Delineating Zinc Influx Mechanisms during Platelet Activation.

Zinc (Zn2+) is released by platelets during a hemostatic response to injury. Extracellular zinc ([Zn2+]o) initiates platelet activation following influx into the platelet cytosol. However, the mechanisms that permit Zn2+ influx are unknown. Fluctuations in intracellular zinc ([Zn2+]i) were measured in fluozin-3-loaded platelets using fluorometry and flow cytometry. Platelet activation was assessed using light transmission aggregometry. The detection of phosphoproteins was performed by Western blotting. [Zn2+]o influx and subsequent platelet activation were abrogated by blocking the sodium/calcium exchanged, TRP channels, and ZIP7. Cation store depletion regulated Zn2+ influx. [Zn2+]o stimulation resulted in the phosphorylation of PKC substates, MLC, and ß3 integrin. Platelet activation via GPVI or Zn2+ resulted in ZIP7 phosphorylation in a casein kinase 2-dependent manner and initiated elevations of [Zn2+]i that were sensitive to the inhibition of Orai1, ZIP7, or IP3R-mediated pathways. These data indicate that platelets detect and respond to changes in [Zn2+]o via influx into the cytosol through TRP channels and the NCX exchanger. Platelet activation results in the externalization of ZIP7, which further regulates Zn2+ influx. Increases in [Zn2+]i contribute to the activation of cation-dependent enzymes. Sensitivity of Zn2+ influx to thapsigargin indicates a store-operated pathway that we term store-operated Zn2+ entry (SOZE). These mechanisms may affect platelet behavior during thrombosis and hemostasis.

Zinc: an endogenous and exogenous regulator of platelet function during hemostasis and thrombosis.

Zinc (Zn2+) is an essential micronutrient and the second most abundant trace metal in the human body. The important role that Zn2+ plays in hemostasis is exemplified by platelet-related bleeding phenotypes coinciding with dietary Zn2+ deficiency. These phenotypes are rectified upon Zn2+ supplementation. Labile (unbound) Zn2+ is present in the plasma at micromolar levels, but is also detected in atherosclerotic plaques, and released from platelet α granules. Therefore, it is likely that localized Zn2+ concentrations are higher at sites of thrombosis and hemostasis. Exogenous Zn2+ is a regulator of the hemostatic responses, with roles during coagulation and platelet activation. Extracellular Zn2+ gains access to the platelet cytosol and induces full platelet activation at high concentrations, and potentiates platelets to activation by conventional agonists at lower concentrations. Zn2+-induced platelet activation is dependent on PKC and integrin αIIbß3, and is associated with tyrosine phosphorylation of platelet proteins. Agonist evoked platelet activation results in intracellular Zn2+ ([Zn2+]i) fluctuations that are sensitive to the platelet redox state. Increases in [Zn2+]i correlate with activation responses, including shape change, granule release, αIIbß3 activation and phosphatidyl-serine exposure, consistent with a role as a second messenger. This review provides insight into the numerous demonstrated and potential roles for Zn2+ in platelet function during thrombosis and hemostasis, highlighting its increasing acceptance as an intracellular and extracellular platelet regulatory agent.

Agonist-Evoked Increases in Intra-Platelet Zinc Couple to Functional Responses.

