aPTT Reagents Research Articles

Effect of Telavancin (Vibativ) on Routine Coagulation Test Results

Telavancin (Vibativ, Astellas Pharma US, Deerfield, IL) is a lipoglycopeptide antibiotic that has activity against gram-positive microorganisms, but also has the ability to bind to artificial phospholipids found in coagulation reagents. Normal pooled plasma was spiked with telavancin to obtain concentrations of 0, 12.5, 25, 50, 75, 100, 125, and 150 μg/mL of drug. Samples were tested using 3 different prothrombin time/international normalized ratio (INR) and activated partial thromboplastin time (aPTT) reagent systems, as well as for fibrinogen level, thrombin time, D-dimer level, dilute Russell viper venom time (DRVVT), protein C activity, and protein S activity. There was no effect of telavancin seen with non-phospholipid-dependent assays: fibrinogen level, thrombin time, and D-dimer testing. All INR and aPTT systems demonstrated concentration-dependent increases in clotting times, with Innovin (Siemens Healthcare Diagnostics, Deerfield, IL) INRs the most dramatic. False-positive DRVVT ratios started at 12.5 μg/mL of telavancin, with no effect on protein C or protein S levels until the telavancin level reached more than 100 μg/mL.

Strategies for Managing Heparin Therapy in Patients with Antiphospholipid Antibody Syndrome

Antiphospholipid antibody syndrome (APS) is a common acquired thrombophilia. The diagnosis of APS is based on both clinical and laboratory criteria. The clinical criteria include vascular thrombosis or pregnancy morbidity. The laboratory criteria include a positive test for lupus anticoagulant, anticardiolipin antibodies, or anti-β(2)-glycoprotein I (anti-β(2)GPI) antibodies on two or more occasions at least 12 weeks apart. Antiphospholipid antibodies with lupus anticoagulant activity may prolong phospholipid-dependent coagulation tests such as the activated partial thromboplastin time (aPTT) and the activated clotting time (ACT). This prolongation adds a level of complexity to monitoring heparin therapy in patients with APS who have thrombosis. A literature search of the PubMed database was conducted for relevant articles published from 1995-April 2011. The usual management approach in nonsurgical patients with APS is to switch to low-molecular-weight heparin. In patients in whom heparin remains the agent of choice, management options include monitoring heparin antifactor Xa levels, determining an individualized therapeutic aPTT range, targeting an aPTT goal of 2 times the baseline aPTT, or using an aPTT reagent insensitive to lupus anticoagulant. An algorithm for anticoagulation management in nonsurgical patients with APS who require heparin is provided. The strategies to monitor intraoperative heparin in patients undergoing cardiac surgery include measuring heparin concentrations by an automated protamine titration device, targeting twice the baseline ACT, using preoperative in vitro heparin-ACT titration curves, and measuring heparin antifactor Xa levels. The available published case reports on the use of these strategies are reviewed. Each institution should determine an approach to managing heparin in patients with APS that best meets its needs and resources.

Selection of a Suitable Rapid Turn Around and Sensitive Test for the Laboratory Monitoring of Newer Oral Anticoagulants, Rivaroxaban, Apixaban and Dabigatran,

Abstract 3363Recently, several of the newer oral anticoagulants such as Rivaroxaban (Bayer Healthcare) (R) Apixaban (BMS/Pfizer) (A) and Dabigatran (Boehringer Ingleheim) (D) have been approved for different indications in the US and European countries. Although the monitoring is not required or promoted to manage the approved dosages of these drugs in different indications, population based bleeding complications with some of these agents have been reported warranting the monitoring of these drugs to optimize therapy. Conventionally the Xa based drugs such as Rivaroxaban and Apixaban are monitorable by following the anti-Xa activity whereas the dabigatran can be followed by anti-IIa activity. The pharmacokinetics of these drugs is reportedly monitored by such sophisticated methods as the liquid chromatographic – mass spectrometric methods. The conventional clotting methods such the PT/INR and APTT assays provide widely variable results which are dependent on the type of reagents used. Heptest exhibits a higher sensitivity to the anti-IIa agents in comparison to the anti-Xa agents. Prothrombinase activated clotting time (PiCT) is a Xa/IIa based clotting assay which can be performed in using a 1 stage and 2 stage method. The 1 stage method is highly sensitive to newer anticoagulants in contrast to the 2 stage PiCT and other tests. For the routine monitoring of these newer oral anticoagulant drugs a simple and rapid assay with the linear sensitivity range of 0–500 ng/ml is optimal for various patient populations. The current study is designed to determine the relative sensitivity of the one stage PiCT test in comparison to 4 PT, 4 APTT, Heptest and thrombin generation assays. Normal human pooled plasma was supplemented with R, A and D at a concentration range of 0–10 μg/ml. PT/INR analysis was carried using Thromboplasin C+, Innovin, Neoplastin and Simplastin reagents. The APTT assays were carried out using Actin FS, Actin FSL, Stago APTT and Platelin reagents. Heptest and one stage and two stage PiCT assays were also performed. All of the clot-based assays were carried out on the ACL analyzer with the exception of Heptest and PiCT tests. The thrombin generation assay was carried out using a flourogenic substrate method (Technoclone). In the PT assay, all reagents showed assay based variabilities in the clotting times and were relatively insensitive to A, however R and D produced a concentration dependent anticoagulant effect. Which were relatively insensitive at concentrations below 1 μg/ml. Similarly in the APTT assays, while R and D produced a concentration dependent anticoagulant effect all 4 reagents were relatively insensitive to A. The Actin FSL reagent was found to be sensitive to both the R and D and concentrations of less than 100 ng/ml were measurable. D produced the strongest anticoagulant effects in both the PT and APTT assays. Heptest was relatively insensitive to R and A, however at concentrations of greater than 1 μg/ml it produced concentration dependent anticoagulant effects. In the one stage PiCT assay, all agents including A prolonged the anticoagulant effect which produced a linear response at submicrogram levels and was found to be highly sensitive to D. The two stage PiCT also exhibited a linear sensitivity in the 0–100 ng/ml range. In the thrombin generation generation assay, R exhibited the strongest inhibitory effect with an IC50 of 160 ng/ml, D at 310 ng/ml and A produced a relatively weaker inhibitory effects with an IC50 >1000 ng/ml. These studies clearly demonstrate the limitations of various laboratory assays in the monitoring of newer oral anticoagulant agents with the exception of 1 stage PiCT test which can be universally used in the monitoring of these agents in the projected clinical ranges. Furthermore the one stage PiCT test can be performed in such matrices as whole blood, PRP and PPP. Moreover this test can also be carried out in point of care settings. Additional results on the assay performance and the clinical utility of some of these tests will be presented with a recommendation for the monitoring of newer oral anticoagulant drugs in such clinical settings as the extended prophylaxis of DVT and atrial fibrillation. Disclosures:No relevant conflicts of interest to declare.

