Plasma Antithrombin Research Articles

Antithrombin significantly influences platelet adhesion onto immobilized fibrinogen in an in-vitro system simulating low flow

BackgroundAdhesion of platelets onto immobilized fibrinogen is of importance in initiation and development of thrombosis. According to a recent increase in evidence of a multiple biological property of antithrombin, we evaluated the influence of antithrombin on platelet adhesion onto immobilized fibrinogen using an in-vitro flow system.MethodsPlatelets in anticoagulated whole blood (29 healthy blood donors) were labelled with fluorescence dye and perfused through a rectangular flow chamber (shear rates of 13 s-1 to 1500 s-1). Platelet adhesion onto fibrinogen-coated slips was assessed using a fluorescence laser-scan microscope and compared to the plasma antithrombin activity. Additionally the effect of supraphysiological AT supplementation on platelets adhesion rate was evaluated.ResultsWithin a first minute of perfusion, an inverse correlation between platelet adhesion and plasma antithrombin were observed at 13 s-1 and 50 s-1 (r = -0.48 and r = -0.7, p < 0.05, respectively). Significant differences in platelet adhesion related to low (92 ± 3.3%) and high (117 ± 4.1%) antithrombin activity (1786 ± 516 U vs. 823 ± 331 U, p < 0.05) at low flow rate (13 s-1, within first minute) have been found. An in-vitro supplementation of whole blood with antithrombin increased the antithrombin activity up to 280% and platelet adhesion rate reached about 65% related to the adhesion rate in a non-supplemented blood (1.25 ± 0.17 vs. 1.95 ± 0.4 p = 0.008, respectively).ConclusionIt appears that antithrombin in a low flow system suppresses platelet adhesion onto immobilized fibrinogen independently from its antithrombin activity. A supraphysiological substitution of blood with antithrombin significantly reduces platelet adhesion rate. This inhibitory effect might be of clinical relevance.

Four-day antithrombin therapy does not seem to attenuate hypercoagulability in patients suffering from sepsis

IntroductionSepsis activates the coagulation system and frequently causes hypercoagulability, which is not detected by routine coagulation tests. A reliable method to evaluate hypercoagulability is thromboelastography (TEG), but this has not so far been used to investigate sepsis-induced hypercoagulability. Antithrombin (AT) in plasma of septic patients is decreased, and administration of AT may therefore reduce the acquired hypercoagulability. Not clear, however, is to what extent supraphysiologic plasma levels of AT decrease the acute hypercoagulability in septic patients. The present study investigates the coagulation profile of septic patients before and during four day high-dose AT therapy.MethodsPatients with severe sepsis were randomly assigned to receive either 6,000 IU AT as a bolus infusion followed by a maintenance dose of 250 IU/hour over four days (n = 17) or placebo (n = 16). TEG, platelet count, plasma fibrinogen levels, prothrombin time and activated partial thromboplastin time were assessed at baseline and daily during AT therapy.ResultsTEG showed a hypercoagulability in both groups at baseline, which was neither reversed by bolus or by maintenance doses of AT. The hypercoagulability was mainly caused by increased plasma fibrinogen, and to a lesser extent by platelets. Plasmatic coagulation as assessed by the prothrombin time and activated partial thromboplastin time was similar in both groups, and did not change during the study period.ConclusionThe current study shows a distinct hypercoagulability in patients suffering from severe sepsis, which was not reversed by high-dose AT treatment over four days. This finding supports recent data showing that modulation of coagulatory activation in septic patients by AT does not occur before one week of therapy. Trial registration: Current Control Trials ISRCTN22931023

