The use of collagen-based model peptides to investigate platelet-reactive sequences in collagen

Simple collagen-like peptides comprising a repeat Gly-Pro-Hyp sequence are highly platelet-reactive when presented to platelets in triple-helical and polymeric form. This activity is not mediated by the platelet collagen receptor integrin alpha 2 beta 1. This may imply the existence of an intrinsic platelet reactivity associated with the collagen triple helix as such or perhaps that the Gly-Pro-Hyp sequence in collagen serves as a specific cell-recognition site. In our view this basic alpha 2 beta 1-independent reactivity is modulated by the presence in collagen of sequences that may either enhance or diminish the interaction with platelets. Inhibition studies with short linear peptides have allowed the tentative identification of sequences in collagen such as XPGEP(Q)GPX and D(N)GE(Q)X that may promote the activation of platelets and so enhance collagen-platelet interaction. Sequences serving as integrin alpha 2 beta 1-binding sites may also promote platelet reactivity by permitting interaction with the collagen receptor. Using triple-helical peptides based on the sequence of the platelet-reactive collagen type III fragment alpha 1(III)CB4, we have been able to locate an alpha 2 beta 1-binding site in collagen type III within a 30-mer sequence representing residues 508-537 of the alpha 1(III) constituent alpha-chain. Despite their alpha 2 beta 1-independent platelet reactivity, signalling by the (Gly-Pro-Hyp)n-based peptides shows many features in common with signalling by collagen fibers, including activation of p72SYK and p125FAK the latter of which has until now been considered a specific consequence of ligand binding to alpha 2 beta 1.     

Identification in collagen type I of an integrin alpha2 beta1-binding site containing an essential GER sequence

The collagen type I-derived fragment alpha1(I)CB3 is known to recognize the platelet collagen receptor integrin alpha2beta1 as effectively as the parent collagen, although it lacks platelet-aggregatory activity. We have synthesized the fragment as seven overlapping peptides that spontaneously assemble into triple helices. On the basis of their capacity to bind purified alpha2 beta1 and the recombinant alpha2 A-domain, and their ability to support alpha2 beta1-mediated cell adhesion, we identified two peptides, CB3(I)-5 and -6, which contain an alpha2 beta1 recognition site. Synthesis of the peptide CB3(I)-5/6, containing the overlap sequence between peptides 5 and 6, allowed us to locate the binding site within the 15-residue sequence, GFP*GERGVEGPP*GPA (where P* represents hydroxyproline), corresponding to residues 502-516 of the collagen type I alpha1 chain. The Glu and Arg residues in the GER triplet were found to be essential for recognition since substitution of either residue with Ala caused a loss of alpha2 A-domain binding. By contrast, substitution of the Glu in GVE did not reduce binding, but rather enhanced it slightly. We were unable to detect significant recognition of alpha2 beta1 by the peptide CB3(I)-2 containing the putative alpha2 beta1 recognition sequence DGEA. Peptides CB3(I)-1 to -6, together with peptide CB3(I)-5/6, exhibited good platelet-aggregatory activity, in some cases better than collagen. However, peptide CB3(I)-7 was inactive, suggesting the presence of an inhibitory element that might account for the lack of aggregatory activity of the parent alpha1(I)CB3 fragment.     

Activation of the fibrinogen binding site on platelets isolated from a patient with the Strasbourg I variant of Glanzmann's thrombasthenia

One proposed ligand binding site on platelet integrin alpha IIb beta 3 is the region of the beta 3 subunit encompassing amino acids 211-221. However, we recently showed that synthetic peptides corresponding to amino acids 211-221 inhibit fibrinogen binding to alpha IIb beta 3 by binding to alpha IIb beta 3 and not to fibrinogen. In this study, we show that AP6, a monoclonal antibody (MoAb) directed against amino acids 214-221 of beta 3, bound to immobilized active alpha IIb beta 3 but did not inhibit fibrinogen binding to the complex. We then determined whether nonfunctional alpha IIb beta 3 on platelets with a beta 3 Arg-214-->Trp mutation (Strasbourg I variant of Glanzmann's thrombasthenia or GTV) could be induced to aggregate after treatment with dithiothreitol (DTT). DTT has been shown to expose the fibrinogen receptor on normal platelets. DTT treatment of GTV platelets did result in the formation of the fibrinogen binding site as indicated by the binding of pI-55, an MoAb that only binds to the activated form of alpha IIb beta 3. Furthermore, DTT-treated GTV platelets aggregated in the presence of fibrinogen and divalent cations. This aggregation was inhibited by EDTA, RGDS, and the selective alpha IIb beta 3 antagonist, Ro 43-5054. These data show that Arg-214 of beta 3 is not required for fibrinogen binding or for platelet aggregation. However, this amino acid appears to be critical for the formation and for the maintenance of the correct tertiary structure of the fibrinogen binding site on alpha IIb beta 3.     

