CalDAG-GEFI deficiency protects mice from FcγRIIa-mediated thrombotic thrombocytopenia induced by CD40L and ß2GPI immune complexes.

INTRODUCTION: Platelet activation via the Fcγ receptor IIa (FcγRIIa) is implicated in the pathogenesis of immune complex (IC)-mediated thrombocytopenia and thrombosis (ITT). We previously showed that ICs composed of antigen and antibodies targeting CD40 ligand (CD40L) or ß2 Glycoprotein I (ß2GPI) induce ITT in mice transgenic for human FcγRIIa (hFcR) but not wild-type controls (which lack FcγRIIa). Here we evaluated the contribution of the guanine nucleotide exchange factor, CalDAG-GEFI, and P2Y12, key regulators of Rap1 signaling in platelets, to ITT induced by these clinically relevant ICs. METHODS: Pre-formed anti-CD40L or anti-ß2GPI ICs were injected into hFcR/Caldaggef1(+/+) or hFcR/Caldaggef1(-/-) mice, with or without clopidogrel pretreatment. Animals were observed for symptoms of shock for 30 min, during which time core body temperature was monitored. Platelet counts were obtained before and 30 min after IC injection. Lungs were assessed for thrombosis by histology or near-infrared imaging. RESULTS: Both CD40L and ß2GPI ICs rapidly induced severe thrombocytopenia, shock and a reduction in body temperature in hFcR/Caldaggef1(+/+) mice. hFcR/Caldaggef1(-/-) mice were protected from CD40L and ß2GPI IC-induced thrombocytopenia and shock, whereas P2Y12 inhibition had only a modest effect on IC-induced ITT. Consistent with these findings, IC-induced integrin activation in vitro and the accumulation of activated platelets in the lungs of IC-challenged mice was strongly dependent on CalDAG-GEFI. CONCLUSIONS: Our studies demonstrate that CalDAG-GEFI plays a critical role in platelet activation, thrombocytopenia and thrombosis induced by clinically relevant ICs in mice. Thus, CalDAG-GEFI may be a promising target for the intervention of IC-associated, FcγRIIa-mediated thrombotic conditions.

Microparticle association and heterogeneity of tumor-derived tissue factor in plasma: is it important for coagulation activation?

BACKGROUND: Tumor-derived tissue factor (TF) activates coagulation in vitro and in vivo in an orthotopic model of human pancreatic cancer. Here, we further characterized tumor-derived TF in this model. METHODS: Conditioned medium (CM) of L3.6pl human pancreatic tumor cells and plasma from nude mice bearing L3.6pl tumors were ultracentrifuged, and the pellets were filtered through membranes with different pore sizes. The size distribution of particles was analyzed in CM or plasma fractions with nanoparticle tracking and dynamic light scattering. Human TF antigen and activity were measured in pellets and supernatants with ELISA and clotting or thrombin generation assays, respectively. Human alternatively spliced TF (asTF) was measured with ELISA. Human TF and thrombin-antithrombin complex (TAT) concentrations were assessed in plasma of mice injected with filtered fractions of CM. RESULTS: Particles in both CM and plasma were < 0.4 µm. TF antigen and activity in the CM were mainly associated with microparticles (MP). Approximately 50% of antigen and 20% of activity were associated with particles of < 0.1 µm. Injection of < 0.1 µm particles into mice caused a 30% drop in platelet counts and an increase in TAT levels. In contrast, ~ 90% of TF antigen in tumor-bearing mice plasmas was non-sedimentable, whereas TF activity was exclusively associated with MP. Particles of < 0.1 µm and the supernatants of both CM and plasma gained TF activity after addition of exogenous phospholipids. Although asTF was found in MP-free CM supernatants, it was also present in CM and plasma pellets. CONCLUSIONS: Tumor-derived particles of < 0.1 µm and non-sedimentable TF are or can become procoagulant in the presence of phospholipids, and may contribute to the procoagulant potential of circulating TF.

