Synthesis and antithrombotic activity of carbolinecarboxyl RGD sequence

3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, RGDS, RGDV, RGDF and their linkers were synthesized. The anti-aggregation and adhesion of platelet indicated that the in vitro activities of the linkers remained at the same level as RGDS, RGDV, and RGDF (p>0.05). The antithrombotic activities in vivo suggested, however, that the potencies of RGDS, RGDV and RGDF were enhanced by the introduction of 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxyl group into their alpha amino group (p<0.05, 0.01 or 0.001).     

Synthesis and bioactivities of nitronyl nitroxide and RGD containing pseudopeptides

1-(1',3'-dioxyl-4',4',5',5'-tetramethyldihydroimidazol-2'-yl)-phenyl-4-yloxylacetic acid (3), and 1-(1',3'-dioxyl-4',4',5',5'-tetramethyldihydroimidazol-2'-yl)-phenyl-4-yloxylacetyl-RGDS (13), -RGDV (14), -RGDF (15) were synthesized. The ESR measurement gave the same spectroscopy for 3 and 13-15. The NO scavenging tests in vitro, anti-platelet aggregation tests in vitro and the anti-thrombosis assay in vivo indicated that introducing 3 into the N-terminal of RGDS, RGDV and RGDF the corresponding bioactivities for both of 3 and RGD peptides can be remained completely. The present combinations provided a beneficial strategy for simultaneous scavenging NO and anti-thrombosis, and for the use of spin label of RGD peptides in the conformational researches.     

Antiplatelet "hybrid" peptides analogous to receptor recognition domains on gamma and alpha chains of human fibrinogen

Platelet receptor recognition domains are located on the gamma and alpha chains of human fibrinogen. The former encompasses residues 400-411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767], and the latter is present in two loci on the alpha chain (alpha 95-97 and alpha 572-574) [Hawiger, J., Kloczewiak, M., Bednarek, M. A., & Timmons, S. (1989) Biochemistry (first of three papers in this issue)]. Peptide gamma 400-411 (HHLGGAKQAGDV) inhibited aggregation of ADP-treated platelets mediated not only by gamma-chain but also by alpha-chain multimers. Peptide alpha 572-575 (RGDS) inhibited aggregation of platelets mediated by alpha-chain as well as gamma-chain multimers. These results indicate that the platelet receptor for fibrinogen is isospecific with regard to the domain present on alpha and gamma chains. Subsequent "checkerboard" analysis of combinations of gamma 400-411 and alpha 572-575 showed that the inhibitory effect toward binding of 125I-fibrinogen was additive rather than synergistic. Next, a series of "hybrid" peptides was constructed in which the alpha-chain sequence RGDF (alpha 95-98) replaced the carboxy-terminal segment of gamma 408-411. The dodecapeptide HHLGGAKQRGDF was inhibitory with concentration, causing 50% inhibition of binding (IC50) at 6 microM, 5 times more potent than gamma 400-411. The shorter peptides AKQRGDF and KQRGDF were also more inhibitory than gamma 400-411. The second series of hybrid peptides was constructed with the alpha-chain sequence RGDS preceding the sequence of gamma 400-411 or sequence RGDV following it.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational modulation of purified glycoprotein (GP) IIb-IIIa allows proteolytic generation of active fragments from either active or inactive GPIIb-IIIa.

