Coordinated responses of natural anticoagulants to allogeneic stem cell transplantation and acute GVHD - A longitudinal study.

BACKGROUND: Allogeneic stem cell transplantation (SCT) enhances coagulation via endothelial perturbation and inflammation. Role of natural anticoagulants in interactions between coagulation and inflammation as well as in acute graft-versus-host disease (GVHD) are not well known. The purpose of this study was to define changes in natural anticoagulants over time in association with GVHD. PATIENTS AND METHODS: This prospective study included 30 patients who received grafts from siblings (n = 19) or unrelated donors (n = 11). Eight patients developed GVHD. Standard clinical assays were applied to measure natural anticoagulants, represented by protein C (PC), antithrombin (AT), protein S (PS), complex of activated PC with its inhibitor (APC-PCI) and by markers of endothelial activation: Factor VIII coagulant activity (FVIII:C) and soluble thrombomodulin (s-TM) at 6-8 time points over three months. RESULTS: Overall, PC, AT and FVIII:C increased in parallel after engraftment. Significant correlations between PC and FVIII:C (r = 0.64-0.82, p<0.001) and between PC and AT (r = 0.62-0.81, p<0.05) were observed at each time point. Patients with GVHD had 21% lower PC during conditioning therapy and 55% lower APC-PCI early after transplantation, as well as 37% higher values of s-TM after engraftment. The GVHD group had also increases of PC (24%), FVIII: C (28%) and AT (16%) three months after transplantation. CONCLUSION: The coordinated activation of natural anticoagulants in our longitudinal study indicates the sustained ability of adaptation to endothelial and inflammatory activation during allogenic SCT treatment. The suboptimal control of coagulation by natural anticoagulants at early stage of SCT may contribute to onset of GVHD.

Initial results of inflammatory response, matrix remodeling, and reactive oxygen species following PCI in acute ischemic myocardial injury in man.

BACKGROUND: Neutrophils and reactive oxygen species (ROS) are suggested to be involved in irreversible myocardial reperfusion injury and stunning. We investigated the relations between circulating biochemical markers and myocardium at risk (MaR), myocardial infarct (MI) size, salvage, and recovery of function in man. METHODS AND RESULTS: In patients undergoing PCI serial blood samples were acquired for markers of inflammatory response (myeloperoxidase [MPO], neutrophil-gelatinase-associated lipocalin [NGAL], interleukins 6 and 8 [IL-6/8], tumor necrosis factor-a [TNF-a], high-sensitive C-reactive protein [hsCRP]), matrix remodeling (matrixmetalloproteinase-9 [MMP-9]) and ROS (malondialdehyde [MDA], isoprostane [IsoP]). Samples were obtained before PCI and 1.5, 3, and 24 hours after reperfusion. Myocardial perfusion SPECT (MPS) was used to assess MaR. Late gadolinum-enhanced cardiac magnetic resonance imaging was performed for regional function in the acute setting, at 1 week and 6 months, and at 1 week also for MI size. Sixteen patients (15 men; 42-78 years) were enrolled, 12 of whom underwent MPS. Peak and cumulative NGAL and cumulative MMP-9 showed inverse correlations to MaR. No correlation was found for MI size. Peak MPO correlated inversely to salvage and to recovery of regional function in the infarcted segments at 1 week and 6 months. CONCLUSIONS: This is the first study in man to show inverse relations between circulating NGAL and MMP-9 and MaR. The current results do not support that ROS has a role in stunning in man. MI size showed no significant correlation to any parameter, challenging inflammatory treatment in reperfusion.

Thrombomodulin as a marker for bleeding complications during warfarin treatment.

BACKGROUND: The major adverse effect of warfarin treatment is hemorrhage. Several risk factors for bleeding complications are also risk factors for thromboembolic events, making the clinical decision to initiate or withhold anticoagulant treatment difficult. Specific markers that solely identify patients at high risk of bleeding would have great clinical impact. This study aimed to test if thrombomodulin (TM) concentrations were associated with bleeding complications, cardiovascular events, or mortality in long-term anticoagulant-treated patients. METHODS: In a longitudinal cohort study we followed up 719 patients receiving warfarin treatment for a mean duration of 4.2 years. All bleeding complications causing hospitalization were registered and classified. Soluble TM antigen (sTM) concentration in plasma was measured with an enzyme-linked immunosorbent assay method. RESULTS: During the follow-up time, 113 clinically relevant bleeding events and 73 major bleeding events occurred. Increased concentration of sTM was associated with both clinically relevant bleeding and major bleeding events after adjustment for age. In the multivariable models, hazard ratios for the highest tertiles compared with the lowest were 2.29 (95% confidence interval, 1.35-3.89) and 2.33 (95% confidence interval, 1.21-4.48), respectively. No association between sTM concentration and nonfatal ischemic cardiovascular events or all-cause mortality was found. CONCLUSIONS: Increased levels of sTM are associated with bleeding complications during warfarin treatment but not with cardiovascular events or all-cause mortality. Soluble TM antigen concentration has potential as a new specific marker to identify patients at high risk of bleeding during warfarin treatment.

