Antibody desensitization therapy in highly sensitized lung transplant candidates.

As HLAs antibody detection technology has evolved, there is now detailed HLA antibody information available on prospective transplant recipients. Determining single antigen antibody specificity allows for a calculated panel reactive antibodies (cPRA) value, providing an estimate of the effective donor pool. For broadly sensitized lung transplant candidates (cPRA ≥ 80%), our center adopted a pretransplant multi-modal desensitization protocol in an effort to decrease the cPRA and expand the donor pool. This desensitization protocol included plasmapheresis, solumedrol, bortezomib and rituximab given in combination over 19 days followed by intravenous immunoglobulin. Eight of 18 candidates completed therapy with the primary reasons for early discontinuation being transplant (by avoiding unacceptable antigens) or thrombocytopenia. In a mixed-model analysis, there were no significant changes in PRA or cPRA changes over time with the protocol. A sub-analysis of the median fluorescence intensity (MFI) change indicated a small decline that was significant in antibodies with MFI 5000-10,000. Nine of 18 candidates subsequently had a transplant. Posttransplant survival in these nine recipients was comparable to other pretransplant-sensitized recipients who did not receive therapy. In summary, an aggressive multi-modal desensitization protocol does not significantly reduce pretransplant HLA antibodies in a broadly sensitized lung transplant candidate cohort.

Tissue factor around dermal vessels has bound factor VII in the absence of injury.

BACKGROUND: 'Idling' or ongoing low-level activity of the tissue factor (TF) pathway is a postulated mechanism by which the coagulation process can become active without a lag period at sites of injury. OBJECTIVE: To determine whether TF around cutaneous vessels has bound factor VIIa in the absence of injury, and thus could participate in the idling process. METHODS: Immunostaining of mouse skin with antibodies against a 15-residue peptide from the sequence of mouse TF, and against the whole extracellular portion of TF. RESULTS: The whole TF antibody recognized TF in squamous epithelium and around vessels in the dermis. By contrast, the monospecific antibody only recognized TF in the squamous epithelium, but not around vessels. We also found that biotinylated, active site-inhibited FVIIa (FVIIai) bound to tissue sections in the same areas in which TF was recognized by the monospecific antibody (squamous epithelium), but did not bind around vessels. Molecular modeling revealed that FVIIa and FX binding to TF masked a significant part of the surface of the target peptide. CONCLUSIONS: In the aggregate, these data are most consistent with the interpretation that TF in perivascular sites has bound FVIIa, even in the absence of any injury. The presence of endogenously bound FVIIa prevents the subsequent binding of the monospecific antibody or exogenous FVIIai to perivascular TF.

Antidote-controlled platelet inhibition targeting von Willebrand factor with aptamers.

Thrombus formation is initiated by platelets and leads to cardiovascular, cerebrovascular, and peripheral vascular disease, the leading causes of morbidity and mortality in the Western world. A number of antiplatelet drugs have improved clinical outcomes for thrombosis patients. However, their expanded use, especially in surgery, is limited by hemorrhage. Here, we describe an antiplatelet agent that can have its activity controlled by a matched antidote. We demonstrate that an RNA aptamer targeting von Willebrand factor (VWF) can potently inhibit VWF-mediated platelet adhesion and aggregation. By targeting this important adhesion step, we show that the aptamer molecule can inhibit platelet aggregation in PFA-100 and ristocetin-induced platelet aggregation assays. Furthermore, we show that a rationally designed antidote molecule can reverse the effects of the aptamer molecule, restoring platelet function quickly and effectively over a clinically relevant period. This aptamer-antidote pair represents a reversible antiplatelet agent inhibiting a platelet specific pathway. Furthermore, it is an important step towards creating safer drugs in clinics through the utilization of an antidote molecule.

The association of anti-platelet factor 4/heparin antibodies with early and delayed thromboembolism after cardiac surgery.

