Intraperitoneal injection of sodium pentobarbital has the potential to elicit pain in adult rats (Rattus norvegicus).

An effective and pain-free killing method is required to achieve the goal of euthanasia, a "good death". Overdose of sodium pentobarbital (PB) by intraperitoneal (IP) injection is a widely accepted technique in laboratory rats, but questions remain regarding pain associated with administration. As PB rapidly causes sedation and loss of consciousness, most studies have relied on indirect evidence of pain. The objective of this study was to assess pain associated with IP PB using an appropriate vehicle control. Adult male and female Sprague Dawley (SD) and female Wistar rats (N = 84) were block randomised by sex and strain to receive one of three treatments: 1) 800 mg/kg PB (pH 11), 2) saline or 3) vehicle controls (pH 11 or 12.5). Behavior (Rat Grimace Scale (RGS), writhing, back arching) was evaluated at baseline, before loss of righting reflex (LORR, PB group), and at 80s, 151s and 10 min post-injection (PI; saline and vehicle control groups). In the PB group, mean time to LORR was 78 ± 7.9 seconds. In the vehicle control groups, RGS scores were increased at 151s PI (SD: p = 0.0002, 95%CI 0.73 to 0.20) from baseline, as was relative frequency of writhing (SD: p < 0.0001; Wistar; p = 0.0004). RGS scores remained elevated 10 mins PI (SD: p = 0.0005, 95%CI 0.71 to 0.18; Wistar: p = 0.0234, 95%CI 0.91 to 0.07) but the relative frequency of writhing did not (p > 0.999). The RGS scores and the relative frequency of writhing remained low in the PB and saline groups (p > 0.05). These results show that, vehicle controls for IP PB result in signs associated with pain, pain may not be experienced following IP PB when LORR occurs quickly, and that the effects of PB limit behavioral pain assessments.

The presence of p16 CDKN2A protein immunostaining within feline nasal planum squamous cell carcinomas is associated with an increased survival time and the presence of papillomaviral DNA.

In humans, oral SCCs are either caused by papillomavirus (PV) infection or by other carcinogens such as tobacco. As these 2 groups of SCCs have different causes they also have different clinical behaviors. Immunostaining using anti-p16(CDKN2A) protein (p16) antibodies is used to indicate a PV etiology in human oral SCCs and p16-positive SCCs have a more favorable prognosis. The present study investigated whether p16 immunostaining within feline nasal planum SCCs was similarly associated with the presence of PV DNA and with a longer survival time. Intense p16 immunostaining was visible in 32 of 51 (63%) SCCs. In 30 cats with nonexcised SCCs, cats with p16-positive neoplasms had a longer estimated mean survival time (643 days) than cats with p16-negative SCCs (217 days, P = .013). Papillomavirus DNA was amplified more frequently from p16-positive nasal planum SCCs (28 of 32) than p16-negative SCCs (5 of 19, P < .001). The different survival times in cats with p16-positive and p16-negative SCCs suggests that p16 could be a useful prognostic indicator in these common feline cancers. As the clinical behavior of the SCCs can be subdivided using p16 immunostaining, the 2 groups of SCCs may be caused by different factors, supporting a PV etiology in a proportion of feline nasal planum SCCs.

Guttural pouch mycosis in six horses in New Zealand.

