Plasma levels of platelet-derived microparticles in patients with obstructive sleep apnea syndrome.

AIM: Obstructive sleep apnea syndrome (OSAS) has been associated with high cardiovascular morbidity and mortality, and patients suffer from repeated episodes of hypoxia. Platelet-derived microparticles (PDMPs) are released via platelet activation by various agonists, including inflammatory cytokines or high shear stress. Plasminogen activator inhibitor -1 (PAI-1) is a fibrinolytic marker and soluble fibrin (SF) is a coagulation activation marker. We examined plasma levels of PDMPs, PAI-1 and SF in patients with OSAS. We also examined the effects of continuous positive airway pressure (CPAP) on plasma levels of PDMPs. METHODS: Full polysomnography (PSG) monitoring was performed on 27 patients. The apneahypopnea index (AHI) of 5 events/h or less than 30 events/h indicated mild to moderate OSAS, and an AHI of 30 events/h or more indicated severe OSAS. Plasma levels of PDMPs were measured using an ELISA kit, and PAI and SF were determined by a latex immunoassay. In addition, the effects of CPAP treatment were studied in 7 patients. RESULT: The plasma level of PDMPs was significantly higher in patients with severe OSAS (15.8±10.4 U/mL) than normal controls (10.8±7.1 U/mL, p < 0.05) and patients with mild to moderate OSAS (9.2±3.5 U/mL, p < 0.05). The plasma levels of PDMPs correlated with the AHI (r = 0.39, p < 0.05). In addition, CPAP treatment decreased the plasma level of PDMPs (11.9±5.6 U/mL to 6.7±3.2 U/mL, p < 0.05). CONCLUSIONS: Patients with OSAS might be at increased cardiovascular risk due to elevated PDMPs. Moreover a decrease in the plasma level of PDMPs by treatment with CPAP might reduce cardiovascular risk.

Two case reports of inherited antithrombin deficiency: a novel frameshift mutation and a large deletion including all seven exons detected using two methods.

An inherited antithrombin deficiency is an autosomal dominant thrombotic disorder. We identified two pedigrees of inherited type I antithrombin deficiency and two responsible mutations in each. A novel 21-22delAA appeared to have caused a frameshift with a premature termination at amino acid +63 in one patient and a large deletion including all seven exons was identified by multiplex ligation-dependent probe amplification in the other. Some asymptomatic relatives of the second patient had the same mutation. The present findings support the value of using more than one method of gene analysis and of studying the families of probands with inherited thrombotic disorders.

Aberrant increase in the immature platelet fraction in patients with myelodysplastic syndrome: a marker of karyotypic abnormalities associated with poor prognosis.

OBJECTIVES: Some patients with myelodysplastic syndrome (MDS) show a marked increase in the percentage of immature platelet fraction (IPF%) despite the absence of severe thrombocytopenia. To determine the significance of such an unbalanced increase in the IPF%, we investigated the IPF% and other laboratory findings of 51 patients recently diagnosed with MDS. METHOD: Subjects consisted of 80 healthy males, 90 healthy females, and 51 patients with MDS and 20 patients with idiopathic thrombocytopenic purpura (ITP). The IPF and IPF% were determined using a Sysmex XE-2100 system loaded with IPF Master software (XE IPF Master, Sysmex). Platelet counts were measured simultaneously. RESULTS: IPF% and platelet counts of these patients ranged from 1.1% to 25.1% (median, 5.3%) and from 6 to 260 x 10(9)/L (median, 71 x 10(9)/L), respectively. Twelve patients showed platelet counts more than 50 x 10(9)/L with 10% or more IPF%. All of the 12 patients had chromosome abnormalities including monosomy 7 and complex abnormalities involving 7 or 5q. In the other 39 patients who did not show the aberrant IPF% increase, chromosomal abnormalities were seen only in seven patients and none of them had chromosome 7 abnormalities. The IPF% of two patients increased to more than 10% in association with the appearance of monosomy 7. CONCLUSIONS: These findings suggest that a high IPF% in MDS patient may be a marker for karyotypic abnormalities with a poor prognosis, including chromosome 7 abnormalities.

Changes in molecular markers of hemostatic and fibrinolytic activation under various sampling conditions using vacuum tube samples from healthy volunteers.

