Clinical and Applied Thrombosis/Hemostasis current issue

Contaminant in the Recalled Unfractionated Heparin Preparations: Where is the Problem?

2008-06-26

Further Insight Into the Heparin-Releasable and Glycosylphosphatidylinositol-Lipid-- Anchored Forms of Tissue Factor Pathway Inhibitor

Ellery, P. E.R., Hardy, K., Oostryck, R., Adams, M. J. — 2008-06-26

The release of tissue factor pathway inhibitor (TFPI) from human umbilical vein endothelial cells (HUVECs) was investigated using heparin and phospholipase C. The experiment included incubating HUVECs with 0, 1, or 10 U/mL heparin diluted in Dulbecco Modified Eagle's Medium plus 5% fetal calf serum for 1 or 24 hours. A statistically significant increase in TFPI activity levels was seen at 1 hour, but not at 24 hours. A 20-fold increase in the release of TFPI after phospholipase C treatment of HUVECs was demonstrated, confirming that it is glycosylphosphatidylinositol-lipid (GPI) anchored. Sequential treatment of HUVECs with phospholipase C and heparin was performed, and a trend was observed where GPI-anchored TFPI levels were increased after 1 hour of pretreatment with heparin but were decreased after 24 hours. Serum is a requirement for the heparin-dependent release of TFPI from HUVECs. Heparin pretreatment of HUVECs may affect levels of GPI anchored TFPI in a time and dose-dependent manner.

Elevated Levels of Prothrombin Fragment 1 + 2 Indicate High Risk of Thrombosis

2008-06-26

Prothrombin fragment 1 + 2 (F1 + 2) is considered to be useful for diagnosis of thrombosis. However, the evidence for a diagnosis of thrombosis by F1 + 2 is still not well established. The plasma concentrations of F1 + 2, soluble fibrin, D-dimer, and thrombin-antithrombin complex were measured in 694 patients suspected of having thrombosis and then were correlated with thrombosis. Plasma concentrations of F1 + 2, soluble fibrin, D-dimer, and thrombin-antithrombin complex were significantly higher in patients with thrombosis, compared with patients without thrombosis. When cutoff values of more than 300 pmol/L for F1 + 2 were used for the diagnosis, more than 50% of the patients were thus found to have thrombosis. The findings showed that F1 + 2, soluble fibrin, D-dimer, and thrombin-antithrombin complex have similar diagnostic ability. The plasma concentration of F1 + 2 closely was well correlated with thrombin-antithrombin complex, soluble fibrin, and D-dimer. Finally, F1 + 2 is one of the most useful parameters for the diagnosis of thrombosis.

Ocular Vascular Thrombotic Events: Central Retinal Vein and Central Retinal Artery Occlusions

Glueck, C. J., Ping Wang, , Hutchins, R., Petersen, M. R., Golnik, K. — 2008-06-26

We prospectively assessed associations of thrombophilia— hypofibrinolysis with central retinal vein occlusion (CRVO) (40 patients) and central retinal artery occlusion (CRAO) (9 patients). We used polymerase chain reaction measures for thrombophilia (factor V Leiden, prothrombin, C677T MTHFR, platelet glycoprotein PlA1/A2) and hypofibrinolysis (plasminogen activator inhibitor-1 4G4G). Serologic thrombophilia measures included protein C, protein S (total and free) and antithrombin III, homocysteine, lupus anticoagulant, anticardiolipin antibodies IgG-IgM, and factors VIII and XI. Serologic hypofibrinolysis measures included Lp(a) and plasminogen activator inhibitor activity. For comparison with 40 CRVO and 9 CRAO patients, 80 and 45 race—gender matched controls were studied. The factor V mutation was more common in CRVO (3/40, 8%) than controls (0/79, 0%), P = .036, as was high (>150%) factor VIII (12/40, 30%) versus (4/77, 5%), P = .0002. Low antithrombin III (<80%) was more common in CRVO (5/39, 13%) than in controls (2/73, 3%), P = .049. Homocysteine was high (≥13.5 µmol/L) in 5/39 (13%) CRVO patients versus 2/78 controls (3%), P = .04. Three of 9 CRAO patients (33%) had low (<73%) protein C versus 2/37 controls (5%), P = .044. Two of 9 CRAO patients (22%) had high (≥13.5 µmol/L) homocysteine versus 0/42 controls (0%), P =. 028. Four of 9 CRAO patients had the lupus anticoagulant (44%) versus 4/33 (12%) controls (P = .050). CRVO is associated with familial thrombophilia (factor V Leiden, factor VIII, low antithrombin III, homocysteinemia), and CRAO is associated with familial and acquired thrombophilia (low protein C, homocysteinemia, lupus anticoagulant), providing avenues for thromboprophylaxis, and triggering family screening.

