Isolation and characterization of a platelet membrane protein related to the vitronectin receptor

Glycoprotein IIb-IIIa is the most prominent Arg-Gly-Asp (RGD)-binding adhesion receptor on platelets. By affinity chromatography on an immobilized RGD peptide, we have investigated the possible existence of other platelet-associated adhesion receptors that bind RGD peptides. When an octyl glucoside extract of surface-radioiodinated platelets was applied to an affinity matrix of KYGRGDS-coupled Sepharose 4B, a 160-kDa-labeled protein (P160) and GPIIb-IIIa bound and were specifically eluted by soluble GRGDSP peptide, but not by the variant GRGESP peptide. Furthermore, a dodecapeptide corresponding to fibrinogen gamma 400-411 eluted only GPIIb-IIIa but not P160 from the RGD affinity matrix. Characterization of P160 by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by the O'Farrell gel electrophoresis system indicated that P160 is a component of platelet GPIc. GoH3, a monoclonal antibody recognizing the alpha subunit of the very late antigen-6, failed to immunoprecipitate P160 from the RGD eluate, indicating that it did not contain the very late antigen-6 alpha subunit. In immunoblots, P160 reacted specifically with a polyclonal anti-peptide antibody recognizing the alpha subunit of the vitronectin receptor (VnR), but not with the monoclonal anti-GPIIb antibody PMI-1, suggesting that P160 is the alpha subunit of platelet VnR. This possibility was further substantiated by the complete identity between the determined amino-terminal sequence of P160 and the known sequence of the VnR alpha subunit. Moreover, direct association of P160 with a beta subunit having an apparent molecular weight similar to that of GPIIIa was demonstrated by immunoprecipitation with LM609, an anti-VnR complex monoclonal antibody. These results indicate that the VnR complex is present on platelets and may play a functional role in platelet adhesive reactions.     

Convulxin binding to platelet receptor GPVI: competition with collagen related peptides

Convulxin (CVX), a potent platelet aggregating protein from the venom of the snake Crotalus durissus terrificus, is known to bind to the platelet collagen receptor, glycoprotein VI (GPVI). CVX binding to human platelets was investigated by flow cytometry, using fluorescein labeled convulxin (FITC-CVX). Scatchard analysis indicated high and low affinity binding sites with Kd values of 0.6 and 4 nM and Bmax values of 1200 and 2000 binding sites per platelet. FITC-CVX binding was inhibited by collagen related peptides (CRPs) comprising a repeated GPO sequence, namely GCO(GPO)(10)GCOGNH(2) and GKO(GPO)(10)GKOGNH(2), which also bind to receptor GPVI. These peptides (monomeric or cross-linked forms) gave a high affinity inhibition of 10-20% for concentrations between 10 ng/ml and 5 microg/ml, followed by a second phase of inhibition at concentrations greater than 5 microg/ml. It was shown also that the inhibition of FITC-CVX binding by CRPs was independent on the time of preincubation of platelets with CRPs, and the same percentage of inhibition was seen with various concentrations of convulxin. Confocal microscopy of the distribution of FITC-CVX binding sites on platelets showed an homogeneous distribution of FITC-CVX bound to GPVI, although some limited clustering may exist.     

Biochemical characterization of VLA-1 and VLA-2. Cell surface heterodimers on activated T cells

The very late antigen complexes VLA-1 and VLA-2 which appear on long-term activated human T cells have been characterized with respect to 1) subunit arrangement, 2) location of monoclonal antibody (MAb) binding sites, 3) carbohydrate content, and 4) protein homology. Cross-linking experiments showed that the VLA-1 complex is a heterodimer composed of an Mr 210,000 subunit (alpha 1) in acid-labile association with an Mr 130,000 subunit (beta). The VLA-2 complex is a heterodimer with an Mr 165,000 subunit (alpha 2) in base-labile association with the Mr 130,000 beta subunit. The subunits of VLA-1 (alpha 1 beta) and VLA-2 (alpha 2 beta) each appear to be arranged with 1:1 stoichiometry. The MAb A-1A5 has been shown to bind to an epitope on the common beta subunit, consistent with its recognition of both the VLA-1 and VLA-2 heterodimers. On the other hand, MAb TS2/7 bound to an epitope of the alpha 1 subunit, thus explaining the specific recognition of the VLA-1 heterodimer by TS2/7. Digestion of the alpha 1, alpha 2, and beta subunits with neuraminidase and with endoglycosidase F revealed that each subunit contains substantial sialic acid and N-linked carbohydrate. By one-dimensional peptide mapping, the alpha 1, alpha 2, and beta subunits were shown to be highly nonhomologous with respect to each other, although each subunit from different T cell sources appeared highly homologous if not identical.     