CASE HISTORIES: Six horses from several geographical locations in New Zealand presented with signs of guttural pouch mycosis. All horses had experienced epistaxis within 14 days of presentation. CLINICAL FINDINGS AND TREATMENT: In five horses with epistaxis, a diagnosis of guttural pouch mycosis was made on endoscopic observation of fungal plaques in the affected guttural pouches. One of these cases died before surgery was attempted. The remaining four cases underwent ligation and balloon catheter occlusion of the internal carotid artery of the affected pouch. Three of these horses survived and were reported to be healthy 1 year after surgery. One case died from haemorrhage 8 weeks after surgery. In a sixth horse, endoscopy was carried out but the affected guttural pouch which had recently haemorrhaged was not entered. This horse underwent ligation of the internal carotid and occipital arteries of the affected side but subsequently died. A diagnosis of guttural pouch mycosis of the maxillary artery was confirmed by post-mortem examination. Histology revealed fungal hyphae within thrombi in the lumen of the maxillary artery in the affected guttural pouch. Two horses displayed signs consistent with cranial nerve damage in the guttural pouch. DIAGNOSIS: Guttural pouch mycosis. CLINICAL RELEVANCE: This is the first report of which we are aware of the diagnosis and treatment of clinical cases of guttural pouch mycosis in horses in New Zealand. Practitioners in New Zealand should be aware of the presence and manifestations of this disease and be prepared to treat or refer horses for surgical treatment before fatal haemorrhage and/or profound neurologic signs occur.

Equine penile squamous cell carcinomas are associated with the presence of equine papillomavirus type 2 DNA sequences.

Forty cases of equine penile disease were screened with polymerase chain reaction for the presence of papillomaviral DNA. Cases consisted of 20 squamous cell carcinomas (average age of horse, 23.9 years) and 20 non-squamous cell carcinoma diseases (average age of horse, 13.3 years). All horses but one originated from the Northeastern United States. Breeds were not recorded. As based on MY09/MY11 consensus primers, DNA sequences from equine papillomavirus type 2 were amplified from 9 of 20 horses (45%) with penile squamous cell carcinoma and only 1 of 20 horses (5%) with non-squamous cell carcinoma penile disease. Equine papillomavirus type 2 DNA was the only papillomaviral DNA amplified from any of the 40 horses. Tissues from the 10 horses in which papillomaviral DNA was detected by polymerase chain reaction were also screened with in situ hybridization and immunohistochemistry. The presence of papillomavirus was demonstrated in a subset of these by in situ hybridization (6 of 10) and immunohistochemistry (1 of 10). This report describes a possible association between equine penile squamous cell carcinomas and equine papillomavirus type 2. This study is also the first report of equine papillomavirus type 2 infection in North American horses.

Testing temperature-induced proteomic changes in the plant-associated bacterium Pseudomonas fluorescens SBW25.

Traits used by bacteria to enhance ecological performance in natural environments are not well understood. Recognizing that the saprophytic plant-colonizing bacterium Pseudomonas fluorescens SBW25 experiences temperatures in its natural environment significantly cooler than the 28°C routinely used in the laboratory, we identified proteins differentially expressed between 28°C and the more environmentally relevant temperature of 14°C. Of 2102 protein isoforms, 32 were temperature responsive and identified by mass spectrometry. Seven of these (OmpR, MucD, GuaD, OsmY and three of unknown function, Tee1, Tee2 and Tee3) were selected for genetic and ecological analyses. In each instance, changes in protein expression with temperature were mirrored by parallel transcriptional changes. The fitness contribution of the genes encoding each of the seven proteins was larger at 14°C than 28°C and included two cases of trade-offs (enhanced fitness at one temperature and reduced fitness at the other -mucD and tee2 deletions). The relationship between the fitness effects of genes in vitro and in vivo was variable, but two temperature-responsive genes -osmY and mucD- contribute substantially to the ability of P. fluorescens to colonize the plant environment.

Comparison of the histology and immunohistochemistry of vaccination-site and non-vaccination-site sarcomas from cats in New Zealand.