Staphylococcus aureus induces platelet aggregation via a fibrinogen-dependent mechanism which is independent of principal platelet glycoprotein IIb/IIIa fibrinogen-binding domains

Platelet aggregation by bacteria is felt to play an important role in the pathogenesis of infective endocarditis. However, the mechanisms involved in bacterium-induced platelet aggregation are not well-defined. In the present study, we examined the mechanisms by which Staphylococcus aureus causes rabbit platelet aggregation in vitro. In normal plasma, the kinetics of S. aureus-induced platelet aggregation were rapid and biphasic. The onset and magnitude of aggregation phase 1 varied with the bacterium-platelet ratio, with maximal aggregation observed at a ratio of 5:1. The onset of aggregation phase 2 was delayed in the presence of apyrase (an ADP hydrolase), suggesting that this later aggregation phase may be triggered by secreted ADP. The onset of aggregation phase 2 was delayed in the presence of prostaglandin I2-treated platelets, and this phase was absent when paraformaldehyde-fixed platelets were used, implicating platelet activation in this process. Platelet aggregation phase 2 was dependent on S. aureus viability and an intact bacterial cell wall, and it was mitigated by antibody directed against staphylococcal clumping factor (a fibrinogen-binding protein) and by the cyclooxygenase inhibitor indomethacin. Similarly, aggregation phase 2 was either delayed or absent in three distinct transposon-induced S. aureus mutants with reduced capacities to bind fibrinogen in vitro. In addition, a synthetic pentadecapeptide, corresponding to the staphylococcal binding domain in the C terminus of the fibrinogen delta-chain, blocked aggregation phase 2. However, phase 2 of aggregation was not inhibited by two synthetic peptides (alone or in combination) analogous to the two principal fibrinogen-binding domains on the platelet glycoprotein (GP) IIb/IIIa integrin receptor: (i) a recognition site on the IIIa molecule for the Arg-Gly-Asp (RGD) sequence of the fibrinogen alpha-chain and (ii) a recognition site on the IIb molecule for a dodecapeptide sequence of the fibrinogen delta-chain. This differs from ADP-induced platelet aggregation, which relies on an intact platelet GP IIb/IIIa receptor with an accessible RGD sequence and dodecapeptide recognition site for fibrinogen. Furthermore, a monoclonal antibody directed against the RGD recognition site on rabbit platelet GP IIb/IIIa receptors failed to inhibit rabbit platelet aggregation by S. aureus. Collectively, these data suggest that S. aureus-induced platelet aggregation requires bacterial binding to fibrinogen but is not principally dependent upon the two major fibrinogen-binding domains on the platelet GP IIb/IIIa integrin receptor, the RGD and dodecapeptide recognition sites.     

Alternative splicing gives rise to two novel long isoforms of Zn-alpha 2-glycoprotein, a member of the immunoglobulin superfamily

The integrin alpha IIb beta 3 (GPIIb/IIIa) mediates platelet aggregation by a change in affinity for the ligand fibrinogen. The amino acids 991-995 (GFFKR) at the NH2-terminus of the cytoplasmic domain are highly conserved in all known integrin alpha subunits. We postulated that the GFFKR-region is important for the inside-out signal transduction and has an influence on the affinity state of integrins. To test this hypothesis, a mutant with a deletion in the GFFKR region was designed. The DNA-constructs were constructed by PCR, sequenced, cotransfected with the beta 3 subunit into CHO cells and cell surface expression was proven with immunoprecipitation and flow cytometry. The GFFKR-deletion mutant demonstrated a high affinity binding of the mAb PAC-1 and I125-labeled fibrinogen. The metabolic inhibitors 2-deoxyglucose and NaN3 did not change the affinity state of the deleted receptor. Neither did the truncation of the cytoplasmic domain of the beta 3 subunit. Additionally, expression of the deleted integrin in the erythropoetic cell line K562 revealed a high affinity state. A deletion of the GFFKR-region in the cytoplasmic domain of the alpha subunit locks integrin alpha IIb beta 3 in a high affinity state. This is an intrinsic property of the deleted receptor since there is no energy dependence and no cell type specifity. Thus, the GFFKR-region is involved in inside-out signaling in alpha IIb beta 3. Furthermore, cell lines expressing this activated alpha IIb beta 3 integrin may be used as models for activated platelets.     

