma of patients and normal volunteers is directly related to their age rather than being a consequence of underlying disease.

Thrombotic thrombocytopenic purpura associated with polyarteritis nodosa.

We present a case of classical polyarteritis nodosa (PN) overlapping thrombotic thrombocytopenic purpura (TTP). A 70-year-old woman was transferred to our hospital because of general fatigue and fever. On admission, laboratory findings revealed leukocytosis, normochromic normocytic anemia and renal dysfunction. About one week later, she developed disturbance of consciousness, and laboratory findings revealed rapidly progressive thrombocytopenia and renal dysfunction. We suspected the presence of microscopic polyangiitis (MPA), based on mild elevation of myeloperoxidase (MPO) anti-neutrophil cytoplasmic antibody (ANCA). On post-admission Day 11, renal biopsy was performed but the diagnosis of MPA could not be confirmed because of the absence of glomerular crescent formation or vasculitis. However, the biopsy specimen showed many collapsed glomeruli and interstitial inflammation, indicating the presence of occlusive lesions, such as vasculitis in larger arteries. We instituted methylprednisolone pulse therapy, cyclophosphamide and plasma exchange, because the clinical symptoms also satisfied the criteria of TTP. Despite the intensive treatment, the patient died on 43rd day of hospitalization due to thalamic hemorrhage. Autopsy showed typical findings of classical PN including disruption of arterial walls and fibrinoid necrosis in the medium-sized arteries of the kidneys and colon. We detected reduced activity of von Willebrand factor-cleaving protease (VWF-CP) and the presence of plasma inhibitory IgG against VWF-CP. A better understanding of the mechanisms would be useful.

Impaired activity of plasma von Willebrand factor-cleaving protease may predict the occurrence of hepatic veno-occlusive disease after stem cell transplantation.

Hepatic veno-occlusive disease (VOD) is a life-threatening complication after stem cell transplantation (SCT), characterized by thrombus formation in hepatic venules leading to a symptom triad of hyperbilirubinemia, hepatomegaly, and ascites. Multifactorial defects in the hemostatic system may contribute to its pathogenesis, but its remains to be investigated. Unusually large VWF multimers (UL-VWFMs), produced in and released from vascular endothelial cells, are most biologically active in the interaction with platelets under a high shear stress. UL-VWFMs are cleaved and degraded into smaller VWFMs by a specific liver producing plasma protease, termed VWF-cleaving protease (VWF-CPase), which has recently been identified as a metalloprotease solely produced in liver, termed ADAMTS13. Herein, we studied the correlation between plasma VWF-CPase activity and UL-VWFMs in 21 patients who received SCT, seven patients with VOD and 14 patients without VOD. In non-VOD patients, activities (mean +/- 1s.d.) of VWF-CPase were 78 +/- 17% of the control before the conditioning regimen, 76 +/- 18% on day 0, 64 +/- 19% on day 7, 57 +/- 23% on day 14, 68 +/- 13% on day 21 and 79 +/- 19% on day 28 after SCT. The respective values in VOD patients were 32 +/- 19%, 27 +/- 15%, 18 +/- 11%, 22 +/- 18%, 26 +/- 22% and 12 +/- 4%. Thus, VWF-CPase activity was significantly reduced in VOD patients, even before the conditioning regimen, and such a difference was not found in other laboratory tests. However, despite such a clear difference, UL-VWFMs were present in plasmas of both patient groups, together with the increase of VWF antigen and ristocetin cofactor activity. These results indicate that the measurement of this enzyme activity is extremely useful in predicting the occurrence of VOD prior to a demonstration of its direct involvement in its pathogenesis.

Ticlopidine-Associated thrombotic thrombocytopenic purpura with an IgG-type inhibitor to von Willebrand factor-cleaving protease activity.

