Ann-Kristin Öhlin

Association of Vitamin K–Dependent Coagulation Proteins and C4b Binding Protein With Triglyceride-Rich Lipoproteins of Human Plasma

The triglyceride (TG) concentration in plasma is an independent risk factor for coronary heart disease. There is evidence that TG-rich lipoprotein (TGRLP), ie, chylomicrons (CMs), chylomicron remnants (CMRs), and VLDLs associate with factor VII and prothrombin and that the association enhances a platelet factor Xa-mediated prothrombin activation when the CM-prothrombin complex is exposed to platelets. In this study, we examined the association of the vitamin K-dependent coagulation factors VII, IX, X, and prothrombin, as well as the anticoagulation protein C and its cofactor protein S, in plasma lipoproteins obtained from human fasting and postprandial plasma. We also analyzed some other proteins that are related to the coagulation system but not to vitamin K-dependent proteins, including factor V, serum amyloid P component (SAP), C4b binding protein (C4BP), and thrombomodulin (TM), and as a control, Ig G. Human TGRLP (d < 1.006 kg/L), LDL (d = 1.006 to 1.063 kg/L), and HDL (d = 1.063 to 1.210 kg/L) were separated from normal subjects both in fasting and 2 to 3 hours after the ingestion of a meal containing 100 g fat. The different coagulation proteins, SAP, C4BP, TM, and Ig G were determined by SDS-polyacrylamide gel electrophoresis combined with Western blotting, using specific polyclonal or monoclonal antibodies, and were visualized by peroxidase staining. All the vitamin K-dependent proteins associate with TGRLP in both fasting and postprandial plasma, but not with LDL or HDL. Factor V, SAP, TM, and Ig G were not found in any lipoprotein classes. C4BP, which is a regulatory protein of the classic pathway of the complement system and which binds protein S in vivo to regulate blood coagulation, was present in TGRLP, especially postprandial, but not in LDL or HDL. The amounts of prothrombin, protein S, and C4BP in postprandial TGRLP were larger than those in fasting TGRLP. Vitamin K-dependent procoagulation and anticoagulation proteins, as well as C4BP, could be associated with TGRLP in vivo. If the association enhances prothrombin activation, this effect may thus be counteracted by simultaneous binding of protein S.

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

Abstract 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 ≤8μmol/L, while 59 had plasma tHcy ≥15.0 μmol/L. The 59 patients were randomized to open therapy with folic acid, 5mg, pyridoxine, 40mg, and cyancobalamin, 1mg once daily for 3months (n=29) or to no vitamin treatment (n=30). Blood samples were obtained from both groups before randomization and 3months 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)μmol/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 3months 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.

Intraoperative autotransfusion in primary hip arthroplasty: A randomized comparison with homologous blood

To study the quality and effect of blood produced by the cell saver compared with homologous blood in total hip arthroplasty, 40 patients were randomly divided into two groups. One group received autologous blood using the cell saver, whereas the second group served as a control, and received homologous bank blood. Hematologic and coagulation parameters of the patients were assessed both preoperatively and postoperatively. Samples from the autologous and the homologous blood were obtained before reinfusion, and were assessed as regards hematologic and biochemical parameters. The autologous blood satisfied all the intraoperative transfusion requirements of the autologous group and 75 percent of the total transfusion requirements. The operative and postoperative blood losses--hence, the total blood loss--were less in the autologous than in the control group. The autologous blood had a high hemoglobin, white blood cell, and plasma hemoglobin content and MCV compared with the homologous blood. Postoperatively, there were no differences as regards the hematologic parameters studied. There was no evidence of intravascular hemolysis in the autologous group. Postoperatively, in both groups, AT III, plasminogen, and protein C decreased. Other coagulation parameters were within normal limits in both groups. Intraoperative autotransfusion is safe and effective, and should be considered in hip arthroplasty to reduce the risks associated with homologous blood transfusion.

Effect of intravenous nitroglycerin on lipid peroxidation after thrombolytic therapy for acute myocardial infarction

Free oxygen radicals are produced after coronary artery occlusion and reperfusion. Polyunsaturated fatty acids are oxidized by free radicals to lipid peroxides. Measurements of plasma malondialdehyde (MDA) formed by the breakdown of lipid peroxides are often used as markers of lipid peroxidation. The effect of intravenous nitroglycerin on plasma MDA levels was studied in 43 patients who received thrombolytic therapy for acute myocardial infarction. Plasma MDA levels in patients were elevated on admission to the hospital compared with healthy controls, and normalized within 48 hours. A greater increase in plasma MDA concentrations after thrombolysis was found in patients with noninvasive signs of reperfusion than in patients judged to have a persistent occlusion. In the 23 patients receiving immediate intravenous nitroglycerin infusion, plasma MDA levels did not change from baseline to 90 minutes (0.92+/-0.22 and 0.92+/-0.23 micromol/L, p=0.99), whereas a significant increase was found in the 20 control patients who did not receive nitroglycerin (from 0.83+/-0.22 to 1.01+/-0.30 micromol/L, p=0.0004) (p=0.036 for the difference between groups). Successful reperfusion after thrombolytic therapy entails increased lipid peroxidation. Intravenous nitroglycerin reduces lipid peroxidation during myocardial ischemia and reperfusion.