Essentials We evaluated antibody status, thromboembolism and survival after cardiac surgery. Positive antibody tests are common - over 50% are seropositive at 30 days. Seropositivity did not increase thromboembolism or impair survival after cardiac surgery. Results show heparin induced thrombocytopenia antibody screening after surgery is not warranted. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is a prothrombotic response to heparin therapy with platelet-activating, anti-platelet factor 4 (PF4)/heparin antibodies leading to thrombocytopenia associated with thromboembolism. Objective We tested the hypothesis that anti-PF4/heparin antibodies are associated with thromboembolism after cardiac surgery. Methods This multicenter, prospective cohort study collected laboratory and clinical data up to 30 days after surgery and longer-term clinical follow-up data. The primary outcome variable combined new arterial or venous thromboembolic complications (TECs) with all-cause death until 90 days after surgery. Laboratory analyses included platelet counts and anti-PF4/heparin antibody titers (GTI ELISA), with a confirmatory excess heparin step and serotonin release assay. Chi-square testing was used to test the relationship between our outcome and HIT antibody seropositivity. Results Initially, 1021 patients were enrolled between August 2006 and May 2009, and follow-up was completed in December 2014. Seropositivity defined by OD > 0.4 was common, being almost 20% preoperatively, > 30% by discharge, and > 60% by day 30. Death (1.7% within 30 days) or TECs (69 in total) were more likely if the partient was seronegative (OD < 0.4), but positivity defined by OD > 1.0 or including an excess heparin confirmatory step resulted in equal incidence of death or TECs, whether the patient was seronegative or seropositive. Incorporating the serotonin release assay for platelet-activating antibodies did not alter these findings. Conclusions Seropositivity for anti-PF4/heparin antibodies does not increase the risk of death or thromboembolism after cardiac surgery. Screening is not indicated, and seropositivity should only be interpreted in the context of clinical evidence for HIT. TRIAL REGISTRATION: Duke IRB Protocol #00010736.

Heparin enhances uptake of platelet factor 4/heparin complexes by monocytes and macrophages.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies to a self-antigen, platelet factor (4) and heparin. The reasons why antibodies form to PF4/heparin, but not to PF4 bound to other cellular glycosaminoglycans are poorly understood. OBJECTIVE: To investigate differences in cellular responses to cell-bound PF4 and PF4/heparin complexes, we studied the internalization of each by peripheral blood-derived monocytes, dendritic cells and neutrophils. METHODS AND RESULTS: Using unlabeled and fluorescently-labeled antigen and/or labeled monoclonal antibody to PF4/heparin complexes (KKO), we show that PF4/heparin complexes are taken up by monocytes in a heparin-dependent manner and are internalized by human monocytes and dendritic cells, but not by neutrophils. Complexes of PF4/low-molecular-weight heparin and complexes composed of heparin and murine PF4, protamine or lysozyme are internalized similarly, suggesting a common endocytic pathway. Uptake of complexes is mediated by macropinocytosis, as shown by inhibition using cytochalasin D and amiloride. Internalized complexes are transported intact to late endosomes, as indicated by co-staining of vesicles with KKO and lysosomal associated membrane protein-2 (LAMP-2). Lastly, we show that cellular uptake is accompanied by expression of MHCII and CD83 co-stimulatory molecules. CONCLUSIONS: Taken together, these studies establish a distinct role for heparin in enhancing antigen uptake and activation of the initial steps in the cellular immune response to PF4-containing complexes.

A mouse model for heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) occurs in 1% to 3% of patients receiving heparin and results from the development of antibodies that recognize heparin-platelet factor 4 (H-PF4) complexes that form on the surface of activated platelets and on the vascular endothelium. With the aim of studying the pathogenic importance of these anti-H-PF4 antibodies in vivo, we attempted to create an animal model of HIT. Such a model was produced by immunization of naive mice with affinity-purified IgG anti-H-PF4 antibodies from two patients with HIT. The immunized mice developed specific antibodies (anti-idiotypic) against the human anti-H-PF4 antibodies and 2 months later, anti-anti-idiotypic antibodies appeared, which functionally resembled the human HIT antibody. Indeed, when the animals bearing anti-anti-idiotypic antibodies were injected with heparin for 4 days, a significant decrease in their platelet counts was observed; however, heparin treatment was not associated with thrombosis in any of the immunized mice. Similar to the observation in HIT patients, injections of equivalent doses of low-molecular-weight (LMW) heparin to the immunized animals did not induce thrombocytopenia. The results of this study support the importance of anti-H-PF4 antibodies in the pathogenesis of HIT. The mouse HIT model may provide a convenient system for studies on the immunoregulation of anti-H-PF4 expression and for evaluation of potential therapeutic modalities.

Rapid detection of the Fc gamma RIIA-H/R 131 ligand-binding polymorphism using an allele-specific restriction enzyme digestion (ASRED).

