Darlene J. Elias

Subcutaneous Enoxaparin Once or Twice Daily Compared with Intravenous Unfractionated Heparin for Treatment of Venous Thromboembolic Disease

Venous thromboembolic disease causes significant morbidity and mortality in both hospitalized and nonhospitalized patients. The mean annual incidence in the United States is 48 per 100 000 for deep venous thrombosis and 23 per 100 000 for pulmonary embolism, according to an epidemiologic study conducted in Massachusetts (1). A similar study in Sweden showed an annual incidence of 160 new cases of deep venous thrombosis per 100 000 inhabitants (2). Five to 10 days of unfractionated heparin is a common recommended initial treatment for deep venous thrombosis. This treatment maintains the activated partial thromboplastin time above 1.5 times its control value (3, 4), as calibrated by protamine titration or an antifactor Xa assay. Another recommended initial treatment is 5 to 10 days of weight-adjusted low-molecular-weight heparin followed by at least 3 months of oral anticoagulant therapy (3-7). Low-molecular-weight heparins are now frequently being used in place of unfractionated heparin for both prevention and treatment of venous thromboembolism (3, 8). Randomized trials and meta-analyses have shown subcutaneously administered low-molecular-weight heparins to have antithrombotic efficacy equal to (9-12) or greater than (13-16) that of continuously infused unfractionated heparin in the initial treatment of deep venous thrombosis and equal to that of unfractionated heparin in the treatment of pulmonary embolism (17, 18). However, many of these studies enrolled small numbers of patients (9-13, 15, 16), used primarily venographic plethysmographic or scintigraphic end points (9-11, 13, 16), and sometimes excluded patients with pulmonary embolism (11, 15). Most trials of twice-daily low-molecular-weight heparin adjusted treatment regimens according to patient weight without laboratory monitoring. However, several studies suggest that once-daily weight-adjusted dosage of a low-molecular-weight heparin is as effective in the treatment of proximal deep venous thrombosis as adjusted dosages of intravenous unfractionated heparin (14, 19) or twice-daily low-molecular-weight heparin (20). Since low-molecular-weight heparins differ in their physicochemical and pharmacologic characteristics, study results that apply to one cannot be extended to another (21, 22). We conducted the present study to determine whether enoxaparin administered subcutaneously once or twice per day is as effective as continuously infused unfractionated heparin in the treatment of patients with acute, symptomatic venous thromboembolic disease. Methods Study Description This parallel-group, randomized, partially blinded, international, multicenter clinical trial compared continuously infused unfractionated heparin (adjusted to maintain activated partial thromboplastin time within a defined range) with two weight-adjusted dosages of enoxaparin administered subcutaneously once or twice daily. The study was conducted in 74 hospitals in 16 countries, including the United States, several European countries, Australia, and Israel, and was approved by the institutional review board or ethics committees at each location. Written informed consent was obtained from each patient. Four committees participated in this study: an Advisory Committee; an Outcome Adjudication Committee, which provided blinded outcome assignments for incidence of recurrent venous thromboembolic disease, major or minor hemorrhage, immune thrombocytopenia, and cause of death; an independent Safety Committee; and a Vascular Imaging Committee, which reviewed all baseline venograms and all vascular imaging studies in a blinded manner to determine whether deep venous thrombosis was present at baseline and whether objective evidence of recurrence existed. Patient Characteristics Patients were required to be at least 18 years of age and willing to remain hospitalized during randomized therapy. The primary inclusion criteria were symptomatic lower-extremity deep venous thrombosis confirmed by venography or ultrasonography (if venography was inconclusive), symptomatic pulmonary embolism confirmed by high-probability ventilationperfusion scanning, or positive pulmonary angiography with confirmation of lower-extremity deep venous thrombosis. All eligible patients underwent baseline lung scanning or angiography. Exclusion criteria were more than 24 hours of previous treatment with heparin or warfarin; need for thrombolytic therapy; known hemorrhagic risk, including active hemorrhage, active intestinal ulcerative disease, known angiodysplasia, or eye, spinal, or central nervous system surgery within the previous month; renal insufficiency (serum creatinine concentration>180 mol/L [2.03 mg/dL]); severe hepatic insufficiency; allergy to heparin, protamine, porcine products (both heparin and enoxaparin are derived from pork intestinal mucosa), iodine, or contrast media; history of heparin-associated thrombocytopenia or heparin- or warfarin-associated skin necrosis; treatment