Laboratory Assays for Monitoring Rivaroxaban: In House in-Vitro Analysis and Review of Literature

Abstract 4320 Introduction:Rivaroxaban an oral direct Xa inhibitor has recently been approved for VTE prophylaxis. It doesn't require monitoring but in special circumstances like bleeds a monitoring tool will be useful. Also, it is prudent to know its effect on various routine coagulation tests. Currently, there is no commercially available method for monitoring of Rivaroxaban. Aim: We conducted an In house- in-vitro analysis to (a)determine the effects of rivaroxaban locally on a range of commercially available coagulation assays, lupus anticoaugulant, factor VIII level, thrombin generation time and (b) measure it by two anti-Xa assays. Materials: Rivaroxaban (Xeralto) tablet, strength-10 mg. Plasma samples: Citrated normal human platelet poor plasma procured and freezed at −70 degree centigrade, Baxter sterile water. Method:The tablet was dissolved over 72 hours in a litre of sterile water to make 10 ug/ml of solution. Despite stirring a fine particulate compound remained which settled on standing. Samples were vortexed prior to each assay. This stock solution was used to spike citrated normal human platelet-poor plasma (PPP) at increasing concentrations of rivaroxaban from 0.1,0.2, 0.3, 0.4, upto 0.5 ug/ml to see the effect on the following tests and to determine rivaroxaban concentration by two 1.anti-Xa methods. All method were run using standard protocols taken from manufacturers' instructions.Anti-Xa assay:a) BIOPHEN DiXaI kit for 2-stage chromogenic assay of Direct Xa inhibitors; Ref 221030 b) Rotachrome using 1:2 dilution and further using 1:10 and 1:20 dilutions; catalogue Number-00612.2.PT & INR (STAGO neoplastin CI Plus MNPT-13.2 s, ISI-1.30 s).3.aPTT (Triniclot aPTT reagent)4.LAC-dRVVT screening and confirm (STAGO).5.Factor VIII assay (Siemens- Factor VIII deficient plasma)6. TT (STAGO-Thrombin)7. Factor VIII assay- Helena SARP.8.TGT: Thrombinoscope 5 picomolar TF and standard CAT method.Values described for Xarelto/ Rivaroxaban on patients with thromboprophylaxis in orthopedic surgery are usually of about 0.1–0.2 ug /ml in plasma.Result & Discussion: Concentration dependent prolongation of PT was observed. We used a single reagent (STAGO neoplastin CI Plus) which was sensitive at 0.1ug/ml concentration. Samama et al found that the results of PT were variable with reagent and not corrected by conversion to INR.We found that aPTT at lower concentrations of Rivaroxaban was not significantly different. Hillarp et al used five different reagents and found a reagent dependent dose response of APTT to rivaroxaban.The effect on Thrombin Time was non concentration dependent with no prolongation of TT at lower levels.There was fall in FVIII level in concentration dependent manner with 35% fall at 0.5ug/ml. We did not come across any literature till date about FVIII levels in Rivaroxaban therapy and our conclusion emphasises the importance of drug history.The LAC by DRVVT test showed positive result at the lowest concentration with an ILR of 1.23 and a dose dependent prolongation of clotting time. Thus making thrombophilia screening difficult.Thrombin generation time showed dose dependent effect on all parameters with almost complete suppression of thrombin generation. The initiation and propagation phase of TGT was affected at the lowest concentration (Green et al).The two anti-Xa tests used for measuring the concentration of Rivaroxaban in spiked plasma showed that Biophen DiXAI method has a broad range of sensitivity (0.1–0.5ug/ml) with calibration curve almost identical to that of manufacturer's with an excellent R value through out.The Rotachrome method at the standard 1:2 dilution was unable to measure concentration beyond 0.2ug/ml and at 1:20 dilution could only measure upto 0.4ug/ml. Thus, this method needs modification to determine a broad range of concentration.There was a variable result with 0.5ug/ml concentration of Rivaroxaban despite repeated vortex with unclear cause. This could be due to instability or settling of non-dissolved particles of the sample.The calibration curves were linear throughout with an acceptable R-value but there was a progressive drop in assay over a period of time. Conclusion:Anti-Xa assay with Biophen DiXAI method is a promising candidate for measurement of concentration of Rivaroxaban in special circumstances. Our findings show a trend towards prolongation of global clotting assays. The tablet is not stable as a calibrator. Disclosures:Pathak: BIOPHEN-DIXAI: Research Funding. McRae:BIOPHEN-DIXAI: Research Funding.