Increased platelet sensitivity toward platelet inhibitors during physical exercise in patients with coronary artery disease

Generalized atherosclerosis and coronary artery disease (CAD) are associated with endothelial dysfunction and during acute myocardial ischemia platelet activation has been reported. Activated platelets exert activated fibrinogen receptors (GP IIb/IIIa) and express CD 62p being regarded as reliable marker for platelet activation. Patients with angiographically proven CAD performed a bicycle exercise test until the onset of angina or ST-segment depression. We studied the ischemia-induced alterations in fibrinogen binding to activated platelet GP IIb/IIIa receptors and CD 62p expression. Therefore, the basal fibrinogen binding to GP IIb/IIIa and CD 62p expression and the thrombin-concentration for half-maximal platelet activation before and after exercise testing were determined. Additionally, inhibition of thrombin-induced platelet activation by increasing concentrations of the prostacyclin-analog iloprost and the NO-donor SIN-1 was examined. In patients with CAD, a significantly reduced basal activation and a highly significant reduction in sensitivity towards thrombin was measured. The thrombin-induced expression of GP IIb/IIIa and CD 62p was significantly diminished in patients with CAD after physical exercise and their platelets were significantly more sensitive towards the inhibitory effects of iloprost and SIN-1. These data demonstrate a significant reduction in platelet activation in response to physical exercise in patients with CAD and advanced atherosclerosis. Despite exercise induced myocardial ischemia as evidenced by angina and ECG-changes, the platelets are not generally activated, as it could be expected. Thus, patients with myocardial ischemia experienced a reduced platelet activity and enhanced sensitivity towards prostacyclin (PGI2) and nitric oxide, probably due to an augmented release of endogenous platelet inhibitory mediators.     

Identification of a novel recognition sequence for integrin alphaM beta2 within the gamma-chain of fibrinogen

The interaction of leukocyte integrin alphaMbeta2 (CD11b/CD18, Mac-1) with fibrinogen has been implicated in the inflammatory response by contributing to leukocyte adhesion to the endothelium and subsequent transmigration. Previously, it has been demonstrated that a peptide, P1, corresponding to residues 190-202 in the gamma-chain of fibrinogen, binds to alphaM beta2 and blocks the interaction of fibrinogen with the receptor and that Asp199 within P1 is important to activity. We have demonstrated, however, that a double mutation of Asp199-Gly200 to Gly-Ala in the recombinant gamma-module of fibrinogen, spanning region 148-411, did not abrogate alphaM beta2 recognition and considered that other binding sites in the gamma-module may participate in the receptor recognition. We have found that synthetic peptide P2, duplicating gamma377-395, inhibited adhesion of alphaM beta2-transfected cells to immobilized D100 fragment of fibrinogen in a dose-dependent manner. In addition, immobilized P2 directly supported efficient adhesion of the alphaM beta2-expressing cells, including activated and non-activated monocytoid cells. The I domain of alphaM beta2 was implicated in recognition of P2, as the biotinylated recombinant alphaMI domain specifically bound to both P2 and P1 peptides. Analysis of overlapping peptides spanning P2 demonstrated that it may contain two functional sequences: gamma377-386 (P2-N) and gamma383-395 (P2-C), with the latter sequence being more active. In the three-dimensional structure of the gamma-module, gamma190-202 and gamma377-395 reside in close proximity, forming two antiparallel beta strands. The juxtapositioning of these two sequences may form an unique and complex binding site for alphaM beta2.     