Psychotropic medication use and risk of adverse cardiovascular events in women with suspected coronary artery disease: outcomes from the Women's Ischemia Syndrome Evaluation (WISE) study.

OBJECTIVE: This study investigated the relation between psychotropic medication use and adverse cardiovascular (CV) events in women with symptoms of myocardial ischaemia undergoing coronary angiography. METHOD: Women enrolled in the Women's Ischemia Syndrome Evaluation (WISE) were classified into one of four groups according to their reported antidepressant and anxiolytic medication usage at study intake: (1) no medication (n = 352); (2) anxiolytics only (n = 67); (3) antidepressants only (n = 58); and (4) combined antidepressant and anxiolytics (n = 39). Participants were followed prospectively for the development of adverse CV events (for example, hospitalisations for non-fatal myocardial infarction, stroke, congestive heart failure and unstable angina) or all-cause mortality over a median of 5.9 years. RESULTS: Use of antidepressant medication was associated with subsequent CV events (HR 2.16, 95% CI 1.21 to 3.93) and death (HR 2.15, 95% CI 1.16 to 3.98) but baseline anxiolytic use alone did not predict subsequent CV events and death. In a final regression model that included demographics, depression and anxiety symptoms, and risk factors for cardiovascular disease, women in the combined medication group (that is, antidepressants and anxiolytics) had higher risk for CV events (HR 3.98, CI 1.74 to 9.10, p = 0.001 and all-cause mortality (HR 4.70, CI 1.7 to 2.97, p = 0.003) compared to those using neither medication. Kaplan-Meier survival curves indicated that there was a significant difference in mortality among the four medication groups (p = 0.001). CONCLUSIONS: These data suggest that factors related to psychotropic medication such as depression refractory to treatment, or medication use itself, are associated with adverse CV events in women with suspected myocardial ischaemia.

Bevacizumab immune complexes activate platelets and induce thrombosis in FCGR2A transgenic mice.

BACKGROUND: Treatment with Bevacizumab has been associated with arterial thromboembolism in colorectal cancer patients. However, the mechanism of this remains poorly understood, and preclinical testing in mice failed to predict thrombosis. OBJECTIVE: We investigated whether thrombosis might be the result of platelet activation mediated via the FcgammaRIIa (IgG) receptor - which is not present on mouse platelets - and aimed to identify the functional roles of heparin and platelet surface localization in Bev-induced FcgammaRIIa activation. METHODS AND RESULTS: We found that Bev immune complexes (IC) activate platelets via FcgammaRIIa, and therefore attempted to reproduce this finding in vivo using FcgammaRIIa (hFcR) transgenic mice. Bev IC were shown to be thrombotic in hFcR mice in the presence of heparin. This activity required the heparin-binding domain of Bev's target, vascular endothelial growth factor (VEGF). Heparin promoted Bev IC deposition on to platelets in a mechanism similar to that observed with antibodies from patients with heparin-induced thrombocytopenia. When sub-active amounts of ADP or thrombin were used to prime platelets (simulating hypercoagulability in patients), Bev IC-induced dense granule release was significantly potentiated, and much lower (sub-therapeutic) heparin concentrations were sufficient for Bev IC-induced platelet aggregation. CONCLUSIONS: The prevailing rationale for thrombosis in Bev therapy is that VEGF blockade leads to vascular inflammation and clotting. However, we conclude that Bev can induce platelet aggregation, degranulation and thrombosis through complex formation with VEGF and activation of the platelet FcgammaRIIa receptor, and that this provides a better explanation for the thrombotic events observed in vivo.

Tissue factor-bearing microparticles derived from tumor cells: impact on coagulation activation.