BACKGROUND: Zinc (Zn2+) is an essential trace element that regulates intracellular processes in multiple cell types. While the role of Zn2+ as a platelet agonist is known, its secondary messenger activity in platelets has not been demonstrated. OBJECTIVES: This article determines whether cytosolic Zn2+ concentrations ([Zn2+]i) change in platelets in response to agonist stimulation, in a manner consistent with a secondary messenger, and correlates the effects of [Zn2+]i changes on activation markers. METHODS: Changes in [Zn2+]i were quantified in Fluozin-3 (Fz-3)-loaded washed, human platelets using fluorometry. Increases in [Zn2+]i were modelled using Zn2+-specific chelators and ionophores. The influence of [Zn2+]i on platelet function was assessed using platelet aggregometry, flow cytometry and Western blotting. RESULTS: Increases of intra-platelet Fluozin-3 (Fz-3) fluorescence occurred in response to stimulation by cross-linked collagen-related peptide (CRP-XL) or U46619, consistent with a rise of [Zn2+]i. Fluoresence increases were blocked by Zn2+ chelators and modulators of the platelet redox state, and were distinct from agonist-evoked [Ca2+]i signals. Stimulation of platelets with the Zn2+ ionophores clioquinol (Cq) or pyrithione (Py) caused sustained increases of [Zn2+]i, resulting in myosin light chain phosphorylation, and cytoskeletal re-arrangements which were sensitive to cytochalasin-D treatment. Cq stimulation resulted in integrin αIIbß3 activation and release of dense, but not α, granules. Furthermore, Zn2+-ionophores induced externalization of phosphatidylserine. CONCLUSION: These data suggest that agonist-evoked fluctuations in intra-platelet Zn2+ couple to functional responses, in a manner that is consistent with a role as a secondary messenger. Increased intra-platelet Zn2+ regulates signalling processes, including shape change, αIIbß3 up-regulation and dense granule release, in a redox-sensitive manner.

Platelet Isolation and Activation Assays.

Platelets regulate hemostasis and are the key determinants of pathogenic thrombosis following atherosclerotic plaque rupture. Platelets circulate in an inactive state, but become activated in response to damage to the endothelium, which exposes thrombogenic material such as collagen to the blood flow. Activation results in a number of responses, including secretion of soluble bioactive molecules via the release of alpha and dense granules, activation of membrane adhesion receptors, release of microparticles, and externalization of phosphatidylserine. These processes facilitate firm adhesion to sites of injury and the recruitment and activation of other platelets and leukocytes, resulting in aggregation and thrombus formation. Platelet activation drives the hemostatic response, and also contributes to pathogenic thrombus formation. Thus, quantification of platelet-associated responses is key to many pathophysiologically relevant processes. Here we describe protocols for isolating, counting, and activating platelets, and for the rapid quantification of cell surface proteins using flow cytometry.

MMP-13 binds to platelet receptors αIIbß3 and GPVI and impairs aggregation and thrombus formation.

BACKGROUND: Acute thrombotic syndromes lead to atherosclerotic plaque rupture with subsequent thrombus formation, myocardial infarction and stroke. Following rupture, flowing blood is exposed to plaque components, including collagen, which triggers platelet activation and aggregation. However, plaque rupture releases other components into the surrounding vessel which have the potential to influence platelet function and thrombus formation. OBJECTIVES: Here we sought to elucidate whether matrix metalloproteinase-13 (MMP-13), a collagenolytic metalloproteinase up-regulated in atherothrombotic and inflammatory conditions, affects platelet aggregation and thrombus formation. RESULTS: We demonstrate that MMP-13 is able to bind to platelet receptors alphaIIbbeta3 (αIIbß3) and platelet glycoprotein (GP)VI. The interactions between MMP-13, GPVI and αIIbß3 are sufficient to significantly inhibit washed platelet aggregation and decrease thrombus formation on fibrillar collagen. CONCLUSIONS: Our data demonstrate a role for MMP-13 in the inhibition of both platelet aggregation and thrombus formation in whole flowing blood, and may provide new avenues of research into the mechanisms underlying the subtle role of MMP-13 in atherothrombotic pathologies.

Stapled peptides as a new technology to investigate protein-protein interactions in human platelets.

Platelets are blood cells with numerous crucial pathophysiological roles in hemostasis, cardiovascular thrombotic events and cancer metastasis. Platelet activation requires the engagement of intracellular signalling pathways that involve protein-protein interactions (PPIs). A better understanding of these pathways is therefore crucial for the development of selective anti-platelet drugs. New strategies for studying PPIs in human platelets are required to overcome limitations associated with conventional platelet research methods. For example, small molecule inhibitors can lack selectivity and are often difficult to design and synthesise. Additionally, development of transgenic animal models is costly and time-consuming and conventional recombinant techniques are ineffective due to the lack of a nucleus in platelets. Herein, we describe the generation of a library of novel, functionalised stapled peptides and their first application in the investigation of platelet PPIs. Moreover, the use of platelet-permeable stapled Bim BH3 peptides confirms the part of Bim in phosphatidyl-serine (PS) exposure and reveals a role for the Bim protein in platelet activatory processes. Our work demonstrates that functionalised stapled peptides are a complementary alternative to conventional platelet research methods, and could make a significant contribution to the understanding of platelet signalling pathways and hence to the development of anti-platelet drugs.

