Fibrinogen-coated Surfaces Research Articles

Effect of strenuous, acute exercise on alpha2-adrenergic agonist-potentiated platelet activation.

Vigorous exercise transiently increases the risk of primary cardiac arrest. Strenuous, acute exercise can also increase the release of plasma epinephrine. Previous investigations have indicated that epinephrine can potentiate platelet activation by activating platelet alpha2-adrenoceptors. This study investigated how strenuous, acute exercise affects alpha2-adrenergic agonist-potentiated platelet activation by closely examining 15 sedentary men who exercised strenuously on a bicycle ergometer. Before and immediately after exercise, platelet adhesiveness on fibrinogen-coated surfaces, [Ca2+]i in platelets, the number and affinity of alpha2-adrenergic sites on the platelet surface, and plasma catecholamine levels were determined. The results of this study can be summarized as follows: (1) The affinity of alpha2-adrenergic receptors on platelets decreases while the maximal binding number significantly increases after strenuous exercise, thereby correlating with the rise in plasma catecholamine levels. (2) Basal, clonidine-treated, ADP-treated, and clonidine plus ADP-treated adhesiveness and [Ca2+]i in platelets increased after strenuous exercise. (3) Strenuous exercise is associated with higher percentages of ADP- and clonidine plus ADP-enhanced platelet adhesiveness and [Ca2+]i than at rest. (4) The synergistic effects of clonidine on ADP-enhanced platelet adhesiveness and [Ca2+]i after strenuous exercise are much greater than those at rest. Therefore, we conclude that strenuous, acute exercise enhances platelet activation, possibly by altering the performance of platelet alpha2-adrenergic receptors, facilitating the ability of ADP-activated fibrinogen receptors, and enhancing fibrinogen binding to platelet fibrinogen receptors.

Enhancement of leukocyte response to lipopolysaccharide by secretory group IIA phospholipase A2.

Secretory nonpancreatic group IIA phospholipase A2 (sPLA2), a lipolytic enzyme found in plasma, is thought to play an important role in inflammation. In patients with sepsis, a strong positive correlation is observed between the plasma level of sPLA2 and poor clinical outcome in sepsis. We have thus asked whether sPLA2 could play a role in enabling responses of cells to bacterial lipopolysaccharide (LPS), a key contributor to sepsis. In the presence of sPLA2, cellular responses to LPS were significantly increased. This was demonstrated in assays of LPS-stimulated interleukin-6 (IL-6) production in whole blood and binding of freshly isolated human polymorphonuclear neutrophils (PMN) to fibrinogen-coated surfaces. We further found that sPLA2 enhanced binding of labeled LPS to PMN, and that the sPLA2-mediated cell responses to LPS were all blocked by monoclonal antibodies directed against membrane CD14. Two properties ofsPLA2 may contribute to its activity to mediate responses to LPS. sPLA2 appears to bind LPS because pre-exposure of sPLA2 to LPS led to a dose-dependent increase in its ability to hydrolyze phospholid substrate, and incubation of sPLA2 with BODIPY-LPS micelles resulted in enhanced fluorescence, presumably from the disaggregation of the LPS aggregates. Additional studies demonstrated that the esterolytic function of sPLA2 is also needed both for the disaggregation of LPS and CD14-dependent cell stimulation. The precise mechanisms by which LPS-binding and esterolytic activity contribute to sPLA2 activity are not clear but our data strongly suggest that these activities result in interaction of LPS with CD14 and subsequent cell activation.

