Platelet storage results in a redistribution of glycoprotein Ib molecules. Evidence for a large intraplatelet pool of glycoprotein Ib.

Platelet membrane glycoprotein (GP) Ib contains receptor for von Willebrand factor and thrombin. Its proteolytic fragment, glycocalicin, circulates in normal plasma. In this study, storage of platelet concentrates for 5 d resulted in a 221% increase in plasma glycocalicin (1.3 times the total amount of glycocalicin present on the surface of all platelets), an 8% overall increase in platelet surface GPIb, and the appearance of a surface GPIb-negative subpopulation of platelets. Total platelet GPIb content of fresh washed platelets, determined by gel electrophoresis and immunoassay of Triton X-100 lysates, averaged 159,740 molecules per platelet. There were 36,360 surface GPIb molecules per platelet, determined by immunoassay of the supernatant of fresh washed platelets whose surface GPIb had been completely plasmin-cleaved. In summary, these studies provide evidence for (a) a redistribution of GPIb molecules with platelet storage, and (b) a large intraplatelet pool of GPIb (approximately threefold larger than the platelet surface pool of GPIb).

A variant of Glanzmann's thrombasthenia with abnormal glycoprotein IIb-IIIa complexes in the platelet membrane.

Patient C.M. presented platelet function defects symptomatic of Glanzmann's thrombasthenia. However, analysis of surface-labeled platelets by SDS-polyacrylamide gel electrophoresis revealed the usual presence of the major glycoproteins, including GP IIb and GP IIIa. Platelet fibrinogen was not detected. Analysis of Triton X-100 extracts of Ca2+-washed C.M. platelets by crossed immunoelectrophoresis (CIE) showed normal amounts of GP IIb-IIIa complexes. However, when samples were electrophoresed through an agarose gel containing 125I-fibrinogen, the usual binding of fibrinogen to GP IIb-IIIa did not occur. Furthermore, the GP IIb-IIIa complexes showed an increased sensitivity to dissociation with EDTA, either after Triton X-100 solubilization or in the intact platelet membrane. For example, after incubation with EDTA at room temperature, the patient's platelets bound little of the monoclonal antibodies AP-2 or T10 (anti-GP IIb-IIIa complex) although normally binding Tab (anti-GP IIb). Patient C.M. appears to represent a subgroup of thrombasthenia where platelets contain unstable GP IIb-IIIa complexes unable to support fibrinogen binding.

Exposure of adhesion molecules on activated platelets in patients with newly diagnosed IDDM is not normalized by near-normoglycemia.

It has been suggested that platelet hyperactivity contributes to the early evolution of diabetic vascular disease per se. This study directly evaluates the level of intravascular platelet activation in newly diagnosed IDDM patients before and after tight metabolic control. Platelet activation was determined by the Duesseldorf-III flow cytometry assay in 21 recent-onset hyperglycemic IDDM patients before insulin, after 3 days of treatment with intravenous insulin, and after 14 and 60 days of intensified conventional insulin therapy. The intravasal platelet activation status was quantified by the percentage of platelets exposing the activation-dependent molecules CD62 (P-selectin), thrombospondin (TSP), and CD63 (GP53) as well as the activated fibrinogen receptor (GPIIB/IIIA). Fifty matched normal subjects served as control subjects. Fourteen patients completed the 60-day study design. After initial recompensation, near-normoglycemic control was achieved after 14 days (fasting blood glucose, 117.0 +/- 19.0 mg/dl), and the HbA1 concentration was 7.6 +/- 1.2% after 60 days. CD62+ (4.0 +/- 4.5%), TSP+ (2.0 +/- 1.8%), CD63+ (11.0 +/- 7.0%), and activated-GPIIB/IIIA+ (7.6 +/- 7.7%) platelet levels were initially 5, 3.3, 5.7, and 2.8 times higher than the mean level of normal. There was no correlation with any of the nearly normalized metabolic parameters. Thus, more activated platelets circulate in newly diagnosed IDDM patients, which supports the assumption of a prethrombotic condition even in disease stages without apparent vascular damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Graded changes in the response of individual human basophils to stimulation: distributional behavior of events temporally coincident with degranulation.

