Treatment of high-risk venous thrombosis patients using low-dose intraclot injections of recombinant tissue plasminogen activator and regional anticoagulation.

Seven patients with venous thrombosis and contraindications to traditional thrombolytic therapy, consisting of recent intracranial surgery, recent pineal or retroperitoneal hemorrhage, active genitourinary or gastrointestinal bleeding, epidural procedures, and impending surgery, were successfully treated with a modified thrombolytic regimen. To improve safety, prolonged continuous infusions of tissue plasminogen activator (tPA) was eliminated in favor of once-daily low-dose intraclot injections of tPA to minimize the amount and duration of tPA in the systemic circulation, and low-therapeutic or regional anticoagulation was used to reduce anticoagulant risks. These modifications may allow thrombolytic treatment for selected patients with severe venous thrombosis who are deemed to be at high risk.

Biochemical dynamics relevant to the safety of low-dose, intraclot alteplase for deep vein thrombosis.

Intraclot tissue plasminogen activator (tPA) has been shown to be an effective treatment for deep vein thrombosis (DVT) (Radiology 2008;246:619 and J Vasc Interv Radiol 2011;22:1107). We sought to correlate pharmacokinetics of tPA, fibrinogen, fibrinolytic inhibitors, and D-dimers with the safety and efficacy of intraclot tPA. Thirty subjects received intraclot tPA for lower extremity DVT by infiltrating the thrombus with ≤10 mg doses tPA in an open-label study, using a pulse-spray catheter. We measured various parameters over 8 h following a first dose of tPA. Mean tPA levels of 75 units per mL (95% confidence interval 19-131 units/mL) were seen immediately after administration of a mean tPA dose of 8.0 mg (SD 1.5 mg). tPA levels returned to baseline within 2 h of completion of treatment. Plasminogen activator inhibitor-1 (PAI-1) was consumed following tPA treatment, but rose to levels significantly greater than baseline (P < 0.001). Fibrinogen decreased slightly, but remained >125 mg/dL for all subjects. α2-antiplasmin decreased from a mean of 115 units/mL to 56 units/mL after tPA administration (P < 0.001) and remained decreased for 8 h. Plasminogen at baseline (112 units/mL) decreased to 89 units/mL immediately after tPA administration (P < 0.001) and was unchanged thereafter. D-dimer levels were >20 µg/mL in all but 4 subjects, one of whom was the only one to fail to achieve clot lysis. The safety of low-dose, intraclot tPA is due to its short persistence in the circulation, lack of hypofibrinogenemia, and a reflexive rise of PAI-1. Subjects whose D-dimers remain <20 µg/mL are at risk of not achieving thrombolysis.

Low-dose, once-daily, intraclot injections of alteplase for treatment of acute deep venous thrombosis.

PURPOSE: To evaluate the safety and efficacy of once-daily intraclot injections of low doses (≤ 10 mg) of tissue plasminogen activator (tPA) for thrombolysis of venous thrombosis. MATERIALS AND METHODS: In prospective studies, 33 patients with subclavian, jugular, and central venous thrombosis (SJ-CVT) (all but two cases associated with central catheters) were treated once a day with ≤ 4 mg/day of tPA, and 30 patients with acute deep vein thrombosis of the lower extremity (DVT-LE) < 14 days old were treated once a day with ≤ 10 mg/leg/day of tPA by intraclot "lacing" of thrombus without continuous infusions of tPA. RESULTS: Patency was restored in 26 (79%) of 33 patients with SJ-CVT using an average total dose of 7.1 mg of tPA/per patient and average of 2.1 treatments or days of therapy. Five patients received thrombolytic therapy for SJ-CVT as outpatients. Initial patency was restored in 29 (97%) of 30 patients with acute DVT-LE using an average total dose of 20 mg of tPA per patient over an average of 2.7 treatments/or days per patient. Follow-up imaging examinations at 6 months showed continued patency in 27 (96%)/of 28 patients. There were no major bleeding complications, and no patient required a blood transfusion. CONCLUSIONS: Intraclot injection of low doses of alteplase is effective for acute venous thrombosis, and pharmacokinetic data suggest potentially greater safety.