Inhibition of 2-p-mercaptophenyl-1,4- naphthoquinone on human platelet function

As widely assumed, platelets and coagulation system heavily influence the pathogenesis and progression of cardiovascular diseases. Some 1,4-naphthoquinone derivatives, such as vitamin K 3, have been reported to increase the synthesis of coagulation proteins. In this study, we examine how 2-p-mercaptophenyl -1,4-naphthoquinone (NTP), a newly synthesized 1,4-naphthoquinone derivative, affects the platelet function in humans. A tapered parallel plate chamber which provided a range of shear stress covering the entire physiological range in human circulation is used to assess platelet adhesiveness on fibrinogen coated-surface. In addition, platelet aggregation and thromboxane B 2 (TXB 2) production by inducers are evaluated by the turbidimetric method and enzyme immunoassay kit, respectively. Moreover, platelets [Ca 2+]i are measured using a dual-wavelength fluorescence spectrophotometer. Analysis results indicate that 1) NTP decreases the percentages of attached platelets at the locations in various shear stresses and the levels of platelet adhesiveness, denoted as the slope; 2) NTP can inhibit the platelet aggregation by ADP (2μM) and collagen (25 μg ml ), and the IC 50 are: 0.32 and 26.83 μg ml , respectively, and 3) NTP markedly inhibits TXB 2 formation and platelet [Ca 2+]i elevation caused by ADP and collagen. Therefore, we conclude that NTP may inhibit platelet adhesiveness on fibrinogen coated-surface, aggregability, [Ca 2+]i, and thromboxane production, and that it may be used as an antiplatelet agent.

Antibacterial action of extracellular mammalian group IIA phospholipase A2 against grossly clumped Staphylococcus aureus.

Fibrinogen-dependent interactions of Staphylococcus aureus are believed to contribute to bacterial virulence by promoting bacterial attachment to fibrinogen-coated surfaces and inducing the formation of bacterial clumps that are likely resistant to phagocytosis. Although S. aureus produces several fibrinogen-binding proteins, the cell wall-associated protein clumping factor (encoded by clfA) appears to be most important in bacterial interactions with immobilized or soluble purified fibrinogen. We have compared bacterial clumping in several strains of S. aureus, including isogenic ClfA+ and ClfA- Newman strains, in the presence of purified rabbit fibrinogen, human plasma, and inflammatory fluid and examined the effect of clumping on bacterial sensitivity to mammalian group IIA phospholipase A2 (PLA2). This enzyme is the major extracellular bactericidal agent in inflammatory fluid active against S. aureus. Both ClfA-dependent and ClfA-independent bacterial clumping was observed, depending on the source and fibrinogen content of the biological fluid. In each case, clumping only partially reduced the antibacterial activity of PLA2, suggesting that this extracellular enzyme can substantially penetrate dense bacterial clumps. Bacterial clumps could be dispersed by added proteases, restoring full antibacterial activity to PLA2. Thus, the extracellular mobilization of group IIA PLA2 during inflammation may provide a mechanism by which the host can control the proliferation and survival of S. aureus even after bacterial clumping.

Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through alphavbeta3 and alpha5beta1 integrins.

Receptors of the Eph family and their ligands (ephrins) mediate developmental vascular assembly and direct axonal guidance. Migrating cell processes identify appropriate targets within migratory fields based on topographically displayed ephrin gradients. Here, EphB1 regulated cell attachment by discriminating the density at which ephrin-B1 was displayed on a reconstituted surface. EphB1-ephrin-B1 engagement did not promote cell attachment through mechanical tethering, but did activate integrin-mediated attachment. In endothelial cells, attachment to RGD peptides or fibrinogen was mediated through alphavbeta3 integrin. EphB1 transfection conferred ephrin-B1-responsive activation of alpha5beta1 integrin-mediated cell attachment in human embryonic kidney cells. Activation-competent but signaling-defective EphB1 point mutants failed to stimulate ephrin-B1 dependent attachment. These findings lead us to propose that EphB1 functions as a 'ligand density sensor' to signal integrin-mediated cell-matrix attachment.