Human basophils, stimulated with either anti-IgE antibody or formyl-methionine-leucine-phenylalanine, were examined by two measures of the cell response that may reflect degranulation. Flow cytometric measurement of either of these two measures, changes in forward scatter intensity (an indirect measure of the basophil size) or changes in the intensity of acridine orange-loaded cells (which labels basophil granules), allowed an assessment of the distribution of single cell responses. With regard to the latter technique, structures that appeared to be basophil granules were shown to metachromatically label with low concentrations of acridine orange, which has little or no effect on histamine release. During stimulation these labeled granules were lost, leading to decreased fluorescence. Changes in either the forward scatter parameter or acridine orange labeling occurred on the same time scale as histamine release, differentiating these measures of the basophil response from early signal transduction events. Challenging basophils with a combination of phorbol myristate acetate and ionomycin caused 100% histamine release and allowed a measurement of the maximum change in forward scatter intensity or loss of acridine orange labeling. The flow cytometric distributions after this treatment were then compared with the distributions obtained by challenging cells with several concentrations of anti-IgE antibody or formyl-methionine-leucine-phenylalanine, which induced various submaximal responses. These flow cytometric distributions demonstrated that single cells could be found in intermediate states of activation, i.e., the response of all cells was graded according to the strength of the stimulus. These studies lead to the general conclusion that all aspects of the basophil response, including those late events in the basophil response we have studied here, as well as early events that we have studied previously, are graded in a continuous manner, according to the magnitude of the stimulus.

P-selectin-dependent leukocyte recruitment and intestinal mucosal injury induced by lactoferrin.

Plasma concentrations of lactoferrin relevant to an inflammatory response are known to elicit leukocyte-endothelial cell adhesion in mesenteric venules. The objectives of this study were (1) to determine whether exogenously administered lactoferrin causes microvascular and mucosal injury in rat intestine and (2) to assess the contribution of adherent leukocytes to a lactoferrin-mediated injury process. Mucosal myeloperoxidase (MPO) activity and vascular protein clearance were monitored in the distal intestine of male Sprague-Dawley rats. Macroscopic erosive lesions of the mucosa and increases in mucosal MPO and intestinal vascular protein were observed 2 h following the lactoferrin infusion, results consistent with granulocyte accumulation and microvascular protein leakage. These lactoferrin-induced alterations were significantly attenuated in animals pretreated with a monoclonal antibody (mAb) directed against P-selectin but not by an E-selectin-specific mAb. In another series of experiments, leukocyte adherence/emigration and leakage of fluorescein isothiocyanate (FITC)-labeled albumin were measured in rat mesenteric venules using intravital video microscopy. Lactoferrin elicited increases in both leukocyte adhesion/emigration and albumin extravasation, which were attenuated by mAbs directed against P-selectin but not E-selectin. These observations indicate that (1) the lactoferrin released by activated neutrophils may lead to significant microvascular and mucosal injury or dysfunction and (2) the lactoferrin-induced injury is related to P-selectin-mediated adhesion of leukocytes to microvascular endothelium. Our results raise the possibility that neutrophil-derived lactoferrin contributes to the inflammatory response by promoting further granulocyte accumulation and activation and that mAbs to P-selectin may be therapeutically beneficial in inflammatory disorders.

Elevated plasma thrombopoietic activity in patients with metastatic cancer-related thrombocytosis.

BACKGROUND AND PURPOSE: High platelet counts are occasionally seen in patients suffering from progressive malignant disorders. While granulocyte colony-stimulating factor (G-CSF) has been implicated in paraneoplastic leukemoid reactions, the stimulus for thrombocytosis is unknown. Our purpose in this study was to determine if plasma from cancer patients with thrombocytosis contains a factor or factors with thrombopoietic activity. METHODS: We tested the effects of plasma obtained from 5 individuals with advanced tumors and high platelet counts and from 4 patients with advanced cancer and normal platelet counts on megakaryocytic differentiation of two megakaryoblastic cell lines (Dami and HEL). Differentiation was evaluated by assessing the expression of the platelet-specific cell-surface antigens CD41 (HUPL-mI) and glycoprotein IIb-IIIa using an immunocytochemical staining score. In addition, plasma samples from 7 of the 9 patients and from 5 additional cancer patients with thrombocytosis were assayed for the levels of interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and IL-1 beta protein using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Expression of platelet-specific cell-surface antigen was increased in HEL cells after exposure to plasma from all 5 of the cancer patients with thrombocytosis, and in Dami cells after exposure to plasma from 4 of the 5. Similar, but less significant, results were found when these cells were incubated with control combinations of recombinant GM-CSF plus IL-6 or of IL-3 plus IL-6. Platelet-specific cell-surface-antigen expression was not increased in HEL or Dami cells after exposure to the plasma from the 4 cancer patients with normal platelet counts or to normal control plasma. ELISA revealed elevated levels of IL-6 in the plasma from 4 patients with thrombocytosis (38, 40, 63, and 99 pg/mL). In addition, GM-CSF concentration was high in 3 of these 4 patients (33, 47, and 127 pg/mL), and the G-CSF level was elevated in 1 (543 pg/mL). IL-1 beta and IL-3 levels were undetectable. CONCLUSIONS: Our data suggest that the thrombocytosis observed in individuals with advanced malignant disease is mediated by a humoral mechanism. Levels of IL-6, GM-CSF, and G-CSF are elevated in some of these patients, but the plasma concentrations are generally lower than those required for in vitro induction of megakaryocytic differentiation. Plasma from patients with paraneoplastic thrombocytosis may therefore contain thrombopoietins that have not yet been identified, and which might have clinical usefulness.

