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The interaction of human blood platelets with collagenase-treated rabbit subendothelium was studied by histochemical ultrastructural methods and by morphometric semi-quantitative analysis. Aortas were deendothelialized and incubated: 1) with a highly purified bacterial collagenase whose specificity was controlled; and 2) with the same collagenase followed by chymotrypsin. For histochemical studies, tannic acid, ruthenium red, and peroxidase-labeled Ricinus communis and concanavalin A were used. Electron microscopy showed that after digestion of fibrillar collagen by collagenase, adherent and aggregated platelets were observed on Ricinus communis-, concanavalin A-, and ruthenium red-positive glycoprotein microfibrils. After successive incubation with collagenase and chymotrypsin, the microfibrils disappeared. No platelets were observed on the remnant amorphous elastin. Morphometric analysis confirmed the interaction of platelets with collagenase-treated subendothelium. In addition, glycoproteins were extracted from collagenase-treated rabbit aortas using 5 M guanidine. Using an in vitro quantitative test, significant platelet adhesion to these glycoproteins was observed. Our results show an interaction between platelets and noncollagenic glycoprotein microfibrils.

Crossed immunoelectrophoresis of Triton X-100 solubilized proteins from normal and abnormal platelets was performed with rabbit antibodies raised against normal platelets. In Bernard-Soulier platelets protein 13 was not detected, and neither the amphiphilic (probably GP Ib) nor the hydrophilic (glycocalicin) glycocalicin-related proteins were seen when monospecific antiglycocalicin antiserum was used. The most prominent precipitate, 16, and platelet fibrinogen, 24 were not detected in platelets of two patients with type I thrombasthenia, whereas in one patient with type II thrombasthenia fibrinogen was clearly detected, but the amount of protein 16 remained severely reduced. Protein 16 was heavily labeled after lactoperoxidase-catalyzed (125)I iodination of normal platelets, and was precipitated by IgG-L, an alloantibody from a polytransfused thrombasthenic patient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or protein 16 cut out from immunoplates showed two (125)I-labeled glycoprotein bands, which migrate as GP IIb and GP IIIa. SDS-PAGE of (125)I-labeled type I thrombasthenic platelets showed no periodic acid-Schiff bands or peaks of radioactivity in the GP IIb and GP IIIa regions, whereas in the GP I region both the periodic acid-Schiff band intensity and the radiolabeling were within the normal range. Autoradiography after crossed immunoelectrophoresis of iodinated thrombasthenic platelets showed that the bulk of radioactivity was bound to protein 17. This glycoprotein, which was also present in normal and Bernard-Soulier platelets, migrates in the GP I region on SDS-PAGE. Thus, the bulk of radioactivity observed in the GP I region after SDS-PAGE is associated with protein 17 and not with glycocalicin.

As excessive monocyte adhesion to blood vessel wall could produce endothelial cell injury, we undertook comparative studies on the monocyte adhesiveness in insulin-dependent diabetics with vascular complications (n = 26) and healthy, normal subjects (n = 36). In the diabetic group, the extent of monocyte adhesion on fibronectin- and autologous plasma-coated surfaces was significantly increased compared with that in the control group (p less than 0.01 on both types of surfaces). Monocytes adhesion to the plasma-coated surface, but not to the fibronectin-coated surface, could be inhibited by monoclonal anti-Mo-1 antibody in a dose-dependent manner. For diabetic monocyte adhesion, a higher amount of anti-Mo-1 antibody was required to produce a similar extent of inhibition as observed with control monocytes. This indication of increased Mo-1 expression on diabetic monocytes was further confirmed by analyzing the fluorescence intensity of monocytes labeled with the antibody anti-Mo-1. The results of the present study suggest that diabetic monocytes have increased adhesiveness as the result of increased expression of fibronectin and Mo-1 receptors.

