Biomedical subjects

E L Yeo

Publications and source records attributed to E L Yeo.

At least 19 recordsLinked to original sources

PubMed2001

Use of statins and the subsequent development of deep vein thrombosis.

BACKGROUND: Some of the benefit of statins for the prevention of cardiovascular disease may be due to their antithrombotic properties. Little is known about the effect of these drugs on the development of deep vein thrombosis. MATERIALS AND METHODS: We conducted a retrospective cohort study over an 8-year period by linking Ontario provincial health care administrative databases covering more than 1.4 million Ontario residents aged 65 years or older. We excluded those with a documented history of atherosclerosis, venous thromboembolism, or cancer within 36 months prior to study enrollment, as well as those prescribed warfarin sodium within 12 months before enrollment. In the primary cohort, we evaluated the subsequent risk of deep vein thrombosis (DVT) among men and women prescribed thyroid replacement therapy, nonstatin lipid-lowering agents, or statins. A second cohort of women only was evaluated in a similar fashion, but estrogen use was added as a third comparison drug group. RESULTS: There were 125 862 men and women in the primary cohort. After adjusting for age; sex; prior hospitalization; newly diagnosed cancer; or prescribed aspirin, warfarin, or estrogen, statin users (n = 77 993) had an associated decreased risk of DVT relative to those prescribed thyroid replacement therapy (n = 35 978) (adjusted hazard ratio [HR], 0.78; 95% confidence interval [CI], 0.69-0.87). Compared with thyroid replacement therapy, users of nonstatin lipid-lowering agents (n = 11 891) did not seem to be at lower risk for deep vein thrombosis (HR, 0.97; 95% CI, 0.79-1.18). In the secondary cohort of 89 508 women, after adjusting for age, prior hospitalization, newly diagnosed cancer, or prescribed aspirin or warfarin, estrogen users (n = 29 165) had an associated increased risk for DVT compared with those receiving thyroid replacement therapy (n = 22 118) (HR, 1.16; 95% CI, 1.01-1.33), while statin users had an associated decreased risk (HR, 0.68; 95% CI, 0.59-0.79). Nonstatin lipid-lowering agents (n = 5155) were not associated with a reduced risk of DVT compared with thyroid replacement therapy (HR, 0.84; 95% CI, 0.63-1.12). CONCLUSION: Among selected individuals aged 65 years or older, statins were associated with a 22% relative risk reduction in the risk of DVT. A randomized clinical trial is needed to evaluate the efficacy of statins for the primary and secondary prevention of DVT.

Aged↗

PubMed2001

Thrombogenicity of radiofrequency ablation procedures: what factors influence thrombin generation?

AIMS: Thromboembolic complications have been reported after radiofrequency ablation but the low incidence of overt clinical events has been a limitation to the study of factors affecting thrombogenic risk. The aim of this study was to determine whether radiofrequency ablation has a procoagulant effect and to examine variables that affect thrombio generation. METHODS AND RESULTS: Thirty-seven consecutive patients who underwent radiofrequency ablation were studied prospectively. Blood samples were assayed for thrombin-antithrombin III (TAT) and d-dimer (DD) at five different time points: (1) baseline; (2) after sheath insertion; (3) after electrophysiological study but before radiofrequency ablation; (4) at completion of the procedure; and (5) 24 h post-procedure. TAT levels were within the normal range at baseline and increased significantly after sheath insertion from 2.1 +/- 1.2 microg l(-1) to 13.3 +/- 16.0 microg l(-1) (P<0.01). Levels increased further to 24.0 +/- 19.9 microg l(-1) (P<0.01) after electrophysiological study but did not increase after radiofrequency ablation. TAT normalized at 24 h. DD increased significantly from baseline values (230.2 +/- 176.8 ng ml(-1)) to 285.4 +/- 237.4 ng ml(-1) (P=0.019) after sheath insertion. There was a further significant increase after electrophysiological study to 423.4 +/- 324.3 ng ml(-1) (P<0.01), and a slight but non-significant increase to 464.4 +/- 307.4 ng ml(-1) after radiofrequency ablation (P=0.159). DD remained elevated at 24 h. Procedure duration was the only variable that correlated with the relative increase in TAT and DD. The patients with the longest procedure durations had more catheters inserted, more radiofrequency applications and largely consisted of accessory bypass tract-mediated tachycardias. Heparin administration significantly blunted the relative increase in TAT after radiofrequency ablation (P=0.005). CONCLUSION: Radiofrequency ablation procedures confer an increased risk of thrombosis. Catheterization and diagnostic study contribute largely to the thrombogenic stimulus. Thrombogenicity is increased in prolonged, complex procedures and is decreased in patients who have been administered heparin during the procedure.