Molecular makers such as thrombin-antithrombin complex (TAT), prothrombin fragment 1+2 (F1+2), soluble fibrin (SF), and D-dimer, are useful markers in the diagnosis and assessment of various thrombotic conditions. These markers are measured in plasma after blood sampling. Difficult blood sampling is known to falsely elevate plasma TAT levels. However, it is not known exactly why this occurs. In the present study, we examined how levels of molecular markers of haemostatic and fibrinolytic activation change under various sampling conditions using vacuum tube samples from healthy volunteers. When blood was sampled continuously by taking 10 consecutive vacuum tube samples following application of a tourniquet, blood sampling resulted in an accurate assessment of these molecular makers. When blood was sampled continuously by taking vacuum tube samples every one minute over a total of 9 minutes to investigate possible changes in the levels of the molecular markers over time, plasma levels of TAT, SF, and F1+2 gradually increased with time. Plasma levels of TAT, F1+2, and SF increased beyond the normal range over the course of nine minutes. When blood was sampled using three alternative methods, which varied in terms of the duration of needle puncture (sampling B), duration of tourniquet use (sampling C), or both (sampling A), plasma TAT and SF levels were significantly increased with all three methods, compared to control samples. Plasma F1+2 levels were significantly increased with sampling methods A and B, compared to control samples, but not with sampling method C. On the other hand, plasma D-dimer levels were not significantly altered by any of the sampling methods. In conclusion, the results suggest that molecular markers of haemostatic and fibrinolytic activation, except for D-dimer, may be affected by sampling method, particularly the duration of needle puncturing. Therefore, care needs to be taken when using TAT, F1+2, and SF levels to diagnose and estimate activation of the coagulation system.

[The positive rate of lupus anticoagulant according to the each understanding disease in our hospital].

We investigated positive rate of lupus anticoagulant (LA) according to the each understanding disease in our hospital. 596 cases (F/M 477/149, 7-87 y.o.) were examined from 2003 to 2004 years. LA tests were performed using 2 methods such as kaolin clotting time (KCT) mixing test and dilute Russell's viper venom time (dRVVT). The LA tests were most frequently ordered in dermatology, and the most common purpose of LA test was the check of existence of antiphospholipid (aPL) in patients with collagen diseases. The LA positive rate was the highest in patients with SLE among the collagen diseases, and in patients with cerebral infarction among the thrombotic diseases. The LA positive rate exceeded 40% in ITP and livedo reticularis. Moreover, LA positive rate was 16% in preoperative tests of the orthopedic patients without any physical diseases. Thus, it was suggested that there were considerable numbers of the asymptomatic LA positive persons. The LA positive cases based on KCT only accounted for about 60% of all the LA positive cases. Among the thrombotic patients, there were not the DVT/PE patients with only KCT positive. On the other hands, the KCT positive rate was higher than the dRVVT positive rate in patients with cerebral infarction. There were not dRVVT single positive cases in patients with recurrent abortion and ITP, but KCT single positive case accounted for about 90%. From these results, it is suggested that there is a difference in KCT and dRVVT about detecting aPL, and that care should be taken to interpret the LA test.

No interplay between the pathways mediating coagulation and inflammation in tissue factor-induced disseminated intravascular coagulation in rats.

OBJECTIVE: Previous reports have suggested an interplay between the pathways mediating coagulation and inflammation in endotoxemia and sepsis. The present study was designed to examine whether cross-signaling between the pathways mediating coagulation and inflammation occurs, as suggested by the pattern of cytokine production observed following tissue-factor (TF)-induced disseminated intravascular coagulation (DIC). DESIGN: Prospective, comparative, experimental study. SETTING: Laboratory at a university hospital. SUBJECTS: Male Wistar rats, aged 6-7 wks, and weighing 160-170 g. INTERVENTIONS: Male Wistar rats were administered TF (3.75 units/kg every 4 hrs), TF, and tranexamic acid (TA; 50 mg/kg every 4.5 hrs) or lipopolysaccharide (30 mg/kg every 4 hrs) via the tail vein, and blood was sampled at 0, 4, 8 and 12 hrs. MEASUREMENTS AND MAIN RESULTS: Subsequent alterations in thrombin-antithrombin complex and fibrinogen levels, as well as platelet counts, indicated that the severity of both types of experimental DIC (TF-induced and lipopolysaccharide-induced) was similar with respect to hemostatic activation and development of consumption coagulopathy. In lipopolysaccharide-induced DIC, a sharp increase in plasma tumor necrosis factor levels was observed at 4 hrs, after which a sharp decline was noted. Plasma levels of interleukin-6 were markedly increased at 4 hrs, after which a sustained elevation was observed for the duration of the experimental period (tumor necrosis factor, 1270 +/- 280, 180 +/- 40, and 120 +/- 30 pg/mL at 4, 8 and 12 hrs, respectively; interleukin-6, 5810 +/- 1320, 4850 +/- 730, and 5230 +/- 1280 pg/mL at 4, 8 and 12 hrs, respectively). On the other hand, tumor necrosis factor and interleukin-6 were not detected following TF-induced DIC (0 +/- 0 at 4, 8, and 12 hrs for both tumor necrosis factor and interleukin-6). In the TF+TA group, significant increases in tumor necrosis factor and interleukin-6 were observed, compared with the TF group. CONCLUSIONS: There is no overt interplay between the pathways mediating coagulation and inflammation in TF-induced DIC, as observed in lipopolysaccharide-induced DIC.