Low plasma activated protein C-protein C inhibitor complex concentration is associated with vascular access failure in hemodialysis patients.

BACKGROUND: Vascular access failure is a common cause of morbidity in patients with end-stage renal failure on hemodialysis (HD). Activation of the coagulation system and formation of a thrombus play important roles in recurrent arteriovenous fistula/graft (AVFG) failure. Thrombin in complex with thrombomodulin (TM) activates the anticoagulant protein C and creates activated protein C (APC), which is subsequently inactivated by the protein C inhibitor (PCI). The plasma concentration of the complex between APC and PCI (P-APC-PCI complex) is increased in hypercoagulable states and is therefore a sensitive indicator of the degree of activation of blood coagulation. METHODS: Thirty-five HD patients dialyzed through a functioning AVFG were studied. The period of patency of AVFGs was recorded. Blood was drawn before and after the HD session for the analysis of the APC-PCI complex, soluble TM concentration and activity, von Willebrand factor antigen and homocysteine. RESULTS: Patients with frequent AVFG failures (n = 8) had a significantly lower level of P-APC-PCI complex (median 0.09 microg/l) than those with less frequent AVFG failures (median 0.18 microg/l; n = 27; p = 0.04). No other significant differences were found between the groups. CONCLUSION: Thus, a low level of P-APC-PCI complex may be associated with an increased risk of AVFG failure in HD patients. Further prospective studies are needed to confirm these results and to evaluate the possibility of prophylactic measures.

Dissociation of the Inflammatory Reaction following PCI for Acute Myocardial Infarction.

OBJECTIVES: Neutrophils are activated and infiltrate the myocardium after ischemia and reperfusion. The involvement of neutrophils in irreversible reperfusion injury is suggested by numerous experimental studies. The aim of this study was to investigate markers of neutrophil activation following reperfusion of acute myocardial infarction (AMI) accomplished with percutaneous coronary intervention (PCI) and their relationship to markers of lipid peroxidation, cytokines and highly-sensitive C-reactive protein (hsCRP). DESIGN: Non-consecutive patients with their first myocardial infarction were evaluated. Setting. University hospital as primary referral center, single center. PATIENTS AND METHODS: Forty-nine patients with AMI were evaluated. All were treated with primary PCI and infusion of abciximab. Reperfusion was verified by angiography. Blood samples for analyses of myeloperoxidase (MPO), neutrophil gelatinase-associated lipocalin (NGAL), matrix metalloproteinase-9 (MMP-9), malondialdehyde (MDA), 8-isoprostane-prostaglandin F2alpha (Iso-P), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factoralpha (TNFalpha), hsCRP, creatine kinase-monobasic fraction (CK-MB) and troponin-T (TnT) were obtained at baseline with the occluded coronary vessel, and subsequently after verified reperfusion at 1.5, 3 and 24 hours. RESULTS: Significant increases in MMP-9, IL-6, IL-8, TNFalpha and hsCRP were observed, and a significant decrease in MPO and MDA was also observed over the same period. No significant changes in Iso-P and NGAL were found. CONCLUSION: We found a dissociation of the inflammatory reaction after PCI for AMI: a decrease of markers of neutrophil activation and MDA, but an increase in cytokines and hsCRP. An antineutrophil effect of the PCI procedure including treatment with abciximab, an antiplatelet drug and a modulator of inflammation, is conceivable.

Venous thromboembolism in carriers of the Factor V Leiden mutation and in patients without known thrombophilic risk factor; prediction of recurrence and APC-PCI complex concentration and/or soluble thrombomodulin antigen and activity.