Adult↗

PubMed2000

A comparison of two regimens of intravaginal misoprostol for termination of second trimester pregnancy: a randomized comparative trial.

A prospective randomized trial was conducted in 148 women to compare the efficacy of two regimens of vaginal misoprostol for termination of second trimester pregnancy. Women aged 16-40 years requesting termination of second trimester pregnancy were randomized into two groups. Women in group 1 were given vaginal misoprostol 400 microg every 3 h for a maximum of five doses in 24 h. Women in group 2 were given vaginal misoprostol 400 microg every 6 h for a maximum of three doses in 24 h. If women did not abort in 24 h, the same regimen was repeated. The median induction-abortion interval in group 1 (15.2 h) was significantly shorter (P < 0.01) than that in the group 2 (19.0 h). The percentage of women who achieved successful abortion within 48 h in group 1 (90.5%) was also significantly higher (P < 0.02) than that in group 2 (75.7%). The incidence of fever was more common in group 1 (P = 0.01). It is concluded that the regimen of vaginal misoprostol 400 microg every 3 h with maximum of five doses in 24 h was more effective than the regimen of misoprostol every 6 h in termination of second trimester pregnancy.

Abortifacient Agents, Nonsteroidal↗

PubMed1999

Flow cytometric study of in vitro neutrophil activation by biomaterials.

Neutrophil activation for adherent and nonadherent cells, as measured by flow cytometry, was not strongly dependent on material surface chemistry. We had hypothesized that material-induced neutrophil activation was an important parameter associated with material failure. All materials tested [cellophane, an acrylonitrile copolymer (AN69), Pellethane, nylon, polyethylene terephthalate, low density polyethylene, and polydimethylsiloxane] activated isolated human neutrophils, which were resuspended in plasma or serum, to similar extents based on L-selectin shedding, CD11b upregulation, and stimulation of the oxidative burst after 30-min exposure. Inhibition of complement activation by sCR1 unexpectedly had little effect if any on nonadherent neutrophils. However, neutrophil adhesion, but not the level of activation of the adherent cells, was strongly dependent on complement activation. Pretreatment with albumin did not inhibit adhesion or reduce neutrophil activation, but plasma pretreatment resulted in increased activation for nonadherent and adherent cells. More adhesion and a higher level of activation of adherent cells was observed following pretreatment with fibrinogen, a ligand of CD11b. Taken together these results suggest that upon contact with a material, neutrophil activation may occur though mechanisms that are not mediated by complement. For example, the presence of plasma proteins such as fibrinogen at the interface may trigger activation and the release of other activating agents. Although the material differences are small, the extent of activation may be significant and warrant further study of the mechanism and consequences of that activation.

Acrylic Resins↗

PubMed1999

CD109 is expressed on a subpopulation of CD34+ cells enriched in hematopoietic stem and progenitor cells.