Further characterization of the thrombasthenia-related idiotype OG. Antiidiotype defines a novel epitope(s) shared by fibrinogen B beta chain, vitronectin, and von Willebrand factor and required for binding to beta 3

A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion after the production of an immunoglobulin G (IgG) isoantibody (Ab1) specific for the integrin subunit beta 3. To determine the frequency at which the OG idiotype is found in the general population and in immune-mediated disease states, we developed a rabbit polyclonal antibody (Ab2) specific for affinity-purified OG anti-beta 3 Fab. The binding of Ab2 to Ab1 is inhibited by purified alpha IIb beta 3. Ab2 als binds to IgG specific for alpha IIb beta 3 obtained from one nonrelated Glanzmann thrombasthenia patient ES who has developed isoantibodies of similar specificity. On the other hand, Ab2 does not recognize alpha IIb beta 3-specific antibodies produced by two Glanzmann thrombasthenia patients, AF and LUC, who have developed isoantibodies with specificities distinct from that of the OG isoantibody. Moreover, Ab2 does not recognize alpha IIb beta 3-specific antibodies developed by three representative patients with (autoimmune) thrombocytopenic purpura or six representative patients with alloimmune thrombocytopenias, nor does it bind to IgG from any of 13 nonimmunized individuals. We have found that Ab2 also binds to selected protein ligands of alpha IIb beta 3 namely, fibrinogen, vitronectin, and von Willebrand factor, but not to other protein ligands or control proteins, such a fibronectin, type I collagen, and albumin. The epitope(s) recognized by Ab2 on each adhesive protein are either very similar or identical since each protein can inhibit the binding of Ab2 to any of the other proteins. The epitope on fibrinogen recognized by Ab2 resides in the B beta chain, and is likely contained within the first 42 amino acids from the NH2 terminus. Since OG IgG inhibits fibrinogen binding to alpha IIb beta 3, the specificity of the OG idiotype defines a novel binding motif for the integrin alpha IIb beta 3 that is shared by fibrinogen, vitronectin, and von Willebrand factor, but distinct from previously described RGD-containing sites on the fibrinogen, A alpha chain or the fibrinogen gamma chain COOH-terminal decapeptide site. Our findings reported here represent an excellent example of molecular mimicry in which an antigen-selected, IgG inhibitor of alpha IIb beta 3 function shares a novel recognition sequence common to three physiologic protein ligands of that receptor.     

SC-49992--a potent and specific inhibitor of platelet aggregation

Fibrinogen binding is required for platelet aggregation and subsequent thrombus formation. SC-49992 (SC), an RGDF mimetic, is a potent and specific inhibitor of the binding of fibrinogen to its receptor on activated platelets, glycoprotein IIb/IIIa (IC50 0.7 microM). SC was more potent (1-5 microM) than either RGDS, RGDF or the gamma chain dodecapeptide in blocking platelet aggregation to a variety of agonists in both dog and human platelet rich plasma. SC was more potent as an inhibitor of GP IIb/IIIa on platelets than it was against other integrin and non-integrin receptors, including the RGD-dependent vitronectin receptor and other non-RGD-dependent integrins such as CDII/CD18. SC had little effect on ristocetin induced agglutination. SC blocked ex vivo collagen induced aggregation in dogs and collagen induced thrombocytopenia in rats. These data suggest that elimination of the Arg-NH2 and the Arg-Gly amide bond of RGDF provided increased inhibitory potency and specificity. This structural modification may be of value in the development of other more potent RGDF mimetics for the inhibition of platelet aggregation.     

alpha IIb beta 3 redistribution triggered by receptor cross-linking

Fibrinogen binding to alpha IIb beta 3 on adherent, spread platelets triggers active, cytoskeletally-directed redistribution of fibrinogen/alpha IIb beta 3 complexes on the platelet surface. Gold-conjugated fibrinogen, unlabeled, soluble fibrinogen, and individual fibrinogen molecules have been demonstrated to trigger receptor redistribution. Here we examine the respective roles of receptor cross-linking and ligand occupancy of receptors in initiating this movement. Monovalent, alpha IIb beta 3-binding fibrinogen fragments RGDS and HHLGGAKQAGDV did not trigger receptor redistribution, suggesting that ligand binding to a single receptor is an insufficient stimulus. Binding of monoclonal antibodies 10E5, AP2, and AP3 to the receptor did not trigger receptor movement. However, cross-linking these receptor-bound monoclonal antibodies by polyclonal anti-mouse IgG or by conjugation of the anti-receptor antibody to large colloidal gold particles triggered receptor redistribution identical in rate, pattern, and final distribution to that previously seen with fibrinogen binding. We conclude that receptor cross-linking provides the signal for initiation of fibrinogen/alpha IIb beta 3 complex redistribution on platelet surfaces.     

