Biomedical subjects
John L Francis
Publications and source records attributed to John L Francis.
Striking back at heparin-induced thrombocytopenia.
This immune reaction to a common anticoagulant can have devastating consequences. Learn how to recognize who's at risk and how to intervene if trouble hits.
Anti-metastatic effect of a non-anticoagulant low-molecular-weight heparin versus the standard low-molecular-weight heparin, enoxaparin.
Low-molecular-weight heparins (LMWH) exhibit potent anticoagulant efficacy via their plasmatic effects on thrombin and factor Xa. These agents are also effective in releasing endothelial tissue factor pathway inhibitor (TFPI), the natural inhibitor of tissue factor, and exhibit significant anti-metastatic effects in experimental animal models. However, the potential for bleeding complications has slowed down the more widespread adoption of LMWH therapy in cancer patients. In this study, the effect of a non-anticoagulant form of LMWH (NA-LMWH) on experimental lung metastasis and tumor cell-induced platelet aggregation in vivo was compared to the LMWH enoxaparin. Using the B16 melanoma mouse model of metastasis, subcutaneous (s.c.) injection of NA-LMWH or enoxaparin (10 mg/kg), three hours before intravenous (i.v.) injection of metastatic melanoma cells, followed by daily doses for 14 days, reduced lung tumor formation by 70% (P < 0.001). I.v. injection of tumor cells resulted in a significant (50-62%, P < 0.01) fall in platelet counts. Pre-injection (i.v.) of enoxaparin completely abolished the tumor cell-induced thrombocytopenia, whereas NA-LMWH had no effect. Four hours after a single s.c. dose, enoxaparin but not NA-LMWH prolonged the clotting time three-fold and delayed the time to clot initiation more than 10-fold as measured by a Sonoclot analyzer and by thromboelastography, respectively. Enoxaparin but not NA-LMWH demonstrated a significant anticoagulant effect in mice. Both NA-LMWH and enoxaparin caused similar TFPI release from endothelial cells in vitro. These data provide evidence to support the potential of NA-LMWH as an anti-metastatic agent without any significant impact on coagulation.
In vitro and in vivo characterization of a novel antibody-like single-chain TCR human IgG1 fusion protein.
We have constructed a protein composed of a soluble single-chain TCR genetically linked to the constant domain of an IgG1 H chain. The Ag recognition portion of the protein binds to an unmutated peptide derived from human p53 (aa 264-272) presented in the context of HLA-A2.1, whereas the IgG1 H chain provides effector functions. The protein is capable of forming dimers, specifically staining tumor cells and promoting target and effector cell conjugation. The protein also has potent antitumor effects in an in vivo tumor model and can mediate cell killing by Ab-dependent cellular cytotoxicity. Therefore, single-chain TCRs linked to IgG1 H chains behave like Abs but possess the ability to recognize Ags derived from intracellular targets. These fusion proteins represent a novel group of immunotherapeutics that have the potential to expand the range of tumors available for targeted therapies beyond those currently addressed by the conventional Ab-based approach.
Detection and significance of heparin-platelet factor 4 antibodies.
Heparin-induced thrombocytopenia (HIT) represents an immune reaction to heparin, which if unrecognized and untreated, has the potential for catastrophic thrombotic events. The demonstration of antibodies directed against the heparin-platelet factor 4 (H-PF4) complex is an important component of the diagnosis of HIT. Typically, test results are not rapidly available, and the diagnosis therefore rests principally on clinical grounds. Nevertheless, the presence of H-PF4 antibodies provides important confirmation of the diagnosis, and may predict for adverse cardiac events even in the absence of HIT. Although more research is certainly required, the use of alternative (non-heparin) anticoagulants for cardiac surgery and other cardiovascular procedures would substantially reduce the incidence of HIT and could possibly improve patient outcomes.
Striking back at heparin-induced thrombocytopenia.
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Effect of argatroban on the activated partial thromboplastin time: a comparison of 21 commercial reagents.
