Viral infection of vascular endothelial cells alters production of colony-stimulating activity.

Viral infections in humans are frequently associated with granulocytopenia and/or granulocytosis. Such changes in myelopoiesis could result from infection of the granulocyte-macrophage colony-forming cell (CFC-GM) or changes in the production of colony-stimulating activity (CSA). Endothelial cells are a known source of CSA and may be transiently or persistently infected during a number of viral infections, including infection with herpes simplex virus type I (HSV-I) and measles virus. Therefore, we examined the effect of endothelial cell infection with these two viruses on the production of CSA. Uninfected passaged endothelial cells produce CSA when stimulated by the continual presence of a factor present in medium conditioned by peripheral blood monocytes (MCM). Within 4 h of infection with HSV-I, endothelial cells no longer produced CSA in response to MCM. In contrast, measles virus infection induced CSA production by passaged endothelial cells even in the absence of MCM. Measles virus-induced CSA production was maximal at 24 h and required the presence of live virus within the endothelial cells. The effects of HSV-I and measles virus on CSA production were not dependent on alterations in the production of alpha- or gamma-interferon by the infected endothelial cells. Infection with HSV-I did not stimulate endothelial cells to release any detectable interferon. In contrast, the supernatants of the measles-infected cells contained only beta-interferon, a known inhibitor of CFC-GM development. These studies suggest that CSA production by endothelial cells is directly altered by infection with HSV-I and measles virus. An alteration in CSA production might contribute to changes in myelopoiesis that frequently accompany viral infection in humans.

Increased fluidity of human platelet membranes during complement-mediated immune platelet injury.

Complement appears to be involved in the destruction of platelets in certain clinical disorders, such as quinidine purpura and post-transfusion purpura. In both disorders, the classical complement sequence is activated by antigen-antibody complexes. It has been suggested that the terminal components of the complement sequence insert into the hydrophobic core of cell surface membranes and that this process leads to cell lysis. Fluidity is a fundamental property of lipids within the membrane's hydrophobic core. To examine the interaction of complement with membranes, we investigated the effect of complement activation on the fluidity of human platelet membranes. Complement was fixed to platelets using a post-transfusion purpura antibody, and membrane lipid fluidity was assessed in terms of fluorescence anisotropy using two fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene and 9-(12-anthroyl) stearic acid. Microviscosity, expressed in poise, was derived from the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene.Post-transfusion purpura antibody plus complement made platelet membranes more fluid as evidenced by a 21% decrease in anisotropy and a 35% decrease in microviscosity of platelets at 37 degrees C, and this was associated with platelet lysis ((51)Cr release). Complement damage to platelets was accompanied by a 10-15% increase in DeltaE, the fusion activation energy for microviscosity, indicating that complement not only decreased membrane microviscosity but also made membrane lipids less ordered. These changes were consistent and rapid, with platelet lysis and the reduction in microviscosity being half-maximal by 6 min. They were prevented by inactivation of complement with heat or with EDTA, and they were not observed when C5-deficient plasma was used as the complement source. Qualitatively similar changes in platelet membrane fluidity were observed when complement was fixed to platelets by a quinidine-dependent anti-platelet antibody rather than by post-transfusion purpura antibody. Post-transfusion purpura antibody plus complement also decreased the microviscosity of isolated platelet membranes. Moreover, the lipids extracted from platelets lysed by complement had a 22% decrease in microviscosity (P < 0.01), with no associated changes in the amount of cholesterol relative to phospholipid or in the amounts of the various phospholipids. These studies demonstrate that lipids within the hydrophobic core of platelet membranes damaged by complement become more fluid, and this is associated with platelet lysis. These findings are consistent with the concept that the insertion of the terminal complement components into the platelet membrane bilayer perturbs lipid-lipid interactions within the membrane's hydrophobic core.

Presence of complement-fixing anti-endothelial cell antibodies in systemic lupus erythematosus.

