Trapidil decreases the aggregation of platelets from heart transplant recipients ex vivo.

Heart transplant recipients show platelet hyperaggregability, which may be related to the incidence of graft vasculopathy. We investigated whether trapidil can inhibit the aggregation of platelets from these patients. Platelet count, mean platelet volume (MPV), and adenosine diphosphate (ADP)-induced platelet aggregation were determined in 18 heart transplant recipients and 12 healthy subjects. Additionally, platelet-rich plasma from the patients was incubated with trapidil or with saline, prior to measuring ADP-induced aggregation. The MPV was significantly greater in patients compared to controls (9.4+/-1.1 vs 8.5+/-0.7 fL; P=.01), and ADP-induced platelet aggregation was significantly increased in patients compared to controls (81.2%+/-13.1% vs 69.6%+/-16.2%; P=.04, respectively). The trapidil-treated samples showed significantly decreased platelet aggregation compared to the control samples (24.2%+/-12.6% vs 66.7%+/-11.7%; P<.001). Platelets from heart transplant recipients showed an increased MPV and increased ADP-induced aggregation. Trapidil effectively reduced the ADP-induced aggregation ex vivo.

Atrial fibrillation at discharge from the hospital in patients undergoing mitral valve repair.

BACKGROUND: Patients undergoing mitral valve repair (MVRr) are often discharged on oral anticoagulation with warfarin. Because the decision about oral anticoagulation is made at discharge from the hospital and because atrial fibrillation (AF) represents the only well-documented indication for oral anticoagulation in these patients, we studied the frequency of AF at discharge after MVRr. METHODS: We reviewed the records of 245 patients who underwent MVRr over the past 5 years and assessed the frequency of AF at discharge from the hospital and the factors that were associated with an increased risk for arrhythmia. RESULTS: The group comprised 95 women and 150 men with a mean age of 62.1 +/- 14 years. Seventy-three (30 %) patients were in and/or had a history of AF on admission. Sixty-five (27 %) patients had AF at discharge. Factors that were associated with AF at discharge were: AF on admission (odds ratio [OR] 57.1; confidence interval [CI] 20.8 - 157.3; p < 0.0001), enlarged left atrium (OR 3.2; CI 1.2 - 8.7; p = 0.025) and intake of ACE inhibitors (OR 3.9; CI 1.2 - 12.3; p = 0.022). The OR for AF at discharge in patients with none of the above risk factors was 0.02 (95 % CI 0.02 - 0.13; p < 0.0001). CONCLUSION: Only a relatively small proportion of the studied patients, especially patients with AF on admission, with larger atria and with a history of ACE inhibitors intake, were in AF at discharge after MVRr. Patients with none of these risk factors were at low risk for AF at discharge after MVRr and the optimal oral anticoagulation regimen for these low-risk patients needs to be determined.

Lipid reductions by low-density lipoprotein apheresis: a comparison of three systems.

There are currently three established low-density lipoprotein (LDL) apheresis systems: immunoadsorption, heparin-induced extracorporeal LDL precipitation (HELP), and dextran sulfate. We treated the same patient with all three systems and compared the lipid reductions achieved. A total of 135 consecutive treatments were studied, 57 with immunoadsorption, followed by 30 with HELP and 48 with dextran sulfate adsorption. The mean plasma volume (mean +/- SD) treated was 4.9 +/- 0.05, 3.08 +/- 0.091, and 3.39 +/- 0.71 L, respectively. The LDL-cholesterol (LDL-C) reduction was 75.5% +/- 7.4%, 61.6% +/- 5.1%, and 57.1% +/- 12.4%, respectively (P < .001 for immunoadsorption vHELP and dextran sulfate). The mean removal efficiency (mass removed/plasma volume treated) for LDL-C was 1.0 +/- 0.12, 1.42 +/- 0.25, and 1.15 +/- 0.21 g/L, respectively (P < .001 for HELP v immunoadsorption and dextran sulfate). The mean LDL-C plasma concentration before apheresis was 199 +/- 23.9, 201 +/- 25.7, and 186 +/- 28 mg/dL, respectively (P < .001 for dextran sulfate adsorption v immunoadsorption and HELP). Among the three LDL apheresis systems, immunoadsorption caused the greatest percent reduction in LDL-C, while HELP eliminated LDL-C from the plasma most efficiently. Dextran sulfate was similar to HELP in terms of LDL-C reduction, and its removal efficiency was similar to immunoadsorption. Dextran sulfate was also associated with the lowest pretreatment plasma LDL-C concentration.

Analysis of the long-term efficacy and selectivity of immunoadsorption columns for low density lipoprotein apheresis.