BACKGROUND: The complex between activated protein C (APC) and the protein C inhibitor (PCI) is a sensitive indicator of the degree of activation of blood coagulation and higher concentrations have been measured in carriers of the FV Leiden mutation who were in the recovery phase after treatment for venous thromboembolism (VTE). OBJECTIVES: The main purpose of this study was to correlate the APC-PCI complex concentration to thrombomodulin activity and antigen concentration in the same group of patients. We also add a prospective clinical follow-up of the VTE recurrence after 1 year to investigate if the markers can predict the risk for a new VTE. PATIENTS/METHODS: Blood samples were collected from 50 patients with the FV Leiden mutation and 132 without any known risk factor for thrombophilia after finished treatment. RESULTS: The APC-PCI complex, s-TM activity and the quotient (s-TM activity)/(s-TM antigen) were higher in VTE patients with FV Leiden. In total, there were 19 VTE recurrences (10%) after 1 year. The OR for recurrence was 1.9 (95% CI 0.68-5.0) in all VTE patients with elevated APC-PCI complex (above 75th percentile) and 3.6 (95% CI 1.1-12) in VTE patients without any known risk factor for thrombophilia and with elevated s-TM activity. CONCLUSION: The APC-PCI complex concentration, s-TM activity and the quotient (s-TM activity)/(s-TM antigen) were higher in VTE patients with FV Leiden. The s-TM activity showed higher OR for recurrence of VTE in patients without known thrombophilic risk factor. Both methods could be sensitive markers of increased risk for venous thrombosis.

Lipid peroxidation is not increased in heart failure patients on modern pharmacological therapy.

BACKGROUND: Previous studies support a role of oxygen-free radicals in the development of congestive heart failure (CHF). The aim of this study was to investigate whether lipid peroxidation is increased in CHF patients on modern pharmacological therapy and whether there is a positive correlation between plasma levels of markers of lipid peroxidation and severity of heart failure (HF). Plasma malondialdehyde (MDA) and isoprostanes are often used as markers of lipid peroxidation and oxidative stress. We also studied whether long-term treatment with isosorbide-5-mononitrate (IS-5-MN) in combination with standard HF therapy affects P-MDA levels in patients with evidence of left ventricular (LV) dysfunction following acute myocardial infarction (AMI). MATERIALS AND METHODS: Ninety-two patients with clinical or echocardiographic evidence of LV-dysfunction following AMI were randomized to treatment with either IS-5-MN or placebo. In a subgroup of 83 patients with available plasma MDA, echocardiography, right-heart catherization, and plasma natriuretic peptides were evaluated. Control subjects were 80 healthy blood donors. A second study group consisted of 56 patients with CHF, evaluated with respect to LV function, brain natriuretic peptide and markers of oxidative stress (P-MDA and 8-isoprostane). The second control group comprised 50 healthy subjects. RESULTS: Lipid peroxidation measured by P-MDA and 8-isoprostane was not increased in patients with LV dysfunction treated with standard HF therapy. No positive correlation was found to the severity of HF. Long-term IS-5-MN therapy did not influence P-MDA concentrations. CONCLUSIONS: Although results from many experimental and clinical studies suggest that oxidative stress is increased in HF, this may not be true for patients treated with beta blockers and inhibitors of the renin-angiotensin system.

Plasma homocysteine and markers for oxidative stress and inflammation in patients with coronary artery disease--a prospective randomized study of vitamin supplementation.

BACKGROUND: Elevated plasma levels of total homocysteine (tHcy) are associated with an increased risk of developing occlusive vascular diseases. To better illustrate the relationship between plasma tHcy concentration, oxidative stress, and inflammation in patients with coronary artery disease (CAD), we measured plasma 8-isoprostane-prostaglandin F 2 (Iso-P), plasma malondialdehyde (MDA), and several markers of inflammation. We also aimed to demonstrate the effects of vitamin supplementation on these markers. METHODS: A total of 93 patients with ischemic heart disease were investigated. Of these, 34 had plasma tHcy < or =8 micromol/L, while 59 had plasma tHcy > or = 15.0 micromol/L. The 59 patients were randomized to open therapy with folic acid, 5 mg, pyridoxine, 40 mg, and cyancobalamin, 1 mg once daily for 3 months (n = 29) or to no vitamin treatment (n = 30). Blood samples were obtained from both groups before randomization and 3 months later. A sample was also obtained from the remaining 34 patients. RESULTS: Plasma Iso-P, serum amyloid A (S-AA), and plasma intercellular adhesion molecule-1 (ICAM-1) concentrations were higher in patients with high plasma tHcy levels than in patients with low to normal tHcy levels. Plasma levels of P-, L-, E-selectins, MDA, C-reactive protein (CRP), and orosomucoid did not differ between the groups. Vitamin therapy reduced plasma tHcy from 17.4 (15.3/20.1) to 9.2 (8.3/10.3) micromol/L (25th and 75th percentiles in parentheses) (p<0.0001). Plasma levels of Iso-P remained unchanged and, of all inflammatory markers, only the S-AA concentrations were slightly reduced by the vitamin treatment, from 5.3 (2.2/7.0) ng/L at baseline to 4.6 (2.1/6.9) ng/L (p<0.05) after 3 months of vitamin supplementation. CONCLUSION: Patients with CAD and high plasma tHcy levels had elevated plasma levels of Iso-P. The increase remained unaffected by plasma tHcy-lowering therapy, suggesting that homocysteine per se does not cause increased lipid peroxidation. Levels of plasma ICAM-1 and S-AA were increased in patients with high plasma tHcy, suggesting an association between homocysteinemia and low-grade inflammation.