Immunoglobulin preparations attenuate organ dysfunction and hemostatic abnormality by suppressing the production of cytokines in lipopolysaccharide-induced disseminated intravascular coagulation in rats.

OBJECTIVE: We attempted to clarify the effect of immunoglobulin concentrates on the rat lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) model. DESIGN: Prospective, comparative, experimental study. SETTING: Laboratory at a university hospital. SUBJECTS: Male Wistar rats, aged 6 to 7 wks and weighing 160 to 170 g. INTERVENTIONS: Two kinds of experiments were performed. In the first, experimental DIC was induced by sustained infusion of 30 mg/kg LPS for 4 hrs via the tail vein, and two doses of immunoglobulin (25 or 100 mg/kg/4.5 hrs) were administered to rats 30 mins before infusion of LPS, after which immunoglobulin infusion was continued for a further 4 hrs. In the second, experimental DIC was induced by sustained infusion (5 mg/kg/1 hr) of LPS for 1 hr, and one dose of immunoglobulin (100 mg/kg/4 hrs) was administered to rats after LPS induction. The parameters were estimated at 4 hrs and 8 hrs in the first experiment and at 1, 5, and 10 hrs in the second one. MEASUREMENT AND MAIN RESULTS: Similar results were observed in the two experiments. Consumption coagulopathy and hemostatic activation were attenuated, especially when immunoglobulin was administered before LPS infusion. Plasma levels of creatinine and alanine aminotransferase were significantly depressed by coadministration of immunoglobulin. Marked glomerular fibrin deposition was observed in the LPS-induced DIC model, but this deposition was reduced by immunoglobulin. In the first stage of the experiment, plasma levels of tumor necrosis factor (TNF) and interleukin (IL)-6 were suppressed by coadministration of immunoglobulin. In the second, plasma levels of IL-6 were significantly suppressed by immunoglobulin. CONCLUSION: It was concluded that plasma levels of TNF and IL-6 could be significantly suppressed by immunoglobulin in the LPS-induced DIC model. Moreover, hemostatic abnormality, organ dysfunction, and glomerular fibrin deposition in this model were all ameliorated by immunoglobulin.

Kasabach-Merritt syndrome associated with giant liver hemangioma: the effect of combined therapy with danaparoid sodium and tranexamic acid.

n patients with Kasabach-Merritt syndrome (KMS), local activation of coagulation commonly results in disseminated intravascular coagulation (DIC). Progress of DIC is associated with 30-40% mortality as a result of uncontrollable hemorrhage. A 39-year-old woman with an enlarging giant liver hemangioma was diagnosed as having KMS with DIC. To control the hemorrhagic diathesis, we commenced combination therapy for DIC with danaparoid (1,250 Ux2/day, intravenously (IV)) and tranexamic acid (0.5 g x 3/day, peros (PO). Rapid improvement of the bleeding tendency and coagulopathy occurred in response to this treatment - that is, DIC was controlled without removing the giant hemangioma. The therapy did not restrict the behavior of the patient by continuous drip and angiography could be performed without bleeding. Such therapy may be beneficial in chronic DIC with activation of fibrinolysis.

Beneficial effects of urokinase on lipopolysaccharide-induced disseminated intravascular coagulation in rats: focus on organ function and endothelin levels.

