Epitope mapping by cDNA expression of a monoclonal antibody which inhibits the binding of von Willebrand factor to platelet glycoprotein IIb/IIIa.
ABSTRACT: In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9). This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment [amino acids (aa) 1366-2050], which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase. Immunological screening of the library with 125I-MAb 9 identified three immunoreactive clones. vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9. vWF-beta-galactosidase fusion protein reacted with 125I-MAb 9 by Western blotting. In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to 125I-MAb 9 by 50%, and this inhibition was dose-dependent between 3.5 and 120 nM. Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.
Project description:A binding domain for Factor VIII (F.VIII) has been previously identified on the N-terminal portion of human von Willebrand Factor (vWF) subunit [amino acids (AA) 1-272]. In order to characterize other possible structures of vWF involved in its capacity to bind and to protect F.VIII against human activated protein C (APC), we used a series of purified vWF fragments overlapping the whole sequence of the subunit. Among those were fragments SpIII (dimer; AA 1-1365), SpII (dimer; AA 1366-2050) and SpI (monomer; AA 911-1365) generated by Staphylococcus aureus V8 proteinase, a P34 species (monomer; AA 1-272) obtained with plasmin, a monomeric 39/34 kDa dispase fragment (AA 480-718) and a tetrameric III-T2 fragment (AA 273-511/674-728) produced from SpIII by trypsin. Three other fragments without precise extremities were located using selected monoclonal antibodies to vWF. Two C-terminal fragments of 270 and 260 kDa, overlapping SpI and SpII, were respectively generated from vWF with trypsin and protease 1 from Crotalus atrox venom. An N-terminal 120 kDa fragment, overlapping P34 and 39/34 kDa fragments, was produced by protease 1. Our results show that vWF bound to F.VIII and protected it from degradation by APC in a dose-dependent way. Among the C-terminal and central vWF fragments (SpII, tryptic 270 kDa, 260 kDa, SpI, 39/34 kDa and III-T2), none had the capacity to bind or to protect F.VIII, even at high concentrations. The three N-terminal fragments (SpIII, 120 kDa and P34) bound to F.VIII in a dose-dependent and saturable fashion. SpIII and the 120 kDa fragment had the capacity to protect F.VIII in a dose-dependent way. In contrast, the P34 species did not significantly protect F.VIII, even when using high concentrations of the fragment. In conclusion, the N-terminal end of vWF subunit (AA 1-272) plays a crucial role in binding to F.VIII, but requires additional structures of the 120 kDa fragment to protect it against APC. In addition, the presence of a secondary binding and/or protecting domain on other portions of the vWF subunit (potentially destroyed during the proteolysis of vWF) is highly unlikely.
Project description:It has been proposed that the receptor for von Willebrand factor (vWF), glycoprotein (GP)Ib-IX-V, signals through the same pathway as the collagen receptor, GPVI, namely via Src kinases, the Fc receptor (FcR) gamma-chain and Syk, leading to tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2). The aim of the present study was to assess the functional significance of this pathway in platelet activation by GPIb-IX-V. In washed platelets, vWF/ristocetin and vWF/botrocetin stimulate weak tyrosine phosphorylation of the FcR gamma-chain, Syk and PLCgamma2, but not the adaptor LAT (linker for activation of T-cells), which is localized to glycolipid-enriched membrane domains. Increases in tyrosine phosphorylation were blocked by the Src family kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1). Under the same conditions, neither stimulus induced activation of PLCgamma2 nor functional responses, such as Ca(2+) elevation, secretion or GPIIb-IIIa-dependent aggregation. In contrast, in platelet-rich plasma (PRP), threshold concentrations of ristocetin or asialo-vWF stimulated GPIb-dependent biphasic aggregation, in which the second phase was blocked by PP1. Importantly, a significant component of the initial phase and the complete second phase of aggregation was blocked by GPIIb-IIIa receptor antagonists in PRP. Higher concentrations of ristocetin stimulated GPIIb-IIIa-independent agglutination in PRP. These results demonstrate that GPIb-IX-V initiates activation of GPIIb-IIIa in PRP through an undefined pathway that is reinforced by a PP1-sensitive pathway. In contrast, activation of GPIbalpha in washed platelets does not promote functional responses.