This study characterized conformational states of platelet glycoprotein IIb-IIIa (GPIIb-IIIa) and regions of the molecule required for fibrinogen binding. Platelet lysates were passed sequentially over concanavalin A and aminoethylglycine (Aeg)RGDS affinity columns. Approximately 10% of the total GPIIb-IIIa bound to the Aeg-RGDS column. The non-binding GPIIb-IIIa was further purified by S300 gel filtration. Only GPIIb-IIIa which recognized immobilized RGDS bound fibrinogen. The functional difference between the Aeg-RGDS binding GPIIb-IIIa (active) and the S300-purified complex (inactive) suggested that the two populations existed in different conformations. This was confirmed immunochemically and in an assay utilizing endoproteinase Arg-C. Active GPIIb-IIIa was heavily degraded by Arg-C, whereas inactive GPIIb-IIIa was highly resistant to degradation. Receptor occupancy by RGDV or peptidomimetic inhibitors prevented degradation of regions of the active complex and stimulated hydrolysis of the inactive receptor such that the two populations yielded fragments of identical electrophoretic mobility. Induction of hydrolysis of inactive GPIIb-IIIa required 15-fold higher concentrations of RGDV than protection of the active complex. Upon removal of inhibitor, fragments generated from either active or inactive GPIIb-IIIa bound fibrinogen. The ability of carboxypeptidase Y to digest inhibitor-protected GPIIb-IIIa was also examined. GPIIb was cleaved to a 58-kDa NH2-terminal fragment, whereas GPIIIa remained essentially intact. The complexed fragments bound fibrinogen with similar affinity as intact GPIIb-IIIa. This binding was inhibited by both RGDV and HHLGGAKQAGDV peptides. These data suggest that: 1) purified active and inactive GPIIb-IIIa exist in different conformations and have different affinities for RGDV; 2) certain peptidomimetic inhibitors (Ro 42-1499 and Ro 43-5054) alter the conformation of inactive GPIIb-IIIa; 3) GPIIIa and a 58-kDa NH2-terminal fragment of GPIIb alpha form a high affinity fibrinogen binding complex.     

Preferred conformations of RGDX tetrapeptides to inhibit the binding of fibrinogen to platelets

The conformational study on Arg-Gly-Asp (RGD)-containing tetrapeptides in the unhydrated and hydrated states has been carried out using the force field ECEPP/3 and the hydration shell model. The tetrapeptides studied here are H-RGDX-OH (X = Trp, Tyr, Phe, Leu, Val, Cys, Gln, and Ser), which show the inhibitory activity for binding of fibrinogen to platelets in the order of RGDW approximately equal to RGDY approximately equal to RGDF approximately equal to RGDL > RGDV > or = RGDC > or = RGDQ > or = RGDS. The backbone conformations with two C(7) backbone-to-backbone hydrogen bonds between Asp and Arg residues and between Xaa and Gly residues are in common most probable for the RGD sequence of RGDX tetrapeptides in the hydrated state. The dominant beta-turns for RGDX are found to be the types V' and IV at Gly-Asp and Asp-Xaa sequences, respectively, which are quite similar to the types II' and I (or II), respectively. However, it cannot be ruled out that the extended conformations are also remarkably feasible for RGDX tetrapeptides in water by peering the distributions of backbone conformations. These calculated results are consistent with the experimental results on RGD-containing proteins and conformationally constrained RGD-containing peptides. The reason why the RGDX becomes more potent as the side chain of the X residue is more hydrophobic may be ascribed to that the more hydrophobic is the residue X, the more populated are beta-turn structures for the Gly-Asp sequence. The hydrophobic side chain of X residue exposed to water is likely to interact with the hydrophobic region of receptor easily.     

Amino acid sequences in fibrinogen mediating its interaction with its platelet receptor, GPIIbIIIa

Platelet membrane GPIIbIIIa is a member of the family receptors named integrins that recognize RGD sequences in their ligands. GPIIbIIIa interacts with at least three different adhesive ligands: fibrinogen, fibronectin, and von Willebrand factor. These interactions are inhibited by RGD-containing peptides and by peptides corresponding to a sequence unique to fibrinogen in the COOH-terminal domain of its gamma chain (HLGGAKQAGDV). Two RGD sequences are present in fibrinogen A alpha chain: an RGDS sequence at A alpha 572-575, and an RGDF sequence at A alpha 95-98. Polyclonal antibodies raised against the RGDF sequence and the gamma COOH-terminal domain both reacted specifically with fibrinogen in solid phase enzyme-linked immunosorbent assays and immunoprecipitated the protein in solution. The Fab fragments prepared from these antibodies inhibited fibrinogen-platelet interaction and aggregation. These results demonstrate that these two sequences are both accessible within the fibrinogen molecule and are both implicated in ligand binding and cell-cell interaction. In addition, by further examining the interaction of the gamma chain peptide with platelets, it was found that RGDF and the gamma peptide produced a similar dose-dependent inhibition of the binding of the labeled gamma peptide to ADP-stimulated platelets. These results provide evidence that the RGDF sequence present at the A alpha 95-98 constitutes with the gamma 401-411 sequence two recognition sites interacting with the same site or with mutually exclusive sites on GPIIbIIIa.     