A role for Bruton's tyrosine kinase (Btk) in platelet activation by collagen

Bruton's tyrosine kinase (Btk) is essential for normal B-cell receptor signalling. The lack of expression of functional Btk in humans leads to the B-cell deficiency X-linked agammaglobulinaemia (XLA). We report here that Btk is also important for signalling via the collagen receptor glycoprotein VI (GPVI) in platelets. GPVI is coupled to the Fc receptor gamma chain (FcRgamma). The FcRgamma-chain contains a consensus sequence known as the immune-receptor tyrosine-based activation motif (ITAM). Tyrosine phosphorylation of the ITAM upon GPVI stimulation is the initial step in the regulation of phospholipase C gamma2 (PLCgamma2) isoforms via the tyrosine kinase p72(Syk) (Syk) in platelets. Here we show that collagen and a collagen-related peptide (CRP), which binds to GPVI but does not bind to the integrin alpha2beta1, induced Btk tyrosine phosphorylation in platelets. Aggregation, dense granule secretion and calcium mobilisation were significantly diminished but not completely abolished in platelets from XLA patients in response to collagen and CRP. These effects were associated with a reduction in tyrosine phosphorylation of PLCgamma2. In contrast, aggregation and secretion stimulated by thrombin in Btk-deficient platelets were not significantly altered. Our results demonstrate that Btk is important for collagen signalling via GPVI, but is not essential for thrombin-mediated platelet activation.     

Integrin alpha IIb beta 3 mediates binding of the Lyme disease agent Borrelia burgdorferi to human platelets

Lyme disease is a chronic, multisystemic infection caused by the tick-borne spirochete Borrelia burgdorferi. Attachment of the spirochete to host cells via specific receptors is likely to be important in the establishment of infection. B. burgdorferi have previously been shown to bind to a variety of mammalian cells in vitro. Here we demonstrate that binding of B. burgdorferi to human platelets is mediated by the integrin alpha IIb beta 3 (glycoprotein IIb-IIIa), a critical receptor in thrombosis and hemostasis. Functional expression of this receptor requires platelet activation, and binding of the spirochete was observed only to activated platelets. Binding was inhibited by a synthetic Arg-Gly-Asp peptide that blocks ligand interaction with many integrins and by a synthetic peptide based on the gamma chain of fibrinogen that blocks binding to alpha IIb beta 3. In addition, attachment of the spirochete to platelets was inhibited by monoclonal antibodies directed against alpha IIb beta 3 that are known to block ligand-receptor interaction. No inhibition was seen with control peptides or with antibodies directed against other platelet receptors. B. burgdorferi bound efficiently to purified alpha IIb beta 3 but did not bind to platelets deficient in this integrin. Efficient platelet binding was displayed by a cloned, infectious B. burgdorferi strain, whereas a cloned noninfectious strain did not bind to platelets. Binding to integrins may be important for the ability of B. burgdorferi to establish infection in the diverse tissues affected by Lyme disease.     

Integrin alpha 2 beta 1 is a positive regulator of collagenase (MMP-1) and collagen alpha 1(I) gene expression

A classical model for studying the effects of extracellular matrix is to culture cells inside a three-dimensional collagen gel. When surrounded by fibrillar collagen, many cell types decrease the production of type I collagen, and the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is simultaneously induced. To study the role of the collagen-binding integrins alpha 1 beta 1 and alpha 2 beta 1 in this process, we used three different osteogenic cell lines with distinct patterns of putative collagen receptors: HOS cells, which express only alpha 1 beta 1 integrin, MG-63 cells, which express only alpha 2 beta 1 integrin, and KHOS-240 cells, which express both. Inside collagen gels, alpha 1 (I) collagen mRNA levels were decreased in HOS and KHOS-240 cells but not in MG-63 cells. In contrast, MMP-1 expression was induced in KHOS-240 and MG-63 cells but not in HOS cells. Transfection of MG-63 cells with alpha 2 integrin cDNA in an antisense orientation reduced the expression level of alpha 2 integrin. These cell clones showed induction and reduction of mRNA levels for MMP-1, respectively. HOS cells normally lacking alpha 2 beta 1 integrin were forced to express it, and this prevented the down-regulation in the levels of alpha 1 (I) collagen mRNA when cells were grown inside collagen gels. The data indicate that the level of MMP-1 expression is regulated by the collagen receptor alpha 2 beta 1 integrin. The down-regulation of collagen alpha 1 (I) is mediated by another receptor. Integrin alpha 2 beta 1 may compete with it and thus be a positive regulator of collagen synthesis.     

