Monitoring treatments with unfractionated heparin: CTAD must be used instead of citrate as the anticoagulant solution when using partial-draw collection tubes. Results of a multicenter evaluation

BackgroundSampling small volumes of blood may be necessary, particularly in pediatric patients, or in case of difficult or recurrent venipunctures.MethodsRoutine hemostasis test results evaluated in partial- and full-draw evacuated polymer tubes obtained in 4 centers were compared.ResultsNo relevant discrepancy (Bland-Altman) was found between test results measured in partial- and full-draw tubes obtained from untreated patients and from patients on vitamin K-antagonist or low molecular weight heparin. In patients on unfractionated heparin (UFH), significantly lower anti-FXa activity [median = 0.29 IU/mL (range:0.04-1.15) vs. 0.39 (0.05-1.25), n = 89, p < 0.0001] and shorter aPTT were measured in partial-draw tubes. This discrepancy was likely to be related to the release of higher amounts of PF4 after increased platelet activation in partial-draw tubes. As CTAD is known to counteract platelet activation, we then collected blood into partial-draw CTAD tube and full-draw citrate tube. Both in patients on UFH and in untreated patients, no relevant difference could be demonstrated for all studied parameters (Bland-Altman), including aPTT and anti-FXa activity, even if analytical comparison showed significantly higher anti-FXa activity in partial-draw CTAD than in full-draw citrated tubes with a mean bias of 0.02 IU/mL, identical throughout the measuring range.ConclusionsThese results suggest that samples collected into partial-draw citrate tubes allow accurate routine coagulation testing in all patients but those requiring UFH assessment, in which their use led to a significant underestimation of anticoagulation. In such cases, partial-draw tubes containing CTAD could be validly used to monitor heparin therapy as well as to perform routine coagulation testing.     

A new plastic collection tube made of polyethylene terephtalate is suitable for monitoring traditional anticoagulant therapy (oral anticoagulant, unfractionated heparin, and low molecular weight heparin)

To improve the safety of blood collection, plastic tubes have been developed but various interactions with the coagulation system and/or antithrombotic drugs were reported with the first generation of such tubes. The aim of this multicentre study was to compare hemostasis test results measured in evacuated plastic tubes made of polyethylene terephtalate (VenoSafe, Terumo Europe) and in siliconized glass tubes containing the same citrate concentration (0.129 M). In addition, the impact of aging of the plastic tube was investigated by collecting blood samples in tubes at 8 months and at 1 month before expiry. Blood was drawn in 3 centres from untreated patients (n=269), patients on oral anticoagulant treatment (OAT, n=221), and patients treated with either unfractionated heparin (UFH, n=73) or a low molecular weight derivative (LMWH, n=48). Prothrombin time (PT) or INR, activated partial thromboplastin time (APTT) and anti-FXa activity were locally performed, when applicable. In untreated patients and in patients on OAT, PT and APTT values were found statistically shorter (p<0.05) when evaluated in plastic tubes than in glass tubes, except when PT was evaluated using a human thromboplastin. Surprisingly, significantly longer APTT and higher anti-FXa activities were obtained when blood from patients on UFH was drawn in plastic than in glass tubes. However, none of the differences had any clinical relevance (Bland-Altman analysis). In patients on anticoagulant treatment, there was no effect of aging of the plastic tubes. These results suggest that the plastic tube VenoSafe is suitable for coagulation testing both in untreated subjects and more interestingly in patients on traditional anticoagulant therapy during the whole shelf life indicated by the manufacturer.     

Evaluation and performance characteristics of the Q Hemostasis Analyzer, an automated coagulation analyzer

