Activation of human platelets by a stimulatory monoclonal antibody

The clinical significance of the interaction of antibodies with circulating platelets is well documented, but the mechanisms underlying these interactions are not fully known. Here we describe the characterization of anti-human platelet membrane protein monoclonal antibody (mAb) termed F11. Interaction of mAb F11 with human platelets resulted in dose-dependent granular secretion, measured by [14C]serotonin and ATP release, fibrinogen binding and aggregation. Analysis of the specific binding of mAb F11 to platelets revealed a high affinity site with 8,067 +/- 1,307 sites per platelet with a dissociation constant (Kd) of 2.7 +/- 0.9 x 10(-8) M. Two membrane proteins of 32,000 and 35,000 daltons, identified by Western blotting, were recognized by mAb F11. Incubation of 32Pi-labeled platelets with mAb F11 resulted in rapid phosphorylation of intracellular 40,000- and 20,000-dalton proteins, followed by dephosphorylation of these proteins. Monovalent Fab fragments or Fc fragments of mAb F11 IgG did not induce platelet aggregation or secretion; however, Fab fragments of mAb F11 IgG blocked mAb F11-induced platelet aggregation and the binding of 125I-mAb F11 to platelets. The addition of an anti-GPIIIa monoclonal antibody (mAb G10), which inhibits 125I-fibrinogen binding and platelet aggregation, completely blocked mAb F11-induced [14C]serotonin secretion and aggregation but not the binding of 125I-mAb F11 to platelets. mAb G10 also inhibited the increase in the phosphorylation of the 40,000- and 20,000-dalton proteins induced by mAb F11. These results implicate the involvement of the GPIIIa molecule in the chain of biochemical events involved in the induction of granular secretion.     

Use of a monoclonal antibody to measure the surface expression of thrombospondin following platelet activation

The radiolabelled monoclonal antibody, 5G11, directed against native thrombospondin, has been used to assess the surface expression of secreted thrombospondin on human blood platelets. Emphasis has been placed on studying the role of fibrinogen in this process. Unstimulated platelets bound low amounts of 5G11 (about 2000 molecules/platelet). Binding increased 2-fold and 5-7-fold after stimulation of platelets with ADP or thrombin (or ionophore A23187) respectively. Unstimulated platelets from patients deficient in alpha-granule proteins (gray platelet syndrome) bound baseline levels of 5G11. However, binding was not increased after activation. Thrombospondin expression on thrombin-stimulated normal platelets was for a large part divalent-cation-dependent and was not affected by AP-2, a monoclonal antibody to GPIIb-IIIa complexes. However, binding of 5G11 was some 50% lower when platelets were stimulated in the presence of Fab fragments of a polyclonal rabbit antibody to fibrinogen. This suggested either a direct binding of thrombospondin to surface-bound fibrinogen or a steric inhibition due to a close proximity of the two proteins. The fact that binding of 5G11 was at the lower limit of the normal range to the stimulated platelets of an afibrinogenaemic patient specifically lacking detectable fibrinogen favoured the latter explanation. Thus, a major fibrinogen-independent pathway for thrombospondin expression must exist.     

Interaction of platelet factor 4 with human platelets

Human washed resting platelets bound 125I-labeled platelet factor 4 in a reaction which was saturable and approached equilibrium within 15-30 min. Scatchard plot analysis of the binding isotherms suggested a single class of specific binding sites. Excess of unlabeled protein and low- and high-affinity heparin competed for platelet factor 4 binding sites on the platelet surface and caused a partial displacement of this molecule. Anti-platelet factor 4 Fab fragments caused inhibition of binding of 125I-platelet factor 4 to platelets. Most of the labeled platelet factor 4 which was bound to intact platelets was recovered in the Triton X-100-insoluble cytoskeletal fraction prepared from the same platelets after their stimulation by thrombin. The association with the cytoskeleton was inhibited by anti-platelet factor 4 Fab fragments and by low-affinity heparin. Anti-platelet factor 4 125I-labeled Fab fragments bound to resting platelets, and this binding was greatly increased following platelet stimulation with thrombin. This suggested that endogenously secreted platelet factor 4 also binds to the platelet surface. No significant binding to platelets of 125I-labeled beta-thromboglobulin and 125I-labeled anti-beta-thromboglobulin Fab fragments was observed. Fab fragments of monospecific anti-human platelet factor 4 antibody raised in rabbits inhibited platelet aggregation and secretion induced by low concentrations of thrombin. Fab fragments of anti-beta-thromboglobulin antibody had no inhibitory effect. We suggest that the binding of alpha-granule-derived platelet factor 4 to the specific sites on the surface of platelets may modulate platelet aggregation and secretion induced by low levels of platelet agonists.     

