Clinical and Applied Thrombosis/Hemostasis recent issues

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

Antigenic (Immunogenic) Profiling of Bovine Thrombin and its Purified Forms

Walenga, J. M., Bick, R. L., Messmore, H. L. — 2008-03-26

Further Removal of Factor V Related Antigen From Bovine Thrombin by Utilizing a Membrane-Filtration Step

Terrab, A., Pawlak, D., Spaay, P., Hoppensteadt, D., Fareed, J. — 2008-03-26

Topical bovine thrombin is commonly used during surgery to maintain hemostasis and is rarely associated with abnormalities in hemostasis, including coagulopathies and bleeding. Coagulopathies may be related to the formation of cross-reactive antibodies to bovine factor V. Effectiveness of a new filtration step to remove factor V/Va from bovine thrombin was evaluated. A highly sensitive and specific Western blot capable of detecting minute amounts of factor V/Va and/or its fragments was developed. Samples were evaluated for bovine factor V related antigens using the Western blot method and a competitive enzyme-linked immunosorbent assay. Factor Va light chain fragment levels were detectable in crude thrombin and chromatographically purified thrombin but not in chromatographically purified and virally filtered preparations. Therefore, inclusion of the viral-filtration step during purification of thrombin is effective in reducing factor V or its fragments to undetectable levels, thus enhancing product purity.

Inflammatory and Hemostatic Activation in Patients Undergoing Off-Pump Coronary Artery Bypass Grafting

2008-03-26

To characterize hemostatic differences imposed by 2 common cardiac surgeries, the authors studied patients undergoing coronary artery revascularization by off-pump (n = 13) or cardiopulmonary bypass on-pump (n = 26) technique. Blood samples collected to 4 days post-surgery were evaluated by flow cytometry and enzyme-linked immunosorbent assay. A significant inflammatory response occurred in both the groups after surgery shown by increased interleukin cytokines and C-reactive protein; however, levels peaked lower and hours later in the off-pump group. Platelets (P-selectin; platelet-leukocyte complexes) and leukocytes (CD11b) were activated only in on-pump patients. Thrombin generation was enhanced in both groups after surgery. Only in the on-pump patients, the thrombin-antithrombin complex, pro-thrombin fragment 1.2, and thrombomodulin (vascular integrity) decreased intraoperatively. Tissue plasminogen activator and plasminogen activator inhibitor-1 were greater in the on-pump patients. Off-pump surgery may place patients at higher risk of postoperative hypercoagulability because of normal platelet function, intraoperative thrombin generation, less fibrinolytic activity, and lack of vascular protection.

Primary Prevention of Venous Thromboembolism in Long-Term Care: Identifying and Managing the Risk

Haas, S., Spyropoulos, A. C. — 2008-03-26

Venous thromboembolism (VTE) is a significant, but underestimated, cause of morbidity and mortality in long-term care settings. VTE risk increases significantly with age and is further increased by comorbidities common to this group; however, advancing age and limited mobility alone are insufficient to warrant pharmacological prophylaxis. Recognizing those at increased VTE risk during an acute illness is crucial for appropriate and timely prophylaxis. Warfarin is used for the long-term secondary prevention of VTE, whereas unfractionated and low-molecular-weight heparins are used for primary prophylaxis. The elderly are at increased risk for bleeding complications, because of the high frequency of comorbidities and comedications. Attention to dosing is recommended for those with severely impaired renal function, low body weight, or perceived to be at high bleeding risk. This review addresses the role of risk assessment in the decision of when to provide prophylaxis to an individual in long-term care and highlights key management issues for those prescribed prophylaxis.

Pulmonary Embolism in Medical Patients: An Autopsy-Based Study

Kakkar, N., Vasishta, R. K. — 2008-03-26

Pulmonary embolism, though treatable, is a devastating disease and an important cause of morbidity and mortality among hospitalized patients. In all, 1000 autopsies were reviewed in adult medical patients. The overall incidence of pulmonary embolism in adult medical autopsies was 15.9% (159/1000). The incidence of pulmonary embolism contributing significantly to the death of the patients (groups 1 and 2) is 126/1000 (12.6%). Thus, pulmonary embolism very significantly contributed to death in 126/159 (79.24%) of group 1 and 2 patients. Pulmonary embolism affected a younger population as 79.87% of the overall patients, 66.67% of the fatal cases (group 1) and 73% of combined group 1 and 2 cases were below the age of 50 years. Sepsis was the primary diagnosis in 32% of total and in 42% of fatal cases. Hence, pulmonary embolism is considered as an important cause of death in patients admitted to the medical wards. It affects a younger population in India and needs to be tackled appropriately.

Hereditary Thrombophilic Risk Factors and Venous Thromboembolism in Istanbul, Turkey: The Role in Different Clinical Manifestations of Venous Thromboembolism

2008-03-26

The aim of this study was to investigate the hereditary thrombophilic risk factors in patients with venous thromboembolism (VTE) and whether these risk factors play a different role in patients with isolated pulmonary embolism (PE) as compared with patients with deep vein thrombosis (DVT) and patients with PE + DVT. The protein C (PC), protein S, antithrombin activities, homocysteine levels, and factor V Leiden (FVL) G1691A and prothrombin G20210A mutations were evaluated in 191 patients with VTE and 191 controls. The prevalence of FVL and PC deficiency were higher in patients (P = .003 and P = .02, respectively). There was no significant difference for the other risk factors. The combination of thrombophilic risk factors was significantly higher in patients with DVT + PE as compared with patients with isolated PE or DVT (P = .04). In conclusion, the most important hereditary risk factors for VTE in this study were the FVL mutation and PC deficiency.