The Q Hemostasis Analyzer (Grifols, Barcelona, Spain) is a fully-automated random-access multiparameter analyzer, designed to perform coagulation, chromogenic and immunologic assays. It is equipped with a cap-piercing system. The instrument was evaluated in a hemostasis laboratory of a University Hospital with respect to its technical features in the determination of coagulation i.e. prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time, fibrinogen and single coagulation factors V (FV) and VIII (FVIII), chromogenic [antithrombin (AT) and protein C activity] and immunologic assays [von Willebrand factor antigen (vWF:Ag) concentration], using reagents from the analyzer manufacturer. Total precision (evaluated as the coefficient of variation) was below 6% for most parameters both in normal and in pathological ranges, except for FV, FVIII, AT and vWF:Ag both in the normal and pathological samples. No carryover was detected in alternating aPTT measurement in a pool of normal plasma samples and in the same pool spiked with unfractionated heparin (> 1.5 IU/mL). The effective throughput was 154 PT, 66 PT/aPTT, 42 PT/aPTT/fibrinogen, and 38 PT/aPTT/AT per hour, leading to 154 to 114 tests performed per hour, depending of the tested panel. Test results obtained on the Q Hemostasis Analyzer were well correlated with those obtained on the ACL TOP analyzer (Instrumentation Laboratory), with r between 0.862 and 0.989. In conclusion, routine coagulation testing can be performed on the Q Hemostasis Analyzer with satisfactory precision and the same apply to more specialized and specific tests.     

Heparin versus prostacyclin in continuous hemodiafiltration for acute renal failure: Effects on platelet function in the systemic circulation and across the filter

Continuous venovenous hemodiafiltration (CVVHDF) is the treatment of choice for critically-ill patients suffering from acute renal failure (ARF). One major problem of extracorporeal circuits is their thrombogenicity, which requires pharmacological blockade of primary (platelet-dependent) or secondary (plasmatic) haemostasis, increasing the patient's bleeding risk.Our study assessed platelet function during CVVHDF, comparing anticoagulant versus antiplatelet pharmacological strategies, commonly used to avoid circuit clotting. Twenty-three critically-ill patients with ARF, requiring CVVHDF were randomized to a prostacyclin analogue (PGI) or to unfractionated heparin (UFH). Ex vivo platelet function, assessed by optical aggregometry (OPA) induced by collagen or ADP, was studied in peripheral blood at baseline, 4 and 24 hrs after starting CVVHDF, and at 4 hrs within the circuit, before and after the filter (n = 9). Coagulation was also monitored.PGI significantly inhibited ADP-induced OPA of peripheral platelets: maximal aggregation (T_max) was reduced at 4 and 24 hrs by 20%, while collagen-induced T_max was significantly reduced at 4 hrs only. In the UFH group, collagen-induced OPA in peripheral platelets was significantly inhibited: slopes of OPA tracings were decreased by 25%, lag time was prolonged by 22%, T_max decreased by 10% already at 4 hrs. ADP-induced OPA showed a similar, but non-significant trend. UFH expectedly prolonged aPTT. In the UFH group, platelet responsiveness to collagen was significantly increased by 30% in post-filter versus pre-filter samples. This effect was blunted in the PGI group.UFH does not protect platelets from filter-induced activation and is associated with a reduced function of systemic platelets. Platelet-inhibiting agents might better prevent the activatory effect of the filter.     

Heparin-induced thrombocytopenia: a stoichiometry-based model to explain the differing immunogenicities of unfractionated heparin, low-molecular-weight heparin, and fondaparinux in different clinical settings

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4 (PF4)/heparin complexes. The frequency of HIT is highly variable in different clinical settings, and is more frequent with unfractionated heparin (UFH) than with low-molecular-weight heparin (LMWH), despite the in vitro observation that HIT antibodies activate platelets similarly well with LMWH as with UFH. An important difference between UFH, LMWH, and fondaparinux is their widely differing plasma concentrations. We aimed to provide a model that included anticoagulant concentrations and PF4 availability as risk factors influencing the anti-PF4/heparin immune response. MATERIALS AND METHODS: By photon correlation spectroscopy we determined the concentrations at which UFH, LMWH, and fondaparinux form complexes optimally with PF4. Plasma concentrations of UFH and LMWH were calculated based on ex vivo pharmacokinetic data, with information on fondaparinux and PF4 concentrations taken from the literature. RESULTS AND CONCLUSIONS: The main features of our model are: optimal complex formation occurs at prophylactic-dose UFH and high PF4 levels, whereas therapeutic-dose LMWH concentrations are too high for optimal complex formation; in contrast, concentrations of fondaparinux are usually below the optimal stoichiometric range. Thus, immunization should occur more often in situations with major rather than minor platelet activation, and--for a given degree of platelet activation (PF4 availability)--as: prophylactic-dose UFH>therapeutic-dose UFH>prophylactic-dose LMWH, fondaparinux>therapeutic-dose LMWH. Our model provides a framework for explaining empirical observations that LMWH induces less anti-PF4/heparin antibodies than does UFH, and that anti-PF4/heparin antibodies are more often found in patients undergoing major surgery than in medical patients.     