The factor VIII inhibitor assays can be standardized: results of a workshop.

The Bethesda and the Nijmegen assays are commonly used for the measurement of inhibitor levels in hemophilia A patients. Despite test innovations, the between-laboratory coefficient of variation (CVb) of factor VIII inhibitor test data in external quality surveys remains very high (40-60%) with a high degree of false-negative and false-positive results resulting in undesired effects on treatment. Organization of a workshop in order to address the causes of this phenomenon and to suggest ways to improve the assays. Fifteen laboratories showing a high CVb in regular surveys and using a variety of methods participated in the wet workshop, which included four different sessions where variables probably contributing to the high CVb (e.g. use of [non-]buffered plasma, FVIII-deficient plasma, sample dilution and APTT reagents) were investigated. The CVb varied from 30% to 70% in the first session of the workshop when the participants used their own test settings and reagents. The use of buffered normal pooled plasma and FVIII-deficient plasma as a reference sample by all participants did not significantly alter the CVb (35-50%) but decreased the number of false positives. However, the use of buffered pooled plasma in combination with standardized sample dilution procedures by all participants showed a significant improvement (CVb, 10-20%). These results may contribute to improvement of FVIII inhibitor testing. However, improved inter-laboratory comparison of factor VIII inhibitor assay results can only be reached when further local standardization is implemented.

Lupus anticoagulant: performance of the tests as recommended by the latest ISTH guidelines

Lupus anticoagulant (LA) is clinically the most relevant among all antiphospholipid antibody tests. Recently, new guidelines for LA detection were published. The objective of this retrospective cohort study was to compare tests recommended under these guidelines with other methods used for LA detection. The study group consisted of 336 subjects suffering from various autoimmune diseases. We used activated partial thromboplastin time (aPTT), diluted Russell viper venom time (dRVVT) and diluted prothrombin time (dPT) tests for LA detection together with a ratio between sensitive and insensitive aPTT reagent. We also tested if LA was dependent on β(2) glycoprotein I (β(2) GPI) using one of the recently described methods. All LA tests performed were associated with a history of thrombosis. The highest odds ratio (OR) for thrombosis was found for β(2) GPI-dependent LA but sensitivity was low (OR = 8.4; specificity/sensitivity = 98%/15%). All LA tests showed a much stronger association with thrombosis than with pregnancy failure. LA tested by aPTT and/or dRVVT (at least one out of two tests positive), as recommended by the guidelines, was associated less strongly with a history of thrombosis (OR = 4.1) than either of these tests separately (OR = 5.0 and 4.3, respectively). With both tests positive ('double LA positivity') the association with thrombosis was stronger (OR = 6.5) compared with only one positive test. In fact, 'double LA positivity', detected by combinations of any of the tests studied, was markedly associated with a history of thrombosis.

Substitution of a heparin correlation value for activated partial thromboplastin time in heparin nomograms

PURPOSE. Use of nomograms based on the "heparin correlation value" (HCV)-a value that corresponds to measured activated partial thromboplastin time (aPTT) and that removes the need to revise nomograms in response to a change in the aPTT reagent or coagulometer used-was evaluated as an alternative to traditional aPTT-based anticoagulation nomograms. SUMMARY. Data were collected on patients receiving heparin therapy for selected indications (thrombotic disorders, cardiac conditions, and acute coronary syndromes) during four-month periods before (n = 59) and after (n = 60) implementation of the HCV-based nomograms. The primary endpoints were the rate at which coagulation laboratory measurements were obtained at the appropriate time and the rate of appropriate dosage adjustment in response to reported laboratory values; secondary endpoints included the time to attainment of the first target anticoagulation value. After implementation of HCV-based nomograms, coagulation laboratory measurements were obtained at the appropriate time in (mean ± S.D.) 92.9% ± 12.8% of patients, compared with 80.1% ± 15.5% of patients who received aPTT-based monitoring (p < 0.0001). After implementation of HCV-based monitoring, the rate of correct heparin dosage adjustments was improved (mean ± S.D. 94.7% ± 7.8% versus 89.3% ± 14.0%, p = 0.01), and the time to attainment of the first target anticoagulation value was shorter (mean ± S.D. 16.4 ± 10.6 hours versus 21.5 ± 14.8 hours, p = 0.03). CONCLUSION. The HCV, which relates measured aPTT values to corresponding antifactor Xa concentrations, was substituted for aPTT in heparin nomograms and appeared to be a viable alternative to the aPTT.