A 41-year-old Japanese man complained of a left-sided visual disturbance. Imaging by magnetic resonance angiography revealed a narrowing of the left internal cervical artery. Thus, ticlopidine (Tc) administration was started at a daily dose of 300 mg. However, 2 weeks later, severe thrombocytopenia, fever, nausea, and psychiatric symptoms developed; Tc was therefore discontinued. Based on the diagnostic hallmark of 5 clinical signs, the patient's disease was diagnosed as thrombotic thrombocytopenic purpura (TTP). Daily plasmapheresis was performed for the first 4 days, and the patient's clinical signs gradually improved. Von Willebrand factor-cleaving protease (vWF-CPase) activity in his plasma was less than 3% of that of the control sample at diagnosis, but that value recovered steadily following plasmapheresis. In addition, immunoglobulin G purified from the patient plasma inhibited vWF-CPase activity in normal plasma with a specific activity of 0.8 Bethesda units/mg. No sign of TTP relapse has been noted following cessation of Tc. Thus, it was concluded that the patient developed TTP by producing an inhibitory autoantibody against vWF-CPase activity that was presumably triggered by Tc administration.

Upshaw-Schulman syndrome revisited: a concept of congenital thrombotic thrombocytopenic purpura.

Upshaw-Schulman syndrome (USS) is a congenital bleeding disorder characterized by repeated episodes of thrombocytopenia and microangiopathic hemolytic anemia that respond to infusions of fresh frozen plasma. Inheritance of USS has been thought to be autosomal recessive, because 2 siblings in the same family are often affected but their parents are asymptomatic. Recently, chronic relapsing thrombotic thrombocytopenic purpura (CR-TTP), reported almost exclusively in adults, was shown to be caused by inherited or acquired deficiency in the activity of a plasma von Willebrand factor-cleaving protease (vWF-CPase). The pathogenesis of USS is unknown, and a relationship between CR-YEP and USS has not been reported. We studied 3 unrelated USS patients (ST, SY, and KI) who presented with severe indirect neonatal hyperbilirubinemia. All 3 patients had undetectable vWF-CPase activity, and the inhibitors to vWF-CPase were all negative. In their parents with no clinical symptoms, vWF-CPase activities as a percentage of control samples (mother/father) were 17/20 for ST, 60/45 for SY, and 36/5.6 for KI. Thus, USS and vWF-CPase activity appear to be coinherited as autosomal recessive traits. Transfusion of fresh frozen plasma in 2 patients (ST and SY) resulted in the expected maximal increment of approximately 7% to 8% in vWF-CPase activity at 1 to 4 hours, but the levels became less than 3% within 2 days. After this decrease, platelet counts increased, plateaued in the normal range at 10 to 12 days, and declined thereafter. Thus, the 2 to 3 weeks of therapeutic benefit from plasma infusions will be discussed in relation to the intravascular lifetime of vWF-CPase.

Plasma of patients with Upshaw-Schulman syndrome, a congenital deficiency of von Willebrand factor-cleaving protease activity, enhances the aggregation of normal platelets under high shear stress.

Upshaw-Schulman syndrome (USS) is an autosomal recessive disorder characterized by repeated episodes of chronic thrombocytopenia and microangiopathic haemolytic anaemia (MAHA) that responds dramatically to infusions of fresh frozen plasma (FFP). Recent studies have provided consistent evidence that USS is a congenital deficiency of plasma von Willebrand factor-cleaving protease (VWF-CPase) activity and, therefore, unusually large VWF multimers (UL-VWFMs) are present in the plasma. However, the molecular mechanism of the clinical symptoms of USS is not well understood. We studied the relationship between UL-VWFMs and thrombocytopenia in two USS patients by analysing platelet aggregation using a mixture of the patient's plasma and normal washed platelets under high shear stress. Our results clearly showed a remarkably enhanced high shear stress-induced platelet aggregation (H-SIPA) by the patient's plasma. At 24 h after FFP infusion (approximately equal to 10 ml/kg body weight), the enhanced H-SIPA became almost completely normalized but, 2 d later, it began to return to the preinfusion level. These results were in accordance with the change in VWFM patterns. The specific effects of enhanced H-SIPA on VWF, platelet glycoprotein Ib and endogenous ADP released from platelets upon stimulation were confirmed using reagents that specifically inhibit their respective functions. Our present results clearly indicate that thrombocytopenia in USS patients is caused by a combination of the presence of UL-VWFMs, platelets and high shear stress generated in the microcirculation.

