The von Willebrand Factor-binding Domain of Platelet Membrane Glycoprotein Ib CHARACTERIZATION BY MONOCLONAL ANTIBODIES AND PARTIAL AMINO ACID SEQUENCE ANALYSIS OF PROTEOLYTIC FRAGMENTS*

The glycoprotein Ib (GPIb), a two-chain integral platelet membrane protein, acts as a receptor for von Willebrand factor. In order to obtain information on the domain involved in this function, as well as on the structural organization of GPIb, the protein has been purified and submitted to limited proteolysis using three different enzymes. The resulting fragments were topographically oriented by means of partial NH2-ter- minal sequence analysis and immunological identifi-cation using monoclonal antibodies. One of these anti- bodies (LJ-Ibl) inhibited the von Willebrand factor-GPIb interaction completely, one (LJ-P3) partially, and one (LJ-IblO) had no inhibitory effect. Three dis- tinct fragments, the 38-kDa fragment produced by Serratia marcescem protease as well as the 45- and 35-kDa fragments produced by trypsin, had the same NHz terminus as the intact GPIb a-chain (apparent molecular mass = 140 kDa). These fragments and the a-chain reacted with the inhibitory antibodies. On the other hand, three fragments produced by Staphylococ- cus aureus VS protease, one of 92 kDa similar to the previously described “macroglycopeptide” and two others of 52 and 45 kDa, had NH2-terminal sequences different from that of the GPIb a-chain and

important role in primary hemostasis by serving as one of the surface receptors that anchor platelets to exposed subendothelium at sites of vascular injury (1)(2)(3). This process is mediated by the adhesive molecule von Willebrand factor, which binds to GPIb as well as to collagen and noncollagenous components in the subendothelial matrix (4)(5)(6)(7)(8)(9)(10)(11).
A limited knowledge of the structural and functional characteristics of GPIb has been attained during the past years (12)(13)(14)(15)(16). This integral membrane protein is composed of two disulfide-linked chains and contains approximately 60% carbohydrate by weight. The structure of the carbohydrate moiety has been determined (17)(18)(19)(20). An endogenous platelet calcium-dependent protease cleaves the a-chain, releasing a large soluble fragment, termed glycocalicin (21-24), which appears to contain the von Willebrand factor-and thrombinbinding domains (25-28). The molecular mass of glycocalicin corresponds to approximately 130 kDa.
In these studies, we have used three murine monoclonal antibodies raised against human GPIb to locate the domain involved in von Willebrand factor binding. For this purpose, several fragments generated by proteolytic cleavage of GPIb have been isolated and characterized in terms of their reactivity with the monoclonal antibodies. Furthermore, the NH2terminal sequences of these proteolytic fragments, as well as of the intact a-chain, have been determined. We found that the two monoclonal antibodies which blocked the von Willebrand factor-GPIb interaction reacted with fragments possessing the same NH, terminus as the intact a-chain. The smallest of these peptides had a molecular mass of 38 kDa. Moreover, a carbohydrate-rich fragment of GPIb, similar to the previously described "macroglycopeptide" (13), had an NH2-terminal sequence distinct from that of the intact achain and reacted only with the antibody that failed to block the von Willebrand factor-GPIb interaction. These studies provide direct immunochemical evidence that the location of the von Willebrand factor-binding domain of GPIb is in proximity of the NH2 terminus of the a-chain and that the carbohydrate-rich region is toward its middle portion.

EXPERIMENTAL PROCEDURES AND RESULTS~
Three enzymes were used to prepare proteolytic fragments of GPIb, namely, Serratia mrcescens protease, trypsin, and * Portions of this paper (including "Experimental Procedures," part of "Results," Figs. 7-11, and Tables I and 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-1090, cite the authors, and include a check or money order for $5.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.  Purified intact GPIb (lane 4 ) and a 6-h digest ( l a n e 5 ) were also analyzed by immunohlot analysis using the monoclonal antibody LI-Ih10. Electroeluted GPIb n-chain (Inne 7) derived from purified GPIb ( l a n e 6 ) and the electroeluted 38-kDa fragment ( l a n e 9) obtained from the whole digest of purified GPIb ( l a n e 8) are also shown after analysis GPIb. The fragments generated by L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin digestion of 12sII-labeled GPIb (incubation times are indicated) were subjected to solid-phase immunoisolation using the monoclonal antibodies LJ-P3 and LJ-IblO. The immunoisolated fragments were then analyzed by 5-15?; gradient SDS-PAGE under nonreducing (N-R) or reducing ( R ) conditions, followed by autoradiography. The molecular mass of the polypeptides is indicated. Note the absence of the 84-kDa fragment in the nonreduced gel (compare with Fig. 10) and the appearance of the 92-kDa fragment after reduction. The latter, as well as the 127-kDa (glycocalicin like) fragment, is present only at the shorter incubation time.
