Baseline and treatment-related changes in thrombin generation in patients with multiple myeloma.

The prothrombotic risk in multiple myeloma (MM) could be potentially assessed by thrombin generation (TG) assays. TG was performed using Calibrated Automated Thrombography with 5 and 1 pM tissue factor. We compared baseline TG among 24 MM patients, 19 MGUS, and 50 healthy controls, and assessed change in TG in MM patients during the initial treatment period at 1, 2, and 3 months. MM subjects demonstrated increased FVIII and VWF:Ag levels pretreatment, and a prothrombotic TG phenotype with increased velocity index, reduced lag time and time-to-peak, and increased resistance to thrombomodulin inhibition. There were no significant changes in TG with treatment for the majority of parameters, however, MM subjects exhibited persistent elevation of velocity index throughout treatment. Two subjects developed thrombosis during the study period despite thromboprophylaxis. This study provides information on the optimal conditions for examining TG as a predictor of thrombotic risk in MM patients.

Evaluation of pre-analytical variables in a commercial thrombin generation assay.

BACKGROUND: There is minimal data on the influence of pre-analytical variables on the use of calibrated automated thrombography (CAT), to measure thrombin generation. OBJECTIVES: To evaluate the impact of centrifugation methods, time after collection, and contact activation inhibition on the CAT assay performed using two commercial reagents. METHODS AND RESULTS: Six different methods of plasma separation were examined. Thrombin generation triggered by a 5 pM tissue factor reagent was not greatly affected by plasma separation method, with similar results obtained with all methods apart from single centrifugation and membrane filtration. Membrane filtration increased APTT and is not recommended. Extended double centrifugation at higher speed was required to minimise the impact of residual phospholipid with 1 pM tissue factor trigger, particularly with inhibition of contact activation. The effect of a delay of up to 24 hours in preparing plasma was assessed. No significant difference in results was observed among samples processed between 0.5 and 6 hours after blood collection into plastic Vacuette® tubes. The presence or absence of corn trypsin inhibitor had a significant impact on all parameters with 1 pM tissue factor trigger, with minor differences seen on Peak and ttPeak results using 5 pM tissue factor. CONCLUSIONS: The impact of pre-analytical variables on thrombin generation results is dependent on the concentration of tissue factor in the trigger reagent used. Results with 1 pM tissue factor are particularly sensitive to centrifugation method and contact activation, and standardisation is required to allow large collaborative studies to be performed.

Diagnostic testing for mild hemophilia a in patients with discrepant one-stage, two-stage, and chromogenic factor VIII:C assays.

In recent years, there has been greater awareness among hemostasis scientists and clinicians that factor VIII coagulant activity (FVIII:C) measured in certain patients with mild hemophilia A can show different results depending on the assay system. A subgroup of mild hemophilia families have a method-related discrepancy in FVIII:C results, whereby the one-stage clotting assay (FVIII:C-1) is significantly higher than the two-stage clotting assay (FVIII:C-2) or the chromogenic assay (FVIII:C-chr). To identify such patients, the routine laboratory can use automated procedures for the FVIII:C-chr to replace the complex, manual FVIII:C-2 method. Laboratories must employ appropriate quality management to ensure accurate and precise results, especially in the abnormal range. This discrepant phenotype of hemophilia A is seen in up to 40% of mild hemophilia A cases and represents a clinically significant bleeding disorder. A small proportion of these cases have FVIII:C-1 within the normal range and risk a missed diagnosis if the FVIII:C-chr is unavailable. Other patients may be mismanaged if FVIII:C-1 gives an overestimate of FVIII:C and their bleeding risk is consequently underestimated. Affected family members in the discrepant group of patients have a limited range of FVIII (F8) gene missense mutations, causing alterations of the structure of the A1, A2, or A3 domains of FVIII. Therefore, both FVIII:C-chr and F8 gene mutation analysis are recommended to confirm the diagnosis of mild hemophilia A and assist with decisions about the patient's phenotype.

Diagnosis and management of adult patients with von Willebrand disease in South Australia.