Gray platelet syndrome: natural history of a large patient cohort and locus assignment to chromosome 3p.

Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by macrothrombocytopenia and absence of platelet α-granules resulting in typical gray platelets on peripheral smears. GPS is associated with a bleeding tendency, myelofibrosis, and splenomegaly. Reports on GPS are limited to case presentations. The causative gene and underlying pathophysiology are largely unknown. We present the results of molecular genetic analysis of 116 individuals including 25 GPS patients from 14 independent families as well as novel clinical data on the natural history of the disease. The mode of inheritance was autosomal recessive (AR) in 11 and indeterminate in 3 families. Using genome-wide linkage analysis, we mapped the AR-GPS gene to a 9.4-Mb interval on 3p21.1-3p22.1, containing 197 protein-coding genes. Sequencing of 1423 (69%) of the 2075 exons in the interval did not identify the GPS gene. Long-term follow-up data demonstrated the progressive nature of the thrombocytopenia and myelofibrosis of GPS resulting in fatal hemorrhages in some patients. We identified high serum vitamin B(12) as a consistent, novel finding in GPS. Chromosome 3p21.1-3p22.1 has not been previously linked to a platelet disorder; identification of the GPS gene will likely lead to the discovery of novel components of platelet organelle biogenesis. This study is registered at www.clinicaltrials.gov as NCT00069680 and NCT00369421.

Individualized treatment for iron-deficiency anemia in adults.

Iron deficiency is one of the most common disorders affecting humans, and iron-deficiency anemia continues to represent a major public health problem worldwide. It is especially common among women of childbearing age because of pregnancy and menstrual blood loss. Additional patient groups include those with other sources of blood loss, malnutrition, or gut malabsorption. Iron-deficiency anemia remains prevalent despite the widespread ability to diagnose the disease and availability of medicinal iron preparations. Therefore, new approaches are needed to effectively manage these patient populations. In this review, the diagnosis and treatment of iron-deficiency anemia are discussed with emphasis placed on consideration of patient-specific features. It is proposed that all patients participate in their own care by helping their physician to identify a tolerable daily iron dose, formulation, and schedule. Dosing cycles are recommended for iron replacement based on the tolerated daily dose and the total iron deficit. Each cycle consists of 5000 mg of oral elemental iron ingested over at least 1 month with appropriate follow-up. This approach should assist physicians and their patients with the implementation of individualized treatment strategies for patients with iron-deficiency anemia.

Increased soluble urokinase plasminogen activator receptor (suPAR) is associated with thrombosis and inhibition of plasmin generation in paroxysmal nocturnal hemoglobinuria (PNH) patients.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired genetic disorder of the bone marrow that produces intravascular hemolysis, proclivity to venous thrombosis, and hematopoietic failure. Mutation in the PIG-A gene of a hematopoietic stem cell abrogates synthesis of glycosylphosphoinositol (GPI) anchors and expression of all GPI-anchored proteins on the surface of progeny erythrocytes, leukocytes, and platelets. Urokinase plasminogen activator receptor (uPAR), a GPI-linked protein expressed on neutrophils, mediates endogenous thrombolysis through a urokinase-dependent mechanism. Here we show that membrane GPI-anchored uPAR is decreased or absent on granulocytes and platelets of patients with PNH, while soluble uPAR (suPAR) levels are increased in patients' plasma. Serum suPAR concentrations correlated with the number of GPI-negative neutrophils and were highest in patients who later develop thrombosis. In vitro, suPAR is released from PNH hematopoietic cells and from platelets upon activation, suggesting that these cells are the probable source of plasma suPAR in the absence of GPI anchor synthesis and trafficking of uPAR to the cell membrane. In vitro, the addition of recombinant suPAR results in a dose-dependent decrease in the activity of single-chain urokinase. We hypothesized that suPAR, prevents the interaction of urokinase with membrane-anchored uPAR on residual normal cells.