Inhibition by heparin and derivatized dextrans of Staphylococcus epidermidis adhesion to in vitro fibronectin-coated or explanted polymer surfaces

The ability of Staphylococcus to recognize several extracellular matrix or plasma proteins (e.g., fibrinogen, fibronectin, and collagen) promotes bacterial attachment to artificial surfaces. Whereas most S. aureus clinical isolates elaborate a wide repertoire of bacterial surface 'receptors' called adhesins, exhibiting specific binding of individual host proteins, S. epidermidis is lacking most of such protein adhesins. To document the interactions between S. epidermidis and various surface-adsorbed proteins, we first compared promotion of bacterial attachment by seven purified human proteins immobilized onto poly(methyl methacrylate) (PMMA) coverslips. Only two of them, namely fibronectin and fibrinogen, exhibited adhesion-promoting activities. In the presence of native heparin or two functionalized dextrans (CMDBS for Carboxy Methyl, Benzylamide sulfonate/sulfate), a dose-dependent inhibition of S. epidermidis adhesion to fibronectin-coated, but not to fibrinogen-coated surfaces was observed. The inhibitory effects of each CMDBS were much stronger than that of native heparin. In contrast, a control highly negatively charged dextran exclusively substituted with carboxy methyl groups exerted no inhibition on S. epidermidis adhesion. To evaluate how CMDBS could interfere with S. epidermidis attachment to coverslips coated in vivo with extracellular matrix components, we also tested PMMA surfaces retrieved from tissue cages Subcutaneously implanted in guinea pigs. Each CMDBS, but not heparin, strongly inhibited S. epidermidis adhesion to explanted coverslips, even in the presence of tissue cage fluid. In conclusion, fibronectin plays an important role in promoting S. epidermidis attachment to implanted biomaterials. Furthermore, S. epidermidis adhesion to fibronectin-coated or implanted biomaterials can be efficiently blocked in vitro by CMDBS.

Adsorption of fibrinogen to droplets of liquid hydrophobic phases. Functionality of the bound protein and biological implications.

Fibrinogen adsorbs spontaneously from aqueous media containing that protein to droplets of liquid hydrophobic phases dispersed in those same media. Examples of such phases include mineral oils, straight-chain hydrocarbons, and various plant- and animal-derived oils. Lecithin preexisting on the surface of oil droplets reduces significantly the amount of fibrinogen that can otherwise bind to them. When bound, fibrinogen remains active in the classic sense of fibrin gelation. As a consequence, oil droplets coated with fibrinogen can participate in a host of biologically important adhesive processes in which the protein would be expected to participate. Certain polyanions, eg, heparin, pentosan polysulfate, dextran sulfate, and suramin, bind to adsorbed fibrin(ogen) and prevent thrombin-dependent adhesion of fibrinogen-coated surfaces. Thus, these polyanions can be used to prevent adhesion between fibrin(ogen)-coated oil droplets and other fibrin(ogen)-coated surfaces. Potential practical applications and biological implications of these phenomena are presented and discussed.

Oleic Acid Increases Cell Surface Expression and Activity of CD11b on Human Neutrophils

Traumatic bone injury frequently results in the release of marrow-derived fatty material into the circulation. This may lead to the syndrome of fat embolism, associated with the generation of free fatty acids, the sequestration of neutrophils in the lungs, and the subsequent development of acute respiratory distress. Neutrophil accumulation in tissues requires their adherence to vascular endothelial cells and involves the beta2 integrin, CD11b/CD18 (Mac-1). We now report that the exposure of isolated human neutrophils to oleic acid causes a rapid increase in the cell surface expression and affinity state of CD11b, particularly under acidic conditions that are typical of inflammatory sites. Oleic acid also triggers neutrophil aggregation and neutrophil adherence to both fibrinogen-coated surfaces and confluent cultures of HUVEC. These processes are blocked by CD11b-specific inhibitors, including neutrophil-inhibitory factor and mAbs to CD11b. These observations may help explain the etiology of so-called fat embolism wherein trauma-induced release of fatty material causes pulmonary neutrophil accumulation and the development of acute respiratory distress.

Preparation of functionally active cell-permeable peptides by single-step ligation of two peptide modules.