with other investigational therapeutic agents within the previous 4 weeks; inferior vena cava interruption; or known pregnancy or lactation. Treatments Within each center, consecutive eligible patients were randomly assigned sequentially to one of three treatment groups. Randomization was done without stratification in blocks of six, according to ascending randomization number. The numbers were affixed to sealed treatment kits that contained study medication and were provided by the study sponsor. Patients assigned to enoxaparin received a weight-adjusted subcutaneous dose. Two blinded regimens were tested: 1.0 mg/kg of body weight twice daily or 1.5 mg/kg once daily. Several clinical trials have shown the twice-daily regimen to be effective and safe (16, 23, 24). The once-daily dosage was chosen on the basis of results of pharmacokinetic studies that showed it to have a suitable pharmacokinetic profile in healthy volunteers and to be well tolerated in the treatment of patients with venous thromboembolism (25, 26). In these previous studies, therapeutic antifactor Xa levels were present for up to 18 hours in both volunteers and patients, and measurable levels were present for up to 24 hours. A total of three injections, study drug and placebo, were given each day to maintain blinding for volume of solutions and frequency of administration. Patients assigned to the nonblinded unfractionated heparin group received an intravenous bolus dose and infusion on the basis of an approved institution-specific nomogram. In most cases, administration was as follows: Six hours after the initial bolus, the activated partial thromboplastin time was measured and the dose was adjusted to maintain the specified value, which was between 55 and 80 seconds in most centers (4-7). Activated partial thromboplastin time was measured at least daily during unfractionated heparin treatment. Enoxaparin and heparin treatments were continued for at least 5 days, and warfarin was started within 72 hours of initial study drug administration. Forty-three patients received phenprocoumon in place of warfarin sodium. Prothrombin time was measured daily, and patients could be discharged from the hospital after the international normalized ratio was found to be between 2.0 and 3.0 on 2 consecutive days. Oral anticoagulation was continued for at least 3 months. Study Assessments Observers who were aware of treatment assignment assessed patients daily and monthly during the 3-month follow-up for worsening or recurrence of deep venous thrombosis or pulmonary embolism, hemorrhage, adverse events, changes in concomitant medications and adequacy of warfarin use, and warfarin adherence. For patients receiving unfractionated heparin, adherence was defined as an activated partial thromboplastin time within or above the therapeutic range on the second day of treatment. For patients receiving enoxaparin, adherence was defined as at least 10 doses of study medication given with no dosing errors. Adherence to warfarin therapy was defined as having at least one international normalized ratio value greater than or equal to 2.0 between day 4 and the last dose of study treatment during the initial treatment period. These definitions of treatment adherence were established before the analysis of the study outcomes. Efficacy Analysis The efficacy analysis was performed on two study samples: all treated patients, who received at least one dose of study medication, and evaluable patients, which excluded all patients who met at least one of the criteria for nonevaluability. These criteria were no confirmed deep venous thrombosis at baseline, insufficient study therapy, placement of an inferior vena cava filter, two random assignments, and no 3-month follow-up. Insufficient study therapy was defined as one or more missed enoxaparin doses among at least eight consecutive enoxaparin doses or less than 4 consecutive days of heparin infusion. The definition of insufficient study therapy was established before analysis of study outcomes. These two study samples were analyzed to strengthen the conclusion of equivalence among the treatment groups. The homogeneity of the results of the two analyses is considered to be more supportive of the conclusion of equivalence than the results of either analysis alone. Primary clinical end points were recurrent deep venous thrombosis or pulmonary embolism within 3 months of randomization. Patients with symptoms of recurrent thrombosis underwent confirmatory testing with venography, ultrasonography, or both. Patients presenting with signs or symptoms of pulmonary embolism underwent lung perfusion scanning, pulmonary angiography, or both. Clinical symptoms and supportive findings on objective tests; extension of existing thrombi or new thrombi for venography, angiography, or ultrasonography; or high-probability defect patterns on perfusion scans were required to confirm recurrent thrombosis. Prespecified subgroup analyses were performed on the basis of patient demog

High-Density Lipoprotein Deficiency and Dyslipoproteinemia Associated With Venous Thrombosis in Men