Argatroban is a direct thrombin inhibitor used for the treatment of heparin-induced thrombocytopenia. The drug is administered by continuous infusion, at a recommended initial dose of 2 microg/kg per min, to achieve activated partial thromboplastin times (aPTTs) 1.5-3.0 times baseline. We evaluated the effect of argatroban, at clinically relevant concentrations, on aPTTs using 21 commercially available reagents. The aPTTs of plasma containing argatroban at 0.125-8.0 microg/ml (final concentration) were assessed using each reagent and an ACL 3000+ coagulation analyzer. Argatroban increased aPTTs (and aPTT ratios relative to control) in a broadly comparable fashion among reagents. Concentration-aPTT ratio profiles linearized well using logarithmic-logarithmic transformation (r > 0.98), with the regression slope taken as the reagent's sensitivity to argatroban. Sensitivity ranged from 0.304 +/- 0.006 to 0.364 +/- 0.007. Only the least and two most sensitive reagents (all now unavailable in the United States) differed significantly in sensitivity from the other reagents (P < 0.05). aPTT ratios of 2.25 occurred for all reagents at 0.41-0.92 mug/ml argatroban, and for 14 (67%) reagents at 0.53-0.67 microg/ml. This corresponds to a approximately 0.5 microg/kg per min dose difference in healthy subjects. We conclude that most aPTT reagents are similarly sensitive to argatroban, and reagent choice is unlikely to significantly affect argatroban monitoring in patients with heparin-induced thrombocytopenia.
The role of CD40 in CD40L- and antibody-mediated platelet activation.
Our initial finding that CD40- and CD40 ligand (CD40L)-deficient mice displayed prolonged tail bleeding and platelet function analyzer (PFA-100) closure times prompted us to further investigate the role of the CD40-CD40L dyad in primary hemostasis and platelet function. Recombinant human soluble CD40L (rhsCD40L), chemical cross-linking of which suggested a trimeric structure of the protein in solution, activated platelets in a CD40-dependent manner as evidenced by increased CD62P expression. CD40 monoclonal antibody (mAb) M3, which completely blocked rhsCD40L-induced platelet activation, also prolonged PFA-100 closure times of normal human blood. In contrast, CD40 mAb G28-5 showed less potential in blocking rhsCD40L-induced CD62P expression and did not affect PFA-100 closure times. However, when added to the platelets after rhsCD40L, G28-5 significantly enhanced the platelet response by causing clustering of, and signaling through, FcgammaRII. Similarly, higher order multimeric immune complexes formed at a 1/3 molar ratio of M90, a CD40L mAb, to rhsCD40L induced strong Fcgamma RII-mediated platelet activation when translocated to the platelet surface in a CD40-dependent manner, including the induction of morphological shape changes, fibrinogen binding, platelet aggregation, dense granule release, microparticle generation and monocyte-platelet-conjugate formation. The results suggest that CD40 may play a role in primary hemostasis and platelet biology by two independent mechanisms: First, by functioning as a primary signaling receptor for CD40L and, second, by serving as a docking molecule for CD40L immune complexes. The latter would also provide a potential mechanistic explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in recent human and animal studies.
Heparin-induced thrombocytopenia in the emergency department.
We describe 3 patients who presented to the emergency department (ED) with stroke, deep venous thrombosis, or pulmonary embolism and renal failure after undergoing cardiac surgery 7 to 17 days earlier. Their onset of thrombosis after previous heparin exposure was temporally plausible for complications of heparin-induced thrombocytopenia, an immune-mediated thrombotic disorder triggered by heparin. The patients had normal platelet counts at presentation, yet each had circulating heparin-induced thrombocytopenia antibodies that were ultimately confirmed. Two patients had heparin reexposure in the ED, 1 of whom developed thrombocytopenia with new thrombosis and died. Alternative parenteral anticoagulation prevented further thrombosis in 2 patients. Because heparin use can be catastrophic in patients with heparin-induced thrombocytopenia, physicians should be vigilant in suspecting heparin-induced thrombocytopenia in patients with thrombosis after recent hospitalization or heparin exposure. Alternative anticoagulants are available for these at-risk patients.
A critical evaluation of assays for detecting antibodies to the heparin-PF4 complex.
Heparin-induced thrombocytopenia (HIT) is a potentially catastrophic complication of heparin therapy. The syndrome is the result of the production of an antibody to the complex that forms between heparin and platelet factor 4 (H-PF4) released from activated platelets. IgG antibodies bind to platelet Fc receptors and cause platelet activation, resulting in thrombocytopenia and greatly increased risk of thrombosis. Tests for the H-PF4 antibody can be classified into functional assays (which rely on the demonstration of platelet activation) and immunoassays (which detect the presence of an antibody without regard for its functional ability). The functional assays have a greater specificity for clinical HIT, but require normal donor platelets and are relatively un-standardized. The immunoassays have the advantage of better standardization and do not require normal platelets, but may give positive results in the absence of clinical HIT. The choice of test is usually dictated by what is possible for a given laboratory in terms of instrumentation, expertise, and interest. For most institutions this will be a commercially available enzyme-linked immunosorbent assay. Although HIT is a true clinicopathological syndrome, there are several reasons why its diagnosis still rests primarily on clinical grounds. First, laboratory tests may not be available locally. Second, they may not be available in a sufficiently timely manner. Finally, available tests are not completely sensitive or specific for the condition. There is, therefore, a continuing need to develop more rapid testing strategies with greater specificity for clinical HIT.