Thrombospondin-1 promotes matrix homeostasis by interacting with collagen and lysyl oxidase precursors and collagen cross-linking sites.

Fibrillar collagens of the extracellular matrix are critical for tissue structure and physiology; however, excessive or abnormal deposition of collagens is a defining feature of fibrosis. Regulatory mechanisms that act on collagen fibril assembly potentially offer new targets for antifibrotic treatments. Tissue weakening, altered collagen fibril morphologies, or both, are shared phenotypes of mice lacking matricellular thrombospondins. Thrombospondin-1 (TSP1) plays an indirect role in collagen homeostasis through interactions with matrix metalloproteinases and transforming growth factor-ß1 (TGF-ß1). We found that TSP1 also affects collagen fibril formation directly. Compared to skin from wild-type mice, skin from Thbs1-/- mice had reduced collagen cross-linking and reduced prolysyl oxidase (proLOX) abundance with increased conversion to catalytically active LOX. In vitro, TSP1 bound to both the C-propeptide domain of collagen I and the highly conserved KGHR sequences of the collagen triple-helical domain that participate in cross-linking. TSP1 also bound to proLOX and inhibited proLOX processing by bone morphogenetic protein-1. In human dermal fibroblasts (HDFs), TSP1 and collagen I colocalized in intracellular vesicles and on extracellular collagen fibrils, whereas TSP1 and proLOX colocalized only in intracellular vesicles. Inhibition of LOX-mediated collagen cross-linking did not prevent the extracellular association between collagen and TSP1; however, treatment of HDFs with KGHR-containing, TSP1-binding, triple-helical peptides disrupted the collagen-TSP1 association, perturbed the collagen extracellular matrix, and increased myofibroblastic differentiation in a manner that depended on TGF-ß receptor 1. Thus, the extracellular KGHR-dependent interaction of TSP1 with fibrillar collagens contributes to fibroblast homeostasis.

Anti-thrombotic efficacy of S007-867: Pre-clinical evaluation in experimental models of thrombosis in vivo and in vitro.

Pharmacological inhibition of platelet collagen interaction is a promising therapeutic strategy to treat intra-vascular thrombosis. S007-867 is a novel synthetic inhibitor of collagen-induced platelet aggregation. It has shown better antithrombotic protection than aspirin and clopidogrel with minimal bleeding tendency in mice. The present study is aimed to systematically investigate the antithrombotic efficacy of S007-867 in comparison to aspirin and clopidogrel in vivo and to delineate its mechanism of action in vitro. Aspirin, clopidogrel, and S007-867 significantly reduced thrombus weight in arterio-venous (AV) shunt model in rats. In mice, following ferric chloride induced thrombosis in either carotid or mesenteric artery; S007-867 significantly prolonged the vessel occlusion time (1.2-fold) and maintained a sustained blood flow velocity for >30 min. Comparatively, clopidogrel showed significant prolongation in TTO (1.3-fold) while aspirin remained ineffective. Both S007-867 and aspirin did not alter bleeding time in either kidney or spleen injury models, and thus maintained hemostasis, while clopidogrel showed significant increase in spleen bleeding time (1.7-fold). The coagulation parameters namely thrombin time, prothrombin time or activated partial thromboplastin time remained unaffected even at high concentration of S007-867 (300 µM), thus implying its antithrombotic effect to be primarily platelet mediated. S007-867 significantly inhibited collagen-mediated platelet adhesion and aggregation in mice ex-vivo. Moreover, when blood was perfused over a highly thrombogenic combination of collagen mimicking peptides like CRP-GFOGER-VWF-III, S007-867 significantly reduced total thrombus volume or ZV50 (53.4 ±â€¯5.7%). Mechanistically, S007-867 (10-300 µM) inhibited collagen-induced ATP release, thromboxane A2 (TxA2) generation, intra-platelet [Ca+2] flux and global tyrosine phosphorylation including PLCγ2. Collectively the present study highlights that S007-867 is a novel synthetic inhibitor of collagen induced platelet activation, that effectively maintains blood flow velocity and delays vascular occlusion. It inhibits thrombogenesis without compromising hemostasis. Therefore, S007-867 may be further developed for the treatment of thrombotic disorders in clinical settings.