Successful Pregnancy Outcome in Women With Bad Obstetric History and Recurrent Fetal Loss Due to Thrombophilia: Effect of Unfractionated Heparin and Low--Molecular Weight Heparin

Ghosh, K., Shetty, S., Vora, S., Salvi, V. — 2008-03-26

Acquired and inherited thrombophilias are known to be associated with unfavorable pregnancy outcome including recurrent fetal loss. There are differences of opinion whether these patients need to be treated with aspirin, unfractionated heparin, low—molecular weight heparin, corticosteroids, or intravenous immunoglobulins. In all, 25 consecutive patients with a history of fetal loss and 7 patients who presented in early pregnancy with deep-vein thrombosis were treated, and their pregnancy outcome was noted. All the women were positive either for a solitary or for a combination of acquired and heritable thrombophilia markers. In all, 23 patients were treated with unfractionated heparin and 9 with low—molecular weight heparin. In all, 16 out of 23 patients (69.6%) treated with unfractionated heparin and 9 out of 9 (100%) treated with low—molecular weight heparin had successful pregnancy outcome. There was a complete resolution of thrombus in all the cases. None of the patients had any adverse reactions such as heparin-induced thrombocytopenia, thrombosis, or fracture. Both unfractionated heparin and low—molecular weight heparin were effective in cases of bad obstetric history and recurrent pregnancy loss due to thrombophilia. However, low—molecular weight heparin was found to be more effective than unfractionated heparin along with other advantages of not requiring laboratory monitoring and easy administration. None of the patients in either group had to interrupt the therapy for any adverse treatment-related complications.

Estrogen Receptor Alpha and Beta Gene Polymorphisms Are Not Risk Factors for Recurrent Miscarriage in a Brazilian Population

2008-03-26

The aim of this study was to determine the prevalence of alpha (ESR1: c.454-397T>C and c.454-351A>G) and beta (ESR2: 1082G>A and 1730G>A) estrogen receptor gene polymorphisms in 2 Brazilian ethnic groups (Caucasian, African Brazilian) and to investigate their association with recurrent miscarriage (RM) in 75 women with a history of 3 or more consecutive pregnancy losses and 139 controls with at least 2 live births and no history of pregnancy loss. Polymerase chain reaction and restriction fragment length polymorphism were used to identify gene polymorphisms. Coagulation methods were used to measure protein C, protein S, and fibrinogen, and a chromogenic method was used for antithrombin quantification. Significantly higher prevalences of 1082G>A and 1730G>A polymorphisms were seen in African Brazilian and Caucasian controls, respectively. There was no association between RM and ESR polymorphisms. There was a difference in the genotype prevalence in the c.454-39T>C polymorphism between RM and control Caucasians, but this finding was not associated with an increased risk of miscarriage. There was no synergistic or additive effect between ESR polymorphisms and thrombophilia in RM patients. A difference in the prevalence of ESR polymorphisms was observed, according to ethnic origin. ESR polymorphisms could not be considered a risk factor for RM.

The Membrane Proteinase 3 Expression on Neutrophils Was Downregulated After Treatment With Infliximab in Patients With Rheumatoid Arthritis

2008-03-26

Proteinase 3 (PR3) expression on neutrophils was examined in rheumatoid arthritis (RA) patients before and after antitumor necrosis factor (TNF)- therapy. Membrane PR3 expression from patients with either an infection or RA significantly increased. Membrane PR3 expression on neutrophils from RA patients treated with infliximab (anti-TNF- antibody) therapy was less than in those without such treatment in a resting state, but the expression later increased after stimulation in vitro. Membrane PR3 expression increased because of the stimulation of TNF, whereas it was significantly suppressed by plasma or ₁-proteinase inhibitor. The condition of patients with RA improved after treatment with infliximab. Membrane PR3 expression on neutrophils in RA patients was downregulated by infliximab. As a result, PR3 might play an important role in the neutrophil-mediated inflammatory reaction in patients with either RA or an infection.

Soluble Fibrin Inhibits Lymphocyte Adherence and Cytotoxicity Against Tumor Cells: Implications for Cancer Metastasis and Immunotherapy

2008-03-26

Circulating soluble fibrin (sFn) is elevated in many cancer patients. It is a marker for ongoing disseminated intravascular coagulation and may have prognostic significance. We have demonstrated that sFn inhibited monocyte adherence and cytotoxicity by a mechanism involving blockade of monocyte Mβ2 and tumor cell CD54. It was, therefore, hypothesized that sFn also inhibits lymphocyte and interleukin-2—activated lymphocyte (LAK) adherence and cytotoxicity against tumor cells. This study sought to identify the lymphocyte subset responsible for adherence and killing of A375 melanoma cells and whether sFn inhibited these parameters. Lymphocyte and LAK cell adherence and cytotoxicity, which was adherence dependent, were inhibited by preincubation with purified or plasma-derived sFn. The lymphocyte and LAK cell activities were primarily a result of CD8⁺ MHC (major histocompatibility complex) unrestricted cytotoxic T cells. These results suggest that elevated levels of circulating sFn may be immunosuppressive and may reduce the efficacy of adoptive immunotherapies.

A Dynamic Comparative Study Concerning the Effects of Angiotensin-Converting Enzyme Inhibitors and Aldosterone Receptor Blockers on the Fibrinolytic System

Usalan, C., Buyukhatipoglu, H. — 2008-03-26

The renin—angiotensin—aldosterone system (RAAS) plays a central role in fibrinolysis. Activation of the RAAS stimulates the expression of plasminogen activator inhibitor-1 (PAI-1), which can be directly implicated in the pathophysiology of thromboembolic events. Our primary aims were to measure (1) the effect of acute RAAS activation on plasma levels of PAI-1, and (2) the inhibitory effect of an angiotensin-converting enzyme (ACE) inhibitor alone, versus a combination of an ACE inhibitor and aldosterone blockade on the increase in PAI-1 usually observed. In the current prospective in vivo study, RAAS was activated by means of phlebotomy, an effective, physiologic means of RAAS activation. Seventeen voluntary prehypertensive, but otherwise healthy, blood donors were included in this study. Renin and PAI-1 levels were measured before and after initial phlebotomy. At the time of the second phlebotomy, 12 of 17 donors randomly were assigned to receive enalapril (5 mg) or a combination of enalapril (5 mg) plus spironolactone (25 mg), beginning 3 days before phlebotomy, and 5 were assigned to be controls, receiving no medications. Plasma renin and PAI levels were significantly increased following initial phlebotomy. At the time of the second phlebotomy, plasma PAI-1 activity was reduced significantly, as compared with the initial phlebotomy, but it did not return to baseline levels. The observed mean reduction in PAI-1 level was greater among the subjects who received both ACE and aldosterone inhibition. Enalapril and the combination of enalapril plus spironolactone efficiently reduce PAI-1 levels, but the reductions are more pronounced with the combined regimen. However, neither treatment appears sufficient to return PAI-1 activity to baseline levels.