Effects of telavancin on coagulation test results

The lipoglycopeptide antibiotic, telavancin, may interfere with some laboratory coagulation tests including prothrombin time (PT) and activated partial thromboplastin time (aPTT). To evaluate the effects of telavancin on PT and aPTT assays in common use. Pooled normal human plasma was spiked with telavancin 10, 20, 100 or 200 μg/ml (equivalent to trough, 2 × trough, peak and 2 × peak clinical plasma concentrations, respectively) or diluent control (0.9% sodium chloride). Samples were analysed using 16 PT reagents and seven aPTT reagents. Telavancin 200 μg/ml (corresponding to 2 × peak clinical plasma concentration), produced significant PT prolongation (> 9% difference vs. diluent control) with all the 16 PT reagents (range 12% to > 600%). At lower telavancin concentrations, PT prolongation was dose-dependent and varied among reagents, but appeared greatest with preparations containing recombinant tissue factor. With telavancin 10 μg/ml (equivalent to trough), PT prolongation was 10% with HemosIL(®) PT-Fibrinogen Recombinant, while ranging from 5% to -1% with all other reagents. Significant (> 34% difference vs. baseline) and dose-dependent aPTT prolongation was observed with all the seven reagents in samples spiked with telavancin 100 or 200 μg/ml (range 65-142% at 200 μg/ml). aPTT reagents containing a silica activator appeared to be more sensitive to telavancin interference. Telavancin 10 μg/ml was not associated with increased aPTT with any of the reagents tested. Telavancin has the potential to prolong both PT and aPTT in vitro. It is recommended that samples for PT or aPTT be obtained just prior to a telavancin dose (trough).

Evaluation of activated partial thromboplastin time (aPTT) reagents for application in biomedical diagnostic device development

The most commonly used test for monitoring heparin therapy is the activated partial thromboplastin time (aPTT). The response of available aPTT reagents to heparin varies significantly. The aim of this study was to highlight the differences between aPTT reagents stored in a dried format to select the most suitable formulations to be used for the development of point-of-care diagnostic devices used for monitoring of unfractionated heparin dose response. Ten reagents were analysed in terms of their performance in liquid and in dried form after storage for 24 h and 14 days. Performance was assessed by measurement of the clotting time (CT) as evidenced by the onset of thrombin formation using a chromogenic thrombin substrate in plasma samples activated with these formulations. Reagents in all of the three forms tested (liquid, 24 h and 14 days) resulted in significant shortening of CTs in comparison with the nonactivated plasma CT. Liquids returned more rapid CTs in comparison with dried reagents. Most of the reagents were more sensitive to heparin in dried, rather than in liquid form. Dried reagents based on kaolin as a surface activator were notably more effective in achieving short CT than others, while dried reagents composed of silica and synthetic phospholipids were the most sensitive to heparin. Two reagents, namely aPTT-SP and SynthASIL both of which are based on synthetic phospholipids and silica, were identified as promising candidates for incorporation into point-of-care diagnostic device platforms as dried reagents.

The Tick Protein Ir-Cpi Efficiently Delays Contact Pathway Induced Thrombin Generation and Displays In Vivo Antithrombotic Activity