Genetic variation in the human thrombomodulin promoter locus and prognosis after acute coronary syndrome.

INTRODUCTION: Endothelial thrombomodulin (TM) plays a critical role in both anticoagulation and anti-inflammation. An impaired TM cofactor function or reduced TM gene expression could constitute a prethrombotic abnormality leading to acute coronary events. Mutations in the TM gene occur, but their functional consequences on the expression and activity of the gene are not yet fully understood. MATERIALS AND METHODS: We performed a prospective study investigating the prevalence of TM mutations in the promoter region in 182 patients with acute coronary syndrome as well as in a control group. The patients were followed-up after 30 days and after 2 years for acute myocardial infarction (MI) and mortality. RESULTS AND CONCLUSIONS: We identified 10 point mutations and 2 small deletions: -1861 C/A, -1852 C/G, -1803 G/C, -1752 G/C, -1213/1212 delTT, -1089 C/G, -1088 C/T, -1083/1082 delCC, -1066 A/C, -801 C/G, -651 A/C and -52 G/A. Two of the mutations, -1752 G/C and -1213/1212 delTT, were frequent in the patients as well as in the controls, while all the others were rare. The only significant finding was that both -1752 G/C and -1213/1212 delTT were associated with a lower than normal risk of suffering a clinical event among smokers at 30 days and 2 years. We did not gain any support for the hypothesis that TM mutations confer an increased risk of MI or mortality.

No increase of plasma malondialdehyde after primary coronary angioplasty for acute myocardial infarction.

OBJECTIVE: Free radicals formed after coronary artery occlusion and reperfusion are assumed to produce myocardial stunning and possibly other forms of reperfusion injury as well. Malondialdehyde (MDA) is an end product in the lipid peroxidation chain reaction and is frequently used as a marker for free oxygen radical production. Increased levels of plasma MDA have been found following successful thrombolytic therapy. The aim of this study was to investigate whether plasma MDA levels also increase after successful primary percutaneous transluminal coronary angioplasty (PTCA) in acute myocardial infarction (AMI). DESIGN: In 23 patients with AMI, treated with primary PTCA, plasma MDA was analysed using a high-performance liquid chromatography method (HPLC). The results obtained with this method were compared with those obtained with a fluorimetric assay of thiobarbituric acid reactive substances (TBARS). This assay measures MDA but with a lower specificity. RESULTS: We found a significant decrease of plasma MDA from baseline 0.99 to 0.87 micro mol/l at 30 min and to 0.90 micro mol/l at 90 min following the primary PTCA (p = 0.048 and 0.014, respectively). No significant changes in TBARS method levels were observed. CONCLUSION: Instead of the expected increase in MDA following reperfusion we found a significant decrease. The results from measurements of MDA and TBARS were significantly incompatible. The results raise serious doubts as to the usefulness of increased plasma levels of MDA as a marker of oxidative stress caused by coronary reperfusion in patients treated with angioplasty.

Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function.

Sporadic mutations in the thrombomodulin (TM) gene occur in patients with both arterial and venous thrombosis, but the effects of these mutations on expression and function are largely unexplored. Full-length wild-type TM complementary DNA (cDNA) was incorporated into vector pcDNA6 for transfection into COS-7 cells for transient expression. Mutagenesis was performed to create 7 TM mutants with natural mutations either previously identified (Ala25Thr, Gly61Ala, Asp468Tyr, Pro477Ser, Pro483Leu) or reported here (an 11-base pair [bp] deletion, del791-801, leading to STOP306, and a missense mutation, Arg385Ser). Four mutations were found to detrimentally affect the level of expression of the TM protein. Of the missense mutations, 3 had reduced expression compared to wild-type TM (100%), Arg385Ser (50.2% +/- 5%, P <.001), Pro477Ser (76.8% +/- 1%, P <.001), Pro483Leu (82.1% +/- 8%, P <.007). No TM protein expression could be detected on the cell surface for mutation del791-801. The cofactor activity of TM in protein C activation was also evaluated. The Michaelis constant (K(m)) for wild-type thrombin-TM complex was 634 +/- 6 nmol/L. Two mutants, with Arg385Ser and Pro477Ser, had increased (P <.0001) K(m), 2967 +/- 283 nM, and 2342 +/- 219 nM, respectively, demonstrating impaired function of the thrombin-TM complex. This work presents biochemical evidence that certain (but not all) natural mutations in the TM gene reduce expression and impair function of the protein on the cell surface, and helps clarify the suggested contribution that these mutations might make to the risk of thromboembolic disease.