Background—Although dyslipoproteinemia is associated with arterial atherothrombosis, little is known about plasma lipoproteins in venous thrombosis patients. Methods and Results—We determined plasma lipoprotein subclass concentrations using nuclear magnetic resonance spectroscopy and antigenic levels of apolipoproteins AI and B in blood samples from 49 male venous thrombosis patients and matched controls aged <55 years. Venous thrombosis patients had significantly lower levels of HDL particles, large HDL particles, HDL cholesterol, and apolipoprotein AI and significantly higher levels of LDL particles and small LDL particles. The quartile-based odds ratios for decreased HDL particle and apolipoprotein AI levels in patients compared with controls were 6.5 and 6.0 (95% CI, 2.3 to 19 and 2.1 to 17), respectively. Odds ratios for apolipoprotein B/apolipoprotein AI ratio and LDL cholesterol/HDL cholesterol ratio were 6.3 and 2.7 (95% CI, 1.9 to 21 and 1.1 to 6.5), respectively. When polymorphisms in genes for hepatic lipase, endothelial lipase, and cholesteryl ester transfer protein were analyzed, patients differed significantly from controls in the allelic frequency for the TaqI B1/B2 polymorphism in cholesteryl ester transfer protein, consistent with the observed pattern of lower HDL and higher LDL. Conclusions—Venous thrombosis in men aged <55 years old is associated with dyslipoproteinemia involving lower levels of HDL particles, elevated levels of small LDL particles, and an elevated ratio of apolipoprotein B/apolipoprotein AI. This dyslipoproteinemia seems associated with a related cholesteryl ester transfer protein genotype difference.

Pulmonary Hypertension Developing after Alglucerase Therapy in Two Patients with Type 1 Gaucher Disease Complicated by the Hepatopulmonary Syndrome