Project description:Arg-Gly-Asp (RGD) is an amino acid sequence in fibrinogen recognized by platelet glycoprotein (GP) IIb/IIIa. Recently, it was found that RGD peptide binding to GPIIb/IIIa leads to conformational changes in the complex that are associated with the acquisition of high-affinity fibrinogen-binding function. In this study, we found that tetrafibricin, a novel non-peptidic GPIIb/IIIa antagonist, induced similar conformational changes in GPIIb/IIIa as did RGD peptides. Tetrafibricin increased the binding of purified inactive GPIIb/IIIa to immobilized pl-80, a monoclonal antibody that preferentially recognizes ligand-occupied GPIIb/IIIa. Exposure of the pl-80 epitope by tetrafibricin was also observed on resting human platelets by flow cytometry. On intact platelets, the conformational changes transformed GPIIb/IIIa into a high-affinity receptor for fibrinogen and triggered subsequent platelet aggregation. Tetrafibricin is the first non-peptidic GPIIb/IIIa antagonist reported that has the capacity to induce conformational changes in GPIIb/IIIa.
Project description:BACKGROUND: The role of genetic risk factors in ischemic stroke is unclear. Platelet glycoprotein IIb/IIIa (GpIIb-IIIa) has been implicated in the pathogenesis of ischemic stroke. We sought to evaluate the relationship between the GpIIb/IIIa complex gene polymorphism and ischemic stroke. MATERIAL/METHODS: We investigated the association of the GpIIb/IIIa complex gene polymorphism with stroke risk in 306 patients with acute ischemic stroke and 266 control subjects by determining the GpIIb and GpIIIa genotype from leukocyte DNA by polymerase chain reaction (PCR) followed by FokI and ScrFI digestion, respectively. RESULTS: Compared with controls, more patients presented with coronary heart disease, hypertension, smoking history, and diabetes. In addition, the patients had higher levels of cholesterol and glucose compared with the control subjects. All donors in the GpIIIa (n=572) group expressed the GpIIIa PlA1 (HPA-1 aa) phenotype. There were no significant differences between the HPA-3 genotype (GpIIb) patient distribution (aa=39.9%, ab=41.4%, bb=28.7%) and healthy control subjects (aa=36.1%, ab=35.0%, bb=28.9%) (P=0.580). Among study participants <60 years, there was a significant difference in the HPA-3 genotype distributions of patients (aa=42.9%, ab=19.8%, bb=37.4%) and healthy control subjects (aa=43.3%, ab=38.8%, bb=17.9%) (P=0.007). Furthermore, HPA-3 b/b increased the risk of ischemic stroke >2-fold (P=0.008). CONCLUSIONS: The GpIIb Ile/Ser843 gene polymorphism is associated with ischemic stroke among young and middle-aged adults (<60 years), especially males. The GpIIIa PlA1 phenotype has no relationship to ischemic stroke.
Project description:Glycoprotein IIb (GPIIb) is a major glycoprotein of the human platelet plasma membrane, which together with glycoprotein IIIa (GPIIIa) forms a Ca2(+)-dependent heterodimer, GPIIb/IIIa, which serves as the major fibrinogen receptor in activated platelets. The precise localization of the epitopes for six anti-GPIIb monoclonal antibodies (M1-M6) has been determined by a combination of enzymic and chemical cleavage procedures, peptide isolation, N-terminal sequence analysis, peptide synthesis and enzyme immunoassay. The following localizations were found: M1, beta 1-16-36, beta 2-4-24; M2, alpha 747-755; M alpha 2, alpha 837-843; M3, alpha 849-857; M4, alpha 143-151; M5, alpha 550-558; M6, alpha 657-665. Besides considerations of the degree of exposure of these epitopes, several remarkable features are readily apparent. The earliest and main chymotryptic cleavage site of GPIIb in whole platelets is between alpha cysteine-545 and alpha phenylalanine-551. The epitope for M3 was located within the same sequence (alpha 842-857) as is the epitope for PMI-1 [Loftus, Plow, Frelinger, D'Souza, Dixon, Lacy, Sorge & Ginsberg (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7114-7118] in spite of the fact that the exposure of the latter in whole platelets is EDTA-dependent whereas that in the former is not. The epitope for M5 shares full homology with the 540-548 peptide stretch of the alpha-subunit of the vitronectin receptor, and this antibody cross-reacts with endothelial cells. The M6 epitope is located in the 25 kDa membrane-bound fragment of GPIIb, which is most epitope is destroyed at an early stage of chymotrypic digestion. This suggests that this region of GPIIb, somewhere between the epitope for M5 (alpha 550-558) and the epitope for M2 (alpha 747-755), may carry the surface of interaction of GPIIb with GPIIIa in the GPIIb/IIIa heterodimer. Finally, the sequence where the epitope for M6 has been located (alpha 657-667) was the only one found to be hydropathically complementary to the gamma 402-411 peptide of fibrinogen within the amino acid sequence of both GPIIb and GPIIIa. This complementariness, the EDTA- or thrombin-dependence of the exposure of the alpha 657-665 stretch in whole platelets to M6 and the ability of this antibody to inhibit platelet aggregation led us to postulate that this peptide stretch is a putative binding site for fibrinogen in the platelet receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
