The basement membrane underlying the vascular endothelium is not thrombogenic: in vivo and in vitro studies with rabbit and human tissue

Studies examining the interaction of platelets with exposed subendothelium in vivo have reported conflicting results. To examine possible explanations for the apparently discrepant findings, we measured the platelet reactivity of subendothelium prepared by a number of methods both in vivo and in vitro. In addition, we examined the possibility that 13-hydroxyoctadecadinoic acid (13-HODE), an endothelial cell-derived chemorepellant, modulates the reactivity of the subendothelium to platelets. In vivo, the subendothelium of segments of rabbit carotid arteries was exposed by removing the endothelial cells by air perfusion or by balloon catheter stripping. Platelet accumulation onto the de-endothelialized segments was assessed by 3H-radioactivity uptake, using 3H-adenine-labelled platelets, and by scanning electron microscopy. In vitro, 3H-adenine-labelled platelet adhesion was measured onto plain plastic discs and onto plastic discs coated with the following purified basement membrane components: collagens type I, III, IV, V, laminin, or fibronectin. In addition, 3H-adenine-labelled platelet adhesion was measured onto plastic discs covered with human endothelial cells or onto the basement membrane underlying the endothelial cells. In vivo, there was marked 3H-platelet accumulation onto the balloon catheter carotid arteries one hour after injury. In contrast, there was no platelet accumulation onto the subendothelium of carotid arteries de-endothelialized by air perfusion. These differences were confirmed by scanning electron microscopy. Transmission electron microscopic examination demonstrated that the extracellular matrix was intact following the air perfusion injury whereas the majority of it was removed by the balloon catheter injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two novel anti-von Willebrand factor monoclonal antibodies

Von Willebrand Factor is a multimer produced by endothelial cells and megakaryocytes, being stored in intracellular organelles, such as the Weibel-Palade bodies and alpha-granules in endothelial cells and platelets, respectively. This molecule acts as a carrier protein for factor VIIIc, involved in the intrinsic pathway of blood coagulation maintaining its stability in circulation. Von Willebrand Factor also plays an important role in platelet aggregation and adhesion to injured vessel wall. It interacts with platelets through two distinct glycoproteins, GPIb and GPIIb/IIIa. We raised two monoclonal antibodies, ECA-3 and ECA-4, against human umbilical vascular endothelial cells that recognize and immunoprecipitate von Willebrand Factor. Interestingly, ECA-4 monoclonal antibody is able to completely inhibit platelet agglutination induced by ristocetin, suggesting that it binds to von Willebrand Factor close to platelet GPIb binding site. The use of monoclonal antibodies to identify von Willebrand Factor binding regions to factor VIII or platelets has been reported by others. In pulmonary hypertension, abnormalities have been detected on the multimeric structure of the molecule as well as on its proteolytic fragments, by using monoclonal antibodies. Moreover, monoclonal antibodies raised against specific regions of von Willebrand Factor molecule may allow studies of functional abnormalities of this protein in inherited and acquired disorders like subtypes of von Willebrand's disease.     

Conjugation of acetylcholine receptor-protecting Fab fragments with polyethylene glycol results in a prolonged half-life in the circulation and reduced immunogenicity

Antibodies to the acetylcholine receptor (AChR) cause AChR loss, resulting in the disease, myasthenia gravis (MG). The majority of the pathogenic antibodies seem to be directed against the main immunogenic region (MIR) of the AChR. In contrast to the intact antibodies, Fab fragments of anti-AChR antibodies are not themselves pathogenic and such fragments of anti-MIR monoclonal antibodies (mAbs) protect the AChR in vitro and in vivo against the pathogenic antibodies. However, Fab fragments have a very short in vivo half-life and are immunogenic, obstacles which must be overcome before their clinical use can be envisaged. We investigated the effect of conjugating Fab fragments to polyethylene glycol (PEG), a method known to increase the in vivo half-life and reduce the immunogenicity of proteins. When the Fab′ fragments of two rat anti-MIR mAbs (nos. 35 and 195) were conjugated to methoxy-PEG-maleimide, the conjugates retained about 10% of their AChR binding activity and efficiently protected the AChR against the binding and modulating activity of myasthenic antibodies. Their in vivo half-life in rats was approximately 15 times longer than that of the unconjugated Fab′ fragment and they were much less immunogenic in mice. This work represents an important step towards the clinical use of AChR-protective anti-MIR Fabs, but further improvements are needed before their clinical use is attempted.     