Certoparin versus UFH to prevent venous thromboembolic events in the very elderly patient: an analysis of the CERTIFY study

Introduction

There is an exponential rise of thromboembolic risk with age because of co-morbidities, immobility and pharmacotherapy. We aimed to investigate the benefits and risks of heparin prophylaxis in very elderly patients ≥ 80 years and the type of heparin used in a subgroup analysis of the CERTIFY trial.

Patients/methods

3,239 patients were randomized to 3,000 U aXa o.d. certoparin or 5,000 IU t.i.d. unfractionated heparin (UFH) for 8-20 days.

Results

Patients ≥ 80 years (n = 1,365) were more likely to be female, had a lower mean bodyweight, were more frequently using antiplatelets and had a GFR below 30 ml/min/1.73 m² more often than patients < 80 years (n = 1,875). The combined endpoint of proximal DVT, symptomatic non-fatal PE and VTE related death was experience by 5.26% of patients ≥ 80 years versus 3.51% in younger patients (OR 1.53; 95%CI 1.05-2.21; p = 0.03). There were no significant differences in both minor (OR 1.11; 95%CI 0.75-1.62) and major (OR 2.53; 95%CI 0.93-6.86) bleeding risks. Certoparin and UFH were equally effective in reducing thromboembolic risk in either age group. The risk of any (OR 0.45; 95%CI 0.26-0.79) and minor bleeding (OR 0.42; 95%CI 0.23-0.78) was reduced with certoparin in the very elderly only. There were more adverse events in elderly patients (OR 1.26; 95%CI 1.1-1.46), but rates were otherwise comparable.

Conclusions

The analysis confirmed the increased thromboembolic risk in very elderly patients, but demonstrated no increased bleeding risk. Certoparin and UFH were equally effective and safe with a reduced risk of minor bleeding complications with certoparin in the very elderly.     

Comparison of the anticoagulant response of a novel fluorogenic anti-FXa assay with two commercial anti-FXa chromogenic assays

Introduction

Fast and accurate monitoring is crucial in the successful regulation of coagulation therapy. For the treatment of venous thromboembolism, both unfractionated heparin (UFH) and low molecular weight heparins (LMWHs) are commonly administered. The chromogenic anti-factor Xa (FXa) assay is currently considered the ‘gold standard’ assay for monitoring LMWH. However different commercial chromogenic methods often differ when tested with the same samples. Fluorogenic anti-FXa assays have the potential to offer greater benefits over chromogenic assays in terms of greater specificity, sensitivity and they are not so influenced by sample opacity or turbidity.

Materials and methods

Commercial plasmas were spiked with pharmacologically relevant concentrations (0–1 U/ml) of UFH, enoxaparin, and tinzaparin. The fluorogenic assay was carried out using previously optimized concentrations of 12 nM FXa and 2.7 μM fluorogenic substrate, in addition to 6 μl of 100 mM CaCl₂ and 44 μl of plasma. The Biophen® and Coamatic chromogenic assays were carried out according to the manufacturer's instructions. Reaction rates and endpoint values were analyzed and statistical analysis by means of one-way analysis of variance (ANOVA) was performed.

Results

The fluorogenic anti-FXa assay was found to have the broadest therapeutic range of 0–1 U/ml with CVs of < 5% for UFH and tinzaparin and CVs < 9% for enoxaparin. Despite their limited measuring range, good assay reproducibility was observed with both chromogenic kits.

Conclusions

This study indicated that the fluorogenic assay is the most sensitive assay with the broadest dynamic range for monitoring LMWH therapy when compared with standard chromogenic assays.     