The effect on platelet function of a monoclonal platelet antibody to platelet membrane glycoprotein I was tested. This antibody, AN51, inhibited ristocetin or bovine factor VIII-induced aggregation but did not modify ADP, collagen type I or type III, thrombin or arachidonic acid induced aggregations. Furthermore, the adhesion-aggregation of platelets induced by microfibrils was also inhibited by the antibody. Platelet adhesion to rabbit aorta subendothelium was impaired by the antibody. The persistent adhesion of platelets to collagenase-treated subendothelium was also inhibited. These findings strongly suggested that platelet membrane glycoprotein I could interact with a non-collagenic microfibrillar component of subendothelium. The binding of factor VIII/von Willebrand factor to platelet membrane in the presence of ristocetin was decreased in the binding site for factor VIII/von Willebrand factor to allow platelet adhesion to subendothelium.

A decreased platelet adhesion to rabbit aorta subendothelium (Baumgartner technique) is confirmed in the Bernard Soulier (giant platelet) syndrome. Electron microscope techniques using a purified antibody against Factor VIII/von Willebrand protein, revealed an apparently normal presence of the Factor VIII/von Willebrand protein on the Bernard Soulier platelets. Electrophoretic characterization of the major protein and glycoprotein components of the Bernard Soulier platelets following sodium dodecyl sulfate solubilization indicated a relatively normal protein content but suggested a reduced content of the 155,000 molecular weight major platelet glycoprotein. This was confirmed by a reduced release of high molecular weight acidic glycopeptides following incubation of washed Bernard Soulier platelets with trypsin. It is proposed that this abnormality may be related to the previously reported reduced sialic acid content and the reduced electrophoretic mobility of the Bernard Soulier platelets and that a glycoprotein reduced or abnormal in the Bernard Soulier platelets is necessary for the normal adhesion of platelets to subendothelium.

Two unrelated patients with "pseudo" ("platelet-type")-von Willebrand's disease (vWD) are described demonstrating thrombocytopenia with a prolonged bleeding time, ristocetin-induced platelet aggregation at low stimulus concentrations, decreased levels of ristocetin-cofactor activity (vWF:RCo), slight cryoprecipitate-induced platelet aggregation in the absence of ristocetin, and a lack of the high molecular weight factor VIII-von Willebrand factor (FVIII/vWF) multimers in plasma. Isolated washed patient platelets bound more FVIII/vWF at high (1 and 0.75 mg/ml) and low (0.5 and 0.25 mg/ml) ristocetin concentrations than control platelets. Fresh or paraformaldehyde-fixed washed platelets from these patients also bound more specific monoclonal antibody to glycoprotein Ib (25,000 binding sites per cell) than normal platelets (15,000 +/- 3,300 binding sites per cell). Results obtained in control patients with thrombocytopenia and increased platelet size (May-Hegglin anomaly and vWD type IIB) excluded a nonspecific increase of glycoprotein Ib in the platelets of the patients with pseudo-vWD. These data indicate that in pseudo-vWD, the primary abnormality lies in the platelet and is related to a quantitative and/or qualitative anomaly of platelet membrane glycoprotein Ib.

Platelet GP IIb-IIIa complex is missing or strongly reduced in thrombasthenia type I or II; in parallel the binding of fibrinogen is either nil or strongly reduced after platelet activation and these platelets do not aggregate. In the platelets of the described variant, GP IIb-IIIa level and PLA1 antigen are around 50% of the normal but the fibrinogen sites are either missing or unavailable. The hypothesis is that the patient's platelets lack the specific receptor site for fibrinogen at the GP IIb-IIIa level. However, platelet fibrinogen is normal as is the clot retraction. The study of this variant allows new concepts on platelet aggregation and clot retraction.