A monoclonal antibody to platelet type III collagen-binding protein (TIIICBP) binds to blood and vascular cells, and inhibits platelet vessel-wall interactions

TIIICBP is a new platelet receptor involved in platelet-type III collagen and platelet-subendothelium interactions. This receptor is composed of a doublet of 72-68 kDa proteins. In this study, the major protein (68 kDa) was purified and used to produce monoclonal antibodies. One of these antibodies, 7F4, binds to platelets as confirmed by flow cytometry. 7F4 inhibited platelet contact, spreading and aggregation induced by type III collagen. Under flow conditions, 7F4 prevented platelet interactions with type III collagen, endothelial cell matrix and the KOGEOGPK type II collagen octapeptide: the specific sequence recognized by TIIICBP. On the other hand, 7F4 had no effect on platelet-type I collagen interactions. TIIICBP was also detected on lymphocytes, granulocytes and monocytes. TIIICBP was expressed on endothelial cells and fibroblasts but not on smooth-muscle cells. These results show that TIIICBP is expressed on several cell types and participates in cell adhesion to the subendothelium.     

The influence of preadsorbed canine von Willebrand factor, fibronectin and fibrinogen on ex vivo artificial surface-induced thrombosis

We have examined the effects of preadsorption of several canine plasma proteins on surface-induced thrombogenesis in a canine ex vivo model. Our technique allowed determination of initial deposition and subsequent embolization of 51Cr-labeled platelets and 125I-fibrinogen onto and from polymeric arterio-venous shunts in non-anticoagulated canines. Segments of the tubing were removed at various time points between 2 and 120 minutes of blood contact for examination of the morphology of the thrombus by scanning electron microscopy. Thrombus deposition was measured on uncoated plasticized poly(vinyl chloride) (PVC) and PVC precoated with canine von Willebrand factor (vWF), fibronectin, partially purified fibrinogen (fibrinogen which contained vWF and fibronectin as impurities), or purified fibrinogen (fibrinogen which had been further purified to remove fibronectin and vWF). Preadsorption of all proteins studied enhanced the thrombogenic response relative to that of the uncoated surface. Precoating with vWF or partially purified fibrinogen resulted in the deposition of the greatest number of thrombi, and embolization was slower than on shunts precoated with canine fibronectin or purified fibrinogen. The deposition-embolization profiles for the fibronectin and purified fibrinogen-coated surfaces were similar. The amount and time sequence of initial adhesion and spreading of platelets was related to the extent and time sequence of peak thrombus formation. The partially purified fibrinogen-coated and vWF-coated surfaces had more adhered and spread platelets at the earliest time points and a greater number of larger thrombi at the peak deposition times. The slowest rate of platelet adhesion and spreading was seen on the purified fibrinogen-coated surface. White blood cells were present very early on surfaces precoated with vWF and partially purified fibrinogen, and were present prior to embolization on all surfaces. Major conclusions from this work indicate that, although fibrinogen and fibronectin promote thrombogenesis when adsorbed to a surface, vWF is even more active in promoting platelet deposition and in anchoring thrombi to the surface of biomaterials. Thus, differences in vWF adsorption to biomaterials may be a determinant of surface-induced thrombogenesis.     

