SyncMRT: a solution to image-guided synchrotron radiotherapy for quality assurance and pre-clinical trials.

In this work, a new image guidance system and protocols for delivering image-guided radiotherapy (IGRT) on the Imaging and Medical Beamline (IMBL) at the ANSTO Australian Synchrotron are introduced. The image guidance methods used and the resulting accuracy of tumour alignment in in vivo experiments are often under-reported. Image guidance tasks are often complex, time-consuming and prone to errors. If unchecked, they may result in potential mis-treatments. We introduce SyncMRT, a software package that provides a simple, image guidance tool-kit for aligning samples to the synchrotron beam. We have demonstrated sub-millimetre alignment using SyncMRT and the small-animal irradiation platform (the DynamicMRT system) on the IMBL. SyncMRT has become the standard for carrying out IGRT treatments on the IMBL and has been used in all pre-clinical radiotherapy experiments since 2017. Further, we introduce two quality assurance (QA) protocols to synchrotron radiotherapy on the IMBL: the Winston-Lutz test and hidden target test. It is shown that the presented QA tests are appropriate for picking up geometrical setup errors and assessing the end-to-end accuracy of the image guidance process. Together, these tools make image guidance easier and provide a mechanism for reporting the geometric accuracy of synchrotron-based IGRT treatments. Importantly, this work is scalable to other delivery systems, and is in continual development to support the upcoming veterinary radiotherapy trials on the IMBL.

Does acute side-alternating vibration exercise enhance ballistic upper-body power?

The aim of this study was to investigate the effects of acute vibration exercise, at 2 different frequencies, on upper body power output. Muscle activity (EMG) and upper-body peak power was measured in 12 healthy males during ballistic bench press throws at 30% of 1-repetition maximum on a Smith machine. Measures were made prior to, 30 s and 5 min after one of 3 conditions performed for 30 s in a press-up position: side-alternating vibration at 20 Hz, 26 Hz and no vibration. EMG was recorded in the anterior deltoid, triceps brachii and pectoralis major during ballistic bench press throws as well as during application of each condition. While peak power output was higher at 5 min post condition across all conditions, compared to baseline measures (P<0.05), only 20 Hz vibration resulted in a significant increase in peak power output (P<0.05) compared to no vibration. EMG was greater during both vibration conditions, compared to no vibration (P<0.001). However, this difference was not evident during bench press throws when no difference was seen in muscle activity between conditions. These findings suggest that 20 Hz vibration has an ergogenic effect on upper-body power that may be due to peripheral, rather than central, mediated mechanisms.

Hypothalamic QRFP: regulation of food intake and fat selection.

QRFP, a member of the RFamide-related peptide family, is a strongly conserved hypothalamic neuropeptide that has been characterized in various species. Prepro-QRFP mRNA expression is localized to select regions of the hypothalamus, which are involved in the regulation of feeding behavior. The localization of the peptide precursor has led to the assessment of QRFP on feeding behaviors and the orexigenic effects of QRFP have been detected in mice, rats, and birds. QRFP acts in a macronutrient specific manner in satiated rats to increase the intake of a high fat diet, but not the intake of a low fat diet, and increases the intake of chow in food-restricted rats. Studies suggest that QRFP's effects on food intake are mediated by the adiposity signal, leptin, and hypothalamic neuropeptides. Additionally, QRFP regulates the expression and release of hypothalamic Neuropeptide Y and proopiomelanocortin/α-Melanocyte-Stimulating Hormone. QRFP binds to receptors throughout the brain, including regions associated with food intake and reward. Taken together, these data suggest that QRFP is a mediator of motivated behaviors, particularly the drive to ingest high fat food. The present review discusses the role of QRFP in the regulation of feeding behavior, with emphasis on the intake of dietary fat.

Autonomic cardiovascular response to acute hypoxia and passive head-up tilting in humans.