Project description:<b>Rationale</b>: Platelets are increasingly recognized as mediators of tumor growth and metastasis. Hypothesizing that activated platelets in the tumor microenvironment provide a targeting epitope for tumor-directed chemotherapy, we developed an antibody-drug conjugate (ADC), comprised of a single-chain antibody (scFv) against the platelet integrin GPIIb/IIIa (scFv<sub>GPIIb/IIIa</sub>) linked to the potent chemotherapeutic microtubule inhibitor, monomethyl auristatin E (MMAE). <b>Methods</b>: We developed an ADC comprised of three components: 1) A scFv which specifically binds to the high affinity, activated integrin GPIIb/IIIa on activated platelets. 2) A highly potent microtubule inhibitor, monomethyl auristatin E. 3) A drug activation/release mechanism using a linker cleavable by cathepsin B, which we demonstrate to be abundant in the tumor microenvironment. The scFv<sub>GPIIb/IIIa</sub>-MMAE was first conjugated with Cyanine7 for <i>in vivo</i> imaging. The therapeutic efficacy of the scFv<sub>GPIIb/IIIa</sub>-MMAE was then tested in a mouse metastasis model of triple negative breast cancer. <b>Results</b>: <i>In vitro</i> studies confirmed that this ADC specifically binds to activated GPIIb/IIIa, and cathepsin B-mediated drug release/activation resulted in tumor cytotoxicity. <i>In vivo</i> fluorescence imaging demonstrated that the newly generated ADC localized to primary tumors and metastases in a mouse xenograft model of triple negative breast cancer, a difficult to treat tumor for which a selective tumor-targeting therapy remains to be clinically established. Importantly, we demonstrated that the scFv<sub>GPIIb/IIIa</sub>-MMAE displays marked efficacy as an anti-cancer agent, reducing tumor growth and preventing metastatic disease, without any discernible toxic effects. <b>Conclusion</b>: Here, we demonstrate the utility of a novel ADC that targets a potent cytotoxic drug to activated platelets and specifically releases the cytotoxic agent within the confines of the tumor. This unique targeting mechanism, specific to the tumor microenvironment, holds promise as a novel therapeutic approach for the treatment of a broad range of primary tumors and metastatic disease, particularly for tumors that lack specific molecular epitopes for drug targeting.
Project description:1. The pharmacological characteristics of solid-phase von Willebrand factor (svWF), a novel platelet agonist, were studied. 2. Washed platelet suspensions were obtained from human blood and the effects of svWF on platelets were measured using aggregometry, phase-contrast microscopy, flow cytometry and zymography. 3. Incubation of platelets with svWF (0.2 - 1.2 microg ml(-1)) resulted in their adhesion to the ligand, while co-incubations of svWF with subthreshold concentrations of ADP, collagen and thrombin resulted in aggregation. 4. 6B4 inhibitory anti-glycoprotein (GP)Ib antibodies abolished platelet adhesion stimulated by svWF, while aggregation was reduced in the presence of 6B4 and N-Acetyl-Pen-Arg-Gly-Asp-Cys, an antagonist of GPIIb/IIIa. 5. Platelet adhesion stimulated with svWF was associated with a concentration-dependent increase in expression of GPIb, but not of GPIIb/IIIa. 6. In contrast, collagen (0.5 - 10.0 microg ml(-1)) caused down-regulation of GPIb and up-regulation of GPIIb/IIIa in platelets. 7. Solid-phase vWF (1.2 microg ml(-1)) resulted in the release of MMP-2 from platelets. 8. Inhibition of MMP-2 with phenanthroline (10 microM), but not with aspirin or apyrase, inhibited platelet adhesion stimulated with svWF. 9. In contrast, human recombinant MMP-2 potentiated both the effects of svWF on adhesion and up-regulation of GPIb. 10. Platelet adhesion and aggregation stimulated with svWF were reduced by S-nitroso-n-acetyl-penicillamine, an NO donor, and prostacyclin. 11. Thus, stimulation of human platelets with svWF leads to adhesion and aggregation that are mediated via activation of GPIb and GPIIb/IIIa, respectively. 12. Mechanisms of activation of GPIb by svWF involve the release of MMP-2, and are regulated by NO and prostacyclin.