CD109 is a monomeric cell surface glycoprotein of 170 kD that is expressed on endothelial cells, activated but not resting T-lymphocytes, activated but not resting platelets, leukemic megakaryoblasts, and a subpopulation of bone marrow CD34+ cells. Observing an apparent association between CD109 expression and the megakaryocyte lineage (MK), we sought to determine whether CD109 was expressed on MK progenitors. In fetal bone marrow (FBM), a rich source of MK progenitors, CD109 is expressed on a mean of 11% of CD34- cells. Fluorescence activated cell sorting (FACS) of FBM CD34+ cells into CD109+ and CD109- fractions revealed that the CD34+CD109+ subset contained virtually all assayable MK progenitors, including the colony-forming unit-MK (CFU-MK) and the more primitive burst-forming unit-MK (BFU-MK). The CD34+CD109+ subset also contained all the assayable burst-forming units-erythroid (BFU-E), 90% of the colony-forming units-granulocyte/macrophage (CFU-GM), and all of the more primitive mixed lineage colony-forming units (CFU-mix). In contrast, phenotypic analysis of the CD34+CD109- cells in FBM, adult bone marrow (ABM) and cytokine-mobilized peripheral blood (MPB) demonstrated that this subset comprises lymphoid-committed progenitors, predominantly of the B-cell lineage. CD109 was expressed on the brightest CD34 cells identifiable not only in FBM, but also in ABM and MPB indicating that the most primitive, candidate hematopoietic stem cells (HSC) might also be contained in the CD109+ subset. In long-term marrow cultures of FBM CD34+ cells, all assayable cobblestone area forming cell (CAFC) activity was contained within the CD109+ cell subset. Further phenotypic analysis of the CD34+CD109+ fraction in ABM indicated that this subset included candidate HSCs that stain poorly with CD38, but express Thy-1 (CD90) and AC133 antigens, and efflux the mitochondrial dye Rhodamine 123 (Rho123). When selected CD34+ cells were sorted for CD109 expression and Rho123 staining, virtually all CAFC activity was found in the CD109+ fraction that stained most poorly with Rho123. CD34+ cells were also sorted into Thy-1 CD109+ and Thy-1 CD109+ fractions and virtually all the CAFC activity was found in the Thy-1+CD109+ subset. In contrast, the Thy-1-CD109+ fraction contained most of the short-term colony-forming cell (CFC) activity. CD109, therefore, is an antigen expressed on a subset of CD34+ cells that includes pluripotent HSCs as well as all classes of MK and myelo-erythroid progenitors. In combination with Thy-1, CD109 can be used to identify and separate myelo-erythroid and all classes of MK progenitors from candidate HSCs.

Adult↗

PubMed1999

The fibrinogen gamma chain dodecapeptide inhibits agonist-induced aggregation of rabbit platelets and fibrinogen binding to rabbit glycoprotein IIb-IIIa.

The HHLGGAKQAGDV (H12) sequence at the carboxyl termini of the y chains and the RGD sequences in the Aalpha chains of human fibrinogen are potential recognition sites for the binding of soluble fibrinogen to glycoprotein IIb-IIIa (GPIIb-IIIa) on activated human platelets. Thus, addition of either H12 or RGD-containing peptides inhibits aggregation of and fibrinogen binding to human platelets. In contrast, we reported previously that RGDS had relatively little inhibitory effect on these functions of rabbit platelets. In the present study, we found that H12 inhibited ADP- and thrombin-induced aggregation of rabbit platelets in a dose-dependent manner. Specificity was demonstrated by the failure of the variant HHLGGAKQAGEV peptide to inhibit ADP-induced aggregation. Furthermore, flow cytometric analyses demonstrated that H12 inhibited the binding of FITC-fibrinogen to ADP-activated rabbit platelets in a dose-dependent manner. To examine the direct interaction of H12 with rabbit GPIIb-IIIa, we performed affinity chromatography by applying an octylglucoside extract of rabbit platelet proteins onto an affinity matrix containing the fibrinogen gamma chain sequence. Proteins of approximately 135 kDa and approximately 95 kDa were specifically eluted by soluble H12, and the 95 kDa protein band was immunoblotted by anti-LIBS1, a monoclonal antibody against human GPIIIa. In control samples, no detectable protein from rabbit platelet lysates was eluted from an RGD affinity matrix by GRGDSP. Collectively, our results demonstrated that H12 inhibits aggregation of and fibrinogen binding to rabbit platelets by directly interacting with rabbit GPIIb-IIIa. These findings suggest that rabbit platelets would serve as a suitable thrombosis model for testing the efficacy of peptide mimetics derived from H12.

Animals↗

PubMed1998

The accuracy of intraoperative frozen section in the diagnosis of ovarian tumors.