Noninvasive cellular import of synthetic peptides can be accomplished by incorporating a hydrophobic, membrane-permeable sequence (MPS). Herein, we describe a facile method that expedites synthesis of biologically active, cell-permeable peptides by site-specific ligation of two free peptide modules: one bearing a functional sequence and the second bearing a MPS. A nonpeptide thiazolidino linkage between the two modules is produced by ligation of the COOH-terminal aldehyde on the MPS and the NH2-terminal 1, 2-amino thiol moiety on the functional sequence. This thiazolidine ligation approach is performed with stoichiometric amounts of fully unprotected MPS and functional peptide in an aqueous buffered solution, eliminating the need for additional chemical manipulation and purification prior to use in bioassays. Two different MPSs were interchangeably combined with two different functional sequences to generate two sets of hybrid peptides. One set of hybrid peptides, carrying the cytoplasmic cell adhesion regulatory domain of the human integrin beta3, inhibited adhesion of human erythroleukemia cells to fibrinogen-coated surfaces. A second set of hybrid peptides, carrying the nuclear localization sequence of the transcription factor NF-kappaB, inhibited nuclear import of transcription factors NF-kappaB, activator protein 1, and nuclear factor of activated T cells in agonist-stimulated Jurkat T lymphocytes. In each assay, these nonamide bond hybrids were found to be functionally comparable to peptides prepared by the conventional method. Cumulatively, this new ligation approach provides an easy and rapid method for engineering of functional, cell-permeable peptides and demonstrates the potential for synthesis of cell-permeable peptide libraries designed to block intracellular protein-protein interactions.

The dipeptide repeat region of the fibrinogen-binding protein (clumping factor) is required for functional expression of the fibrinogen-binding domain on the Staphylococcus aureus cell surface.

Clumping factor of Staphylococcus aureus is a fibrinogen-binding protein that is located on the bacterial cell surface. The protein has an unusual repeat domain (region R) comprising mainly the dipeptide aspartate and serine. To determine if region R has a role in the surface display of the fibrinogen-binding region A domain, deletions lacking the region R encoding region of the clfA gene were generated. To determine the minimum length of region R required for wild-type levels of ClfA expression, variants with truncated region R domains were constructed. S. aureus cells expressing mutated clfA genes were tested for (i) proteins released by lysostaphin treatment that reacted with antisera specific for region A, (ii) clumping in soluble fibrinogen, (iii) adherence to immobilized fibrinogen and (iv) expression of the ClfA antigen on the cell surface by fluorescent activated cell sorting analysis. Each construct expressed three major immunoreactive proteins, two of which were putative N-terminal degradation products. Region R residues greater than 40 were required between region A and W (72 residues between region A and the LPDTG sorting signal) for wild-type levels of clumping in fibrinogen. A stepwise decrease in clumping titre was observed as the distance between region A and LPDTG was decreased from 72 to 4 residues. Similarly, a decrease in binding of anti-ClfA serum and in binding to fibrinogen-coated plastic surfaces was observed with cells expressing ClfA with 40 region R residues or less. Nevertheless, low levels of adherence to fibrinogen and binding to anti-ClfA serum occurred with ClfA derivatives that lacked region R altogether. This indicates that a small proportion of the ClfA molecules are linked to peptidoglycan very close to the cell surface but that residues greater than 72 are needed to allow sufficient ClfA molecules to span the entire cell wall and to display the biologically active A domain in a form that can participate fully in fibrinogen binding.

Inhibition of human neutrophil beta2-integrin-dependent adherence by hyperbaric O2.