Digestion with S. marcescens Protease-Two of the fragments generated by S. marcescens protease, one of 127 kDa and one of 38 kDa, reacted with all three monoclonal antibodies used, i.e. LJ-P3 and LJ-Ibl ( Fig. 1) and LJ-IblO (not shown). None of the antibodies reacted with an S. marcescens protease-generated fragment of 92 kDa. The reactivity of LJ-IblO, the only one of the three antibodies that reacted with denatured reduced a-chain, was also confirmed by immunoblotting of reduced polypeptides (Fig. 1). The 38-kDa polypeptide was isolated by electroelution from polyacrylamide gels (Fig. l ) , and the NH2-terminal sequence was determined.
The sequence of the first three amino acid residues was His-Pro-Ile and was identical to that of the intact a-chain. As with the a-chain, the yield of PTH-derivatives was 15-20% of the protein applied.
Digestion with Trypsin-Immunoisolation of the tryptic fragments demonstrated that LJ-P3 and LJ-IblO (Fig. 2) as well as LJ-Ibl (not shown) reacted with the same species. Analysis of the isolated polypeptides under nonreducing conditions demonstrated fragments of 127 and 45 kDa, but not the 84-kDa one. Analysis after reduction of disulfide bonds demonstrated, in addition, fragments of 92 and 35 kDa (Fig.  2). Both the 45-and 35-kDa tryptic fragments were isolated by electroelution. When analyzed by immunoblotting, only the 45-kDa fragment reacted with antibody LJ-IblO (Fig. 3). Both polypeptides were submitted to NH2-terminal sequence analysis. The same sequence, His-Pro-Ile, was determined in both cases, identical to that of the intact a-chain and the 38-  kDa fragment generated by S. marcescem protease. The initial yield of PTH-derivatives was 15-20% of the applied protein in these cases also. Digestion with S. a u r e u s V8 Protease-Immunoisolation of proteolytic fragments generated by S. a u r e u s V8 protease demonstrated that W-P3 (Fig. 4)  The 92-, 52-, and 45-kDa polypeptides were isolated by elect roelutiqn (Fig. 5) and submitted to NH2-terminal sequence analysis. The 92-and 45-kDa fragments gave the same sequence, and the first three amino acid residues were Asn-Ser-Leu. The first 2 residues of the 52-kDa peptide, on the other hand, were identified as Ser-Ile. The third cycle gave no PTH-derivative, and the fourth residue was phenylalanine. The NH2-terminal sequences of these three peptides, therefore, were different from that of the intact a-chain. The yield of PTH-derivatives of amino acids was greater than 50% of applied protein in these cases.

DISCUSSION
These studies demonstrate that all GPIb fragments of molecular mass between 38 and 45 kDa that reacted with two monoclonal antibodies inhibiting von Willebrand factor binding to GPIb had the same NH,-terminal sequence as the intact a-chain. This finding provides strong immunochemical evidence that the von Willebrand factor-binding domain is located in a region near the NH2 terminus of the a-chain. This possible localization had been previously suggested (46), but alternative models had also been proposed whereby this functional domain was assigned to the "tail" region of the (Ychain (3,16,47). The latter hypothesis is clearly in contrast with the present results. In studies not reported here, we also found that glycocalicin, a soluble proteolytic fragment released from platelets by an endogenous calcium-dependent protease, has the same NH2-terminal sequence as the intact a-chain, thus demonstrating that this is the distal portion of of the major proteolytic fragments generated by digestion of the GPIb a-chain with S. mrcescem protease (SP), trypsin (7'). and S. aurew V8 protease (SV8) is indicated by the corresponding NH,-terminal sequence and reactivity with three distinct monoclonal antibodies. Localization of the epitopes corresponding to these antibodies (LJ-P3, LJ-Ibl, LJ-IblO) is tentative, but within regions of the GPIb a-chain that can be defined with certainty.
The localization of the NH2 terminus of the fragments generated by S. a w e u s V8 protease digestion is indicated with broken lines because it cannot be established on the basis of these results. Since all these fragments react with W-IblO, however, their NH, termini must be distal to the W-IblO epitope. The approximate molecular masses of the fragments are indicated. The von Willebrand factor (uWF)-binding domain lies within the NHZterminal region of the a-chain. The COOH terminus of the GPIb a-chain is indicated with a broken line because it is not yet known whether it has a transmembrane portion. One-letter abbreviation for amino acids indicates the NHZ-terminal sequence of fragments. the molecule. On the other hand, all proteolytic fragments of GPIb reacting only with the noninhibitory antibody LJ-IblO had an NHZ-terminal sequence distinct from that of the intact a-chain. These included polypeptides that were rich in carbohydrate, thus demonstrating that the previously described macroglycopeptide derived from GPIb does not extend into the NHz-terminal region of the a-chain, as suggested by some authors (16,47).