Evidence that plasma fibrinogen and platelet membrane GPIIb-IIIa are involved in the adhesion of platelets to an artificial surface exposed to plasma

We investigated the molecular mechanism(s) by which platelets adhere to an artificial surface exposed to plasma, using polystyrene microtiter plates pretreated with plasma. Washed platelets labelled with ⁵¹Cr were incubated with the plates under static conditions. Prostaglandin E₁(PGE₁) was added to the platelets to prevent platelet-platelet interactions. Adhesion required the presence of a divalent cation such as Mg⁺⁺ or Ca⁺⁺. Polyclonal anti-fibrinogen antibody inhibited adhesion by 70%. Polyclonal antibodies against fibronectin, vitronectin, von Willebrand's Factor, and the Fc portion of human IgG, had no effect on adhesion. Platelets adhered normally to a surface pretreated with plasma from a patient with severe von Willebrand's disease. No platelet adhesion occurred when the surface was pretreated with an afibrinogenemic plasma. Monoclonal antibodies against platelet membrane GPIIb-IIIa, potent inhibitors of ADP-induced fibrinogen binding to platelets, completely inhibited adhesion. Monoclonal antibodies against the GPIb subunit and GPIc(VLA ₅) showed no inhibitory effects on adhesion. Platelets from a patient with Glanzmann's thrombasthenia (type 1) did not adhere to the surface pretreated with normal plasma. These results suggest that plasma fibrinogen adsorbed onto the surface and that platelet membrane glycoprotein(GP)IIb-IIIa were responsible for adhesion in an activation-independent manner.     

Immunoelectrophoretic analysis of membrane glycoproteins in cultured human endothelial cells

Human endothelial cells isolated from umbilical cords were solubilized in Triton X-100 and examined by crossed immunoelectrophoresis using rabbit antiserum against endothelial cells. Endogenous labelling of the endothelial cell proteins with 14C-mannose followed by crossed immunoelectrophoresis and autoradiography revealed about 10 immunoprecipitates. Four of these endothelial cell glycoproteins were labelled by lactoperoxidase catalyzed iodination and thus were surface located. Three of the surface located glycoproteins showed reduced electrophoretic mobility after incubation of the endothelial cells with neuraminidase and were therefore sialoglycoproteins. Amphiphilicity of endothelial cell glycoproteins was studied by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose in the intermediate gel. Amphiphilic proteins also show increasing electrophoretic migration velocity with decreasing concentration of Triton X-100 in the first dimension gels. Five of the endothelial cell glycoproteins were shown to be amphiphilic using these two techniques. Two monoclonal antibodies against the platelet glycoprotein complex IIb-IIIa and glycoprotein IIIa, respectively, reacted with the same precipitate of endothelial cells. When a polyclonal antibody against the platelet glycoprotein complex IIb-IIIa was incorporated into the intermediate gel the position of two endothelial cell precipitates were lowered. One of these was a sialoglycoprotein.     

Monoclonal antibodies for diagnosis and research in enteric nervous system pathology. A review

Monoclonal antibodies (MAb) directed against neuron-specific epitopes are valuable tools in the diagnosis of congenital and acquired enteric nervous system anomalies. MAb raised against cytoskeleton proteins (neurofilaments) revealed a characteristic staining pattern in patients with various motility disorders of the gut. Application of MAb in the study of the development of the enteric nervous system in the chicken embryo provided new insights into the fate of migrating neural crest cells. The relationship between mesenchymal target cells in the gut and proliferating neural crest cells was studied by means of MAb raised against cell surface markers (HNK-1) in combination with characterization of the microenvironment using monoclonal antibodies raised against cell adhesion molecules (N-CAM).     

Thrombin binding to GPIbalpha induces integrin alphaIIbbeta3 dependent platelet adhesion to fibrin in ex vivo flowing whole blood

We have investigated the role of the thrombin/GPIbalpha interaction in the adhesion of platelets to fibrin in a whole blood ex vivo perfusion model at a shear rate of 280 s(-1). Blood was perfused through parallel-plate chambers containing coverslips coated with cells expressing tissue factor, leading to the generation of thrombin and thus, deposition of fibrin onto the exposed cells. Adhesion of platelets to fibrin and thrombus growth were analyzed. Interestingly, when GPIbalpha was removed from the platelet surface by action of mocarhagin, platelet adhesion on fibrin was inhibited. Furthermore, a monoclonal antibody, VM16d, directed against the thrombin binding site on GPIbalpha also inhibited platelet adhesion on fibrin, showing the importance of the thrombin/GPIbalpha interaction.We then looked at the involvement of alphaIIbbeta3 and showed that platelet adhesion and thrombus growth on fibrin were inhibited by the dodecapeptide, whereas lamifiban only inhibited the growth of the platelet thrombus. These results indicated that binding of thrombin to GPIbalpha induced an intracellular signaling leading to the interaction of the platelet integrin alphaIIbbeta3 with the fibrin-dodecapeptide sequence.     