Animal and clinical investigations have reported that exposure to hyperbaric O(2) improved the outcome of some reperfusion injuries. Animal studies have suggested that this may be due to an inhibition of leukocyte adherence to injured endothelium. This investigation tested the hypothesis that exposure to hyperbaric O(2) would inhibit beta2-integrin-dependent adherence of human neutrophils. Subjects were exposed to O(2) at partial pressures of up to 3 atmospheres absolute (ATA; 1 ATA = 0.1 MPa) for 45 min, and neutrophil binding to nylon columns and to fibrinogen-coated surfaces was measured. Exposure to O(2) at 2.8 or 3.0 ATA inhibited beta2-integrin-dependent neutrophil adherence but had no effect on the cell-surface expression of beta2-integrins, respiratory burst in response to phorbol ester, or non-beta2-integrin-dependent adherence to plastic plates coated with a fibronectin-like protein. beta2-Integrin adherence was restored by incubating blood with 8-bromoguanosine 3',5'-cyclic monophosphate (cGMP) and hyperbaric O(2) inhibited synthesis of cGMP by neutrophils stimulated with N-formyl-Met-Leu-Phe (FMLP). In studies of cell fractions, the activity of membrane guanylate cyclase was found to be increased by incubation with FMLP as well as by atrial natriuretic peptide (ANP) plus ATP. Hyperbaric O(2) had no effect on the basal activity of soluble or membrane-bound guanylate cyclase. However, hyperbaric O(2) inhibited the function of both the extracellular binding domain of membrane guanylate cyclase as well as intracellular catalytic activity. There are approximately 7,300 membrane guanylate cyclase molecules per cell, based on binding studies with ANP, with a dissociation constant of approximately 450 pM. Hyperbaric O(2) inhibits the function of human neutrophil beta2-integrins by a process linked to impaired synthesis of cGMP.

Signaling by adhesion in human neutrophils: activation of the p72syk tyrosine kinase and formation of protein complexes containing p72syk and Src family kinases in neutrophils spreading over fibrinogen.

Src family kinases are implicated in signaling by integrins in polymorphonuclear neutrophils (PMN). To identify proteins present in complexes with Src family kinases, we subjected p58(c-fgr) or p53/56(lyn) immunoprecipitates from Triton X-100 lysates of PMN incubated on fibrinogen-coated surfaces to in vitro kinase assays. Assays on p58(c-fgr) or p53/56(lyn) immunoprecipitates from Triton lysates of PMN induced to spread over fibrinogen in response to TNF-alpha showed that several proteins form complexes with Src family kinases and can be phosphorylated in vitro. Immunoblot analysis showed that the p72(syk) tyrosine kinase is one of these proteins. Formation of protein complexes containing Src family kinases and p72(syk) required PMN spreading because p72(syk) was not detected in p58(c-fgr) or p53/56(lyn) immunoprecipitates from PMN, which were stimulated with TNF-alpha, in suspension. In addition, induction of PMN spreading with Mn2+ in the absence of TNF-alpha promoted the formation of protein complexes containing Src family kinases and p72(syk). We also found that p72(syk)-autophosphorylating kinase activity was enhanced, and a fraction of p72(syk) was translocated to a Triton-insoluble cytoskeletal fraction in PMN induced to spread over fibrinogen by TNF-alpha or Mn2+. In vitro kinase assays on CD18 (beta 2 integrin subunit) immunoprecipitates from Triton lysates of spread PMN showed that several proteins formed complexes with CD18 and could be phosphorylated in vitro. Immunoblot analysis of CD18 immunoprecipitates allowed us to identify p72(syk) as one of these proteins. These findings show that PMN spreading is accompanied by activation of p72(syk) and formation of multimolecular complexes in which Src family kinases and p72(syk) colocalize.

Cholesterol enhances the adhesion of human platelets to fibrinogen: studies using a novel fluorescence based assay

This communication reports investigations on the effect of platelet cholesterol content on adhesion of platelets to a fibrinogen coated surface. The adhesion of platelets stimulated with thrombin or ADP was dramatically increased when the platelet cholesterol content was enriched by incubation with cholesterol containing phosphatidylcholine vesicles. In contrast, ADP failed to promote the adhesion of platelets to fibrinogen after they had been depleted of cholesterol, either by incubation with phosphatidylcholine vesicles or by brief exposure to cholesterol oxidase. By comparison, the adhesion of resting platelets to fibrinogen coated surface was unaltered following either enrichment or depletion of cholesterol. These data were obtained using a novel method of measuring the adhesion of platelets to a protein coated surface based upon the fluorescent detection of platelets containing the fluorescent probe octadecyl rhodamine (R membranes using standard ethanol injection techniques at room temperature for 30 min. The platelets were introduced into fibrinogen coated wells of a 96-well microtiter plate in the presence of various cations and stimulatory or inhibitory ligands. The plate was then incubated at room temperature without agitation for various periods of time. Adhesion measured in this manner had characteristics similar to those reported using other methods. Thus the extent of adhesion ranged from 1-4% under basal conditions, and was increased in a dose-dependent manner by Mg2+ and Ca2+, increased further by ADP, collagen or thrombin and not affected by prostacyclin. ). R was incorporated into platelet 18 18