An in vitro study on the mechanisms of coagulation activation in acute myelogenous leukemia (AML): role of tissue factor regulation by cytotoxic drugs and GM-CSF.
AML patients may suffer from a disseminated coagulopathy, which can aggravate a pre-existing bleeding tendency due to thrombocytopenia and platelet dysfunction. The cellular and molecular mechanisms underlying this coagulopathy, however, are not completely understood. Indeed, the broad and increasing therapeutic use of cytotoxic drugs and growth factors is likely to contribute to the complexity of hemostatic abnormalities encountered in this hematologic malignancy. The nature of coagulation activation in AML was therefore investigated in vitro using the human leukemic cell line, HL60. Tissue factor (TF) was almost entirely located on the cell surface and bound factor VIIa, but only 15-25% of this TF was primarily functionally active. Treatment with increasing concentrations of daunorubicin or cytosine-beta-D-arabinofuranoside, two cytotoxic drugs commonly used in AML therapy, induced apoptosis and secondary necrosis of HL60 cells and resulted in marked decryption of TF PCA independent of de novo protein synthesis. This PCA-modulating effect was concomitant with and functionally dependent on the exposure of phosphatidylserine on the outer membrane leaflet. Similar observations were made in analogous ex vivo studies on patient-derived myeloblasts. Incubation of HL60 cells with GM-CSF, a cytokine expressed in the bone marrow microenvironment and used as an adjunct to AML treatment, evoked a cellular response, which included both enhanced TF production and release of VEGF-A and uPA into the culture medium. We conclude that both decryption of pre-formed TF PCA by chemotherapeutic drugs and de novo induction of TF by cytokines such as GM-CSF can regulate the pro-coagulant phenotype of HL60 cells in vitro.
Heparin overview and issues.
Unfractionated heparin (UFH), which has been available commercially for over half a century, has been the most widely used agent for quickly suppressing thrombosis. When given intravenously, UFH quickly binds to and activates antithrombin, which then inhibits several activated factors in the clotting cascade. For decades, UFH was invaluable for treating arterial and venous thrombosis, and no alternative was available. The short half-life of UFH and the fact that its action could be reversed readily with protamine made it an almost ideal antithrombotic agent. However, variable pharmacokinetics, together with problems of inaccuracy and unreliability of the activated partial thromboplastin time, have made it difficult to use this drug optimally. In addition, side effects such as osteoporosis, heparin-induced thrombocytopenia (HIT), and delayed HIT have led to increased concerns about the use of UFH in view of the advantages offered by newer agents. Fractionating heparin into low-molecular-weight heparins that still retain the pentasaccharide active site provided a way to achieve the same type of therapeutic effect with more predictable dosing and fewer adverse effects. Similarly, a pentasaccharide has been synthesized and marketed as fondaparinux. Although these advances have improved our therapeutic options, continued advances on the horizon raise the question of whether the use of UFH will soon be abandoned.
Challenges in variation and responsiveness of unfractionated heparin.
Unfractionated heparin (UFH) has been in clinical use for more than half a century. Despite its undoubted contribution to the treatment and prevention of thrombosis, heparin is significantly limited by its variable biochemical composition and unpredictable pharmacokinetics. The situation is compounded by the fact that methods for monitoring heparin do not necessarily reflect its therapeutic effect. The activated partial thromboplastin time (aPTT) is a method for monitoring heparin therapy that is simple, cheap, and readily available. However, it is also poorly standardized and is affected by numerous factors-both analytic and preanalytic-that are unrelated to the heparin effect. Establishing an appropriate therapeutic range for the aPTT is challenging for smaller clinical laboratories, and the antifactor Xa method of measuring heparin levels is not widely available. The College of American Pathologists published consensus guidelines in an effort to improve the laboratory monitoring of UFH therapy. However, it seems unlikely that the laboratory problems associated with monitoring UFH will be resolved. Unfractionated heparin is highly antigenic and carries a significant risk of heparin-induced thrombocytopenia (HIT). Even in the absence of thrombocytopenia or thrombosis, the presence of heparin-associated antibodies may predict adverse clinical outcomes and strengthen the rationale for the ultimate replacement of UFH. Fortunately, alternatives to UFH, such as low-molecular-weight heparins, direct thrombin inhibitors, and more specific factor Xa inhibitors, are becoming available for clinical use. The pharmacokinetics of these agents are more predictable and rely much less on laboratory monitoring. Nonheparin agents also eliminate the risk of HIT. The emergence of these newer anticoagulants makes the continued use of UFH increasingly difficult to justify.