AIMS: To compare the histology and immunohistochemistry of vaccination-site sarcomas (VSSs) with non-vaccination-site sarcomas (NVSSs) in cats in New Zealand. To determine whether VSSs in cats in New Zealand have similar histological and immunohistochemical features to those previously described in feline vaccine-associated sarcomas (VASs) in North American studies. METHODS: A retrospective survey of skin biopsies submitted between 2004 and 2006 was performed to identify cutaneous sarcomas from both vaccination and non-vaccination sites in cats. Vaccination sites included the interscapular, shoulder, or dorsal or lateral cervical and thoracic regions. All sarcomas were examined histologically, and smooth muscle actin and desmin were assessed immunohistochemically. Features previously described in VASs were assessed and compared. RESULTS: Sarcomas from 34 cats were identified, 10 of which occurred at vaccination sites. Compared with NVSSs, VSSs were more likely to be located in the hypodermis and have greater cellular pleomorphism, higher mitotic rates, more frequent peripheral lymphocytic aggregates and multinucleated giant cells. VSSs were also more likely than NVSSs to show partial myofibroblastic differentiation, demonstrable using immunohistochemistry. The histological and immunohistochemical features of VSSs in cats in New Zealand are consistent with those previously described in VASs in cats in North America. CONCLUSIONS: The results of this study suggest that VASs occur in cats in New Zealand. CLINICAL RELEVANCE: The occurrence of VASs in cats in New Zealand would provide further support for restriction of the vaccination of cats to the minimum necessary to protect health, and adoption of the New Zealand Veterinary Association guidelines on vaccination.

Robot-assisted minimally invasive Kasai portoenterostomy: a survival porcine study.

BACKGROUND: Major enhancements offered by robotic surgery for minimally invasive procedure include tremor filtration, motion scaling, and the addition of a wrist to the instrument. Minor enhancements include indexing as well as safe and rapid instrument exchange. A benefit associated with any endoscopic procedure is magnification. It was hypothesized that these enhancements would allow the performance of complex gastrointestinal surgery. METHODS: Eight survival pigs (weight, 2.5-8 kg) underwent a robotically assisted minimally invasive portoenterostomy. The procedure was analogous to the Kasai portoenterostomy for biliary atresia usually performed for human patients at the age of 4 to 12 weeks. RESULTS: Five of the eight animals survived for more than 1 month after the operation, returning to normal eating and bowel habits in 2 to 3 days. None were jaundiced. All laboratory values were normal. At 1 month, the animals were killed. There was no anastomotic stenosis at either the end-to-side enteroenterostomy or the portoenterostomy. Histologically, the anastomoses were well healed. CONCLUSION: Computer-assisted robot-enhanced technology allows complex gastrointestinal surgery to be performed using minimally invasive techniques.

Testing life-history pleiotropy in Caenorhabditis elegans.

Much life-history theory assumes that alleles segregating in natural populations pleiotropically affect life-history traits. This assumption, while plausible, has rarely been tested directly. Here we investigate the genetic relationship between two traits often suggested to be connected by pleiotropy: maternal body size and fertility. We carry out a quantitative trait locus (QTL) analysis on two isolates of the free-living nematode Caenorhabditis elegans, and identify two body size and three fertility QTLs. We find that one of the fertility QTLs colocalizes with the two body size QTLs on Chromosome IV. Further analysis, however, shows that these QTLs are genetically separable. Thus, none of the five body size or fertility QTLs identified here shows detectable pleiotropy for the assayed traits. The evolutionary origin of these QTLs, possible candidate loci, and the significance for life-history evolution are discussed.

Integrin-independent tyrosine phosphorylation of p125(fak) in human platelets stimulated by collagen.