Heterogeneity in Glanzmann's Thrombasthenia

The activation-independent platelet adhesion to the fibrinogen-coated surface under static conditions was examined in patients with Glanzmann's thrombasthenia (type I: 5 cases; type II: 1 case). Among five type I patients, platelet adhesion in three patients was found to be less than 20% of that in normal controls, while two showed 66% and 89% adhesion, respectively. Platelet adhesion in one type II patient was normal. In all patients, platelet adhesion to fibronectin or collagen type I was much the same as that in normal controls.Binding of an anti-GPIIb/IIIa complex monoclonal antibody LJ-P9 to platelets, which completely inhibits platelet adhesion to fibrinogen, was measured by flow cytometry. Antibody binding in the three type I patients, who exhibited severely defective adhesion, was found to be less than 5%, while that in the two type I patients, who showed 66% and 89% adhesion, was 9% and 24%, respectively.These results demonstrate that the level of GPIIb/IIIa complex on the surface of patient s platelets may account for the residual function of platelet adhesion to fibrinogen in type I thrombasthenia, suggesting the heterogeneity of this congenial disease.

Adhesion of Platelets to Surface-Bound Fibrinogen Under Flow

After platelet activation, fibrinogen mediates platelet-platelet interactions leading to platelet aggregation. In addition, fibrinogen can also function as a cell adhesion molecule, providing a substratum for adhesion of platelets and endothelial cells. In this report, we studied the adhesion of platelets to surface-immobilized fibrinogen under flow in different shear rates. Heparinized whole blood containing mepacrine-labeled platelets was perfused for two minutes at various wall shear rates from 250 to 2,000 s-1 in a parallel plate flow chamber. The number of adherent fluorescent platelets was quantitated every 15 seconds with an epifluorescent videomicroscope and digital image processing system. When compared with platelet adhesion and aggregation seen on glass surfaces coated with type I bovine collagen, a significant increase in platelet adhesion was observed on immobilized fibrinogen up to wall shear rates of 800 s-1. The adherent platelets formed a single layer on fibrinogen-coated surfaces. Under identical conditions, no significant adhesion was observed on fibronectin- or vitronectin-coated surfaces. Although platelet adhesion to collagen was substantially inhibited by the platelet inhibitors prostaglandin E1 and theophylline, these inhibitors had no effect on platelet adhesion to fibrinogen. Platelets adhered to recombinant homodimeric wild-type (gamma 400-411) fibrinogen, but not to the recombinant homodimeric gamma' variant of fibrinogen. Platelet adhesion to recombinant fibrinogen with RGD to RGE mutations at positions alpha 95-97 and alpha 572-574 was similar to that with plasma-derived fibrinogen. These results show that platelets adhere to fibrinogen-coated surfaces under moderate wall shear rates, that the interaction is mediated by the fibrinogen 400-411 sequence at the carboxy-terminus of the gamma chain, and that the interaction is independent of platelet activation and the RGD sequences in the alpha chain.