Unfractionated heparin: focus on a high-alert drug.
Unfractionated heparin (UFH) is associated with a high rate of drug-related problems due to either its inherent pharmacologic properties or an extension of these properties often caused by medication errors. The drug-related problems associated with UFH can significantly hinder the success of therapy and negatively affect the overall cost of care. Unfractionated heparin has been classified as a high-alert drug by the Institute for Safe Medication Practices. Approximately 2.1% of the total records submitted to the MedMARx national error database were related to UFH; 4.5-5.5% of these errors reported were harmful. With this high potential for error, it is essential that all health care providers adopt a collaborative or systems approach to identify solutions to reduce the occurrence of these medication errors. The Joint Commission on Accreditation of Healthcare Organizations has published national patient safety goals for improving the safety of patient care, many of which are applicable to UFH therapy. Unfractionated heparin drug-related problems not necessarily related to medication errors include heparin-induced thrombocytopenia, bleeding events, and osteopenia. Heparin-induced thrombocytopenia is a serious complication of heparin therapy and remains seriously undiagnosed. Bleeding events often occur with therapeutic as well as prophylactic UFH administration even when monitoring indexes are within the therapeutic range. However, due to the variability associated with UFH monitoring methods, definitive guidelines are lacking to assist in avoiding such serious events. Osteopenia has been associated with long-term UFH therapy; one third of patients experience reductions in bone density, potentially leading to fractures. Today, safer alternative anticoagulation therapies are available, such as the low-molecular-weight heparins. When compared with UFH, these alternative therapies provide equivalent or superior efficacy for numerous indications.
A soluble single-chain T-cell receptor IL-2 fusion protein retains MHC-restricted peptide specificity and IL-2 bioactivity.
Antibody-based targeted immunotherapy has shown promise as an approach to treat cancer. However, many known tumor-associated antigens are not expressed as integral membrane proteins and cannot be utilized as targets for antibody-based therapeutics. In order to expand the limited target range of antibodies, we have constructed a soluble single-chain T-cell receptor (TCR) fusion protein designated 264scTCR/IL-2. This fusion protein is comprised of a three-domain HLA-A2-restricted TCR specific for a peptide epitope of the human p53 tumor suppressor protein, which is overexpressed in a broad range of human malignancies. The 264scTCR/IL-2 fusion protein has been expressed at high levels in mammalian cells, and milligram quantities have been purified. MHC-restricted antigen-specific binding properties are maintained in the single-chain, three-domain TCR portion of the fusion protein, and the IL-2 portion retains bioactivity similar to that of free recombinant IL-2. Moreover, this fusion protein is capable of conjugating target and effector cells, remains intact in the blood and substantially increases the half life of the IL-2 portion of the molecule. Finally, the 264scTCR/IL-2 fusion protein can be used to stain tumor cells and is capable of reducing lung metastases in an experimental model of metastasis. Thus, TCR-based fusion proteins may provide a novel class of targeted immunotherapeutics for cancer.
Comparison of bovine and porcine heparin in heparin antibody formation after cardiac surgery.
BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a potentially devastating complication of heparin therapy. The incidence of clinical HIT after cardiovascular surgery is less than 2%, although asymptomatic antibodies to heparin-platelet factor 4 (PF4) occur more frequently. Bovine heparin is thought to cause more HIT than porcine heparin, although this has never been established for heparin use during coronary artery bypass grafting. We therefore undertook a randomized, prospective study of heparin-PF4 antibody formation in patients undergoing first-time CABG given intraoperative bovine or porcine heparin. METHODS: Two hundred seven patients (108 porcine, 99 bovine) completed the study. Heparin given pre- or postoperatively was always porcine. Platelet counts and heparin-PF4 antibody tests (enzyme-linked immunosorbent assays) were performed preoperatively and daily until postoperative day 7 or discharge if earlier. RESULTS: The overall incidence of heparin-PF4 antibody formation was 42%. Six patients (2.9%) were positive preoperatively, of which, 1 developed clinical HIT. When these were excluded, seroconversion rates were 44 of 99 (44.4%) and 33 of 108 (30.6%) for bovine and porcine heparin, respectively (p = 0.041). Among patients who produced antibodies, most (90% bovine, 85% porcine) seroconverted after postoperative day 2. There were no differences in postoperative platelet counts; only 1 patient developed thrombosis associated with seroconversion, but without developing thrombocytopenia. The seroconversion rates for patients having cardiopulmonary bypass or off-pump surgery were not significantly different. CONCLUSIONS: This study confirms the high frequency of heparin-PF4 antibodies after coronary artery bypass grafting and demonstrates a significantly higher incidence after bovine heparin. However, because some patients may seroconvert after discharge, our study may underestimate the true incidence.