Low-Density Lipoprotein Specifically Binds Glycoprotein IIb/IIIa: A Flow Cytometric Method for Ligand-Receptor Interaction

Tetik, S., Uras, F., Eksioglu-Demiralp, E., Turay Yardimci, K. — 2008-03-26

Primary platelet aggregation requires agonist-mediated activation of membrane receptor glycoprotein (GP) IIb/IIIa, binding of fibrinogen to GpIIb/IIIa, and cellular events after fibrinogen binding. This study investigated whether fibrinogen receptor GpIIb/IIIa is also the binding site for low-density lipoprotein (LDL) in platelets by using GpIIb/IIIa-coated polystyrene microbeads incubated with various concentrations of fluorescein isothiocyanate (FITC)-labeled ligands. Binding was assayed by flow cytometry. Binding of fibrinogen (Fg)-FITC and LDL-FITC to GpIIb/IIIa coated microbeads was concentration dependent, reaching saturation. Binding of LDL-FITC to GpIIb/IIIa coated microbeads was inhibited by fibrinogen. Binding of LDL-FITC or Fg-FITC to freshly isolated platelets gave similar results as those of GpIIb/IIIa coated microbeads. Glycoprotein IIb/IIIa, the fibrinogen receptor on platelets is also one of the binding sites of LDL on platelets. This rapid and reliable flow cytometric technique using coated microbeads may be used as a first step for the study of all ligand receptor interactions.

Distribution of --844 G/A and Hind III C/G PAI-1 Polymorphisms and Plasma PAI-1 Levels in Mexican Subjects: Comparison of Frequencies Between Populations

2008-03-26

Several polymorphisms have been described in the PAI-1 gene including the —844 G/A and Hind III C/G polymorphisms. These polymorphisms have been associated with different diseases such as preeclampsia and cardiovascular diseases. The allele and genotype frequencies of both PAI-1 polymorphism where investigated in Mexican subjects and compared with other healthy worldwide populations. The hematological and biochemical parameters where classified according each genotype in our studied group. One hundred Mexican subjects were recruited. Demographic data and hematological and biochemical parameters were collected, and genomic DNA isolation was performed in all the participants. Screening of both polymorphisms studied was made by polymerase chain reaction and restriction analysis. Levels of plasminogen activator inhibitor-1 in plasma were measured by ELIS-ARA plasminogen activator inhibitor antigen kit. The —844 and Hind III genotypes frequencies were as follows: 49% (G/G), 40% (G/A), 11% (A/A) and 50% (C/C), 44% (C/G), 6% (G/G), respectively. The wild-type genotypes (G/G and C/C) were significantly higher with respect to the compared populations. In addition, a significant increase of apolipoprotein A1 in the carriers of G/A —844 and C/G Hind III genotypes was observed. However, when the plasma plasminogen activator inhibitor levels were analyzed with respect to each genotype and haplotype, no significant differences were found.

Soluble but Not Platelet P-selectin Correlates With Spontaneous Platelet Aggregation: A Pilot Study

2008-03-26

Background. P-selectin (PS) is a marker of platelet activation measured on the platelet surface as platelet PS (pPS) or in serum as soluble PS (sPS). Controversy remains over the exact relationship between sPS, pPS, and other markers such as spontaneous platelet aggregation (SPA). Objective. To investigate correlations between pPS, sPS, and SPA in patients with peripheral arterial disease. Methods. SPA, pPS, and sPS levels were measured in venous blood sampled from patients following intermittent claudication (n = 18) or an acute stroke (n = 18). Results. SPA and sPS correlated significantly in the claudicants (Pearson correlation coefficient, r = 0.661; P = .0020) and stroke patients (r = 0.514; P = .020). No significant correlation was identified between pPS and SPA, or sPS and pPS. Conclusions. The 2 methods of assessing PS are not comparable. Although pPS is accepted as a platelet activation marker, sPS may be a better indicator of aggregation represented by SPA.

In Vitro Simulation of Thrombolysis Inhibition

Stief, T. W. — 2008-03-26

Hyperfibrinolysis is a serious clinical complication. The inhibitory concentrations 50% of antifibrinolytics were analyzed in the microtiter plate clot lysis assay, using 50 µL of plasma clots, 10 µL of antifibrinolytic drug, 10 µL of 354 IU/mL (final) urokinase, 4.46 µg/mL (final) tissue-type plasminogen activator or 0.6 mg/mL plasmin, and 50 µL of pooled normal plasma as clot supernatant. The inhibitory concentrations 50% of lysine against urokinase or tissue-type plasminogen activator is 2.0 or 4.2 mM, against -amino-caproic acid 0.7 or 1.5 mM, against tranexamic acid 0.03 or 0.17 mM, respectively. The inhibitory concentrations 50% of lysine, -amino-caproic acid, or tranexamic acid against plasmin is 7.4, 0.4, or 0.04 mM. The inhibitory concentrations 50% of aprotinin against urokinase or tissue-type plasminogen activator is about 60 KIU/mL, against plasmin 19 KIU/mL. Lysine might be a new antifibrinolytic drug with a clinically interesting rapid pharmacokinetic. This data help correct dosing of antifibrinolytics to patients with hyperfibrinolysis.

In Vitro Simulation of Extremely Activated Thrombolysis

Stief, T. W. — 2008-03-26

A life-threatening thrombus in massive pulmonary embolism has to be eliminated within minutes. Extremely activated plasmatic fibrinolysis destroys such thrombi in time: 50 µL plasma clots were incubated with urokinase or tissue-type plasminogen activator and 50 µL pooled normal plasma. The microtiter plate clot lysis assay was performed. The time point at which 50% of the clot has been lysed is 4 minutes for 8333 IU/mL urokinase or an equimolar concentration of tissue-type plasminogen activator (52498 IU/mL = 105 µg/mL). The effective dose 50% at 5 minutes lysis time is about 800 nM (4320 IU/mL) urokinase or (27220 IU/mL = 54 µg/mL) tissue-type plasminogen activator. Addition of plasminogen to the plasmatic clot supernatant improves thrombolysis if 65 IU/mL of urokinase acts for 10 minutes. The risk for severe intracranial hemorrhage in massive thrombolysis might be much lower than the lethality of a massive pulmonary embolism. Extremely activated plasmatic thrombolysis could be clinically indicated.