Acute hypoxia may alter autonomic cardiovascular reflexes during orthostasis. Heart rate variability (HRV), arterial blood pressure (MAP), and respiratory sinus arrhythmia (RSA) were recorded during supine (SUP) and passive head up tilt (HUT) in eight healthy humans, spontaneously breathing either room air or 10% O2 in N2. In the time domain, heart rate increased and variability decreased with HUT in both trials, with no difference between trials. In the frequency domain, normalized low frequency HRV increased, and normalized high frequency HRV decreased with HUT in both trials, with no difference between trials. MAP was 74.9 (8.6) and 77.5 (11.7) mmHg when SUP in the room air and hypoxia trials, respectively. A significant increase in MAP occurred with HUT in the room air trial but not in the hypoxia trial. In both trials, end tidal CO2 decreased with HUT, with no difference between trials. In the room air trial, end tidal O2 increased with HUT, whereas during the hypoxia trial, end tidal O2 decreased with HUT. The distribution of heart beats relative to the phase of ventilation (%HBIN and %HBOUT) was similar in both trials: the %HBIN was 43.5 (3.3) % and %HBOUT was 56.5 (4.2) % breathing room air when SUP, and 45.5 (3.0) and 54.5 (3.2) when hypoxic and SUP. For both trials, this distribution did not change with HUT. As both HRV and RSA showed similar responses to HUT when spontaneously breathing either room air or 10% O2 in N2, we suggest that autonomic cardiovascular reflexes are preserved during acute hypoxia.

Does intermittent pneumatic leg compression enhance muscle recovery after strenuous eccentric exercise?

Intermittent pneumatic compression (IPC) has gained rapid popularity as a post-exercise recovery modality. Despite its widespread use and anecdotal claims for enhancing muscle recovery there is no scientific evidence to support its use. 10 healthy, active males performed a strenuous bout of eccentric exercise (3 sets of 100 repetitions) followed by IPC treatment or control performed immediately after exercise and at 24 and 48 h post-exercise. Muscular performance measurements were taken prior to exercise and 24, 48 and 72 h post-exercise and included single-leg vertical jump (VJ) and peak and average isometric [knee angle 75º] (ISO), concentric (CON) and eccentric (ECC) contractions performed at slow (30° · s⁻¹) and fast (180° · s⁻¹) velocities. Plasma creatine kinase (CK) samples were taken at pre- and post-exercise 24, 48 and 72 h. Strenuous eccentric exercise resulted in a significant decrease in peak ISO, peak and average CON (30° · s⁻¹) at 24 h compared to pre-exercise for both IPC and control, however VJ performance remained unchanged. There were no significant differences between conditions (IPC and control) or condition-time interactions for any of the contraction types (ISO, CON, ECC) or velocities (CON, ECC 30° · s⁻¹ and 180° · s⁻¹). However, CK was significantly elevated at 24 h compared to pre-exercise in both conditions (IPC and control). IPC did not attenuate muscle force loss following a bout of strenuous eccentric exercise in comparison to a control. While IPC has been used in the clinical setting to treat pathologic conditions, the parameters used to treat muscle damage following strenuous exercise in healthy participants are likely to be very different than those used to treat pathologic conditions.

Training injury incidence in an amateur women's rugby union team in New Zealand over two consecutive seasons.

OBJECTIVES: To describe the training injury incidence in amateur women's rugby union in New Zealand over two consecutive seasons. DESIGN: A prospective cohort observational study METHODS: A total of 69 amateur women's rugby 15s team playerswere observed. Training exposure and training injury incidence were calculated. RESULTS: The 38 training injuries resulted in a total injury incidence of 11.4 (8.3-15.6) per 1,000 training-hours. There were 12 injuries that resulted in a time-loss injury incidence of 3.6 (95% CI: 2.0-6.3) per 1,000 training-hours. Forwards recorded more total (RR: 1.8 [95% CI: 0.9-3.5]; p=0.0516) and time-loss (RR: 2.0 [95% CI: 0.6-6.6]; p=0.2482) injuries than Backs. The tackle was the most common injury cause for total (3.0 [95% CI: 1.6-5.6] per 1,000 training-hours.) injuries, but collisions (1.5 [95% CI: 0.6-3.6] per 1,000 training-hours.) with the ground or another person were the most common cause for time-loss injuries.The training injuries occurred most often to the lower limb and during the latter part of training sessions. These injuries were mostly minor in nature resulting in minimal time-loss away from training. DISCUSSION: The time-loss injury incidence (3.6 per 1,000 training-hours.) for the amateur women's rugby 15s team players was higher than that reported for National (1.2 per 1,000 training-hours.) and Rugby World Cup for women (0.2 to 3.0 per 1,000 training-hours.) competitions. CONCLUSION: The training injury incidence in amateur women's rugby union in New Zealand was higher than that reported for national and international rugby union injury incidences.