Pharmacokinetics and Pharmacodynamics of Enoxaparin in Multiple Trauma Patients

Enoxaparin is the only low molecular-weight heparin (LMWH) with documented efficacy for the prevention of venous thromboemobolism (VTE) following trauma, and it is currently considered the treatment of choice. Recent reports have suggested that the pharmacokinetics (PK) and pharmacodynamics of LMWH products may be altered in critically ill patients. Two cohorts of critically ill multiple trauma patients were enrolled in this study: A (nonedematous) and B (edematous, defined as the presence of peripheral edema and an increase in body weight of > or =10 kg). All patients received at least four doses of enoxaparin 30 mg subcutaneously every 12 hours before the study dose. Blood samples were collected before and 0.5, 1, 2, 3, 4, 6, 8, and 12 hours following a morning dose. Plasma anti-Xa and antithrombin (AT) activities were determined using chromogenic assays. A compartmental PK analysis model was defined for the data. PK parameters for the two cohorts were compared using a Mann-Whitney Rank Sum test. The area under the curve (AUC)0-12 hour, maximal plasma anti-activated Factor Xa (anti-Xa) activity (Amax), and AT activity were significantly lower in the edematous trauma patients (p < 0.05). The AUC0-12 hour for plasma anti-Xa activity was highly variable in both study cohorts. Seven of the 10 edematous patients had barely quantifiable anti-Xa results. Activity levels were too low to reliably estimate the PK parameters for most patients in cohort B. The standard dose of enoxaparin recommended for the prevention of VTE following multiple trauma provides unreliable and highly variable anti-Xa activity in critically ill trauma patients, and is strongly affected by the presence of significant peripheral edema.

Heparin Chain Length Dependence of Factor Xa Inhibition by Antithrombin in Plasma.

Heparin anticoagulants function by enhancing the inhibition of coagulation proteases by the serpin antithrombin (AT). Evaluation of the specific anti-factor Xa (fXa) activity of therapeutic heparins in the physiologically relevant plasma-based clotting assays has not been feasible since thrombin, the final protease of the cascade, is the primary target for inhibition by AT in the presence of heparin. To circumvent this problem, we developed an assay in which the native AT in plasma was replaced with an AT mutant which exhibits identical affinity for heparin and near normal reactivity for fXa, but does not react with thrombin and other coagulation proteases in either the absence or presence of heparin. This assay was used to distinguish the anti-fXa activity of different molecular weight heparins from their anti-thrombin activity in clotting assays which were initiated by triggers of either extrinsic or intrinsic pathways of coagulation. The results suggest that the acceleration of fXa inhibition by AT exhibits a marked heparin chain-length dependence, with fondaparinux (a pentasaccharide) having the lowest and unfractionated heparin having the highest effect. Interestingly, comparative studies revealed that the fondaparinux-catalyzed acceleration of thrombin inhibition by AT also makes a significant contribution to prolongation of the clotting time, possibly suggesting that the anticoagulant function of the therapeutic pentasaccharide is mediated though the inhibition of both fXa and thrombin.

Modulation of Platelet Adhesion by Antithrombin.

In this study, we evaluated the influence of antithrombin on platelet adhesion onto immobilized fibrinogen using an in vitro system simulating venous and arterial flow. Platelets in anticoagulated whole blood (PPACK, 40μM) were labeled with mepacrine (10μM). Adhesion of platelets onto fibrinogen-coated glass cover slips was assessed in a rectangular flow chamber (shear rates of 13 s−1 to 1500 s−1). Platelets were visualized at 15 sec, 1 and 5 min following perfusion using a fluorescence laser-scan microscope. In parallel, the effects of supraphysiological supplementation of blood with antithrombin (2.8 IU/ml of blood) on platelet adhesion rates was evaluated. During perfusion, platelet adhesion onto fibrinogen linearly increased with exposure time and shear rates. Within the first min of perfusion, an inverse correlation between platelet adhesion and plasma antithrombin activity was observed at shear rates of 13 s−1 and 50 s−1 (r=−0.48 and r=−0.7, p each <0.05). Significant differences in platelet adhesion (1786±516 U vs. 823±331 U, p<0.05) related to low (92±3.3%) and high (117±4.1%) antithrombin activity was observed at a flow rate of 13s−1 within first minute. Further supplementation of anticoagulated whole blood with antithrombin (activity up to 280 %) decreased the rate of platelets adhesion (ratio of adhesion at 1 and 5 min) about 35% when compared to nonsupplemented blood (1.25 ± 0.17 vs. 1.95 ± 0.4, p=0.008). Application of heparine as anticoagulant did not enhance the antiadhesion properties of antithrombin. Our findings are in accordance with the “low shear phenomenon” of arterial thrombus progression, i.e. thrombus enlargement at distal areas with reduced flow or even stasis. Moreover, the observation that antithrombin significantly suppressed platelet adhesion onto immobilized fibrinogen under low flow conditions is of therapeutic interest and needs further evaluation.

Low Protein Z Levels and the Risk of Venous Thrombosis.

