Biomedical subjects

M J Barnes

Publications and source records attributed to M J Barnes.

At least 37 records · Page 2Linked to original sources

PubMed1999

Comparison of fluorescence in situ hybridization and immunohistochemistry for the evaluation of HER-2/neu in breast cancer.

PURPOSE: To compare fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in the determination of HER-2/neu status of breast cancers. MATERIALS AND METHODS: FISH and IHC for HER-2/neu were performed on formalin-fixed paraffin sections of 100 consecutive invasive breast cancers. FISH was performed at Beth Israel Deaconess Medical Center, Boston, MA, using the Oncor/Ventana INFORM kit (Ventana Medical Systems, Tucson, AZ; formerly sold by Oncor, Inc, Gaithersburg, MD) in a laboratory certified as proficient in this procedure. IHC was performed at PhenoPath Laboratories, Seattle, WA, using a polyclonal antibody to the HER-2/neu protein. FISH and IHC were analyzed in a blinded fashion, and the results were then compared. Procedure and interpretation times and reagent costs for FISH and IHC were also compared. RESULTS: HER-2/neu was amplified by FISH in 26% of cases, and 23% were HER-2/neu-positive by IHC. FISH and IHC were both assessable in 90 cases. Concordance between FISH and IHC results was seen in 82 of these cases (91%, P <.001). The FISH procedure required more technologist time and more interpretation time per case for the pathologist than IHC. Reagent costs were substantially higher for FISH than for IHC. CONCLUSION: There is a high level of correlation between FISH and IHC in the evaluation of HER-2/neu status of breast cancers using formalin-fixed paraffin-embedded specimens. Although the choice of which assay to use should be left for individual laboratories to make based on technical and economic considerations, our results may make it difficult to justify the routine use of FISH for determination of HER-2/neu status in breast cancer.

Adult↗

PubMed1999

Specificity of HercepTest in determining HER-2/neu status of breast cancers using the United States Food and Drug Administration-approved scoring system.

PURPOSE: To evaluate the specificity of the HercepTest for Immunoenzymatic Staining (Dako Corp, Carpinteria, CA) for determining HER-2/neu protein expression in breast cancer. MATERIALS AND METHODS: Forty-eight invasive breast cancers previously found to be HER-2/neu-negative by two different immunohistochemical (IHC) assays and not amplified for the HER-2/neu gene by fluorescence in situ hybridization were studied using the HercepTest kit. HercepTest was performed according to the manufacturer's guidelines, and the results were scored on a 0 to 3+ scale using the United States Food and Drug Administration (FDA)-approved grading system. In this system, cases scored as 2+ or 3+ are considered HER-2/neu-positive. RESULTS: Among these 48 cases, the IHC score using the FDA-approved scoring system was 0 in four cases (8.3%), 1+ in 16 (33.3%), 2+ in 21 (43.8%), and 3+ in seven (14.6%). Therefore, 58.4% of these cases were categorized as HER-2/neu-positive, and the specificity of the HercepTest kit for HER-2/neu expression was 41.6%. However, with the use of a modified scoring system that took into account the level of staining of nonneoplastic epithelium, the specificity increased to 93.2%. CONCLUSION: Our results indicate that the HercepTest kit, when used in accordance with the manufacturer's guidelines and the FDA-approved scoring system, results in a large proportion of breast cancers being categorized as positive for HER-2/neu protein expression and that many of these seem to be false-positives. Consideration of the level of staining of nonneoplastic epithelium resulted in improved specificity. The current FDA-approved scoring system for HercepTest results should be reevaluated before its widespread use in clinical practice.

Adult↗

PubMed1999

Function of glycoprotein VI and integrin alpha2beta1 in the procoagulant response of single, collagen-adherent platelets.