Reversible inhibition of human platelet activation by hypothermia in vivo and in vitro.

A hypothermia-induced hemorrhagic diathesis is associated with cardiopulmonary bypass, major surgery, and multiple trauma, but its pathophysiological basis is not well understood. We examined the hypothesis that hypothermia reversibly inhibits human platelet activation in vitro and in vivo. Platelet activation was studied in normal volunteers by whole blood flow cytometric analysis of modulation of platelet surface GMP-140 and the glycoprotein (GP) Ib-IX complex in: a) shed blood emerging from a standardized in vivo bleeding time wound; b) peripheral blood activated in vitro with either thrombin (in the presence of gly-pro-arg-pro, an inhibitor of fibrin polymerization) or the stable thromboxane (TX) A2 analogue U46619. Platelets in peripheral whole blood were activated at temperatures between 22 degrees C and 37 degrees C. the forearm skin temperature was maintained at temperatures between 22 degrees C and 37 degrees C prior to and during the bleeding time incision. Platelet aggregation was studied in shed blood by flow cytometry and in peripheral blood by aggregometry. Generation of TXB2 (the stable metabolite of TXA2) was determined by radioimmunoassay. In vitro, hypothermia inhibited both thrombin- and U46619-induced upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregation, and TXB2 generation. These inhibitory effects of hypothermia were all completely reversed by rewarming the blood to 37 degrees C. In vivo, platelet activation was inhibited by hypothermia as shown by 5 independent assays of shed blood: upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregate formation, TXB2 generation, and the bleeding time.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody to the human platelet glycoprotein IIb/IIIa complex induces platelet activation.

The platelet glycoprotein (GP) IIb/IIIa complex functions as the receptor for fibrinogen on activated platelets. The effects of two anti-GPIIb/IIIa monoclonal antibodies on platelet function were studied. These antibodies, 6C9 and C17, recognized different epitopes, which were exclusively present on the undissociated GPIIb/IIIa complex. Whereas C17 inhibited the binding of fibrinogen to platelets and platelet aggregation induced by adenosine diphosphate (ADP) or collagen, 6C9 caused irreversible aggregation of platelets, both in the presence and absence of extracellular fibrinogen. When incubated with unstirred (non-aggregating) platelets, 6C9 induced release of alpha and dense granule-constituents as well as binding of 125I-fibrinogen to platelets. The latter was evidently mediated in part by platelet-derived ADP, since it was inhibited to a large extent by apyrase, the ADP-hydrolyzing enzyme. F(ab')2 fragments of 6C9 did not induce platelet-release reactions but caused (slow) aggregation of platelets in the presence of extracellular fibrinogen. These results indicate that binding of an antibody to a specific site on the platelet GPIIb/IIIa complex may cause fibrinogen-mediated aggregation. The Fc part of the platelet-bound antibody appears to be involved in the induction of platelet release.

The platelet glycoprotein IIb/IIIa complex is involved in the adhesion of activated platelets to leukocytes.