The Effect of Tirofiban on Fibrinogen/Agonist-Induced Platelet Shape Change and Aggregation

Jagroop, I. A., Mikhailidis, D. P. — 2008-06-26

There is evidence linking raised plasma fibrinogen (fib) and platelet hyperactivity with vascular events. One way to inhibit platelets is to block the platelet membrane glycoprotein (GP) IIb/IIIa receptor, which binds circulating fib or von Willebrand factor and cross-links platelets at the final common pathway to platelet aggregation. Tirofiban is a potent and specific fib receptor antagonist, used in the treatment of unstable angina. The authors assessed the effect of tirofiban on spontaneous platelet aggregation (SPA), fib-induced, serotonin (5HT)-induced, and adenosine diphosphate (ADP)-induced aggregation in whole blood by calculating the percentage free platelet count. These various agonists were used alone and in combination. The authors also measured the effect of tirofiban on agonists-induced (ADP, 5HT) platelet shape change (PSC). The effect of fib on PSC was also evaluated in platelet-rich plasma using a high-resolution (0.07 fL) channelyzer. Tirofiban significantly inhibited SPA, fib (2, 4, 8 g/L), ADP, ADP + fib combination, and 5HT-induced aggregation. Tirofiban had no effect on agonist-induced PSC. There was no apparent change in platelet volume with fib. In conclusion, tirofiban does not appear to have an effect on PSC, an early phase of platelet activation. Tirofiban seems to be a nonspecific and an effective inhibitor of platelet aggregation (a later phase of platelet activation) in whole blood. The clinical significance of these findings remains to be established.

The Extrinsic Coagulation Activity Assay

Stief, T. W., Wieczerzak, A., Renz, H. — 2008-06-26

A chromogenic assay for the tissue factor—mediated thrombin generation was developed, the extrinsic coagulation activity assay: 50 µL citrated plasma is incubated with 5 µL tissue factor in 6% albumin and 250 mM CaCl₂. After 1-minute (37°C) coagulation reaction time, (extrinsic coagulation activity assay with 1-minute coagulation reaction time; generating normally about 1 IU/mL thrombin) 100 µL 2.5 M arginine is added to stop hemostasis activation. Generated thrombin is then chromogenically quantified. The normal extrinsic coagulation activity assay range is 100% ± 20%. Extrinsic coagulation activity assay in plasma of patients on heparin or coumarines is about 10-fold lower. Advantages of extrinsic coagulation activity assay: normal range of extrinsic hemostasis is truly represented, patients prone to hyper-activated extrinsic pathway are detected, anticoagulants result in respective test inhibition, fibrinogen/fibrin concentration does not artefactually alters the test result, plasma matrix is not changed significantly in the assay, and assay results are IU/mL thrombin or % of normal, which can be measured by every normal photometer.

