Platelet glycocalicin. I. Orientation of glycoproteins of the human platelet surface.

The orientation of proteins and glycoproteins of the platelet surface has been studied using various surface probes and labeling reagents. A fourth major glycoprotein has now been detected in platelet plasma membranes by sodium dodecyl sulfate-gel electrophoresis in addition to the previously recognized glycoproteins I, II, and III. Glycoprotein IV Mr, = approximately 87,000) appears to be present on the inner aspect of the membrane or buried within it since it is not accessible to surface probes such as lactoperoxidase-catalyzed iodination, radiolabeling with transglutaminase and [14C]glycine ethyl ester, or proteolytic enzymes. The ratio of these four major membrane-bound glycoproteins is approximately 10:4:2:3. Contrary to previous reports, only one glycoprotein, glycoprotein III, is accessible to lactoperoxidase-catalyzed iodination in intact platelets. Differences in the rate of destruction of glycoprotein II in intact platelets by trypsin suggests that two components may be migrating in this region. Examination of the soluble fraction obtained following platelet homogenization showed the presence of a single soluble glycoprotein of molecular weight 148,000 comprising about 10% of total platelet sialic acid. Treatment of intact platelets with neuraminidase resulted in the quantitative loss of siliac acid from the soluble glycoprotein, and it was strongly labeled in the intact platelet by [14C]glycine ethyl ester in the presence of transglutaminase. Treatment of intact platelets with chymotrypsin which does not cause the platelet release reaction, caused the rapid conversion of the soluble glycoprotein to a macroglycopeptide. These results indicate a surface origin for the soluble glycoprotein rather than a cytoplasmic or granular origin. The term glycocalicin is suggested for this glycoprotein in view of its origin in the platelet glycocalyx.

, the modification of platelet properties by treatment with neuraminidase (3,4) and the isolation of defined glycopeptides from intact platelets (5) and isolated platelet membranes (6,7). This glycoprotein coat appears to be involved in the reaction of platelets with lectins (8,9) and may also play a role in their reaction with thrombin (10) and with ADP and serotonin (11  (15) or after homogenization by the use of ultrasound (Branson Sonifer, Branson Sonic Power Co., Plainview, N. Y.; output control setting 6) followed by sedimentation in the same way onto a sucrose cushion.
In most cases, polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate, was carried out under standard conditions (16) using either 6% polyacrylamide and 2.5% cross-linking reagent or, preferably, 10% polyacrylamide with 0.67% cross-linking agent for 2.5 h at 2.5 mA/gel for the first 15 min increased to 5 mA/gel for the remaining period; gel size, 0.5 x 7.5 cm. In experiments designed to obtain a higher resolution of the glycoprotein components, the content of P-mercaptoethanol was increased as was the temperature of incubation: platelet membranes, prepared from 1 unit of fresh platelet-rich plasma by the glycerol-lysis method, were dissolved in 1 ml of 0.01 M Tris buffer (pH 7.4) containing 0.2 mM EDTA together with SDS' (1%) and &mercaptoethanol (5%), and containing bromphenol blue (0.002%) as a marker.
The mixture was incubated for 15 min at 60" and subjected to electrophoresis as above for 5 h. Gels were stained for protein with Coomassie blue and for carbohydrate by a modified periodic acid-Schiff (PAS) reagent.' The gels were steeped in 100. to 150.ml volumes of 40% aqueous ethanol containing 5% acetic acid, the wash solution being changed after 1, 3, and 6 h or alternatively, the gel was allowed to steep in the second solution overnight.
The gel was then transferred to a solution of sodium metaperiodate (0.7%) in 5% acetic acid for a period of 1 to 2 h, and then to a 1% solution of sodium metabisulfite or sodium bisulfite until the brown color disappeared (about 45 min). The gels were then placed in Schiff's reagent; the colored band appeared after 30 to 45 min and was stable for more than 2 days. x g for 20 min and then resuspended in 10 ml of the same buffer; 3. The mixture was incubated at 37" for 1 h with occasional gentle agitation.
The suspension was diluted with 30 ml of Phillips' buffer containing apyrase, and washed three times by centrifugation at 480 x g for 15 min. The final pellet was suspended in 3 ml of 0.05 M Tris, 1 mM EDTA (pH 7.4), and homogenized at ice bath temperature with 4 x 10-s bursts of ultrasound followed by centrifugation onto a sucrose cushion.