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.     

F(ab')2 reagents are not required if goat, rather than rabbit, antibodies are used to detect human surface immunoglobulin.

The present report provides evidence that whole goat anti-human immunoglobulin, unlike similar reagents produced in the rabbit, binds both to the same number of and the same individual cells as the F(ab')2 fragments of rabbit or goat anti-human immunoglobulin. These results suggest that goat IgG has a lower affinity for the Fc receptors of human lymphocytes and monocytes than rabbit IgG. Because of this property, whole goat antibodies against human immunoglobulin can be used as simple, convenient relatively inexpensive reagents for the routine detection of immunoglobulin on cell surfaces by immunofluorescence microscopy. The preparation of F(ab')2 fragments of anti-immunoglobulin, which are necessary when rabbit antibodies are used, does not appear to -e required if goat antibodies can be empolyed. This observation has multiple practival applications in cellular immunology.     

Characterization of a monoclonal antibody which is an activator of rabbit platelets

A monoclonal antibody (MAb) raised against rabbit platelet membranes was shown to be a strong agonist to induce platelet aggregation and secretion. This MAb, designated 19CB-1, was identified as an IgM and purified to near homogeneity by ammonium sulfate precipitation and Q-sepharose column chromatography. Aggregation induced by 19CB-1 was only slightly affected in the presence of creatine phosphate/creatine phosphokinase and aspirin, indicating that it was not mediated through the cyclooxygenase pathway and the release of ADP. 19CB-1 Fab fragments did not induce platelet aggregation. However, 19CB-1-induced aggregation was inhibited by these Fab fragments. 19CB-1 also elicited a rise in cytoplasmic calcium concentration in fura-2 loaded platelets. In the absence of external calcium, a substantial calcium signal remained to be observed, suggesting the release of calcium from intracellular stores in response to 19CB-1. This MAb reacted primarily with a polypeptide of Mr = 57,000, as revealed by immunoblotting. These results suggest that the 57 kDa antigen is one of the platelet surface proteins directly involved in the activation of rabbit platelets.     

Redistribution of alpha-granules and their contents in thrombin- stimulated platelets

The redistribution of beta-thromboglobulin (beta TG), platelet Factor 4 (PF4), and fibrinogen from the alpha granules of the platelet after stimulation with thrombin was studied by morphologic and immunocytochemical techniques. The use of tannic acid stain and quick-freeze techniques revealed several thrombin-induced morphologic changes. First, the normally discoid platelet became rounder in form, with filopodia, and the granules clustered in its center. The granules then fused with one another and with elements of the surface-connected canalicular system (SCCS) to form large vacuoles in the center of the cell and near the periphery. Neither these vacuoles nor the alpha granules appeared to fuse with the plasma membrane, but the vacuoles were connected to the extracellular space by wide necks, presumably formed by enlargement of the narrow necks connecting the SCCS to the surface of the unstimulated cell. The presence of fibrinogen, beta TG, and PF4 in corresponding large intracellular vacuoles and along the platelet plasma membrane after thrombin stimulation was demonstrated by immunocytochemical techniques in saponin-permeabilized and nonpermeabilized platelets. Immunocytochemical labeling of the three proteins on frozen thin sections of thrombin-stimulated platelets confirmed these findings and showed that all three proteins reached the plasma membrane by the same pathway. We conclude that thrombin stimulation of platelets causes at least some of the fibrinogen, beta TG, and PF4 stored in their alpha granules to be redistributed to their plasma membranes by way of surface-connected vacuoles formed by fusion of the alpha granules with elements of the SCCS.     