Extracellular chloride is required for efficient platelet aggregation.

Anion channels perform a diverse range of functions and have been implicated in ATP release, volume regulation, and phosphatidylserine exposure. Platelets have been shown to express several anion channels but their function is incompletely understood. Due to a paucity of specific pharmacological blockers, we investigated the effect of extracellular chloride substitution on platelet activation using aggregometry and flow cytometry. In the absence of extracellular chloride, we observed a modest reduction of the maximum aggregation response to thrombin or collagen-related peptide. However, the rate of aggregation was substantially reduced in a manner that was dependent on the extracellular chloride concentration and aggregation in the absence of chloride was noticeably biphasic, indicative of impaired secondary signaling. This was further investigated by targeting secondary agonists with aspirin and apyrase or by blockade of the ADP receptor P2Y12. Under these conditions, the rates of aggregation were comparable to those recorded in the absence of extracellular chloride. Finally, we assessed platelet granule release by flow cytometry and report a chloride-dependent element of alpha, but not dense, granule secretion. Taken together these data support a role for anion channels in the efficient induction of platelet activation, likely via enhancement of secondary signaling pathways.

Differential integrin activity mediated by platelet collagen receptor engagement under flow conditions.

The platelet receptors glycoprotein (Gp)VI, integrin α2ß1 and GpIb/V/IX mediate platelet adhesion and activation during thrombogenesis. Increases of intracellular Ca2+ ([Ca2+]i) are key signals during platelet activation; however, their relative importance in coupling different collagen receptors to functional responses under shear conditions remains unclear. To study shear-dependent, receptor-specific platelet responses, we used collagen or combinations of receptor-specific collagen-mimetic peptides as substrates for platelet adhesion and activation in whole human blood under arterial flow conditions and compared real-time and endpoint parameters of thrombus formation alongside [Ca2+]i measurements using confocal imaging. All three collagen receptors coupled to [Ca2+]i signals, but these varied in amplitude and temporal pattern alongside variable integrin activation. GpVI engagement produced large, sustained [Ca2+]i signals leading to real-time increases in integrins α2ß1- and αIIbß3-mediated platelet adhesion. αIIbß3-dependent platelet aggregation was dependent on P2Y12 signalling. Co-engagement of α2ß1 and GpIb/V/IX generated transient [Ca2+]i spikes and low amplitude [Ca2+]i responses that potentiated GpVI-dependent [Ca2+]i signalling. Therefore α2ß1, GpIb/V/IX and GpVI synergise to generate [Ca2+]i signals that regulate platelet behaviour and thrombus formation. Antagonism of secondary signalling pathways reveals distinct, separate roles for αIIbß3 in stable platelet adhesion and aggregation.

Zinc is a transmembrane agonist that induces platelet activation in a tyrosine phosphorylation-dependent manner.

Following platelet adhesion and primary activation at sites of vascular injury, secondary platelet activation is induced by soluble platelet agonists, such as ADP, ATP, thrombin and thromboxane. Zinc ions are also released from platelets and damaged cells and have been shown to act as a platelet agonist. However, the mechanism of zinc-induced platelet activation is not well understood. Here we show that exogenous zinc gains access to the platelet cytosol and induces full platelet aggregation that is dependent on platelet protein tyrosine phosphorylation, PKC and integrin αIIbß3 activity and is mediated by granule release and secondary signalling. ZnSO4 increased the binding affinity of GpVI, but not integrin α2ß1. Low concentrations of ZnSO4 potentiated platelet aggregation by collagen-related peptide (CRP-XL), thrombin and adrenaline. Chelation of intracellular zinc reduced platelet aggregation induced by a number of different agonists, inhibited zinc-induced tyrosine phosphorylation and inhibited platelet activation in whole blood under physiologically relevant flow conditions. Our data are consistent with a transmembrane signalling role for zinc in platelet activation during thrombus formation.