In vitro studies with the platelet-reactive antibody 50H.19 and its fragments

The platelet-reactive monoclonal antibody 50H.19, its F(ab')2 fragments, and 99m-technetium (99m-Tc)-labeled fragments used in thrombus imaging were evaluated for their ability to cause platelet aggregation. The intact antibody caused a dose-dependent platelet aggregation in either platelet-rich plasma (PRP) or defined buffer solutions. The F(ab')2 fragments did not cause platelet aggregation except at high calcium concentrations. Neither stannous-ion-treated antibody fragments nor 99m-Tc-labeled antibody fragments caused platelet aggregation. The antibody-induced platelet aggregation was completely inhibited by 8-bromo-cAMP, caffeine, theophylline diltiazem, and staurosporin; partially inhibited by EDTA, EGTA, cytochalasin B, colchicine, aspirin, or indomethacin. Treatment of platelets with the intact 50H.19 antibody resulted in phosphorylation of a 40K dalton platelet protein, similar to that caused by treatment with phorbol myristate acetate (PMA). Phosphorylization of the 40K protein was not observed after treatment with either the 50H.19 F(ab')2 fragments or the calcium ionophore A23187.     

The species distribution of nervous system antigens that react with anti-myelin-associated glycoprotein antibodies

The reactivity of monoclonal and polyclonal antibodies directed against human central nervous system (CNS) myelin-associated glycoprotein (MAG) was investigated in a number of animal species. The antibodies included mouse monoclonal antibodies obtained by immunization with human MAG; HNK-1, a mouse monoclonal antibody raised against a human lymphoblastoma and used to identify a subset of lymphocytes with natural killer function; human IgM paraproteins associated with neuropathy; and polyclonal antibodies obtained from rabbits immunized with rat or human MAG. Following polyacrylamide gel electrophoresis of CNS and peripheral nervous system (PNS) tissue from human, bovine, cat, rabbit, guinea pig, rat, mouse, frog, gold fish and chicken, proteins were electrophoretically transferred onto nitrocellulose. The immune-staining of electroblots showed distinct interspecies variation in the reactivity of the antibodies with MAG. In addition, the species distribution of several low molecular weight glycoproteins present in PNS tissue that cross-react with anti-MAG antibodies was determined. These low molecular weight antigens are not present in CNS homogenates or in purified human CNS myelin. It was also shown that IgM from a patient with peripheral neuropathy and paraproteinemia associated with anti-MAG antibodies recognized these low molecular weight antigens. The results suggest that IgM paraproteins, HNK-1 and some mouse monoclonal antibodies react with carbohydrate determinants shared by MAG and several lower molecular weight glycoproteins present only in human, bovine, cat and chicken PNS. Rabbit polyclonal anti-rat MAG antisera and mouse monoclonal antibodies reacting with peptide epitopes of MAG are much more specific for detecting MAG than antibodies reacting with carbohydrate epitopes of human MAG. The results are discussed in relation to human demyelinating peripheral neuropathy associated with IgM paraproteinemia.     

The adhesion molecule on glia (AMOG) incorporated into lipid vesicles binds to subpopulations of neurons

