Clinical and Applied Thrombosis/Hemostasis

Heparin has been conventionally used as an anticoagulant for medical and surgical indications. Because factor Xa is an essential component of the prothrombinase complex and leads to the generation of thrombin, its inhibition has become a focus of newer antithrombotic drug development. The in vitro anticoagulant profile of DX-9065a, a synthetic direct factor Xa inhibitor, was studied using activated clotting time assay, thrombelastography, and global clotting tests, such as prothrombin time (PT), activated partial thromboplastin time (aPTT), diluted aPTT, Heptest, Heptest-HI, dilute Russell's viper venom time (dRVVIT), thrombin time, ecarin clotting time, and amidolytic anti-Xa assay. In addition, the effect of DX-9065a on platelet aggregation and inhibition of thrombin generation markers (FPA, Fl + 2, and TAT) were studied. The pharmacokinetic and pharmacodynamic profiles of DX-9065a were also studied in a non-human primate (Macaca mulatta) model. DX-9065a produced a concentration-dependent increase in the Hemochron celite ACT and HemoTec ACT. Clotting times of 538 ± 19 and 401 ± 12, respectively, were reached at a concentration of 25 μg/mL signifying that DX-9065a may be useful in interventional cardiological procedures. DX-9065a prolonged the r-time on thrombelastography. DX-9065a did not show any effect on adenosine diphosphate (ADP)-, collagen-, epinephrine-, and arachidonic acid-induced platelet aggregation at concentrations up to 10 μg/mL. DX-9065a exhibited a concentration-dependent prolongation of the PT, aPTT, diluted aPTT, Heptest, dRVVT, and reached the clotting times of 51.6, 132, 193, 47.9, 129.9 seconds, respectively, at a final concentration of 12.5,tg/mL; compared to a control value of 10.6, 30.2, 41.9, 14, 32.2 seconds, respectively. DX9065a did not affect the ecarin clotting time and thrombin time at concentrations up to 12.5 μg/mL. Because DX-9065a prolonged the dRVVT, this may impact diagnostic screening of patients with systemic lupus erythematosus.

This study evaluated the protective effects of Venoruton on the development of flight edema in normal subjects travelling in economy in long-haul flights (8-9 hours). Edema is a relevant aspect of long-haul flights affecting both venous patients and normal subjects. Edema and flight microangiopathy were studied in a group of 164 subjects randomized (after informed consent) into two groups to evaluate prophylaxis in 8 to 9-hour flights. The treatment group received Venoruton (hydroxyethyl rutosides) 1 g twice daily for 3 days (2 days before the flight and the day of the flight). The control group received comparable placebo. Edema was evaluated with a composite edema score based on the edema tester, on the measurements of ankle circumference, volume measurements, subjective swelling, and on a discomfort score. Items 1, 4, and 5 are based on an analogue scale line (I to 10) directly defined by the subjects before and after the flights. Of the included subjects, 151 completed the study. Dropouts were due to poor compliance, travelling, or connection problems. Age and gender distribution were comparable in the two groups as were risk factor distributions. The level of edema at inclusion was comparable in the two groups of subjects. After the flight there was an average score of 6.7 (SD 1.7) in the control group, while in the Venoruton group the score was on average 3 (SD 1.1) (p < 0.05). In the control group 77% of the subjects had an evident increase in ankle circumference and volume, which was clearly visible at inspection and associated with discomfort. In the Venoruton group, edema was clearly present only in 8% of subjects (associated with discomfort rated between 3 and 6 on the analogue scale line) and it was mild, not associated with symptoms. Therefore the control of flight edema with Venoruton was clear both considering parametric data (circumference and volume) and non-parametric (analogue scale lines) measurements. The combined evaluation of the edema score is significantly favorable for subjects under prophylaxis with Venoruton.