Platelets interact with soluble and insoluble collagens through characteristically different reactions

Platelet interaction with soluble and insoluble collagens was characterized through binding studies. In contrast to resting platelets, cells reacted with activators, TS2/16 (integrin alpha2 beta1-activating antibody), thrombin, collagen-related peptide, or ADP, exhibited specific soluble collagen binding that is Mg2+-dependent, but inhibited by prostaglandin I2, Ca2+, and Gi9 (anti-integrin alpha2 beta1 antibody). Each platelet has 1500-3500 soluble collagen binding sites, with a dissociation constant of 3. 5-9 x 10(-8) M. This is the first study to show the specific binding of soluble collagen to platelets; our data strongly suggest that the receptor is integrin alpha2 beta1 after it becomes activated upon platelet activation. These results suggest that activation of platelets transforms integrin alpha2 beta1 to a state with higher affinity binding sites for soluble collagen. The soluble collagen-platelet interaction was compared with the platelet interaction with fibrillar collagen, which has until now not been demonstrated to bind specifically to platelets. Here, we demonstrated specific, biphasic fibrillar collagen binding. One phase is rapid and metal ion-independent, and accounts for most of the binding. The other phase is slow and Mg2+-dependent. The characteristic differences in the specific bindings of soluble and fibrous collagens demonstrate the different contributions of two different collagen receptors.     

Involvement of integrins with adhesion-promoting, heparin-binding peptides of type IV collagen in cultured human corneal epithelial cells

PURPOSE: The aim of this work was to identify the integrin subunits present on the cell surface of human corneal epithelial cells. The authors determined to show whether type IV collagen, heparin-binding peptides of type IV collagen (Hep-I, Hep-II, and Hep-III), fibronectin, and GRGDSP promote cell adhesion of human corneal epithelial cells. Type IV collagen and heparin-binding peptides of type IV collagen may be important in corneal epithelial cell adhesion in normal and pathologic conditions and reepithelialization. The authors assess the role of cell surface integrins in mediating cell adhesion to these proteins and peptides. METHODS: Fluorescence-activated cell sorter (FACS) analysis was used to determine the integrin subunits expressed at the cell surface of the cultured human corneal epithelial cells. Cell adhesion was assessed with type IV collagen, heparin-binding peptides of type IV collagen, fibronectin, and GRGDSP: Antibodies to the integrin subunits were used to determine the potential role of integrins in cell adhesion to the above proteins and peptides. RESULTS: FACS analysis identified the beta 1, beta 4, alpha 2, alpha 3, alpha 5, alpha 6, and alpha v integrin subunits on human corneal epithelial cells grown as primary cultures. The anti-beta 1 antibody inhibited cell adhesion to heparin-binding peptides of type IV collagen, type IV collagen, fibronectin, and GRGDSP: Antibodies to the alpha 2 integrin subunit inhibited cell adhesion to the heparin-binding peptides of type IV collagen and slightly inhibited cell adhesion to intact type IV. Antibodies to the alpha 3 integrin subunit exhibited a somewhat lesser effect compared to the anti-alpha 2 integrin antibody. CONCLUSIONS: These data show that the alpha 2 beta 1 integrin of human corneal epithelial cells recognize heparin-binding peptide sequences derived from human type IV collagen. It seems likely that these sequences play an important role in integrin-mediated corneal epithelial cell adhesion. In addition, the alpha 3 beta 1 integrin may mediate similar events.     