AbstractAbstract 3336 Introduction:The search for new anticoagulants is a major challenge in medicine. Contact factors have never been considered as interesting targets for the development of new anticoagulant agents since they are not required for in vivo coagulation (i.e. deficiencies affecting these factors do not cause excessive bleeding). The discovery that FXI and FXII deficiency protects against thrombosis without causing spontaneous bleeding in mice makes FXII a unique and ideal target for drug design. We demonstrated in vitro that Ir-CPI, a 67 amino acids recombinant Kunitz-type protein from Ixodes ricinus, specifically interacted with activated human contact phase factors (FXIIa, FXIa, and kallikrein). Aims:The goal of this study was to investigate the potential anticoagulant and antithrombotic efficacy of Ir-CPI. Methods:The effects of Ir-CPI were investigated on the thrombin activity during the coagulation process in human plasma using the CAT method. Three different inducers were employed to specifically trigger the coagulation pathways and to generate thrombin. A standard concentration of 5 pM TF with 4 μM PL and 16.7 mM CaCl2 was used to activate the TF pathway whereas a lower TF concentration of 1 pM with 4 μM PL and 16.7 mM CaCl2 was selected for stimulating the TF pathway in combination with the contact phase pathway through a thrombin-mediated positive feedback. The contact phase pathway was also triggered alone by ellagic acid, PL and 16.7 mM CaCl2 (25-fold diluted APTT reagent Actin FS). The effect of Ir-CPI on venous thrombus formation was assessed using a rat thrombosis model induced by complete venous stasis in the posterior vena cava and FeCl3 topical application on the outer vessel wall. Results:When stimulating plasma coagulation trough the contact pathway, Ir-CPI caused a concentration-dependent prolongation of the lag time and the Tmax and a concentration-dependent decrease in the Cmax compared to the control curve (i.e. without inhibitor). When the coagulation cascade was triggered by the TF pathway (5 or 1 pM TF), only a slight concentration-dependent decrease of the Cmax and a concentration-dependent prolongation of the lag time and the Tmax were observed. For comparison purpose, the effects of the specific competitive FXIIa inhibitor corn trypsin inhibitor (CTI) were also investigated using the three same inducers than for Ir-CPI. For the contact pathway, a concentration-dependent decrease of the Cmax and a concentration-dependent prolongation of the lag time and the Tmax were found. When stimulating the TF pathway (5 or 1 pM TF), no modification of the thrombin generation curves was observed with the tested concentrations of CTI. When comparing the results of the two inhibitors acting trough the delay of the contact pathway, we found that Ir-CPI was about 30-fold more potent than CTI.We further evaluated the antithrombotic effect of Ir-CPI on a rat venous thrombosis model induced by endothelial damage and vessel ligation, close to the physiological venous thrombus formation in humans. We showed that Ir-CPI reduced thrombosis in a dose-dependent manner with an ED50 close to 65 μg/kg. A maximum effect starting from 0.5 mg/kg was observed with a mean reduction in the clot weight/body weight of 75 ± 7%. This antithrombotic effect of Ir-CPI was exclusively mediated through the inhibition of thrombin generation since it did not interference with collagen-induced platelet aggregation. This is the first time that an inhibitor of the coagulation contact phase was shown to protect against the formation of venous thrombi. The antithrombotic effect of Ir-CPI was also confirmed using other venous and arterial thrombosis models. We also showed that the effective antithrombotic dose of Ir-CPI in these tests did not promote bleeding or impair blood coagulation parameters. Conclusions:The present study demonstrated that Ir-CPI, a recombinant protein from the tick Ixodes ricinus targeting the contact factors (FXII, FXI and kallikrein), displayed an anticoagulant activity mainly through the delay of the contact pathway induced thrombin generation. This drug also exhibited an antithrombotic activity in our venous and arterial thrombosis model. This drug may thus provide an interesting and innovative therapeutic tool for the prevention and the treatment of thromboembolic diseases with a minimal risk of therapy-associated bleeding. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The responsiveness of different APTT reagents to mild factor VIII, IX and XI deficiencies

The sensitivity of APTT reagents to deficiencies of factors VIII, IX, XI and XII varies because of their composition. The APTT is used as a screening test for these factors, and a deficiency should manifest with a prolongation to the APTT, which may trigger the need for specific factor assays to be performed. The suitability of APTT reagents to detect mild deficiencies can be assessed by the analysis of the APTT of plasma, which has an increasing concentration of the factor in question. The APTT responsiveness can be determined from the intersection of the curve and the upper limit of the APTT normal reference range for that APTT reagent. We assessed the APTT responsiveness (in U/dl) to factors VIII, IX and XI of four APTT reagents; Actin FS (Siemens), Synthasil (IL), STA-PTTA (Stago) and Dapttin (Technoclone). Actin FS was the most sensitive reagent to mild reductions of factors VIII, IX and XI [Correction added on 26 October 2010, after first online publication: Synthasil was corrected to Actin FS]. STA-PTTA showed less sensitivity than Synthasil and Actin FS; Dapttin was insensitive to mild deficiencies of factors IX and XI and should not be used as a screening test. Both Synthasil and Actin FS are acceptable reagents to screen for reduced factors VIII, IX and XI, and the number of mildly reduced factors not diagnosed will be limited.

Screening coagulation testing using the APTT: Which reagent to choose?

APTT testing is integral to hemostasis testing. A prolonged result, however, can be difficult to interpret, depending on the APTT reagent's sensitivity to the lupus anticoagulant. This often generates additional laboratory testing for both factor deficiencies and the presence of a lupus anticoagulant, and in so doing, delays patient management. We have found it useful to provide APTT testing with both a lupus anticoagulant sensitive and insensitive reagent, to facilitate the rapid exclusion of significant factor deficiencies. The following case report illustrates the utility of this approach and provides a backdrop for necessary discussions between laboratories and clinicians regarding which APTT reagent best meets their clinical need for screening hemostasis testing.

Factor VIII Lacking the C2 Domain Retains Cofactor Activity in Vitro

Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains). The activated form of FVIII, FVIIIa, functions as a cofactor for FIXa in catalyzing the membrane-dependent activation of FX. Whereas the FVIII C2 domain is believed to anchor FVIIIa to the phospholipid surface, recent x-ray crystal structures of FVIII suggest that the C1 domain may also contribute to this function. We constructed a FVIII variant lacking the C2 domain (designated DeltaC2) to characterize the contributions of the C1 domain to function. Binding affinity of the DeltaC2 variant to phospholipid vesicles as measured by energy transfer was reduced approximately 14-fold. However, the activity of DeltaC2 as measured by FXa generation and one-stage clotting assays retained 76 and 36%, respectively, of the WT FVIII value. Modest reductions ( approximately 4-fold) were observed in the functional affinity of DeltaC2 FVIII for FIXa and rates of thrombin activation. On the other hand, deletion of C2 resulted in significant reductions in FVIIIa stability ( approximately 3.6-fold). Thrombin generation assays showed peak thrombin and endogenous thrombin potential were reduced as much as approximately 60-fold. These effects likely result from a combination of the intermolecular functional defects plus reduced protein stability. Together, these results indicate that FVIII domains other than C2, likely C1, make significant contributions to membrane-binding and membrane-dependent function.