We have analyzed the databases for von Willebrand disease (VWD) from the hemophilia center for adult patients with bleeding disorders in South Australia. We define the prevalence of types of VWD to determine the proportion of who would be treated with factor (F) VIII/von Willebrand factor (VWF) concentrate to prevent or control hemorrhage. In severe or moderately severe patients, we use plasma-derived FVIII/VWF concentrate, and for mild to severe cases, we use desmopressin plus tranexamic acid. There are 103 patients with VWF ristocetin (RCo) ≤50 IU/dL: 38 (37%) severe (VWF:RCo <10 IU/dL), 28 (27%) moderate (VWF:RCo 10 to 29 IU/dL), and 37 (36%) mild (VWF:RCo 30 to 50 IU/dL). Hence in 66 (64%), FVIII/VWF concentrate is the mainstay of treatment. The prevalence of VWD in our region according to data from our center is ~1 per 12,000. A total of 52% of patients are type 1, 44% type 2, and 5% type 3. In our experience, type 2M (45% of type 2) is much more common than types 2A and 2B (each 9% of type 2). Mutation detection is useful for identifying some subtypes of VWD.

Latest medical treatment strategies for venous thromboembolism.

Anticoagulant therapy with unfractionated heparin (UFH) followed by warfarin prevents thrombus extension, reduces the risk of recurrent thrombosis and prevents death in patients with venous thromboembolism (VTE). Low-molecular-weight heparin (LMWH) has replaced UFH as the preferred initial anticoagulant therapy for VTE because it is as effective and safe as UFH, but does not require laboratory monitoring and is less likely to cause immune thrombocytopenia and osteoporosis. More recently, fondaparinux has been shown to be an effective and safe alternative to LMWH and several new parenteral anticoagulants are being evaluated. The most important unmet need in the anticoagulant management of VTE is a replacement for warfarin. New oral anticoagulants that selectively target individual steps in the coagulation cascade have been shown to be effective for the long-term treatment of VTE in Phase II and III trials and are likely to become available in the near future.

In the presence of phospholipids, glycosaminoglycans potentiate factor Xa-mediated protein C activation by modulating factor Xa activity.

Although the thrombin/thrombomodulin complex is considered the physiological activator of protein C, factor Xa (f.Xa) can also activate protein C in a reaction that is potentiated by glycosaminoglycans. To explore this phenomenon, we first examined the effect of glycosaminoglycans of varying degrees of sulfation on the kinetics of protein C activation by f.Xa in the presence of Ca2+ and phosphatidylcholine-phosphatidylserine vesicles (PCPS). Heparin increased the rate of protein C activation by f.Xa by 4-fold. In contrast, N-desulfated heparin had no effect on activation, whereas dextran sulfate, which is more sulfated than heparin, increased catalytic efficiency 21-fold. These data suggest that the capacity of glycosaminoglycans to catalyze protein C activation by f.Xa depends on their degree of sulfation. The affinities of individual glycosaminoglycans for protein C and f.Xa were measured in the absence or presence of PCPS by monitoring changes in extrinsic fluorescence when fluorescein-labeled f.Xa or protein C was titrated with the various glycosaminoglycans. Heparin binds protein C with low affinity in the absence or presence of PCPS. In contrast, the affinity of heparin for f.Xa is 86-fold higher in the presence of PCPS compared to that in the absence of PCPS. Similar results were obtained using surface plasmon resonance. These findings suggest that a high affinity glycosaminoglycan binding site is exposed when f.Xa binds to PCPS. The observation that heparin promotes f.Xa-mediated activation of prethrombin 1 only in the presence of phospholipid suggests that glycosaminoglycan binding modulates the active site of f.Xa. This study reveals that when f.Xa interacts with anionic phospholipids, glycosaminoglycans bind f.Xa more tightly, allosterically modulate its active site, and enhance its capacity to activate protein C.

Update in the diagnosis of deep-vein thrombosis and pulmonary embolism.

PURPOSE OF REVIEW: Diagnostic strategies for venous thromboembolism must both accurately diagnose thrombus when present, and safely exclude it when absent. This review summarizes recent data on diagnostic strategies for venous thromboembolism. RECENT FINDINGS: Noninvasive diagnostic strategies have emerged to limit the need for invasive testing for deep-vein thrombosis and pulmonary embolism. D-Dimer testing combined with clinical assessment can be used to safely exclude deep vein thrombosis, limiting the need for further testing. Extended lower limb ultrasonography also shows promise although requires further data. Spiral computed tomography has become widely used for the diagnosis of pulmonary embolism. Evidence either for the use of single-detector spiral computed tomography combined with ultrasound or for multidetector spiral computed tomography as a safe and stand-alone test, for the purpose of excluding pulmonary embolism, is finally catching up with current practice. SUMMARY: Invasive testing for venous thromboembolism can be safely avoided in the majority of patients, using diagnostic strategies combining noninvasive tests. Initial evidence suggests that multidetector spiral computed tomography is a safe stand-alone test for pulmonary embolism. Local cost and expertise with separate diagnostic tests will influence the appropriate choice of diagnostic strategies for venous thromboembolism at individual institutions.