Plasma Plasminogen Activator Inhibitor-1 Levels and Nonalcoholic Fatty Liver in Individuals With Features of Metabolic Syndrome

2008-06-26

Fatty liver represents the liver component of metabolic syndrome and may be involved in plasminogen activator inhibitor-1 (PAI-1) synthesis. We studied plasma PAI-1 levels and relationships with risk factors for metabolic syndrome, including fatty liver, in 170 patients. Liver ultrasound scan was performed on all patients, and a liver biopsy was performed on those patients with chronically elevated transaminase levels. Plasma PAI-1 levels correlated significantly (P < .05) with body mass index, degree of steatosis, insulin resistance, insulin level, waist circumference, triglycerides, and high-density lipoprotein (HDL) -cholesterol. However, only body mass index (β = .455) and HDL-cholesterol (β = .293) remained predictors of PAI-1 levels. Liver biopsy revealed a significant correlation (P < .05) between insulin resistance (r = 0.381) or insulin level (r = 0.519) and liver fibrosis. In patients presenting features of metabolic syndrome, plasma PAI-1 levels were mainly conditioned by the whole-body fat content.

Transition From Argatroban to Oral Anticoagulation With Phenprocoumon or Acenocoumarol: Effect on Coagulation Factor Testing

2008-06-26

Treatment with the thrombin inhibitor argatroban is often followed by vitamin K-antagonist treatment. In this study, the behavior of coagulation factors measured under these treatment regimens is shown. Healthy subjects received infusions of 1.0, 2.0, or 3.0 µg/kg/hr argatroban before and during phenprocoumon or acenocoumarol dosing. Quantitation of factors II, VII, IX, and X by clot-based assays resulted in dose dependent, approximately 20%, lower than expected values in the presence of argatroban. On the contrary, values for the inhibitors, protein C and protein S, were higher. Cotherapy exaggerated the effect by vitamin K-antagonist alone. However, testing by immunologic and chromogenic assays did not show any effect by argatroban. Coupled with a lack of bleeding in the subjects, these data suggests that argatroban does not affect coagulation proteins and that the observations are only an assay artifact. Assay interferences must be considered when measuring coagulation proteins in patients receiving thrombin inhibitors.

The Activated Seven Lupus Anticoagulant Assay Detects Clinically Significant Antibodies

Moore, G. W., Rangarajan, S., Savidge, G. F. — 2008-06-26

Lupus anticoagulants are a heterogeneous group of autoantibodies detected by their effects on phospholipid-dependent coagulation assays. Persistent lupus anticoagulants are associated with thrombotic disease, but not all are clinically significant. Antibody heterogeneity and reagent and test variability dictate that at least 2 tests, of different types, should be used to screen lupus anticoagulants. The objective of this study was to investigate whether the activated seven lupus anticoagulant assay detects clinically significant antibodies. Eighty-two patients with antiphospholipid syndrome (APS) and 32 with systemic lupus erythematosus + positive for activated seven lupus anticoagulant and who were without thrombosis, who were positive by activated seven lupus anticoagulant assay, were investigated for lupus anticoagulants by dilute Russell's viper venom time, dilute activated partial thromboplastin time, and Taipan snake venom time, and for anticardiolipin antibodies. Fifty-seven of the APS patients were positive for lupus anticoagulants in multiple assays, 25 in activated seven lupus anticoagulant alone. Fourteen of the latter group were previously positive in other antiphospholipid antibodies assays, and 11 had only been positive for lupus anticoagulants by activated seven lupus anticoagulant. Twenty-eight had elevated anticardiolipin antibodies, 6 of whom were from the group that was positive in activated seven lupus anticoagulant only. Eight of the systemic lupus erythematosus + lupus anticoagulants (without thrombosis) patients were positive for lupus anticoagulant by activated seven lupus anticoagulant alone and had only been positive in activated seven lupus anticoagulant previously, and none had elevated anticardiolipin antibodies. The remaining 24 patients were lupus-anticoagulant positive in multiple assays, and 9 had elevated anticardiolipin antibodies. Dilute Russell's viper venom time and Dilute activated partial thromboplastin time are widely used to detect lupus anticoagulants and are considered to detect clinically significant antibodies. Activated seven lupus anticoagulant detected antibodies in APS patients who were positive by these assays and also lupus anticoagulants undetectable by the dilute Russell's viper venom time/dilute activated partial thromboplastin time reagents used, demonstrating its utility as a first-line or second-line assay.