Activated platelets form protected zones of adhesion on fibrinogen and fibronectin-coated surfaces

Leukocytes form zones of close apposition when they adhere to ligand- coated surfaces. Because plasma proteins are excluded from these contact zones, we have termed them protected zones of adhesion. To determine whether platelets form similar protected zones of adhesion, gel-filtered platelets stimulated with thrombin or ADP were allowed to adhere to fibrinogen- or fibronectin-coated surfaces. The protein- coated surfaces with platelets attached were stained with either fluorochrome-conjugated goat anti-human fibrinogen or anti-human fibronectin antibodies, or with rhodamine-conjugated polyethylene glycol polymers. Fluorescence microscopy revealed that F(ab')2 anti- fibrinogen (100 kD) did not penetrate into the contact zones between stimulated platelets and the underlying fibrinogen-coated surface, while Fab antifibrinogen (50 kD) and 10 kD polyethylene glycol readily penetrated and stained the substrate beneath the platelets. Thrombin- or ADP-stimulated platelets also formed protected zones of adhesion on fibronectin-coated surfaces. F(ab')2 anti-fibronectin and 10 kD polyethylene glycol were excluded from these adhesion zones, indicating that they are much less permeable than those formed by platelets on fibrinogen-coated surfaces. The permeability properties of protected zones of adhesion formed by stimulated platelets on surfaces coated with both fibrinogen and fibronectin were similar to the zones of adhesion formed on fibronectin alone. mAb 7E3, directed against the alpha IIb beta 3 integrin blocked the formation of protected adhesion zones between thrombin-stimulated platelets and fibrinogen or fibronectin coated surfaces. mAb C13 is directed against the alpha 5 beta 1 integrin on platelets. Stimulated platelets treated with this mAb formed protected zones of adhesion on surfaces coated with fibronectin. These protected zones were impermeable to F(ab')2 antifibronectin but were permeable to 10 kD polyethylene glycol. These results show that activated platelets form protected zones of adhesion and that the size of molecules excluded from these zones depends upon the composition of the matrix proteins to which the platelets adhere. They also show that formation of protected zones of adhesion by platelets requires alpha IIb beta 3 integrins while the permeability properties of these zones of adhesion are regulated by both alpha IIb beta 3 and alpha 5 beta 1 integrins.     

Regulatory control of complement on blood platelets. Modulation of platelet procoagulant responses by a membrane inhibitor of the C5b-9 complex

Antibody against a membrane inhibitor of the C5b-9 complex has been used to investigate regulatory control of the terminal complement proteins on blood platelets. Monospecific rabbit antibody (alpha-P18) was raised against the purified 18-kDa erythrocyte membrane inhibitor of C5b-9 (Sugita, Y., Nakano, Y., and Tomita, M. (1988) J. Biochem. (Tokyo) 104, 633-637). In addition to its interaction with erythrocytes, this antibody (and its Fab) bound specifically to platelet membranes. In immunoblots of cell membrane proteins prepared under non-reducing conditions, alpha-P18 bound specifically to an 18-kDa erythrocyte membrane protein and to a 37-kDa platelet membrane protein. Absorption of this antibody by platelet membranes competed its binding to the purified 18-kDa erythrocyte protein, suggesting that epitopes expressed by the erythrocyte 18-kDa C5b-9 inhibitor are common to the platelet. When bound to the platelet surface, the Fab of alpha-P18 increased C9 activation by membrane C5b-8, monitored by exposure of a complex-dependent C9 neo-epitope. Although alpha-P18 caused little increase in the cytolysis of platelets treated with C5b-9 (total release of lactate dehydrogenase less than 5%), it markedly increased the cell stimulatory responses induced by these complement proteins, including, secretion from platelet alpha- and dense granules, conformational activation of cell surface GP IIb-IIIa, release of membrane microparticles from the platelet surface, and exposure of new membrane binding sites for components of the prothrombinase enzyme complex. Prior incubation of C5b67 platelets with 100 micrograms/ml alpha-P18 (Fab) lowered by approximately 10-fold the half-maximal concentration of C8 required to elicit each of these responses (in the presence of excess C9). Incubation with alpha-P18 (Fab) alone did not activate platelets, nor did incubation with this antibody potentiate the stimulatory responses of platelets exposed to other agonists. These data indicate that a membrane inhibitor of the C5b-9 complex normally serves to attenuate the procoagulant responses of blood platelets exposed to activated complement proteins, and suggest the mechanism by which a deletion or inactivation of this cell surface component would increase the risk of vascular thrombosis.     