A retrospective study of 316 ovarian neoplasms which had frozen section evaluation between January 1, 1990 to December 31, 1995 was conducted to determine the accuracy of frozen section diagnosis of ovarian neoplasms. The frozen section results were compared with final diagnoses from paraffin sections. The frozen section diagnosis was accurate in 95.2% of all cases and inaccurate in 4.8%. The positive predictive value of a positive (or malignant) frozen section was 100%, the negative predictive value of a negative (or benign) frozen section was 98.2%. The sensitivity for malignant tumors as 87%. For tumors of borderline malignancy, the sensitivity and specificity were 60% and 98.6% respectively. Of the false negative frozen section diagnoses, 73% (8 cases) occurred in tumors of borderline malignancy. We concluded that with the exception of the sensitivity for the diagnosis of tumors of borderline malignancy, the sensitivity and specificity of frozen section diagnosis for benign and overtly malignant ovarian neoplasms are high.

Adolescent↗

PubMed1998

Ethanol inhibits thrombin-induced secretion of the contents of human platelet dense and alpha-granules and lysosomes.

Moderate consumption of alcoholic beverages is associated with a reduction in thromboembolic complications of coronary artery disease, possibly partially attributable to inhibition by ethanol of platelet responses to some aggregating agents. Although ethanol is known to inhibit thrombin-induced secretion of platelet dense granule contents, the effect of ethanol on secretion of alpha-granule and lysosomal contents has not been studied. Using suspensions of washed platelets, and a range of thrombin concentrations (up to 0.1 U/ml), we examined the effect of 87 mM ethanol on secretion of [14C]serotonin from prelabelled platelets as a measure of secretion of dense granule contents. Secretion of alpha-granule and lysosomal contents was examined by flow cytometric measurement of the surface expression of CD62P (P-selectin) and CD63, respectively. Secretion of the lysosomal enzyme, beta-N-acetylglucosaminidase was also quantified. Results were expressed as % of maximum response induced by 1 U/ml thrombin. Ethanol inhibited the thrombin-induced secretion of both dense and alpha-granule contents (P <0.001, 2-way ANOVA), and of lysosomal contents (P <0.005 for CD63 expression and P <0.001 for beta-N-acetylglucosaminidase secretion). When platelets were pretreated with aspirin, thrombin-induced secretion of storage granule and lysosomal contents was slightly inhibited, but secretion was inhibited by ethanol to the same extent as the untreated platelets, indicating that this inhibition was independent of thromboxane A2. Surface expression of CD63 occurred at lower thrombin concentrations than those required for secretion of beta-N-acetylglucosaminidase, possibly due to the presence of some CD63 on granule membranes. Although the role of lysosomal contents in thrombus formation is not established, some constituents of storage granules are known to augment thrombus formation; ethanol's inhibition of their secretion by stimulated platelets may contribute to its beneficial effect on thromboembolism.

Blood Platelets↗

PubMed1997

Flow cytometric analysis of material-induced platelet activation in a canine model: elevated microparticle levels and reduced platelet life span.

Assessment of material-induced platelet activation is important given that it is thought to be a major mechanism of biomaterials thrombogenicity. We monitored, by flow cytometry, platelet microparticle (MP) levels in the circulation during the connection of polyvinyl alcohol (PVA) hydrogel and polyethylene (PE) test segments (3.18 mm ID, 20 and 50 cm L) to our chronically shunted beagle dogs. We report that circulating microparticle levels were dependent on test segment material, length, and time. The connection of 50-cm lengths of PVA hydrogel test segments led to MP levels two to three times greater than background at 48 h, while the connection of polyethylene test segments did not lead to elevated microparticle levels. MP levels were near background 24 h after removal of the PVA test segment. To determine platelet life span during the connection of test segments, platelets were labeled in vivo with biotin and their disappearance monitored flow cytometrically. While platelet life span for shunted dogs (no test segment) was 4.7 +/- 0.2 days, the connection of PVA hydrogel test segments led to a platelet life span of < 2 days.

Animals↗

PubMed1996

Conditions influencing release of granule contents from human platelets in citrated plasma induced by ADP or the thrombin receptor activating peptide SFLLRN: direct measurement of percent release of beta-thromboglobulin and assessment by flow cytometry of P-selectin expression.