CASE REPORT: Patient with Passovoy Defect with An Olfactory Meningioma Who Underwent Craniotomy without Bleeding Complications.

Abstract 4204 A 58 year old male with a known clinical history of Passovoy defect was evaluated for a craniotomy for an olfactory meningioma. His family history revealed that this defect was present in multiple family members (siblings, parent and child). In the past, he had had several successful and several complicated surgeries. According to his past history, bleeding did not occur intra-operatively but typically 24-72 hours post-operatively. There was no evidence of mucosal bleeding, petechiae, rashes or ecchymosis on examination. There was no past history of spontaneous hemorrhage, epistaxis, hemoptysis, hematemesis, melena, petechiae or ecchymosis. Past surgical history revealed that when bleeding occurred and FFP was transfused post-operatively, the bleeding subsided. When Fresh Frozen Plasma (FFP) was transfused pre-operatively, intra-operatively and post-operatively there was no significant bleeding. Laboratory tests showed normal ProthrombinTime (PT), activated Partial Thromboplastin Time (aPTT), ThrombinTime (TT), Fibrinogen, Factor VIII and Factor XIII, vWFAntigen and vWF multimers. Platelet Aggregation studies were normal except for a decreased response to epinephrine. Thromboelastogram studies revealed a mild coagulation factor deficiency. Our workup did not show any of the common coagulation defects. There was no evidence of a prolonged Partial Thromboplastin Time (aPTT) as reported in the literature. Due to normal aPTT results (average 27.7 secs, range 25.5– 38.1 secs), mixing studies were not performed. Due to his significant past clinical history, the patient was transfused with Fresh Frozen Plasma (FFP) pre-operatively, intra-operatively and post- operatively for 72 hrs. The PT/aPTT and platelet counts remained within normal range throughout the patient's entire hospital stay with no evidence of bleeding post-operatively. Passovoy Defect is a rare autosomal disorder and is a coagulation abnormality affecting the intrinsic coagulation system characterized by a prolonged aPTT. The defect is associated with a clinical bleeding tendency characterized by easy bruising and undue blood loss following trauma and/or surgery. Fresh Frozen Plasma appears to provide effective prophylaxis during surgery. The bleeding phenotype is variable. There may be no bleeding after major surgeries. Major bleeding after minor surgeries is not uncommon. Our case represents a rare type of a defect in the intrinsic system that may not be encountered in the daily setting with a normal active Partial Thromboplastic Time (aPTT). The Passovoy Defect in our patient appears to be transmitted as an autosomal dominant trait. Despite a very significant clinical and family history, the aPTT was within normal range throughout his hospitaization. No diagnostic laboratory findings were noted. It has been reported that the prolongation of aPTT in some of the patients may be relatively mild. However this was not the case in our patient. We do not know whether our patient has a deficiency of an unknown coagulation factor or that our aPTT reagent (HemosIL SynthASIL Catalogue # 20006800, DiaPharma Group Inc, West Chester, OH, USA) was not sensitive to the Passovoy Defect. Disclosures: No relevant conflicts of interest to declare.

Asymptomatic Familial Factor XI Deficiency.

Abstract 4449 BackgroundFactor XI deficiency (known as Rosenthal syndrome or hemophilia C) is an autosomal disorder affecting both sexes which results in a bleeding disorder of variable severity. This condition is very uncommon among the non-Jewish population and consists mostly of sporadic cases, although an occasional familial cluster has been described (Bolton-Maggs et al. J Thromb Haemost 2004 Jun;2(6):918-24). We present two asymptomatic siblings with coagulation Factor XI deficiency. Case ReportIn February 2004 a healthy 33 year-old non-Jewish white female without personal or family medical history presented with an elevated partial thromboplastin time (PTT) incidentally found during a routine pre-operative evaluation. Her prothrombin time (PT) was normal. Her PTT was 45 seconds (reference interval 23-37 seconds), and normalized with 1:1 mixing studies of patient and normal plasma. Preliminary laboratory evaluation included negative tests for the presence of a Lupus Anticoagulant, a normal serum albumin level, and a normal urinalysis. Additional laboratory testing of coagulation and included FXI and XII activities, measured by means of a one stage APTT-based clotting assay using APTT reagent (Automated APTT, Trinity Biotech, Bray Ireland), congenitally depleted deficient plasma (HRF, Inc, Raleigh NC) using the MDA II Trinity Biotech, Bray Ireland). FXI was 46% (reference range 60-150) and FXII was 42% (reference range 50-150). In March 2004, a repeated the laboratory evaluation showed a FXI of 41% and a FXII of 46%. In April 2006, FXI was 49%, and FXII was 40%. Is to be noted that the patient never had abnormal bleeding episodes despite abdominal and breast surgical procedures. In 2009, her only brother (aged 29 and otherwise healthy) underwent hematological evaluation that showed normal levels of PT, PTT, platelet count, fibrinogen, and factor XII (88% activity). His factor XI was 51% (decreased activity). ConclusionAlthough modern laboratory methods are able to identify unusual coagulation defects, their clinical significance requires additional investigation. Disclosures:No relevant conflicts of interest to declare.