BACKGROUND: Tissue factor (TF)-bearing microparticles (MP) from different origins are thought to be involved in the pathogenesis of cancer-associated thrombosis. However, the role of circulating tumor cell-derived TF is not well understood. METHODS: TF antigen and activity were measured in MP generated in vitro from human TF-expressing cancer cells by ELISA and clotting or thrombin generation assays, respectively. TF antigen and activity were also measured in vivo in cell-free plasmas from mice previously injected with in vitro-generated MP or in cell-free plasmas from nude mice bearing orthotopically injected human cancer cells. RESULTS: Tumor cell-derived MP (TMP) exhibited strong TF-dependent procoagulant activity (PCA) in vitro and in vivo. Injection of TMP into mice was associated with acute thrombocytopenia and signs of shock, which were prevented by prior heparinization. Human TF antigen and activity could be detected in mouse cell-free plasmas up to 30 min after TMP injections. Human TF was detected in the spleen of injected mice and its clearance from circulation was delayed in splenectomized mice, suggesting the involvement of the spleen in the rapid clearance of circulating MP in vivo. Detectable levels of TF-dependent PCA and thrombin-antithrombin complex were found in cell-free plasmas from mice growing pancreatic human tumors, suggesting that circulating tumor-derived TF causes coagulation activation in vivo. CONCLUSIONS: MP derived from certain cancer cells exhibit TF-dependent PCA both in vitro and in vivo. These results provide new information about the specific contribution of tumor-derived MP to the hypercoagulable state observed in cancer.

Enumeration of leukocyte infiltration in solid tumors by confocal laser scanning microscopy.

BACKGROUND: Leukocytes commonly infiltrate solid tumors, and have been implicated in the mechanism of spontaneous regression in some cancers. Conventional techniques for the quantitative estimation of leukocyte infiltrates in tumors rely on light microscopy of immunostained thin tissue sections, in which an arbitrary assessment (based on low, medium or high levels of infiltration) of antigen density is made by the pathologist. These estimates are relatively subjective and often require the opinion of a second pathologist. In addition, since thin tissue sections are cut, no data regarding the three-dimensional distribution of antigen can be obtained. RESULTS: To overcome these problems, we have designed a method to enumerate leukocyte infiltration into tumors, using confocal laser scanning microscopy of fluorescently immunostained leukocytes in thick tissue sections. Using image analysis software, a threshold was applied to eliminate unstained tissue and residual noise. The total antigen volume in the scanned tissue was calculated and divided by the mean cell volume (calculated by "seeding" ten individual cells) to obtain the cell count. Using this method, we compared the calculated leukocyte counts with those obtained manually by ten laboratory personnel. There was no significant difference (P > 0.05) between the cell counts obtained by either method. We then compared leukocyte infiltration into seven tumors and matched non-malignant tissue obtained from the periphery of the resected tissue. There was a significant increase in the infiltration of all leukocyte subsets into the tumors compared to minimal numbers in the non-malignant tissue. CONCLUSION: From these results we conclude that this method may be of considerable use for the enumeration of cells in tissues. Furthermore, since it can be performed by laboratory technical staff, less time input is required by the pathologist in assessing the degree of leukocyte infiltration into tumors.

Experimental metastasis and primary tumor growth in mice with hemophilia A.

During experimental lung metastasis, tumor cells adhere to the pulmonary microvasculature and activate coagulation via surface-expressed tissue factor (TF), leading to local fibrin deposition and platelet aggregation. While interventional studies have demonstrated great efficacy of anticoagulants and antiplatelet agents in inhibiting metastasis, no information is available on how tumor biology may be affected by congenital bleeding disorders such as hemophilia A. We therefore used a syngeneic model to study experimental metastasis and primary tumor growth in factor VIII (FVIII)-deficient mice. By conventional reverse transcription-polymerase chain reaction, flow cytometry, and one-stage clotting assays, we demonstrated constitutive expression of TF mRNA, antigen, and procoagulant activity in the murine B16F10 melanoma cell line. In hemophilic mice, B16F10 lung metastasis was significantly (P < 0.001) enhanced by a single dose of human FVIII (100 U kg(-1)), suggesting that FVIII played a critical role during the early blood-borne phase of the metastatic cascade. In contrast, lung seeding was significantly (P < 0.05) reduced by lepirudin, a direct thrombin inhibitor, suggesting that thrombin generation contributed to pulmonary metastasis even in the absence of FVIII. Consistent with this finding, intravenous injection of B16F10 cell-evoked laboratory changes of a hemolytic thrombotic microangiopathy and consumptive coagulopathy in both hemophilic and non-hemophilic mice. Subcutaneous implantation of B16F10 cells into mice with hemophilia A gave rise to primary tumors in an exponential growth pattern similar to that observed in non-hemophilic mice. Although TF expression by B16F10 cells may promote thrombin-dependent metastasis in mice with hemophilia A, amplification of coagulation by host FVIII appears to be necessary for maximum lung seeding.