Vasculitis in systemic lupus erythematosus (SLE) is associated with the deposition of IgG and complement in blood vessel walls. However, it is not known whether immune injury to endothelial cells is a part of this process. Therefore, we used a solid phase radioimmunoassay to study the ability of IgG from normal human sera and sera from patients with SLE to bind to endothelial cells. In this assay, cultured human umbilical venous endothelial cells were sequentially incubated with normal or SLE sera, goat anti-human IgG, and 125I-labeled staphylococcal protein A (*SPA). After exposure to normal sera, 2.5 +/- 0.5% (mean +/- SD) of the added *SPA bound to the cells, whereas after exposure to SLE sera 13.8 +/- 7.6% of the added *SPA bound to these cells. This difference in binding was highly significant (P less than 0.001). Binding was partially reduced when SLE sera were preincubated with B-lymphocytes or monocytes, but not after exposure to erythrocytes, platelets, or T lymphocytes. Incubation of endothelial cells with the 7S fraction of SLE sera or with the F(ab')2 fragment of SLE-IgG resulted in the deposition of greater than 80% as much IgG as was deposited on endothelial cells by whole serum. However, since higher molecular weight fractions (greater than 7S) of SLE sera were also active, we tested the capacity of endothelial cells to bind IgG complexes. Endothelial cells bound heat-aggregated IgG (HA-IgG) in a saturable manner at one log concentration below the binding of normal monomeric IgG. Binding of HA-IgG to endothelial cells was markedly enhanced by preincubation with a serum source of complement. Both HA-IgG and SLE-IgG also bound to freshly obtained endothelial cells in suspension, as detected by automated fluorescence flow cytometry. Binding of SLE-IgG and HA-IgG to endothelium initiated complement activation, deposition of the third component of complement, and disruption of the monolayer. In addition, SLE-IgG and HA-IgG caused endothelial cells to secrete prostacyclin and caused the adherence of platelets, confirmed by scanning electron microscopy. These studies demonstrate that IgG anti-endothelial antibodies are present in the sera of patients with active SLE. These sera may also contain IgG complexes that are capable of binding to endothelial cells. The association of IgG and complement with endothelial cells may initiate vascular injury in SLE and other human disorders.

Platelet antibodies of the IgM class in immune thrombocytopenic purpura.

The clinical course and response to therapy of patients with immune thrombocytopenic purpura (ITP) are not completely determined by the level of IgG present on the platelet surface. It is possible that antibodies of other immunoglobulin classes also play a role in platelet destruction in some of these patients. Therefore, we studied 175 patients with ITP for the presence of IgM anti-platelet antibodies using radiolabeled polyclonal or monoclonal anti-IgM. We observed that 57% of patients with clinical ITP had increased levels of IgM on their platelets, compared with normal controls and patients with thrombocytopenia who did not have ITP (less than 10%), (P less than 0.01). We obtained similar results using either radiolabeled polyclonal or monoclonal anti-IgM, reagents whose integrity was first characterized using erythrocytes coated with defined amounts of IgM antibody. Among patients with increased platelet-IgM there was a significant correlation both with the presence of increased platelet-C3 as well as the amount of platelet-C3 (P less than 0.01, r = 0.53). We demonstrated the presence of warm-reacting IgM anti-platelet antibodies in the plasma of two of these patients who were further studied. The isolated IgM fraction from these two plasmas was able to activate complement and place 3H-C3 on normal platelets. These studies demonstrate the presence of warm-reacting IgM anti-platelet antibodies in some patients with ITP. They suggest that the binding of complement to platelets by IgM antibodies may initiate platelet clearance as well as enhance the effect of IgG antibodies in ITP.

Fc and C3 receptors induced by herpes simplex virus on cultured human endothelial cells.

The mechanism by which immune complexes deposit in vascular tissue is uncertain. Several human viruses, including herpes simplex virus, have recently been demonstrated to replicate in human endothelial cells. Such viruses may injure vascular tissue and could play a role in the pathogenesis of immune complex deposition. Therefore, we studied the expression of receptors for immune complexes containing IgG and C3 on endothelial cells after infection with herpes simplex virus type I.Human umbilical vein endothelial cells were incubated with (51)Cr-labeled sheep erythrocytes sensitized with IgG, IgM, or IgM plus complement. Preferential binding of IgG or complement-coated erythrocytes to uninfected endothelial monolayers was not observed. In contrast, significant binding of erythrocytes coated with IgG or IgM plus complement was observed after viral infection. Phase-contrast and scanning electron microscopy demonstrated erythrocyte adherence around the infected endothelial cells in a rosette pattern. Binding of IgG-coated erythrocytes was fully inhibited by Fc (0.31 mg/ml) but not Fab' fragments of nonimmune IgG. Binding of complement-coated cells was unaffected by the presence of IgG (1 mg/ml). With purified individual components, binding of complement-coated erythrocytes depended on the presence and was proportional to the concentration of C3. Binding of IgG-or C3-coated cells was detected beginning 4 h after infection. These studies indicate that herpes simplex virus type I infection can induce IgG and C3 receptors on human endothelial cells. These receptors may promote the deposition of immune complexes in vascular tissue after certain viral infections.

