Kazuyo Hiyoyama

Hemostatic Molecular Markers Before the Onset of Disseminated Intravascular Coagulation

We retrospectively measured various hemostatic markers in 240 patients with disseminated intravascular coagulation (DIC) before the onset of DIC and in 110 non‐DIC patients, and examined their usefulness for the diagnosis of pre‐DIC. Changes in prothrombin time ratio and fibrinogen levels were not significant before the onset of DIC. The plasma levels of fibrinogen and fibrin degradation products before the onset of DIC were increased and the platelet count was gradually reduced in nonleukemic patients; these changes were already significant in the non‐DIC state. The plasma levels of thrombin–antithrombin complex (TAT), plasmin–plasmin inhibitor complex (PPIC), D‐dimer, and soluble fibrin monomer (sFM) were increased before the onset of DIC. In leukemic patients, the plasma levels of sFM on day 5, those of TAT on day 3, and D‐dimer on day 1, were significantly increased before the onset of DIC. The levels of most hemostatic markers 7 days before the onset of DIC were not different from those observed in the non‐DIC state. In nonleukemic patients, only D‐dimer, sFM, and TAT levels were significantly increased 7 days before the onset of DIC compared with values in the non‐DIC state. The positive rate of hemostatic markers for the diagnosis of DIC, TAT, and PPIC were high during the pre‐DIC and non‐DIC groups. The plasma levels of sFM and D‐dimer were low in non‐DIC and increased gradually during the pre‐DIC state. These findings suggest that hemostatic molecular markers such as sFM, D‐dimer, and TAT are useful for the diagnosis of pre‐DIC, although their cutoff values were different among various diseases. Am. J. Hematol. 60:273–278, 1999.

Increased serum levels of thrombopoietin in patients with thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura, or disseminated intravascular coagulation

The serum levels of thrombopoietin (TPO) were measured in 16 patients with thrombotic thrombocytopenic purpura (TTP), 12 with hemolytic uremic syndrome (HUS), 10 with aplastic anemia (AA), 10 with disseminated intravascular coagulation (DIC), and 71 with idiopathic thrombocytopenic purpura (ITP). The serum TPO levels were measured with a sensitive sandwich enzyme-linked immunosorbent assay. The serum TPO level in the ITP group (1.68 ± 0.85 fmol/ml) were not significantly increased compared with those of the normal subjects. The TPO levels in the TTP (2.77 ± 1.38 fmol/ml) and HUS groups (5.77 ± 4.41 fmol/ml) were higher than those of the normal subjects. The patients with AA (12.7 ± 8.0 fmol/ml) and those with DIC (13.3 ± 5.7 mol/ml) had significantly higher serum TPO levels than did the normal subjects and ITP patients. The TPO levels were well correlated with the platelet counts in the TTP patients, and were negatively correlated with the platelet counts in the ITP patients. These results suggest that the serum TPO levels in some thrombocytopenic diseases are regulated not only by the platelet count and the megakaryocyte mass, but also by other factors.

Changes of plasma hemostatic markers during percutaneous transluminal coronary angioplasty in patients with chronic coronary artery disease

Changes of hemostatic parameters during percutaneous transluminal coronary angioplasty (PTCA) in 75 patients with chronic coronary artery disease were evaluated. Plasma levels of D‐dimer, soluble fibrin monomer, plasmin‐α2 antiplasmin inhibitor complex, and tissue factor (TF) were significantly increased in all patients with chronic coronary artery disease. The activity of antithrombin and protein C and the levels of protein C antigen were significantly decreased 1 hr after PTCA, but they returned to normal range 1 day after PTCA. There was no significant difference in the level of plasma APC‐PCI complex before and 1 hr after PTCA. The plasma levels of D‐dimer, soluble fibrin monomer, thrombomodulin, TF and PPIC were significantly decreased 1 hr, and the plasma levels of plasmin‐α2 antiplasmin inhibitor complex 1 day after PTCA. These findings suggest that the decrease of protein C and antithrombin resulted in activation of the coagulation system. One hour after PTCA, the plasma levels of (total‐free) TF pathway inhibitor (TFPI) were significantly decreased, but the plasma levels of total and free‐TFPI were significantly increased, suggesting that comsumption of (total‐free) TFPI occurs during PTCA. Overall, these findings suggest that the hypercoagulable state improves during PTCA and that transient decrease of antithrombin, protein C, (total‐free) TFPI or plasmin‐α2 antiplasmin inhibitor complex may cause restenosis of coronary artery. Am. J. Hematol. 61:238–242, 1999.