Dextran sulfate included in factor Xa assay reagent overestimates heparin activity in patients after heparin reversal by protamine

A lack of correlation between activated partial thromboplastin time (aPTT), thrombin time (TT) and anti-factor Xa (AXa) activity was observed in patients after cardiac surgery with cardiopulmonary bypass (CBP). Indeed, AXa activity measured by the chromogenic assay, Coamatic Heparin, was higher than expected with regard to results obtained in coagulation assays. To account for this discrepancy, another AXa chromogenic assay was tested. First, AXa activity was measured with two chromogenic assays (Coamatic Heparin and Rotachrom Heparin) in plasma samples of 25 patients undergoing cardiac surgery at two time points after heparin reversal by protamine. AXa activity was significantly higher when measured with Coamatic Heparin than with Rotachrom Heparin in samples collected just after protamine infusion (p<0.01). Next, since Coamatic( Heparin contains dextran sulfate (DXS) to reduce the influence of heparin antagonists such as platelet factor 4 (PF4), whereas Rotachrom Heparin does not, we hypothesized that the dextran sulfate contained in the reagent might explain this discrepancy. We therefore performed in vitro studies consisting in neutralizing unfractionated heparin (UFH) with protamine and measuring AXa activity with the two chromogenic assays. An AXa activity was still measurable with Coamatic Heparin after neutralization, thus strongly suggesting that dextran sulfate dissociates protamine/heparin complexes. We conclude that Coamatic Heparin assays should be avoided when measuring AXa activity in plasma samples immediately after protamine infusion, as inaccurate results may lead to inadequate management of heparin reversal.     

Ex vivo reversal of the anticoagulant effects of edoxaban

Introduction

Edoxaban is an oral, once-daily direct factor Xa (FXa) inhibitor. Although rapidly cleared, strategies to reverse edoxaban-mediated effects on anticoagulation are needed in cases of excessive bleeding or emergency. This study evaluated the effect of two prohemostatic agents, recombinant factor VIIa (rFVIIa) and factor VIII inhibitor bypass activity (FEIBA), on the anticoagulatory effects of supratherapeutic concentrations of edoxaban in human whole blood ex vivo.

Materials and Methods

Blood samples were collected from six healthy volunteers. Edoxaban (500 or 1000 ng/mL), alone or followed by rFVIIa (0.8 or 1.8 μg/mL) or FEIBA (0.75 or 1.5 U/mL), was added to an aliquot of each sample. Biomarkers, including prothrombin time (PT), activated partial thromboplastin time (aPTT), extrinsic FXa activity (anti-FXa), intrinsic factor X activity, and D-dimer, were assessed at 0.25, 0.5, 1, 2, and 4 hours after adding rFVIIa or FEIBA.

Results

Decreases in measures of PT (p < 0.0001), aPTT (p < 0.0001), and anti-FXa (p < 0.0001) were observed when rFVIIa or FEIBA was added to edoxaban-containing blood samples. Intrinsic FX activity was increased up to 20% and 31% of normal in the presence of edoxaban by rFVIIa and FEIBA, respectively. The impact of these agents on the anticoagulant effects of edoxaban were observed within 15 minutes and remained relatively unchanged at each timepoint thereafter.

Conclusions

The findings of this ex vivo study suggest that rFVIIa and FEIBA rapidly reversed edoxaban-mediated anticoagulation effects based on PT and aPTT, but had minimal effect based on intrinsic FX activity. No dose response was observed for rFVIIa or FEIBA.     

Effects of argatroban and heparin on thrombus formation and tissue plasminogen activator-induced thrombolysis in a microvascular thrombosis model