Immunoadsorption low density lipoprotein (LDL) apheresis is performed with reusable columns containing anti-apolipoprotein B(ApoB) antibodies. We analyzed their long-term efficacy and selectivity. Performance over 60 treatment sessions of six pairs of immunoadsorption LDL apheresis columns was evaluated by analysis of variance using the removal of total cholesterol and ApoB to assess efficacy and the ratio of total cholesterol/high density cholesterol removed to assess selectivity. The removal of cholesterol did not vary significantly with treatment number. The mass of ApoB removed increased significantly (p = 0.002), and the mass of ApoB removed per volume unit of processed plasma showed a trend (p = 0.065) toward an increase with treatment number. Both parameters correlated with the serum ApoB concentration before treatment, which also increased significantly (p = 0.0007) with treatment number. No significant variation of selectivity was found. The efficacy of the LDL apheresis immunoadsorption columns did not decrease after 60 treatment sessions. The columns' selectivity also remained unchanged.

Changes in blood coagulation and fibrinolysis associated with maximal exercise and physical conditioning in women taking low dose oral contraceptives.

Blood coagulation parameters (thromboplastin time. PT; activated partial thromboplastin time, aPTT; fibrinogen; antithrombin III, ATIII; von Willebrand factor-concentration, vWF; factor VIII-activity, FVIII) and fibrinolytic parameters (plasminogen; (alpha-antiplasmin; euglobulin-lysis-time, Elt; tissue plasminogenactivator-antigen, tPA-antigen; plasminogenactivator-1-antigen, PAI-1-antigen) were evaluated in 34 women on low-dose oral contraceptives (OC) twice at intervals of 12 weeks each time before and after maximal exercise. During the 12 weeks, 24 women took part in an aerobic conditioning program and 10 women were requested to avoid any kind of sports activity for this period. Blood samples were taken before training and before and after maximal treadmill exercise. This procedure was repeated after the training program. After maximal exercise we found a significant reduction of aPTT and PT (increase in %), a decrease in ATIII, vWF, fibrinogen, plasminogen and alpha2-antiplasmin but an increase in fibrinolytic activity (all p<0.05). Maximal exercise is associated with an increase in blood coagulation and fibrinolysis also in women taking OC. After the physical conditioning program an increase in fibrinolytic activity at rest was noted in the training group. Opposed to that the fibrinolytic activity at rest decreased in the control group after abstinence of sports activity over this period (p<0.05, MANOVA).

The 536C-->T transition in the human tissue factor pathway inhibitor (TFPI) gene is statistically associated with a higher risk for venous thrombosis.

Tissue factor pathway inhibitor (TFPI) is an important regulator in the extrinsic blood coagulation pathway. Although the regulatory biochemical role of TFPI is evident, the clinical significance of this proteinase inhibitor remains to be elucidated. The definition of a clinical TFPI deficiency seems to be more complex than that of other coagulation inhibitors because the activity and concentration of circulating TFPI can not be considered a true measure of in vivo levels. Its determination in plasma samples by immunological methods or functional assays has been shown to be inadequate in the detection of a clinical deficiency. Therefore, we screened genomic DNA samples of blood donors and thrombotic patients for alterations in the TFPI gene to assess the influence of a modified TFPI in venous thromboembolic diseases. We detected a single nucleotide substitution in exon 7 (536C-->T) leading to a proline to leucine exchange at amino acid position 151 of the protein ([P151L]TFPI) and found the prevalence of heterozygous carriers in German unrelated blood donors to be 0.2% (n = 5120). Four unrelated persons out of 14 probands carrying the genetic variation could be linked to venous thrombosis. For calculation of a potential risk for venous thrombosis for carriers of the mutation we investigated healthy blood donors about thrombotic events. 7 out of 308 blood donors were found to have a history of venous thrombosis, one of them carried the TFPI mutation. Statistical calculation showed a significant relative risk for venous thrombosis for individuals with the trait (odds ratio, 9.3; confidence interval, 1.8-48.6; p <0.01).

State-of-the-art patient self-management for control of oral anticoagulation.

Currently, in Germany, there is a successful program where patients monitor their own coagulation status through self-testing. The advent of a new generation of coagulometers has allowed more and more patients to use self-testing to monitor their coagulation status. The development of a structured training program by the Association of Self-Management of Anticoagulation (ASA), and the effective cost reimbursement system by health insurance companies has furthered the success of this program. The reliability of the coagulometers is quite important to the success of this program, and has been extensively evaluated. These systems are characterized by high accuracy and precision, and low intra-/interassay variation. They also exhibit excellent recovery of the therapeutic range. Several clinical studies have shown that patients performing self-management remain in the therapeutic range a greater percentage of the time when compared to conventional testing, and tended to have less incidences of bleeding or thromboembolic complications. It is estimated that about 50 to 60% of all patients on anticoagulant therapy in Germany are suitable candidates for self-management.