In vitro characterization of a monoclonal IgG(kappa) from a patient with planar xanthomatosis.

OBJECTIVE: To characterize a monoclonal IgG(kappa) (MAb) from a patient with planar xanthoma that precipitated with serum lipids at reduced temperature. METHODS: The molecular weight (Mr), sensitivity to proteases, and glycosylation of the purified MAb were analyzed. The specificity of the MAb was tested by measuring cryoprecipitation with pure high- (HDL) and low-density (LDL) lipoproteins. The effect of choline, phosphocholine, and glycerol 3-phosphate on the precipitation temperature of LDL by the MAb was studied. RESULTS: The MAb was larger than normal IgG due to hyperglycosylation of the MAb light chain. It formed cryoprecipitates with pure HDL and LDL as well as the lipids extracted from these lipoproteins. Fab fragments of the MAb lowered the temperature of its precipitation with LDL. Choline, phosphocholine, and glycerol 3-phosphate also lower the precipitation temperature. CONCLUSION: This is the first human IgG reported with apparent specificity for phosphocholine antigens. Its precipitation with lipids at reduced temperature suggests that it recognizes conformations of phospholipid head groups that develop below core body temperature.

Deep vein thrombosis of lower extremity: direct intraclot injection of alteplase once daily with systemic anticoagulation--results of pilot study.

PURPOSE: To prospectively evaluate the outcome of patients with acute deep vein thrombosis (DVT) of the lower extremity treated with "lacing" of the thrombus with alteplase (recombinant tissue plasminogen activator, or rTPA). MATERIALS AND METHODS: This HIPAA-compliant study was approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute and was funded by the National Institutes of Health. After giving written consent, 20 patients with first-onset acute DVT were treated with direct intraclot lacing of the thrombus with alteplase (maximum daily dose, 50 mg per leg per day; maximum of four treatments) and full systemic anticoagulation. Alteplase was chosen because its high fibrin affinity obviates continuous infusion of this thrombolytic agent. Ventilation-perfusion (V/Q) scans were performed for evaluation of embolic risks, and clinical and imaging examinations were supplemented with pharmacokinetic studies to enable further assessment of treatment outcomes. RESULTS: The 20 patients included 13 men and seven women aged 18-79 years. Antegrade blood flow was restored throughout the deep venous system in 16 patients (80%) during thrombolytic therapy, with complete resolution of symptoms in 18 patients (90%) after 6 months of anticoagulation. Pharmacokinetic studies showed rapid clearance of circulating alteplase and recovery of plasminogen activator inhibitor-1 levels within 2 hours after termination of alteplase treatment. V/Q scans revealed a 40% incidence of pulmonary embolism before treatment and a 15% incidence of asymptomatic pulmonary embolism during thrombolytic therapy. There were no cases of clinically important pulmonary embolism or serious bleeding during thrombolytic therapy. During a mean follow-up period of 3.4 years, no patient developed a postthrombotic syndrome or recurrent thromboembolism. CONCLUSION: Intraclot injection or lacing of the thrombus with a fibrin-binding thrombolytic agent such as alteplase is an alternative to continuous-infusion thrombolytic regimens and minimizes the duration of systemic exposure to thrombolytic agents.

Cardiovascular manifestations of hypereosinophilic syndromes.

The hypereosinophilic syndromes (HESs) are characterized by persistent marked eosinophilia (>1500 eosinophils/mm(3)), the absence of a primary cause of eosinophilia (such as parasitic or allergic disease), and evidence of eosinophil-mediated end organ damage. Cardiovascular complications of HES are a major source of morbidity and mortality in these disorders. The most characteristic cardiovascular abnormality in HES is endomyocardial fibrosis. Patients who have an HES also may develop thrombosis, particularly in the cardiac ventricles, but also occasionally in deep veins. Because of the rarity of these disorders, specific guidelines for the management of the cardiac and thrombotic complications of HES are lacking. This article reviews the diagnosis and management of the cardiovascular manifestations of HES.