Heparin-induced thrombocytopenia (HIT) is a rare but dangerous complication of heparin prophylaxis or treatment. The present laboratory tests to measure heparin-associated antibodies are not specific. The diagnosis of HIT mainly depends on the decrease in platelet count and on clinical symptoms. To evaluate clinical outcome, bleeding complications and platelet counts were evaluated in 45 patients with HIT type II (HIT II) treated prophylactically (subcutaneous injections) or therapeutically (intravenous infusion) with danaparoid. Group I included 24 patients with HIT II without thromboembolic complications who received danaparoid twice daily subcutaneously (10 IU/kg) for a mean of 16 days. Group II included 21 patients with thromboembolic complications. They were treated with intravenous danaparoid (2.6 IU/kg/h ± 1.1) for a mean of 17 days. During subcutaneous prophylaxis, mean anti-Xa levels of 0.2 U/mL and during intravenous treatment, mean anti-Xa levels of 0.4 U/mL were reached. No deaths, amputations, or serious bleeding complications occurred, and no new thromboses were observed in both patient groups.Treatment with danaparoid led to a fast normalization of the platelet counts. This normalization occurred earlier and the concentration of platelets was higher in patients treated with intravenous doses. Danaparoid with subsequent vitamin K-antagonist treatment effectively prevents thromboembolic complications in patients with HIT.

Oral contraceptive therapy (OCT) is associated with a fourfold increase in the risk for venous thromboembolism (VTE). Women with congenital defects have a much higher risk. Because it is impossible to screen all women undergoing OCT, it is advisable to adopt some guidelines in an attempt to define the group of women who may be particularly exposed to the risk of VTE. On the basis of experience and that in the literature, two flow charts are used that are simple and reliable. The two flow charts are based on an accurate family and personal history so that an extensive investigation can be carried out only for women who show a positive family or personal history for idiopathic thrombosis. The definition of idiopathic thrombosis may be difficult, particularly in the case of family members. In case of doubt the thrombotic event should be considered as idiopathic. The two flow charts are not intended to eliminate the OCT-related VTE but represent only an attempt to decrease the prevalence of this complication.

Plasma-reduced platelet concentrates are commonly administered to prevent febrile transfusion reactions and to avoid fluid overload in neonates. Because little is known about the influence of centrifugation and resuspension on functional aspects of platelets, we examined the effects of plasma-reduction on platelet aggregation and platelet-dependent thrombin generation. Our results show that plasma reduction and resuspension of the platelet pellet in saline or plasma results in a significant reduction in platelet aggregation to a combination of the platelet agonists adenosine diphosphate and epinephrine (p < 0.001). In contrast, when a combination of the more potent agonists collagen and thrombin was used, platelet aggregation was maintained. Likewise, no decline was observed in plateletdependent thrombin generation as measured by the functional prothrombinase assay or Annexin V binding. We conclude that centrifugation and resuspension of platelets to render the concentrate plasma-free, as a routine procedure in blood banking, variably affects in vitro platelet aggregability but does not significantly affect plateletdependent thrombin generation.

Since their introduction, oral contraceptives have been linked to an increased incidence of thromboembolic events. Epidemiologic studies have shown that women who use third-generation oral contraceptives containing desogestrel, gestodene, or norgestimate have a higher risk of venous thrombosis than women who use second-generation oral contraceptives containing levonorgestrel. Women who use oral contraceptives are significantly less sensitive to activated protein C. From January 1996 to December 2001, 17,577 patients were hospitalized in our department, 177 of them (1%) had confirmed diagnosis of venous thromboembolism and 15 of those (177) were women on oral contraceptive therapy. Oral contraceptives were taken from 28 days to 18 months. No other potential congenital or acquired causes of thrombosis were present before thromboembolic events occurred in these women. The discontinuation of the pill combined with usual heparin (in one patient thrombolysis) and coumarin therapy was effective in all cases. After the discontinuation of coumarin (3-6 months), every patient was screened for hereditary thrombophilia. All women on oral contraceptive therapy had confirmed hereditary risk factors for venous thrombosis. Acquired resistance to activated protein C may explain the epidemiologic observation of increased risk of venous thrombosis in oral contraceptive users, especially in women using third-generation oral contraceptives who had other risk factors (hereditary or acquired) for venous thrombosis.