Various collagen-based materials were used to assess the structural requirements of collagen for inducing the procoagulant response of adhering platelets, as well as the collagen receptors involved. Cross-linked or monomeric collagen-related peptide (CRP), Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly was highly adhesive for platelets in a glycoprotein VI-(GpVI-)dependent manner. Adhesion was followed by a prolonged increase in cytosolic [Ca2+]i, formation of membrane blebs, exposure of phosphatidylserine (PS) and generation of prothrombinase-stimulating activity. Fibrils of type-I collagen were less adhesive but, once adhered, many of the platelets presented a full procoagulant response. Monomeric type-I collagen was unable to support adhesion, unless Mg(2+)-dependent integrin alpha2beta1 interactions were facilitated by omission of Ca2+ ions. With all surfaces, however, post-addition of CaCl2 to adhering platelets resulted in a potent Ca(2+)-influx signal, followed by PS exposure and bleb formation. The procoagulant response elicited by binding to CRP was inhibited by anti-GpVI Fab fragments, but not by impeding integrin alpha2beta1-mediated events. With fibrillar collagen, it was inhibited by blocking either the GpVI- or integrin alpha2beta1-mediated interactions. This suggests that the triple-helical Gly-Pro-Hyp repeat in CRP and analogous sequences in fibrillar collagen stimulate the procoagulant response of adherent platelets by acting as ligands for GpVI. Influx of Ca2+ is required for this response, and adhesion via integrin alpha2beta1 serves to potentiate the signaling effects of GpVI.

Binding Sites↗

PubMed1999

Adhesive domains in the collagen III fragment alpha1(III)CB4 that support alpha2beta1- and von Willebrand factor-mediated platelet adhesion under flow conditions.

Seven overlapping peptides derived from the bovine alpha1(III)CB4 fragment of collagen III support static platelet adhesion, and an integrin alpha2beta1-recognition site has been assigned within this fragment to residues 522-528 of the collagen alpha1(III) chain; (25). In this study we found that two of the peptides, CB4(III)-6 and -7, were able to support platelet adhesion under flow conditions, whereas the other peptides showed either very little (CB4(III)-1 and -4) or no platelet adhesion at all (CB4(III)-2, -3 and -5). Using the recombinant leech anti-platelet protein (rLAPP), known to prevent both alpha2beta1 integrin- and von Willebrand factor (vWF)-binding to collagen, we observed almost complete inhibition of platelet adhesion to peptides CB4(III)-6 and -7. In solid-phase binding assays rLAPP bound to CB4(III)-6 and -7 and to CB4(III)-6/7, containing the peptide 6/7 overlap sequence, and not to any other peptide. Our results suggest that the overlap sequence GPP*GPRGGAGPP*GPEGGK (single-letter amino acid code, P* = hydroxyproline), corresponding to residues 523-540 of the alpha1(III) collagen chain, contains a binding site for rLAPP. Monoclonal antibodies (MoAbs) directed against the alpha2 subunit of integrin alpha2beta1 inhibited platelet adhesion to both CB4(III)-6 and -7 by about 50%, showing that the alpha2beta1-recognition site in this locality in alpha1(III)CB4 detected under static conditions is of sufficient affinity to withstand shear forces. Solid-phase binding studies indicated that vWF binds to CB4(III)-7 and to a lesser extent to CB4(III)-4. Furthermore, rLAPP competed with vWF in binding to CB4(III)-7. Our results indicate that residues 541-558 of the alpha1(III)-chain may contain one of the critical vWF-binding sites involved in the initial phase of platelet adhesion to collagen III. MoAbs against vWF (A1 and A3 domain) and glycoprotein (GP)Ib confirmed that vWF is involved in adhesion to CB4(III)-7 and showed that vWF is also involved in adhesion to CB4(III)-6 despite the absence of direct binding of vWF to the peptide. The existence of alpha2beta1-, vWF- and rLAPP-binding sites all in close proximity in alpha1(III)CB4 testifies to the importance of this locus in collagen III for its platelet reactivity.

Amino Acid Sequence↗

PubMed1999

Impact of polyglutamation on sensitivity to raltitrexed and methotrexate in relation to drug-induced inhibition of de novo thymidylate and purine biosynthesis in CCRF-CEM cell lines.