Background. Protein Z (PZ) is a vitamin-K dependent protein that serves as cofactor for PZ-dependent protease inhibitor, inactivating factor Xa. Therefore, deficiency of PZ could led to a prothrombotic phenotype. Contrasting data are available on the relationship between PZ and venous thromboembolism (VTE).Patients and Methods. We carried out a case-control study on 443 patients with deep vein thrombosis of the lower limbs and/or pulmonary embolism who discontinued oral anticoagulant therapy and 394 controls, similar for age and sex. PZ was measured with an enzyme immuno-assay (Diagnostica Stago, France). A thrombophilia screening including DNA testing for factor V Leiden and G20210A prothrombin mutation and measurements of plasma antithrombin, protein C, protein S, and homocysteine (fasting and post-methionine load) was performed. Pregnancy, oral contraceptive use, liver or renal disease were exclusion criteria.Results. 443 patients and 394 healthy controls who underwent thrombophilia screening were investigated. Median PZ levels (range) were 1.95 μg/mL (0.22–6.26 μg/mL) in patients and 1.79 μg/mL (0.25–7.82 μg/mL) in controls (p=0.07). The odds ratios (adjusted for age, sex and the presence of thrombophilia) for VTE in patients with PZ levels in the lowest quartile was 0.8 (95% CI 0.5–1.1), and remained statistically non significant considering PZ levels below the 10th percentile. However, low PZ levels (lowest quartile) increased the risk of VTE associated with factor V Leiden (odds ratio 14.6, 95% CI 1.9–113), prothrombin G20210A (odds ratio 3.2, 95% CI 1.0–10.3), and hyperhomocysteinemia (odds ratio 4.7, 95% CI 1.7–17.0). These interactions remained, apart for prothrombin G20210A, when PZ levels below the 10th percentile were considered.Conclusion. Low PZ levels are not an independent risk factor for VTE, but may enhance the relative risk due to already established risk factors, particularly factor V Leiden.

Ximelagatran: An Oral Direct Thrombin Inhibitor for Anticoagulant Therapy

Ximelagatran is a prodrug that is rapidly converted to melagatran, a direct, competitive and reversible inhibitor of the active site of human α-thrombin. Its effect is independent from diet, plasma antithrombin levels, obesity or ethnicity. It has little effect on bleeding time at doses up to twice those required, and interference with other drugs is avoided by the absence of interaction with cytochrome P450. Ximelagatran has been documented in a large clinical programme with proven efficacy in prevention and treatment of venous thromboembolism, prevention of stroke in patients with atrial fibrillation and prevention of major cardiovascular events following a recent myocardial infarction. Bleeding has not been a problem, but increase in liver enzymes is a cause of concern for long term use until more information is available.

Prevalence of inherited prothrombotic abnormalities and central venous catheter-related thrombosis in haematopoietic stem cell transplants recipients

In this prospective study, we assessed the incidence of central venous catheter (CVC)-related thrombosis in haematopoietic stem cell transplant (HSCT) recipients. We determined the contribution of inherited prothrombotic abnormalities in blood coagulation to CVC-related thrombosis in these patients. The study was conducted between May 2002 and September 2004. CVCs were externalized, nontunneled, polyurethane double lumen catheters. Before catheter insertion, laboratory prothrombotic markers included factor V Leiden, the prothrombin gene Gly20210A mutation, plasma antithrombin levels, and protein C and S activity. All patients were systematically examined by ultrasonography just before, or <24 h after, catheter removal, and in case of clinical signs of thrombosis. A total of 171 patients were included during the 28-month study period. Five (2.9%) and three (1.7%) patients had evidence of protein C and protein S deficiency, respectively. Only one patient had an antithrombin deficiency (0.6%). In total, 10 patients (5.8%) were heterozygous for the factor V Leiden mutation, and one patient had heterozygous prothrombin G20210A mutation (0.6%). We observed a CVC-related thrombosis in 13 patients (7.6%). Thrombosis was diagnosed in four out of 20 patients (20%) with a inherited prothrombotic abnormality compared to nine of 151 patients (6%) who did not have a thrombophilic marker (relative risk 3.3 CI 95% 1.1-9.9). Our results suggest that inherited prothrombotic abnormalities contribute substantially to CVC-related thrombosis in HSCT recipients. In view of physicians' reluctance to prescribe prophylactic anticoagulant treatment in these patients, a priori determination of inherited prothrombotic abnormalities may form a basis to guide these treatment decisions.