Gaucher disease is an autosomal-recessive disorder that is caused by a deficiency of the enzyme glucocerebrosidase and results in accumulation of an insoluble glucocerebroside in macrophages. Type 1 (chronic non-neuropathic or adult) Gaucher disease is usually characterized by hepatosplenomegaly, thrombocytopenia, and skeletal complications (including bone pain, osteonecrosis, and pathologic fractures) [1, 2]. With the exception of splenectomy, few options were available for treating this disorder until enzyme replacement therapy with alglucerase became available [3]. Pulmonary involvement has long been recognized as a rare complication of Gaucher disease. Pulmonary hypertension, severe hypoxemia, and infiltration of the lungs with Gaucher cells have been described in case reports and small case series. We report the results of therapy with alglucerase in two patients with severe hypoxemia and intrapulmonary vascular dilatations that appeared on agitated saline contrast echocardiography [4]. Case Reports Patient 1 A previous article [3] and two case series [2, 5] described patient 1. We first evaluated this patient in 1984 when she was 23 years of age. Progressive dyspnea began when the patient was about 20 years of age; by 1990, she would become breathless after walking a few steps. The patient was small (height, 159 cm; weight, 40 kg) and deeply cyanotic with clubbing of the fingers and a large liver. Bronchoalveolar lavage fluid and transbronchial lung biopsy specimens showed no Gaucher cells. Results of studies of lung function are shown in Table 1. Table 1. Pulmonary Function, Liver Volume, and Pulmonary Hemodynamics Therapy with alglucerase began in January 1990. The patient initially received alglucerase, 30 U/kg of body weight per month at 3.3 U/kg twice weekly. After 7 months, the dose was reduced to 2.3 U/kg twice weekly. After 33 months, the patient no longer needed administration of oxygen and was working full-time, but chest radiography showed enlarged pulmonary arteries (Figure 1, left and middle). Because the patient had no tricuspid regurgitation, we could not estimate pulmonary artery pressure from the echocardiogram [7]. After 6 years of therapy, no further progression of pulmonary hypertension has been seen on physical examination or chest radiography. Figure 1. Left. Middle. arrows Right. Patient 2 Patient 2 was 41 years of age at his first evaluation. He had had splenectomy at 13 years of age but remained otherwise asymptomatic for Gaucher disease until 38 years of age, at which time vertebral osteomyelitis, hypoxemia, and a cerebral abscess developed. Thoracotomy with ligation of a suspected arteriovenous malformation did not alleviate hypoxemia. Examination of open lung biopsy specimens showed no Gaucher cells in the lungs. However, medial hypertrophy of small muscular arteries and arterioles was seen (Figure 1, right), and some large pulmonary arteries showed intimal proliferation of the type occurring in the early stages of pulmonary hypertension. Patient 2 was thin and muscular (height, 178 cm; weight, 68.5 kg) and had moderate finger clubbing and a markedly enlarged liver. Results of studies of lung function are shown in Table 1. Therapy with alglucerase (120 U/kg per month for 7 months as part of a dose-ranging protocol, followed by 30 U/kg per month) was started in September 1991. After 20 months of treatment (total dose, 1230 U/kg), the patient had no symptoms and was following a vigorous exercise program. His chest radiograph showed an increase in the size of the main pulmonary artery, and progressive pulmonary hypertension was seen after catheterization of the right side of the heart. Discussion We describe two patients with type 1 Gaucher disease who presented with hepatomegaly, severe hypoxemia, and intrapulmonary vascular dilatations. Therapy with alglucerase decreased the size of the liver and almost completely eliminated the hypoxemia. However, the patients then developed pulmonary hypertension. Until recently, pulmonary involvement has been considered to be a rare complication of Gaucher disease. When lung tissue is available, examination of the tissue frequently shows that the lungs are infiltrated with Gaucher cells [8, 9]. Pulmonary hypertension has occasionally been reported with Gaucher disease; in some cases, the pulmonary capillaries were obstructed by Gaucher cells [9, 10]. In one patient, however, few Gaucher cells were found in the lungs, and the pathologic findings resembled those of primary pulmonary hypertension [11]. More recent studies have shown pulmonary involvement in Gaucher disease. In a large autopsy series [12] that included patients with type 2 and type 3 Gaucher disease, more than one third of the cases had interstitial infiltration, plugging of the capillaries, or filling of the air spaces with Gaucher cells. Kerem and colleagues [13] found that 68% of patients in a clinic for Gaucher disease had some abnormality of lung function. Our patients had no evidence of parenchymal lung infiltration with Gaucher cells. No Gaucher cells were seen in patient 1 on examinations of transbronchial biopsy specimens of the lung or bronchoalveolar lavage fluid. Patient 2 had an open lung biopsy, and no Gaucher cells were found on examination of the specimens. No lung infiltrates were seen on the chest radiograph of either patient. The hypoxemia thus seemed to be caused by intrapulmonary vascular dilatations. The hepatopulmonary syndrome is defined as the triad of liver disease, hypoxemia, and intrapulmonary vascular dilatations [14]. Our patients had little evidence of hepatocellular dysfunction, but both had a very enlarged liver. Improvement in the hypoxemia and disappearance of the intrapulmonary vascular dilatations were presumably related to the decreased liver size seen with treatment. Severe hypoxemia has been reversed after successful liver transplantation and with medical therapy; in some cases, intrapulmonary vascular dilatations are reversible if the underlying liver disease is treated [15]. Pelini and colleagues [16] described a patient with type 1 Gaucher disease and severe hypoxemia that showed remarkable improvement after therapy with alglucerase. Although this patient may have had the hepatopulmonary syndrome, Pelini and colleagues did not test for intrapulmonary vascular dilatations. The abnormalities in the patients lung function and the time taken to respond to treatment were similar to those of our patients. It is difficult to account for the progression of pulmonary hypertension in our patients, whose response to alglucerase was otherwise favorable. Pulmonary hypertension does not seem to be a feature of the hepatopulmonary syndrome. In fact, patients with severe hypoxemia and liver disease usually have low pulmonary artery pressure and pulmonary vascular resistance [17] that tend to increase after such patients receive liver transplants [18]. Patient 2 developed pulmonary hypertension when his intrapulmonary vascular dilatation decreased. Patient 1 had moderate pulmonary hypertension after treatment, but evidence that her pulmonary artery pressure increased after therapy is somewhat unconvincing. The size of the main pulmonary artery in patient 1 definitely increased, but only one catheterization of the right side of the heart was done for confirmation. The increase in pulmonary artery pressure may be partly explained by diminished dilatation of intrapulmonary vessels in a vascular bed that was already abnormally restricted. This explanation was suggested by the appearance of the small vessels in the lung biopsy specimens of patient 2 and by the low diffusing capacity that remained diminished after therapy in both patients. Decreased intrapulmonary vascular dilatation in patient 2 cannot be the only cause of pulmonary hypertension because his pulmonary artery pressure continued to increase after hypoxemia resolved. Progressive infiltration of the lung with Gaucher cells, leading to obstruction of small pulmonary arteries, also does not seem to explain the worsening pulmonary hypertension because the severe hepatomegaly improved remarkably after alglucerase therapy. Treatment of severe hypoxemia may have allowed patient 2 to survive long enough for progressive changes in the pulmonary vascular bed to become clinically evident. We also cannot exclude the possibility that alglucerase therapy aggravates pulmonary hypertension despite its beneficial effect on other manifestations of Gaucher disease. We cannot explain the reappearance of intrapulmonary vascular dilatation in patient 2, but the dilatation must have been relatively minor because the arterial oxygen tension remained normal. Alglucerase saved the lives of these two patients and allowed them to return to a normal lifestyle. The increase in pulmonary artery pressure seen in these patients was not expected. Our first suspicion of increasing pressure was based on changes in the physical examination findings and the chest radiograph; therefore, screening does not have to be expensive. For these reasons, we believe that patients treated for Gaucher disease should be carefully examined for signs of pulmonary hypertension. Dr. Garver: Department of Radiology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, CA 92037. Dr. Kay: Sandoz Pharmaceutical Corporation, Building 419, Room 2111, 59 Route 10, East Hanover, NJ 07936. Dr. Bloor: Department of Pathology (8320), University of California at San Diego Medical Center, 225 Dickinson Street, San Diego, CA 92103. Dr. Beutler: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037.