The aim of this study was to investigate the influence of folylpolyglutamyl synthetase (FPGS) activity on the cellular pharmacology of the classical antifolates raltitrexed and methotrexate (MTX) using two human leukemia cell lines, CCRF-CEM and CCRF-CEM:RC2Tomudex. Cell growth inhibition and drug-induced inhibition of de novo thymidylate and purine biosynthesis were used as measures of the cellular effects of the drugs. CCRF-CEM:RC2Tomudex cells had <11% of the FPGS activity of CCRF-CEM cells, whereas MTX uptake and TS activity were equivalent. In CCRF-CEM:RC2Tomudex cells, MTX polyglutamate formation was undetectable after exposure to 1 microM [3H]MTX for 24 h. After exposure to 0.1 microM raltitrexed, levels of total intracellular raltitrexed-derived material in CCRF-CEM:RC2Tomudex cells were 30- to 50-fold lower than in the CCRF-CEM cell line. CCRF-CEM: RC2Tomudex cells were >1000-fold resistant to raltitrexed and 6-fold resistant to lometrexol but sensitive to MTX and nolatrexed when exposed to these antifolates for 96 h. After 6 h of exposure, CCRF-CEM cells retained sensitivity to MTX and raltitrexed but were less sensitive to lometrexol-mediated growth inhibition. In contrast, CCRF-CEM: RC2Tomudex cells were markedly insensitive to raltitrexed, lometrexol, and to a lesser degree, MTX. Simultaneous measurement of de novo thymidylate and purine biosynthesis revealed 90% inhibition of TS activity by 100 nM MTX in both cell lines, whereas inhibition of de novo purine synthesis was only observed in CCRF-CEM cells, and only after exposure to 1000 nM MTX. Ten nM raltitrexed induced >90% inhibition of TS activity in CCRF-CEM cells, whereas in CCRF-CEM:RC2Tomudex cells, there was no evidence of inhibition after exposure to 1000 nM raltitrexed. These studies demonstrate that polyglutamation is a critical determinant of the cellular pharmacology of both raltitrexed and MTX, markedly influencing potency in the case of raltitrexed and locus of action in the case of MTX.

Biological Transport↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

Simple collagen-like peptides support platelet adhesion under static but not under flow conditions: interaction via alpha2 beta1 and von Willebrand factor with specific sequences in native collagen is a requirement to resist shear forces.

The aim of this study was to define the need for specific collagen sequences and the role of their conformation in platelet adhesion to collagen under both static and flow conditions. We recently reported that simple triple-helical collagen-related peptides (CRPs), GCP*(GPP*)10GCP*G and GKP*(GPP*)10GKP*G (single-letter amino acid code, P* = hydroxyproline; Morton et al, Biochem J 306:337, 1995) were potent stimulators of platelet activation and were able to support the adhesion of gel-filtered platelets examined under static conditions. The present study investigated whether these same peptides were able to support platelet adhesion under more physiologic conditions by examining static adhesion with platelet-rich plasma (PRP) and adhesion under flow conditions. In the static adhesion assay, we observed 20% surface coverage with platelet aggregates. In marked contrast, there was a total lack of adhesion under flow conditions examined at shear rates of 50 and 300 s-1. Thus, the interaction of platelets with the CRPs is a low-affinity interaction unable on its own to withstand shear forces. However, the addition of CRPs to whole blood, in the presence of 200 micromol/L D-arginyl-glycyl-L-aspartyl-L-tryptophan (dRGDW) to prevent platelet aggregation, caused an inhibition of about 50% of platelet adhesion to collagens I and III under flow. These results suggest that the collagen triple helix per se, as defined by these simple collagen sequences, plays an important contributory role in the overall process of adhesion to collagen under flow. The monoclonal antibody (MoAb) 176D7, directed against the alpha2 subunit of the integrin alpha2 beta1, was found to inhibit static platelet adhesion to monomeric but not fibrillar collagens I and III. However, under flow conditions, anti-alpha2 MoAbs (176D7 anf 6F1) inhibited adhesion to both monomeric and fibrillar collagens, indicating that alpha2 beta1 is essential for adhesion to collagen under flow, independent of collagen conformation, whether monomeric or polymeric. To obtain further insight into the nature of the different adhesive properties of CRPs and native collagen, we investigated the relative importance of von Willebrand factor (vWF) and the integrin alpha2 beta1 in platelet adhesion to collagen types I and III, using the same shear rate (300 s-1) as used when testing CRPs under flow conditions. Our results, together with recent data of others, support a two-step mechanism of platelet interaction with collagen under flow conditions. The first step involves adhesion via both the indirect interaction of platelet glycoprotein (GP) Ib with collagen mediated by vWF binding to specific vWF-recognition sites in collagen and the direct interaction between platelet alpha2 beta1 and specific alpha2 beta1-recognition sites in collagen. This suffices to hold platelets at the collagen surface. The second step occurs via another collagen receptor (thought to be GPVI) that binds to simple collagen sequences, required essentially to delineate the collagen triple helix. Recognition of the triple helix leads to strengthening of attachment and platelet activation.

Adult↗

PubMed1998

Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not.