To investigate the functional role of the novel adhesion molecule on glia (AMOG) in cell surface interactions, immunoaffinity-purified AMOG was incorporated into liposomes and measured for its ability to bind to cells in monolayer cultures. AMOG could be incorporated into liposomes in functionally active form after solubilization from membranes in 1% cholate buffer containing soybean lecithin, elution from the AMOG monoclonal antibody column with 4 M MgCl2, containing 1% octylglucoside, and removal of detergent for liposome incorporation by gel filtration. AMOG-containing liposomes bound to neurons, but not to oligodendrocytes, astrocytes, or fibroblasts in early postnatal cerebellar cultures. AMOG-containing liposomes also bound to the pheochromocytoma cell line PC12, but not to neurons in cultures of spinal cord and dorsal root ganglia after various times in vitro. Fab fragments of monoclonal AMOG antibodies, but not of L3 monoclonal antibodies directed against a carbohydrate structure on AMOG, inhibited binding of liposomes. Liposome binding was not reduced by preincubation of cerebellar cells with antibodies to AMOG, to the neuron adhesion molecule L1, the neural cell adhesion molecule N-CAM, or the L3 carbohydrate structure, nor with 2 monoclonal antibodies reacting with neuronal cell surface glycoproteins related to the L2/HNK-1 family. These results show that AMOG is indeed a ligand in adhesion and binds to particular subpopulations of neurons in L1- and N-CAM-independent mechanisms.     

Transforming growth factor beta-1 released from platelets contributes to hypercoagulability in veno-occlusive disease following hematopoetic stem cell transplantation

BACKGROUND: Hepatic veno-occlusive disease (VOD) is one of the most disastrous complications after allogeneic hematopoetic stem cell transplantation (HSCT). Thrombocytopenia with refractoriness to platelet transfusions suggests an increased platelet consumption in these patients. Interactions between platelets and endothelial cells might contribute to the hypercoagulable state at the sinusoidal endothelium as a central mechanism in the pathogenesis of VOD. STUDY DESIGN: The influence of activated platelets on cultured human endothelial cells was investigated in vitro. We focused on the release of plasminogen activator inhibitor-1 (PAI-1) from endothelial cells which has earlier been found to be significantly elevated in plasma of VOD patients. Endothelial cells isolated from human umbilical cords (HUVEC) were incubated with activated platelets. The release of PAI-1 in the presence or absence of specific antibodies was determined by ELISA technique. Tissue factor (TF) expression on endothelial cells was observed by flowcytometric analysis. RESULTS: HUVEC incubated with activated platelets were found to release significantly more PAI-1 compared to untreated cultures. The endothelial PAI-1-secretion after incubation of HUVEC with activated platelets was completely inhibited by an IgG monoclonal antibody against human transforming growth factor beta-1 (TGF beta-1). In contrast, PAI-1 production was not suppressed after inhibition of HUVEC-platelet-interaction by an IgG monoclonal antibody against CD154 (CD40L) expressed on the surface of activated platelets. An increased release of PAI-1 and an increased expression of tissue factor (TF) on the endothelial cell surface were observed after stimulation with TGF beta-1. CONCLUSION: TGF beta-1 released from activated platelets contributes to the hemostatic imbalance at the sinusoidal endothelium in patients with hepatic VOD by increase of endothelial cell PAI-1 production and TF expression. As a potent profibrotic cytokine, TGF beta-1 might further be involved in phlebosclerosis and sinusoidal fibrosis occurring in VOD.     

The Neural Cell Adhesion Molecule (N-CAM) Modulates K+ Channels in Cultured Glial Precursor Cells

Application of antibodies against the neural cell adhesion molecule (N-CAM) to O4-positive murine glial precursor cells in vitro results in a reduction of two distinct K+ currents measured using the whole cell patch clamp technique. Both the A-type and delayed rectifier K+ currents are reduced in amplitude within a few minutes of the application of poly- or monoclonal antibodies against N-CAM. This effect is not due to the binding of any antibody to the surface of the glial precursor cells because monoclonal antibody directed against the O4 surface antigen, or polyclonal antibodies directed against liver cell membranes (which also bind to the surface of glial precursor cells), do not affect membrane currents. Activators of protein kinase C, such as phorbol esters or diacylglycerol, also induce changes in potassium currents that appear, both in magnitude and kinetics, to be similar to those induced by antibodies against N-CAM. In contrast, activation of G proteins upregulates K+ currents. Glial precursor cells thus respond to triggering of N-CAM by altering channel properties. These observations suggest that adhesive events between neural cells can influence the intracellular ionic milieu.     