A collagen-like immunodeterminant on the surface of Streptococcus sanguis induces platelet aggregation

The basis of similarities in the mechanism of human platelet aggregation induced by soluble collagen and the dental plaque bacterium Streptococcus sanguis was analyzed. Structural and functional comparisons were made by using molecular probes, including rabbit antibody fractions reactive with components on S. sanguis and a synthetic, collagen-like octapeptide mimicking segments from cyanogen bromide fragments 6 and 4 of types I and III collagen, respectively. When platelets were pretreated with tryptic peptides or class II antigen of S. sanguis or with the synthetic, collagen-like octapeptide, the onset of aggregation in response to S. sanguis and collagen was prolonged. When compared to other peptides of similar size and charge, the collagen-like peptide's action towards platelets was shown to be selective. Indeed, absorption of antiserum to S. sanguis cells with particulate type I collagen removed specificities directed at a single S. sanguis antigen. These observations suggested that a common platelet-interactive immunodeterminant on soluble types I and III collagens, particulate type I collagen, and S. sanguis cells was present. Selective inhibition by antibody was used to show structural similarities between the S. sanguis surface proteins and collagen. When either agonist was pretreated with anti-S. sanguis IgG or Fab fragments, the lag time to onset of platelet aggregation was increased. Greater increases in the lag time to aggregation was seen when S. sanguis cells or collagen were pretreated with anti-S. sanguis IgG or Fab fragments made relatively specific for the class II antigen. Neutralization of the platelet-interactive action of the octapeptide by anti-S. sanguis antibody fractions showed that the immunodeterminant common to S. sanguis and collagen triggered platelets in plasma to aggregate. Although the anti-S. sanguis antibodies could inhibit fibrillogenesis, this action was apparently independent of interactions with platelets. In contrast, S. sanguis could bind or adhere to platelets by different determinants. Our data suggest that platelets have at least two distinct sites that bind collagen or S. sanguis. One of these may be a common site for collagen and S. sanguis agonists.     

Fibrinogen distribution on surfaces and in organelles of ADP stimulated human blood platelets

The fibrinogen distribution in platelet organelles after ADP-stimulation was investigated with anti-human fibrinogen using protein A-gold applied to serial sections. Fibrinogen was detected in the so-called alpha-granules of platelets and also in granule protrusions which were observed after ADP-stimulation. The ends of these protrusions were formed as coated membranes and the tips were often in apposition to the surface connected membranes or the plasmalemma. At such places fusion events and hence signs of an exocytosis could be demonstrated by means of cryofixation and cryosubstitution. Examination of serial sections revealed fibrinogen on all these granule profiles. Surface connected membranes, free surfaces and the characteristic structure of the contact zones of aggregated platelets were also labelled by gold particles but less than anticipated. On the platelet surfaces and surface connected membranes fibrinogen was rarely demonstrable with ferritin-labelled anti-human fibrinogen on washed or thrombin-stimulated, almost fibrinogen free platelets. After addition of human fibrinogen to the thrombin stimulated and disaggregated platelets a part of the platelets aggregated spontaneously and formed characteristic contact zones. Anti-human fibrinogen was observed on the free surfaces, in filamentous bridges between the contact spaces and in a tubular surface connected membrane system with involvement of coated membranes at the central ends of these structures. The results indicate the following: all alpha-granules contain fibrinogen; after ADP-stimulation secretion takes place with involvement of coated membranes; during aggregation fibrinogen binds to platelet surfaces and forms contact spaces; fibrinogen is taken up by the surface connected system with involvement of coated membranes.     