Roles of the Ser146, Tyr159, and Lys163 residues in the catalytic action of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli

Adhesion to collagens by most cell types is mediated by the integrins alpha1beta1 and alpha2beta1. Both integrin alpha subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified alpha1beta1 and alpha2beta1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the alpha1 and alpha2 I domains in specific collagen adhesion. We find that introducing the alpha2 I domain into alpha1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (alpha1-2-1beta1) is similar to the adhesion profile conferred by alpha2beta1, not alpha1beta1. The presence of alpha2 or alpha1-2-1 results in preferential binding to collagen I, whereas alpha1 expressing cells bind better to collagen IV. In addition, alpha1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas alpha2 or alpha1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by alpha2beta1 or alpha1-2-1beta1, but not by alpha1, requires the presence of Mn2+ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins.     

Differential expression of platelet activation markers in aspirin-sensitive asthmatics and normal subjects

BACKGROUND: Activation of platelets and expression of adhesion molecules (e.g. CD62P and CD63) which mediate interactions between platelets and other cells may be important in the pathogenesis of aspirin-sensitive asthma. OBJECTIVE: To determine the expression of CD62P and CD63 on platelets from aspirin-sensitive asthmatic (ASA+), aspirin-tolerant asthmatic (ASA-) and normal subjects and to assess the modulatory effect of aspirin on platelet CD62P and CD63 expression following stimulation with either platelet-activating factor (PAF), arachidonic acid (AA) or collagen (COL). METHODS: Platelet-rich plasma was obtained from 10 ASA+, 10 ASA- and 10 normal control subjects, and expression of CD62P and CD63 was measured by flow cytometry. Platelets were stimulated with PAF (10, 80 nM), AA (0.1, 1 mM) or COL (80, 800 micrograms/mL) with or without aspirin (concentration range 0.4-4 mg/mL). RESULTS: In the absence of aspirin, CD62P expression induced by AA and COL was greater in ASA+ patients compared with control subjects (P < 0.001) while CD62P expression with PAF, AA and COL was reduced in ASA- when compared with ASA+ and control subjects (P < 0.001). CD63 expression with PAF and AA was reduced in both ASA+ and ASA- patients compared with control subjects (P < 0.001). Aspirin inhibited the expression of both CD62P and CD63 after agonist stimulation. Greater inhibition of CD62P expression was observed in ASA+ compared with ASA- patients (P < 0.001) and normal subjects (P < 0.05) while greater inhibition of CD63 expression was observed in normal subjects compared with both ASA+ and ASA- patients (P < 0.05). In ASA+ patients and normal subjects, stimulation with PAF and COL resulted in only one platelet population while in contrast with 1 mM AA two populations were observed. CONCLUSIONS: Enhanced AA- and collagen-induced platelet CD62P expression in ASA+ patients compared with normal subjects and greater inhibition by aspirin of CD62P expression in ASA+ may be relevant to the pathogenesis of this syndrome. Reduced expression of CD62P and CD63 in platelets of ASA- patients following stimulation with PAF and AA may also have implications for the role of platelets and these mediators in the pathogenesis of other forms of asthma.     

Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis"

The murine/human chimeric monoclonal antibody fragment (c7E3 Fab) blocks GPIIb/IIIa and alpha v beta 3 receptors, inhibits platelet aggregation, and decreases the frequency of ischemic events after coronary artery angioplasty in patients at high risk of suffering such events. Although inhibition of platelet aggregation is likely to be the major mechanism of c7E3 Fab's effects, since activated platelets facilitate thrombin generation, it is possible that c7E3 Fab also decreases thrombin generation. To test this hypothesis, the effects of c7E3 Fab and other antiplatelet agents were tested in a thrombin generation assay triggered by tissue factor. c7E3 Fab produced dose-dependent inhibition of thrombin generation, reaching a plateau of 45-50% inhibition at concentrations > or = 15 micrograms/ml. It also inhibited thrombin-antithrombin complex formation, prothrombin fragment F1-2 generation, platelet-derived growth factor and platelet factor 4 release, incorporation of thrombin into clots, and microparticle formation. Antibody 6D1, which blocks platelet GPIb binding of von Willebrand factor, had no effect on thrombin generation, whereas antibody 10E5, which blocks GPIIb/IIIa but not alpha v beta 3 receptors decreased thrombin generation by approximately 25%. Combining antibody LM609, which blocks alpha v beta 3 receptors, with 10E5 increased the inhibition of thrombin generation to approximately 32-41%. The platelets from three patients with Glanzmann thrombasthenia, who lacked GPIIb/IIIa receptors but had normal or increased alpha v beta 3 receptors, supported approximately 21% less thrombin generation than normal platelets. We conclude that thrombin generation initiated by tissue factor in the presence of platelets is significantly inhibited by c7E3 Fab, most likely in part through both GPIIb/IIIa and alpha v beta 3 blockade, and that this effect may contribute to its antithrombotic properties.     