Contrary to a recent report [Rinder et al.: Blood 82:505, 1993], aspirin does inhibit the release of alpha-granule contents as well as inhibiting the release of dense granule contents by human platelets during ADP-induced aggregation in citrated platelet-rich plasma (PRP). Measurements were: percent release of 14C-serotonin from prelabeled platelets, radio-immunoassay of beta-thromboglobulin (beta TG), and expression on the platelet surface of the alpha-granule constituent, P-selectin, by flow cytometry. During the second phase of ADP-induced aggregation, 69.0 +/- 8.3% of beta TG and 54.1 +/- 4.6% of 14C-serotonin were released (mean +/- SEM, n = 13); aspirin treatment reduced these values to 6.0 +/- 1.2 and 1.0 +/- 0.3%, respectively. In contrast, incubation of platelets with ADP without stirring caused only 6.7 +/- 1.7% release of beta TG and 2.1 +/- 0.4% release of 14C-serotonin; these low values were not appreciably affected by aspirin. During ADP-induced primary aggregation in PRP anticoagulated with FPRCH2CI (PPACK), only 4.7 +/- 0.9% release of beta TG and no detectable release of 14C-serotonin occurred; aspirin had no effect. In both stirred and unstirred PRP, the thrombin receptor activating peptide, SFLLRN (50 microM), caused at least 75% release of the contents of both granules, which was partially inhibited by aspirin. Upon incubation of platelets with ADP (2-10 microM), the mean fluorescence intensity due to P-selectin was < 14% of that induced by SFLLRN. In this unstirred system used for flow cytometry, aspirin treatment caused no significant inhibition of P-selectin expression. Thus, under conditions in which ADP does not cause secondary aggregation (physiological Ca2+ concentration or unstirred citrated PRP) release of the contents of both types of granules is less than 7% and aspirin is not inhibitory; the P-selectin expression associated with this low percent release is also unaffected by aspirin. However, aspirin does strongly inhibit the extensive release of both alpha-granule and dense granule contents during ADP-induced secondary aggregation in citrated PRP.

Adenosine Diphosphate↗

PubMed1996

Material-induced up-regulation of leukocyte CD11b during whole blood contact: material differences and a role for complement.

Material-induced thrombogenicity is in part a consequence of leukocyte activation. To evaluate and compare material-induced platelet damage, we have expanded our in vitro flow cytometric immunoassay to include assessment of leukocyte activation. We have used a very simple system whereby fresh, heparinized whole blood contacts materials for 1 h at 37 degrees C under low shear. Unlike other tests that focus on adherent leukocytes, this assay evaluates the leukocytes in the whole blood drained from the tube (1.57 mm internal diameter, 25 cm length) after material contact. We demonstrate that whole blood contact with a polyvinyl alcohol (PVA) hydrogel surface leads to a twofold up-regulation in CD11b surface expression of all monocytes and neutrophils. The activation is metal-ion dependent and highly material dependent in that blood contact with polyethylene and Silastic surface leads to minimal activation. The shedding of L-selection as a marker of leukocyte activation was found to be unsuitable in our assay given it ease of shedding in resting heparinized whole blood. Further, plasma levels of complement components Bb and sC5b-9 (ELISA assays) were significantly elevated only after blood contact with PVA hydrogel surfaces (9.4 micrograms/mL sC5b-9 and 9.6 micrograms/mL Bb). Use of recombinant soluble human CR1 (sCR1) to inhibit the action of the C3 and C5 convertases completely inhibited sC5b-9 levels in whole blood after contact with PVA hydrogel surfaces and inhibited CD11b up-regulation by over 70%, suggesting that material-induced leukocyte activation is partially mediated by C5a production.

Biocompatible Materials↗

PubMed1996

Identification of CD34+ subsets after glycoprotease selection: engraftment of CD34+Thy-1+Lin- stem cells in fetal sheep.