We have studied the fibrinogen binding to the platelets of four type I thrombasthenic two type II thrombasthenic patients and 15 normal subjects. The amount of [125I]fibrinogen bound to washed platelets in the presence or absence of 10 micrometers ADP was measured and the results expressed as the percentage of the total radioactivity added. The mean value for specific binding to the platelets of normal subjects was 3.7 (range 1--7.25) at 15 min after the addition of ADP. Type I thrombasthenic patients lack glycoproteins (GP) IIb and IIIa in the platelet membrane and intra-platelet fibrinogen. In all four patients studied, little or no specific fibrinogen binding was detected. Platelets of type II thrombasthenic patients contain detectable, although markedly reduced amounts of GP IIb and IIIa, and subnormal or normal levels of fibrinogen. In these platelets, significant specific fibrinogen binding was observed, the values being slightly below or at the lower end of the normal range. Fibrinogen binding to normal platelets was also measured after the addition of an IgG purified from the serum of a multi-transfused thrombasthenic type I patient. This antibody was previously shown to be directed against the GP IIb/IIIa complex as located by crossed immunoelectrophoresis. Specific fibrinogen-binding was blocked and the extent of the inhibition was directly proportional to the concentration of the antibody added.

A large reduction in the staining capacity of a glycoprotein of 155 000 M.W. was observed in the platelets of 2 macrogiant platelets syndrome patients, associated with a reduced platelet adhesion to rabbit aorta subendothelium and with the presence of von Willebrand protein on these platelets as revealed by an antihuman factor VIII-Von Willebrand protein rabbit antibody. These results led the authors to propose a strong hypothesis on the role of this glycoprotein rich in sialic acid - in platelet adhesion to subendothelium.

Washed human platelets were labeled with 125I by the lactoperoxidase-catalyzed method and solubilized in 1% Triton X-100. The soluble proteins were analyzed by crossed-immunoelectrophoresis in 1% agarose, employing a rabbit antibody raised against whole human platelets. Analysis of autoradiograms developed from dried agarose gels led to the establishment of a normal reference pattern that was consistent for platelets obtained from more than 50 normal individuals. Six platelet membrane glycoprotein antigens contained in four distinguishable precipitates were identified. Each identification was based on direct sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 125I-antigens contained in individually excised precipitates. These platelet antigens include major membrane glycoproteins previously designated la, lb, lla, llb, llla, and lllb. Glycoproteins llb and llla were shown to be contained in a single immunoprecipitate, while glycoproteins la and lla were routinely detected in a single different immunoprecipitate. Analysis of soluble proteins from platelets of five patients with Glanzmann's thrombasthenia demonstrated either a complete absence or a marked reduction of only one radiolabeled precipitate, that containing membrane glycoproteins llb and llla. Platelet samples from two patients with Bernard-Soulier syndrome were devoid of a different precipitate, that containing membrane glycoprotein lb.

The gray platelet syndrome is a rare inherited platelet disorder characterized by the absence of alp ha-granules as observed by electron microscopy. Analysis of the glycoprotein composition of the platelets of 2 such patients by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed decreased or absent staining for carbohydrate of several high molecular weight glycoproteins. The major periodic acid Schiff (PAS) staining membrane glycoproteins were normally detected and were normally labeled with 125I during lactoperoxidase-catalyzed iodination. Analysis of the protein composition of gray platelets by single or two-dimensional SDS-PAGE followed by Coomassie blue staining revealed an apparent absence of GP Ig (thrombospondin), markedly reduced platelet fibrinogen and albumin concentrations, and severely reduced levels of 2 low molecular weight polypeptides exhibiting identical rates of migration on SDS-PAGE as platelet factor 4 and beta-thromboglobulin. SDS-PAGE profiles similar to those of the gray platelets were observed with normal human platelets that had undergone the release reaction induced by thrombin. Analysis of gray platelet proteins by crossed immunoelectrophoresis using a rabbit anti-human platelet antibody preparation and rocket immunoelectrophoresis using monospecific antisera confirmed the above findings and showed additional severe deficiencies of factor VIIIR:Ag and cold-insoluble globulin. In contrast, factor XIII (subunit A), a cytoplasmic protein, was normally detected. Our studies provide further evidence that circulating gray platelets specifically lack, or have markedly decreased concentrations of, the alpha-granule proteins.