The impact of major surgery on blood coagulation factors and thrombin generation.

We studied the blood coagulation system of 14 patients with metastatic malignancies before and after they had undergone major surgery. In addition to measuring a battery of coagulation factors, we assessed the function of the system with assays of whole blood thrombin generation. With the exceptions of factor VIII (fVIII), which increased, and fibrinogen and fIX, which did not change, the activities of all the pro- and anticoagulant proteins were significantly lower postoperatively. However, the thrombin generating capacity of the system was relatively preserved. Although the integral of thrombin activity over time was lower after surgery, the mean peak thrombin concentration was unchanged and the time to clot formation was shortened. Similar changes could be reproduced by lowering the concentrations of pro- and anticoagulant factors together in control blood samples. Therefore, simultaneous reductions in pro- and anticoagulant proteins postoperatively worked to maintain the functional integrity of the blood coagulation system.

PF4 ENHANCED assay for the diagnosis of heparin-induced thrombocytopenia in complex medical and surgical patients.

OBJECTIVE: To evaluate the sensitivity and specificity of the PF4 ENHANCED (GTI Diagnostics, Waukesha, WI) enzyme-linked immunosorbent assay for heparin-induced thrombocytopenia using the carbon-14 serotonin-release assay as the reference method. DESIGN: A total of 34 patients were prospectively enrolled with a variety of diagnoses and suspected heparin-induced thrombocytopenia. They were clinically scored and underwent testing with the (14)C-serotonin-release assay. Enzyme-linked immunosorbent assay and (14)C-serotonin-release assay results were also available from 21 medical and surgical patients who had previously been tested. MAIN RESULTS: With the (14)C-serotonin-release assay as the reference method, the sensitivity and specificity of the enzyme-linked immunosorbent assay were 93% and 65%, respectively. There was only one false-negative enzyme-linked immunosorbent assay. The clinical scores were frequently misleading, largely because of difficulty excluding other causes of thrombocytopenia. CONCLUSION: Because of its high sensitivity, we believe the PF4 ENHANCED enzyme-linked immunosorbent assay should be used to identify heparin-induced thrombocytopenia in patients with multiple potential causes of thrombocytopenia, although false-positive results will not be uncommon.

Pulsed-high intensity focused ultrasound enhanced tPA mediated thrombolysis in a novel in vivo clot model, a pilot study.

INTRODUCTION: Thrombotic disease continues to account for significant morbidity and mortality. Ultrasound energy has been investigated as a potential primary and adjunctive treatment for thrombotic disease. We have previously shown that pulsed-high intensity focused ultrasound (HIFU) enhances thrombolysis induced by tissue plasminogen activator (tPA) in vitro, including describing the non-destructive mechanism by which tPA availability and consequent activity are increased. In this study we aimed to determine if the same effects could be achieved in vivo. MATERIALS AND METHODS: In this study, pulsed-HIFU exposures combined with tPA boluses were compared to treatment with tPA alone, HIFU alone and control in a novel in vivo clot model. Clots were formed in the rabbit marginal ear vein and verified using venography and infrared imaging. The efficacy of thrombolytic treatment was monitored via high resolution ultrasonography for 5 h post-treatment. The cross-sectional area of clots at 4 points along the vein was measured and normalized to the pre-treatment size. RESULTS: At 5 h the complete recanalization of clots treated with pulsed-HIFU and tPA was significantly different from the partial recanalization seen with tPA treatment alone. tPA treatment alone showed a significant decrease in clot versus control, where HIFU was not significantly different than control. Histological analysis of the vessel walls in the treated veins showed no apparent irreversible damage to endothelial cells or extravascular tissue. CONCLUSIONS: This study demonstrates that tPA mediated thrombolysis can be significantly enhanced when combined with non-invasive pulsed-HIFU exposures.