Isolation and characterization of a collagen binding domain in human von Willebrand factor

von Willebrand factor binds to fibrillar type I collagen in a rapid, temperature-independent, reversible, specific, and saturable manner. Evaluation of binding isotherms by Scatchard-type analysis demonstrated that 6-18 micrograms of von Willebrand factor bind per mg of collagen, with Ka between 2 and 8 X 10(8) M-1. Five distinct tryptic fragments, purified under denaturing and reducing conditions and representing over 75% of the molecular mass of the von Willebrand factor subunit, were tested for their capacity to inhibit the von Willebrand factor-collagen interaction. Complete inhibition was obtained with a 52/48-kDa fragment at a concentration of approximately 1 microM. The location of this fragment in the subunit was established to be between Val-449 and Lys-728. Fifteen monoclonal antibodies against the 52/48-kDa fragment inhibited von Willebrand factor binding to collagen. Six antibodies against other portions of the von Willebrand factor subunit had no inhibitory effect. The tryptic fragment was a competitive inhibitor of von Willebrand factor binding to collagen and, therefore, recognizes the same interaction site as the intact molecule. These studies precisely define a domain in the von Willebrand factor subunit that interacts with type I collagen.     

The collagen binding specificity of bone and platelet osteonectin is related to differences in glycosylation

In this study we report that bone and platelet osteonectin are structurally and functionally heterogeneous in terms of glycosylation and collagen binding capacity. The relative sensitivity of bone and platelet osteonectin to specific glycosidases was used to evaluate potential differences in glycosylation. Although native bone and platelet osteonectin are electrophoretically nonidentical, N-glycanase treatment yielded products with the same apparent molecular weight. Bone osteonectin was also susceptible to cleavage by endo H but not to neuraminidase, while platelet osteonectin was susceptible to neuraminidase but not to endo H. In lectin blotting experiments of bone and platelet osteonectin, concanavalin A bound specifically to bone osteonectin but not to platelet osteonectin. However, Lens culinaris agglutinin bound to platelet osteonectin but not to bone osteonectin. These data suggest that bone and platelet osteonectin differ in their oligosaccharide side chain structures, with bone osteonectin possessing a high mannose-type and platelet osteonectin, a complex-type structure. Solid-phase binding techniques were used to functionally evaluate bone and platelet osteonectin in terms of collagen binding. Although bone osteonectin bound specifically to types I, III, and V collagen, platelet osteonectin had no apparent affinity for these collagen types suggesting that the two proteins are also functionally distinct.     

Divalent cation-dependent structure in the platelet membrane glycoprotein Ia-IIa (VLA-2) complex

Recent studies have shown that the platelet membrane glycoprotein Ia-IIa (VLA-2) complex mediates the Mg(++)-dependent adhesion of platelets to collagen and that this adhesion is inhibited by Ca++ in a simple, linear, noncompetitive manner. These findings suggested that separate binding sites for Mg++ and Ca++ stabilize different divalent cation-dependent structures within the receptor complex. To provide evidence for the existence of such structures purified platelet Ia-IIa complex was subjected to limited proteolytic digestion in the presence of Mg++, Ca++, Mg++ and Ca++, or EDTA and the resulting peptides mapped by SDS-PAGE using both one and two-dimensional techniques. Unique patterns of tryptic peptides were produced under each of the conditions. The results indicate that Mg++ and Ca++ stabilize different structures within the Ia-IIa (VLA-2) complex and that these structures influence both the collagen binding activity and proteolytic susceptibility of the complex.     

Isolation and characterization of a chymotryptic fragment of platelet glycoprotein IIb-IIIa retaining Arg-Gly-Asp binding activity.