Baboon fibrinogen adsorption and platelet adhesion to polymeric materials

The role of fibrinogen in mediating platelet adhesion to polymers exposed to blood plasma was studied by comparison of the effect of plasma dilution on fibrinogen adsorption and platelet adhesion, and by the use of coagulation factor deficient plasmas. Polyetherurethane substrates were first preadsorbed with dilute plasma, then contacted with washed platelets suspended in a modified, apyrase containing Tyrode's buffer. Platelet adhesion was studied under static conditions in Multiwell dishes, and also under shearing conditions using a parallel plate perfusion chamber. Fibrinogen adsorption and platelet adhesion were measured using 125I radiolabeled baboon fibrinogen and 111In radiolabeled baboon platelets, respectively. Surfaces were characterized by electron spectroscopy for chemical analysis (ESCA). When fibrinogen adsorption to Biomer was measured after 2 h contact with a series of dilute plasma solutions under static conditions, a peak in adsorption was observed from 0.26% plasma, i.e., adsorption was greater from 0.26% plasma than from either more or less dilute plasma. A peak in subsequent platelet adhesion to the plasma preadsorbed surfaces, measured after 2 h static incubation with washed platelets, was also observed but occurred on Biomer preadsorbed with 1.0% plasma. When fibrinogen adsorption was measured after 5 min contact under shearing conditions, the fibrinogen adsorption peak occurred on surfaces that had been exposed to 1.0% plasma. A peak in platelet adhesion to these preadsorbed surfaces, measured after 5 min contact with the platelet suspensions under shearing conditions, was observed on Biomer preadsorbed with 0.1% plasma. Shifts between the positions of the peaks in protein adsorption and platelet adhesion occurred on other polymers tested as well. Platelet adhesion was almost completely inhibited when baboon and human plasmas lacking fibrinogen (i.e., serum, heat defibrinogenated plasma, and congenitally afibrinogenemic plasma) were used. Platelet adhesion was restored to near normal when exogenous fibrinogen was added to fibrinogen deficient plasmas. Adhesion was also inhibited completely when a monoclonal antibody directed against the glycoprotein IIb/IIIa complex was added to the platelet suspension. Platelet adhesion to surfaces preadsorbed to von Willebrand factor deficient plasma was the same as to surfaces preadsorbed with normal plasma. While it appears that surface bound fibrinogen does mediate the initial attachment of platelets to Biomer, the observation that the fibrinogen adsorption and platelet adhesion maxima do not coincide exactly also suggests that the degree of subsequent platelet adhesion is dictated not only by the amount of surface bound fibrinogen but also by its conformation.     

The action of Lonomin V (Lonomia achelous) on fibronectin functional properties

Lonomia achelous caterpillar, Lepidoptera distributed along some South American countries, induces a hemorrhagic syndrome in people who come into contact with its bristles. A clinical characteristic in these patients is that fresh healed wounds are re-opened and bleed. In order to explain this symptomatology, we evaluated the effect of Lonomin V (a protein isolated from L. achelous hemolymph), on some functional properties of fibronectin, which in turn plays an important role in the hemostasis. The effect of Lonomin V on fibronectin was studied by SDS-PAGE in reduced condition, binding to gelatin and heparin, crosslinking to fibrin and platelet adhesion. Formation of degradation products of 120, 66, 50, 40 and 29 kDa, some of which retain affinity to heparin and gelatin were observed; however, the fibronectin degradation fragments presented a significant decrease of crosslinking capacity to fibrin and platelet adhesion, suggesting that the proteolysis of fibronectin by Lonomin V induces changes in its crosslinking sites and on platelet receptors. These findings might partially explain the wound dehiscence observed in the patients. Due to its effect on adhesive proteins with concomitant impairment of some functional properties, Lonomin V might be useful for cellular adhesion studies involved in hemostasis such as platelet adhesion.     

Dipyridamole induces changes in the thrombogenic properties of extracellular matrix generated by endothelial cells in culture.