Synthesis of RGD containing peptides and their vasodilation effect

APLRV, SLRR, RGDS, and RGDF were synthesized by use of the solution method via the corresponding protective intermediates. After fragment condensation and deprotection, APLRVRGDS, APLRVRGDF, SLRRRGDS, and SLRRRGDF were obtained. The effect of these RGD containing peptides on rat aortic strips pretreated with noradrenaline (NE) were observed. The relaxing extents of contracted strips for them at three doses (10(-5) mol/L, 10(-6) mol/L and 10(-7) mol/L) indicated that, in a few cases, this kind of combination of these fragments may enhance the desirable activity.     

Platelet receptor recognition domains on the alpha chain of human fibrinogen: structure-function analysis

We have previously shown that the alpha chain of human fibrinogen interacts directly with ADP-activated human platelets [Hawiger, J., Timmons, S., Kloczewiak, M., Strong, D. D., & Doolittle, R. F. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2068]. Now, we report that platelet receptor recognition domains are localized on two CNBr fragments of the human fibrinogen alpha chain. They encompass residues 92-147 and 518-584, which inhibit 125I-fibrinogen binding to ADP-stimulated platelets. The inhibitory CNBr fragment alpha 92-147 contains the RGD sequence at residues 95-97. Synthetic peptides encompassing this sequence were inhibitory while peptide 99-113 lacking the RGD sequence was inactive. The synthetic peptide RGDF, corresponding to residues alpha 95-98, inhibited the binding of 125I-fibrinogen to ADP-treated platelets (IC50 = 2 microM). However, the peptides containing sequence RGDF, with residues preceding Arg95 or following Phe98, were less inhibitory. It appears that the sequence alpha 95-98 constitutes a platelet receptor recognition domain which is constrained by flanking residues. The second inhibitory CNBr fragment, alpha 518-584, also contains the sequence RGD at positions 572-574. Synthetic peptides overlapping this sequence were inhibitory, while peptides lacking the sequence RGDS were not reactive. Thus, another platelet reactive site on the alpha chain encompasses residues 572-575 containing sequence RGDS. In conclusion, the platelet receptor recognition domains on the human fibrinogen alpha chain in the amino-terminal and in the carboxy-terminal zones contain the ubiquitous cell recognition sequence RGD shared with other known adhesive proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

A class of novel nitronyl nitroxide labeling basic and acidic amino acids: synthesis, application for preparing ESR optionally labeling peptides, and bioactivity investigations

Aimed at optional ESR label 2-(4'-hydroxyl)phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl was introduced into the guanido of L-Arg-OH, the omega-amino group of L-Lys-OH with methylcarboxyl as a linker, and into the beta-carboxyl of L-Asp-OH and the gamma-carboxyl of L-Glu-OH with ethylamino as a linker. It was explored that the synthetic 30 novel ESR labeling amino acid derivatives were stable enough to the reaction conditions of peptide synthesis. Their incorporation led to 12 novel ESR optionally labeling PAK, RGDS, RGDV, and ECG. A series of NO related chemical tests, the in vitro and in vivo assays of these peptides confirmed that this strategy was practical.     

Hairpin RNA derived from the gene for Pns9, a viroplasm matrix protein of Rice gall dwarf virus, confers strong resistance to virus infection in transgenic rice plants

The nonstructural Pns9 protein of Rice gall dwarf virus (RGDV) accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in host cells infected by viruses in the family Reoviridae. An RNA interference construct was designed to target the gene for Pns9 of RGDV, namely Trigger_G9. The resultant transgenic plants accumulated short interfering RNAs specific for the construct. All progenies from self-fertilized transgenic plants had strong and heritable resistance to RGDV infection and did not allow the propagation of RGDV. By contrast, our transgenic plants remained susceptible to Rice dwarf virus, another phytoreovirus. There were no significant changes in the morphology of our transgenic plants compared with non-inoculated wild-type rice plants, suggesting that genes critical for the growth of rice plants were unaffected. Our results demonstrate that the resistance to RGDV of our transgenic rice plants is not due to resistance to the vector insects but to specific inhibition of RGDV replication and that the designed trigger sequence is functioning normally. Thus, our strategy to target a gene for viroplasm matrix protein should be applicable to plant viruses that belong to the family Reoviridae.     