Prothrombotic factors enhance heparin-induced thrombocytopenia and thrombosis in vivo in a mouse model.

BACKGROUND: Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common cause of life- and limb-threatening thrombosis. The development of antibodies that react with complexes of heparin and platelet factor 4 (PF4) is fundamental to the development of the disease. However, anti-PF4/heparin antibodies are far more common than is HIT/T and there is less understanding of the factors that contribute to thrombosis in only a subset of patients. OBJECTIVES: Both qualitative and quantitative differences in multiple factors (e.g. antibodies, heparin and platelets) may influence the clinical course of patients who develop anti-PF4/heparin antibodies. We examined the hypothesis that host-specific factors, such as comorbid prothrombotic conditions, would exacerbate the pathologic effects of anti-PF4/heparin antibodies. METHODS AND RESULTS: A mouse model transgenic for human Fcgamma RIIa and PF4 and null for mouse PF4 was used to study the influence of prothrombotic conditions on the effects of anti-PF4/heparin antibodies in vivo. To simulate a prothrombotic milieu, mice were fed a hypercholesterolemic diet (HD). HD-fed mice had elevated plasma cholesterol, increased platelet reactivity and increased endothelial activation relative to mice fed a standard diet (SD). Age- and sex-matched mice from each diet group were treated with an anti-PF4/heparin antibody and heparin. HD-fed mice developed more severe thrombocytopenia than similarly treated SD-fed mice. Mice with moderate to severe thrombocytopenia had elevated plasma levels of thrombin-antithrombin complexes, indicative of increased thrombin generation in vivo. Platelet-fibrin thrombi were observed in multiple organs of HD-fed mice that developed severe thrombocytopenia. CONCLUSIONS: Host-specific factors, such as prothrombotic changes in platelet reactivity and/or endothelial activation, may influence the development of thrombosis in a subset of patients who develop anti-PF4/heparin antibodies.

Overexpression of urokinase receptor increases matrix invasion without altering cell migration in a human osteosarcoma cell line.

Proteolysis triggered by receptor-bound urokinase-type plasminogen activator (uPA) involves a cascade of species-specific molecular interactions. To study the role of the uPA receptor (uPAR) in such interactions, a human osteosarcoma cell line (HOS), which normally expresses low levels of uPAR, was transfected with human uPAR complementary DNA. One of several stably transformed clonal cells lines, designated 2A2, was characterized and compared to the parental HOS, revealing the following: (a) stable incorporation of uPAR complementary DNA into the genome demonstrated by Southern blot analysis; (b) a 10-fold increase in steady state mRNA levels of uPAR assessed by Northern blot analysis; (c) a 2-fold increase in the surface expression of glycosylphosphatidylinositol anchored uPAR protein determined by enzyme-linked immunosorbent assay and by the specific binding of radiolabeled single chain uPA; (d) a 2-fold increase in internalization and degradation of radiolabeled uPA/PAI-1 complexes; and (e) a 2-fold increase in receptor-bound uPA-mediated plasmin generation measured by the cleavage of a chromogenic substrate and degradation of 125I-labeled laminin. The involvement of uPAR in cellular processes was determined by comparing 2A2 and HOS cells in in vitro migration and invasion assays. The migration of 2A2 cells were slower on fibronectin-coated surfaces in a linear under-agarose assay, but both cell lines migrated at the same rate on uncoated polycarbonate filters in Boyden chamber assays. In the invasion experiments, 4 times more 2A2 than HOS cells penetrated through the barrier of reconstituted basement membrane Matrigel. These data suggest that uPAR does not potentiate random cell migration but facilitates matrix degradation and subsequent cell invasion.

Abciximab readministration: results of the ReoPro Readministration Registry.