A polymorphism of the gene for Fc gamma RIIA, arginine (R) or histidine (H) at position 131, alters the ability of the receptor to bind certain IgG subclasses. Identification of the Fc gamma RIIA-H/R 131 genotype has assumed increasing importance in disorders of host defense, immunohematologic diseases and systemic autoimmune disorders. We report a new method for determination of this genotype in which an allele-specific restriction enzyme site is introduced into an Fc gamma RIIA PCR product from genomic DNA, and polymorphism assignment is determined by restriction enzyme digestion followed by agarose gel electrophoresis. This method is more rapid, more reliable and less expensive than currently available methods.

Comparison of PF4/heparin ELISA assay with the 14C-serotonin release assay in the diagnosis of heparin-induced thrombocytopenia.

The diagnosis of heparin-induced thrombocytopenia (HIT) may be affirmed by demonstrating heparin-dependent anti-platelet antibodies using the 14C-serotonin release assay (SRA). In this study, results of the SRA was compared with the recently described platelet factor 4 (PF4)/heparin enzyme-linked immunosorbent assay (ELISA). Compared with the SRA, the sensitivity and specificity of a PF4/heparin ELISA was 87% and 92%, respectively, using an assay developed in our laboratory; and 90% and 98%, respectively, using a commercially developed kit (Diagnostica Stago, Asnieres, France). However, antibodies to PF4/heparin were also detected in up to 8% of patients whose plasma was negative by SRA, and 23% of patients receiving heparin who were not thrombocytopenic. These data indicate that results obtained with the PF4/heparin ELISA and the SRA are generally in accord in patients with a clinical diagnosis of HIT. However, discrepant results occur in approximately 20% of cases because of the greater sensitivity of ELISA and the possible involvement of other heparin-binding proteins. The fact that each assay contributes independent information in some cases must be considered in the sequence of test performance and in providing consultation to the practicing hematologist.

Heparin-induced thrombocytopenia: bovine versus porcine heparin in cardiopulmonary bypass surgery.

BACKGROUND: Studies have demonstrated a high incidence of antibodies to heparin/platelet factor 4 complexes, the antigen in heparin-induced thrombocytopenia, in patients after cardiopulmonary bypass surgery. In many hospitals, beef lung heparin has been used historically for cardiopulmonary bypass, and there has been reluctance to change to porcine heparin despite concerns of an increased incidence of heparin-induced thrombocytopenia in patients receiving bovine heparin. METHODS: A prospective randomized trial comparing bovine and porcine heparin in cardiopulmonary bypass surgery was conducted. Presurgery and postsurgery heparin antibody formation was studied using the serotonin release assay and a heparin/platelet factor 4 enzyme-linked immunosorbent assay. RESULTS: Data available on 98 patients, randomized to receive either bovine or porcine heparin, revealed no significant difference in patient positivity by serotonin release assay (12% in both groups) or by the heparin/platelet factor 4 enzyme-linked immunosorbent assay (29% with porcine and 35% with bovine heparin) postoperatively. There were no significant differences between preoperative and postoperative platelet counts or thromboembolic complications. CONCLUSIONS: Our study does not support the belief that bovine heparin is more likely than porcine heparin to induce the development of antibodies to heparin/platelet factor 4.

Late onset of isolated central nervous system metastasis of liposarcoma--a case report.

Metastatic soft-tissue sarcoma of the central nervous system (CNS) is exceedingly rare. We report a case of a 56-year-old male treated for a right lower extremity liposarcoma at the age of 30 years, whose first recurrence was an intracerebral metastasis occurring 26 years after resection of the primary tumor. Initial treatment of the metastasis with surgical resection and adjuvant radiotherapy was followed rapidly by CNS recurrence in 3 months. Further debulking and interstitial brachytherapy were unsuccessful in controlling disease progression. Clinical presentation and treatment of brain metastases in soft-tissue sarcoma are discussed.

Kaposi's sarcoma mimicking cellulitis.

Although Kaposi's sarcoma is not the most common cause of death in AIDS patients, it is often one of the initial opportunistic illnesses associated with human immunodeficiency virus infection. Extensive plaque formation and edema in the lower extremities may take on the appearance of cellulitis, and in dark-skinned persons, the lesions of the neoplasm may not be noticeable. Treatment is palliative; therapy for local effect is appropriate unless lesions are extensive or systemic involvement is present.

Disruption of PF4/H multimolecular complex formation with a minimally anticoagulant heparin (ODSH).