Type II Cryoglobulinemia and Brain Hemorrhage

Wei Hsi Chen, , Hung Sheng Lin, , Kao, Y.-F. — 2008-03-26

By virtue of an understanding of hemostasis and coagulopathy using modern techniques, the exact role of individual serum protein in vascular thrombosis or hemorrhage becomes more apparent. Cryoglobulin causes vasculitude and thrombosis in various vascular beds, but its role in brain hemorrhage is unknown. We encountered a cryoglobulinemic patient to have cryoglobulinemia, hypocomplementia, and cerebellar hemorrhage during a reactivation of cytomegalovirus infection. Because cryoglobulin is harmful to vessel and hemostasis, and often increases nonspecifically in response to incitement, its weight in vascular syndrome must seriously be reviewed. Coagulopathy in a reactivation of latent virus such as cytomegalovirus should be cautioned in older patients.

The Safety Profile of Yasmin Is Similar to Other Combined Oral Contraceptives

Cronin, M., Korner, P. — 2008-03-26

To the Editor

de Miranda, P. A. P., Reiter, P. D. — 2008-03-26

The Immunogenic Potential of Generic Version of Low-Molecular-Weight Heparins May Not be the Same as the Branded Products

2008-01-08

Homocysteine Activates Platelets In Vitro

Mohan, I. V., Jagroop, I. A., Mikhailidis, D. P., Stansby, G. P. — 2008-01-08

The mechanism of thrombogenicity in hyperhomocysteinemia remains controversial. The authors investigated the association between elevated plasma homocysteine levels, platelet function, and blood coagulation. Blood was collected from healthy subjects and patients with critical limb ischemia. Basal platelet counts and platelet aggregation as well as flow cytometry were performed to assess spontaneous- and agonist-induced platelet aggregation as well as P-selectin and Glycoprotein IIb/IIIa expression at different homocysteine concentrations. Thromboelastography was performed, and platelet shape change was assessed, using a channelyzer, by measuring median platelet volume. Lactate dehydrogenase was measured, to indirectly assess red blood cell membrane integrity, after homocysteine exposure. The study results suggest that platelet activation and hypercoagulability occur after exposure to homocysteine, especially in patients with critical limb ischemia. Homocysteine concentrations of approximately 50 µmol/L appear to be the level at which these changes occur in vitro, and this effect on platelets appears to be indirect.

Mean Maternal Second-Trimester Hemoglobin Concentration and Outcome of Pregnancy: A Population-Based Study

2008-01-08

Both anemia and the lack of physiological maternal plasma volume expansion during the second trimester are associated with higher maternal morbidity and poor fetal outcome. Mean hemoglobin levels between the 14th and 30th gestational weeks were calculated in 4985 consecutive pregnant women and were correlated with outcome data of pregnancy. It was found that 9.4% of participants (n = 3959) had normal pregnancy outcome. Mean maternal hemoglobin levels were significantly lower in women with a normal pregnancy (11.96 ± 0.94 g/dL) compared with women who had adverse outcome events (preeclampsia, n = 423, 12.5 ± 1.0 g/dL, P < .0001; early birth, n = 464, 12.2 ± 1.01 g/dL, P < .0001; low birth weight newborn, n = 473, 12.2 ± 1.10 g/dL, P < .0001; intrauterine growth retardation, n = 250, 12.2 ± 1.0 g/dL, P < .0001). The risk for any adverse outcome event was lowest with a mean hemoglobin between 11.0 and 12.0 g/dL (odds ratio, 0.625; 95% confidence interval, 0.43-0.89) and highest between 13.0 and 15.0 g/dL (odds ratio, 2.24; 95% confidence interval, 1.54-3.31). In this population-based study from a community in Western Germany, impaired plasma volume expansion was an independent risk factor for the development of an adverse outcome of pregnancy.

Variant CYP2C9 Alleles and Warfarin Concentrations in Patients Receiving Low-Dose Versus Average-Dose Warfarin Therapy

2008-01-08

This study compared the frequency of variant cytochrome P450 2C9 (CYP2C9) alleles and warfarin S/R concentration ratio in patients who required low-dose (<2.5 mg/day) and average-dose (5 ± 0.5 mg/day) warfarin. Patients who achieved a therapeutic international normalized ratio were recruited from the Atlanta Veterans Affairs Medical Center anticoagulation clinic. CYP2C9*2 and *3 alleles were determined by validated Taqman allelic discrimination assays. Warfarin S and R concentrations were determined by chiral capillary electrochromatography with electrospray ionization mass spectrometry. At least 1 variant allele was found in 66.7% and 22.2% of patients in the low-dose and average-dose groups, respectively (P = .001, ²). The warfarin S/R concentration ratio was 0.665 (range, 0.162-3.58) and 0.452 (range, 0.159-2.36) for patients receiving low-dose and average-dose therapy, respectively ( P = .097). A warfarin requirement of <2.5 mg/day and an elevated warfarin S/R concentration ratio were each associated with a higher frequency of variant CYP2C9 alleles.

Correction of the Bleeding Time With Lyophilized Platelet Infusions in Dogs on Cardiopulmonary Bypass

Bode, A. P., Lust, R. M., Read, M. S., Fischer, T. H. — 2008-01-08

Lyophilized canine platelets were infused in a single large bolus dose into splenectomized dogs after 2 hours' perfusion on cardiopulmonary bypass to test their possible efficacy in restoring hemostasis after compromise of platelet function. The vessel bleeding time (VBT) was monitored by venipuncture of the exposed jugular vein. During cardiopulmonary bypass, platelet counts fell quickly and the VBTs became prolonged over baseline. Infusion of lyophilized platelets reconstituted in normal saline occurred just before or immediately after weaning from the cardiopulmonary bypass pump. The results showed consistent and persistent lowering of the VBTs by the infused lyophilized platelets. Controls showed continuously prolonged VBTs. The weighted average VBT in infused subjects was significantly lower than the average in controls: 3 minutes 10 seconds versus 6 minutes 59 seconds, respectively (t test, P = .01). These results in this setting indicate the possible effectiveness of similar human lyophilized platelet preparations in reducing postoperative bleeding in open heart surgery.