Three-dimensional analysis of the association of viral particles with mitochondria during the replication of Rice gall dwarf virus

Examination of cultured insect vector cells that had been infected with Rice gall dwarf virus (RGDV), using transmission electron microscopy and confocal microscopy, revealed the presence of clusters of virus-coated mitochondria around viroplasms in which replication and assembly of RGDV occurred, suggesting a role for mitochondria in supplying the energy required for viral morphogenetic processes. Electron tomography revealed that RGDV particles on the surface of mitochondria are arrayed in an orderly but loose manner, unlike tightly packaged particles in vesicular compartments, suggesting the presence of counterpart molecules on the surface of mitochondria. The viral particles in close proximity to mitochondria were aligned along intermediate filaments, which might serve as scaffolds for the anchorage of these particles. RGDV has a putative mitochondrion-targeting sequence on the outer surface of the outer-capsid protein P8. The arrangement of RGDV particles around mitochondria suggests that the region of the P8 protein containing the mitochondrion-targeting sequence might attach to a molecule like a receptor on the outer mitochondrial membrane. Our analysis demonstrates the three-dimensional arrangement and molecular basis for the mitochondrial proximity of RGDV particles during viral replication.     

Transcapsidation and the conserved interactions of two major structural proteins of a pair of phytoreoviruses confirm the mechanism of assembly of the outer capsid layer

The strongly conserved amino acid sequences of the P8 outer capsid proteins of Rice dwarf virus (RDV) and Rice gall dwarf virus (RGDV) and the distribution of electrostatic potential on the proteins at the interfaces between structural proteins suggested the possibility that P8-trimers of RGDV might bind to the 3-fold symmetrical axes of RDV core particles, with vertical interaction between heterologous P3 and P8 proteins and lateral binding of homologous P8 proteins, thereby allowing formation of the double-layered capsids that are characteristic of viruses that belong to the family Reoviridae. We proved this hypothesis using chimeric virus-like particles composed of the P3 core capsid protein of RDV and the P8 outer capsid protein of RGDV, which were co-expressed in a baculovirus expression system. This is the first report on the molecular biological proof of the mechanism of the assembly of the double-layered capsids with disparate icosahedral lattices.     

水稻瘤矮病毒P8蛋白的结构分析及其表达

为揭示水稻瘤矮病毒(RGDV)外层衣壳蛋白P8的结构特点及其在大肠杆菌中的表达特性。利用生物学软件和在线分析工具,对RGDV P8蛋白结构特征进行了生物信息学分析;同时将通过RT-PCR扩增的P8基因克隆到pGEX-4T-1上,构建pGP8转化大肠杆菌E.coli BL21(DE3),在IPTG诱导下表达。利用表达的融合蛋白免疫家兔,制备了抗血清,并以RGDV、水稻矮缩病毒(RDV)、水稻条纹病毒（RSV）、水稻锯齿叶矮缩病毒(RRSV)4种病毒作为供试材料进行特异性检测。分析显示P8蛋白在C端位置有一个典型的跨膜螺旋区,整个蛋白也具有Phytoreo P8家族共有的典型结构域Phytoreo P8 domain。SDSPAGE和蛋白印记分析表明,RGDV P8融合蛋白分子量约为73kDa,以包涵体的形式获得了高效表达。获得的效价高、特异性强的抗血清,为水稻瘤矮病毒的快速检测及P8蛋白的功能研究奠定了基础。     

Characterization of zinc-alpha(2)-glycoprotein as a cell adhesion molecule that inhibits the proliferation of an oral tumor cell line.