Platelet membrane glycoprotein (GP)IIb-IIIa exists as a divalent cation-dependent heterodimer which recognizes the Arg-Gly-Asp (RGD) sequence of adhesive proteins. To isolate the RGD binding domain of GPIIb-IIIa we performed proteolysis of GPIIb-IIIa with alpha-chymotrypsin. GPIIb-IIIa was bound to an affinity matrix of GRGDSPK-coupled Sepharose 4B and was then treated with chymotrypsin. After washing the unbound fragments, two discrete polypeptides of 55 and 85 kDa remained bound to the RGD affinity matrix and were specifically eluted by soluble HHLGGAKQAGDV (H12) or by GRGDSP, but not by GRGESP. Immunoblotting with subunit-specific polyclonal antibodies showed that the 55- and 85-kDa fragments were derived from GPIIb and GPIIIa, respectively. Amino-terminal sequencing and immunoblotting using site-specific antibodies indicated that these fragments contained the amino termini of their parent molecules. In the presence of 1 mM Ca2+ and 1 mM Mg2+, these two fragments were maintained as a heterodimer inasmuch as both fragments were immunoprecipitated by the polyclonal anti-GPIIIa antibodies. In contrast, chelating the divalent cations with 5 mM EDTA resulted in the lack of co-immunoprecipitation of the 55-kDa GPIIb fragment. After removal of the H12 peptide, the 55/85-kDa heterodimer bound to immobilized fibrinogen in an enzyme-linked immunosorbent assay by an RGD-dependent mechanism. These findings suggest that the RGD binding domain and structures required for heterodimer maintenance are present within the 55/85-kDa chymotryptic fragment of GPIIb-IIIa.     

Characterization of the cell surface heterodimer VLA-4 and related peptides

A monoclonal antibody (B-5G10) was produced which specifically recognizes the Mr 150,000/130,000 VLA-4 complex on the surface of human cells. Cross-linking studies indicated that the Mr 150,000 alpha 4 subunit of VLA-4 is in noncovalent 1:1 association with the Mr 130,000 VLA beta subunit. In the absence of cross-linking, the VLA-4 alpha 4 beta subunit complex was easily dissociated, especially in Nonidet P-40 detergent, or at elevated pH (above 8.0). Studies of dissociated subunits showed that B-5G10 recognizes an epitope on the Mr 150,000 alpha 4 subunit of VLA-4, whereas the beta subunit is immunologically identical to the Mr 130,000 beta subunit common to all VLA heterodimers. VLA-4 is widely distributed on hematopoietic cells, including thymocytes, peripheral blood lymphocytes, monocytes, activated T cells, T and B lymphoblastoid cell lines, and myeloid cell lines. However, VLA-4 is only weakly expressed on most adherent cell lines tested. Immunoprecipitates of VLA-4 often contain additional proteins of Mr 80,000 and Mr 70,000. These are probably derived from the Mr 150,000 alpha 4 subunit because: 1) they are both recognized by anti-alpha 4 sera, but not anti-beta sera; 2) the sum of their sizes is equal to the size of alpha 4; 3) they are selectively coexpressed with alpha 4 and not other VLA alpha subunits; 4) the Mr 80,000 protein has an identical NH2-terminal sequence to alpha 4; 5) like alpha 4, the Mr 70,000 and 80,000 peptides can variably associate with the VLA beta subunit; and 6) trypsin appears to cleave the Mr 150,000 alpha 4 subunit into products of Mr 70,000 and 80,000.     

Isolation and partial characterization of a novel basement membrane collagen

A guanidine-HCl extraction of lens capsule basement membrane dissolves collagenous material. This material was fractionated on an Agarose A-5M column. Fractions 1, 2 and 3 were further purified and partially characterized immunochemically and by amino acid analysis. Fraction 3 has a molecular weight of 55,000 when compared with collagen type I standard. The CNBr peptide pattern and composition of fraction 3 are different from those of alpha 1 (IV) 95K and alpha 2 (IV) 95K chains. The results described suggest the presence of a new chain in lens capsule basement membrane.     

The membrane glycoprotein Ia-IIa (VLA-2) complex mediates the Mg++- dependent adhesion of platelets to collagen

We have purified the platelet membrane glycoprotein Ia-IIa complex by detergent solubilization and sequential affinity chromatography on Concanavalin A-Sepharose and collagen-Sepharose. The complex, which is identical to the VLA-2 complex of lymphocytes and other cells and contains subunits of 160 and 130 kD on SDS-PAGE, was labeled with 125I and incorporated into phosphatidyl choline liposomes. The liposomes, like intact platelets, adhered to collagenous substrates in an Mg++-dependent manner with a K'a(Mg++) of 3.5 mM. Little adhesion of the liposomes to collagen occurred when Mg++ was replaced by Ca++ or EDTA. Calcium ions inhibited the Mg++-dependent adhesion with a K'i(Ca++) of 5.5 mM. Liposomes containing the Ia-IIa complex adhered to substrates composed of types I, II, III, and IV collagen, but did not effectively adhere to substrates composed of type V collagen or gelatin. Adhesion to collagen was specific. The liposomes did not adhere to fibronectin, vitronectin, laminin, thrombospondin, fibrinogen, or von Willebrand factor substrates. The monoclonal antibody P1H5, which specifically immunoprecipitated the Ia-IIa complex, also specifically inhibited the Mg++-dependent adhesion of both platelets and Ia-IIa-containing liposomes to collagen substrates. These findings provide additional evidence that the platelet membrane Ia-IIa complex is the mediator of Mg++-dependent platelet adhesion to collagen and suggest that the VLA-2 complex may also function as an Mg++-dependent collagen receptor in other cells.     