The initial yield of PTH-derivatives was low for the intact a-chain and for the fragments that gave the same NH,terminal sequence of His-Pro-Ile. Whereas we have no definitive explanation at present for this poor initial yield, it is noteworthy that its possible cause may be related to the presence of histidine and proline residues in the first two positions (48-50). The possibility of partial blockage at the NH, terminus should also be considered, particularly in view of the fact that others have not been able to obtain an unambiguous NH,-terminal sequence for glycocalicin (46).
Our findings indicate the presence of at least one intrachain disulfide bond within the NH2-terminal region of the GPIb a-chain (Fig. 6) as also suggested by others? A 35-kDa trypsin-generated fragment that was detected only after reduction of disulfide bonds possessed the same NH,-terminal sequence as a fragment of 45 kDa, seen both under nonreducing and reducing conditions, and as the intact a-chain. Therefore, the 35-kDa fragment derives from the 45-kDa one, and the cleavage that generates it must occur within a disulfide loop (Fig.  6). The two inhibitory antibodies LJ-P3 and LJ-Ibl, unlike the noninhibitory antibody W-IblO, failed to react with GPIb or GPIb fragments maintained under reducing conditions. Therefore, intrachain disulfide bond(s) within the NHp-terminal region play an important role in providing the appropriate conformation of epitopes corresponding to antibodies that block the von Willebrand factor-GPIb interaction. It remains to be demonstrated whether the same applies to the function of the von Willebrand factor-binding domain itself.
It is also noteworthy that only the 45-kDa trypsin fragment, but not the 35-kDa one, reacted with the noninhibitory antibody LJ-IblO. Thus, the epitope recognized by U-IblO can be localized with certainty in the region representing the portion of the 38-kDa S. mrcescens protease-generated fragment extending beyond the 35-kDa trypsin-generated fragment (Fig. 6). In fact, these two fragments exhibited the same NH, terminus, but only the 38-kDa polypeptide reacted with the antibody. All three enzymes used in these studies generated, among others, fragments of approximately 127 and 92 kDa. The 127-kDa fragments, which may differ slightly from one another, are likely to derive from cleavages within a protease-sensitive region corresponding to the area where a cleavage by the platelet calcium-dependent protease results in the generation of glycocalicin (Fig. 6). All these "glycocalicin-like" molecules reacted with the three monoclonal antibodies used, including the two inhibitory ones. Thus, they are likely to have the same NH, terminus as glycocalicin (the same as the intact achain) and comprise the von Willebrand factor-binding domain.
The fragments of approximately 92 kDa were found to be different from each other, but exhibited the common property of having the NH,-terrninal sequence distinct from that of the a-chain. This was established for the s. marcescens Protease-generated fragment because it did not react with antibody W-IblO and therefore had no relationship with the 38-kDa fragment having the same NH, terminus as the a-chain (Fig. 6). The 92-kDa fragment generated by trypsin, on the other hand, reacted with L J -I b l O . This antibody failed to react with the 84and 35-kDa tryptic fragments, but reacted with the 45-kDa fragment (having the same NH, terminus as the intact a-chain and the 35-kDa fragment). Thus, the NH, terminus of the 92-kDa fragment can be assigned to the cleavage site indicated as Tz in Fig. 6 and must be different from that of the S. mrcescens protease-generated fragment of similar size that does not react with LJ-IblO. Finally, the 92-kDa fragment generated by S. aureus V8 protease digestion was clearly distinct from that generated by S. mrcescens protease digestion, as shown by the different reactivity with W-IblO; but its relationship to the fragment of similar size generated by trypsin remains unclear. Nevertheless, we have determined the NH,-terminal sequence of the S. aureus V8 protease-generated 92-kDa polypeptide and found that it is distinct from that of the a-chain.
In conclusion, the present studies provide immunochemical evidence for localization of the von Willebrand factor-binding domain of GPIb in the proximity of the NHp-terminal region of the cy-chain, where at least one intrachain disulfide bond is also located. In addition, they provide information on the orientation of several proteolytic fragments of the a-chain, including glycocalicin, and represent a firm basis for further characterization of the structural and functional organization of GPIb.