Sensitivity to the satiating effects of exendin 4 is decreased in obesity-prone Osborne-Mendel rats compared to obesity-resistant S5B/Pl rats.

BACKGROUND: Osborne-Mendel (OM) rats are prone to obesity when fed a high-fat diet, whereas S5B/Pl (S5B) rats are resistant to diet-induced obesity when fed the same diet. OM rats have a decreased satiation response to fatty acids infused in the gastrointestinal tract, compared to S5B rats. One possible explanation is that OM rats are less sensitive to the satiating hormone, glucagon-like peptide 1 (GLP-1). GLP-1 is produced in the small intestine and is released in response to a meal. The current experiments examined the role of GLP-1 in OM and S5B rats. METHODS: Experiment 1 examined preproglucagon mRNA expression in the ileum of OM and S5B rats fed a high-fat (55% kcal) or low-fat (10% kcal) diet. Experiment 2 investigated the effects of a 2 h high-fat meal after a 24 h fast in OM and S5B rats on circulating GLP-1 (active) levels. Experiment 3 examined the effects of exendin-4 (GLP-1 receptor agonist) administration on the intake of a high-fat or a low-fat diet in OM and S5B rats. RESULTS: Preproglucagon mRNA levels were increased in the ileum of OM rats compared to S5B rats and were increased by high-fat diet in OM and S5B rats. OM and S5B rats exhibited a similar meal-initiated increase in circulating GLP-1 (active) levels. Exendin-4 dose dependently decreased food intake to a greater extent in S5B rats compared to OM rats. The intake of low-fat diet, compared to the intake of high-fat diet, was more sensitive to the effects of exendin-4 in these strains. CONCLUSIONS: These results suggest that though OM and S5B rats have similar preproglucagon mRNA expression in the ileum and circulating GLP-1 levels, OM rats are less sensitive to the satiating effects of GLP-1. Therefore, dysregulation of the GLP-1 system may be a mechanism through which OM rats overeat and gain weight.

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.

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.

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.

Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen.

OBJECTIVE: Peptides consisting of a repeat Gly-Pro-Hyp sequence are potent platelet agonists. The aim of this study was: (1) to examine the specificity of this sequence for platelet activation; (2) to confirm its recognition by platelet glycoprotein VI; and (3) to assess with suitable peptides the relative importance of glycoprotein VI and integrin alpha 2 beta 1 in platelet activation by collagen. METHODS: Peptides were synthesized by standard Fmoc chemistry and tested for their ability to support adhesion of human platelets and HT 1080 cells, induce platelet aggregation, bind integrin alpha 2 subunit A-domain and to cause tyrosine phosphorylation of platelet proteins. RESULTS: (1) Peptides consisting of a repeat Gly-Pro-Pro, Gly-Pro-Ala or Gly-Pro-Arg sequence exhibited little if any platelet-reactivity. (2) The platelet-reactive peptide consisting of a repeating Gly-Pro-Hyp sequence failed to induce tyrosine phosphorylation in glycoprotein VI-deficient platelets. Platelet adhesion to this peptide was inhibited by intact anti-glycoprotein VI antibody and its Fab fragment. The latter inhibited aggregation by the peptide and fibres of both collagens I and III. (3) A peptide containing a 15-mer alpha 2 beta 1-binding sequence in a repeat Gly-Pro-Pro structure supported alpha 2 beta 1-mediated platelet and HT 1080 cell adhesion and bound alpha 2 A-domain, but failed to activate platelets or to induce tyrosine phosphorylation. Conversely, a peptide containing this sequence but with an essential Glu replaced by Ala and inserted in a repeat Gly-Pro-Hyp structure did not recognize alpha 2 beta 1, but was highly platelet activatory. CONCLUSIONS: Platelet activation by collagen involves the highly-specific recognition of the Gly-Pro-Hyp sequence by platelet glycoprotein VI. Recognition of alpha 2 beta 1 is insufficient to cause activation. Interaction between collagen and glycoprotein VI is unique since Gly-Pro-Hyp is common in collagens but occurs rarely in other proteins, and glycoprotein VI may be expressed solely by platelets. This sequence could provide a basis for a highly-specific anti-thrombotic reagent to control thrombosis associated with plaque rupture.

Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo.

Replication-deficient adenoviruses are known to induce acute injury and inflammation of infected tissues, thus limiting their use for human gene therapy. However, molecular mechanisms triggering this response have not been fully defined. To characterize this response, chemokine expression was evaluated in DBA/2 mice following the intravenous administration of various adenoviral vectors. Administration of adCMVbeta gal, adCMV-GFP, or FG140 intravenously rapidly induced a consistent pattern of C-X-C and C-C chemokine expression in mouse liver in a dose-dependent fashion. One hour following infection with 10(10) PFU of adCMVbeta gal, hepatic levels of MIP-2 mRNA were increased >60-fold over baseline. MCP-1 and IP-10 mRNA levels were also increased immediately following infection with various adenoviral vectors, peaking at 6 hr with >25- and >100-fold expression, respectively. Early induction of RANTES and MIP-1beta mRNA by adenoviral vectors also occurred, but to a lesser degree. The induction of chemokines occurred independently of viral gene expression since psoralen-inactivated adenoviral particles produced an identical pattern of chemokine gene transcription within the first 16 hr of administration. The expression of chemokines correlated as expected with the influx of neutrophils and CD11b+ cells into the livers of infected animals. At high titers, all adenoviral vectors caused significant hepatic necrosis and apoptosis following systemic administration to DBA/2 mice. To investigate the role of neutrophils in this adenovirus-induced hepatic injury, animals were pretreated with neutralizing anti-MIP-2 antibodies or depleted of neutrophils. MIP-2 antagonism and neutrophil depletion both resulted in reduced serum ALT/AST levels and attenuation of the adenovirus-induced hepatic injury histologically, confirming that this early injury is largely due to chemokine production and neutrophil recruitment. Our findings further clarify the early immune response against replication-deficient adenoviral vectors and suggest a strategy to prevent adenovirus-mediated inflammation and tissue injury by interfering with chemokine or neutrophil function.

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.

Collagen polymorphism in the normal and diseased blood vessel wall. Investigation of collagens types I, III and V.

Estimation of collagens types I and III in pepsin digests and by analysis of specific cyanogen-bromide derived peptides by SDS-polyacrylamide gel electrophoresis, has indicated that both the undiseased human aortic media and the atherosclerotic plaque of the diseased intima contain more type I collagen than type III. There was only a relatively small shift in composition in favour of type I collagen in the diseased compared to the undiseased tissue. Diffusely thickened intima was similar in composition to the atherosclerotic plaque. These results suggest that both atherogenesis and diffuse intimal thickening may involve primarily smooth muscle cell hyperplasia with increased overall collagen production but little alteration in cell phenotype as regards the relative proportions of the individual collagens produced. They do not support the contention that atherosclerosis involves a 'transformation' of smooth muscle cells to fibroblast in type, whereby a major switch in synthesis occurs from largely type III collagen to mainly type I in disease. Type V collagen(s) containing both alpha A- and alpha B-chains has been detected throughout the vessel wall in diffusely thickened intima, media and adventitia, as well as in the plaque where, in the latter case, a marked enrichment relative to interstitial collagens was noted. This is presumed to reflect the relatively cellular nature of the atherosclerotic lesion. The alpha C-chain of type V collagen was detected in porcine but not human aorta.

The interaction of fibronectin (fn) with native, polymeric collagen (collagen fibres): comparison with von Willebrand factor (vWf)-binding by collagen.