In a rat model of lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC), we used urokinase (UK) in an attempt to clarify the role of fibrinolysis and to investigate changes in plasma endothelin levels. Two kinds of experiment were performed. The first one: experimental DIC was induced by sustained infusion of 30 mg/kg LPS for 4 h via the tail vein, and two doses of UK (2.0 or 10.0 IU/g/4.5 h) were administered to rats 30 min before infusion of LPS, after which UK infusion was continued for a further 4 h. The second one: experimental DIC was induced by sustained infusion of 1 mg/kg/10 min LPS for 10 min, and two doses of UK (2.0 or 10.0 IU/g/4 h) were administered to rats at 30 min after LPS infusion. The parameters described below were determined at 4 h in the first experiment, at 4 h and 8 h in the second one. The similar results were observed in both kinds of experiment. There were no significant differences in plasma thrombin-antithrombin complex, fibrinogen or platelet number among the three DIC groups, in both kinds of experiment. Plasma levels of D-dimer were significantly increased in the LPS + higher dose of UK group when compared with the LPS group. The increased plasma plasminogen activator inhibitor (PAI) activity seen in the LPS group was significantly suppressed in the groups receiving UK (especially higher dose of UK). In addition, the increased plasma levels of creatinine and alanine aminotransferase seen in the LPS group were significantly suppressed in the groups receiving UK (especially higher dose of UK). Plasma levels of endothelin, known to be a potent vasoconstrictive agent, were markedly elevated by LPS infusion, and were significantly suppressed in the groups receiving UK of both kinds of experiment, in a dose-dependent fashion compared with LPS group. Glomerular fibrin deposition was significantly suppressed in the groups receiving UK when compared with the LPS group. No manifestations of bleeding were observed in any of the groups. Enhanced fibrinolysis and depressed endothelin induced by UK thus appear to play an important role in preventing the development of organ failure in the LPS-induced DIC model.

Beneficial effect of low-molecular-weight heparin against lipopolysaccharide-induced disseminated intravascular coagulation in rats is abolished by coadministration of tranexamic acid.

OBJECTIVE: We examined the role of coagulation and fibrinolysis in lipopolysaccharide (LPS) induced disseminated intravascular coagulation (DIC) in rats, studying their contribution to fibrin deposition and organ failure in rats with LPS-induced DIC by concurrent administration of low molecular weight heparin (LMWH) with or without tranexamic acid (TA). METHODS: DIC was induced in male Wistar rats by a 4-h infusion of LPS (30 mg/kg) via the tail vein (LPS group). In the LPS+LMWH group LMWH (200 u/kg) was administered to rats from 30 min before the infusion of LPS for 4.5 h. In the LPS+LMWH+TA group LMWH (200 microg/kg) and TA (50 mg/kg) were administered to rats from 30 min before the infusion of LPS for 4.5 h. RESULTS: In the LPS+LMWH group lower plasma levels of TAT, D dimer, creatinine, and alanine aminotransferase were observed, along with less glomerular fibrin deposition and improved survival over rats administered LPS alone. However, these effects of LMWH were completely eliminated and damage beyond that observed in rats administered LPS alone resulted from combined administration of TA (LPS+LMWH+TA group), except that TAT and D dimer levels remained lower than in the group administered LPS alone. CONCLUSIONS: Suppression of fibrinolysis by TA (despite coadministration of LMWH) resulted in increased organ damage in this study, suggesting that depressed fibrinolysis plays a large role in organ failure resulting from LPS-induced DIC, even though hemostatic activation is moderately suppressed by LMWH.

Relationship between endothelin and the pathophysiology of tissue factor-induced and lipopolysaccharide-induced disseminated intravascular coagulation in rats: a study examining the effect of an endothelin receptor antagonist.

We investigated the relationship between endothelin, a potent vasoconstrictor peptide, and the pathophysiology of disseminated intravascular coagulation (DIC), using two models of DIC. Experimental DIC was induced by sustained infusion of 50 mg/kg lipopolysaccharide (LPS), or 3.75 U/kg thromboplastin, for 4 h via the rat tail vein. The effect of administration of a non-selective endothelin receptor antagonist (TAK-044) (2, 10, or 50 mg/kg, from -0.5 to 4 h) on thromboplastin-induced DIC was not significant. However, LPS-induced elevation of alanine aminotransferase, creatinine and glomerular fibrin deposition was significantly suppressed by co-administration of TAK-044 in a dose-dependent manner, although no effect of TAK-044 was observed on the platelet count, fibrinogen concentration or the level of thrombin-antithrombin complex. Moreover, plasma levels of D-dimer, which reflect the grade of fibrinolysis of cross-linked fibrin, were significantly increased by co-administration of each dose of TAK-044 in the LPS-induced DIC model in rats. Our results suggest that vasoconstriction, as well as depressed fibrinolysis, contribute to severe organ dysfunction in LPS-induced, but not thromboplastin-induced, DIC, and that endothelin plays a role in the development of organ injury in LPS-induced DIC in rats.