The adhesion of activated platelets to leukocytes (rosette formation) seems to be mediated by CD62 on platelets and its counter-receptor (CD15 or a sialic acid-containing glycoprotein) on polymorphonuclear leukocytes (PMNL). However, neither treatment of platelets with an anti-CD62 antibody or fucoidan nor treatment of PMNL with anti-CD15 antibody or neuraminidase are able to inhibit completely the adhesion. Therefore, we have studied the platelet GPIIb/IIIa complex (CD41a) for its involvement in the adhesion of activated platelets to PMNL. The following evidences point to a participation of CD41a in the adhesion of activated platelets to leukocytes: a) inhibition of adhesion by monoclonal antibodies (mab) raised toward CD41a, b) inhibition of adhesion by peptides such as RGDS and echistatin, c) inhibition of adhesion by dissociation of the CD41a complex with EGTA, and d) inhibition of rosette formation using platelets from a thrombasthenic patient which have almost no CD41a in the surface membrane but a normal expression of CD62. It is likely that fibrinogen is involved in the adhesion of platelets to PMNL via CD41a, since fibrinogen increases the rosette formation of ADP-stimulated platelets. Furthermore, the incubation of unstimulated platelets with fibrinogen and an antibody raised against glycoprotein IIIa which stimulates fibrinogen binding to the platelet surface results in an enlarged rosette formation.

Amyloid beta-protein precursor-rich platelet microparticles in thrombotic disease.

We investigated the association of amyloid beta-protein precursor (APP) and platelet derived microparticles in 20 normal controls and 91 patients with various diseases causing a thrombotic tendency. Compared with the controls, the mean percentage of APP-positive microparticles was significantly greater in the patients with cerebral infarction (39.1 +/- 17.7%, p < 0.001), diabetes (31.1 +/- 12.6%, p < 0.001), and uremia (30.1 +/- 14.7%, p < 0.01), but not in those with hypertension (8.2 +/- 6.3%, p = NS). Sixteen patients with cerebral infarction, 20 with diabetes, and 11 with uremia had microparticles with very high APP levels. In normal controls, 7.2 +/- 3.7% of the microparticles were positive for P-selectin, while the percentage in cerebral infarction, diabetes, uremia, and hypertension was respectively 43.5 +/- 15.1%, 40.0 +/- 12.8%, 31.8 +/- 12.2%, and 11.6 +/- 7.3%. There was a significant correlation between P-selectin and APP positivity of microparticles. Our results suggest that microparticle APP may have a regulatory influence on coagulation abnormalities.

Evaluation of whole blood flow cytometric detection of platelet bound fibrinogen on normal subjects and patients with activated platelets.

Activated platelets can be detected by measuring platelet-bound fibrinogen in a whole blood, flow cytometric assay, using a fluorescently-conjugated polyclonal antibody. Fibrinogen binding to unstimulated platelets from normal subjects was low in this assay, as was expression of the CD63 antigen. Single cell counting of samples prepared for flow cytometric analysis showed platelet aggregates do not form during the assay procedure. Immune complexes were not seen, and fibrinogen binding to the platelets was unaffected by the CD32 MAb, IV.3. Artefactual activation of the unfixed samples could be minimised by control of phlebotomy, time and temperature of incubation. Variations in platelet count in the range 140-430 x 10(9) 1(-1) and in plasma fibrinogen in the range 2-6 g 1(-1) did not affect the assay results. Comparison of fibrinogen binding with expression of CD63 antigen on normal platelets, stimulated with agonists in vitro, demonstrated that fibrinogen binding detects an earlier stage of platelet activation. Platelet bound fibrinogen was shown to be sensitive in detecting small numbers of activated platelets in clinical samples in twelve patients on intensive care, four undergoing haemofiltration. The patients had a significantly higher median percentage of circulating platelets with bound fibrinogen (p < 0.005), but fibrinogen binding was significantly lower (p < 0.02) in response to 10(-5) M ADP, compared to twelve age-matched normal controls.

Rapid detection of plasma glycocalicin by a latex agglutination test. A useful adjunct in the differential diagnosis of thrombocytopenia.

To aid in the rapid differential diagnosis of thrombocytopenia, the authors developed a latex agglutination test for glycocalicin, a proteolytic fragment of platelet membrane glycoprotein Ib. Plasma glycocalicin determinations were performed for 34 patients with thrombocytopenia. Plasma samples from four patients with aplastic anemia and ten patients with myelodysplastic syndromes, all with glycocalicin levels less than 0.6 mg/L by an enzyme-linked immunosorbent assay, all had negative results by the latex test. In contrast, positive latex agglutination titers were obtained for all 12 patients with idiopathic thrombocytopenic purpura. Eight patients with liver cirrhosis and splenomegaly had elevated levels of plasma glycocalicin, and all of their plasma samples produced agglutination. This latex agglutination test for glycocalicin allows a rapid discrimination of thrombocytopenia caused by impaired platelet production from that caused by increased platelet destruction; thus, it is suitable for use as a screening test in a routine clinical laboratory.