Adipokines, Linking Adipocytes and Vascular Function in Hemodialyzed Patients, May Also Be Possibly Related to CD146, a Novel Adhesion Molecule

Malyszko, J., Malyszko, J.S., Kozminski, P., Pawlak, K., Mysliwiec, M. — 2008-06-26

Possible correlations between adiponectin, leptin, CD146, a novel adhesion molecule localized at the endothelial junction, and other markers of endothelial cell injury, von Willebrand factor, thrombomodulin, vascular cell adhesion molecule, and intracellular adhesion molecule, and markers of inflammation, tumor necrosis factor-, interleukin-6, and high-sensitivity C-reactive protein in nondiabetic hemodialyzed patients with and without coronary artery disease were studied. Markers of endothelial dysfunction were elevated in hemodialyzed patients, predominantly with coronary artery disease. In multivariate analysis, kinetic urea modeling and plasminogen activator inhibitor-1 remained the only positive predictors of adiponectin. In multivariate analysis, predictors of leptin were triglycerides, tissue plasminogen activator, CD146, and coronary artery disease. In multivariate analysis, predictors of CD146 were age, hemoglobin, and adiponectin. Elevated adiponectin correlated to CD146 may be the expression of a counterregulatory response aimed at mitigating the consequences in endothelial damage and increased cardiovascular risk in renal failure. The data provide further support for a link between adipocytokines, endothelial dysfunction, cardiovascular risk, and renal failure.

Platelet Aggregation and Activation in Thalassemia Major Patients in Indonesia

Setiabudy, R., Wahidiyat, P. A., Setiawan, L. — 2008-06-26

Thromboembolic events and hypercoagulable state have been reported in patients with thalassemia. As platelets play an important role in the pathogenesis of thrombosis, the authors aimed to find the pattern of changes in platelet count, function and activation, and evidence of coagulation activation in patients with thalassemia major in Indonesia. A total of 31 patients with splenectomized and 35 patients with nonsplenectomized thalassemia major were enrolled in this study. Platelet count, platelet aggregation, β-thromboglobulin, and D-dimer levels were measured. All measured parameters were significantly higher in splenectomized than in nonsplenectomized patients. β-thromboglobulin level was increased, but D-dimer level was within normal range. The authors concluded that there was an increase in platelet activation in patients with β-thalassemia major. Platelet activation was higher in splenectomized than in nonsplenectomized patients.

Human Immunodeficiency Virus Infection and Acute Deep Vein Thromboses

Louw, S., Jacobson, B. F., Buller, H. — 2008-06-26

Abnormalities that predispose to a hypercoagulable state with an increased incidence of venous thrombosis have been described in human immunodeficiency virus (HIV) infections and are associated with an increased mortality. A recent systematic review by Klein et al concluded that further studies are essential to elucidate the link between HIV infection and deep vein thrombosis (DVT). We prospectively evaluated 24 consecutive, active people presenting with an acute DVT; 13 consented to HIV testing, revealing an HIV prevalence of 84% (95% confidence interval [CI], 0.65-1.04). In a matched healthy control group, the HIV prevalence was 4% (95% CI, 0.039-0.041). The high HIV prevalence in the DVT group that consented to testing was also significantly higher compared to that in the South African population, estimated to be 10% in 2005. Although the study numbers were low, a statistically significant increased prevalence of HIV infection was found in patients with acute DVTs.

The Importance of Thrombotic Risk Factors in the Development of Idiopathic Sudden Hearing Loss