Changes in plasma tissue factor pathway inhibitor levels during the clinical course of disseminated intravascular coagulation

In healthy volunteers, the plasma total tissue factor pathway inhibitor (TFPI) level was 68.7 ± 14.1 ng/ml; the plasma free TFPI level, 17.7 ± 5.4 ng/ml; the lipoprotein-associated TFPI (LP-TFPI), 51.1 ± 12.0 ng/ml; the free TFPI/total TFPI ratio 0.26 ± 0.07; and the plasma tissue factor levels were 149 ± 46 pg/ml.Plasma tissue factor levels in patients with disseminated intravascular coagulation (DIC) were significantly higher than those in pre-DIC patients or in non-DIC patients. Plasma total-TFPI, free-TFPI and LP-TFPI levels were significantly higher in DIC patients than those in pre-DIC patients or in non-DIC patients. Before the onset of DIC, the plasma levels of tissue factor gradually increased, and 3 days before the onset of DIC they were significantly higher than those in non-DIC patients. The plasma levels of tissue factor reached their highest level 1 day before the onset of DIC and gradually decreased after the onset of DIC. Plasma levels of total-TFPI, free-TFPI, and LP-TFPI gradually increased before the onset of DIC, and the total-TFPI and LP-TFPI reached their highest levels at the onset of DIC. Plasma free-TFPI reached highest level one day after the onset of DIC. During the clinical course of DIC, the plasma level of tissue factor was the first to increase, then that of LP-TFPI and finally the free-TFPI plasma levels. These differences in the peak plasma levels of tissue factor, free-TFPI, and LP-TFPI might be related to the clinical course of DIC.

Hemostatic Abnormalities in Patients With Thrombotic Complications on Maintenance Hemodialysis

Before hemodialysis (HD), plasma levels of tissue factor (TF), free-TF pathway inhibitor (TFPI) and thrombo modulin (TM) were significantly higher in patients with HD than in healthy volunteers. Plasma levels of (T-F) TFPI and plasmin plasmin inhibitor complex (PPIC) were significantly higher in patients with HD than in healthy volunteers. During HD, plasma levels of TF and (T-F) TFPI were not significantly increased, but plasma levels of total TFPI and free TFPI at 1 hour after and at the end of HD were significantly increased, compared with levels before start of HD. Plasma level of PPIC 1 hour after start of HD was significantly higher than before start of HD, and plasma levels of thrombin antithrombin com plex (TAT). PPIC. D-dimer. TM, and protein C (PC) at the end of HD were significantly higher than before start of HD. In patients with thrombosis complications, plasma TF levels were significantly higher than in patients without thrombotic com plications during HD. Plasma levels of PC were significantly lower in patients with thrombotic complications than in patients without thrombotic complications. There was no significant difference between both groups during HD in hemostatic pa rameters, with the exception of TF and PC. Hemostatic abnormalities existed in patients with HD; espe cially, increased TF and decreased PC might cause thrombotic complications.

Increased soluble fibrin monomer and soluble thrombomodulin levels in non-insulin-dependent diabetes mellitus.

We measured the plasma levels of fibrinogen, D-dimer, thrombin-antithrombin complex (TAT), plasmin-plasmin inhibitor complex (PPIC), soluble fibrin monomer (SFM), tissue-type plasminogen activator (t-PA) and thrombomodulin (TM) in patients with non-insulin-dependent diabetes mellitus (NIDDM). There were no significant differences in the hemostatic parameters between the 77 patients with NIDDM and healthy control subjects, although the plasma levels of fibrinogen, D-dimer, TAT, and PPIC in the NIDDM patients were slightly higher than those in the healthy controls. Among the NIDDM patients divided into three groups by the urinary albumin excretion (UAE) level, there was no significant difference in age or sex among the normo-, micro-, and macroalbuminuria groups, and the HbA1C level in the micro- and macroalbuminuria groups were slightly higher than those in the normoalbuminuria group. There was no significant difference in activated partial thromboplastin time, prothrombin time, fibrinogen, TAT, PPIC, D-dimer, or t-PA among these three groups. The plasma SFM and TM levels in the macroalbuminuria group were significantly higher than those in the normo- and microalbuminuria groups. The relationships between HbA1C and the hemostatic parameters were poor, but the plasma TM and SFM levels were significantly correlated with the urine albumin index.

Plasma sFas and sFas ligand levels in patients with thrombotic thrombocytopenic purpura and in those with disseminated intravascular coagulation

Fas, a member of the tumor necrosis receptor superfamily, is 36 kD surface protein containing a single transmembrane region and induces apoptosis by Fas–Fas ligand binding. Soluble Fas (sFas) is produced as the form lacking 21 amino acid residues containing the transmembrane domain by alternative splicing. We found that the plasma sFas levels of 33 patients with thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), 19 patients with disseminated intravascular coagulation (DIC), and 10 non‐DIC patients with multiple organ failure (MOF) were significantly higher than those of 21 non‐DIC patients without organ failure and those of 25 healthy volunteers. The plasma sFas ligand levels of the TTP/HUS patients, the DIC patients, and the non‐DIC patients with MOF were significantly higher than those of the non‐DIC patients without organ failure and those of the healthy volunteers. The plasma sFas levels were significantly correlated with the plasma sFas ligand levels in all subjects. The plasma thrombomodulin (TM) levels were increased significantly in the TTP/HUS patients, the DIC patients, and the non‐DIC patients with MOF compared with the levels of the non‐DIC patients without organ failure and the healthy volunteers. The plasma sFas antigen levels were correlated significantly with the plasma TM levels in all subjects. These findings suggest that the increases of sFas and sFas ligand that cause apoptosis might be related to the vascular endothelial cell injuries in TTP and DIC with organ failure. Am. J. Hematol. 61:21–25, 1999.