Dynamic analysis of platelet deposition to resolve platelet adhesion receptor activity in whole blood at arterial shear rate.

Platelet activation is traditionally quantified using turbidimetric aggregometry, which reflects integrin αIIbß3 activity, an important determinant of platelet function during pathophysiological thrombus formation. However, aggregometry does not recreate the shear conditions prevailing during thrombosis in vivo. Here we describe novel whole-frame analysis of real-time video microscopy to quantify platelet adhesion receptor activity under shear in parallel-plate flow chambers. We demonstrate that the rate of change of surface coverage (designated Platelet Population Mobility, PM) quantifies platelet mobility. On collagen, PM falls exponentially to a low level, corresponding to firm platelet adhesion, while on other substrates, PM remains high. Different receptor-specific thrombogenic surfaces reveal that the PM time constant reflects real-time changes in integrins αIIbß3 and α2ß1 activity. This ensemble kinetic analysis has the potential to provide valuable diagnostic information about platelet thrombus formation with both academic and clinical applications.

Identification of platelet function defects by multi-parameter assessment of thrombus formation.

Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6ß1, αIIbß3>α2ß1>CD36, α5ß1, αvß3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

Pericellular Ca(2+) recycling potentiates thrombin-evoked Ca(2+) signals in human platelets.

We have previously demonstrated that Na(+)/Ca(2+) exchangers (NCXs) potentiate Ca(2+) signaling evoked by thapsigargin in human platelets, via their ability to modulate the secretion of autocoids from dense granules. This link was confirmed in platelets stimulated with the physiological agonist, thrombin, and experiments were performed to examine how Ca(2+) removal by the NCX modulates platelet dense granule secretion. In cells loaded with the near-membrane indicator FFP-18, thrombin stimulation was observed to elicit an NCX-dependent accumulation of Ca(2+) in a pericellular region around the platelets. To test whether this pericellular Ca(2+) accumulation might be responsible for the influence of NCXs over platelet function, platelets were exposed to fast Ca(2+) chelators or had their glycocalyx removed. Both manipulations of the pericellular Ca(2+) rise reduced thrombin-evoked Ca(2+) signals and dense granule secretion. Blocking Ca(2+)-permeable ion channels had a similar effect, suggesting that Ca(2+) exported into the pericellular region is able to recycle back into the platelet cytosol. Single cell imaging with extracellular Fluo-4 indicated that thrombin-evoked rises in extracellular [Ca(2+)] occurred within the boundary described by the cell surface, suggesting their presence within the open canalicular system (OCS). FFP-18 fluorescence was similarly distributed. These data suggest that upon thrombin stimulation, NCX activity creates a rise in [Ca(2+)] within the pericellular region of the platelet from where it recycles back into the platelet cytosol, acting to both accelerate dense granule secretion and maintain the initial rise in cytosolic [Ca(2+)].

Constitutive dimerization of glycoprotein VI (GPVI) in resting platelets is essential for binding to collagen and activation in flowing blood.

The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (K(d)) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)(10)-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ~29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ~39 and ~44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation.

Mapping of potent and specific binding motifs, GLOGEN and GVOGEA, for integrin α1ß1 using collagen toolkits II and III.