Antimetastatic effect of tinzaparin, a low-molecular-weight heparin.

The importance of coagulation activation in cancer patients is suggested by the clinical finding of hypercoagulability, experimental enhancement of metastasis and angiogenesis by coagulation factors such as tissue factor (TF) and thrombin and the possible antitumor effects of anticoagulant agents. Tinzaparin is a low-molecular-weight heparin (LMWH) with a relatively high molecular weight distribution and high sulfate to carboxylate ratio. In addition to its ability to inhibit thrombin and factor Xa, tinzaparin is particularly effective at releasing endothelial tissue factor pathway inhibitor (TFPI), the natural inhibitor of both procoagulant and non-coagulant effects of TF. The present study was undertaken to investigate the effect of tinzaparin on lung metastasis using a B16 melanoma model in experimental mice. Tinzaparin's anticoagulant effect in mice and its ability to release TFPI from human endothelial cells at various time points were demonstrated. Subcutaneous (s.c.) injection of tinzaparin (10 mg kg-1) 4 h before intravenous administration of melanoma cells (2.0 x 105) markedly (89%) reduced lung tumor formation (3 +/- 2) compared with controls (31 +/- 23; P < 0.001). In a second group of animals, tinzaparin (10 mg kg-1, s.c.) administered daily for 14 days following the initial (pretumor cell) dose, before assessment of lung seeding, reduced tumor formation by 96% (P < 0.001). No bleeding problems were observed in any of the tinzaparin-treated animals, despite a 4-fold prolongation of the whole blood clotting time after a single s.c. dose of tinzaparin (10 mg kg-1). Administration of tumor cells (2 x 106) caused a rapid and significant fall in platelet count 15 min after injection (a sensitive marker of intravascular coagulation) in controls (939 +/- 37 vs. 498 +/- 94 x 106 mL-1, P < 0.01), but this was prevented by tinzaparin treatment (921 +/- 104 x 106 mL-1). These data provide further experimental evidence to support the potential for LMWH as antimetastatic agents.

Platelet-CD40 ligand interaction with melanoma cell and monocyte CD40 enhances cellular procoagulant activity.

Platelet-tumor cell interactions are believed to be important in tumor metastasis. Tumor cell tissue factor (TF) expression enhances metastasis and angiogenesis, and is primarily responsible for tumor-induced thrombin generation and the formation of tumor cell-platelet aggregates. Activated platelets express and release CD40 ligand (CD40L), which induces endothelial TF expression by ligation to CD40. We investigated the effect of platelet-derived CD40L on the TF activity of human CD40-positive melanoma cells and monocytes by incubating supernatants from activated or resting platelets with tumor cells or monocytes, and by bringing resting or activated platelets into close apposition with tumor cell monolayers. CD40L was present on the surface of activated (but not resting) platelets and was also released following platelet activation. Both recombinant soluble CD40L (rsCD40L) and activated platelet supernatants increased procoagulant activity (PCA) and TF antigen in tumor cells and monocytes. The increase in TF activity induced by both rsCD40L and activated platelet supernatants was inhibited by anti-CD40L antibody. Furthermore, contact of activated platelets with tumor cells increased cellular PCA, and this effect was also inhibited by anti-CD40L. In malignancy, the increase in cellular TF activity via CD40 (tumor cell)-CD40L (platelet) interaction may possibly enhance intravascular coagulation and hematogenous metastasis.

Hemoglobin binds melanoma cell tissue factor and enhances its procoagulant activity.