Increased activated protein C-protein C inhibitor complex level in patients positive for lupus anticoagulant.

Activated protein C (APC)-protein C inhibitor (PCI) complex and APC-α1antitrypsin (α1AT) complex levels were measured in 29 patients positive for lupus anticoagulant (LA). LA was considered positive if two of the following three criteria were fulfilled: (1) prolongation of the activated partial thromboplastin time, (2) prolongation of the kaolin clotting time (KCT) and KCT mixing test, and (3) prolongation of the dilute Russells viper venom time (DRWT) and DRWT/DR WT with high lipid concentration. Plasma thrombin-antithrombin III (AT-III) complex and plasmin-α2-antiplasmin inhibitor complex levels in patients positive for LA were increased slightly, but not significantly, and FDP-d-dimer and t-PA levels were not markedly increased. Plasma PAI-1 level in the LA-positive patients was significantly increased compared with normal volunteers. AT-III activity, protein C antigen, PCI antigen, and protein S antigen levels in the LA-positive patients were virtually normal, while protein C activity was slightly, but not significantly, decreased. APC-PCI complex level was increased in all LA-positive patients, and was not detectable in patients with systemic lupus erythematosus and normal volunteers. APC-α1AT complex was increased slightly, in only two LA-positive patients; it was not detectable in the other patients or in the normal volunteers. These findings suggest that patients positive for LA are in a hypercoagulable state and that protein C activity in such patients is decreased, due to the activation of this protein.

Plasma levels of activated FVII in various diseases.

Plasma activated factor VIIa (FVIIa) levels were measured in various diseases using mutant tissue factor (TF). FVIIa levels in thrombotic patients and patients with idiopathic thrombocytopenic purpura were significantly higher than those in healthy control subjects. The plasma FVIIa levels in thrombotic patients treated with warfarin were similar to those of control subjects. The plasma FVIIa levels in pregnant women and patients with systemic lupus erythematosus, infection or malignancies were high. However, the levels in patients with disseminated intravascular coagulation (DIC) were not significantly increased. DIC patients are in a severe hypercoagulable state, and exhibit severe consumption of coagulation factors. The slightly increased FVIIa level in the DIC patients observed is probably considered to be caused by consumption of coagulation factors. The plasma FVIIa level was poorly correlated with other hemostatic parameters except for protein C in our analysis of all cases. In the analysis of DIC and thrombotic patients treated without warfarin, the plasma FVIIa level was negatively correlated with TF antigen. Plasma FVIIa levels might reflect hypercoagulability in thrombotic diseases, and a normalized FVIIa level in patients with thrombotic diseases should be considered to be associated with DIC.

Hemostatic markers in Japanese patients undergoing anticoagulant therapy under thrombo-test monitoring.

The objective of this study was to evaluate several molecular markers of hemostasis in 84 patients with hypercoagulable state, treated with warfarin under thrombo-test (TT) monitoring; TT was expressed as percent of control (TT%). In all patients, the average values of international normalized ratio (INR) of prothrombin time (PT;PT-INR) was 1.68+/-0.49; this increase in PT-INR was not, however, significant in patients under TT% monitoring. There were no thrombotic or severe bleeding complications in these patients during a period of 2 years. Plasma levels of thrombin-antithrombin complex (TAT), plasmin-plasmin inhibitor complex (PPIC), D-dimer, and soluble fibrin monomer (sFM) were slightly increased, suggesting that anticoagulant therapy was not completely effective in our Japanese patients based on the values of TT%. Activated partial thromboplastin time, PT-INR, TT% and protein C activity were significantly correlated with the dose of warfarin; fibrinogen, activated thromboplastin, TAT, PPIC, D-dimer, sFM, protein S and thrombomodulin were not significantly correlated with the dose of warfarin. The PT-INR was negatively correlated with TT%, protein C and protein S, and the correlation between PT-INR and TT-INR was better than that between PT-INR and TT%. The range of TT% was not correlated with the plasma levels of TAT, PPIC, D-dimer or sFM, but the range of PT-INR was correlated with the plasma level of TAT, D-dimer and sFM. The percentage of TAT, D-dimer and sFM within normal range was significantly low in patients with high PT-INR. These finding showed that PT-INR is better than TT% for monitoring the anticoagulant therapy with warfarin, and that TT should be expressed as INR. The values of PT-INR should be more than 1.7 during the anticoagulant therapy with warfarin in Japanese patients with high risk of thrombosis.