Effects of (2R,4R)-4-methyl-1-[N(2)-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl]-2-piperidine-carboxylic acid monohydrate (argatroban) and unfractionated heparin (UFH) were compared with respect to thrombus formation and tissue-type plasminogen activator (t-PA)-induced thrombolysis in a microvasculature thrombosis model. The antithrombotic activities of anticoagulants were evaluated with respect to the time required for the initiation of thrombus formation (T(i)) and the time required for the thrombus to stop blood flow (T(s)). The effects of anticoagulants administered with t-PA were evaluated by percent stenosis of the vessel and percent area of the thrombus. Argatroban (1-3 mg/kg/bolus) significantly prolonged T(i) and T(s) in a dose-dependent fashion compared to control. Argatroban (3 mg/kg/bolus) significantly prolonged both the T(i) and T(s) more effectively than UFH (100 anti-XaU (a-XaU)/kg/bolus), despite equivalent prolongation of the activated partial thromboplastin time (aPTT). Higher doses of UFH (300-500 a-XaU/kg) were required to significantly prolong T(i) and T(s), but at these doses, UFH caused over-prolongation of aPTT (>180 s), which might consequently cause bleeding complications. Argatroban (0.1-0.3 mg/kg/h) significantly accelerated thrombolysis by t-PA in both a dose- and time-dependent fashion. Although argatroban (0.1-0.2 mg/kg/h) did not significantly prolong the aPTT and bleeding time (BT) as compared with control, it significantly accelerated thrombolysis by t-PA at these doses of lower bleeding risk. Argatroban (0.3 mg/kg/h) significantly enhanced thrombolysis by t-PA, while UFH (12.5 anti-XaU/kg/h) attenuated it again, despite equivalent prolongation of the aPTT and BT. We conclude that argatroban seems to be a more efficient and safer anticoagulant than UFH for the prevention of thrombus formation and acceleration of t-PA-induced thrombolysis.     

Heparin affinity of factor VIIa: implications on the physiological inhibition by antithrombin and clearance of recombinant factor VIIa

Factor VIIa (FVIIa), a trypsin-like serine protease, plays an essential role in haemostasis by initiating the coagulation in complex with its cofactor, tissue factor (TF). The TF pathway inhibitor is the main physiological inhibitor of FVIIa-TF complex, but FVIIa can also be inhibited by antithrombin, although little is known about this process.Functional analyses by second order kinetic determination and identification of FVIIa-antithrombin complex by electrophoresis, evaluating the effect of different cofactors: pentasaccharide, low molecular weight heparin (LMWH) and unfractionated heparin (UFH), confirmed that any activation of antithrombin significantly enhanced the inhibition of FVIIa. The analysis of the binding of FVIIa to heparin by surface plasmon resonance identified a high affinity interaction under physiologic conditions (K_D = 3.38 μM, with 0.15 M of ionic strength) strongly dependent on Ca²⁺ and ionic strength. This interaction was verified in cell models, indicating that FVIIa also binds to the surface of endothelial cells with similar requirements. Structural modeling suggests the presence of a potential exosite II in FVIIa. However, the binding of heparin did not display significant changes on both the intrinsic fluorescence and the associated functional consequences of FVIIa.These results indicate that FVIIa binds to exposed glycosaminglycans of the endothelium through an exosite II, structurally similar to that reported for thrombin and suggested for FIXa. This binding may favor its inhibition by antithrombin in the absence of TF, contributing to the physiological control of this protease. This process may also play an important role in the clearance of recombinant FVIIa administered to patients.     

A shortened activated partial thromboplastin time predicts the risk of catheter-associated venous thrombosis in cancer patients

Introduction

Hypercoagulability due to high coagulation factor levels resulting from host inflammatory response to cancer contributes to an increased risk of venous thromboembolism (VTE) in cancer patients. Central venous catheters (CVCs) further heighten this risk. Activated partial thromboplastin time (aPTT) can be used to broadly screen for elevated levels of relevant coagulation factors. Our objective was to determine if a shortened aPTT ratio (coagulation time of test- to- reference plasma) was a predictor of CVC-associated VTE in cancer patients.

Materials and Methods

We performed a retrospective case–control study on cancer patients undergoing tunneled CVC insertion at our center from 1999 to 2006 and identified 40 patients who had CVC-associated VTE. VTE was confirmed with color duplex ultrasonography or computed tomography scan. For each case, we obtained 5 controls that had the same cancer diagnosis and were matched on the following factors: age, chemotherapy, hormone therapy (if applicable), tobacco use, TNM staging and year of diagnosis. All patients had aPTT testing within 30 days prior to surgery. We compared aPTT and aPTT ratio between cases and controls using Wilcoxon two sample test.

Results

aPTT ratio was significantly shorter in patients with CVC-related VTE as compared to controls [0.86 (95% confidence interval (CI) 0.78, 0.94) vs. 0.98 (0.94, 1.01), p = 0.0003]. Mean aPTT was also significantly shorter. [25.6 seconds (95% CI 23.2, 27.9) vs. 28.1 (26.9, 29.3), p = 0.001] aPTT ratios of the controls tended to spread across larger aPTT ratio values whereas those of cases tended to clustered around the mean.