New anticoagulants for venous thromboembolic disease.

PURPOSE OF REVIEW: In this paper, recent advances in new anticoagulants with the potential to be used for prevention or treatment of venous thrombosis are reviewed. RECENT FINDINGS: Numerous novel anticoagulants targeting specific stages of the coagulant pathway are in various stages of development. Fondaparinux, an indirect activated factor VII inhibitor, has been shown to be effective for initial treatment and prevention of venous thromboembolism, but still requires parenteral administration. Ximelagatran, an oral direct thrombin inhibitor, has also been shown to effective for treatment and prevention of venous thrombosis. Both agents are associated with bleeding, however, and ximelagatran is associated with hepatic toxicity with long-term use. Direct activated factor X inhibitors, orally available forms of heparin, and other direct thrombin inhibitors remain in early stages of development. Further data on the clinical utility of these agents are likely to emerge in the next few years, and uptake of their use will be affected by the cost considerations. SUMMARY: Numerous alternative anticoagulants are in varying stages of development. Clinical data have yet to show that these agents have a clearly superior risk-benefit ratio compared with currently used antithrombotics. Many drugs remain in initial stages of development. The ideal anticoagulant agent is being sought but has yet to be discovered.

The diagnostic evaluation of pulmonary embolism.

Due to the morbidity and mortality associated with either untreated disease or inappropriate anticoagulant therapy, accurate diagnosis of pulmonary embolism is essential. Pulmonary angiography, the current gold standard test for diagnosing pulmonary embolus, is both invasive and costly; therefore, noninvasive diagnostic strategies have been developed. Noninvasive tests often have to be combined to either raise the posttest probability of disease to a level justifying treatment or lower it to a level at which withholding treatment is warranted. Diagnostic algorithms involving clinical assessment; venous ultrasonography; D-dimer testing; ventilation-perfusion lung scanning; and, more recently, computed tomography have been validated in management trials of patients with a suspected pulmonary embolism. The optimal strategy at individual institutions is dependent on local availability, expertise, and cost. Magnetic resonance imaging and combined computed tomographic pulmonary angiography and venography possess the potential to be used as stand-alone tests for pulmonary embolism but require further evaluation.

New anticoagulants for the prevention and treatment of venous thromboembolism.

Anticoagulant therapy is effective at preventing the development of venous thromboembolism in high-risk patients, and reduces morbidity and mortality in individuals with established thromboembolic disease. Vitamin K antagonists and heparins are currently the most commonly used anticoagulant drugs, but they have practical limitations. Therefore, new antithrombotic agents with predictable dose-responses (thereby decreasing the need for monitoring without compromising efficacy or safety), ideally available in an oral formulation and with a rapidly reversible anticoagulant effect, are needed. New drugs fulfilling some of the above criteria have been developed and have proven to be effective agents for the treatment and prevention of venous thromboembolism.

The diagnostic evaluation of deep vein thrombosis.

Due to the morbidity and mortality associated with either untreated disease or inappropriate anticoagulant therapy, accurate diagnosis of venous thromboembolism is essential. As venography, the current gold standard test for deep vein thrombosis (DVT), is both invasive and costly, noninvasive diagnostic strategies for diagnosing DVT have been developed. Noninvasive tests often have to be combined to either raise the post-test probability of disease to a level justifying treatment or lower it to a level at which withholding treatment is warranted. Diagnostic algorithms involving clinical assessment, venous ultrasonography, and D-dimer testing have been validated in management trials of patients with DVT. The optimal strategy at individual institutions is dependent on local expertise and cost. Magnetic resonance venography has the potential to be used as a stand-alone test for DVT but requires further evaluation.

Initial treatment of venous thromboembolism.

Adequate initial anticoagulant therapy of deep venous thrombosis (DVT) is required to prevent thrombus growth and pulmonary embolism (PE). Intravenous unfractionated heparin (UFH) is being replaced by low-molecular-weight heparin (LMWH) as the anticoagulant of choice for initial treatment of venous thromboembolism (VTE). Both agents are relatively safe and effective when used to treat VTE, with LMWH suitable for outpatient therapy because of improved bioavailability and more predictable anticoagulant response. Serious potential complications of heparin therapy, such as heparin-induced thrombocytopenia (HIT) and osteoporosis, seem less common with LMWH. The potential for fetal harm and changes in maternal physiology complicate the treatment of VTE during pregnancy. Although systemic thrombolysis is used in patients with massive PE and in some patients with proximal DVT, controversy persists with respect to appropriate patient selection for this intervention.