Hemostasis Activation in Thrombophilic Subjects With or Without a History of Venous Thrombosis

Cuderman, T. V., Bozic, M., Peternel, P., Stegnar, M. — 2008-01-08

Thrombophilia is considered to increase the risk of venous thrombosis (VT) due to hemostasis activation. To determine the level of hemostasis activation in thrombophilic subjects with or without a history of VT, hemostasis activation markers prothrombin fragment 1 and 2 (F1+2), thrombin—antithrombin complex (TAT), and cross-linked fibrin degradation products (D-dimer) were measured in 94 subjects with (patients) and 101 subjects without a history of VT (controls). A total of 34.8% of patients and 14.8% of controls (P = .002) had at least 1 thrombophilic defect (protein C deficiency, activated protein C [APC] resistance, presence of lupus anticoagulants, or prothrombin G20210A polymorphism). The subjects were divided into 4 subgroups: patients with (TF⁺ patients) and without (TF^– patients) thrombophilia, and controls with (TF⁺ controls) and without (TF^– controls) thrombophilia. Hemostasis activation was comparable between all patients and controls (TAT: 2.1 vs 2.6 µg/L; F1+2: 1.0 vs 0.9 nmol/L; D-dimer: 36 vs 37 µg/L, respectively) and between TF⁺ and TF^– patients. However, TF⁺ controls had a significantly higher prevalence of increased hemostasis activation markers compared with TF^– controls (TAT > 4.4 µg/L, 38.4 vs 7.3%; F1+2 > 1.1 nmol/L, 53.8 vs 22.0%; D-dimer > 78 µg/L, 30.7 vs 8.8% of subjects, respectively; all P < .05). After stratification for thrombophilic defects, hemostasis activation was associated with APC resistance in controls and with protein C deficiency in patients. To conclude, thrombophilia was associated with hemostasis activation in controls. We assumed that, in patients, the differences in hemostasis activation between subjects with or without thrombophilia were blurred due to undetermined and unidentified thrombophilic defects.

Outpatient-Based Primary and Secondary Thromboprophylaxis With Low-Molecular-Weight Heparin

Spyropoulos, A. C. — 2008-01-08

Although oral vitamin K antagonists such as warfarin have been the mainstay of thromboprophylaxis in the outpatient setting, warfarin has potential disadvantages, including food and drug interactions, the need for drug monitoring, intolerance, failure, and hypersensitivity syndromes. The use of low-molecular-weight heparin as a primary or secondary thromboprophylactic drug in the outpatient setting for extended prophylaxis or as outpatient bridging therapy has been addressed less extensively. Available evidence shows that low-molecular-weight heparin can be used as extended outpatient-based primary thromboprophylaxis for major orthopedic and cancer surgery and is a safe and effective alternative to warfarin in long-term secondary thromboprophylaxis, especially in cancer patients and in pregnant women. Low-molecular-weight heparin can also be used as an alternative to unfractionated heparin as outpatient-based bridging therapy. In addition to good clinical outcomes and financial benefits, mainly resulting from a reduction in the length of hospital stay, the use of extended-duration low-molecular-weight heparin in the outpatient setting appears to be feasible, with high patient compliance.

Evaluation of the Effectiveness and Safety of Bemiparin in a Large Population of Orthopedic Patients in a Normal Clinical Practice

2008-01-08

The authors conducted a prospective, open, multicenter, observational study to audit the utilization patterns of bemiparin in orthopedic patients in daily clinical practice. They analyzed rates of documented symptomatic venous thromboembolism (VTE) (deep vein thrombosis and pulmonary embolism) confirmed by objective methods, major bleeding, death, thrombocytopenia, and other adverse events. It was also intended to analyze the influence of concomitant factors (bemiparin dose, concomitant medications, age, and obesity) on VTE and bleeding rates. A total of 7959 patients were included and received bemiparin for 28 days (median). Bemiparin 3500 IU/d was used in 84.9% of patients, whereas bemiparin 2500 IU/d was administered to 15.1% of patients. Reason for prophylaxis (number of cases [%]) included cast immobilization of the leg (2052 [25.8%]), knee replacement (1082 [13.6%]), hip replacement (876 [11.0%]), hip fracture surgery (437 [5.5%]), other lower limb surgery (1569 [19.7%]), knee arthroscopy (769 [9.7%]), and spine surgery (231 [2.9%]). A total of 943 patients with insufficient data on reason for prophylaxis and 560 patients with no outcome assessment were excluded from the analysis of clinical outcomes. Among 6456 assessable patients, the authors found a low rate of documented symptomatic VTE (0.91%), major bleeding (0.17%), deaths (0.37%), and mild to moderate thrombocytopenia (0.51%). None of the major bleedings was fatal or occurred in a critical organ. There were 3 deaths in which fatal pulmonary embolism (PE) could not be ruled out. There were no cases of severe type-II thrombocytopenia. VTE rates were not increased in obese patients, and major bleeding rates were not increased in elderly patients or in patients taking nonsteroidal anti-inflammatory drugs. In conclusion, bemiparin prophylaxis, given for 3 to 4 weeks in cast immobilization of the leg and other orthopedic procedures, was associated with low rates of VTE, bleeding, and other adverse events in normal clinical practice.

The Fibrinogen Functional Turbidimetric Assay

Stief, T. W. — 2008-01-08

Hitherto, clinical fibrinogen methods were based on coagulation seconds, with assay conditions not similar to a plasma milieu. The fibrinogen functional turbidimetric assay included 50 µL citrated plasma + 100 µL 300 mIU/mL thrombin, 400 µg/mL polybrene, and 6% albumin—phosphate-buffered saline; an increase in absorbance at 405 nm/5 min at room temperature (or 2 minutes at 37°C) was observed. In all, 6% albumin in the fibrinogen functional turbidimetric assay reagent abolishes falsely elevated fibrinogen to fibrin turbidity in hypoproteinemic plasma samples. This assay can detect fibrinogen activity of 250% to 300% of normal, the lower detection limit being 7% of normal (0.2 g/L). The normal range of this assay is 100% ± 20% (mean value ± 1 SD; coefficient of variations <4%). This assay imitates fibrinogen to fibrin conversion in clotting blood plasma; it is independent of plasmatic albumin or heparin and can be performed everywhere. This assay has a diagnostic value in pathology-disseminated intravascular coagulation and in assessing risk for atherothrombosis.