A receptor-induced binding site in fibrinogen elicited by its interaction with platelet membrane glycoprotein IIb-IIIa.

The interaction of fibrinogen with membrane glycoprotein GPIIb-IIIa regulates platelet aggregation. This ligand:integrin receptor interaction elicits conformational changes in GPIIb-IIIa as evidenced by the induction of ligand-induced binding sites which are recognized by antibodies that react selectively with the occupied receptor. The dynamic nature of these conformational changes is now demonstrated by the identification and characterization of a receptor-induced binding site (RIBS) elicited in fibrinogen bound to GPIIb-IIIa. A monoclonal antibody to fibrinogen, anti-Fg-RIBS-I, failed to bind to nonstimulated platelets in the presence or absence of fibrinogen. However, when platelets were stimulated with an agonist, the antibody reacted with platelet-bound fibrinogen even in the presence of a marked excess of unbound fibrinogen. A key element of the RIBS epitope has been precisely localized to residues 373-385 of the gamma chain of fibrinogen. Conformational elements also are important in defining the epitope. Fab fragments of the antibody inhibited platelet aggregation. As these fragments also inhibited fibrin polymerization, a commonality between these two diverse functions of fibrinogen in thrombus formation is indicated. In general, antibodies to RIBS and ligand-induced binding site provide unique probes for characterizing ligand:receptor interactions.     

The platelet open canalicular system: a final common pathway.

Channels of the surface-connected, open canalicular system (OCS) of human platelets serve as the pathway for transport of substances into the cells and as conduits for the discharge of alpha granule products secreted during the platelet release reaction. The purpose of the present study was to determine if both functions of the OCS can take place simultaneously. Suspensions of washed platelets were exposed to thrombin at 1 U/ml for 5, 60, or 180 seconds in the presence of fibrinogen molecules coupled to particles of colloidal gold (Fgn/Au). The samples were fixed in a low concentration of glutaraldehyde and embedded in L.R. White resin to preserve antigenicity. Thin sections were exposed to a rabbit polyclonal antibody to human fibrinogen followed by an anti-rabbit IgG coupled to 5-nm gold beads. Thrombin caused Fgn/Au particles to bind to platelets and enter channels of the surface-connected OCS. Endogenous fibrinogen detected by immunogold 5-nm beads were localized to alpha granules in resting platelets and 5 seconds after thrombin stimulation. At 60 seconds and 3 minutes Fgn/Au particles were present in swollen alpha granules, as well as OCS channels. Fibrinogen gold beads were evident in alpha granules and OCS channels connected to the platelet surface. The 18- to 20-nm Fgn/Au particles were in the same channels of the OCS as fibrinogen gold beads. The OCS is a final common pathway for uptake of particulates and discharge of secretory products in thrombin-activated human platelets.     

Identification of glycoprotein IV (CD36) as a primary receptor for platelet-collagen adhesion

The role of glycoprotein IV (GPIV) in platelet activation processes has been examined by several different approaches: (i) Fab fragments of a monospecific polyclonal antibody to purified platelet GPIV (approximately 20 micrograms/ml) completely inhibited platelet shape change, aggregation, and secretion induced by collagen. Aggregation and secretion by ADP (but not shape change) and by epinephrine were also inhibited, but there was no effect on platelet activation induced by thrombin, arachidonate, or ionophore A23187. (ii) Purified GPIV was able to compete completely with membrane-bound GPIV to inhibit platelet activation induced by collagen, including shape change, but not in activation induced by any of the other platelet agonists. 50% inhibition of collagen-induced activation and secretion were obtained at GPIV concentrations of approximately 10 nM (1 micrograms/ml). (iii) Purified GPIV bound rapidly and reversibly to collagen Type I fibrils, and binding was not inhibited by adhesive proteins such as denatured collagen, fibronectin, fibrinogen, or von Willebrand factor. The direct binding of purified GPIV to collagen Type I fibrils fit best to a single site model with Kd 0.34 +/- 0.10 nM. (iv) Using a microtiter assay, platelet adhesion to collagen was shown to be inhibited by Fab fragments of monospecific polyclonal anti-GPIV antibodies, but adhesion to other adhesive proteins was unaffected. (v) When anti-GPIV was added at various times during adhesion the time dependence of inhibition was seen to be biphasic. Anti-GP antibody was able to reverse adhesion that occurred within the first 5-8 min and to inhibit adhesion occurring thereafter. These results demonstrate that GPIV mediates the early stages of platelet recognition by and attachment to collagen but that there may be a second GPIV-independent mechanism that mediates the subsequent anchorage of these adherent platelets.     