Collagen fibers or a glycoprotein VI-specific collagen-related peptide (CRP-XL) stimulated tyrosine phosphorylation of the focal adhesion kinase, p125(fak) (FAK), in human platelets. An integrin alpha(2)beta(1)-specific triple-helical peptide ligand, containing the sequence GFOGER (single-letter nomenclature, O = Hyp) was without effect. Antibodies to the alpha(2) and beta(1) integrin subunits did not inhibit platelet FAK tyrosine phosphorylation caused by either collagen fibers or CRP-XL. Tyrosine phosphorylation of FAK caused by CRP-XL or thrombin, but not that caused by collagen fibers, was partially inhibited by GR144053F, an antagonist of integrin alpha(IIb)beta(3). The intracellular Ca(2+) chelator, BAPTA, and the protein kinase C inhibitor, Ro31-8220, were each highly effective inhibitors of the FAK tyrosine phosphorylation caused by collagen or CRP-XL. These data suggest that, in human platelets, 1) occupation or clustering of the integrin alpha(2)beta(1) is neither sufficient nor necessary for activation of FAK, 2) the fibrinogen receptor alpha(IIb)beta(3) is not required for activation of FAK by collagen fibers, and 3) both intracellular Ca(2+) and protein kinase C activity are essential intermediaries of FAK activation.

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

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

Micromolar Ca2+ concentrations are essential for Mg2+-dependent binding of collagen by the integrin alpha 2beta 1 in human platelets.

Integrin receptor alpha(2)beta(1) requires micromolar Ca(2+) to bind to collagen and to the peptide GPC(GPP)(5)GFOGER(GPP)(5)GPC (denoted GFOGER-GPP, where O represents hydroxyproline), which contains the minimum recognition sequence for the collagen-binding alpha(2) I-domain (Knight, C. G., Morton, L. F., Peachey, A. R., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (2000) J. Biol. Chem. 275, 35-40). Platelet adhesion to these ligands is completely dependent on alpha(2)beta(1) in the presence of 2 mm Mg(2+). However, we show here that this interaction was abolished in the presence of 25 microm EGTA. Adhesion of Glanzmann's thrombasthenic platelets, which lack the fibrinogen receptor alpha(IIb)beta(3), was also inhibited by micromolar EGTA. Mg(2+)-dependent adhesion of platelets was restored by the addition of 10 microm Ca(2+), but millimolar Ca(2+) was inhibitory. Binding of isolated alpha(2)beta(1) to GFOGER-GPP was 70% inhibited by 50 microm EGTA but, as with intact platelets, was fully restored by the addition of micromolar Ca(2+). 2 mm Ca(2+) did not inhibit binding of isolated alpha(2)beta(1) to collagen or to GFOGER-GPP. Binding of recombinant alpha(2) I-domain was not inhibited by EGTA, nor did millimolar Ca(2+) inhibit binding. Our data suggest that high affinity Ca(2+) binding to alpha(2)beta(1), outside the I-domain, is essential for adhesion to collagen. This is the first demonstration of a Ca(2+) requirement in alpha(2)beta(1) function.

The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3.

A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described 'metallosheddase' cleavage sites of tumour necrosis factor alpha, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.

Cloning of a mucin-desulfating sulfatase gene from Prevotella strain RS2 and its expression using a Bacteroides recombinant system.

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into a Bacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.

Structural basis of collagen recognition by integrin alpha2beta1.

We have determined the crystal structure of a complex between the I domain of integrin alpha2beta1 and a triple helical collagen peptide containing a critical GFOGER motif. Three loops on the upper surface of the I domain that coordinate a metal ion also engage the collagen, with a collagen glutamate completing the coordination sphere of the metal. Comparison with the unliganded I domain reveals a change in metal coordination linked to a reorganization of the upper surface that together create a complementary surface for binding collagen. Conformational changes propagate from the upper surface to the opposite pole of the domain, suggesting both a basis for affinity regulation and a pathway for signal transduction. The structural features observed here may represent a general mechanism for integrin-ligand recognition.

A sequential study of the pathology associated with the infection of sheep with adult and larval Ostertagia circumcincta.