Dipyridamole (DIP) is a drug widely used as an antiplatelet agent, which also has effects on endothelial cells. In this study, the effects of treating confluent endothelial cell monolayers (EC) with DIP on EC viability (trypan blue exclusion test) and metabolic activity (3H-thymidine incorporation) were examined. Platelet reactivity of the extracellular matrix (ECM) produced by untreated and DIP-treated ECs was determined morphometrically by a perfusion technique. Levels of ECM-associated von Willebrand factor (vWF) and fibronectin (FN) were also quantified (ELISA). The present results indicate that treatment of EC with 10 microM DIP did not reduce EC viability but that the incorporation of labelled nucleotides was significantly decreased (p less than 0.01). Platelet deposition onto the ECM generated by DIP-treated cells, perfused at a shear rate of 1300 sec-1, differed significantly with respect to controls (p less than 0.05), and platelet adhesion was also reduced (25% less, p less than 0.05). This effect was shear rate dependent, as no differences were noted when the ECMs were perfused at 300 sec-1 shear rate. Levels of VWF and FN associated with ECM remained unchanged with respect to controls. These results suggest that treatment with DIP alters EC metabolic activity, which in turn, influences the reactivity of the ECM generated by treated cells.     

The Dami cell possesses the platelet collagen receptor VLA-2, but does not mobilize calcium

Flow cytometric and Western blot analysis showed that Dami cells possess the major platelet collagen adhesion receptor, the integrin VLA-2, and that VLA-2 was expressed in higher levels in a time-dependent manner in DMSO-induced Dami cells. Both control and DMSO-induced Dami cells were able to adhere to collagen as measured in a microtiter-based adhesion assay. It appeared that collagen adhesion was solely mediated by VLA-2, since inclusion of a monoclonal antibody directed against the alpha-2 subunit of VLA-2 in the adhesion assay was able to totally inhibit adhesion. Although Dami cells possess a variety of platelet markers, and are able to mobilize intracellular calcium in response to ADP, U-46619, and thrombin, they were unable to respond to collagen challenge. We concluded that Dami cells may lack some key transducing element present in platelets that prevents them from being activated by collagen.     

Identification of platelet antigens by monoclonal antibodies using crossed immunoelectrophoresis with immunoblotting of the monoclonal antibody

A method is described for the identification of antigens by monoclonal antibodies. This is applicable whenever precipitating antibodies to the same antigens from a different species are available. The method is based upon: Separation and immunoprecipitation of cellular proteins with a polyspecific antiserum in crossed immunoelectrophoresis in the presence of the non-denaturing detergent Triton X-100 and the monoclonal antibody. Coprecipitation of the monoclonal antibody with its antigen. Subsequent passive transfer of the monoclonal antibody in the antibody-antigen complex onto a nitrocellulose membrane. Visualization of the blotted antibody using an enzyme-linked secondary antibody and a chromogenic substrate. Identification of the corresponding antigen by comparisons to the immunoprecipitate pattern of the original immunoplate. To test this method we have analyzed the detection of the antigens recognized by six previously described monoclonal antibodies against platelet membrane proteins and von Willebrand factor. Specific immunoblots were obtained in each case using small amounts of monoclonal antibodies. Thus, the technique provides an alternative when epitopes are denatured by SDS, and avoids the use of radioactively labelled monoclonal antibodies.     

Changes in platelet function and reactivity induced by quinine in relation to quinine (drug) induced immune thrombocytopenia

Quinine, a drug known to induce immune mediated thrombocytopenia, has been postulated to mediate binding of drug dependent antibodies to a range of platelet membrane glycoproteins. Quinine may not act solely as a hapten however, as we have shown that it inhibits platelet aggregation ( and ) and release and modifies the ability of activated platelets to bind the adhesive proteins fibrinogen and fibronectin in a dose dependent fashion. Studies on the effect of quinine on the binding of monoclonal antibodies HuPlml (GpIIIa) FMC25 (GpIX) and AN51 (Gplb) to platelets shows a selective reduction in AN51 binding. In addition quinine induced platelet antibodies from thrombocytopenic patients, in the presence of quinine, have been shown to inhibit binding of these monoclonal antibodies to platelets to varying degrees. These observations suggest that quinine causes widespread but specific conformational changes in platelet membrane antigens which may expose neoantigens resulting in the production of quinine induced antibodies.