Study of portal vein thrombosis in patients with idiopathic portal hypertension in Japan

The aim of this study was to elucidate the incidence and clinical manifestations of portal vein thrombosis (PVT) in patients with idiopathic portal hypertension (IPH) in Japan during long-term follow-up. Twenty-two patients with IPH were examined for PVT by sonography during a follow-up of 12+/-6 years. Clinical manifestations and patient outcome related to PVT were studied. Seventy patients with liver cirrhosis were examined by sonography as an incidence control of thrombosis. Nine IPH patients had portal thrombosis (9/22, 41%), a higher incidence than in liver cirrhosis patients (7/70, 10%). Those with thrombosis showed ascites, marked hypersplenism, and low serum albumin. Four patients with thrombosis died. Patients without thrombosis showed less clinical problems after long-term follow-up. Plasma antithrombin III and protein C activity decreased in almost half of the patients. However, there were no differences in these parameters between patients with and without thrombosis. In Japan, IPH patients had a high incidence of portal thrombosis, a significant factor for poor prognosis. Whether the management of PVT contributes to an improvement of a clinical course of IPH or not should be clarified in further study.

Successful therapy with argatroban for superior mesenteric vein thrombosis in a patient with congenital antithrombin deficiency

A 38-year-old woman was admitted with superior mesenteric vein (SMV) thrombosis, which was refractory to anticoagulation therapy. The plasma antithrombin activity was decreased and hardly compensated by concentrated antithrombin preparation due to high consumption rate. However, successful anticoagulation was achieved by administration of direct thrombin inhibitor, argatroban. Family studies of antithrombin activity revealed that she had type I congenital antithrombin deficiency. A novel heterozygous mutation in the gene for antithrombin (single nucleotide T insertion at 7916 and 7917, Glu 272 to stop in exon 4) was identified. Argatroban administration would be effective in the treatment of congenital antithrombin deficiency with SMV thrombosis.

Modified score for disseminated intravascular coagulation in the critically ill

To assess the value of the diagnosis of overt disseminated intravascular coagulation (DIC) according to the International Society on Thrombosis and Haemostasis (ISTH) criteria and that of the parameters included in the ISTH score for overt DIC in predicting day 28 mortality in intensive care patients. Also, to assess the value of the components of the score in the diagnosis of overt DIC. Retrospective clinical study in a university hospital intensive care unit. 494 consecutive patients admitted in the ICU between January 2002 and October 2003. Clinical and laboratory data, including hemostatic parameters, were collected from computerized databases and patient files. Altogether 19% (95/494) of the patients fulfilled the criteria for overt DIC. Their day 28 mortality rate was higher than that of patients without overt DIC (40% vs. 16%). The lowest platelet count (area under curve, AUC, 0.910), highest plasma D-dimer (AUC 0.846), lowest antithrombin (AUC 0.823), and Owren-type prothrombin time activity (AUC 0.797) discriminated well the patients with and without overt DIC, whereas plasma fibrinogen (AUC 0.690) had poor discriminative power. No patient with the diagnosis of overt DIC had decreased plasma fibrinogen. Day-1 SOFA and APACHE II score, the first CRP measurement, and the lowest antithrombin were independent predictors of day 28 mortality. The diagnosis of overt DIC was not an independent predictor of day 28 mortality. In ICU patients plasma antithrombin seems a promising candidate in the panel of indicators for overt DIC whereas the value of plasma fibrinogen is in doubt.

Phosphatidylserine/Phosphatidylcholine Microvesicles Can Induce Preeclampsia-Like Changes in Pregnant Mice