Mechanisms underlying the age-related decrement in the human sweating response.

To examine the mechanisms underlying the age-related decrement in the ability to sweat, seven older (64-76 years) and seven younger (20-24 years) men participated in a 60-min sweating test. The test consisted of placing the subject's lower legs in a water bath at 42 degrees C while sitting in a controlled environment of 35 degrees C ambient temperature and 45% relative humidity. The rectal (Trc) and skin temperatures, local sweating rates (m(sw): on the forehead, chest, back, forearm and thigh) and the frequency of sweat expulsion (f(sw)) were measured during the test. No group difference was observed in the mean body temperature (Tb) throughout the passive heating, although the older men had a higher Tre and a lower mean skin temperature during the last half of the 60-min test. There were no group differences in the Tb threshold for sweating, although the time to the onset of sweating tended to be longer for the older men regardless of body site. The m(sw) increased gradually for approximately 35 min after the start of heat exposure in the older men and for 30 min in the younger men and then reached a steady state. During the first half of the test, the older men had a significantly lower m(sw) at all sites. During the last half of the test, only m(sw) on the thigh was significantly lower in the older men than in the younger men. There was no group difference in the slope of f(sw) versus Tb (an indicator of the change in the central sudomotor response to thermal input). The slope of m(sw) versus f(sw) (an indicator of the change in peripheral activity in response to central sudomotor changes) was significantly lower on the thigh in the older men, but there were no differences for the other sites. These results suggest that in older men the lower thigh m(sw) observed during the last half of the heat test was possibly due to age-related modifications of peripheral mechanisms involving the sweat glands and surrounding tissues. It was not due to a change in the central drive to sudomotor function. Furthermore, the sluggish m(sw) responses in the older men appear to have been related to age-related modifications of the sensitivity of thermoreceptors in various body regions to thermal stimuli. They may also involve lower sweat glands' sensitivity to cholinergic stimulus or sluggish vasodilatation, and do not reflect age-related changes in the central drive.

Effect of exercise on endotoxin shock with special reference to changes in concentration of cytokines.

The effects of exercise on susceptibility to endotoxin shock and the serum levels of interleukin-1 (IL-1) and interleukin-6 (IL-6) were studied in mice primed with Propionibacterium acnes (P. acnes, 1.0 mg). Endotoxin shock was induced by injection of lipopolysaccharide (LPS, 0.2 mg) 7d after the priming injection of P. acnes. The exercise-loaded mice performed voluntary exercise for 120 min on a running-wheel and LPS was injected into the tail vein immediately after the end of exercise. Two of the nine animals in the exercised group survived, whereas all the mice in the control group died. The mean survival time after LPS injection in the exercised group (10.75 +/- 4.51 h) was significantly longer than that in the control group (3.3 +/- 1.34h). The serum concentrations of IL-1 (2,883 +/- 1,542 U/ml) and IL-6 (966 +/- 619 ng/ml) in the exercised group, sampled 2h after LPS challenge, were significantly lower than those in the control group (6,571 +/- 2,766 U/ml and 2,428 +/- 1,228 ng/ml, respectively). It is suggested that the exercise-induced inhibition of endotoxin shock may be caused, at least partly, by the decreased production of IL-1 and IL-6.

Effect of muscular exercise by bicycle ergometer on erythrocyte purine nucleotides.