Vascular events caused by atherosclerosis are the major cause of death in patients undergoing hemodialysis (HD). The relationship between the tests of atherosclerosis and hemostasis in 84 patients with HD was examined. Abnormal test results indicating the occurrence of atherosclerosis were found in 66% by the Fontaine score, in 33% by ankle blood pressures, and in 79% by aortic calcification index (ACI). When HD was prolonged, the mean Fontaine score and ACI were further increased. Particularly, the ACI tended to correlate with HD duration. The ankle-brachial index (ABI) was decreased in patients with HD duration of more than 10 years. Before HD, the plasma levels of fibrinogen, plasmin-plasmin inhibitor complex (PIC), thrombomodulin (TM), and D-dimer were increased, while the plasma levels of protein C (PC), antithrombin (AT), thrombin-antithrombin complex (TAT), and tissue plasminogen activator (tPA)-plasminogen activator inhibitor-I (PAI-I) complex (tPA-PAI-1 complex) were decreased. With prolonged HD, the plasma levels of AT and PC were decreased, while those of D-dimer were increased. The plasma levels of TAT and TPA-PAI-1 complex were significantly increased and those of PIC, soluble fibrin (SF) and D-dimer tended to be high in patients with less than 0.7 of ABI. The plasma levels of Ddimer, TPA-PAI-1 complex, TAT, PIC, and SF tended to be high in patients with more than 0.5 in ABI. These findings suggest that patients undergoing HD have progressive atherosclerosis and that this is associated with some hemostatic abnormalities.

Protein S deficiency is one of the principal congenital thrombophilias reported. The objective of this study was to assess the prevalence of protein S deficiency in patients with repetitive superficial thrombophlebitis. Thirty-six consecutive patients, 28 female and eight male, who presented with repetitive superficial thrombophlebitis (two or more cases); age range, 17 to 58 years, mean, 38.2 years; were evaluated. Coagulometry was used to determine the biologic activity of protein S. In 5.5% of the patients with repetitive superficial thrombophlebitis, protein S deficiency was detected.

Tissue type plasminogen activator (t-PA) is released from endothelium in both a constitutive and regulated fashion. In healthy subjects, an association between net t-PA release rate and a few t-PA gene polymorphisms, including the Alu-repeat I/D polymorphism in intron 8, was described. The possible influence of the Alu-repeat polymorphism on t-PA release was evaluated after a venous occlusion test (VO) in 82 patients showing an impaired fibrinolytic capacity associated with different arterial disease or with previous venous thrombosis, and in 50 healthy controls. Euglobulin lysis time, t-PA antigen (t-PA:Ag) and activity, PAI-1 antigen and activity plasma levels were assayed before and 20 minutes after VO; the Alurepeat I/D polymorphism was determined by PCR. Defective fibrinolysis was due to reduced t-PA release in 40 patients (t-PA group) and to PAI-1 excess in 42 patients (PAI group). No differences in both genotype distribution and allele frequencies were observed between patients and controls. The t-PA:Ag increase after VO (20/0-minute levels ratio adjusted for hematocrit) was considerably higher both in controls and in PAI group patients carrying the I allele than in the DD genotype carriers (II, ID, DD: 3.77∓0.62, 3.43∓0.44, 2.06∓0.32 in controls, and 3.67∓0.23, 2.80∓0.50, 1.62∓0.29 in PAI group, respectively). The difference was significant between the DD and both the ID and II genotypes in controls (p<0.05), and between the DD and II genotypes in PAI-I group (p<0.05). A slight and nonsignificant trend of association between genotype and t-PA:Ag 20/0 ratio was seen in the t-PA group patients. In conclusion, these data suggest a possible genetic modulation of t-PA-regulated secretion.