The binding of fn to collagen (type I) fibres has been found to resemble that of vWf in the following respects: Binding is rapid, specific, saturable, similar at 4 and 37 degrees C, and reduced by increasing ionic strength. Binding is not inhibited by native, monomeric collagen, suggesting a multivalent mechanism of interaction. Binding of fn occurs to a variety of collagen fragments (after their renaturation and polymerization), including, for example, the collagenase-derived TCA and TCB 3/4 and 1/4 molecular fragments and the peptides alpha 1(I)CB3, 6b, 7 and 8 obtained by cleavage with cyanogen bromide (CB), suggesting a wide distribution of binding sites on the native collagen molecule. As judged by the effect of heat-treatment, the native conformation of fn is required. Chemical modification indicates the involvement of arginyl residues in collagen and carboxyl groups in fn. However, fn and vWf did not compete with one another in binding to collagen, suggesting the participation of different collagen arginyl residues in the two interactions. Fn-binding differed from that of vWf in that the former was inhibited by denatured monomeric collagen (gelatin). Fn-binding was also inhibited by the fragment TCA in denatured form. The inhibitory activity was lost after chemical modification of arginyl residues in gelatin. Our results suggest that fn binding to collagen fibres and gelatin involves the same widely-distributed spectrum of binding sites.

The platelet reactivity of collagen type I: evidence for multiple platelet-reactive sites in the type I collagen molecule.

In this study, the ability of peptides, obtained by fragmentation of the collagen type I molecule, to induce platelet aggregation has been examined. In order to satisfy requirements for tertiary and quaternary structure, peptides were first renatured (where necessary) to restore triple-helical configuration and then polymerised. Fragmentation with mammalian collagenase indicated the presence of platelet-reactive sites in both the N-terminal three-quarter and C-terminal one quarter fragment of the collagen molecule. Cleavage with cyanogen bromide indicated the presence in the constituent alpha 1(I)-chain of at least four platelet-reactive sites. Our results suggest a relatively wide distribution of platelet-binding sites situated throughout the length of the collagen (type I) molecule, each probably of relatively low affinity and low structural specificity, at least in terms of amino acid sequence, and probably of a similar nature to those that might be expected to exist in any collagen-like species.

The interaction of von Willebrand factor (vWf) with collagen: investigation of vWf-binding sites in the collagen molecule.

Following fragmentation of the collagen molecule, we have examined the ability of the isolated fragments to bind vWf. In view of the importance of collagen tertiary and quaternary structure for binding, fragments were first renatured to restore triple-helical conformation and then polymerized. Results indicate the presence of specific vWf-binding sites in both the alpha 1(I)- and alpha 2(I)-chains of type I collagen. Cleavage of the alpha 1(I)-chain with cyanogen bromide suggests the presence of at least four (conceivably several more) binding sites implying a wide distribution of sites along the length of the collagen type I molecule. Collagen type III appears to possess a similar wide distribution of sites. Chemical modification of specific amino acid residues indicates that interaction involves arginyl residues in collagen and carboxyl groups in vWf. Although interaction between fibronectin and collagen fibres also involves collagen arginyl residues and carboxyl groups in fibronectin (authors' unpublished results), fibronectin does not compete with vWf in the binding to collagen fibres.

The tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, inhibit the release of (3H)arachidonate from human platelets stimulated by thrombin or collagen.

We have investigated the effects of the tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, on [3H]arachidonic acid release from human platelets. Both tyrosine kinase inhibitors blocked, in a dose-dependent manner, the release of arachidonic acid stimulated by thrombin or suspensions of collagen fibres. Blockade by the tyrosine kinase inhibitors occurred early in the arachidonate release time course. Both genistein and methyl 2,5-dihydroxycinnamate also inhibited tyrosine phosphorylation in platelets. The inhibitors were specific in that they did not affect protein kinase C activity, ATP levels or mobilization of Ca2+ from internal stores. These findings suggest a role for tyrosine kinase activity in the regulation of phospholipase A2 in platelets stimulated by the physiological ligands, thrombin and collagen.

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.

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.