Activated platelets induce tissue factor expression on human umbilical vein endothelial cells by ligation of CD40.

CD40 is a type I member of the tumour necrosis factor (TNF) receptor superfamily of proteins, and is present on a wide variety of cells including vascular endothelial cells. Ligation of this receptor on endothelial cells is known to increase expression of inflammatory adhesion molecules. We have recently demonstrated that platelets express the ligand of CD40 (CD154) within seconds of exposure to agonist, and interact with endothelial cells to participate directly in the induction of an inflammatory response. Here we show that activated platelets induce tissue factor (TF) expression on endothelial cells in a CD40/CD154-dependent manner, and that the magnitude of this response can equal that induced by TNFe. Moreover, CD40 ligation on endothelial cells downregulates the expression of thrombomodulin. We also show that CD40-mediated TF expression is less sensitive to inhibition with the oxidative radical scavenger pyrrolidine dithiocarbamate than is that mediated by TNFalpha, indicating that CD40 has a distinct signalling pathway. Tissue factor is a cell membrane protein which functions as the main trigger of the extrinsic pathway of blood coagulation, and its expression on endothelial cells is implicated in wound healing and angiogenesis. Since platelets are among the first cells involved in haemostasis following tissue injury, our data showing that ligation of CD40 by CD154 induces a procoagulant phenotype on vascular endothelial cells suggests that platelets may play an important role in the induction of wound healing.

Thromboembolic complications associated with the use of prothrombin complex and factor IX concentrates.

In 1994, shortly after a heat-treated prothrombin complex concentrate (PCC) had been withdrawn from the German market due to transmission of hepatitis B, the license of another brand was withdrawn, due to 3 acute fatalities associated with the use of this product. We report on the clinical data of altogether 5 patients, who died during a 3 month period in Germany after having received this brand of PCC. All patients had surgery, acquired deficiencies of coagulation factors, and underlying diseases predisposing for thrombosis or disseminated intravascular coagulation. PCC was administered for the prevention of bleeding. In three patients, a drug interaction of PCC with aprotinin may also have played a role. Several points, however, are suspicious of a major causative effect of the respective product, (a) the close temporal correlation between administration of the drug and the subsequent clinical as well as laboratory deterioration, (b) the accumulation of these adverse events in a short period of time, when the use and market share of this brand increased due to the shortage of other products, and (c) laboratory abnormalities of this brand which have been consistently observed in several in vitro studies.

CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells.

CD40 ligand (CD40L, CD154), a transmembrane protein structurally related to the cytokine TNF-alpha, was originally identified on stimulated CD4+ T cells, and later on stimulated mast cells and basophils. Interaction of CD40L on T cells with CD40 on B cells is of paramount importance for the development and function of the humoral immune system. CD40 is not only constitutively present on B cells, but it is also found on monocytes, macrophages and endothelial cells, suggesting that CD40L has a broader function in vivo. We now report that platelets express CD40L within seconds of activation in vitro and in the process of thrombus formation in vivo. Like TNF-alpha and interleukin-1, CD40L on platelets induces endothelial cells to secrete chemokines and to express adhesion molecules, thereby generating signals for the recruitment and extravasation of leukocytes at the site of injury. Our results indicate that platelets are not only involved in haemostasis but that they also directly initiate an inflammatory response of the vessel wall.

Monitoring of recombinant hirudin: assessment of a plasma-based ecarin clotting time assay.

Assay conditions of a plasma-based ecarin clotting time (ECT) were evaluated and the precision of the ECT in monitoring plasma levels of r-hirudin assessed. The snake venom enzyme ecarin converts prothrombin to meizothrombin possessing only weak coagulant but strong esterase activity. In r-hirudin containing plasma samples, meizo thrombin is rapidly neutralized by r-hirudin resulting in a dose-dependent prolongation of the clotting times. Among the different assay conditions tested, addition of 50 microliters of ecarin (4 U/ml) to 100 microliters of undiluted citrate-anticoagulated plasma gave optimal results with respect to precision and reproducibility. The measuring range of the ECT performed in this way is about 0.02-5.0 micrograms/ml r-hirudin. In vitro studies performed on r-hirudin-spiked plasma samples of 50 healthy individuals demonstrate remarkably low interindividual differences in r-hirudin responsiveness as indicated by CV-values below 5% and 7% at r-hirudin concentrations between 0-3 micrograms/ml and 4-5 micrograms/ml, respectively. The specificity for r-hirudin of the ECT is further demonstrated by the strong correlation (r = 0.94) between the results of a chromogenic assay and the ECT-measured r-hirudin concentrations obtained on 67 ex vivo blood samples. Depending on the concentration of r-hirudin the ECT is sensitive to plasma levels of prothrombin. In the absence of r-hirudin the critical prothrombin concentration was found to be 20% but increasing to 60% in the presence of 2.0 micrograms/ml r-hirudin. A fibrinogen concentration of 50 mg/dl was found to be the minimal concentration independent of the r-hirudin concentration. The precision of the ECT in measuring plasma levels of r-hirudin is not influenced by treatment with heparin, aprotinin or oral anticoagulants. The data demonstrate that the ECT is a rapid and easily perfor mable clotting assay which allows accurate measurement of r-hirudin plasma levels. ECT-monitored hirudin treatment will help to establish optimal dose regimens that are more efficacious but still as safe as heparin.