Recent studies have shown that ultra-large complexes (ULCs) of platelet factor 4 (PF4) and heparin (H) play an essential role in the pathogenesis of heparin-induced thrombocytopenia (HIT), an immune-mediated disorder caused by PF4/H antibodies. Because antigenic PF4/H ULCs assemble through non-specific electrostatic interactions, we reasoned that disruption of charge-based interactions can modulate the immune response to antigen. We tested a minimally anticoagulant compound (2-O, 3-O desulfated heparin, ODSH) with preserved charge to disrupt PF4/H complex formation and immunogenicity. We show that ODSH disrupts complexes when added to pre-formed PF4/H ULCs and prevents ULC formation when incubated simultaneously with PF4 and UFH. In other studies, we show that excess ODSH reduces HIT antibody (Ab) binding in immunoassays and that PF4/ODSH complexes do not cross-react with HIT Abs. When ODSH and unfractionated heparin (UFH) are mixed at equimolar concentrations, we show that there is a negligible effect on amount of protamine required for heparin neutralisation and reduced immunogenicity of PF4/UFH in the presence of ODSH. Taken together, these studies suggest that ODSH can be used concurrently with UFH to disrupt PF4/H charge interactions and provides a novel strategy to reduce antibody mediated complications in HIT.

The HIT Expert Probability (HEP) Score: a novel pre-test probability model for heparin-induced thrombocytopenia based on broad expert opinion.

BACKGROUND: The diagnosis of heparin-induced thrombocytopenia (HIT) is challenging. Over-diagnosis and over-treatment are common. OBJECTIVES: To develop a pre-test clinical scoring model for HIT based on broad expert opinion that may be useful in guiding clinical decisions regarding therapy. PATIENTS/METHODS: A pre-test model, the HIT Expert Probability (HEP) Score, was constructed based on the opinions of 26 HIT experts. Fifty patients referred to a reference laboratory for HIT testing comprised the validation cohort. Two hematology trainees scored each patient using the HEP Score and a previously published clinical scoring system (4 T's). A panel of three independent experts adjudicated the 50 patients and rendered a diagnosis of HIT likely or unlikely. All subjects underwent HIT laboratory testing with a polyspecific HIT ELISA and serotonin release assay (SRA). RESULTS: The HEP Score exhibited significantly greater interobserver agreement [intraclass correlation coefficient: 0.88 (95% CI 0.80-0.93) vs. 0.71 (0.54-0.83)], correlation with the results of HIT laboratory testing and concordance with the diagnosis of the expert panel (area under receiver-operating curve: 0.91 vs. 0.74, P = 0.017) than the 4 T's. The model was 100% sensitive and 60% specific for determining the presence of HIT as defined by the expert panel and would have allowed for a 41% reduction in the number of patients receiving a direct thrombin inhibitor (DTI). CONCLUSION: The HEP Score is the first pre-test clinical scoring model for HIT based on broad expert opinion, exhibited favorable operating characteristics and may permit clinicians to confidently reduce use of alternative anticoagulants. Prospective multicenter validation is warranted.

Optimization of a murine immunization model for study of PF4/heparin antibodies.

SUMMARY BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a life-threatening thrombotic illness caused by drug-dependent antibodies recognizing complexes of platelet factor 4 (PF4) and heparin. Little is known about the immune pathogenesis of HIT, in particular factors influencing PF4/heparin antibody formation. To gain insight into the biologic basis of heparin sensitization, we have recently developed an animal model using wild-type (WT) mice in which murine PF4/heparin antibodies (anti-mPF4/H) arise de novo after antigen challenge. OBJECTIVES AND METHODS: This report describes technical refinements to the murine model and describes additional biologic features of the immune response to mPF4/heparin. RESULTS: Our studies indicate that antibody responses to mPF4/heparin are dependent on murine strain, injection routes and doses of mPF4 and heparin. C57BL/6 mice are more immunologically responsive to mPF4/heparin antigen than BALB/c mice and robust immunization can be achieved with intravenous, but not intraperitoneal, administration of antigen. We also observe a direct relationship between initial concentrations of mPF4 and antibody levels. Additionally, we demonstrate that mPF4/H immune response in mice decays with time, is not associated with thrombocytopenia and displays characteristics of immune recall on re-exposure to antigen. CONCLUSIONS: These studies describe and characterize a murine model for studying the immunologic basis of PF4/heparin sensitization.

Platelet and monocyte antigenic complexes in the pathogenesis of heparin-induced thrombocytopenia (HIT).