Hemoglobin induces the production and release of matrix metalloproteinase-9 from human malignant cells.
Matrix metalloproteinase-9 (MMP-9) plays a crucial role in both angiogenesis and tumor invasion. Vascular endothelial growth factor (VEGF) has been shown to up-regulate the expression of MMP-9 in vascular smooth muscle cells. We recently reported that hemoglobin (Hb) enhances the expression of tissue factor (TF) and VEGF on TF-positive human malignant cells. Therefore, to explore the relationship between tumor cell angiogenic protein VEGF and MMP-9, we studied the effect of Hb on MMP-9 production in human A375 malignant melanoma and J82 bladder carcinoma (TF+) cells and in KG1 myeloid leukemia (TF-) cells. Malignant cells were incubated with varying concentrations (0-1.0 mg/ml) of Hb and analyzed for released MMP-9 by gelatin zymography, dot immunoblotting, enzyme-linked immunosorbent assay, and Western blotting. Hb (0.50 mg/ml) induced an almost two-fold increase of MMP-9 in both A375 malignant melanoma (398 +/- 62 versus 233 +/- 61.0 ng/ml, P = 0.027) and J82 bladder carcinoma cells (1.55 +/- 0.12 versus 0.80 +/- 0.004 ng/ml, P = 0.004), compared with cells incubated without Hb. This release of MMP-9 was significantly inhibited by cycloheximide (95%) and by the specific inhibitors of protein tyrosine kinase, genistein (70 +/- 3.0%, P = 0.00027 and 67 +/- 1.0%, P = 0.00005) and mitogen-activated protein (MAP)-kinase, PD98059 (56 +/- 2.0%, P = 0.0001 and 62 +/- 1.0%, P = 0.00003) in A375 and J82 cells, respectively. In contrast, Hb (2.0 mg/ml) did not increase MMP-9 in KG1 cells. We conclude that Hb-induced synthesis of active MMP-9 in TF-bearing malignant cells is due to de novo synthesis of newly formed protein and is mediated by protein tyrosine kinase and by mitogen-activated protein kinase pathways.
Inhibition of tumor cell-induced platelet aggregation and lung metastasis by the oral GpIIb/IIIa antagonist XV454.
Platelets are known to play a role in blood borne metastasis. Previous experimental studies have suggested that platelet GpIIb/IIIa may be a therapeutic target. However, the need for intravenous administration limits the potential application of current GpIIb/IIIa inhibitors to cancer therapy. The aim of the present study was to assess the efficacy of a novel, non-peptide oral GpIIb/IIIa antagonist (XV454) on tumor cell-induced platelet aggregation in vivo and on experimental metastasis. A Lewis lung carcinoma (LL2) mouse model of experimental metastasis was used in this study. XV454 (100 micro g) was administered intravenously (via tail vein) or orally (gavages) to 20 g mice. To determine the effect of XV454 on platelet aggregation, blood samples were collected by cardiac puncture 10 minutes after intravenous and 1-24 hrs after oral XV454, and platelet function was assessed by aggregometry, thrombelastography and the Platelet Function Analyzer (PFA100). The effect of XV454 on tumor cell-induced thrombocytopenia was determined 10 minutes after intravenous and 3 hrs after oral XV454 administration. Tumor cells (2 x 10(6)) were injected intravenously and 15 minutes after cell injection, platelet count was measured and compared to baseline (pre-injection) counts. To assess the effect on metastasis, XV454 was administered intravenous or orally 10 minutes and 3 hrs before tumor cell injection, respectively. Eighteen days later, surface lung tumor nodules were counted and the total lung tumor burden assessed. In a fourth group, in addition to the initial oral dose (before tumor cell injection), oral XV454 was given daily for the first week and three times in the second week. Administration of XV454 (5 mg/kg) completely inhibited platelet aggregation and this effect persisted for at least 24 hrs after oral delivery. Both intravenous and oral XV454 significantly inhibited tumor cell-induced thrombocytopenia (P < 0.01), the number of surface lung tumor nodules (80-85%; P < 0.001) and total tumor burden (83% for intravenous group; 50% oral [single treatment] group; 91% oral [multiple treatment] group, P < 0.001). Overall, these data provide further evidence for the effect of oral and intravenous GpIIb/IIIa antagonism on tumor cell-platelet interaction and metastasis.