Bruce S. Sachais

Evidence-based practice guidelines for plasma transfusion

BACKGROUND: There is little systematically derived evidence‐based guidance to inform plasma transfusion decisions. To address this issue, the AABB commissioned the development of clinical practice guidelines to help direct appropriate transfusion of plasma.

Platelet factor 4 localization in carotid atherosclerotic plaques: correlation with clinical parameters

Emerging evidence supports a role for platelets in the progression of atherosclerosis in addition to an involvement in thrombotic vascular occlusion. Platelet Factor 4 (PF4), a chemokine released by activated platelets, stimulates several pro-atherogenic processes. Therefore, we examined the localization of PF4 and the homologous protein, Neutrophil Activating Protein-2 (NAP-2) in lesions representing the evolution of human atherosclerotic plaques. Carotid plaques from 132 patients with critical carotid stenosis and 6 autopsy specimens were studied. Clinical, histologic and immunohistochemical data were analyzed using a chi(2)-test. PF4 was detected in the cytoplasm of luminal and neovascular endothelium, in macrophages and in regions of plaque calcification. The presence of PF4 in macrophages and neovascular endothelium correlated with lesion grade (p = 0.004; p = 0.044). Staining of macrophages for PF4 correlated with the presence of symptomatic atherosclerotic disease (p = 0.028). In early lesions, PF4 was commonly found in macrophages of early lesions (Grade I/II), whereas NAP-2 was rarely present. In conclusion, correlation between PF4 deposition, lesion severity and symptomatic atherosclerosis suggests that persistent platelet activation may contribute to the evolution of atherosclerotic vascular lesions. These studies support the rationale for the chronic use of anti-platelet therapy in patients at risk for developing symptomatic atherosclerosis.

A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo

Urokinase plasminogen activator (uPA) plays an important role in the progression of several malignancies including breast cancer. We have identified a noncompetitive antagonist of the uPA‐uPAR interaction derived from a nonreceptor binding region of uPA (amino acids 136‐143). This 8‐mer capped peptide (Å6) inhibited breast cancer cell invasion and endothelial cell migration in a dose‐dependent manner in vitro without altering cell doubling time. Intraperitoneal administration of Å6 resulted in a significant inhibition of tumor growth and suppressed the development of lymph node metastases in several models of breast cancer cell growth and metastasis. Large areas of tumor necrosis and extensive positive staining by TUNEL were observed on histological and immunohistochemical analysis of experimental tumor sections from Å6‐treated animals. Å6 treatment also resulted in a decrease in factor VIII‐positive tumor microvessel hot‐spots. These results identify a new epitope in uPA that is involved in the uPA‐uPAR interaction and indicate that an antagonist based on this epitope is able to inhibit tumor progression by modulating the tumor microenvironment in the absence of direct cytotoxic effects in vivo.—Guo, Y., Higazi, A. A., Arakelian, A., Sachais, B. S., Cines, D., Goldfarb, R. H., Jones, T. R., Kwaan, H., Mazar, A. P., Rabbani, S. A. A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo. FASEB J. 14, 1400–1410 (2000)

Elimination of platelet factor 4 (PF4) from platelets reduces atherosclerosis in C57Bl/6 and apoE-/- mice

Activated platelets, which release platelet factor 4 (PF4) are present in patients with atherosclerosis. To date, no direct in-vivo evidence exists for the involvement of PF4 in atherogenesis. In the current study, we tested the hypothesis that PF4 is atherogenic, and that genetic elimination of PF4 would protect mice from atherosclerosis. We have bred PF4(-/-) mice onto two athero-susceptible backgrounds, WT-C57Bl/6(WT) and apoE(-/-) to examine the importance of PF4 in atherogenesis. In order to induce atherosclerosis, WT and PF4(-/-) mice were fed an atherogenic diet for 30 weeks, while apoE(-/-) and apoE(-/-) PF4(-/-) mice were fed a high-fat Western-style diet for 10 weeks. Examination of lesions in the aortic roots of atherogenic diet fed mice demonstrated reduced atherosclerosis in PF4(-/-) (20% compared to WT). Examination of apoE(-/-) mice demonstrated similar changes, with apoE(-/-) PF4(-/-) mice demonstrating 37% of the aortic atherosclerotic burden compared to apoE(-/-) mice. Although we found similar levels of total and non-HDL cholesterol in WT and PF4(-/-) mice, HDL-cholesterol levels were increased in PF4(-/-) on both backgrounds. These data demonstrate, for the first time, that the platelet specific chemokine PF4 promotes atherosclerotic lesion development in vivo.