Heparin-induced thrombocytopenia (HIT) is an iatrogenic disorder that occurs in a small subset of patients receiving heparin. Twenty-five per cent (or higher) of affected patients develop limb or life-threatening thrombosis. The effectiveness of therapy is incomplete and may be complicated by bleeding. HIT is caused by antibodies that recognize the platelet chemokine, Platelet Factor 4 (PF4), complexed to heparin or to cellular glycosaminoglycans (GAGs). However, antibodies with the same apparent specificity are found in many more patients without clinical disease and the reason why so few develop HIT is uncertain. We propose that HIT antibodies recognize cell surface PF4/GAG complexes on intravascular cells, including platelets and monocytes that are dynamic and mutable. Heparin removes cell surface-bound PF4 in most individuals, but removal is incomplete in those with high pre-exposure surface-bound PF4 levels. Such individuals retain critically localized cellular antigenic complexes at the time antibodies develop and are at risk to develop HIT. This article reviews the scientific basis for this model and its clinical implications.

Platelet functions beyond hemostasis.

Although their central role is in the prevention of bleeding, platelets probably contribute to diverse processes that extend beyond hemostasis and thrombosis. For example, platelets can recruit leukocytes and progenitor cells to sites of vascular injury and inflammation; they release proinflammatory and anti-inflammatory and angiogenic factors and microparticles into the circulation; and they spur thrombin generation. Data from animal models suggest that these functions may contribute to atherosclerosis, sepsis, hepatitis, vascular restenosis, acute lung injury, and transplant rejection. This article represents an integrated summary of presentations given at the Fourth Annual Platelet Colloquium in January 2009. The process of and factors mediating platelet-platelet and platelet-leukocyte interactions in inflammatory and immune responses are discussed, with the roles of P-selectin, chemokines and Src family kinases being highlighted. Also discussed are specific disorders characterized by local or systemic platelet activation, including coronary artery restenosis after percutaneous intervention, alloantibody-mediated transplant rejection, wound healing, and heparin-induced thrombocytopenia.

Antibodies from patients with heparin-induced thrombocytopenia stimulate monocytic cells to express tissue factor and secrete interleukin-8.

Thrombosis is a life-threatening complication that occurs in a subset of patients with heparin-induced thrombocytopenia (HITT). The pathogenic mechanisms underlying the variable occurrence of thrombosis in HITT is poorly understood. It was hypothesized that monocyte activation leading to tissue factor expression may play a role in promoting a thrombogenic state in HITT. This study demonstrates that a human platelet factor 4 (PF4)/heparin-specific murine monoclonal antibody (KKO) binds to peripheral blood-derived human monocytes in a PF4-dependent manner. KKO and antibodies from patients with HITT induce monocytes to synthesize and secrete interleukin-8 and induce cell-surface procoagulant activity, which is abrogated following treatment with antihuman tissue factor antibody. The findings suggest a novel mechanism by which PF4/heparin antibodies may promote a hypercoagulable state in patients with HITT. (Blood. 2001;98:1252-1254)

Pathogenicity of human anti-platelet factor 4 (PF4)/heparin in vivo: generation of mouse anti-PF4/heparin and induction of thrombocytopenia by heparin.

Heparin-induced thrombocytopenia/thrombosis (HIT) is a severe thrombotic disorder that occurs in approximately 1% of patients treated with heparin. Affected patients commonly develop antibodies that recognize PF4/heparin complexes that may form on the surface of activated platelets and on the endothelium. However, it has not been established that anti-PF4/heparin antibodies are responsible for the clinical manifestations of HIT. To address this issue, we employed a recently developed model of active immunity to study the effect of IgG anti-PF4/heparin antibody in vivo. In previous studies we have shown that it is possible to induce autoimmune diseases such as systemic lupus erythematosus (SLE), anti-phospholipid syndrome (APS) or vasculitis in naive mice by active immunization with anti-DNA, anti-cardiolipin and anti-neutrophil cytoplasmic antibodies, respectively. Immunized animals develop anti-idiotypic antibodies (Ab2) and, after 2-4 months, anti-anti-idiotypic antibodies (Ab3). Ab3s generated in this manner often simulate the binding activity of Ab1 and their expression correlates with the development of specific clinical manifestations typical of the respective human disease. Based on this experience, naive BALB/c mice were immunized with IgG anti-PF4/heparin antibodies isolated from two patients with HIT. The actively immunized mice developed mouse anti-PF4/heparin antibody (Ab3). Administration of unfractionated heparin, but not low molecular weight heparin (LMWH), to the actively immunized animals induced thrombocytopenia by day 4 of drug exposure. There was no evidence of thrombosis. The results of this study support the importance of anti-PF4/heparin antibodies in the pathogenesis of HIT. Further, this model may help to elucidate the factors responsible for thrombosis as well as providing means to assess new treatment options for patients with this disorder.