Conclusions

Cancer patients undergoing catheter placement who develop CVC-associated VTE have a shorter aPTT and aPTT ratio than those who do not develop VTE. aPTT, a simple and inexpensive test might be useful as a predictor of CVC-associated VTE risk in cancer patients.     

Effects of heparin and a semi-synthetic heparin analogue on platelet aggregation, lipoprotein lipase and other laboratory tests in surgical patients

Platelet aggregation, lipoprotein lipase activity, coagulation parameters and routine blood chemistry were measured in a randomised study of 21 surgical patients before, immediately after and 3 months after operation. Sodium heparin 5000 IU was given subcutaneously to 11 patients every 12 hours for 7 days, the first injection 2 hours preoperatively; 10 patients received a semi-synthetic heparin analogue (SSHA 75 mg) in the same manner. The groups were sex and age matched. No conclusive changes were found in platelet aggregation. The increase in lipoprotein lipase activity in SSHA patients 2 hours after injection was significantly greater than in heparin patients. Neither of the two drugs induced significant changes in coagulation parameters or routine blood chemistry. The results indicate a difference in the effect on lipoprotein lipase release between heparin and SSHA at the used dosage schedules.     

Potentiation of platelet aggregation by heparin in human whole blood is attenuated by P2Y12 and P2Y1 antagonists but not aspirin

INTRODUCTION: Unfractionated heparin (UFH) potentiates platelet aggregation induced by some agonists. P2Y12 and P2Y1 receptors play a major role in amplifying platelet aggregation. We assessed the ability of cangrelor, a selective P2Y12 antagonist, A2P5P, a selective P2Y1 antagonist, and aspirin to block the potentiating effects of heparin. MATERIALS AND METHODS: Whole blood from healthy human volunteers was anticoagulated with either hirudin or UFH 10 IU/ml. Some tubes anticoagulated with hirudin also contained UFH 1 or 10 IU/ml. The low-molecular-weight heparin dalteparin was also assessed. Platelet aggregation was performed using whole blood single-platelet counting. Dense granule release was assessed using 14C-5HT-labelled platelets. RESULTS: UFH and, to a lesser extent, dalteparin potentiated platelet aggregation induced by ADP, PAF, 5HT, U46619, epinephrine and TRAP in a concentration-dependent manner but inhibited aggregation induced by collagen. Cangrelor effectively opposed the potentiating effects of heparins on sustained aggregation induced by ADP, PAF, 5HT, U46619 and TRAP but had less effect on epinephrine-induced aggregation, whereas A2P5P was more effective at blocking both the initial phase of ADP-induced aggregation and the aggregation response to epinephrine, reflecting the differences in G protein coupling between the agonist receptors. Aspirin had no effect on potentiation by heparin. Heparins did not increase ADP- or TRAP-induced 14C-5HT release. CONCLUSIONS: Heparins potentiate platelet responses to ADP and numerous other agonists. This potentiation is attenuated by cangrelor and A2P5P, and is not mediated by increased dense granule release. ADP receptor antagonists but not aspirin may have potential therapeutic benefits in counteracting the pro-thrombotic effects of heparins.     

Analysis of unfractionated heparin dose requirements to target therapeutic anti-Xa intensity during pregnancy

Introduction

Unfractionated heparin (UFH) does not cross the placenta and has demonstrated utility in the prevention and treatment of thrombosis during pregnancy. Limited information is available to guide initiation and monitoring of therapeutic UFH targeting an anti-Xa concentration of 0.3-0.7 u/ml during pregnancy. The objective of this study was to describe UFH doses and monitoring strategies required to achieve and maintain therapeutic anti-Xa intensity in a cohort of women treated with UFH during pregnancy.

Materials/Methods

Patients prescribed anti-Xa adjusted UFH during pregnancies occurring between January 1998 and March 2005 were included.