Glycoproteins present at the external surface of cells probably play specific roles in cellular function. Increasing evidence suggests that the glycoproteins span the plasma membrane with the bulk of the bound carbohydrate asymmetrically distributed on the outer surface. Micellar association of glycoproteins in membranes leads to pore formation and functional roles in transport through the membrane, while surface glycoproteins have been shown to be enzymes, to determine cell specificity and contribute to the cell surface change. The platelet plasma membrane contains 3 major glycoproteins, glycoproteins I, II and III as characterized in order of their decreasing molecular weight. Glycoprotein I appears to have the highest sialic acid content and to give rise to a platelet specific acidic macroglycopeptide on trypsin digestion. Specific glycoprotein abnormalities in the platelets of patients with Glanzmann's thrombasthenia suggest that the glycoproteins play a role in the mechanism of platelet aggregation. A much reduced content of glycoprotein I in the platelets of 2 patients with the Bernard Soulier syndrome may be associated with their defective adhesion to subendothelium and indicates a possible relationship on the platelet surface with the von Willebrand factor protein. Preliminary evidence suggests that in common with other plasma membranes the platelet membrane has a fluid structure and that the organization of the glycoproteins on the platelet surface is extremely sensitive to stimuli and susceptible to change.

Previous reports have described conflicting results concerning the glycoprotein (GP) and protein composition of Bernard-Soulier platelets. In view of this controversy we have analyzed the platelets of four Bernard-Soulier patients using improved single and two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis procedures. An absence of staining for carbohydrate of membrane GP Ib was characteristic for the platelets of each patient. Major periodate-Schiff staining bands corresponding to membrane GP IIb, IIIa, and IIIb were clearly detected and their presence was confirmed by two-dimensional SDS-polyacrylamide gel electrophoresis. The protein content of the Bernard-Soulier platelets was increased two- to fourfold. However, analysis of their protein composition using 7-12% acrylamide gradient gels showed normal polypeptide profiles. Lactoperoxidase-catalyzed 125I-labeling of the Bernard-Soulier platelet surface proteins was followed by SDS-polyacrylamide gel electrophoresis and autoradiography. No labeling in the Ib position was detected whereas the other major membrane GP, including Ia and IIa, were normally located. In contrast, GP Ib was clearly detected by periodate-Schiff staining and autoradiography when normal human platelets that had been exhaustively treated with neuraminidase before the lactoperoxidase-catalyzed iodination were analysed. No abnormalities were detected in the GP patterns of membranes isolated from the patients' erythrocytes. Only a severe molecular abnormality or possible deletion of GP Ib could account for this major platelet lesion in the Bernard-Soulier syndrome.

The present case of constitutional thrombasthenia is unique. It demonstrates the importance of the three major steps of human platelet aggregation: 1. binding of agonists to their respective membrane sites; 2. transmission of a message probably membrane Ca++ dependent, 3. exposure of the preformed GP IIb-III a complex allowing fibrinogen binding. We postulate that in this case the second step is impaired while in the classical thrombasthenia, the third one is abnormal by absence, strong reduction or functional abnormality of the GP II b-III a complex.

An IgG antiplatelet antibody found in a multitransfused patient with Bernard-Soulier syndrome (BSS), reacted with a normal platelet surface antigen of 150 000 daltons which was similar to the glycoprotein missing from BSS platelets. The BSS platelet antibody (BSS-Pab) aggregated all control platelets which then released ADP and 5-HT and synthesized thromboxane. When mixed with the antibody, BSS platelets did not aggregate, did not release ADP and 5-HT and failed to synthesize thromboxane. The BSS-Pab was not inactivated by incubation with BSS platelet stroma. While the antibody did not aggregate thrombasthenic platelets, its aggregating activity was lost after incubation with their stroma. The BSS-Pab did not provoke ADP or 5-HT release or thromboxane synthesis in thrombasthenic platelets or in the platelets of a patient with platelet cyclooxygenase deficiency or in normal platelets treated with indomethacin. The aggregating, release and synthetic responses of platelets after binding of BSS-Pab to its membrane antigen (probably glycoprotein I) requires the presence of glycoprotein IIb and/or IIa and the normal metabolism of arachidonic acid.