The membrane and soluble glycoprotein fractions were subjected to electrophoresis in 6% polyacrylamide as described above. After electrophoresis the gel was stained by the PAS method, optically scanned, and then sliced into 2-mm sections which were solubilized in 1 ml of NCS tissue solubilizer (Amersham/Searle) containing 10% water by heating at 55" for 15 h. Bruno's scintillation liquid (10 ml) (18) was added, and the radioactivity of each slice determined.
lodination of Intact Platelets-This was carried out by standard procedures (13). to Neuraminidase-Intact platelets were treated with neuraminidase and the lysate was separated into soluble and membrane fractions. When subjected to electrophoresis in the absence of SDS, the rate of migration of glycocalicin in the soluble fraction was greatly reduced compared with that of the control ( Fig. 2A). That the decrease in electrophoretic mobility was due to charge alone and did not arise from contaminating activities such as proteases in the neuraminidase was shown by the fact that both the desialylated glycocalicin and the control had identical mobilities in the presence of SDS (Fig. 2B). The decreased reaction of glycocalicin in each of the gels with the PAS-stain following treatment with neuraminidase is a reflection of the sensitivity of this procedure to the presence of sialic acid in glycoproteins, since similar amounts of native and desialylated glycocalicin were applied to the gels in each case.
Accessibility of Glycocalicin to Chymotrypsin-In order to determine whether glycocalicin was present on the outer surface of the platelet or in the platelet cytoplasm, experiments were carried out with chymotrypsin, which does not induce the platelet release reaction. Electrophoresis of the membrane fraction obtained after chymotrypsin treatment of intact platelets showed the glycoprotein I had been completely removed and that only glycoprotein IV remained in the membrane (Fig. 3A). Similarly, no glycoprotein corresponding to glycocalicin could be detected in the soluble fraction remaining after lysis of the washed chymotrypsin-treated platelets (Fig. 3B).

Lactoperoxidase-catalyzed
Iodination-When subjected to lactoperoxidase-catalyzed iodination and SDS-gel electrophoresis under standard conditions, the pattern of radiolabeling was generally similar to that found by others (12, 13) with the maximum incorporation being in the area of glycoprotein  8. Immunochemical comparison of macroglycopeptides and glycocalicin. Center well, chicken antiglycocalicin antiserum; well I, glycocalicin; well 2 and well 5, macroglycopeptide from isolated membranes; well 3, macroglycopeptide from purified glycocalicin; well 4, (non-glyco)peptide from glycocalicin.
showed reactions of immunological identity with each other when tested against a chicken antiserum to glycocalicin (19) (Fig. 8) but each gave a reaction of only partial identity with the purified glycocalicin itself. In addition, the macroglycopeptides from membrane-bound glycoprotein I and from glycocalitin gave reactions of only partial identity (Fig. 8) with the (non-glyco)peptide fraction obtained by proteolysis of glycocalicin itself (19).
Chicken antiserum to the purified glycocalicin caused the aggregation of intact washed platelets at a dilution of 1:200, while a microtiter assay showed that isolated platelet plasma membranes also. were agglutinated by the antiserum at the same dilution.

DISCUSSION
The blood platelet is an intensely reactive cell which responds to a variety of stimuli to undergo shape change, adhesion, primary and secondary aggregation, the release of lysomal components through the convoluted channels of a surface connected system and, finally, a process known as viscous metamorphosis in which membrane fusion occurs between adjacent platelets with the loss of membrane integrity. Many of these events are thought to be mediated at the platelet surface and studies on membrane architecture in the platelet have been facilitated by the use of SDS-gel electrophoresis in combination with the periodic acid-Schiff reagent.
The present work demonstrates that four major glycoprotein bands can be detected in isolated platelet plasma membranes. Three of these appear to correspond to glycoproteins I, II, and III which have been previously described. Glycoprotein IV, which has been resolved from glycoprotein III under improved electrophoretic conditions, appears to be inaccessible to a variety of surface probes and reagents. Similarly, the band corresponding to glycoprotein II appears to contain two components differing in their accessibility to proteolytic enzymes. Moreover, unlike previous reports that indicated that all the surface glycoproteins of the platelet were iodinated in the presence of lactoperoxidase we find that only glycoprotein III is accessible to this reagent.
In addition to these four membrane-bound glycoproteins. we find that homogenization of intact platelets leads to the release of a soluble glycoprotein containing about 10% of the total platelet sialic acid.
The subcellular location of this glycoprotein is of particular importance. The present experiments show that it is accessible to neuraminidase and to chymotrypsin in intact platelets and, in addition, it is rapidly degraded by trypsin and chymotrypsin. Taken together, these data strongly support a surface location for this glycoprotein in intact platelets. Finally, this surface glycoprotein is labeled with ["Clglycine ethyl ester in the presence of transglutaminiase.