T cell receptor biases and clonal proliferations among lung transplant recipients with obliterative bronchiolitis.

Obliterative bronchiolitis (OB) is the most serious late complication of lung transplantation, but the pathogenesis of this disorder has not been elucidated. We sought evidence that OB is mediated by a cellular immunologic response by characterizing T cell antigen receptor beta-chain variable gene (TCRBV) repertoires in lung allograft recipients. Expression levels of 27 TCRBV among recipients were determined by multiprobe RNase protection assay after PCR amplification. In comparison to recipients with no evidence of rejection (n = 9), the PBL TCRBV repertoires of OB subjects (n = 16) exhibited more frequent expansions (16 vs. 9% of all measured TCRBV, P < 0.02), and the magnitudes of these abnormalities were greater (8.2 +/- 0.8 vs. 4.5 +/- 0.3 SD from mean normal values, P < 0.01). TCRBV sequencing showed these expansions were composed of clonal or oligoclonal populations. Thus, T cell responses in the recipients are marked by highly selective clonal expansions, presumably driven by indirect recognition of a limited number of immunodominant alloantigens. These processes are exaggerated among allograft recipients with OB, implying that cognate immune mechanisms are important in the pathogenesis of the disorder. Furthermore, the prominence of finite, distinct TCR phenotypes raise possibilities for development of novel diagnostic modalities and targeted immunotherapies for OB and other manifestations of chronic allograft rejection.

Prevalence and Risk Factors for Hypertension in Hemophilia

Hypertension (HTN) is a major risk factor for intracranial hemorrhage. We, therefore, investigated the prevalence, treatment, and control of HTN in adult patients with hemophilia (PWH). PWH≥18 years (n=458) from 3 geographically different cohorts in the United States were evaluated retrospectively for HTN and risk factors. Results were compared with the nationally representative sample provided by the contemporary National Health and Nutrition Examination Survey (NHANES). PWH had a significantly higher prevalence of HTN compared with NHANES. Overall, the prevalence of HTN was 49.1% in PWH compared with 31.7% in NHANES. At ages 18 to 44, 45 to 64, 65 to 74, and ≥75 years, the prevalence of HTN for PWH was 31.8%, 72.6%, 89.7%, and 100.0% compared with 12.5%, 41.2%, 64.1%, and 71.7% in NHANES, respectively. Of treated hypertensive PWH, only 27.1% were controlled, compared with 47.7% in NHANES (all P<0.05). Age, body mass index, diabetes mellitus, and renal function were independently associated with HTN. Among patients with moderate or severe hemophilia there was a trend (≈1.5-fold) for higher odds of having HTN compared with patients with mild hemophilia. On the basis of these results, new care models for adult PWH and further studies for the causes of HTN in hemophilia are recommended.