Changes in Cytosolic Calcium Concentrations and Cell Morphology in Single Platelets Adhered to Fibrinogen-Coated Surface Under Flow

Changes in intracellular calcium concentration [Ca2+]i of fura-2-loaded human platelet during its adhesion to a fibrinogen-coated surface were studied, using a flow chamber mounted on an epifluorescence microscope equipped with digital-ratio imaging. Adherent platelets were individually mapped under a scanning electron microscope to establish the possible correlation between adhesion-associated shape alterations and [Ca2+]i changes. We found that 1) there was no immediate [Ca2+]i elevation on platelet adhesion; 2) [Ca2+]i changes varied drastically platelets with a lag time ranging 10 to 200 s, averaging about 1 minute; 3) the pattern of [Ca2+]i changes varied drastically among individual adherent platelets; 4) the degree of [Ca2+]i elevation appeared to correlate with the extent of morphology change, with the vast majority ( > 90%) of spread platelets showed detectable [Ca2+]i changes; 5) neither morphological nor [Ca2+]i changes correlated with the lag time; 6) platelets treated with dimethyl-BAPTA (15 mumol/L) underwent normal shape change without [Ca2+]i elevation; 7) cytochalasin D (10 mumol/L) inhibited both shape change and [Ca2+]i elevation; 8) colchicine (1 mmol/L) was ineffective in both regards. We conclude that although platelet adhesion-associated shape changes may be accompanied with heterogeneous [Ca2+]i changes that are microfilament-dependent, [Ca2+]i changes do not happen immediately after platelet-surface contact and they are not required for adherent platelets to undergo postcontact morphological changes.

Channel catfish, Ictalurus punctatus rafinesque, neutrophil adhesion to selected extracellular matrix proteins, lipopolysaccharide and catfish serum

Adhesion of leukocytes to endothelium and extracellular matrix proteins is an important step in the inflammatory process. Therefore, the adhesion of channel catfish neutrophils to a surface coated with extracellular matrix proteins, LPS, and non-immune catfish serum was evaluated. Stimulation of neutrophils with phorbol dibutyrate (PDBU) resulted in at least two-fold increases in cellular adhesion to all substrates tested except laminin. When EDTA was included during or after PDBU stimulation, neutrophil adhesion to fibrinogen and LPS coated surfaces was reduced to the level of unstimulated neutrophils or to 50–60% of that for stimulated neutrophils. Similarly, EDTA and Ca 2+ Mg 2+ deficient medium reduced homotypic aggregation of PDBU stimulated neutrophils to background levels. Adhesion of stimulated neutrophils to fibrinogen coated surfaces was inhibited 44, 33, and 50% when soluble fibrinogen, fibronectin, and serum, respectively, were used to block the adhesion assay. The tripeptide integrin adhesion recognition sequence, Arg-Gly-Asp (RGD), caused 83% reduction and the fibrinogen-binding inhibitor protein caused 10% reduction in binding of stimulated neutrophils to fibrinogen coated surfaces. Two hexapeptides tested did not reduce neutrophil adhesion to fibrinogen. The binding of channel catfish neutrophils to the matrices used in the present study is suggestive that integrin mediated adhesion occurs during biological and pathological processes of teleosts.

Flow-induced detachment of adherent platelets from fibrinogen-coated surface

A study of the shear forces under which adherent platelets of different morphologies can be detached from surfaces was carried out using a newly designed tapered flow chamber, which covered the entire shear range of physiological circulation. Platelets that naturally settled on a fibrinogen-coated surface were exposed to shear flow and were subsequently processed for scanning electron microscopic observation. We found that 1) the density of platelets remaining after flow exposure decreased with local shear stress, 2) adherent platelets of different morphologies withstood different levels of shear stress: most round cells and 40% of the cells that had a few short pseudopods were detached at < 10 dyn/cm2, whereas most spread cells could withstand 50 dyn/cm2, 3) pulsatile flow was more effective in removing adherent platelets than equivalent steady flow, 4) cytochalasin D and colchicine retarded platelet shape change and made them more easily detached by shear forces, and 5) metabolic energy-depleted platelets spread readily and formed shear-resistant clumps. Our observations indicated that adherent platelets of different morphologies on a fibrinogen-coated surface could withstand different levels of flow shear stress.