Project description:<h4>Background</h4>We previously described a mouse model in which platelet immunization between selected strains leads to production of alloantibodies and severe autoimmune thrombocytopenia and mimics the human condition posttransfusion purpura (PTP). This report describes studies defining epitopes recognized by these alloantibodies.<h4>Study design</h4>Hybridomas were produced from spleen cells of immunized mice. Glycoprotein (GP) targets of resulting monoclonal antibodies were characterized by immunoprecipitation using platelets from the immunizing strains. Antigens defined by single amino acid (AA) polymorphisms recognized by monoclonal antibodies were identified by mutagenizing target glycoproteins expressed in Chinese hamster ovary cells and observing the effects on antibody binding.<h4>Results</h4>Three monoclonal antibodies (417.1, 417.3, 425.1) were produced that recognized GPIIb on immunizing platelets. Monoclonal antibodies 417.1 and 417.3 both required G111 and 425.1 required V37, located on the beta propeller domain of GPIIb, for binding to platelets from the immunizing strains C57 and PWK, respectively. Injection of 417.3 and 425.1 into mice caused platelet destruction only in mice with GPIIb containing the targeted AAs.<h4>Conclusions</h4>Findings made provide evidence that alloantibodies produced by mice experiencing thrombocytopenia in a mouse model of PTP are specific for single AA polymorphisms that differ in GPIIb/IIIa integrin of the immunizing and immunized strains and therefore closely resemble the potent alloantibodies found in patients with PTP. The observations show that naturally occurring single AA differences in GPIIb/IIIa integrin of various mouse strains are highly immunogenic in the mouse strains studied and readily induce antibodies comparable to human platelet antigen-specific antibodies found in transfused and pregnant humans.
Project description:The interaction of human thrombospondin (TSP) with GPIa-IIa and GPIIb-IIIa was studied. The binding for both proteins became time-independent after 60 min. A 7-fold excess concentration of unlabelled GPIa-IIa added either initially, or after time-dependent binding, resulted in a 50% inhibition of GPIa-IIa bound to TSP. GPIa-IIa and GPIIb-IIIa specifically bound TSP since: (a) the binding of GPIIb-IIIa to TSP was dependent on the presence of 1 mM MgCl2 and 1 mM CaCl2, whereas binding of GPIa-IIa was ion-independent. (b) The binding was saturable, with dissociation constants of 0.69 +/- 0.17 microM and 3.77 +/- 1.02 microM for GPIa-IIa and GPIIb-IIIa respectively. (c) GPIIb-IIIa and GPIa-IIa did not significantly bind to BSA. (d) GPIIb-IIIa bound fibrinogen ion-specifically, whereas little or no binding of GPIa-IIa was detectable. (e) Both GPIIb-IIIa and GPIa-IIa bound collagen in an ion-independent manner. (f) GPIIb-IIIa did not compete with GPIa-IIa for binding to TSP. (g) Binding of GPIa-IIa to TSP was inhibited with anti-(GPIa-IIa) (6F1), whereas mouse IgG and anti-(GPIIb-IIIa) (AP-2) had no effect. (h) The interaction of GPIa-IIa with TSP is 5.5-fold more favourable than that of GPIIb-IIIa suggesting that GPIa-IIa may be a preferred binding protein for TSP-mediated platelet adhesion.
Project description:A mutual relationship exists between metastasizing tumor cells and components of the coagulation cascade. The exact mechanisms as to how platelets influence blood-borne metastasis, however, remain poorly understood. Here, we used murine B16 melanoma cells to observe functional aspects of how platelets contribute to the process of hematogenous metastasis. We found that platelets interfere with a distinct step of the metastasis cascade, as they promote adhesion of melanoma cells to the endothelium in vitro under shear conditions. Constitutively active platelet receptor GPIIb/IIIa (integrin ?IIb?3) expressed on Chinese hamster ovary cells promoted melanoma cell adhesion in the presence of fibrinogen, whereas blocking antibodies to a??3 integrin on melanoma cells or to GPIIb/IIIa significantly reduced melanoma cell adhesion to platelets. Furthermore, using intravital microscopy, we observed functional platelet-melanoma cell interactions, as platelet depletion resulted in significantly reduced melanoma cell adhesion to the injured vascular wall in vivo. Using a mouse model of hematogenous metastasis to the lung, we observed decreased metastasis of B16 melanoma cells to the lung by treatment with a mAb blocking the a? subunit of a??3 integrin. This effect was significantly reduced when platelets were depleted in vivo. Thus, the engagement of GPIIb/IIIa with a??3 integrin interaction mediates tumor cell-platelet interactions and highlights how this interaction is involved in hematogenous tumor metastasis.