The carbohydrate moiety of human platelet glycocalicin: the structures of the major Asn-linked sugar chains.

Glycocalicin, a predominant glycoprotein on the human platelet surface, has been purified from a platelet suspension by means of sonication, ammonium sulfate precipitation and acid treatment followed by chromatography on columns of wheat germ agglutinin-Sepharose and Mono Q. Asparagine-linked (N-linked) oligosaccharides were released by hydrazinolysis, and then N-acetylated and reduced with NaBH4 or NaB3H4. The released carbohydrate chains were found to be of the complex-type from their interaction with immobilized lectin columns. The structures of the two major oligosaccharide-alditols separated by ion-exchange chromatography on a Mono Q column were investigated by means of methylation analysis, glycosidase digestion, and Smith periodate degradation, and they were assigned as typical di- and trisialylated complex-type oligosaccharide-alditols with two and three peripheral chains consisting of Gal-GlcNAc sequences, respectively.

Structures of the carbohydrate chains of membrane glycoproteins IIb and IIIa of human platelets.

Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.

Steroid receptor-mediated modulation of CD4+CD62L+ cell homing. Implications for drug abusers.

Naive human T cells home to peripheral lymph nodes via the leukocyte endothelial cell adhesion molecule-1 (LECAM-1, l-selectin, CD62L, Leu8 antigen) they express. We enriched populations of CD4+CD62L+ cells (attachment of Leu8+ T cells to flasks coated with anti-mouse IgG (AIS); Leu8+ T cells, 82.3% pure (+/- 2.3%), enriched for CD4+ cells by incubation over flasks coated with anti-CD4 antibody--this 3-4-day procedure yields an 88 +/- 1.4% recovery. Cells were treated with dexamethasone in vitro for 24-48 h, and monitored by flow cytometry. We found severe toxicity by this steroid at high concentration (10(-6) M: 35% decrease in CD62L+ T cells, 22% drop specifically in CD4+CD62L+ cells), suggesting the onset of receptor-mediated apoptotic events. The toxicity was dose dependent (5% and 7% drop in CD62L+ T and CD4+CD62L+ cells, respectively, at 10(-9) M, the concentration found in plasma 10 h following the administration of 1 mg dexamethasone). One mg of dexamethasone given to normal subjects leads to a 15-20% decrease in circulating CD4+CD62L+ cells at 10 h. This tends to be correlated with a drop in the number of glucocorticoid cytosolic receptors. Thus, steroids seem to modulate CD4+CD62L+ cell homing by means of receptor-mediated mechanisms.

Flow cytometric analysis of surface membrane proteins on activated platelets and platelet-derived microparticles from healthy and thrombasthenic individuals.

We used flow cytometry to investigate surface membrane protein expression by platelets and platelet-derived microparticles from normal individuals and a patient with Glanzmann's thrombasthenia. Microparticles were detected by both forward scatter and side scatter using FACScan. The binding of coagulation factors on microparticles was investigated by using monoclonal anti-Factor IX (IXa) and anti-Factor X (Xa) antibodies. Furthermore, the procoagulant activity of microparticles was measured with a chromogenic substrate (S-2222) using a microtiter enzyme-linked immunosorbent assay. Both types of platelets showed similar release of microparticles. Microparticles released from platelets after activation with the calcium ionophore A23187 did not bind factors IXa and Xa, but when purified factors Va and Xa were added to the incubation buffer, factor Xa binding increased markedly in both normal and thrombasthenic platelets. Both normal and thrombasthenic platelets showed a similar time-dependent release of microparticles when activated with A23187. However, the binding of an antibody to granule membrane protein-140 also increased time-dependently in normal microparticles, but was little increased in thrombasthenic microparticles. These findings suggest that glycoprotein IIb/IIIa does not participate in the expression of prothrombinase activity on the surface of activated platelets and microparticles, whereas this glycoprotein appears to have an important role in the movement of granule membrane protein-140 from platelets to microparticles.

Granulocyte colony-stimulating factor enhances the expression of CD62 on platelets in vivo.