Results

A total of 39 pregnancies for 37 women were identified. Unfractionated heparin doses were titrated to achieve a mid-interval anti-Xa level of 0.3-0.7 u/ml. Patients required a median 6.5 days and a mean UFH dose of 403.5 u/kg/day to achieve therapeutic anti-Xa levels. Most anti-Xa levels were within the target range (59%). The final UFH dose/kg required at the end of pregnancy was similar to that at the first therapeutic level (P > 0.05); however some patients did require dose modification. Patients required a mean 14.1 anti-Xa determinations and 4.6 dose modifications during a mean 23.9 weeks of antenatal UFH therapy. Patient weight and UFH dose at the first therapeutic anti-Xa level were correlated (r = 0.383, P = 0.018).

Conclusions

Pregnant women required a mean UFH dose of 403.5 u/kg/day to achieve midinterval anti-Xa levels of 0.3-0.7 u/ml. The required dose was correlated with patient weight and most anti-Xa measurements were within the target range.     

Effect of Pre-Analytical Conditions on the Thrombodynamics Assay

Introduction

Standardization of pre-analytical conditions is the obligatory step for all potential diagnostic tests. Spatial clot growth (Thrombodynamics) is a new global hemostasis assay that considers spatial organization of coagulation. The principal parameter is rate of fibrin clot growth from the tissue-factor coated surface. In this work we studied the pre-analytical variables of Thrombodynamics assay that include conditions of blood collection, sample preparation and storage.

Materials and Methods

Blood of apparently healthy volunteers was used. Eight types of citrate blood collection tubes were tested, centrifugation conditions for plasma preparation were evaluated and impact of plasma freezing/thawing was tested.

Results

Among the blood collection tubes tested, BD Vacutainer glass tubes showed a significantly higher clot growth rate compared to plastic tubes. There was no difference between 3.2% and 3.8% of sodium citrate. For plasma preparation, a single 15 min centrifugation at 1 600 g shows significantly increased clot growth rate compared to plasma obtained by two sequential centrifugations (15 min 1 600 g, 5 min 10 000 g). There was no significant difference between 1 600 g and 2 100 g if the second centrifugation was performed. For the second centrifugation there was no difference between 20 min at 1 600 g and 5 min at 10 000 g. Frozen-thawed plasma showed increased clot growth rate compared to fresh plasma.

Conclusion

The data represent the necessary steps for the standardization of Thrombodynamics assay and for the formulation of the operating guide.     

Unfractionated heparin attenuates lung vascular leak in a mouse model of sepsis:Role of RhoA/Rho kinase pathway

Introduction

Excessive vascular permeability is a characteristic feature of ALI. We have previously demonstrated that UFH prevents LPS-induced disruption of endothelial barrier function in vitro. It was the objective of this study to determine whether UFH may attenuate endotoxin-induced lung vascular leak in mice and to further explore the possible underlying mechanisms.

Methods

C57BL/6J mice were randomly divided into the control, LPS and LPS plus UFH groups. Sepsis was induced by intraperitoneal injection of LPS at a dose of 30 mg/kg. Mice in the LPS plus UFH group were intravenously received 8 units UFH (heparin sodium) diluted in 20 μl sterile saline at 0.5 h before the injection of LPS.

Results

1) UFH pretreatment attenuated LPS-induced histopathological changes in Lung at 6 h; 2) Pretreatment of mice with UFH ameliorated LPS-induced lung edema and lung vascular leak at 6 h; 3) UFH pretreatment dramatically inhibited RhoA and ROCK activation in the lung tissues of LPS-treated mice (3 and 6 h). 4) UFH pretreatment significantly down-regulated ROCK1 gene expression, but did not affect the increased expression of ROCK2 mRNA in the lung tissues of LPS-treated mice at 3 or 6 h.

Conclusion

These data suggest that UFH may attenuate endotoxin-induced lung vascular leak by regulating RhoA/Rho kinase pathway.     

Physiological changes in membrane-expressed platelet factor 4: Implications in heparin-induced thrombocytopenia

Background

Many heparin-induced thrombocytopenia (HIT) antibodies cause platelet activation in the serotonin release assay (SRA) in the absence of heparin. This in vitro observation may help unravel the mechanism of delayed-onset HIT, where seropositive patients develop thrombocytopenia and associated thrombosis after cessation of heparin.

Objective

Studies were conducted to examine the relationship between platelet environment, surface PF4 expression, and the extent of heparin-independent platelet activation in the SRA.