The effect of muscular exercise by bicycle ergometer on erythrocyte purine nucleotides was investigated in 6 athletes. Muscular exercise increased the concentration of inosine monophosphate from 5.9 +/- 1.1 to 7.3 +/- 1.3 nmol/ml in venous erythrocytes and from 5.7 +/- 1.0 to 6.8 +/- 1.4 nmol/ml in arterial erythrocytes, respectively, while it decreased the concentrations of adenosine diphosphate and adenosine monophosphate from 189.3 +/- 42.7 to 141.2 +/- 26.9 and from 26.0 +/- 7.8 to 15.7 +/- 4.3 nmol/ml in venous erythrocytes and also decreased their concentrations from 195.1 +/- 51.0 to 141 +/- 29.2 and from 26.5 +/- 9.6 to 14.8 +/- 3.0 nmol/ml in arterial erythrocytes, respectively. The muscular exercise also increased the concentration of inorganic phosphate in venous plasma from 1.12 +/- 0.12 to 1.46 +/- 0.22 mmol/l, that of NH3 in blood from 41.90 +/- 6.91 to 150.22 +/- 50.80 mumol/l, that of lactic acid in blood from 7.90 +/- 1.71 to 61.03 +/- 18.43 mg/dl and that of hypoxanthine in venous plasma from 1.32 +/- 0.36 to 18.14 +/- 4.87 mumol/l, respectively. Therefore, in vitro study was performed to investigate whether inorganic phosphate, NH4Cl, lactic acid or hypoxanthine affects nucleotides in erythrocytes. After 2 hour-incubation, 2 mM inorganic phosphate increased the erythrocyte concentration of inosine monophosphate 1.6 fold but decreased the erythrocyte concentrations of adenosine monophosphate and adenosine diphosphate 0.72 and 0.89 fold, respectively, in the suspension (pH 7.35), as compared with 1 mM inorganic phosphate. However NH4Cl, lactic acid or hypoxanthine did not affect erythrocyte purine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Role and regulation of interleukin (IL)-2 receptor alpha and beta chains in IL-2-driven B-cell growth.

Substantial proportions of resting B cells constitutively express low levels of IL-2 receptor (IL-2R) alpha and/or beta chains. The expression of these chains is differentially regulated by anti-IgM and IL-2/IL-4. The anti-IgM induces IL-2R alpha chain expression, whereas each of the two cytokines induces IL-2R beta chain expression in a dose-dependent manner. Moreover, IL-2 induces the growth of B cells, when the cells were pretreated with IL-2 or IL-4 for 24 h. The magnitude of this IL-2-driven B-cell growth depends upon the level of IL-2R beta chain expression. Costimulation of the B cells with IL-2 and anti-IgM shifts the dose-response curve, and the cells proliferate at an IL-2 concentration as low as 40 pM. These results indicate that the levels of anti-IgM-induced IL-2R alpha chain and IL-2-induced IL-2R beta chain determine the sensitivity of the cells to IL-2.

Estimation of total oxygen uptake in women during exercise by total heart beats and aerobic fitness.

The present study was performed to find a practical method for estimating total O2 uptake (TVO2) of women during exercise based on total heart beats (THB) and aerobic fitness level, and examine the influence of the type of exercise on the estimation. After 60 observations on 20 female subjects tested by the cycle ergometer, the following formula was derived. TVO2 (ml.kg-1) = SR125 X (61.0 X mean HR + 2543) X THB X 10(-4), where mean HR is mean heart rate (beats.min-1) in exercise, and SR125 is the slope of the regression line between accumulated heart beats and accumulated O2 uptake during exercise at 125 beats.min-1 of mean HR. SR125 was significantly correlated not only to VO2max but also each score (X) in any simple endurance tests, such as the step test for 3 min, yielding a formula, SR125 = -0.00115X + 0.3081. Both formulae indicate that the TVO2 of any exercising person can be estimated from THB and mean HR when SR125 was determined by the simple endurance test. The discrepancy between both TVO2 as estimated by our method and measured directly by the Douglas bag method during walking on a treadmill was not significant with that during the cycling on an ergometer. Accordingly, our method may possibly be used for estimating TVO2 in exercise mainly using the leg muscles such as in cycling and walking.