Tissue factor (TF), the membrane-bound glycoprotein that normally initiates the coagulation pathway, is expressed on the surface of various cells including endothelial cells, fibroblasts, monocytes and tumor cells. We recently reported that hemoglobin (Hb) enhances TF expression and procoagulant activity on TF-bearing human A375 malignant melanoma cells. To elucidate the mechanism of Hb-induced TF expression, we studied the interaction between purified TF from human A375 malignant melanoma cells and Hb. Selective binding of highly purified melanoma cell TF-apoprotein to Hb was demonstrated under native conditions using a dot-immunobinding assay and under denaturing conditions by Western blotting. The complex formation between purified melanoma cell TF-apoprotein and Hb was also demonstrated by the binding of fluid-phase Hb to immobilized TF-apoprotein (0-2.0 microg/ml) in an enzyme-linked immunosorbent assay. The binding was specific, concentration-dependent, saturable and inhibited significantly (60%) by Concanavalin-A. Hb enhanced the factor X-activating procoagulant activity of melanoma cell TF in a concentration-dependent manner, but had no effect on recombinant human TF. Concanavalin-A and wheat germ agglutinin significantly (60%) inhibited the Hb-induced procoagulant activity of malignant cell TF. We conclude that TF-apoprotein selectively binds Hb, most probably via the carbohydrate moieties (alpha-d-glucosyl; alpha-d-mannosyl and N-acetyl-beta-d-glucosaminyl residues) of TF, and enhances its procoagulant activity. The physiological significance of this interaction remains to be established.

Purification and properties of human melanoma cell tissue factor.

Tissue factor (TF) is a transmembrane glycoprotein that acts as a receptor for nonactivated and activated factor VII (FVII) and triggers the coagulation cascade. TF plays an important role in hemostasis, but may also have noncoagulation functions in vascular development, angiogenesis, and tumor cell metastasis. In tumor cells, analysis of the role of TF has been hampered by the lack of purified TF. In this study, TF antigen was identified on human A375 malignant melanoma cells using flow cytometry. We further purified TF apoprotein 2,000-fold to homogeneity from A375 melanoma cells using immunoaffinity chromatography. On SDS-polyacrylamide gel electrophoresis under reduction, purified TF apoprotein gave two major protein bands corresponding to molecular weights of 53 and 34 to 36 KD. The identity of these forms of TF was confirmed by Western blotting using a polyclonal antibody against human brain TF. Under reduction, the TF antibody bound with a monomeric form of TF (53 KD), and without reduction, to several forms of TF (34 to 128 KD). Preliminary carbohydrate analysis suggested that TF is a glycoprotein and contains about 22% total carbohydrates. The coagulant activity of the purified apoprotein was reconstituted by the addition of phospholipids. The effects of varying concentrations (0 to 8 microg) of polyclonal antibodies to TF and FVII on TF procoagulant activity were studied. Both antibodies inhibited more than 70% of the procoagulant activity of TF in an FX activation assay. The complex formation between purified TF apoprotein and FVIIa was demonstrated by using an enzyme-linked immunosorbent assay. TF formed a complex with FVIIa in a concentration-dependent and saturable manner. We conclude that in human melanoma cells, TF occurs in monomeric and heterodimeric forms and appears to have similar properties as reported for TF from other sources.

Hemoglobin enhances tissue factor expression on human malignant cells.