Kallikrein Activates Prothrombin

Stief, T. W. — 2008-01-08

Kallikrein is a multitalented enzyme in hemostasis and inflammation. Normally, kallikrein is formed in intrinsic hemostasis and activates factor XII. A total of 10 µL of 0 to 100 µg/mL human plasma kallikrein in 6% human albumin—PBS were incubated with 90 µL 111.1 µg/mL prothrombin in 6% human albumin in absence and presence of 23 mM Ca⁺⁺. After 0 to 64 minutes (37°C), 100 µL of 2.5 M arginine, pH 9, were added. Fifty microliters of 0.72 mM HD-CHG-Ala-Arg-pNA in 1.36 M arginine were added and increase in absorbance at 405 nm was determined. Within 8 minutes (37°C), 1 µg/mL kallikrein, ie, 2.5% of the normal plasmatic prekallikrein concentration, generates approximately 3 mIU/mL thrombin in absence and 27 mIU/mL thrombin in presence of Ca⁺⁺. Kallikrein can directly activate prothrombin; there is a shortcut in the intrinsic hemostasis system that generates catalytic amounts of thrombin without following the known intrinsic clotting pathway.

Correlation Between Thrombomodulin and Severe Preeclampsia: A Summary

Wiwanitkit, V. — 2008-01-08

Thrombomodulin is a critical cofactor in the initiation of the protein C anticoagulant pathway. Plasma levels of thrombomodulin are regulated on a genetic basis, but more important is the dependence on a series of other atherosclerotic risk factors, such as hypertriglyceridemia, stroke, cancer, and diabetes. There is considerable controversy regarding the clinical role of thrombomodulin level as a risk factor of severe preeclampsia. A retrospective analysis of recent reports on the thrombomodulin level and its correlation to preeclampsia was performed to assess the correlation between the pattern of thrombomodulin level and preeclampsia. From the available 4 case-control studies, 149 patients and 120 controls are evaluated. The overall average thrombomodulin level for the patients and controls is 66.7 ± 11.9 ng/mL and 45.7 ± 7.3 ng/mL, respectively, which is significantly higher in patients than in controls (P < .05). In addition, the author reports a significant correlation between population ethnicity and thrombomodulin level (r = .96; P < .05).

Recurrent Abortions in Asian Indians: No Role of Factor V Leiden Hong Kong/Cambridge Mutation and MTHFR Polymorphism

2008-01-08

Recurrent fetal loss is a frequent health problem. Data accumulated over the past few years have suggested a possible correlation between thrombophilia and fetal loss. Although a clear association has been established between fetal loss and certain thrombophilic states, such as antiphospholipid antibody syndromes, antithrombin deficiency, and combined defects, reports on the prevalence of inherited prothrombotic defects such as factor V Leiden mutation and methylene tetrahydrofolate reductase C677T polymorphism in fetal loss are contradictory. The prevalence of these 2 mutations in Asian Indians with recurrent fetal loss has not yet been studied. In light of this, the present study looked at the prevalence of these mutations in 85 patients with spontaneous recurrent abortion and 31 controls. The authors did not find any significant role of these mutations in the development of recurrent abortion.

Heparin-Induced Thrombocytopenia Complicating Hemodialysis

2008-01-08

Hemodialysis complicated by heparin-induced thrombocytopenia (HIT) is a rare event requiring anticoagulation with direct-thrombin inhibitors. Contaminant calcific uremic arteriolopathy (calciphylaxis) further complicates this situation due to the possibility that warfarin anticoagulation may exacerbate skin necrosis. The authors report a patient with renal failure and calciphylaxis who developed HIT after starting hemodialysis. She was successfully treated with Argatroban.

Optimal Management of an Aneurysmal Subarachnoid Hemorrhage in a Patient With Known Factor XI Deficiency: A Case Report

Siao, D., Seetapah, A., Ryman, A., Guerin, V., Mesli, A., Maurette, P. — 2008-01-08

The authors report a rare case of an acute cerebral aneurysm rupture in a patient with a known factor XI deficiency. Aneurysmal subarachnoid hemorrhage (SAH) accounts for a high mortality and morbidity rate. When SAH is associated with an inherited coagulation disorder such as hemophilia C, an unexpected and possible increase in hemorrhagic stroke and increase in bleeding during surgery and in the postoperative period could lead to an extremely bad outcome. Clinical management consists of rapid correction of the coagulation disorder before undergoing any invasive intracranial procedure. Such an optimal therapeutic strategy must be under the care of a multidisciplinary medical and surgical team. Human factor XI concentrate (Hemoleven, Laboratoire Français du Fractionnement et des Biotechnologies [LFB], Les Ulis, France) was used successfully in this case report. New treatment using recombinant factor VIIa is discussed.

Multifactorial Thrombophilia in a Pregnancy: A Case Report

Gumus, I. I., Uslu, H., Bavbek, N., Turhan, N. — 2008-01-08

Thrombophilias are inherited or acquired conditions that predispose individuals to thromboembolism. Thrombophilic disorders increase obstetric complications, such as early pregnancy loss, fetal growth retardation, placental abruption, and preeclampsia. Recurrent pregnancy loss affects 1% to 3% of women of reproductive age, and a large proportion of these losses remain unexplained. Thrombophilic defects were found in 49% to 65% of women with pregnancy complications compared with 18% to 22% of women with normal pregnancies, suggesting a 3- to 8-fold increase in risk. We report a case of a pregnant woman who had a history of recurrent pregnancy losses that was complicated with protein S deficiency, factor V Leiden mutation, methylene tetrahydrofolate reductase mutation, and antiphospholipid syndrome in her pregnancy.