Epitopes on the CD34 molecule detected by some CD34 antibodies can be cleaved by a unique glycoprotease from Pasteurella haemolytica, which cleaves only glycoproteins rich in O-linked glycans. A method to isolate CD34+ cells from adult bone marrow was developed subsequently, in which CD34+ cells were isolated in high purity and yield following immunomagnetic bead selection and detachment with the glycoprotease. Using a variety of other cell-surface markers shown here to be insensitive to glycoprotease, committed progenitors of T lymphoid, B lymphoid, monomyeloid, megakaryoblastic, or erythroid lineages could be identified. Significantly, candidate hematopoietic stem cells (HSC) that are contained within a CD34+Lin- (CD2-, CD14-, CD15-, CD16-, CD19-) (or CD34+CD38-) subset expressing the Thy-1 antigen (CDw90), c-kit receptor (CD117), and CDw109 but lacking expression of CD71 and HLA-DR antigens also were detected. Functionally distinct subsets of glycoprotease-selected CD34+ cells were identified and subfractionated using flow cytometry and fluorescence-activated cell sorting (FACS). These subsets included candidate HSCs expressing the CD34+Thy-1+Lin- phenotype, which were sorted from a CD34+ fraction of a mobilized peripheral blood (MPB) sample. In a fetal sheep model, when CD34+Thy-1+Lin- cells were injected intraperitoneally, they were capable of homing to the marrow, where they generated long-term multilineage hematopoiesis and maintained human CD34+ cells, indicating that candidate HSC subsets of CD34+ cells selected with this highly specific enzyme were capable of engraftment in vivo. The ability to identify and purify virtually any phenotypically defined subset of glycoprotease-selected CD34+ stem/progenitor cells should facilitate the study of hematopoiesis in vitro and in animal models in vivo as well as the development of novel genetic techniques for the correction of specific blood cell disorders in humans.

Adult↗

PubMed1995

Intravenous cocaine induces platelet activation in the conscious dog.

BACKGROUND: Cocaine consumption has been associated with thrombosis of coronary and peripheral arteries. Since cocaine has been found to induce platelet activation in vitro, we sought to establish whether cocaine induced platelet activation in vivo. METHODS AND RESULTS: Chronically instrumented, conscious dogs were infused with cocaine (1 mg/kg), norepinephrine (0.2 to 0.4 mg/kg), or saline intravenously over 1 minute. Activated canine platelets were identified in whole blood collected from an indwelling aortic catheter by flow cytometric detection of the binding of a monoclonal antibody directed against the activation-dependent antigen P-selectin. Infusion of cocaine resulted in an elevation of mean arterial pressure (91 +/- 3 to 128 +/- 7 mm Hg [P < .01]) and heart rate (87 +/- 9 to 125 +/- 11 beats per minute [P < .01]). A similar change (P = NS) in mean arterial pressure followed norepinephrine infusion (100 +/- 5 to 137 +/- 13 mm Hg [P < .04]), whereas saline infusion had no effect. Cocaine resulted in a substantial but delayed increase in platelet P-selectin expression (14 +/- 7% [P < .08], 31 +/- 12% [P < .04], and 55 +/- 22% [P < .04] at 17, 22, and 27 minutes after drug infusion, respectively). The magnitude of this increase was similar to that found in blood treated ex vivo with the agonists ADP or PAF (23 +/- 7% and 53 +/- 15%, respectively). No significant increase in P-selectin expression was detected in the blood of animals that received norepinephrine or saline. Serum cocaine concentrations were highest immediately after infusion (538 +/- 55 ng/mL at 2 minutes) but declined rapidly (185 +/- 22 and 110 +/- 25 ng/mL at 17 and 32 minutes after infusion); in contrast, the increase in benzoylecgonine concentrations was delayed (from < 25 ng/mL in all but one animal [34 ng/mL] at 2 minutes to 46 +/- 4 and 71 +/- 11 ng/mL at 17 and 32 minutes, respectively, after infusion). CONCLUSIONS: Intravenous cocaine induces activation of individual circulating platelets; this effect is not reproduced by infusion of norepinephrine at doses sufficient to exert similar hemodynamic effects. The delay in detection of activated platelets after treatment with cocaine may result from the adhesion and subsequent detachment of activated platelets; alternatively, cocaine metabolites, rather than the drug itself, may induce platelet activation.