Simple collagen-related peptides (CRPs) containing a repeat Gly-Pro-Hyp sequence are highly potent platelet agonists. Like collagen, they must exhibit tertiary (triple-helical) and quaternary (polymeric) structure to activate platelets. Platelet signaling events induced by the peptides are the same as most of those induced by collagen. The peptides do not recognize the alpha 2 beta 1 integrin. To identify the signaling receptor involved, we have evaluated the response to the CRP, Gly-Lys-Hyp(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly of platelets with defined functional deficiencies. These studies exclude a primary recognition role for CD36, von Willebrand factor (vWF), or glycoprotein (GP) IIb/IIIa. Thus, both CD36 and vWF-deficient platelets exhibited normal aggregation, normal fibrinogen binding, and normal expression of CD62 and CD63, measured by flow cytometry, in response to the peptide, and there was normal expression of CD62 and CD63 on thrombasthenic platelets. In contrast, GPVI-deficient platelets were totally unresponsive to the peptide, indicating that this receptor recognizes the Gly-Pro-Hyp sequence in collagen. GPVI-deficient platelets showed some fibrinogen binding in response to collagen but failed to aggregate and to express CD62 and CD63. Collagen, but not CRP-XL, contains binding sites for alpha 2 beta 1. Therefore, it is possible that collagen still induces some signaling via alpha 2 beta 1, leading to activation of GPIIb/IIIa. Our findings are consistent with a two-site, two-step model of collagen interaction with platelets involving recognition of specific sequences in collagen by an adhesive receptor such as alpha 2 beta 1 to arrest platelets under flow and subsequent recognition of another specific collagen sequence by an activatory receptor, namely GPVI.

Binding Sites↗

PubMed1998

The collagen-platelet interaction.

Collagen-platelet interaction, occurring in hemostasis but also a cause of thrombosis, is a two-step process of adhesion and activation involving the sequential recognition of distinct receptors. Adhesion involves first the reversible recognition of collagen-bound von Willebrand factor by the platelet receptor complex Gp Ib/IX/V, followed by direct interaction between collagen and the platelet integrin receptor alpha2beta1, which binds to specific sequences in collagen in which the GER motif appears important. Platelet activation then follows from the recognition by the receptor Gp VI of GPP* sequences in collagen, culminating in signalling events unique to collagen as a platelet agonist: Gp VI leads via the novel platelet Fc receptor gamma-chain to p72syk and phospholipase Cgamma2.

Animals↗

PubMed1997

The platelet reactivity of synthetic peptides based on the collagen III fragment alpha1(III)CB4. Evidence for an integrin alpha2beta1 recognition site involving residues 522-528 of the alpha1(III) collagen chain.

The platelet-reactive collagen III-derived fragment alpha1(III)CB4 has been synthesized as seven overlapping peptides, each as a homotrimeric triple-helical species covalently linked at the C terminus. Additional Gly-Pro-Hyp triplets were introduced at each end of the peptide sequence to ensure a stable triple-helical conformation at 20 degrees C, the temperature at which cell reactivity was measured. A Cys-containing triplet was included at each end to allow intermolecular cross-linking. All seven peptides in triple-helical, cross-linked form were able to cause platelet aggregation. Peptide 6, the most reactive species, was more aggregatory than collagen fibers. Platelet adhesion occurred to all peptides immobilized on plastic in monomeric form. Adhesion was integrin alpha2beta1-independent except in the case of peptide 6, adhesion to which was partially reduced by anti-integrin alpha2beta1 monoclonal antibodies. The presence of an alpha2beta1 recognition site in peptide 6 was confirmed using HT 1080 cells, which express alpha2beta1 as their major or sole collagen receptor. HT 1080 adhesion to both peptide 6 and collagen was strongly inhibited by anti-integrin alpha2beta1 monoclonal antibodies. These cells did not adhere to any of the other peptides. Comparison of the structure of peptide 6 with that of adjacent peptides indicates that the sequence Gly-Gly-Pro-Hyp-Gly-Pro-Arg, residues 522-528 of the collagen alpha1(III) chain, represents the minimum structure required for the recognition of alpha2beta1. Our findings support the view that the collagen triple helix possesses an intrinsic platelet reactivity that can be expressed independently of integrin alpha2beta1 and the precise level of which is governed by the exact nature of the primary sequence. Sequences such as those recognizing alpha2beta1 may potentiate the activity, whereas others may have the opposite effect.

Amino Acid Sequence↗

PubMed1997

A collagen-like peptide stimulates tyrosine phosphorylation of syk and phospholipase C gamma2 in platelets independent of the integrin alpha2beta1.