Dynamic antibody-binding properties in the pathogenesis of HIT

Rapid laboratory assessment of heparin-induced thrombocytopenia (HIT) is important for disease recognition and management. The utility of contemporary immunoassays to detect antiplatelet factor 4 (PF4)/heparin antibodies is hindered by detection of antibodies unassociated with disease. To begin to distinguish properties of pathogenic anti-PF4/heparin antibodies, we compared isotype-matched monoclonal antibodies that bind to different epitopes: KKO causes thrombocytopenia in an in vivo model of HIT, whereas RTO does not. KKO binding to PF4 and heparin is specifically inhibited by human HIT antibodies that activate platelets, whereas inhibition of RTO binding is not differentially affected. Heparin increased the avidity of KKO binding to PF4 without affecting RTO, but it did not increase total binding or binding to nontetrameric PF4(K50E). Single-molecule forced unbinding demonstrated KKO was 8-fold more reactive toward PF4 tetramers and formed stronger complexes than RTO, but not to PF4(K50E) dimers. KKO, but not RTO, promoted oligomerization of PF4 but not PF4(K50E). This study reveals differences in the properties of anti-PF4 antibodies that cause thrombocytopenia not revealed by ELISA that correlate with oligomerization of PF4 and sustained high-avidity interactions that may simulate transient antibody-antigen interactions in vivo. These differences suggest the potential importance of epitope specificity in the pathogenesis of HIT.

Transfusion of 28-day-old leucoreduced or non-leucoreduced stored red blood cells induces an inflammatory response in healthy dogs

Studies in mice suggest that rapid transfusions of red blood cells (RBCs), refrigerator stored for longer durations, induce a pro‐inflammatory cytokine response. Studies in human neonates confirm these findings; however, to date, adult human studies have failed to replicate these findings. We used healthy research dogs to begin to examine the factors affecting the cytokine response to transfusion.

Chemokines and thrombogenicity

Thrombosis is an important clinical entity, and pathologic thrombosis, in the form of atherosclerosis, is a major cause of morbidity and mortality. Recent research points to the role of chemokines, normally key factors in inflammation, in thrombogenesis. Many recent studies in murine transgenic and knockout models show that chemokines and their receptors are important modulators of the process of thrombus formation, particularly in atherosclerosis. Platelet-released chemokines can potentiate or inhibit thrombosis and inflammation. This review focuses on the role of chemokines in platelet activation and thrombosis, particularly as it relates to atherosclerosis. Further studies to define this complex interaction are underway.

Blood transfusion costs by diagnosis-related groupsin 60 university hospitals in 1995

BACKGROUND: Transfusion services are frequently challenged to initiate efforts to reduce blood transfusion costs. One approach is to analyze blood transfusion costs for individual medical and surgical Diagnosis‐Related Groups (DRGs). Rank ordering of DRGs by transfusion costs and interinstitutional comparisons of these costs may lead to the selection of DRGs for further analysis of the process of blood transfusion.

Urokinase-derived peptides regulate vascular smooth muscle contraction in vitro and in vivo

We examined the effect of urokinase (uPA) and its fragments on vascular smooth muscle cell contraction. Single-chain uPA inhibits phenylepherine (PE) -induced contraction of rat aortic rings, whereas two-chain uPA exerts the opposite effect. Two independent epitopes mediating these opposing activities were identified. A6, a capped peptide corresponding to amino acids 136-143 (KPSSPPEE) of uPA, increased the EC(50) of PE-induced vascular contraction sevenfold by inhibiting the release of calcium from intracellular stores. A6 activity was abolished by deleting the carboxyl-terminal Glu or by mutating the Ser corresponding to position 138 in uPA to Glu. A single-chain uPA variant lacking amino acids 136-143 did not induce vasorelaxation. A second epitope within the kringle of uPA potentiated PE-induced vasoconstriction. This epitope was exposed when single-chain uPA was converted to a two-chain molecule by plasmin. The isolated uPA kringle augmented vasoconstriction, whereas uPA variant lacking the kringle had no procontractile activity. These studies reveal previously undescribed vasoactive domains within urokinase and its naturally derived fragments.

Novel interactions between urokinase and its receptor

Urokinase-type plasminogen activator (uPA) binds to its receptor (uPAR) with a K d of about 1 nm. The catalytic activity of the complex is apparent at uPA concentrations close to K d . Other functions of the complex, such as signal transduction, are apparent at much higher concentrations (35–60 nm). In the present study, we show that uPA and recombinant soluble uPAR (suPAR), at concentrations that exceed the K d and the theoretical saturation levels (10–80 nm), establish novel interactions that lead to a further increase in the activity of the single-chain uPA (scuPA)/suPAR and two-chain uPA (tcuPA)/suPAR complexes. Experiments performed using dynamic light scattering, gel filtration, and electron microscopy techniques indicate that suPAR forms dimers and oligomers. The three techniques provide evidence that the addition of an equimolar concentration of scuPA leads to the dissociation of these dimers and oligomers. Biacore data show that suPAR dimers and oligomers bind scuPA with decreased affinity when compared with monomers. We postulate that uPAR is present in equilibrium between oligomer/dimer/monomer forms. The binding of uPA to suPAR dimers and oligomers occurs with lower affinity than the binding to monomer. These novel interactions regulate the activity of the resultant complexes and may be involved in uPA/uPAR mediated signal transduction.