The expression of CD41, CD42 and CD62 on platelets was determined in ten patients with and without G-CSF treatment. CD41 and CD42 is expressed in nearly 100% of the platelets without change after G-CSF treatment. The expression of CD62 on the platelets' surface is significantly enhanced by G-CSF indicating a depletion of the alpha-granules. No platelet aggregation was observed. The enhanced secretion of thrombocyte-specific proteins does not induce aggregation but may promote ADP-induced aggregation. Furthermore, the surface-bound and soluble CD62 binds specifically to macrophages and endothelial cells and is involved in the regulation of inflammatory processes. Thus indirect mechanisms may supplement direct effects of G-CSF after chemotherapy.

Platelet activation in warm and cold heart surgery.

Recent studies suggest that patients undergoing warm heart surgical procedures have reduced postoperative bleeding. To determine if this is due to differences in platelet activation, we measured platelet membrane glycoproteins (GPIb, GPIIb/IIIa, GMP 140), platelet fragments, and platelet counts before, during, and after normothermic (37 degrees C) or hypothermic (28 degrees to 30 degrees C) cardiopulmonary bypass. Cardiopulmonary bypass was associated with a significant decrease in platelet count, platelet membrane GPIb, and platelet fragments, and an increase in GMP 140 (p < 0.05). Normothermic cardiopulmonary bypass induced an early significant increase in granulocytes, whereas this was delayed until after rewarming in the hypothermic group. Mean 24-hour postoperative blood loss was 786 +/- 226 mL in the cold group versus 547 +/- 56 mL in the warm group (p = not significant). We conclude that cardiopulmonary bypass affects platelet activation and integrity and that these changes are similar in direction and magnitude for hypothermic and normothermic techniques.

Effect of cardiopulmonary bypass on the circulating level of soluble GMP-140.

Soluble GMP-140 can prevent the adhesion of activated neutrophils to endothelium in vitro. Because cardiopulmonary bypass causes neutrophil-endothelial interaction, the plasma level of soluble GMP-140 was measured using an enzyme immunoassay system in 32 children undergoing intracardiac repair of congenital heart disease. They had either a high, low, or normal pulmonary blood flow (n = 13, 12, and 7 respectively). Because activated platelets are a source of GMP-140, the plasma beta-thromboglobulin level was also measured. Blood was sampled before, during, and for 24 hours after cardiopulmonary bypass. Plasma levels of both soluble GMP-140 and beta-thromboglobulin increased after the onset of cardiopulmonary bypass in all patients but for both substances the increase was greater in those with a low pulmonary blood flow (p < 0.05 for all comparisons). The sum total of soluble GMP-140 values during and after operation was correlated negatively with the preoperative mean pulmonary arterial pressure (p < 0.05 for all time intervals). GMP-140 level correlated with the plasma beta-thromboglobulin level (r = 0.5, p < 0.05). This work supports the contention that soluble GMP-140 is released from activated platelets during cardiopulmonary bypass, the level being particularly high in those who had intrinsically abnormal platelets preoperatively in association with a low pulmonary blood flow. Patients with a high pulmonary blood flow, who are more susceptible to endothelial cell injury, may be less well protected by soluble GMP-140.

Adhesion of activated platelets to polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNL) are components of the blood which as such interact extensively with other blood cells, with endothelial cells or with plasma. Here, we consider the interaction between PMNL and platelets which is efficient during adhesion of platelets to PMNL and which can be studied in vitro using the rosette formation assay. The adhesion of activated platelets to PMNL seems to be mediated mainly by a protein of platelets (CD62) and its counterreceptor on PMNL, but also other platelet receptors are involved. Here we demonstrate the participation of the glycoprotein IIb-IIIa complex (CD41a) in the adhesion of activated platelets to PMNL due to the following findings: a) inhibition of adhesion by monoclonal antibodies raised against CD41a, b) inhibition of adhesion by peptides such as RGDS and echistatin, c) inhibition of adhesion by dissociation of CD41a with EGTA and d) inhibition of adhesion using platelets from a thrombasthenic patient which have almost no CD41a in the surface membrane but a normal expression of CD62 upon activation. The adhesion of activated platelets to PMNL via CD41a seems to be mediated by fibrinogen due to the following findings: a) addition of fibrinogen to ADP-stimulated and fixed platelets increases significantly the rosette formation and b) the incubation of unstimulated platelets with fibrinogen and an antibody raised against glycoprotein IIIa which stimulates fibrinogen binding to the platelet surface results in an enlarged rosette formation.