Rectus Sheath Hematoma Due to Inappropriate Warfarin Usage

Gungor Kaya, M., Poyraz, F., Cengel, A. — 2008-01-08

Anti factor Xa Assay: An Effective Method to Determine the Appropriate Dose of Heparin in Pregnant Women

Vineeta, S., Ved Prakash, C., Renu, S. — 2008-01-08

Book Review: Cardiovascular Disease, Methods and Protocols, Volumes 1 & 2. Qing K. Wang, Editor. Humana Press, 999 Riverview Drive, Suite 208, Totowa, NJ 07512-10121, USA. 2006. $99.50 and $125

Wehrmacher, W. H., Messmore, H. — 2008-01-08

Book Review: Wintrobe's Atlas of Clinical Hematology. D. C. Tkachuk and J. V. Hischman, Editors. Lippincott, Williams and Wilkins, Philadelphia, PA, USA. 2007. 349 pp. illus, with DVD. $199.00

Wehrmacher, W. H., Messmore, H. — 2008-01-08

Argatroban Therapy for Heparin-Induced Thrombocytopenia in Acutely Ill Patients

Gray, A., Wallis, D. E., Hursting, M. J., Katz, E., Lewis, B. E. — 2007-10-02

Heparin-induced thrombocytopenia (HIT) is a prothrombotic, immune-mediated adverse reaction to heparin therapy. To evaluate clinical outcomes and effects of argatroban therapy in acutely ill HIT patients. Retrospective analysis. Hospital in-patient. Acutely ill patients with clinically diagnosed HIT from previous multicenter, historically controlled studies of argatroban therapy in HIT. Argatroban, adjusted to maintain activated partial thromboplastin times 1.5 to 3 times baseline, or historical control therapy (ie, no direct thrombin inhibition). We identified 488 patients who received argatroban (N = 390; mean dose of 1.9 µg/kg/min for a mean 6 days) or historical control therapy (N = 98) for HIT. The primary all-cause composite endpoint of death, amputation, or new thrombosis within 37 days occurred in 133 (34.1%) argatroban-treated patients and 38 (38.8%) controls (P = .41). Argatroban, versus control, significantly reduced the primary thrombosis-related composite endpoint of death because of thrombosis, amputation secondary to ischemic complications of HIT, or new thrombosis (17.7% vs 30.6%, P = .007). Significant reductions also occurred in new thrombosis and death because of thrombosis. Major bleeding was similar between groups (7.7% vs 8.2%; P = .84). Adverse outcomes were more likely to occur in patients who were initially diagnosed with HIT and thrombosis, had undergone cardiac surgery, were not white, or had more severe thrombocytopenia. In acutely ill HIT patients, argatroban, versus historical control, provides effective antithrombotic therapy without increasing major bleeding. Patients with more severe thrombocytopenia or HIT-related thrombosis on HIT diagnosis have a poorer prognosis, emphasizing the importance of prompt recognition/ treatment of HIT in acutely ill patients.

Plasma Thrombin Activatable Fibrinolysis Inhibitor and Tissue Factor Pathway Inhibitor Changes Following Sepsis

2007-10-02

Sepsis-induced systemic inflammation results in coagulation abnormalities that may be different in gram-positive and gram-negative infections. We used ciprofloxacin to induce a predominantly gram-positive Enterococcus faecalis polymicrobial sepsis in rats. Ciprofloxacin-untreated rats exhibited a predominantly gram-negative polymicrobial sepsis. Rats were subjected to 30% body surface area burn (B), cecal ligation puncture (CLP) with a 22-gauge needle, and B + CLP. Ciprofloxacin-treated B + CLP rats showed a significant decrease in plasma thrombin activatable fibrinolysis inhibitor (TAFI) levels compared with sham rats. However, plasma tissue factor pathway inhibitor (TFPI) levels decreased significantly in B, CLP, and B + CLP groups compared with sham rats. The ciprofloxacin-untreated group showed a significant decrease in plasma TAFI levels in CLP and B + CLP and plasma TFPI levels decreased in all 3 groups compared with sham rats. Histological changes in the liver and kidney included vascular congestion and parenchyma bleed following B + CLP in ciprofloxacin-untreated rats. These results suggest that plasma TAFI and TFPI levels differ depending on the type of bacteria involved in the septic process.

Singlet Oxygen Enhances Intrinsic Thrombolysis: The Intrinsic Oxidative Clot Lysis Assay (INOXCLA)

Stief, T. W. — 2007-10-02

Granulocytes are important cells of inflammation and cellular thrombolysis. They produce urokinase (u-PA) and chloramines. In this study, u-PA/chloramine—mediated fibrinolysis is imitated in a microtiter-plate. Seventy-five microliters plasma are incubated with 50 µL 50% Pathromtin SL, 6% BSA, and 38 mM CaCl2 for 30 minutes (37°C). Then, 50 µL 10 mM chloramine-T in PBS are added. After 30 minutes (37°C), 50 µL 0, 100, or 10 IU/mL u-PA in 6% BSA-PBS are added and the turbidity is determined at 405 nm after 0, 3, or 16 hours. Clot lysis was increased more than tenfold by 0.5 to 1 µmoles chloramine (ED50 after 3h = about 0.25 µmoles = 2mM final concentration). The normal range for the present intrinsic oxidative clot lysis assay (INOXCLA) is 100% ± 25% (MV ± SD; 100 relative % of norm; the normal lysis being 60 absolute %; CVs < 10%). Fifty percent lysis of adherent microclots occurred after 0.75 hours, 2 hours, 14 hours, 13 days, or 17 days when using 1000, 100, 10, 1, or 0 IU/mL u-PA reagent. If the u-PA activity is quenched by PAI-2, no clot lysis appears. Chloramines are important physiologic generators of nonradical excited singlet oxygen and enhance u-PA—mediated lysis of plasma clots. Based on the u-PA/chloramines coaction, a new global fibrinolysis assay has been derived.