Animals↗

PubMed1995

Platelet activation in whole blood by artificial surfaces: identification of platelet-derived microparticles and activated platelet binding to leukocytes as material-induced activation events.

Because the lack of thromboresistant vascular biomaterials is in part due to platelet activation, we have attempted, by using fluorescence-activated flow cytometry, to fully characterize the platelet population after in vitro material contact with whole blood. We have used a very simple, near-physiologic system whereby whole blood, anticoagulated with D-phenylalanyl-L-prolyl-arginyl chloromethyl ketone (thrombin inhibitor), contacts materials for 1 hour at 37 degrees C, under low shear. Unlike other tests of platelet compatibility that focus on adherent platelets, this assay evaluates the platelets in the whole blood drained from the tube (1.57 mm internal diameter, 25 cm length) after material contact. We demonstrate for the first time significant materials-induced microparticle formation. One-hour contact with Silastic, polyethylene, and polyvinyl alcohol hydrogel surfaces lead to 30 +/- 1, 33 +/- 4, and 43 +/- 4 x 10(9) microparticles/L, respectively, whereas resting blood samples contained only 10 +/- 1 x 10(9) microparticles/L. In addition, significant increases in activated platelet(s) binding to neutrophils/monocytes after material contact were noted for all surfaces tested. For polyvinyl alcohol hydrogel surfaces a greater than 500% increase in the fluorescent intensity over that of resting whole blood was attained. The addition of monoclonal antibodies to GPIIb/IIIa (A2A9), the tetrapeptide adhesion ligand RGDS (arginine-glycine-aspartate-serine), or the calcium ion chelator ethyleneglycol-bis-(B-aminoethyl-ether)- N,N,N',N'-tetraacetic acid to the whole blood before material contact fully inhibited all platelet reactivity noted for all surfaces--platelet microparticles, platelet P-selectin expression, loss of platelets from bulk, and the formation of platelet/leukocyte aggregates--thereby indicating that material-induced platelet activation is a calcium-dependent process involving GPIIb/IIIa receptors.

Antibodies↗

PubMed1995

Elevated platelet-derived microparticle levels during unstable angina.

OBJECTIVE: To use fluorescent-activated flow cytometry coupled with activation-dependent and -independent platelet-specific monoclonal antibodies in a pilot study to assess the degree and time course of platelet activation events in patients presenting within 24 h of onset of Canadian Cardiovascular Society class 4 angina. BACKGROUND: Although activated platelets play a key role in the pathogenesis of unstable angina, the development of simple assays to quantify platelet activation events directly is lacking. METHODS: Blood samples were drawn from six unstable angina patients from the coronary care every 4 h over a 24 h period into a fixative and analyzed the following day. All patients were on acetylsalicylic acid and heparin. Comparisons were made with six healthy, medication-free volunteers. Platelets were defined by flow cytometry as positive for fluorochrome-labelled monoclonal antibody to glycoprotein Ib (AP1) and within the single intact platelet window defined by scatter characteristics. The presence of the fluorochrome-labelled activation-specific monoclonal antibody (KC4.1 for anti-P-selectin, PAC-1 for activated glycoprotein IIb/IIIa) was used to determine the percentage of activated platelets. Platelet activation-dependent microparticles were identified by gating on AP1-positive events and defining microparticles (percentage of total platelet events) as being smaller (forward size scatter) than single intact platelets. RESULTS: There was a marked, sixfold increase in microparticle generation (17 +/- 7% versus 2.8 +/- 1.4%) in the unstable angina patients (P = 0.001) compared with healthy volunteers. Further assessment of six coronary care unit patients with nonischemic cardiac disorders demonstrated a highly variable intermediate level of microparticle generation (11 +/- 7%). No differences in activated glycoprotein IIb/IIIa expression were noted for the various groups and P-selectin expression was lower in the unstable angina patients (6 +/- 2% versus 12 +/- 3%, (P = 0.007). CONCLUSIONS: This pilot study suggests that measuring circulating platelet microparticle levels is a simple yet useful parameter for the assessment of platelet activation in unstable angina compared with activation markers on intact whole platelets. Despite antiplatelet and antithrombin therapy, significant platelet activation occurred in these patients over the initial 24 h. Flow cytometry may be a useful tool in assessing the efficacy of newer therapeutic modalities.