Failure to validate association of gene polymorphisms in EPCR, PAR-1, FSAP and protein S Tokushima with venous thromboembolism among Californians of European ancestry

Failure to validate association of gene polymorphisms in EPCR, PAR-1, FSAP and protein S Tokushima with venous thromboembolism among Californians of European ancestry -

T-Cell Receptor Biases and Clonal Proliferations in Blood and Pleural Effusions of Patients with Lung Cancer

We sought evidence that pulmonary carcinomas mediate a cellular immunologic response by analyzing T-cell antigen receptor beta-chain variable gene (TCRBV) repertoires of lymphocytes from peripheral blood (PBL) and malignant pleural effusions (PEL) of five lung cancer patients. Expression levels of 27 TCRBV were quantitated by multiprobe RNase protection assay (RPA), and clonal expansions were identified by sequence enrichment nuclease assay (SENA) and junctional region sequencing. Abnormal TCRBV expansions were identified in all subjects by RPA (mean 6.9 +/- 1.7/patient), and their number closely correlated with elapsed time since initial diagnosis (r = 0.97). SENA, performed in specimens from three patients, confirmed the presence of mono or oligoclonality in 48% of abnormal RPA expansions, and further identified T-cell clones among TCRBV with normal expression levels. The majority of clonal expansions were among PEL, and were nearly equally divided between CD4 and CD8. These data show that T-cell repertoires of lung cancer patients are characterized by marked abnormalities and frequent clonal expansions, most likely representing responses to unique, tumor-specific antigens (TSA). Moreover, this process appears exaggerated among PEL, further suggesting that malignant effusions include local proliferations of tumor reactive T cells. These findings imply the presence of lung cancer TSA capable of eliciting cellular immune responses and raise the possibility that selective immunotherapies can ultimately be developed.

Arteriovenous Blood Metabolomics: A Readout of Intra-Tissue Metabostasis

The human circulatory system consists of arterial blood that delivers nutrients to tissues, and venous blood that removes the metabolic by-products. Although it is well established that arterial blood generally has higher concentrations of glucose and oxygen relative to venous blood, a comprehensive biochemical characterization of arteriovenous differences has not yet been reported. Here we apply cutting-edge, mass spectrometry-based metabolomic technologies to provide a global characterization of metabolites that vary in concentration between the arterial and venous blood of human patients. Global profiling of paired arterial and venous plasma from 20 healthy individuals, followed up by targeted analysis made it possible to measure subtle (<2 fold), yet highly statistically significant and physiologically important differences in water soluble human plasma metabolome. While we detected changes in lactic acid, alanine, glutamine, and glutamate as expected from skeletal muscle activity, a number of unanticipated metabolites were also determined to be significantly altered including Krebs cycle intermediates, amino acids that have not been previously implicated in transport, and a few oxidized fatty acids. This study provides the most comprehensive assessment of metabolic changes in the blood during circulation to date and suggests that such profiling approach may offer new insights into organ homeostasis and organ specific pathology.

Warfarin Pharmacogenomics: A big step forward for individualized medicine: enlightened dosing of warfarin

Warfarin Pharmacogenomics: A big step forward for individualized medicine: enlightened dosing of warfarin

Engineered virus-like nanoparticles reverse heparin anticoagulation more consistently than protamine in plasma from heparin-treated patients

Heparin is widely used for anticoagulation, often requiring the subsequent administration of a reversal agent. The only approved reversal agent for heparin is protamine sulfate, which induces well described adverse reactions in patients. Previously we reported a novel class of heparin antagonists based on the bacteriophage Qβ platform, displaying polyvalent cationic motifs which bind with high affinity to heparin. Here we report heparin reversal by the most effective of these virus-like particles (VLP) in samples from patients who were administered heparin during cardiac procedures or therapeutically for treatment of various thrombotic conditions. The VLP consistently reversed heparin in these samples, including those from patients that received high doses of heparin, with greater efficiency than a negative control VLP and with significantly less variability than protamine sulfate. These results provide the first step towards validation of heparin antagonist VLPs as viable alternatives to protamine.