Activation of platelets by collagen is mediated through a tyrosine kinase-dependent pathway that is associated with phosphorylation of the Fc receptor gamma chain, the tyrosine kinase syk, and phospholipase C gamma2 (PLC gamma2). We recently described a collagen-related triple-helical peptide (CRP) with the sequence GCP*(GPP*)GCP*G (single letter amino acid code: P* = hydroxyproline; Morton et al, Biochem J306:337, 1995). The cross-linked peptide is a potent stimulus of platelet activation but, unlike collagen, does not support alpha2beta1-mediated, Mg2+-dependent adhesion, suggesting that its action is independent of the integrin alpha2beta1. This finding suggests the existence of a platelet receptor other than alpha2beta1 that underlies activation. In the present study, we show that CRP stimulates tyrosine phosphorylation of the same pattern of proteins in platelets as collagen, including syk and PLC gamma2. Protein tyrosine phosphorylation induced by CRP is not altered in the absence of Mg2+ or the presence of monoclonal antibodies (MoAbs) to the integrin alpha2beta1 (MoAb 6F1 and MoAb 13), conditions that prevent the interaction of collagen with the integrin. In contrast, phosphorylation of syk and PLC gamma2 by collagen is partially reduced by MoAb 6F1 and MoAb 13 or by removal of Mg2+. This may reflect a direct role of alpha2beta1 in collagen-induced signaling events or an indirect role in which the integrin facilitates the binding of collagen to its signaling receptor. The results show an alpha2beta1-independent pathway of platelet activation by CRP that involves phosphorylation of syk and PLC gamma2. This pathway appears to contribute to platelet activation by collagen.

Collagen↗

PubMed1997

Ethnicity, drug user status and academic performance.

The impact of drug use and race on school performance was investigated. Data were collected retrospectively from a sample of 100 students in Grades 9 through 12, using a questionnaire devised by the authors. Most recent grades in English, mathematics, social studies, and science, and percentile scores on the mathematics portion of a recent Metropolitan Achievement Test, constituted achievement. Sufficient data were obtained for 38 students (11 black, 18 white, and 9 Latino). Results included significant main effects for both race (p < .05) and user status (p < .05). An interaction of race and user status was suggested. While black users and nonusers did not differ across academic areas, large differences for the two other ethnic groups emerged.

Adolescent↗

PubMed1996

The A-domain of integrin alpha 2 binds specifically to a range of collagens but is not a general receptor for the collagenous motif.

Integrin alpha 2 beta 1 is a major cellular receptor for collagens, but the molecular basis of its function is unknown. The alpha 2 subunit contains a von Willebrand factor A-domain (I-domain) in its N-terminal region, and it has been demonstrated recently that this domain binds specifically to collagen I. This interaction requires divalent cations (e.g., Mg2+) and native collagen conformation, as does binding of the parent integrin to collagen. The alpha 2 A-domain therefore has a number of functional similarities to the parent integrin, alpha 2 beta 1. However, while sequence specificity has been demonstrated for the parent integrin, no such observations have been made for the A-domain. In particular, it is not known whether the A-domain is responsible for sequence-specific recognition of collagens or whether it binds to the genetic collagenous motif. To investigate the ligand specificity of the alpha 2 A-domain, its binding to a range of collagenous ligands has been studied, with cation dependence, collagen triple-helicity, and inhibition by function-blocking antibodies as criteria for specificity. Binding of the parent integrin was examined for comparison. The alpha 2 A-domain was found to bind specifically to collagens I, II, IV and XI. The complement component C1q has a collagenous domain but this was unable to support specific binding of alpha 2 A-domain or alpha 2 beta 1. Furthermore, synthetic triple-helical collagenous peptides failed to act as specific ligands. In conclusion, the alpha 2 A-domain binds specifically to a range of extracellular matrix collagens, but it is not a receptor for all collagenous triple helices. By inference, these findings indicate the existence of an integrin-specific sequence motif within collagenous ligands recognised by the alpha 2 A-domain.