Tissue Factor (TF) is a transmembrane glycoprotein that complexes with factor VII/activated factor VII to initiate blood coagulation. TF may be expressed on the surface of various cells including monocytes and endothelial cells. Over-expression of TF in human tumor cell lines promotes metastasis. We recently showed that hemoglobin (Hb) forms a specific complex with TF purified from human malignant melanoma cells and enhances its procoagulant activity (PCA). To further study this interaction, we examined the effect of Hb on the expression of TF on human malignant (TF+) cells and KG1 myeloid leukemia (TF-) cells. Human melanoma A375 and J82 bladder carcinoma cells, which express TF at moderate and relatively high levels, respectively, were incubated with varying concentrations (0-1.5 mg/ml) of Hb. After washing, cells were analyzed for Hb binding and TF expression using flow cytometry and confocal microscopy. Hb bound to the cells in a concentration-dependent manner, and increased both TF expression and PCA. The human A375 malignant melanoma cells incubated with Hb (1 mg/ml) expressed up to six times more TF antigen than cells without Hb. This increase in TF expression and PCA of intact cells incubated with Hb was significantly inhibited by cycloheximide at a concentration of 10 microg/ml (P < 0.01). An increase in total cellular TF antigen content was demonstrated by specific immunoassay. In contrast, Hb (5 mg/ml) did not induce TF expression and PCA on KG1 cells as determined by flow cytometry and TF (FXAA) activity. We conclude that Hb specifically binds to TF-bearing malignant cells and increases their PCA. This effect seems to be at least partly due to de novo synthesis of TF and increased surface expression. However, the exact mechanism by which Hb binds and upregulates TF expression remains to be determined.

The relationship between insight and social skill in persons with severe mental illness.

The relationship of insight with the social behaviors of outpatients with severe mental illness (SMI) was investigated. Participants' engaged in two social interactions (i.e., stigmatizing and nonstigmatizing), each with a different research confederate. The participant's behavior was later coded for the presence of various self-presentation and social skill variables. Results indicated that greater insight was associated with better overall social skill, less observed strangeness, and greater self-disclosure of one's mental illness. Furthermore, the three measures of insight, one based on self-report and two interview-based, were all highly intercorrelated, suggesting that they are measuring a similar construct. Finally, consistent with previous research in the area, greater insight was associated with less severe psychiatric symptoms. Implications of these findings for future research are discussed.

One year in Antarctica: mucosal immunity at three Australian stations.

The effect of a year's isolation in Antarctica on the human mucosal immune system was assessed during the winter of 1992 at three Australian Antarctic stations: Casey, Davis and Mawson. Saliva samples were collected from each expeditioner prior to their departure from Australia and during each month in Antarctica. The concentrations of salivary immunoglobulins IgA and IgG were significantly different between the three stations, but there were no differences for salivary IgM and albumin. The mean concentrations of IgA were higher at Mawson (P < 0.008), and the mean concentrations of IgG were lower at Davis (P < 0.001) compared with the other stations. Ranges of values observed at the stations over the 12-13 months were similar. The variability of values within individuals showed station differences for salivary IgM and IgG only. The study revealed significant changes in salivary immunoglobulin values over the period in Antarctica, with similar patterns at the three Australian stations. The salivary IgA and IgM levels were lower in the first 4 months in Antarctica (January-April) and increased to maximum values in July-August, before returning to mean levels when isolation was broken in October-November. The patterns of salivary IgA and IgM suggest that stressors due to isolation may play a role in alterations of mucosal immunity in expeditioners in Antarctica.

Validation of the ISAAC video questionnaire (AVQ3.0) in adolescents from a mixed ethnic background.

BACKGROUND: The International Study of Asthma and Allergies in Childhood (ISAAC) has developed an international version of the asthma video questionnaire (AVQ3.0) to measure asthma prevalence. This questionnaire has not been validated in adolescents from a mixed ethnic background. OBJECTIVE: The aims of this study were to compare the video questionnaire with a written questionnaire in the detection of airway hyperresponsiveness to hypertonic saline in a population of adolescents from a mixed ethnic background, and to establish the repeatability and psychometric properties of the asthma video questionnaire. METHODS: The study was conducted in four secondary schools in Sydney, an area with a high proportion of people from a non-English speaking background. Four hundred and seventy-five students from schools 1 and 2 completed the video questionnaire and a subgroup of these students (n = 170) completed the written questionnaire and a hypertonic saline inhalation challenge. Reproducibility of the questionnaire was evaluated by administering the questionnaire to a subsample of students 2 weeks later. The psychometric properties of the video questionnaire were examined in 852 students at two other schools (schools 3 and 4). RESULTS: One hundred and sixty-nine students aged 13.5 (sd 1.3) years completed both written and video questionnaires, and the hypertonic saline challenge. The students had widely different cultural backgrounds including Asian, South Pacific, Middle Eastern, European and African countries. There was good agreement between the questionnaires for wheeze (kappa 0.42). Questions on the video questionnaire concerning wheezing had good sensitivity (90%) and specificity (68%) for airway hyperresponsiveness to hypertonic saline. The video questionnaire was reproducible (kappa 0.82), had good internal consistency (Cronbach's alpha 0.81) and each question pertained to a single construct explaining 58% of the variance in total score. CONCLUSION: This study has validated the international version of the ISAAC video questionnaire against airway hyperresponsiveness to hypertonic saline in adolescents from a mixed ethnic background, and identified that the questionnaire has good psychometric properties. The ISAAC video has proved to be a valuable tool for the assessment of asthma prevalence in populations of ethnic diversity.