Neocarzinostatin: controlled release of chromophore and its interaction with DNA

A nonagglutinating derivative of wheat germ agglutinin has been prepared that binds to platelets and precipitates an antibody to the lectin. Platelets treated with this inactive derivative released serotonin when exposed to bivalent F(ab′)₂, but not monovalent Fab, fragments of the lectin antibody. Bridging of platelet-bound Fab by an antibody again induced secretion. The F(ab′)₂ or Fab fragments plus IgG, without the derivative, did not induce secretion. This secretion was not affected by indomethacin showing a direct activation of platelets. Platelets treated with con A followed by F(ab′)₂ to con A did not secrete. In addition, lentil lectin failed to release platelet serotonin. The receptors of the lectin derivative are mobile on the platelet surface and their redistribution may lead to secretion.     

Intracellular distribution and degradation of immunoglobulin G and immunoglobulin G fragments injected into HeLa cells

Intact rabbit immunoglobulin G molecules (IgGs) and their papain or pepsin fragments were radio-iodinated and injected into HeLa cells. Whole IgGs, Fab2, and Fc fragments were degraded with half-lives of 60- 90 h, whereas half-lives of Fab fragments were 110 h. These results indicate that proteolytic cleavage in the hinge region of the IgG molecule is not the rate-limiting step in its intracellular degradation. The hingeless human myeloma protein, Mcg, was degraded at the same rate as bulk human IgG, providing further evidence that the proteolytically susceptible hinge region is not important for intracellular degradation of IgG molecules. SDS acrylamide gel analysis of injected rabbit IgG molecules revealed that heavy and light chains were degraded at the same rate. Injected rabbit IgGs and rabbit IgG fragments were also examined on isoelectric focusing gels. Fab, Fab2, and Fc fragments were degraded without any correlation with respect to isoelectric point. Positively charged rabbit IgGs disappeared more rapidly than their negative counterparts, contrary to the trend reported for normal intracellular proteins. The isoelectric points of two mouse monoclonal antibodies were essentially unchanged after injection into HeLa cells, suggesting that the altered isoelectric profile observed for intact rabbit IgG resulted from degradation and not protein modification. The intracellular distributions of IgG fragments and intact rabbit IgG molecules were determined by autoradiography of thin sections through injected cells. Intact IgG molecules were excluded from HeLa nuclei whereas both Fab and Fc fragments readily entered them. Thus, for some proteins, entry into the nuclear compartment is determined primarily by size.     

Washed human platelets prevent ischemia-reperfusion edema in isolated rabbit lungs

Washed human platelets prevent edema formation in isolated rabbit lungs infused with xanthine oxidase, an enzyme that injures endothelial membranes by generating extracellular oxidants. We hypothesized that platelets would similarly preserve membrane permeability in isolated lungs exposed to ischemia-reperfusion injury, a model that perturbs endothelial cells by the generation of intracellular oxidants. Isolated perfused rabbit lungs (IPL) were exposed to warm ischemia-reperfusion to cause lung edema. The infusion of washed human platelets (1.05 +/- 0.02 x 10(10) cells) prevented edema formation as measured by lung weight gain, wet-to-dry lung weight ratios, histological edema, and preservation of paraendothelial cell tight junctions. Inhibition of the platelet glutathione redox cycle with 1,3-bis(2-chloroethyl)-1-nitrosourea, dehydroepiandrosterone, or 1-chloro-2,4-dinitrobenzene interfered with platelet protective effects. In contrast, inhibition of platelet catalase with aminotriazole and H2O2 had no effect on platelet protection. Lung tissue malonyldialdehyde concentrations were similar in isolated lungs exposed to ischemia-reperfusion with or without the infusion of platelets. These results indicate that platelet attenuation of ischemia-reperfusion lung edema depends on platelet glutathione redox cycle antioxidants but not platelet catalase.     

Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: an isolated defect in platelet procoagulant activity

Activation of human platelets by complement proteins C5b-9 is accompanied by the release of small plasma membrane vesicles (microparticles) that are highly enriched in binding sites for coagulation factor Va and exhibit prothrombinase activity. We have now examined whether assembly of the prothrombinase enzyme complex (factors VaXa) is directly linked to the process of microparticle formation. Gel-filtered platelets were incubated without stirring with various agonists at 37 degrees C, and the functional expression of cell surface receptors on platelets and on shed microparticles was analyzed using specific monoclonal antibodies and fluorescence-gated flow cytometry. In addition to the C5b-9 proteins, thrombin, collagen, and the calcium ionophore A23187 were each found to induce formation of platelet microparticles that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa. These microparticles were enriched in binding sites for factor Va, and their formation paralleled the expression of catalytic surface for the prothrombinase enzyme complex. Little or no microparticle release or prothrombinase activity were observed when platelets were stimulated with epinephrine and ADP, despite exposure of platelet fibrinogen receptors by these agonists. When platelets were exposed to thrombin plus collagen, the shed microparticles contained activated GP IIb-IIIa complexes that bound fibrinogen. By contrast, GP IIb-IIIa incorporated into C5b-9 induced microparticles did not express fibrinogen receptor function. Platelets from a patient with an isolated defect in inducible procoagulant activity (Scott syndrome) were found to be markedly impaired in their capacity to generate microparticles in response to all platelet activators, and this was accompanied by a comparable decrease in the number and function of inducible factor Va receptors. Taken together, these data indicate that the exposure of the platelet factor Va receptor is directly coupled to plasma membrane vesiculation and that this event can be dissociated from other activation-dependent platelet responses. Since a catalytic membrane surface is required for optimal thrombin generation, platelet microparticle formation may play a role in the normal hemostatic response to vascular injury.     

Erythrocytes infected by Plasmodium falciparum activate human platelets

Blood platelets are involved in Plasmodium falciparum malaria pathology as shown by thrombocytopenia and increased plasma level of two alpha granule proteins: beta thromboglobulin (beta TG) and platelet factor 4 (PF4). In this study we demonstrate that Plasmodium falciparum parasitized erythrocytes activate directly the secretion of beta TG and PF4 by human platelets. This secretion is related to parasitemia and occurs immediately after contact. Treatment of parasited erythrocytes by trypsin and diffusion chamber experiments suggest that platelet activation is triggered by parasitic substances shed on erythrocyte membrane and released in the culture medium.     

The role of ligand-ligand interactions in competition by fibrinogen and fibrin degradation products for fibrinogen binding to human platelets

Binding to human platelets of radioiodinated human fibrinogen and fragments X, Y, D, D₁ dimer and E was studied to determine the domain of the fibrinogen molecule responsible for binding to the platelet receptor. Although the fragments did not bind, some wer able to complete for the binding of fibrinogen to platelets. It was postulated that the fragments bound to fibrinogen and subsequently interfered with its binding to the receptor. Two approaches were developed to test this hypothesis. In the first technique, molecular exclusion on Sephacryl S-200 superfine was utilized to examine the interaction of radiolabeled fragments with fibrinogen. In the second seties of studies, fibrinogen-Sepharose was prepared and the binding of degradation products directly determined. A spin dialysis apparatus was employed in each case to achieve rapid separation of bound and free radioligand. These studies demonstrated that fragments D and E bind to fibrinogen. Therefore, the mechanism by which degradation products interfere with fibrinogen binding to the platelet receptor is ligand-ligand interaction rather than binding of the fragments to the receptor. Since none of the radiolabeled degradation products bound to platelets, it appears that receptor recognition requires the intact molecule.