Characterization of a homozygous Gly11Val mutation in the Gla domain of coagulation factor X

Factor (F) X deficiency is a rare inherited autosomal recessive trait. We report on a patient affected by a severe bleeding diathesis. Mutations were sought by F10 sequence analysis. The consequences of the mutation were characterized by measuring thrombin and FXa formation after triggering the clotting cascade with activated partial thromboplastin time (aPTT) reagent or with phospholipid vesicles plus either tissue factor (TF) or FIXaβ, or with the FX activator from Russell's viper venom (RVV-X). The patient was found to be homozygous for a novel FX p.G51V mutation (G11V of the mature protein) within the ω-loop of the γ-carboxyglutamic-rich domain. FX activity was markedly reduced (FX:C < 1%) in prothrombin time and aPTT assays, and was 15% of normal in the RVV-X assay. The antigen level (FX:Ag) was 75%. TF, alone or in combination with recombinant FVIIa, failed to trigger detectable FXa or thrombin activity in the patient's plasma. FIXaβ also failed to trigger measurable FXa or thrombin production, but activation with RVV-X was only 4-fold less effective in the patient's plasma than in normal plasma. Supplementation with normal FX suggested that FX G11V and/or FXa G11V might slow the clotting cascade by competition. Overall, the patient's phenotype appears to be due to a very low rate of FX G11V activation by TF/FVIIa and FVIIIa/FIXa complexes rather than to FXa G11V activity within prothrombinase.

Combined Coagulation Factor XI and Factor XII Deficiency in An Asymptomatic Female

Background: Multiple combined coagulation factor deficiencies have been reported, although the genetic defects associated with these conditions remain largely unknown. We present an asymptomatic female with a persistent deficiency of both coagulation Factor XI (FXI) and Factor XII (FXII). To our knowledge, this particular combination of coagulation factor deficiency has not been previously reported.Case Report: In February 2004 a healthy 33 year-old white female without personal or family medical history presented with an elevated partial thromboplastin time (PTT) incidentally found during a routine pre-operative evaluation. Her prothrombin time (PT) was normal. Her PTT was 45 seconds (reference interval 23–37 seconds), and normalized with 1:1 mixing studies of patient and normal plasma. Preliminary laboratory evaluation included negative tests for the presence of a Lupus Anticoagulant, a normal serum albumin level, and a normal urinalysis. Additional laboratory testing of coagulation and included FXI and XII activities, measured by means of a one stage APTT-based clotting assay using APTT reagent (Automated APTT, Trinity Biotech, Bray Ireland), congenitally depleted deficient plasma (HRF, Inc, Raleigh NC) using the MDA II Trinity Biotech, Bray Ireland). FXI was 46% (reference range 60–150) and FXII was 42% (reference range 50–150). In March 2004, a repeated the laboratory evaluation showed a FXI of 41% and a FXII of 46%. In April 2006, FXI was 49%, and FXII was 40%. Is to be noted that the patient has never had a documented thromboembolic episode or excessive bleeding, despite the exposure, respectively, to estrogen supplementation for seven years, to a full-term pregnancy, and to an abdominal surgical event.Conclusion: Although modern laboratory methods are able to identify novel combinations of coagulation defects, their clinical significance requires additional investigation.

Recombinant Antidote for Reversal of Anticoagulation by Factor Xa Inhibitors.