BACKGROUND: Platelet glycoprotein IIb/IIIa blockade with abciximab (ReoPro) improves the clinical outcomes of percutaneous coronary intervention. This registry was conducted to characterize the effects of repeated administration of abciximab during intervention. METHODS AND RESULTS: We recruited 500 consecutive patients at 22 centers in the United States who were receiving abciximab for at least a second time during percutaneous coronary intervention. Safety was measured as the incidence of hypersensitivity reactions, major bleeding, and thrombocytopenia. Efficacy was assessed as event-free clinical success. Human antichimeric antibody (HACA) responses were also characterized. There were no cases of hypersensitivity (95% upper confidence bound, 0.3%), major bleeding, or death. Clinical success was 94.4%. Thrombocytopenia occurred in 23 patients (4.6%; 95% CI, 2.8% to 6.4%), including 12 (2.4%; 95% CI, 1.1% to 3.7%) who developed profound thrombocytopenia (<20x10(9) cells/L). In 2 patients (0.4%), profound thrombocytopenia did not develop until after hospital discharge; in 4 (0.8%), profound thrombocytopenia recurred despite platelet transfusion. Before a first readministration, a positive HACA titer was present in 22 of 454 patients (4.8%); after a first readministration, an additional 82 of 432 (19.0%) became HACA-positive. HACA did not neutralize the in vitro inhibition of platelet aggregation by abciximab or correlate with clinical events. CONCLUSIONS: The results, including overall rates of thrombocytopenia, were consistent with randomized clinical trials of first abciximab treatment. However, there was a shift from mild to profound thrombocytopenia, and cases of delayed presentation and of recurrent thrombocytopenia were seen. These findings suggest that indications and guidelines for first-time use apply to retreatment, particularly the systematic monitoring for thrombocytopenia.

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.

Thermotaxis of human trophoblastic cells.

The objective of this study was to determine whether cultured human trophoblasts migrate in response to changes in oxygen tension or temperature. Human trophoblastic cells distributed homogenously within individual wells of standard culture plates were subjected to oxygen and thermal gradients. The redistribution of cells was determined 90 min to 18 h after these gradients had been established. Trophoblastic cells did not migrate in response to gradients of oxygen or carbon dioxide applied in this manner. In contrast, the cells migrated in response to thermal gradients of less than 1 degree C in the direction of the warmer temperature. The response began within minutes, was reversed by a change in the direction of the thermal gradient, and was inhibited at high cell concentrations. Migration was independent of proliferation or protein synthesis, but required microfilament assembly. The capacity of trophoblasts to migrate in response to small difference of temperature within the physiologic range may contribute to the initiation of placental development before contact with the maternal circulation has been established.

Antibodies reactive with surface membranes of cellular elements in the blood.

Autoreactive antibodies or immune complexes may accelerate clearance of mature erythrocytes, leukocytes, and platelets from the circulation in patients with rheumatologic and immunologic disorders. The most compelling evidence for immune injury to hematopoietic cells exists in patients with systemic lupus erythematosus and patients with Felty's syndrome and its variants. These disorders may also cause tissue inflammation, which in turn commonly results in underproduction of erythrocytes and development of thrombocytosis. However, recent evidence indicates that underproduction of hematopoietic cells may also result from immune injury to cellular elements in the bone marrow. In many laboratories, sensitive techniques are now clinically available for the detection of cell-associated immunoglobulin and complement. These assays have helped confirm the role of antibody in the pathogenesis of autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura. However, recent data indicate that there is probably a continuum between the amount of immunoglobulin and complement found on normal cells and that found in a variety of disease states. In several of these disorders, additional evidence will be required to establish that the increase in cell-bound immunoglobulin leads to a decrease in the life-span of the cell. In order to provide significant help to the clinician managing an individual patient, these serologic tests must be capable of identifying the portion of the cell-associated protein actually involved in the destructive process. The availability of monoclonal reagents capable of identifying restricted regions on cell-bound immunoglobulin may help identify molecules bound specifically as antibody and may help identify the antigens involved in autoimmune disorders.

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.

Isolation and characterization of monoclonal antibodies reactive with endothelial cells.

Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function.

Clot penetration and retention by plasminogen activators promote fibrinolysis.