Markedly increased vitamin B12 concentrations attributable to IgG-IgM-vitamin B12 immune complexes.

BACKGROUND: High serum vitamin B12 concentrations have been reported in patients with hepatic disease, disseminated neoplasia, myeloproliferative disorders, and hypereosinophilic syndromes. We recently discovered an extraordinarily increased vitamin B12 concentration in a patient without these underlying conditions. METHODS: Affinity and size-exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and ELISA methods were used to determine the cause of the increased vitamin B12 concentrations in this patient's serum. RESULTS: The protein G column eluates from 2 apparently healthy volunteers and 2 patients with recent vitamin B12 treatment for anemia had vitamin B12 concentrations of <74 pmol/L, whereas the vitamin B12 concentration in the protein G column eluate from the patient was 7380 pmol/L. The elution profile from size-exclusion chromatography of vitamin B12-binding proteins in the patient's serum revealed an abnormal vitamin-B12-binding protein. SDS-PAGE analysis of the concentrated eluates from the protein G column, under reducing conditions, revealed an additional band with an apparent molecular mass of 76 kDa, which was not present in control column eluates. MALDI-TOF MS identified this band as an IgM heavy chain. By use of a modified ELISA, we determined that the IgM present in the patient's eluates was associated with the IgG to form IgG-IgM immune complexes. CONCLUSIONS: This case demonstrates the unusual circumstance of a patient with markedly increased vitamin B12 concentrations attributed to immune complexes composed of IgG, IgM, and vitamin B12 and illustrates techniques that can be used to identify this occurrence.

Use of heparin versus lepirudin flushes to prevent withdrawal occlusion of central venous access devices.

STUDY OBJECTIVE: To determine whether lepirudin flushes are more effective than heparinized saline in preventing withdrawal occlusion of central venous access devices. DESIGN: Randomized, double-blind clinical trial. SETTING: Research institution-tertiary referral center. PATIENTS: Forty-nine adults undergoing bone marrow transplantation for hematologic malignancies or metastatic solid tumors. INTERVENTION: Twenty-four patients received heparin and 25 received lepirudin flushes. The heparin dose was 3 ml of porcine heparin 100 U/ml (300 U) per catheter lumen at least once/day; the lepirudin dose was 3 ml of lepirudin 100 microg/ml (300 microg) per catheter lumen at least once/day. After 3-4 weeks, all 49 patients received the heparin flushes. MEASUREMENTS AND MAIN RESULTS: Efficacy was assessed by the frequency with which the patients were treated with alteplase instillations for withdrawal occlusion of their central venous access devices during the first 4 months of catheterization. Three (12.5%) patients treated with heparin alone and five (20%) treated initially with lepirudin required alteplase instillations for an estimated relative risk with lepirudin versus heparin of 1.6 (95% confidence interval [CI] 0.40-13.86, p=0.70). CONCLUSION: Lepirudin was not more effective than heparin, which may have been related to the conservative dose of lepirudin administered. However, higher lepirudin doses are likely to incur an unacceptable risk of systemic anticoagulation.

Venlafaxine-induced ecchymoses and impaired platelet aggregation.

OBJECTIVE: To describe a case of venlafaxine-induced ecchymoses. METHODS: A patient with a history of ecchymoses coincident with venlafaxine therapy was rechallenged with the drug. Her platelet function was assessed with aggregation and ATP release studies before the rechallenge and after she developed ecchymoses. In addition, the effect of venlafaxine on platelet aggregation and ATP release was studied in vitro by adding the drug to platelet-rich plasma from normal donors. RESULTS: After 4 wk of treatment with venlafaxine our patient developed extensive ecchymoses. At that time her platelet aggregation and release responses to epinephrine, ADP, collagen, and arachidonic acid were markedly suppressed. Adding venlafaxine to normal platelet-rich plasma also dramatically reduced the aggregation and release responses to the same agonists as well as to serotonin, but the concentrations of venlafaxine required were 1000-fold greater than those normally achieved clinically. CONCLUSIONS: Our patient demonstrated an idiosyncratic hypersensitivity to the platelet inhibitory effects of venlafaxine. Because venlafaxine is an inhibitor of serotonin uptake by platelets and neurons, this mechanism may contribute to the impact of this drug on platelet function. However, our in vitro studies suggest that this hypothesis is inadequate to explain the observations completely.