Isolation, quantitation, and characterization of a stable complex formed by Lp[a] binding to triglyceride-rich lipoproteins.

Lipoprotein [a] (Lp[a]) is a cholesterol-rich lipoprotein resembling LDL to which a large polymorphic glycoprotein, apolipoprotein [a] (apo[a]), is covalently coupled. Lp[a] usually exists as a free-standing particle in normolipidemic subjects; however, it can associate noncovalently with triglyceride-rich lipoproteins in hypertriglyceridemic (HTG) subjects. In this study, 10-78% of the Lp[a] present in five HTG subjects was found in the triglyceride-rich lipoprotein (TRL) fraction. The Lp[a]-TRL complex was resistant to dissociation by ultracentrifugation (UCF) alone, but was quantitatively dissociated by UCF in the presence of 100 mM proline. Of this dissociated Lp[a], 70-88% was in the form of a lipoprotein resembling conventional Lp[a]. Incubation of Lp[a]-depleted TRL with native Lp[a] resulted in a reconstituted Lp[a]-TRL complex that closely resembled the native isolates in all examined properties. Complex formation was inhibited by several compounds in the order proline > tranexamate > epsilon-aminocaproate > arginine > lysine. Neither plasminogen nor LDL inhibited binding of Lp[a] to TRL. We observed the preferential binding of Lp[a] containing higher apparent molecular weight apo[a] polymorphs to TRL both in native and reconstituted Lp[a]-TRL complexes. A disproportionate amount of Lp[a] was bound to the larger TRL particles. Although most apo[a] bound to TRL was in the form of conventional Lp[a] particles, lipid-free recombinant apo[a] was observed to bind TRL.These results provide unequivocal evidence of the existence of an Lp[a]-TRL complex under pathophysiologic conditions. The metabolic fate of the Lp[a]-TRL complex, which is more abundant in hypertriglyceridemia, may be different from that of conventional Lp[a], and may contribute uniquely to the progression or severity of cardiovascular disease.     

The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.     

Functional analysis of a recombinant glycoprotein Ib alpha polypeptide which inhibits von Willebrand factor binding to the platelet glycoprotein Ib-IX complex and to collagen.

By deletion mutagenesis and transient expression in COS cells, a 96-amino acid hydrophilic sequence in the glycoprotein Ib alpha polypeptide located between L220 and L318 was identified which appeared to contain its von Willebrand factor- (vWF) binding site. The cDNA encoding this fragment was then expressed in Escherichia coli and purified from the bacterial cell lysate. The recombinant polypeptide, rGpIb alpha Q221-L318, was monomeric and had an apparent molecular weight of 14,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It inhibited both ristocetin-induced binding of 125I-vWF to fixed washed platelets and ristocetin-induced platelet agglutination. The recombinant polypeptide also inhibited the binding of 125I-vWF to immobilized type I and III collagen. Inhibition of 125I-vWF binding to platelets and collagen was dose-dependent, with IC50 values of 500 and 200 nM rGpIb alpha Q221-L318, respectively. Fifty % inhibition of ristocetin-induced platelet agglutination required 500 nM rGpIb alpha Q221-L318. Although rGpIb alpha Q221-L318 inhibited vWF binding to collagen it did not, itself, bind to collagen-coated surfaces. Reduction of the disulfide bond between C248 and C264 abolished activity. 125I-rGpIb alpha Q221-L318 bound directly to GpIb/IX sites on multimeric vWF. These studies document that a portion of the sequence between Q221 and L318 is needed for recognition and binding to vWF and that binding requires an intact disulfide bond between C248 and C264. The binding of this recombinant polypeptide to vWF multimers inhibits vWF interaction with two important substrates, platelet GpIb/IX and collagen.     

Isolation and characterization of collagen messenger RNA*.