Monitoring of r-hirudin anticoagulation during cardiopulmonary bypass--assessment of the whole blood ecarin clotting time.

The use of recombinant (r) hirudin as an anticoagulant in performing extracorporeal circulation systems including cardiopulmonary bypass (CPB) devices requires a specific and easy to handle monitoring system. The usefulness of the celite-induced activated clotting time (ACT) and the activated partial thromboplastin time (APTT) for r-hirudin monitoring has been tested on ex vivo blood samples obtained from eight patients treated with r-hirudin during open heart surgery. The very poor relationship between the prolongation of the ACT and APTT values and the concentration of r-hirudin as measured using a chromogenic factor IIa assay indicates that both assays are not suitable to monitor r-hirudin anticoagulation. As an alternative approach a whole blood clotting assay based on the prothrombin-activating snake venom ecarin has been tested. In vitro experiments using r-hirudin-spiked whole blood samples showed a linear relationship between the concentration of hirudin added and the prolongation of the clotting times up to a concentration of r-hirudin of 4.0 micrograms/ml. Interassay coefficients (CV) of variation between 2.1% and 5.4% demonstrate the accuracy of the ecarin clotting time (ECT) assay. Differences in the interindividual responsiveness to r-hirudin were analyzed on r-hirudin-spiked blood samples obtained from 50 healthy blood donors. CV-values between 1.8% and 6% measured at r-hirudin concentrations between 0.5 and 4 micrograms/ml indicate remarkably slight differences in r-hirudin responsiveness. ECT assay results of the ex vivo blood samples linearily correlate (r = 0.79) to the concentration of r-hirudin. Moreover, assay results were not influenced by treatment with aprotinin or heparin. These findings together with the short measuring time with less than 120 seconds warrant the whole blood ECT to be a suitable assay for monitoring of r-hirudin anticoagulation in cardiac surgery.

Recombinant hirudin as an anticoagulant during cardiac operations: experiments in a pig model.

OBJECTIVE: The efficacy and safety of recombinant hirudin (r-hirudin) compared with heparin as an anticoagulant during open-heart surgery has been studied in a pig model. METHODS: A total of 18 Göttingen minipigs were randomly divided into three treatment groups and subjected to cardiopulmonary bypass for 1 h. Heparin-treated animals received a bolus of unfractionated heparin of 400 IU/kg body weight. Recombinant hirudin was given by a bolus injection of 1 mg/kg body weight, followed by a 1 h lasting infusion of 1 mg/kg body weight per h. The heparin-anticoagulated animals and one group of the hirudin-treated animals additionally received aprotinin at a dosage of 17500 KIU/kg body weight (KIU, kallikrein inhibitory units). In the second group of r-hirudin-treated animals, the aprotinin was replaced by saline. RESULTS: The extracorporeal circuit remained patent for a 1 h pump period in all of the animals studied. There was no evidence of vascular occlusion or clot formation in the r-hirudin-treated animals. The anticoagulant efficacy of the hirudin protocol used is further demonstrated by the results of electron-microscopical scans of the pump-line filters. Fibrin deposits were visible only in the heparin-treated animals and not in r-hirudin-treated pigs. Despite this strong anticoagulant effect, there was no evidence of an increased bleeding tendency in r-hirudin-treated pigs. Moreover, histological studies showed a statistically significant (P < 0.05) higher incidence of tissue bleeding in the heparin/aprotinin-treated animals compared with the r-hirudin/aprotinin-treated pigs. Studying the platelet function, a statistically significant (P < 0.01) better preserved ADP- and collagen-induced platelet aggregation was seen in the r-hirudin/aprotinin-treated animals when compared with heparin/aprotinin-treated animals. CONCLUSIONS: These data demonstrate that r-hirudin can be used successfully as an alternative anticoagulant to heparin during cardiac operations including cardiopulmonary bypass. The better preservation of platelet function suggests that r-hirudin may reduce the postoperative risk of bleeding.