We established a phosphatidylserine (PS)/phosphatidylcholine (PC) microvesicles-induced preeclampsia-like model in mice. PS/PC were prepared by mixing 80% PC and 20% PS, and suspended in 0.05 M Tris-HCl at a concentration of 10 mg/mL. One hundred microliters of PS/PC (n = 6) and saline as a control (n = 10) were injected in tail veins of Institute of Cancer Research (ICR) mice every day from days 5.5 to 16.5 of pregnancy. Systolic blood pressure (SBP) was measured by means of the tail-cuff method. On day 17.5, the mice were anesthetized by diethyl ether and euthanized with the collapse of the circulation by drawing blood from the heart. The animals were dissected and the fetuses and placentas removed. Fetal weight and placental weight were evaluated. Plasma antithrombin activity (AT), thrombin-antithrombin complex (TAT), platelet counts, and proteinuria were measured on day 17.5. Placentas were fixed in 4% paraformaldehyde for histologic studies. Statistical analysis was evaluated by analysis of variance and Welch's t-test. Mice injected with PS/PC showed a significant elevation in SBP (124 versus 101 mm Hg; p < 0.001), a significant increase in TAT levels (23 versus 6.6 mug/L; p < 0.05), a significant decrease in platelet counts (88 versus 102 x 10 (10)/L; p < 0.05), a decrease in AT, an increase in proteinuria, and a significant reduction in fetal weight (1.2 versus 1.3 g; p < 0.0001) and placental weight (0.13 versus 0.15 g; p < 0.001), compared with controls. Placentas of mice injected with PS/PC showed diffuse fibrin depositions in the labyrinth layer. We have demonstrated that the artificial PS/PC vesicles induce intrauterine growth restriction with elevations of SBP. The elevation of plasma TAT and the diffuse fibrin depositions in the placentas indicate enhanced thrombin formation, and the significant elevations of SBP indicate preeclampsia-like changes that can be induced by hypercoagulation in the placenta.

Sepsis-associated coagulation disorders

Coagulation activation with intravascular fibrin formation is a general finding in patients with sepsis. Low coagulation factors may be caused by disseminated intravascular coagulation, as well as by loss of plasma and impaired hepatic synthesis in the course of sepsis. The leading clinical symptom in consumption coagulopathy is bleeding. Therefore, treatment mainly consists of substitution of coagulation factors and platelets. Meningococcal and pneumococcal, as well as some other infections may lead to sepsis-induced purpura fulminans, a condition associated with microvascular thrombosis, necrosis, and haemorrhage. A typical laboratory sign is a very low plasma protein C level. Treatment with protein C concentrate or recombinant activated protein C (Drotrecogin alfa, activated) has been shown to be beneficial in sepsis-induced purpura fulminans. Unfractionated heparin or low molecular weight heparin has been recommended for prophylaxis of venous thrombosis, but there are no clinical studies specifically on patients with sepsis. Antithrombin concentrate is used in patients with antithrombin deficiency treated with heparin for acute venous thrombosis or embolism, extracorporeal circulation procedures or other invasive procedures. There is no indication for general use of antithrombin concentrate in patients with sepsis even in patients with low plasma antithrombin levels. Drotrecogin alfa, activated, is used for treatment of patients with severe sepsis. Its use is not limited to patients with sepsis-induced disseminated intravascular coagulation, although these patients appear to benefit especially from this therapy.

Pre-treatment of donor with 1-deamino-8-d-arginine vasopressin could alleviate early failure of porcine xenograft in a cobra venom factor treated canine recipient☆

Unlike cardiac or renal xenotransplants, the depletion of complement using cobra venom factor (CVF) does not improve pulmonary xenograft survival. Several cases suggest that the swine von Willebrand factor (vWF) may play a major role in presenting a different pathogenesis of pulmonary xenograft dysfunction from other organs. To evaluate the role of vWF and the complement system in mediating hyperacute vascular injury of pulmonary xenografts and elucidate pathogenesis of the injury, we performed swine-to-canine orthotropic single lung xenotransplantation after pre-treatment of 1-deamino-8-d-arginine vasopressin (DDAVP) and CVF. We set up three groups for lung xenotransplantation: group I served as the control group; group II, recipients pre-treated with CVF; group III, donors pre-treated with DDAVP (9 mg/kg, 3 days)/recipients pre-treated with CVF (60 u/kg). Hemodynamic data, coagulation and complement system parameters, and grafted lung pathologies were examined serially for 3h after transplantation. DDAVP infusion reduced the vWF content in swine lung tissue in vivo (7.7+/-2.4 AU/mg vs 16.0+/-5.6 AU/mg, P < 0.0001). Infusion of CVF 24 h prior to transplantation effectively depleted the recipient's serum C3 and complement hemolytic activity below the detectable range. Regardless of the use of CVF, both groups I and II transplanted with unmodified grafts showed an immediate drop in leukocytes and platelet counts after transplantation. However, in group III, in recipients transplanted with DDAVP pre-treated swine lung, the platelet count did not decrease after transplantation (P = 0.0295). The decrease of plasma antithrombin and fibrinogen tended to be attenuated in group III. Light microscopic examination revealed extensive vascular thromboses in both capillary and larger vessels, as well as early pulmonary parenchymal damage in groups I and II, but were rarely observed in group III. Complement inhibition alone was not enough to alleviate intravascular thrombosis, the main pathology in pulmonary xenotransplantation. Pre-infusion of DDAVP to the donor animal was effective in preventing platelet sequestration and attenuated intravascular thrombosis. It is suggested that the strategies targeting vWF would be promising for successful pulmonary xenotransplantation.