Chick embryo collagen-synthesizing polysomes were isolated by differential centrifugation. RNA extracted from these particles was chromatographed in oligo(dT)-cellulose solumns and the mRNA thus obtained characterized as collagen mRNA by its electrophoetical mobility in acrylamide gels (equivalent to 1.05 x 10-6 daltons) and its effect upon a cell-free system derived from Krebs ascites tumor cells. The incorporation of 3H-proline was markedly dependent upon rabbit reticulocyte initiation factors and inhibited by initiation inhibitors such as aurintricaboxilate and pyrocatechol violet. The incorporation product was characterized as collagen by its lack of tryptophan, digestibility by purified bacterial collagenase, and by its co-chromatography with unlabled chick collagen in Sephadex G-200 and CM-cellulose columns.     

Human platelet glycoprotein V: a surface leucine-rich glycoprotein related to adhesion

Human platelet glycoprotein V (Mr 82,000) is a surface glycoprotein and a substrate for thrombin, undergoing proteolytic cleavage by thrombin and releasing a soluble fragment, glycoprotein Vfl (Mr 69,000). It does not appear to be the receptor for thrombin's agonist effect on platelets. A congenital platelet disorder, Bernard-Soulier syndrome, is marked by a deficiency of glycoprotein V and two other surface glycoproteins, Ib-IX. The latter two, Ib-IX, constitute the platelet receptor for von Willebrand factor, mediate arterial platelet adhesion, and contain unique 24-amino acid sequences, termed "leucine-rich glycoprotein" segments. The segments relate to adhesive function and distinguish the leucine-rich glycoprotein family. Surface glycoprotein V is not physically associated with Ib-IX nor does it bind to von Willebrand factor. To date, no common denominator has been found that explains the combined deficiency of glycoproteins V and Ib-IX in Bernard-Soulier syndrome. This study describes the isolation of glycoprotein V/anti-glycoprotein V antibody and the analysis of three glycoprotein V peptides that contain "leucine-rich" sequences. Therefore, glycoprotein V shares the "leucine-rich" structure with platelet glycoproteins Ib-IX and belongs to the family of leucine-rich glycoproteins.     

Isolation and partial characterization of human platelet vinculin

A 130,000 Mr protein was isolated from human platelets by sequential DEAE-Sephacel and Sepharose Cl-4B chromatography. Low shear viscometric measurements showed that the enriched protein after DEAE-Sephacel chromatography inhibited actin polymerization. This effect was somewhat greater in the presence of EGTA than in the presence of calcium. Further purification by Sepharose Cl-4B chromatography resulted in a complete loss of this inhibitory effect. Studies with fluorescent actin detected no nucleation or "+" end capping activity in either the DEAE-Sephacel- or Sepharose Cl-4B-purified vinculin. Antibodies raised in mice against the 130,000-mol-wt protein were shown to cross-react with chicken gizzard vinculin and a similar molecular weight protein was detected in WI38 cells and, Madin-Darby canine kidney cells. Lysis experiments with the Madin-Darby canine kidney cells indicated that most of the vinculin was soluble in Triton X-100, although some was found associated with the insoluble cytoskeletal residue. By immunofluorescence, vinculin in WI38 cells was localized to adhesion plaques as described by others. Discrete localization in platelets was also detected and appeared to depend on their state of adhesion and spreading. The results of these experiments suggest that human platelets contain a protein similar to vinculin. It is not clear if platelet vinculin is associated with structures analogous to adhesion plaques found in other cell types. The data indicate that the previously reported effects of nonmuscle vinculins on actin polymerization may be due to a contaminant or contaminants.     

Isolation and characterization of human platelet glycoprotein IX

Human platelet glycoprotein IX is a small (Mr 17,000) surface glycoprotein present in the plasma membrane as a 1:1 non-covalent complex with glycoprotein Ib (Mr 165,000). Glycoprotein IX was purified to near homogeneity by sequential wheat germ agglutinin and immuno-affinity chromatography, followed by gel filtration chromatography under denaturing conditions. Purified glycoprotein IX was characterized by determination of its amino acid composition and its NH2-terminal amino acid sequence (thr-lys-asp-xxx-pro-ser-xxx-leu-thr-(thr)2-arg-ala-(leu)-(glu)-xxx-met- gly), and monospecific anti-glycoprotein IX antibody was prepared. The study outlines a useful approach for separating and characterizing glycoprotein IX, free from other members of the glycoprotein Ib complex.