The specific type IV phosphodiesterase inhibitor rolipram combined with adenosine reduces tumor necrosis factor-α-primed neutrophil oxidative activity

Monocytes and macrophages produce tumor necrosis factor-α (TNFα) in response to microbial products including endotoxin. TNFα is a potent primer of neutrophil (PMN) oxidative activity. Certain xanthine phosphodiesterase (PDE) inhibitors such as pentoxifylline have been shown to inhibit stimulated oxidative activity in PMN. In the present study, the non-xanthine PDE type IV inhibitor rolipram (4-[3′-cyclopentyloxy-4′-methoxyphenyl]-2-pyrrolidone) alone and in combination with adenosine is examined as a potential modulator of TNFα-primed PMN oxidative activity. Attainable in vivo concentrations of rolipram and physiological concentrations of adenosine alone and together synergistically decreased rhTNFα-primed suspended PMN oxidative activity stimulated by the chemoattractant f-met-leu-phe. The rolipram effect was reversible by washing, and rolipram had a comparable effect if added before or after priming, indicating that its effect was on the primed response rather than on priming per se. In addition, rolipram especially when combined with adenosine, decreased rhTNFα-stimulated PMN adherence to a fibrinogen-coated surface, and the oxidative burst of rhTNFα-stimulated adherent PMN. The specific adenosine A 2a receptor agonists CGS 21680 and WRC-0474 had comparable activity to adenosine in these experiments. Adenosine (or CGS 21680) combined with rolipram synergistically increased f-met-leu-phe-stimulated PMN cAMP content. The effects of both adenosine and rolipram with adenosine could be only partly counteracted by treatment of the PMN with the protein kinase A inhibitor KT 5720, indicating that protein phosphorylation is only partially involved. Rolipram activity was about 1000x (by molar concentration) greater than pentoxifylline in comparable assays. Thus, rolipram, especially when combined with adenosine, has potent modulating effects on PMN activation and may be useful in decreasing inflammatory tissue damage in patients with sepsis.

FcγRII, FcγRIII, and CD18 receptors mediate in part neutrophil activation on a plasma coated expanded polytetrafluoroethylene surface*

A biomaterial-induced polymorphonuclear neutrophil (PMN) defect may predispose the implanted vascular graft to infection. PMNs bind, activate, and undergo morphologic changes when exposed to uncoated or plasma coated expanded polytetrafluoroethylene (ePTFE) surfaces. The purpose of this study was to investigate whether the CD18 integrin receptor or the immunoglobulin receptors Fc gamma RII and Fc gamma RIII mediate either PMN binding or activation on ePTFE. PMN binding and activation were determined after incubation of these cells on human immunoglobulin (IgG) or fibrinogen coated surfaces and uncoated or plasma coated ePTFE. PMN activation was measured by using the ferricytochrome reduction assay. Binding was determined with chromium 51-labeled PMNs. To block the Fc gamma RII, Fc gamma RIII, and CD18 receptors, PMNs were preincubated with the monoclonal antibodies (mAbs) IV.3, 3G8, and IB4, respectively. Irrelevant isotype matched mAbs were used as control. Monoclonal antibody IB4 inhibited binding of activated PMNs to fibrinogen coated surfaces. Binding to IgG was affected by either mAb IB4 or IV.3, but the greatest degree of inhibition was achieved when mAbs IB4 and IV.3 were used in combination. IgG-induced activation was partially inhibited by mAb IV.3 but was fully inhibited by a combination of mAbs IB4 and IV.3 The mAbs did not affect PMN binding to uncoated or plasma coated ePTFE, nor was PMN activation on the uncoated ePTFE surface inhibited. PMN activation on the plasma coated ePTFE surface was, however, partially inhibited by the combination of mAb IB4 with either mAb IV.3 or 3G8. A synergistic interaction between the PMN Fc gamma RII receptor and the CD18 integrin receptor accounts for surface bound IgG-induced cell activation. Both receptors also play a role in mediating PMN activation on the plasma-coated ePTFE surface.