Methods

Ex vivo platelets were washed and labeled for SRA, then used either before or after 45 minutes of recovery at 37 °C. HIT antibody-mediated serotonin release in the absence of heparin was compared to the extent of surface staining of the platelets with fluorescent anti-human PF4 antibodies.

Results

Handling of platelets for in vitro studies resulted in transient expression of surface PF4, and it was during this interval that platelets were most sensitive to activation by HIT antibodies in the absence of heparin. Heparin-independent platelet activation was attenuated when SRA-positive specimens were retested after platelets were incubated 45 minutes at 37 °C. Surface PF4 expression was diminished on the rested platelets, compared to the same platelets labeled immediately after handling. Thus compared to rested platelets, mildly activated platelets had elevated surface PF4 expression and a higher level of HIT antibody-mediated, heparin-independent platelet activation.

Conclusion

Surface expression of PF4 reflects HIT antigen presentation, and varies with the physiological state of platelets. Thus there can be differences in HIT antibody target availability among patients which may explain the variability in consequences of HIT antibody seropositivity.     

An open-label, comparative study of the efficacy and safety of once-daily dose of enoxaparin versus unfractionated heparin in the treatment of proximal lower limb deep-vein thrombosis

BACKGROUND: Treatment of deep-vein thrombosis (DVT) with a once-daily regimen of enoxaparin, rather than a continuous infusion of unfractionated heparin (UFH) is more convenient and allows for home care in some patients. This study was designed to compare the efficacy and safety of these two regimens for the treatment of patients with proximal lower limb DVT. METHODS: 201 patients with proximal lower limb DVT from 13 centers in Brazil were randomized in an open manner to receive either enoxaparin [1.5 mg/kg subcutaneous (s.c.) OD] or intravenous (i.v.) UFH (adjusted to aPTT 1.5-2.5 times control) for 5-10 days. All patients also received warfarin (INR 2-3) for at least 3 months. The primary efficacy endpoint was recurrent DVT (confirmed by venography or ultrasonography), and safety endpoints included bleeding and serious adverse events. The rate of pulmonary embolism (PE) was also collected. Hospitalization was at the physician's discretion. RESULTS: Baseline patient characteristics were comparable between groups. The duration of hospital stay was significantly shorter with enoxaparin than with UFH (3 versus 7 days). In addition, 36% of patients receiving enoxaparin did not need to be hospitalized, whereas all of the patients receiving UFH were hospitalized. The treatment duration was slightly longer with enoxaparin (8 versus 7 days). There was a nonsignificant trend toward a reduction in the rate of recurrent DVT with enoxaparin versus UFH, and similar safety. CONCLUSIONS: A once-daily regimen of enoxaparin 1.5 mg/kg subcutaneous is at least as effective and safe as conventional treatment with a continuous intravenous infusion of UFH. However, the once daily enoxaparin regimen is easier to administer (subcutaneous versus intravenous), does not require aPTT monitoring, and leads to both a reduced number of hospital admissions and an average 4-day-shorter hospital stay.     

A whole blood flow cytometric determination of platelet activation by unfractionated and low molecular weight heparin in vitro

The influence of unfractionated (Heparin–Natrium) and low-molecular heparin (Fragmin®) on platelet activation in whole blood was investigated by FACS analysis in vitro using antibodies against glycoprotein (gp) IIb/IIIa (CD 41), GMP 140 (CD 62P), gp 53 (CD 63) and fibrinogen. Samples were also labeled with anti-gp Ib (CD 42b). Neither unfractionated heparin (UFH) nor low molecular weight heparin (LMWH) led to significant (i.e., p<0.05) changes in fluorescence intensities of platelets labeled with anti-gp IIb/IIIa or anti-gp 53. Significant platelet activation due to unfractionated heparin could be observed by labeling with anti-GMP 140 (UFH: p=0.009; LMWH: p=0.16). The proportion of platelets with surface-bound fibrinogen was significantly increased (UFH: p=0.00006; LMWH: p=0.008). After incubation with heparins, activation ability of platelets by adenosine diphosphate (ADP) was significantly increased. The potentiating action of unfractionated heparin was larger. Therefore, flow cytometric results of platelet activation in patients receiving heparin should be interpreted carefully.