Yildiz, Z., Ulu, A., Incesulu, A., Ozkaptan, Y., Akar, N. — 2008-06-26

Impaired cochlear blood circulation has been suggested to cause sudden hearing loss. In this study, the role of factor V 1691 G-A (FV 1691 G-A), prothrombin 20210 G-A (PT 20210 G-A), methylene tetrahydrofolate reductase 677 C-T (MTHFR 677 C-T), factor V 4070 A-G (FV 4070 A-G), endothelial cell protein C receptor (EPCR) gene 23-bp insertion, and plasminogen activator inhibitor-1 (PAI-1) 4G/5G mutation was assessed. Fifty-three patients with idiopathic sudden sensorineural hearing loss and 80 individuals comprising the control group were included in this study. The frequency for FV 1691 A was 6.2% in the patient group and 3.7% in the control group, PT 20210 G-A was 1.2% in the patient group and 1.9% in the control group, and FV 4070 A-G was 7.5% in the patient group and 11.3% in the control group. The frequency of MTHFR 677 C-T was significantly higher in the patient group than in the control group, with a P value of .03. PAI-1-675 4G/5G polymorphism was found to be 71.2% and 69.8%, in the control group and the patient group, respectively. The EPCR 23-bp insertion was 0% in the control group and was found in 3 patients (3.7%), which needs further study.

Fatal Bleeding Due to a Heparin-Like Anticoagulant in a 37-Year-Old Woman Suffering From Systemic Mastocytosis

2008-06-26

A 37-year-old female patient with systemic mastocytosis who was admitted with severe unexplained bleeding symptoms is studied. Laboratory procedures established the diagnosis of a patient-derived—heparin-like anticoagulant as a very rare hemostatic abnormality predisposing to bleeding. The patient died from refractory disease despite therapy with protamine, initiation of chemotherapy, and supportive measures. The case illustrates the clinical presentation and diagnosis of heparin-like anticoagulants. Etiology, pathophysiology, and therapeutic options are discussed.

Hyperhomocysteinemia Due to Pernicious Anemia Leading to Pulmonary Thromboembolism in a Heterozygous Mutation Carrier

Kupeli, E., Cengiz, C., Cila, A., Karnak, D. — 2008-06-26

Pulmonary thromboembolism is a life-threatening condition resulting mostly from lower extremity deep-vein or pelvic-vein thrombosis. A 46-year-old woman was admitted to hospital with pain on the right side of the chest and hemoptysis. On laboratory analysis, D-dimer level was elevated. Computed tomographic pulmonary angiography revealed intravascular filling defects due to thrombi in right lower lobe pulmonary segmental arteries. Screening for thrombophilic states was normal except for heterozygous mutations of both prothrombin and methylene tetrahydrofolate reductase (MTHFR 677) genes. Homocysteine level was high, and vitamin B12 level and serum ferritin level were reduced. Serum antiparietal antibody was positive, and therefore, pernicious anemia was diagnosed along with iron-deficiency anemia. After the diagnoses were established, enoxaparin followed by warfarin was started in addition to oral vitamin B12, pyridoxine, thiamine, folic acid, and ferroglycine sulfate supplementation. At the end of 8 weeks of the replacement therapy, vitamin B12, folate, and homocysteine levels and red cell volume were found to be normal, with complete resolution of the thrombus confirmed by repeat computed tomographic pulmonary angiography. We conclude that hyperhomocysteinemia due to vitamin B12 deficiency associated with pernicious anemia might have decreased the threshold for thrombosis. In addition, the presence of heterozygous prothrombin and methylene tetrahydrofolate reductase mutations might serve as synergistic cofactors triggering pulmonary thromboembolism.

Severe Arterial Thrombophilia Associated With a Homozygous MTHFR Gene Mutation (A1298C) in a Young Man With Klinefelter Syndrome

2008-06-26

Klinefelter syndrome (KS) is the most common sex chromosome disorder in men. It may be associated with an increased risk for venous thrombosis and thromboembolism, which is partially explained by hypofibrinolysis due to androgen deficiency. Additional genetic or acquired thrombophilic states have been shown in KS patients complicated with venous thrombosis as isolated case reports. Arterial thrombotic events had not been previously reported in KS. In this study, a young man with KS who developed acute arterial thrombosis during testosterone replacement therapy is presented. He was homozygous for the A1298C mutation of the methylenetetrahydrofolate reductase (MTHFR) gene.

Increased Factor VIII Level Lends Diversity to Ischemic Stroke Etiology

Erol, O., Kara, G., Ozcakar, L., Haznedaroglu, I. C. — 2008-06-26