Significance of decreased plasma D-dimer levels following lipopolysaccharide-induced disseminated intravascular coagulation in rats.

Plasma D-dimer (DD) is considered to be one of the most useful markers in the diagnosis and assessment of disseminated intravascular coagulation (DIC). The present study was performed to clarify the role of DD in a rat model of lipopolysaccharide (LPS)-induced DIC in which low-molecular-weight heparin (LMWH) and tranexamic acid (TA) were used. We investigated whether a relationship exists between plasma DD levels and severity of DIC. Experimental DIC was induced in rats by a sustained 4-hour infusion of 30 mg/kg LPS administered via the tail vein (LPS group). Rats received either LPS alone (LPS group) or LPS combined with 200 U/kg LMWH (LPS+LMWH group) or 50 mg/kg TA (LPS+TA group) from -30 minutes to 4 hours. Blood was drawn from each rat at 4, 8, and 12 hours. Plasma levels of thrombin-antithrombin complex (TAT) and creatinine were suppressed in the LPS+LMWH group, and less glomerular fibrin deposition was observed compared with the LPS group. On the other hand, an increased level of creatinine and increased glomerular fibrin deposition were observed in the LPS+TA group compared with the LPS group. LMWH demonstrated a protective effect against LPS-induced DIC, resulting in increased survival at 12 hours, whereas TA had the opposite effect. From these results, it appears that LMWH protects against LPS-induced DIC, but TA exacerbates LPS-induced DIC. It was interesting that plasma levels of DD were almost completely suppressed by concurrent administration of either TA or LMWH in this LPS-induced DIC model. This finding suggested that plasma levels of DD were suppressed by inhibition of coagulation (reduced deposition of fibrin) in the LPS+LMWH group and that DD levels were also suppressed by inhibition of fibrinolysis (reduced degradation of fibrin by plasmin) in the LPS+TA group. Thus care should be taken when evaluating the significance of plasma DD levels, because suppressed levels can occur with progressive fibrin deposition and worsening organ dysfunction or improvement in the course of DIC.

Antithrombotic role of nitric oxide in rats under physiological conditions.

Although sepsis-induced release of nitric oxide (NO) is known to have an antithrombotic effect, it is unknown if NO exerts this same effect under physiological conditions. We have there-fore attempted to determine whether or not NO protects against thrombus formation in normal Wistar rats injected with various amounts (0.8, 4.0, 20.0 and 100 mg/kg/4 hr) of L-NAME (N (omega)-nitro-l-arginine methyl ester), an NO synthase inhibitor, via the tail vein. Plasma levels of D-dimer fragments of fibrin were significantly increased in rats receiving L-NAME (0.21+/-0.04, 0.22+/-0.05, 0.26+/-0.07, 0.59+/-0.17 micro g/mL, means+/-SE; p<0.05, 0.05, 0.05, 0.01: L-NAME 0.8, 4, 20, 100, respectively, compared with control levels: <0.06 micro g/mL), and thrombin-anti-thrombin complex (TAT) levels were significantly increased in rats receiving 20mg/kg/4 hr or greater doses of L-NAME (4.5+/-1.1, 4.7+/-1.4, 18.7+/-4.9, 42.5+/-4.0 ng/mL, NS, NS, p<0.05, 0.01, respectively, compared with control levels: 3.8+/-1.2 ng/mL). Glomerular fibrin deposition was increased in a dose-dependent manner in rats receiving L-NAME (6.8+/-1.5, 13.9+/-1.6, 32.4+/-2.6, 49.2+/-5.2%, p<0.05, 0.05, 0.01, 0.01, respectively, com-pared with control levels: 0.0+/-0.0%). Renal dysfunction and hepatic dysfunction were observed in rats receiving 20mg/kg/4 hr or greater, or 100mg/kg/4 hr, doses of L-NAME, respectively. Mean blood pressure was also elevated in rats receiving L-NAME in a dose-dependent manner. These findings suggest that NO, in addition to regulating blood pressure, is involved in prevention of thrombus formation under physiological circumstances.