Platelet dysfunction detected at high shear in patients with heart valve disease.

Whether patients with valvular heart disease have a defect of platelet function has been unclear. Despite evidence that these individuals have an abnormality detectable only under conditions of high shear stress, no methods have been widely available to adequately assess platelet function under such conditions. The Platelet Function Analyzer (PFA)-100 measures platelet function in a high shear environment and is well suited to the detection of platelet dysfunction in the clinical laboratory. The instrument records the time for platelets to occlude a membrane coated with collagen and either epinephrine (CEPI) or ADP (CADP). We studied the PFA-100 in 398 patients before open heart surgery; 308 for coronary artery bypass grafting (CABG) and 90 for aortic or mitral valve replacement (VR). Patients were classified as normal (CEPI < or = 153 s); 'aspirin effect' (CEPI > 153 s but CADP < or = 109 s) or abnormal (CEPI > 153 s and CADP > 109 s). In the CABG group, 41.2% were classified as normal, 43.2% as 'aspirin effect' and 15.6% as abnormal. In contrast, in patients undergoing VR, these values were 6.7, 11.1 and 82.4%, respectively. Patients with valvular disease had significantly longer closure times for both CEPI and CADP tests (P < 0.001). In addition, the valvular disease group had a significantly higher proportion of patients with markedly prolonged (> 150 s) closure times in the CADP cartridge (43.3 vs. 3.6%, respectively). Only one (0.3%) patient in the CABG group had non-closure (> 300 s) in the CADP test compared to seven (7.8%) in the valvular disease group. Three of six patients in the latter group bled excessively during surgery. We conclude that abnormal CADP closure is much more frequent among patients with aortic or mitral valve disease compared to those with coronary artery disease. This may reflect pre-existing high-shear damage to platelets that renders them refractory to subsequent shear activation and aggregation in the PFA-100 system. Further studies are needed to more precisely define the platelet defect in these patients. Markedly prolonged CADP closure in patients with valvular disease may indicate an increased likelihood of intra-operative bleeding, although an appropriately designed prospective study is needed to adequately address this hypothesis.

Immune status and respiratory illness for elite swimmers during a 12-week training cycle.

The impact of a 12-week training program by elite swimmers on systemic and mucosal immunity was studied prospectively to examine the relationship between changes in immune parameters and the incidence of respiratory illness. Saliva was collected before and after selected training sessions at 2 weekly intervals. There were significant decreases in salivary IgA (p=0.05) and salivary IgM (p < 0.0001) concentrations after individual training sessions, but no significant changes in salivary IgG or albumin concentrations. Over the 12-week training program there were small but statistically significant increases in pre-exercise concentrations of salivary IgA (p<0.001), IgM (p=0.015) and IgG (p=0.003) and post-exercise salivary IgA (p <0.001). There were no significant trends over the 12 weeks for any class of serum immunoglobulins but a significant fall in NK-cell numbers (p<0.001). There were no associations between serum or salivary immunoglobulin levels or NK-cell numbers and upper respiratory tract illness (URTI) during the 12-week program. The data indicated that despite changes in some immune parameters during this final training program prior to competition there were no associations detected with URTI for this cohort of elite swimmers.