Factor V Leiden as a common genetic risk factor for venous thromboembolism.

PURPOSE: To increase nurses' knowledge of the Factor V Leiden (FVL) genetic trait for venous thromboembolism. ORGANIZING FRAMEWORK: An overview of the history, prevalence, and predisposition of the FVL genetic mutation, including who should be tested and how and in what circumstances people with FVL should be treated. FINDINGS: FVL is the most commonly recognized genetic trait associated with venous thrombosis. It is found predominantly in Caucasian populations. Biochemically it causes "activated protein C resistance (APCR)." The decision to test for FVL depends on whether the information gained will potentially improve the health care of the person or family. For people who have had deep venous thrombosis, testing for FVL will likely not alter treatment approaches. Currently the advantage for testing is primarily limited to asymptomatic family members who carry FVL and who have had deep vein thrombosis. Close relatives who also carry the mutated gene might benefit from prophylactic anticoagulation when their risk of thrombosis is increased by temporary factors such as surgery. CONCLUSIONS: Nurses are in a unique position to provide accurate information and counseling when patients and their family members are presented with the results of thrombophilia testing.

The effect of red blood cells on thrombin generation.

We have developed an assay of thrombin generation in clotting whole blood. Because our goal was to study patients with red blood cell diseases that are associated with thrombosis, the initial evaluation of this assay analysed the effect of the haematocrit and haemolysate on the total amount of thrombin activity generated and the maximal concentration of thrombin achieved. Both of these parameters were proportional to the haematocrit throughout a wide range of clinically relevant cell concentrations. Red cell lysate also augmented thrombin generation. Because this effect was removed by filtration of the lysate, we propose that it was related to red cell microparticles.

Pulsed high-intensity focused ultrasound enhances thrombolysis in an in vitro model.

PURPOSE: To evaluate the use of pulsed high-intensity focused ultrasound exposures to improve tissue plasminogen activator (tPA)-mediated thrombolysis in an in vitro model. MATERIALS AND METHODS: All experimental work was compliant with institutional guidelines and HIPAA. Clots were formed by placing 1 mL of human blood in closed-off sections of pediatric Penrose tubes. Four experimental groups were evaluated: control (nontreated) clots, clots treated with pulsed high-intensity focused ultrasound only, clots treated with tPA only, and clots treated with pulsed high-intensity focused ultrasound plus tPA. The focused ultrasound exposures (real or sham) were followed by incubations of the clots in tPA with saline or in saline only. Thrombolysis was measured as the relative reduction in the mass of the clot. D-Dimer assays also were performed. Two additional experiments were performed and yielded dose-response curves for two exposure parameters: number of pulses per raster point and total acoustic power. Radiation force-induced displacements caused by focused ultrasound exposures were simulated in the clots. A Tukey-Kramer honestly significant difference test was performed for comparisons between all pairs of experimental groups. RESULTS: The clots treated with focused ultrasound alone did not show significant increases in thrombolysis compared with the control clots. The clots treated with focused ultrasound plus tPA showed a 50% ([30.2/20.1]/20.1) increase in the degree of thrombolysis compared with the clots treated with tPA only (P < .001), further corroborating the d-dimer assay results (P < .001). Additional experiments revealed how increasing both the number of pulses per raster point and the total acoustic power yielded corresponding increases in the thrombolysis rate. In the latter experiment, simulations performed at a range of power settings revealed a direct correlation between increased displacement and observed thrombolysis rate. CONCLUSION: The rate of tPA-mediated thrombolysis can be enhanced by using pulsed high-intensity focused ultrasound exposure in vitro.