Tissue-type plasminogen activator (tPA) remains the sole thrombolytic approved by the FDA for the treatment of pulmonary embolism (PE). tPA has not been replaced by third generation plasminogen activators, e.g. Reteplase (Ret) and Tenecteplase (TNK) that circulate with longer life-spans and in theory should have more extended potency in vivo. One reason for this paradox is the inability to assign units of activity to plasminogen activators based on specific biologically relevant standards, which impairs objective comparison. Here, we compare clot permeation, retention and fibrinolytic activities of tPA, TNK and Ret in vitro and clot composition over time with outcome in a mouse model of disseminated pulmonary microembolism (ME). When clots were incubated in the continuous presence of drug, tPA, TNK and Ret lysed fibrin clots identically in the absence of PA inhibitor-1 (e.g. PAI-1). Ret, which has lower fibrin affinity and greater susceptibility to inhibition by PAI-1 than tPA, was less effective in lysing plasma clots, while TNK was less effective when the fibrin content of the clots was enhanced. However, when clots were afforded only brief exposure to drug, as occurs in vivo, Ret showed more extensive clot permeation, greater retention and lysis than tPA or TNK. These results were reproduced in vivo in a mouse model of ME. These studies indicate the need for more relevant tests of plasminogen activator activity in vitro and in vivo and they show that clot permeation and retention are important potential predictors of clinical utility.

Interaction of high-molecular-weight kininogen with endothelial cell binding proteins suPAR, gC1qR and cytokeratin 1 determined by surface plasmon resonance (BiaCore).

The physiologic activation of the plasma kallikrein-kinin system requires the assembly of its constituents on a cell membrane. High- molecular-weight kininogen (HK) and cleaved HK (HKa) both interact with at least three endothelial cell binding proteins: urokinase plasminogen activator receptor (uPAR), globular C1q receptor (gC1qR,) and cytokeratin 1 (CK1). The affinity of HK and HKa for endothelial cells are KD=7-52 nM. The contribution of each protein is unknown. We examined the direct binding of HK and HKa to the soluble extracellular form of uPAR (suPAR), gC1qR and CK1 using surface plasmon resonance. Each binding protein linked to a CM-5 chip and the association, dissociation and KD (equilibrium constant) were measured. The interaction of HK and HKa with each binding protein was zinc-dependent. The affinity for HK and HKa was gC1qR>CK1>suPAR, indicating that gC1qR is dominant for binding. The affinity for HKa compared to HK was the same for gC1qR, 2.6-fold tighter for CK1 but 53-fold tighter for suPAR. Complex between binding proteins was only observed between gC1qR and CK1 indicating that a binary CK1-gC1qR complex can form independently of kininogen. Although suPAR has the weakest affinity of the three binding proteins, it is the only one that distinguished between HK and HKa. This finding indicates that uPAR may be a key membrane binding protein for differential binding and signalling between the cleaved and uncleaved forms of kininogen. The role of CK1 and gC1qR may be to initially bind HK to the membrane surface before productive cleavage to HKa.

The spatial dynamics of fibrin clot dissolution catalyzed by erythrocyte-bound vs. free fibrinolytics.

SUMMARY BACKGROUND: Coupling fibrinolytic plasminogen activators to red blood cells (RBCs) has been proposed as an effective, yet safe method of thromboprophylaxis, because of increased circulation lifetime and reduced propensity to induce hemorrhage by selectivity for nascent thrombi rather than pre-formed hemostatic clots. OBJECTIVES AND METHODS: We used confocal microscopy of fluorescently labeled fibrin and erythrocytes in plasma-derived clots to study the spatial dynamics of lysis catalyzed by RBC-coupled vs. free plasminogen activators (RBC-PA vs. PA). RESULTS: Clot lysis catalyzed by free PA progressed gradually and uniformly. In contrast, distinct holes formed surrounding RBC-PA while the rest of the clot remained intact until these holes enlarged sufficiently to merge, causing sudden clot dissolution. Compared with naïve RBCs within clots lysed by free PA, RBC-PA moved faster inside the fibrin network prior to clot dissolution, providing a potential mechanism for spatial propagation of RBC-PA induced lysis. We also showed the focal nature of fibrinolysis by RBC-PA as dense loading of PA onto RBCs initiates more efficient lysis than equal amounts of PA spread sparsely over more RBCs. In an in vitro model of clots exposed to buffer flow, incorporated RBC-PA increased permeability and formed channels eventually triggering clot dissolution, whereas clots containing free PA remained intact. CONCLUSIONS: Clot lysis by RBC-PA begins focally, has a longer lag phase when measured by residual mass than homogeneous lysis by PA, is propagated by RBC-PA motility and provides more effective clot reperfusion than free PA, making RBC-PA attractive for short-term thromboprophylaxis.

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.