Changes of hemostatic markers in 226 patients with disseminated intravascular coagulation (DIC) and hematopoietic disorders were examined after treatment of DIC. The changes in prothrombin time (PT) ratio, fibrinogen, fibrin and fibrinogen degradation products (FDP), antithrombin, and protein C, thrombin-antithrombin complex (TAT), plasmin-plasmin inhibitor complex (PPIC), and soluble fibrin monomer complex (SFMC) in all patients with DIC were significant during the clinical course of DIC, but those of D-dimer, thrombomodulin (TM), tissue factor (TF), and tissue factor pathway inhibitor (TFPI) were not. Activated partial thromboplastin time (aPTT) and PT were significantly longer in the poor response group than in good response group. Plasma levels of FDP, TAT, PPIC, SFMC, TM, and DIC score were significantly higher in poor response group than in good response. Protein C and antithrombin levels were significantly lower in poor response group than in good response group. The changes of PT ratio, fibrinogen, FDP, DIC score, antithrombin, plasmin inhibitor, and protein C were significant in the good response group, but these levels were not significant in the poor response group. The changes in plasma TAT and SFMC levels were significant in the good response group but were not in poor response group. The changes in D-dimer, TM, TF, or TFPI were not significant in both groups. These findings suggest that anticoagulant agents should be administered at levels below TAT 40 ng/mL or SFMC 300 μg/mL in patients with DIC and hematopoietic disorders.

The purpose of this study was to investigate the pharmacodynamics of PEG-Hirudin and its potential interactions with acetylsalicylic acid (ASA 325 mg once daily from days 1-3). In a randomized, 2-way cross-over trial, 6 healthy volunteers received PEG-Hirudin (IV bolus of 0.2 mg/kg + 0.02 mg/kg/h for 24 hours) and placebo (IV bolus + 24-hour infusion). In a further randomized, 3way cross-over trial another 9 healthy volunteers received ASA (325 mg) or oral placebo from days 1 to 3 and PEG-Hirudin (0.2 mg/kg + 0.02 mg/kg/h for 24 h) or IV placebo on day 3. Assessments included bleeding time (BT), collagen (1 μg ml^-1)-induced platelet aggregation (CIPA), platelet adhesion, ecarin clotting time (ECT), activated clotting time (ACT), plasma anti-factor Ila activity (aIla), and activated partial thromboplastin time (aPTT). Ten minutes after the PEG-Hirudin injection/starting the infusion, mean plasma concentration was 3.1 μg/mL and aPTT, ECT, and ACT were prolonged up to 80, 309, and 233 seconds, respectively. During the last 8 hours of the 24-hour infusion mean PEG-Hirudin plasma concentration was 1.3 μg/mL. In the interaction study, ASA significantly inhibited CIPA. At 6 hours after administration, on day 3 mean BT was 6.5 minutes after PEG-Hirudin alone, 18.2 minutes after ASA alone, and 32.9 minutes after combined administration of ASA and PEG-Hirudin. PEG-Hirudin (0.2 mg/kg + 0.02 mg/kg/h for 24 hours) administered alone or together with 325 mg ASA proved to be safe in healthy volunteers. Combined use of PEG-Hirudin and ASA significantly increased the mean bleeding time compared to ASA or PEG-Hirudin monodrug administration. None of the clotting parameters or platelet function tests correlated with the prolongation of the bleeding time.

Failure of anticoagulant therapy in a patient with a St. Jude heart valve in the mitral position is reported. After 8 years of optimal anticoagulation at an International Normalized Ratio between 2.5 and 3.5, a floating thrombus was discovered by chance during transthoracic echocardiography and then surgically removed. A study for congenital or acquired thrombophilia disclosed a positivity for activated protein C resistance, due to the presence of factor V Leiden. The intensity of warfarin anticoagulation was increased to provide more protection for the patient.