Differential effects of estrogens and progestins on the anticoagulant tissue factor pathway inhibitor in the rat

Oral contraceptives (OC) and postmenopausal hormone therapy (HT) modulate plasma levels of proteins that regulate blood coagulation. It remains unclear whether the progestin component contributes to these changes. The present study was designed to determine whether progestins modulate two essential plasma anticoagulants, antithrombin (AT) and tissue factor pathway inhibitor (TFPI), in an animal model. Ovariectomized rats were treated orally with three progestins, norethindrone acetate (NETA), trimegestone (TMG), or drospirenone (DSP), either alone or combined with 17α-ethyinylestradiol (EE). Plasma AT levels were unchanged. However, TFPI activity was reduced by EE alone (10–100 μg/kg/day) in a dose-dependent manner; NETA (3 or 10 mg/kg/day) reduced TFPI by ∼40 or ∼80%, respectively, while TMG and DSP had no effect. NETA and EE effects were blocked by co-administration of ICI-182,780, an estrogen receptor antagonist, suggesting that both responses were likely estrogen receptor-mediated. Reduced TFPI after NETA or EE treatment was not accompanied by changes in TFPI mRNA levels in tissues that express TFPI, but there was a positive correlation between plasma TFPI and total cholesterol. Sex hormone effects on TFPI in this model and as reported in women may help to shift the coagulation balance to a more prothrombotic state. Progestins such as TMG and DSP that lack estrogenic activity could potentially have an improved clinical profile.

The inhibition of antithrombin by peptidylarginine deiminase 4 may contribute to pathogenesis of rheumatoid arthritis

The gene for peptidylarginine deiminase 4 (PADI4) has been found to be closely associated with rheumatoid arthritis (RA). Peptidylarginine deiminase (PADI) catalyses the post-translational modification of peptidylarginine to citrulline, a reaction known as citrullination. PADI extracted from rabbit muscle has been reported to citrullinate antithrombin, a principal plasma inhibitor of thrombin. Thrombin is known to induce angiogenesis, fibrin formation and inflammation, the primary events of the RA joint. Here, we investigate whether human PADI4 can inhibit antithrombin by catalysing antithrombin citrullination and how the enzyme is involved in RA pathogenesis. Antithrombin was incubated with recombinant PADI4 protein, and the inactivation of antithrombin was determined by reduction of its thrombin-inhibiting activity. Citrullination of antithrombin was detected by western blotting and enzyme-linked immunosorbent assay (ELISA). In addition, the citrullination level, activity and concentration of antithrombin in RA plasma were investigated by sandwich ELISA. Incubation of antithrombin with PADI4 resulted in loss of thrombin-inhibitory activity and in citrullination of antithrombin. RA plasma showed higher levels of citrullinated antithrombin than controls with non-arthritis disease and healthy individuals. The results indicate that PADI4 could inactivate antithrombin through citrullination. The abnormal expression or activation of PADI4 in RA synovium is suggested to be responsible for the high level of citrullinated antithrombin in RA plasma. Local inhibition of antithrombin activity in RA synovium might lead to the excessive angiogenesis, fibrin deposition and inflammation of the tissue.

Conformational Asn187Asp/Lys antithrombin variants and thrombosis

Antithrombin Rouen VI (N187D) is a rare conformational thermolabile variant. The unique symptomatic carrier reported in the literature developed 3 thrombotic events during pregnancy, in each case in a context of pyrexial infection. In fresh plasma, antithrombin activity and antigen level were normal but in vitro experiments demonstrated the presence of a thermolabile variant, suggesting that fever could be a trigger for thrombosis in N187D carriers. The RouenVI variant was further found in two asymptomatic brothers. In these subjects, it was associated with normal antigen level but reduced activity. In order to better delineate the functional and clinical consequences of the N187 variants, we have studied a series of seven subjects from two distinct families heterozygous for the Rouen VI mutation. Antithrombin levels were normal or borderline in these patients. Thermostability of plasma antithrombin was normal. We have also studied six subjects heterozygous for a new mutation, 6462C>G,which results in an asparagine to lysine substitution at residue 187. In these patients, the N187K mutation is associated with a clear type II deficiency and decreased thermostability of the plasma protein has been demonstrated. That the N187D mutation has milder consequences on plasma antithrombin activity than the N187K mutation is in agreement with structural predictions. About 50% of the N187 carriers studied have suffered venous thrombotic events, strongly suggesting that both mutations are risk factors for thrombosis, but none occurred during pyrexial infections.