Zn-alpha(2)-glycoprotein (Znalpha(2)gp) is a soluble protein widely distributed in body fluids and glandular epithelia. We have found it to be expressed in stratified epithelia as well. Znalpha(2)gp is clinically correlated with differentiation in various epithelial tumors, including oral and epidermal tumors. We have cloned epidermal Znalpha(2)gp and report the preparation of the recombinant protein in a Baculovirus expression system. Like the native molecule, recombinant Znalpha(2)gp has RNase activity. Znalpha(2)gp functions as a matrix protein for the Tu-138 oral squamous cell carcinoma cell line. Cell attachment to Znalpha(2)gp is comparable to that for fibronectin and is inhibited by the synthetic RGD peptides RGD, RGDV, and RGDS. Attachment is also inhibited by the antibody to integrin alpha(5)beta(1) (the fibronectin receptor), but not by antibodies to integrins alpha(v)beta(3), alpha(3)beta(1), and alpha(2)beta(1). We find that the proliferation of Tu-138 cells is inhibited on a Znalpha(2)gp matrix, as compared with other matrix proteins (fibronectin, vitronectin, laminin, and collagens I and IV) on which growth resembles that on the BSA control. We believe that the role of Znalpha(2)gp in differentiation and its RNase activity are two likely suspects as agents of the inhibition of proliferation.     

Anti-(Arg-Gly-Asp-Ser) antibody and its interaction with fibronectin, fibrinogen and platelets

An antibody population recognizing the sequence Arg-Gly-Asp-Ser (RGDS) in fibronectin, anti-(RGDS)N, was isolated by immunoadsorption. Between 2.5% and 4.9% of antibodies were obtained from two different anti-fibronectin sera indicating that this region represents an antigenic epitope in native fibronectin. Complete inhibition of binding of 125I-fibronectin to anti-(RGDS)N was produced only by nonreduced and reduced fibronectin. Fibrinogen and synthetic RGDS tetrapeptide, each at concentration of 10 microM, showed only a slight inhibition of 22% and 17%, respectively. Measurements of the conformational constant, the equilibrium constant for the interconversion of the non-native and native conformations of this epitope, showed that less than 0.0001% of the RGDS molecules adopt the native conformation in aqueous solutions. It indicates that long-range interactions in fibronectin and fibrinogen result in different conformations of the RGDS sequence in both proteins. Anti-(RGDS)N antibodies purified from anti-fibronectin serum had a strong inhibitory effect on thrombin-stimulated platelet aggregation. They also inhibited binding of fibronectin and fibrinogen to thrombin-stimulated platelets, supporting the primary role of the RGDS sequence in the direct interaction of these proteins with platelet membrane receptors.     

Sequence Analysis of Segment 8 of Five Chinese Isolates of Rice Gall Dwarf Virus and Expression of a Main Outer Capsid Protein in Escherichia coli

The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8(S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%-98.8% and 97.3%-99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%-95.6% and 95.0%-96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b(+) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta(DE3)Ⅱcells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression.     

The effect of glycoprotein IIb-IIIa receptor occupancy on the cytoskeleton of resting and activated platelets

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), serves as the receptor for fibrinogen. This study examined what effect GPIIb-IIIa receptor occupancy had on the cytoskeleton of resting and activated platelets. Triton X-100-insoluble residues (cytoskeletons) were isolated from resting washed platelets incubated with either 500 microM RGDS or 500 microM RGES and examined for protein content. RGDS did not increase the amount of GPIIb-IIIa associated with the cytoskeletal residues which sedimented at either 15,800 x g or 100,000 x g. To determine the effect of receptor occupancy on the formation of the activated platelet cytoskeleton, stirred and nonstirred RGDS-treated platelets in plasma were activated with ADP. Triton X-100-insoluble residues were isolated and examined for both protein content and retention of GPIIb-IIIa. Further, morphological studies were performed on the RGDS-ADP-stimulated platelets. The results of this study suggest that 1) RGDS peptide receptor occupancy does not lead to GPIIb-IIIa linkage to the cytoskeleton, 2) ADP-stimulated platelet shape change, polymerization of actin, and association of myosin with the cytoskeleton are unaffected by RGDS peptide receptor occupancy. 3) RGDS inhibits an aggregation-dependent incorporation of ABP, alpha-actinin, talin, and GPIIb-IIIa into the Triton-insoluble residue.     

Attachment, spreading and locomotion of avian neural crest cells are mediated by multiple adhesion sites on fibronectin molecules.