Integrins are well characterized cell surface receptors for extracellular matrix proteins. Mapping integrin-binding sites within the fibrillar collagens identified GFOGER as a high affinity site recognized by α2ß1, but with lower affinity for α1ß1. Here, to identify specific ligands for α1ß1, we examined binding of the recombinant human α1 I domain, the rat pheochromocytoma cell line (PC12), and the rat glioma Rugli cell line to our collagen Toolkit II and III peptides using solid-phase and real-time label-free adhesion assays. We observed Mg(2+)-dependent binding of the α1 I domain to the peptides in the following rank order: III-7 (GLOGEN), II-28 (GFOGER), II-7 and II-8 (GLOGER), II-18 (GAOGER), III-4 (GROGER). PC12 cells showed a similar profile. Using antibody blockade, we confirmed that binding of PC12 cells to peptide III-7 was mediated by integrin α1ß1. We also identified a new α1ß1-binding activity within peptide II-27. The sequence GVOGEA bound weakly to PC12 cells and strongly to activated Rugli cells or to an activated α1 I domain, but not to the α2 I domain or to C2C12 cells expressing α2ß1 or α11ß1. Thus, GVOGEA is specific for α1ß1. Although recognized by both α2ß1 and α11ß1, GLOGEN is a better ligand for α1ß1 compared with GFOGER. Finally, using biosensor assays, we show that although GLOGEN is able to compete for the α1 I domain from collagen IV (IC(50) ∼3 µm), GFOGER is much less potent (IC(50) ∼90 µm), as shown previously. These data confirm the selectivity of GFOGER for α2ß1 and establish GLOGEN as a high affinity site for α1ß1.

Crosslinking and composition influence the surface properties, mechanical stiffness and cell reactivity of collagen-based films.

This study focuses on determining the effect of varying the composition and crosslinking of collagen-based films on their physical properties and interaction with myoblasts. Films composed of collagen or gelatin and crosslinked with a carbodiimide were assessed for their surface roughness and stiffness. These samples are significant because they allow variation of physical properties as well as offering different recognition motifs for cell binding. Cell reactivity was determined by the ability of myoblastic C2C12 and C2C12-α2+ cell lines (with different integrin expression) to adhere to and spread on the films. Significantly, crosslinking reduced the cell reactivity of all films, irrespective of their initial composition, stiffness or roughness. Crosslinking resulted in a dramatic increase in the stiffness of the collagen film and also tended to reduce the roughness of the films (R(q) = 0.417 ± 0.035 µm, E = 31 ± 4.4 MPa). Gelatin films were generally smoother and more compliant than comparable collagen films (R(q) = 7.9 ± 1.5 nm, E = 15 ± 3.1 MPa). The adhesion of α2-positive cells was enhanced relative to the parental C2C12 cells on collagen compared with gelatin films. These results indicate that the detrimental effect of crosslinking on cell response may be due to the altered physical properties of the films as well as a reduction in the number of available cell binding sites. Hence, although crosslinking can be used to enhance the mechanical stiffness and reduce the roughness of films, it reduces their capacity to support cell activity and could potentially limit the effectiveness of the collagen-based films and scaffolds.

The properties conferred upon triple-helical collagen-mimetic peptides by the presence of cysteine residues.

Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)(10). With Gly-Pro-Cys triplets appended to both of its termini, designated CRP(cys), chemical cross-linking using heterobifunctional reagents generates CRP(cys)-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRP(cys) antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze-thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays.

The impact of factor Xa inhibition on axial dependent arterial thrombus formation triggered by a tissue factor rich surface.

This study was designed to assess the effect of Factor Xa antagonists on thrombus formation at various axial positions on a tissue factor rich surface under arterial blood flow conditions. Non-anticoagulated, flowing human blood, drawn directly from an antecubital vein, was perfused over a tissue factor coated cover slip in a parallel-plate perfusion chamber. Thrombus surface coverage, thrombus mean height and fibrin surface coverage were measured at six different axial positions by confocal microscopy. Both thrombus surface coverage and mean height decreased along the cover slip axis whereas the fibrin surface coverage increased. Pre-chamber treatment of blood with the direct Factor Xa inhibitors Razaxaban and 813893 resulted in significantly reduced thrombus and fibrin formation at all axial positions investigated (P < 0.05). Thrombus and fibrin deposition in a laminar flow chamber changed with axial position with surface coverage measurements being more reproducible than thrombus mean height. Data were more reproducible towards the centre of the flow chamber than at the extremities. Razaxaban and 813893 inhibited thrombus and fibrin formation at the highest concentrations tested. No difference in drug effect was apparent at different axial positions. In conclusion, axial position influences the degree of thrombus and fibrin deposition with measurements being less reproducible at the extremities of the flow chamber. This technique may prove useful for analysing anti-thrombotic drug effects before progression to clinical trials.