The high molecular weight of this enzyme (85,000;Ref. 20) and the fact that it labels only the exterior of the PM2 virion, for which this method was originally developed (171, makes it unlikely that internal labeling of the platelet occurs with this technique and further supports the presence of glycocalicin in a site exterior to the platelet membrane itself. Glycocalicin and the membrane-bound glycoprotein I appear to be similar in terms of electrophoretic mobility in the presence of SDS. In addition, chicken antiglycocalicin antiserum is able to cause the aggregation of both isolated platelet membranes which lack glycocalicin and washed platelets. Furthermore, the macroglycopeptides derived from the membrane-bound and soluble glycoproteins give reactions of immunological identity. Although these data might suggest that glycocalicin and glycoprotein I are identical, resolution of this question will have to await the isolation of the membranebound and soluble components in pure form: the purification of soluble glycocalicin is reported in the following paper (19).
Four possibilities seem to exist: one is that the soluble and membrane-bound glycoproteins are identical and that the soluble form is released from the membrane by the effects of platelet homogenization and membrane isolation. However, further amounts of the membrane-bound glycoprotein were not solubilized following prolonged sonication of isolated membranes. The second possibility is that the macroglycopeptide portions of the membrane bound and soluble forms are identical but that the (non-glyco)peptide "tail" portions differ so that this portion of the soluble glycoprotein is more loosely bound to the membrane and therefore more readily displaced during homogenization and isolation. The third possibility, which is closely related to the second, is that the structural differences in the soluble and membrane-bound forms are sufficiently great that the tail portion is not inserted within the membrane itself but that the soluble glycoprotein is loosely adsorbed to the platelet surface. The fourth possibility is that the soluble and membrane-bound forms are structurally identical but that individual molecules are bound to regions of somehow differing affinity in the membrane perhaps reflecting regions capable of varying degrees of lipophilic interaction.
The two latter possibilities are, perhaps, supported by the fact that the ratio of radiolabeling with ["C]glycine ethyl ester to PAS stainability is much higher in the soluble glycoprotein than in the equivalent membrane-bound glycoprotein, suggesting that these portions of the molecule are more accessible in the former case. On the other hand, both glycocalicin and glycoprotein I appear to be completely inaccessible to lactoperoxidase-catalyzed iodination in the intact platelet but the former can be readily iodinated in both the peptide and macroglycopeptide portions when isolated in pure form. This question of accessibility of surface components is particularly important since widespread use of the lactoperoxidase-catalyzed iodination procedure has tended to indicate that only glycoproteins are accessible at the surface of platelets (13) and other cells (21). The application to platelets of the transglutaminase reaction with ['%]glycine ethyl ester, originally developed for analysis of a viral coat protein (17), shows that a number of other components, both glycoprotein and non-glycoprotein in nature, are available at the cell surface. This observation emphasizes that the orientation of individual membrane components cannot be determined by a single labeling procedure and that differences in chemical composition and protein conformation must be taken into account as well as accessibility to the reagent itself.
While the actual radioactive counts incorporated with the transglutaminase procedure is relatively low in the present work, this is due to the much lower specific activity of ['Clglycine ethyl ester used (8 mCi/mmol) compared with "'1 used for the iodination procedure (12 Ci/mmol). Taking this into account, the transglutaminase procedure is about 50 times more efficient in modifying accessible components of the platelet surface.
The fluid mosaic model of membrane structure (22) characterizes membrane surface proteins as intrinsic or extrinsic depending on the degree to which they are bound to the membrane bilayer. Although these proteins are thought to differ in their structural and functional characteristics in the fluid mosaic model, the present findings suggest that closely similar, if not identical, glycoproteins may have the properties of both intrinsic and extrinsic molecules.
A tentative model for the orientation of the various components of the platelet surface may be suggested on the basis of the present work. The most loosely-bound components, which are released from the platelet surface in soluble form following platelet homogenization, include glycocalicin and another component both of which are labeled by the transglutaminiase reagent in the intact platelet. However, glycocalicin is not accessible in its entirety to membrane-labeling reagents, since differences have been found in the susceptibility to radiolabeling of the glycoprotein before and after solubilization.
Glycoproteins I, II, and III appear to be integral components of the platelet membrane and to be present on its outer surface, based on their accessibility in varying degrees to proteolytic enzymes. Glycoprotein III appears to be the only glycoprotein labeled with lactoperoxidase reagent. On the other hand, glycoprotein II is most strongly labeled by the transglutaminase method in the intact platelet. Glycoprotein I is only slightly labeled although its analog, glycocalicin, is strongly labeled under the same conditions. Four other membranebound proteins also appear to be accessible to this reagent.
Glycoprotein IV is not radiolabeled by either the transglutaminase or lactoperoxidase methods. It is not accessible to trypsin or chymotrypsin in the intact platelet, but is susceptible to proteolytic digestion in isolated plasma membranes, suggesting that it is present on the inner aspect of the membrane, or is buried within the membrane of the intact platelet but is exposed during isolation procedures. It may be noted that the membrane changes which are associated with the platelet release reaction induced by trypsin are not sufficient to expose glycoprotein IV to proteolytic attack in intact platelets.