Type IIB von Willebrand Disease: Role of Qualitative Defects in Atherosclerosis and Endothelial Dysfunction

2007-10-02

Objective. To verify whether a hereditary bleeding tendency, such as von Willebrand disease (vWD) type IIB, protects against the onset of atherosclerosis. Participants and Methods. Twenty-four patients with vWD type IIB and 24 healthy controls, matched for common atherosclerotic risk factors. All patients were evaluated by color Doppler ultrasound of the common carotid, carotid bifurcation, common femoral artery, brachial artery, and abdominal aorta, investigating intima-media thickness (IMT) and presence of plaques in each arterial district. Flow mediated dilation (FMD) of the brachial artery was used to test endothelial function. Results. vWD type IIB patients presented no significant difference in IMT in any arterial district. FMD showed no differences between the 2 groups. Conclusions. The quantitative clotting defect characteristic of vWD type IIB does not seem to protect against atherosclerosis.

Elevated Concentrations of Soluble Adhesion Molecules and Large Platelets in Diabetic Patients: Are They Markers of Vascular Disease and Diabetic Nephropathy?

Bavbek, N., Kargili, A., Kaftan, O., Karakurt, F., Kosar, A., Akcay, A. — 2007-10-02

P-selectin, E-selectin, and mean platelet volume are markers associated with platelet reactivity that have been demonstrated to be increased in diabetes. We were particularly interested to see if there was a difference in mean platelet volume and selectins between diabetics and nondiabetics, and in diabetics with and without nephropathy, and whether there was a correlation between mean platelet volume and selectins. One hundred and fourteen diabetic patients and 31 healthy controls were investigated. Plasma levels of P-selectin and E-selectin were higher in the diabetic group than in controls (P = .001 and P = .007, respectively) and in diabetic patients with proteinuria than in patients without proteinuria (P = .002 and P = .004, respectively). Protein excretion was lower in patients with low mean platelet volume values (P = .004). In conclusion, elevated platelet volume and high selectin values may play a role in the development of vasculopathies and complications in diabetes mellitus. Further studies are needed to prove these results.

Hyperhomocysteinemia Relates to the Subtype of Antiphospholipid Antibodies in Non-SLE Patients

Wei Hsi Chen, , Hung Sheng Lin, , Yi Fen Kao, , Min Yu Lan, , Jia Shou Liu, — 2007-10-02

Abnormal increases of antiphospholipid antibody and plasma homocysteine levels are recently emerging as nonlipidic risk factors for cerebral atherogenesis and thrombosis. Both antiphospholipid antibody and homocysteine share many similar bioeffects in hemostasis, but their interaction is still inconsistent. In this study, we examined the relation between the plasma homocysteine level and lupus anticoagulant, anticardiolipin antibody, and anti-β2-glycoprotein I antibody in patients with noncardiac cerebral ischemia. Systemic lupus erythrematosus patients were excluded. The results showed a higher frequency of moderate hyperhomocysteinemia in patients with an abnormal increase of lupus anticoagulant only. Neither the serum folate and cobalamin levels nor methylenetetrahydrofolate reductase allele mutation contributes to this result. Accordingly, homocysteine interacts with lupus anticoagulant to promote cerebral atherosclerosis and ischemia. The role of vasculopathic or prothrombotic autoantibody generation in response to specific pathological change such as hyperhomocysteinemia warrants further investigation.

Variability in Anticardiolipin Antibody Detection: Role of Nonspecific IgG Binding and Different Microtiter Plates

Pellegrino, N. M., Caccavo, D. — 2007-10-02

There are many studies that are available on the Internet that attempt to standardize the assay for anticardiolipin antibody evaluation because of the variability of results. The aim of this study was to evaluate simultaneously the role of different microplates and the importance of sample nonspecific binding in determining different results in anticardiolipin antibody detection. Sera from 8 patients with raised levels of IgG anticardiolipin antibodies and 10 control sera were assayed by enzyme-linked immunosorbent assay in the presence (specific binding) or in the absence of cardiolipin (sample blank) with four different microplates, that is, NUNC PolySorp, FALCON ProBIND, Greiner 655061 (high binding), and Greiner 655001 (medium binding). Results were expressed as optical densities or net-optical densities (following sample blank subtraction) as well as international IgG anticardiolipin units (GPL) or net-GPL. A wide interplate variability of optical densities was found. When results were expressed as GPL, significant differences were only found between Greiner 655061, FALCON ProBIND, and NUNC PolySorp (P < .05 and P < .001, respectively) whereas differences were not statistically significant if interplate variability was analyzed as net-GPL. Results expressed as categorical variables (ie, positive/negative, according to a GPL cut-off and net-GPL cut-off, obtained with sera from 100 apparently healthy blood donors) showed a good or excellent Cohen's coefficient of concordance among plates when positivity was evaluated on net-GPL. Our data strongly suggest that quantification and subtraction of sample blank may improve both interlaboratory agreement and reliability of anticardiolipin assay and minimize false-positive results.

Soluble P-Selectin During a Single Hemodialysis Session in Patients With Chronic Renal Failure and Erythropoietin Treatment

Stasko, J., Galajda, P., Ivankova, J., Holly, P., Rozborilova, E., Kubisz, P. — 2007-10-02

In several studies, hemodialysis (HD) patients treated with recombinant human erythropoietin (rHuEPO) because of renal anemia showed increased levels of soluble adhesion molecules. The purpose of the study was to investigate the changes of soluble P-selectin (sSELP) and its relationship to platelet activation during a single HD session in patients with long-term rHuEPO treatment. Fifty-two HD patients with chronic renal failure were involved—26 with rHuEPO treatment (EPO group) and 26 without (non-EPO group). Thirty healthy subjects served as the control group. The sSELP, β-thromboglobulin, and platelet factor 4 plasma levels were measured before and after a single 4-hour HD session on a cuprophane dialyzer. The basal β-thromboglobulin and platelet factor 4 plasma levels were significantly increased in both HD groups compared with healthy controls but did not change after a single HD session, except for a significant decrease of platelet factor 4 in the non-EPO group. The predialysis sSELP plasma levels did not differ significantly compared with those of the healthy controls, but there was a significant increase of sSELP levels after a single HD session in both groups (EPO, P < .005; non-EPO, P < .05, respectively). These results suppose that the increased sSELP level was released from platelets during the course of a single HD session. The more significant increase of the sSELP plasma levels in EPO group during HD indicates that platelets are more activated in patients with long-term rHuEPO treatment, and this fact could partially explain the suspected tendency for thrombosis in these patients.