Disturbances in the physiology of the abomasa of sheep infected with either adult Ostertagia circumcincta given via abomasal cannulae, or larvae (L3) given intraruminally were matched by pathological changes in tissues collected by repeated mucosal biopsy. Within 2-3 days of the transplant of adult worms, abomasal pH had increased markedly in five out of six animals, and there also had been rapid increases in serum gastrin and pepsinogen concentrations in all animals. Reductions in parietal cell number were recorded as early as 1 day after the transplant of adults and were associated with the rapid accumulation of many neutrophils and eosinophils. Mucosal hyperplasia, with increased numbers of cells closer in appearance to mucous/mucous neck cells, was a relatively late development, being most pronounced in the latter part of the infection. In sheep given larvae, changes in secretory physiology were again matched by a concurrent fall in parietal cell number and by the accumulation of inflammatory cells. Changes became maximal when most worms could be expected to be present as adults, confirming the role of adults in the natural disease. Some abnormalities were detected in biopsies collected from animals maintained free of parasites and, although milder in degree, there were similarities to those observed in parasitised tissues, there being fewer parietal cells, a modest degree of mucous cell hyperplasia and inflammatory infiltrates of predominantly neutrophils. These changes were the likely result of trauma to the tissues in the immediate vicinity of the cannula, due either to the presence of the cannula itself or to the frequent collection of biopsy material from areas close to it.

The collagen-binding A-domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens.

We have previously assigned an integrin alpha(2)beta(1)-recognition site in collagen I to the sequence, GFOGERGVEGPOGPA (O = Hyp), corresponding to residues 502-516 of the alpha(1)(I) chain and located in the fragment alpha(1)(I)CB3 (Knight, C. G., Morton, L. F., Onley, D. J., Peachey, A. R., Messent, A. J., Smethurst, P. A., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (1998) J. Biol. Chem. 273, 33287-33294). In this study, we show that recognition is entirely contained within the six-residue sequence GFOGER. This sequence, when in triple-helical conformation, readily supports alpha(2)beta(1)-dependent cell adhesion and exhibits divalent cation-dependent binding of isolated alpha(2)beta(1) and recombinant alpha(2) A-domain, being at least as active as the parent collagen. Replacement of E by D causes loss of recognition. The same sequence binds integrin alpha(1) A-domain and supports integrin alpha(1)beta(1)-mediated cell adhesion. Triple-helical GFOGER completely inhibits alpha(2) A-domain binding to collagens I and IV and alpha(2)beta(1)-dependent adhesion of platelets and HT 1080 cells to these collagens. It also fully inhibits alpha(1) A-domain binding to collagen I and strongly inhibits alpha(1)beta(1)-mediated adhesion of Rugli cells to this collagen but has little effect on either alpha1 A-domain binding or adhesion of Rugli cells to collagen IV. We conclude that the sequence GFOGER represents a high-affinity binding site in collagens I and IV for alpha(2)beta(1) and in collagen I for alpha(1)beta(1). Other high-affinity sites in collagen IV mediate its recognition of alpha(1)beta(1).

Colorimetric and fluorimetric microplate assays for legumain and a staining reaction for detection of the enzyme after electrophoresis.

The cysteine endopeptidase legumain was recently discovered in mammalian cells, predominantly localized in the lysosomal system where it is believed to contribute to antigen processing for MHC class II. Here we describe rapid assay procedures for the enzyme in 96-well microplates with two substrates, a novel compound, succinyl-Ala-Ala-Asn-4-methoxy-2-naphthylamide, and benzyloxycarbonyl-Ala-Ala-Asn-4-methyl-7-coumarylamide. Both substrates are suitable for fluorimetric assays, but the naphthylamide also allows colorimetric detection of legumain activity, since the released 4-methoxy-2-naphthylamine gives a red product when coupled with the Fast Garnet color reagent. We show that the naphthylamide substrate can be used to visualize active legumain after electrophoresis in polyacrylamide gel. Both substrates provide assays that are reproducible and sufficiently sensitive to allow the assay of legumain in crude samples such as tissue homogenates, although the coumarylamide is the more sensitive. The specificity of both assay methods for legumain was verified by the lack of inhibition by E-64 and total inhibition by egg white cystatin.

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.

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.