Cellular adhesion to fibronectin (FN) can be mediated by several sequences located in different portions of the molecule. In human FN, these are: (i) the bipartite RGDS domain containing the RGDS cell-binding sequence functioning in synergy for full cellular adhesion with a second site (termed here the synergistic adhesion site) and (ii) the recently characterized CS1 and REDV adhesion sites within the alternatively-spliced type III homology-connecting segment. Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in the adhesion, spreading, and motility of avian neural crest cells in vitro. Both the RGDS domain and the CS1 adhesion site were found to promote attachment of neural crest cells, but only the RGDS domain supported their spreading. However, the RGDS sequence could mediate both attachment and spreading efficiently only when it was associated with the synergistic adhesion site. In migratory assays, it was found that both the RGDS domain and the CS1 site are required in association, each with functional specificity, to permit effective locomotion of neural crest cells. The REDV adhesion site was apparently not recognized by avian neural crest cells, presumably because this sequence is absent from chicken FN. Finally, it was found that recognition of both the RGDS domain and CS1 binding site by neural crest cells involved receptors belonging to the integrin family. From these results, we conclude that neural crest cells can interact with several binding sites of FN molecules, and use them for distinct functions. Our results also suggest the possibility of an instructive role for FN in the control of adhesive and migratory events during embryonic development.     

The fibrinogen-derived peptide (RGDS) prevents proteolytic degradation of protein kinase C in platelets by inhibiting platelet aggregation

The effects of the fibrinogen-derived tetrapeptide, Arg-Gly-Asp-Ser (RGDS), on platelet activation processes was studied. At concentrations of 100-300 microM, RGDS completely prevented platelet aggregation induced by all the common platelet agonists, 'weak' and 'strong'. In agreement with earlier views on the aggregation-dependency of weak agonist-induced thromboxane synthesis and 5-hydroxytryptamine (5HT) secretion, RGDS (100-300 microM) inhibited these events induced by ADP, adrenaline and low concentrations of thrombin and collagen but not that induced by high concentrations of thrombin and collagen. 5HT secretion induced by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), was also not affected by RGDS, but proteolytic degradation of the translocated membrane-bound enzyme in PMA-treated platelets, due to the actions of the Ca2+-dependent protease (Ca-DP), was completely prevented such that in the presence of RGDS, sustained increases in membrane-bound PKC activity were observed. PMA alone caused only transient increases in membrane-bound PKC. This effect of RGDS was similar to the effect of E64-d, a recently described inhibitor of Ca-DP in platelets, or the effects seen with PMA in unstirred non-aggregating platelets. It is concluded that RGDS inhibits the actions of Ca-DP in platelets via inhibition of aggregation.     

RGD-containing peptides inhibit fibrinogen binding to platelet alpha(IIb)beta3 by inducing an allosteric change in the amino-terminal portion of alpha(IIb)

To determine the molecular basis for the insensitivity of rat alpha(IIb)beta(3) to inhibition by RGD-containing peptides, hybrids of human and rat alpha(IIb)beta(3) and chimeras of alpha(IIb)beta(3) in which alpha(IIb) was composed of portions of human and rat alpha(IIb) were expressed in Chinese hamster ovary cells and B lymphocytes, and the ability of the tetrapeptide RGDS to inhibit fibrinogen binding to the various forms of alpha(IIb)beta(3) was measured. These measurements indicated that sequences regulating the sensitivity of alpha(IIb)beta(3) to RGDS are located in the seven amino-terminal repeats of alpha(IIb). Moreover, replacing the first three or four (but not the first two) repeats of rat alpha(IIb) with the corresponding human sequences enhanced sensitivity to RGDS, whereas replacing the first two or three repeats of human alpha(IIb) with the corresponding rat sequences had little or no effect. Nevertheless, RGDS bound to Chinese hamster ovary cells expressing alpha(IIb)beta(3) regardless whether the alpha(IIb) in the heterodimers was human, rat, or a rat-human chimera. These results indicate that the sequences determining the sensitivity of alpha(IIb)beta(3) to RGD-containing peptides are located in the third and fourth amino-terminal repeats of alpha(IIb). Because RGDS binds to both human and rat alpha(IIb)beta(3), the results suggest that differences in RGDS sensitivity result from differences in the allosteric changes induced in these repeats following RGDS binding.