Effect of ligand conformation on melanoma cell alpha3beta1 integrin-mediated signal transduction events: implications for a collagen structural modulation mechanism of tumor cell invasion

The importance of three-dimensional interactions between receptors with their respective ligands has been extensively explored during the binding process, but considerably less so for postbinding events such as induction of signaling pathways. Tumor cell receptor association with basement membrane proteins is believed to facilitate the metastatic process. Melanoma and ovarian carcinoma cells have been shown to utilize the alpha3beta1 integrin to bind to models of the alpha1(IV)531-543 sequence from basement membrane (type IV) collagen [Miles, A. J., et al. (1994) J. Biol. Chem. 269, 30939-30945; Miles, A. J., et al. (1995) J. Biol. Chem. 270, 29047-29050]. In the present study, the effects of ligand three-dimensional structure on possible signal transduction pathways induced by alpha3beta1 integrin binding have been evaluated. Human melanoma cell binding to type IV collagen resulted in Tyr phosphorylation of p125(FAK), consistent with prior studies correlating beta1 integrin subunit binding to collagen and p125(FAK) Tyr phosphorylation. Cross-linking of an anti-alpha3 integrin subunit monoclonal antibody also induced p125(FAK) Tyr phosphorylation. Incubation of melanoma cells with single-stranded or triple-helical peptide models of alpha1(IV)531-543 induced Tyr phosphorylation of intracellular proteins. Immunoprecipitation analysis identified one of these proteins as pp125(FAK). Induction of p125(FAK) Tyr phosphorylation was enhanced and the time of induction was shortened when the ligand was used in triple-helical conformation. Subsequent clustering of either the single-stranded or the triple-helical ligand also increased the level of p125(FAK) phosphorylation compared to unclustered ligand. The clustered triple-helical peptide ligand induced more rapid paxillin Tyr phosphorylation than the single-stranded ligand. In addition, the induction of activated proteases was found to be more rapid due to ligand triple helicity. Overall, these studies have shown that (i) a model of an isolated sequence from type IV collagen, alpha1(IV)531-543, can induce alpha3beta1 integrin-mediated signal transduction in melanoma cells and (ii) ligand conformation (secondary, tertiary, and/or quaternary structure) can directly influence several alpha3beta1 integrin-mediated signal transduction events. The effects of ligand conformation suggest that a "collagen structural modulation" mechanism may exist for tumor cell invasion, whereby triple-helical collagen promotes cell binding and induction of signal transduction, subsequently leading to collagen dissolution by proteases, decreased signal transduction, and enhanced tumor cell motility.     

Binding of collagen alpha1 chains to human platelets

We previously reported that purified alpha1 chains of type 1 chick skin collagen induce platelet aggregation. We now describe immunological and biochemical evidence that the peptide binds to intact platelets as an early event in the induction of platelet aggregation and the release reaction. Antibody against alpha1 (I) was obtained by immunizing rabbits with complete Freund's adjuvant mixed with purified alpha1. Immunofluorescence studies showed that alpha1(I)-treated platelets exhibited strong immunofluorescence. The intensity of fluorescence was markedly decreased by the pretreatment of platelets with alpha1-CB5 and glucosylgalactosylhydroxylysine. Dose-response curves of platelet aggregation induced by alpha1 and the binding of alpha1 by washed intact platelets are correlated. The biochemical studies showed that the binding of the alpha1 chain to washed intact platelets was platelet concentration and temperature dependent, and that it reached a maximum in 10 min. The process was reversible and specific, with an association constant of 1.7 muM. The inhibitor of alpha1-induced platelet aggregation, glucosylgalactosyl hydroxylysine, inhibited the alpha1 binding. These results suggest that alpha1(I) chains bind to specific receptor site(s) on platelet membranes to trigger aggregation and the release reaction.