Antigens, CD↗

PubMed1996

Role for p38 mitogen-activated protein kinase in platelet aggregation caused by collagen or a thromboxane analogue.

p38 mitogen-activated protein kinase (MAPK) was identified in platelets on the basis of (a) its reactivity with antibodies to C-terminal and N-terminal peptides, and (b) its ability to activate MAPK-activated protein kinase-2, which phosphorylates the small heat shock protein, hsp27. p38 MAPK was activated in platelets by collagen fibers, a collagen-related cross-linked peptide, thrombin, or the thromboxane analogue U46619. A highly specific inhibitor of p38 MAPK, a pyridinyl imidazole known as SB203580, inhibited the platelet enzyme in vitro (IC50 approximately 0.5 microM). At similar concentrations it also inhibited agonist-stimulated phosphorylation of hsp27 in platelets, and platelet aggregation and secretion induced by minimal aggregatory concentrations of collagen or U46619, but not thrombin. Inhibition of aggregation was overcome by increasing agonist dose. SB203580 might act by inhibiting thromboxane generation, but this was only inhibited by 10-20% at low agonist concentrations. p38 MAPK provides a crucial signal, which is necessary for aggregation caused by minimal concentrations of collagen fibers or U46619. Thrombin or high doses of these agonists generate signals that bypass the enzyme, or render the enzyme no longer rate-limiting.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1996

Beta 1 integrins and osteoclast function: involvement in collagen recognition and bone resorption.

The extracellular matrix of bone is composed mainly of type I collagen. In this report we studied the role and collagen-binding properties of osteoclast integrins (alpha v, alpha 2, beta 1, and beta 3). Cell adhesion assays with rat osteoclasts and affinity chromatography/SDS-PAGE analysis with purified human osteoclast membranes demonstrated adhesion of osteoclasts to native type I collagen in a divalent cation and Arg-Gly-Asp (RGD)-dependent way via alpha 2 beta 1 integrin, whereas osteoclast adhesion to denatured collagen predominantly involved alpha v beta 3. In receptor-binding assays, the involvement of human recombinant alpha v beta 3 in adhesion to denatured collagen was confirmed. Additionally, osteoclasts adhered to type I collagen fibers and to monomeric types II-V collagen with characteristics similar to those on native monomeric type I collagen. Osteoclastic bone resorption in vitro was inhibited (> 40%) in the presence of alpha 2 and beta 1 antibodies. Using scanning laser confocal microscopy, alpha v beta 3, alpha 2, and beta 1 integrin were detected within podosomes in nonresorbing osteoclasts and in the ruffled border area and basolateral membrane in resorbing osteoclasts, but not in the sealing zone of resorbing osteoclasts. These results demonstrate that alpha 2 beta 1, in addition to alpha v beta 3, has an important role in osteoclast function and acts as a receptor for native, but not denatured, collagen.

Amino Acid Sequence↗

PubMed1996

Signals elicited from human platelets by synthetic, triple helical, collagen-like peptides.

Synthetic collagen-like peptides, of general structure [Gly-Pro-HyP]n, adopt the triple-helical structure which is essential for the platelet-reactivity of native collagens. These peptides are potent activators of platelets, stimulating platelet aggregation at much lower dose than collagen fibres, but, unlike collagen fibres, they are not recognised by the integrin alpha 2 beta 1. We have examined the ability of the synthetic peptides to activate the various signalling pathways which regulate human platelet function. The peptides are potent activators of Ca2+ mobilisation and of protein kinase C, and they stimulate tyrosine phosphorylation of some substrates preferentially. However, in contrast with native type I collagen fibres, they are unable to inhibit platelet adenylate cyclase. This suggests a mode of action for the synthetic peptides which substantially overlaps, but which is not entirely identical with, that of native collagen.

Apyrase↗

PubMed1995

Integrin alpha 2 beta 1-independent activation of platelets by simple collagen-like peptides: collagen tertiary (triple-helical) and quaternary (polymeric) structures are sufficient alone for alpha 2 beta 1-independent platelet reactivity.

The platelet reactivities of two simple collagen-like synthetic peptides, Gly-Lys-Hyp-(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly and Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly, were investigated. Both peptides adopted a stable triple-helical conformation in solution. Following cross-linking, both peptides proved to be highly platelet-aggregatory, more active than collagen fibres, inducing aggregation at concentrations as low as 20 ng/ml. These peptides formed microaggregates in solution, and cross-linking was thought to stabilize these structures, allowing expression of their platelet reactivity at 37 degrees C. Like collagen fibres, the peptides caused platelet secretion and release of arachidonate from platelet membrane lipids as well as activation of integrin alpha IIb beta 3 culminating in aggregation. Monoclonal antibodies directed against the integrin alpha 2 beta 1 failed to prevent aggregation release of arachidonate or platelet adhesion to the peptides. Our results indicate that collagen can activate platelets by a mechanism that is independent of integrin alpha 2 beta 1 and for which collagen tertiary and quaternary structures are sufficient alone for activity without the involvement of highly specific cell-recognition sequences.

Amino Acid Sequence↗