Glanzmann's thrombasthenia and the Bernard-Soulier syndrome are inherited blood disorders characterized by abnormalities in different aspects of platelet function during haemostasis. Platelets from patients with thrombasthenia do not aggregate in response to the normal physiological platelet aggregation inducing stimuli, while Bernard-Soulier platelet have a reduced capacity to adhere to exposed subendothelium. Deficiencies of different membrane glycoproteins have been located in the platelets of both disorders and suggest specific roles for membrane glycoproteins in different aspects of platelet function.

A method is described for the study of the interaction between human blood platelets and intercellular matrix macromolecules of connective tissues such as those found in the sub-endothelium. The method is based on the qualitative and quantitative evaluation of the elution pattern of a platelet suspension from a mixed Sepharose 2B-fibrous protein column containing standardized amounts of fibrous proteins such as elastin or structural glycoprotein-microfibrils (SGP-MF). No interaction could be demonstrated with this method between platelets and SGP-MF but there was a very distinct interaction between platelets and elastin fibers. This could be confirmed by direct microscopical demonstration of platelet clusters around elastin fibers in cryostat sections of the affinity column.

The authors report here the results of fracture-labeling of wheat germ agglutinin (WGA) and concanavalin A (Con A) receptors on the plasma membranes of normal, Bernard-Soulier, and thrombasthenic platelets. In all cases, virtually all of the label was confined to the exoplasmic half of the membrane. Despite the absence of GP Ib in Bernard-Soulier platelets and the absence or strong reduction of Gp IIb and GP IIIa in thrombasthenic platelets, their plasma membranes were strongly labeled by both Con A and WGA. These results are best accounted for by the presence of other glycoproteins and/or glycolipids at the platelet surface.

A monoclonal hybridoma antibody specific for platelet glycoprotein I complex is described. The nature od the antigen was determined by demonstration that it was chymotrypsin sensitive and gave a peak at 150 000 daltons on SDS-PAGE after immunoprecipitation. The expression of the antigen is restricted to platelets and megakaryocytes with at least 1.6 x 10(4) molecules of antigen per platelet. The antibody failed to bind to platelets from patients with Bernard Soulier syndrome, where there is known to be a deficiency of glycoprotein Ib/Is expression. Binding to platelets from patients with Glanzmann's thrombasthenia was normal.

The autosomal recessive transmission of Glanzmann's thrombasthenia (GT) and Bernard-Soulier syndrome (BSS), together with requests of families who already had children with these diseases, prompted us to investigate the feasibility of their antenatal diagnosis. The preliminary step leading to the early detection of GT or BSS was to characterize, in the normal human fetus, the platelet antigens and glycoproteins (GPs) and to define their normal amounts on the membrane surface. Blood samples from 32 fetuses between 18 to 26 weeks of gestation were collected by direct puncture of the umbilical vein using an ultrasound-guided needle. Polyclonal antibodies from human origin directed against PLA1, Leka antigens, and the GPIIb IIIa complex (IgGL), or murine monoclonal antibodies specific for GPIb (AN51, 6D1), GPIIIa (AP-3), or GPIIb IIIa (AP-2) were studied using platelet suspension immunofluorescence tests. The binding of each antibody was quantified using a cytofluorograph (Ortho 50H). PLA1 and Leka antigens were expressed in normal amounts on fetal platelets as early as 16 weeks of intrauterine life. The GPIIb IIIa complex quantified by polyclonal or monoclonal antibodies was in the same range in fetuses (IgGL = 427 +/- 23 AUF, AP-2 = 459.5 +/- 8.5; AP-3 = 536 +/- 14) and in adults (IgGL = 420 +/- 30; AP-2 = 498 +/- 11; AP-3 = 515 +/- 13). The platelet binding of antibodies that recognized GPIb was higher in fetuses (AN51 = 491.5 +/- 14; 6D1 = 479 +/- 15) than in adults (AN51 = 426.5 +/- 9; 6D1 = 449 +/- 8.7). These results suggest that immunological techniques can be applied as early as 18 weeks of gestation for the antenatal diagnosis of GT and BSS.