Gerinnungsstörungen bei Sepsis

ZusammenfassungBei Patienten mit Sepsis findet sich in den meisten Fällen eine Gerinnungsaktivierung mit intravasaler Fibrinbildung. Ein vermindertes Hämostase-und Inhibitorpotenzial ist oft nachweisbar und kann zu Blutungen im Rahmen einer Verbrauchskoagulopathie führen. Ursache des Gerinnungsfaktoren-und Inhibitorenmangels ist nur zum Teil ein Vebrauch durch die Gerinnungsaktivierung. Relevant sind auch Plasmaverlust und hepatische Synthesestörung. Bestimmte Formen (z.B. Meningokokken-und Pneumokokkensepsis) können auch zu einer Purpura fulminans führen, die sich durch mikrovaskuläre Thrombosen mit Nekrosen und Blutungen sowie sehr niedrige Protein-C-Konzentrationen auszeichnet.Bei der Verbrauchskoagulopathie-induzierten Blutung zielt die Therapie primär auf eine Substitution von hämostatischen Komponenten. Bei Sepsis-induzierter Purpura fulminans ist das Ziel eine Unterbrechung der Gerinnungsaktivierung und Verbesserung der Fibrinolyse (z. B. durch Gabe von Protein C oder rekombinantem aktiviertem Protein C).Unfraktioniertes oder niedermolekulares Heparin wird zur Thromboembolieprophylaxe empfohlen, aber Ergebnisse klinischer Studien zum Einsatz von Heparin speziell bei Sepsis liegen nicht vor. Die Indikation für Antithrombinkonzentrate ist primär die Substitution bei nachgewiesenem Antithrombinmangel, beispielsweise im Rahmen der Therapie venöser Thromboembolien mit Heparin oder -derivaten oder bei extrakorporalen Zirkulationsverfahren unter Verwendung von Heparin. Zugelassene Indikation für rekombinantes aktiviertes Protein C (Drotrecogin alfa, aktiviert) ist die schwere Sepsis, unabhängig vom Vorhandensein einer Sepsis-induzierten Gerinnungsstörung oder Verbrauchskoagulopathie.

Coagulation status and complications of pregnancy

Introduction There is much interest in the relationship between coagulation status and complications of pregnancy. The thrombelastograph (TEG) has been proposed as a useful, inexpensive tool to screen for patients with hypercoagulable states. Materials and methods We investigated 588 unselected pregnant women at booking, obtaining blood samples for TEG and thrombophilia investigation. Pregnancy outcome data was recorded. Results We found significant correlations between TEG parameters and the Prothrombin time (PT) and Activated Partial Thromboplastin time (APTT) ( p<0.01) and with plasma Antithrombin level ( p<0.01). There was no correlation between TEG and other thrombophilic defects (protein C, protein S, Factor V Leiden mutation, Prothrombin G20210A mutation, MTHFR C677T mutation and Lupus Anticoagulant). There was a significant association of TEG parameters with mid-trimester loss (MTL) but not with other adverse pregnancy outcomes. Conclusions The correlation between TEG and PT, APTT and antithrombin level supports its value in providing a global measure of haemostasis. Coagulation status at booking is associated with increased risk of MTL but not with complications occurring later in pregnancy.

Effect of a Single Plasma Transfusion on Thromboembolism in 13 Dogs With Primary Immune-Mediated Hemolytic Anemia

Thirteen dogs with primary immune-mediated hemolytic anemia received fresh-frozen plasma within 12 hours of admission, in addition to unfractionated heparin and other therapies, such as prednisone, azathioprine, and packed red blood cell transfusion. Antithrombin activity was quantified prior to transfusion and at 30 minutes and 48 hours after transfusion. Plasma antithrombin activity did not change significantly after a single plasma transfusion. There were no deaths in the first 48 hours of treatment. Thromboembolism was identified at necropsy in six of 10 dogs that died within 12 months of admission. There was no significant difference in the incidence of thromboembolism between the current treatment group and a historical control group.