Individuals anticoagulated with warfarin or heparin are typically treated with specific antidotes such as vitamin K or protamine, respectively, if they bleed or require surgery. In contrast, specific and effective antidotes are not available for the reversal of the anticoagulant effects of the low molecular weight heparins (LMWH) or the new oral anticoagulants targeting factor Xa (fXa) which are predictably only marginally affected by standard treatments using rfVIIa or fresh frozen plasma. The present study was designed to test the hypothesis that plasma-derived or recombinant fXa, modified to lack catalytic and membrane binding activities, could neutralize the anticoagulant activities of small molecule fXa inhibitors and LMWH. Plasma derived antidote (pd-Antidote) was prepared by chemical modification of the active site serine of fXa followed by chymotryptic removal of the Gla domain. Preliminary experiments showed that pd-Antidote dose dependently reversed the activity of rivaroxaban, apixaban or betrixaban, three small molecule fXa inhibitors currently in clinical trials. In a fXa amidolytic assay with 3nM enzyme, half maximal reversal of inhibitory activities (EC50) was attained at the following pd-Antidote concentrations: rivaroxaban =49 nM, apixaban = 122 nM, betrixaban = 41 nM. The pd-Antidote had no effect on the activity of fXa in the absence of inhibitors. Thus, active site inactivated pd-Antidote retained the ability to bind small molecule inhibitors of fXa. The activity of pd-Antidote was not limited to reversal of purified fXa catalytic activity. In a tissue factor-initiated thrombin generation assay in plasma, while pd-Antidote did not interfere with the normal function of prothrombinase complexes, it dose-dependently and completely reversed the inhibition produced by the small molecule fXa inhibitors. Pd-Antidote also demonstrated reversal of the in-vitro anticoagulant activity of the LMWH, enoxaparin. Addition of pd-Antidote (515nM) produced a 37% reduction of the clotting activity of enoxaparin (1.25Units/ml). In an activated partial thromboplastin time assay (aPTT), pd-Antidote also dose-dependently reversed the inhibitory effects of the fXa inhibitors. For example, aPTT prolongation by betrixaban (400nM) was reversed with an estimated EC50=650nM. Baseline aPTT was not altered upon addition of pd-Antidote up to a concentration of 2.5 μM, the highest concentration studied. Recombinant antidote (r-Antidote) was expressed in mammalian cells (Chinese hamster ovary) using a construct for human fXa with the S195A mutation and lacking the Gla-domain. The EC50's for purified r-Antidote in reversing 7.5 nM fXa inhibitors in the fXa catalytic activity assay were: rivaroxaban =17 nM, apixaban = 45 nM, betrixaban = 15 nM. As expected, there was no inhibition of fXa activity by r-Antidote alone. Purified r-Antidote also reversed anticoagulant activity in plasma clotting assays. Prothrombin time (PT) extensions achieved by supratherapeutic concentrations of rivaroxaban (1 μM) or apixaban (1 μM) were completely normalized upon addition of r-Antidote (1.5 μM). Ex vivo clotting prolongation by an excess of betrixaban (300 nM) was essentially reversed (88% correction) by pre-incubation with r-Antidote (570 nM) prior to addition of aPTT reagents. The concentration of r-Antidote required for corrective activity was related to the inhibitory potency of the oral fXa inhibitors. PT or aPTT baselines did not change upon addition of r-Antidote alone at 1.9 μM, the highest concentration tested. We also examined the ability of both pd-Antidote and r-Antidote to alleviate inhibitory activities produced upon oral dosing of fXa inhibitors in mice. Perturbation of whole blood PT/INR in dosed animals was followed as a marker of anticoagulation. The effect following dosing of a fXa inhibitor could be reversed by a single intravenous injection of antidote. INR measurements in blood samples of treated animals showed a >50% reduction of observed inhibitory activity compared to control animals. Our results suggest that these plasma derived or recombinant proteins have the potential to act as universal antidotes for reversal of anticoagulation of all current fXa inhibitors, both small molecule and antithrombin dependent, in patients with bleeding related medical emergencies or those requiring cessation of anticoagulation prior to surgery.

The Direct Thrombin Inhibitor Argatroban: A Review of Its Pharmacokinetics and Pharmacokinetic/Pharmacodynamic Relationships

The direct thrombin inhibitor argatroban exhibits pharmacological properties that are distinctly different from those of other thrombin inhibitors. Within its clinically relevant dose range, argatroban shows linear pharmacokinetic behavior and concentrations that are well correlated with anticoagulant effects. The pharmacokinetic profile can be described by a two-compartment model with first-order elimination. Whereas argatroban does not require dosage modification in patients with renal dysfunction and has been demonstrated to be safe and well tolerated in hemodialysis, dosing precautions are recommended in hepatically impaired patients. The effects of high-dose or prevention therapy can be monitored by various coagulation parameters like activated clotting time (ACT), activated partial thromboplastin time (aPTT), or ecarin clotting time (ECT). Whereas the aPTT response to argatroban varies with aPTT reagent, ECT seems to be a more specific monitoring parameter for the direct thrombin inhibitor. Argatroban influences the prothrombin time, expressed as the international normalized ratio (INR), which complicates the transition to oral anticoagulation with coumarin derivatives. An overlap period with warfarin for a minimum of 4 to 5 days, aimed at an INR of > 4 on a Quick-type prothrombin time assay, is recommended before discontinuation of argatroban therapy. The good predictability of the anticoagulant effect of argatroban, together with its fast clearance and lack of influence by renal dysfunction, makes argatroban an ideal antithrombotic agent in the intensive care unit setting and in interventional cardiology.

Assessment of the Coag-a-Pet Dual Channel in the routine haemostatic laboratory*

The Coag-a-Pet Dual Channel is an instrument which automatically records coagulation test end-points utilizing a photo-optical clot detection system. The instrument and its operation are described in detail. The capability of the instrument to perform tests of oral anticoagulant control, basic coagulation profiles and one-stage factor assays is assessed. In terms of precision and accuracy, the instrument performs well in carrying out the one-stage prothrombin time, Thrombotest, activated partial thromboplastin time using an automated APTT reagent but not kaolin, and one-stage factor assays. The thrombin clotting time can not be measured on this instrument. The instrument is most suitable for batched repetitive tests, reducing observer error and improving laboratory efficiency.