Adult↗

PubMed1994

Role of P-selectin and leukocyte activation in polymorphonuclear cell adhesion to surface adherent activated platelets under physiologic shear conditions (an injury vessel wall model).

Carbohydrate moieties on leukocytes adhere to activated platelets via P-selectin under static binding condition studies. We characterize polymorphonuclear cell (PMN) surface interactions with surface adherent platelets and the PMNs response, under physiologic flow conditions corresponding to a shear of 100 s-1, in an in vitro flow chamber. Fluorescent labeled PMNs with red blood cells were drawn through a transparent flow channel and visually quantitated over 30 minutes, interacting with a confluent monolayer of activated, shear-spread platelets expressing P-selectin. PMN adhesion was saturable (2,250 +/- 350/mm2), and time and cation (Ca2+, Mg2+) dependent, and PMNs did not bind to the experimental surface in the absence of a platelet monolayer. P-selectin antibodies completely abolished PMN adhesion in a concentration-dependent manner with half inhibition at 70 micrograms/mL. Antibodies to a putative P-selectin receptor CD15 (80H5 and MMA) maximally inhibited PMN adhesion by 73% and 10%, respectively. Adherent PMNs appeared morphologically activated and flow cytometric analysis of adherent PMNs confirmed activation because CD11b and CD18 surface expression was upregulated (100% and 27%, respectively), whereas L-selectin was downregulated (55%) compared with control nonadherent PMNs. In the presence of the metabolic inhibitor sodium azide (0.02% and 0.1%) there was a 23% +/- 9% and 51% +/- 3% decrease, respectively, in PMN adhesion at 100 s-1. Thus, P-selectin is required for PMN adhesion to a pathophysiologic surface of activated adherent platelets at physiologic shear rates. Furthermore, a secondary step involving PMN activation after platelet binding appears necessary for complete (irreversible) adhesion to occur. This unique flow cell provides a model to explore, under controlled conditions, biologic mechanisms and ligands involved in leukocyte-platelet binding that play important roles in PMN localization at sites of thrombosis and vascular injury.

Antibodies, Monoclonal↗

PubMed1993

Platelet-derived microparticle formation involves glycoprotein IIb-IIIa. Inhibition by RGDS and a Glanzmann's thrombasthenia defect.

While the physiologic role of platelet microparticles may include a stable, physical dispersion of concentrated surface procoagulant activity the mechanism(s) of platelet vesiculation remains unknown. We demonstrate using flow cytometric methods a central role for the beta 3 integrin glycoprotein (GP) IIb-IIIa complex and its ligand tetrapeptide Arg-Gly-Asp-Ser (RGDS) binding site in platelet vesiculation. Time- and calcium-dependent vesiculation of platelets in response to ADP, collagen, thrombin, phorbol myristate acetate, and the thrombin peptide SFLLRN were dramatically inhibited, in a concentration-dependent manner, by monoclonal antibodies to GPIIb-IIIa (A2A9, 7E3, PAC1) and RGDS. Complete inhibition with A2A9 and RGDS occurred at 7.5 micrograms/ml and 75 microM, respectively, while control antibodies and a mock peptide had no effect. Platelet vesiculation requires intact GPIIb-IIIa and is fully supported by the intracellular pool of GPIIb-IIIa alone since de-complexing of this heterodimer by calcium chelation completely abolished microparticle formation in response to collagen (no alpha-granule release) but not to thrombin or SFLLRN. A central role for GPIIb-IIIa is supported by the near total inability of Glanzmann's thrombasthenic (type I) platelets to vesiculate in response to thrombin, ADP, collagen, and phorbol 12-myristate 13-acetate. This extends the biologic roles of GPIIb-IIIa to include platelet vesiculation and suggests that one or all of its binding ligands play a role.

Amino Acid Sequence↗

PubMed1989

PADGEM protein.

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Antibodies↗