Soluble thrombomodulin antigen in plasma is increased in patients with acute myocardial infarction treated with thrombolytic therapy.

Thrombomodulin (TM) is an integral endothelial cell membrane protein that functions as a cofactor for thrombin mediated activation of protein C. The anticoagulant functions of the protein C system are important in contributing to a hemostatic balance and prevention of thromboembolic disease. It has been suggested that impaired TM cofactor function could also constitute a prothrombotic abnormality leading to thromboembolic diseases. TM exists not only on the surface of endothelial cells but also as soluble fragment(s) circulating in plasma. The concept of a thrombotic occlusion as the critical event in acute myocardial infarction (AMI) forms the rationale for thrombolytic therapy. After successful reperfusion, patients remain at substantial risk for recurrent infarctions due to rethrombosis. The balance between procoagulant and anticoagulant mechanisms in the postthrombolytic phase have not been studied in detail. We have studied whether the plasma levels of soluble TM are influenced by thrombolytic therapy with streptokinase in patients suffering from AMI. Soluble TM concentrations increased significantly by 24 to 48 h after thrombolytic treatment, simultaneously with an increase in C-reactive protein (CRP, a marker of the inflammatory component of the cell damage) and in thio-barbituric acid reactive substances (TBARS, an indirect marker of lipid peroxidation).

Autologous blood transfusion in revision hip arthroplasty. A prospective, controlled study of 30 patients.

In a prospective, randomized study of the efficacy and effects of autologous blood transfusion in revision hip arthroplasty, 30 patients were randomly allocated into two groups. The Control Group received homologous blood transfusion. The Study Group deposited 2-3 units of blood preoperatively, intraoperative blood salvage was used, and no homologous blood was transfused intraoperatively. There was a smaller postoperative blood loss in the Study Group. The preoperative hemoglobin values were lower in the Study Group, but one week postoperatively they were higher than in the Control Group. The decrease in the values of AT III and protein C was lower in the Study Group. The combination of preoperative blood donation and intraoperative blood salvage reduced blood loss and homologous blood transfusion in revision hip arthroplasty.

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 µmol/l at 30 min and to 0.90 µmol/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.

Binding of prothrombin to chyle chylomicrons: Effects of temperature and calcium ions, and role of surface phospholipids

The ability of chyle chylomicrons to bind prothrombin has been studied. Rat chyle chylomicrons were incubated with human 125I-prothrombin and binding was examined by separating the chylomicrons from free 125-I-prothrombin by density-gradient ultracentrifugation, and by gel filtration on Sepharose CL-2B. A significant binding of prothrombin to chyle chylomicrons occurred. The complex formation was calcium dependent, and decreased markedly when the temperature was lowered from 37 degrees C to 20 degrees C and when PH was raised above 8. The time course for the binding at 37 degrees C in presence of 2 mmol/L CaCl2 exhibited an initial lag phase at about 10 minutes. Thereafter most of the binding occurred within 30 minutes. Bound prothrombin could not be removed from chyle chylomicrons by treatment with EDTA, suggesting that this binding is not a simple Ca2+ dependent association between prothrombin and chyle chylomicrons. Inclusion of 1% purified human serum albumin caused a 50% decrease in binding, half of which was reversed by increasing the Ca2+ ion concentration. Addition of pancreatic phospholipase A2 (PLA2) in doses sufficient to hydrolyze more than 95% of the phosphatidylethanolamine (PE) and 37% of the phosphatidylcholine (PC) decreased the binding by 50%. Doses of PLA2 that hydrolyze more than 95% of the phosphatidylethanolamine (PE) and 37% of the phosphatidylcholine (PC) decreased the binding by 50%. Doses of PLA2 that hydrolyzed 60-80% of (PE and 4-10% of the PC decreased the binding by only 7-15%. It is suggested that the binding of prothrombin to chyle chylomicrons is in part mediated by negatively charged phospholipids of the chylomicron surface, although a specific role of the PE could not be demonstrated.

Epidermal growth factor-like domains in the vitamin K-dependent clotting factors. Some structure-function relationships

The serine proteases of blood coagulation and fibrinolysis, in conjunction with their respective cofactors, accomplish a regulation of the hemostatic response that is remarkably precise. Bleeding is promptly arrested, without propagation of the thrombus into the undamaged blood vessel. Furthermore, thrombotic occlusion of the capillary system does not occur under normal conditions in spite of the very large surface-

Low soluble thrombomodulin activity and antigen is associated with a family history of heart disease while a high level is associated with a personal history of heart disease in type 2 diabetes

Low soluble thrombomodulin activity and antigen is associated with a family history of heart disease while a high level is associated with a personal history of heart disease in type 2 diabetes -