Ras-like small GTPases in platelet biology

In a human body, the behaviour of each cell is controlled by signals like hormones or growth factors. A cell receives such messages by means of specific receptors. These transmit the messages to other proteins in the cell and so on. Such cascades control for example the pattern of active genes or metabolic responses. In this way, proliferation, differentiation, growth and cell death are tightly regulated. The complex of biochemical reactions that occurs in the cell upon signals is called signal transduction. Members of the Ras-like small GTPases protein family function as molecular switches in signalling pathways. Activating signals induce the GTP-bound form of Ras proteins. In this conformation these proteins pass the signals on to other proteins. Hydrolysis of GTP returns them into the inactive, GDP-bound version and signalling is terminated. Certain mutations in the prototype family member Ras are known which render the protein continuously active. This contributes to oncogenic transformation. The function of the family members Rap1 and Ral is largely unknown. However, they are abundant and rapidly activated upon a variety of signals in platelets. This suggests these GTPases participate in signalling that controls platelet functions. Upon activation, platelets change their shape and concomitantly start sticking to each other (adhesion) by activation of adhesion molecules (integrins). This results in the formation of a blood clot which stops bleeding. Activation under inproper conditions may result in thrombosis, heart or brain infarct. Knowledge and insight with respect to signal transduction is thus required to understand platelet behaviour (and cells in general) under normal and pathological circumstances. In this thesis, the focus has been on how Ras-like small GTPases are involved in signalling pathways as they occur in platelets. In chapter 1 the Ras proteins and their behaviour and function in blood platelets are discussed. In chapter 2 the calcium-induced Ras-independent Ral activation mechanism in platelets is studied. This research describes the identification and characterization of the Ral-specific guanine nucleotide exchange factor RalGEF2. In chapter 3, new insight is provided on the role of Rap1 in integrin-mediated cell adhesion. Rap1 is required for ?1 and ?2 integrin-mediated adhesion induced by Mn2+- or integrin activating antibodies. Treatment with Mn2+ or the activating antibodies did not induce Rap1 activation. Rap1 may fulfil a facilitating function. In chapter 4 the proposed Rap1 function in cAMP-induced PKA-independent elevation of the intracellular calcium concentration is investigated in megakaryocytes. Also in platelets Rap1 has been connected with the regulation of intracellular calcium concentration. However, no evidence was found for the involvement of Rap1 in this process. The experiments described in chapter 5 show Rap1 activation in a diversity of human blood cell lines representing megakaryocytes, monocytes, B and T lymphocytes as the consequence of shear stress. Moreover, Rap1 activity was required for ?IIb?3-mediated adhesion of human megakaryocytes. These findings suggest a connection between shear stress, Rap1 and integrin regulation, but also link Rap1 with the most important platelet integrin. Proper control of this integrin is inevitable for the correct functioning of blood platelets. This connects Rap1 with one of the most elementary processes in platelet activation

A humanized monoclonal antibody that inhibits platelet-surface ERp72 reveals a role for ERp72 in thrombosis

Background: Within the endoplasmic reticulum, thiol isomerase enzymes modulate the formation and rearrangement of disulphide bonds in newly folded proteins entering the secretory pathway to ensure correct protein folding. In addition to their intracellular importance, thiol isomerases have been recently identified to be present on the surface of a number of cell types where they are important for cell function. Several thiol isomerases are known to be present on the resting platelet surface including PDI, ERp5 and ERp57 and levels are increased following platelet activation. Inhibition of the catalytic activity of these enzymes results in diminished platelet function and thrombosis. Aim: We previously determined that ERp72 is present at the resting platelet surface and levels increase upon platelet activation, however its functional role on the cell surface was unclear. We aimed to investigate the role of ERp72 in platelet function and its role in thrombosis. Methods: Using HuCAL technology, fully humanised Fc-null anti-ERp72 antibodies were generated. Eleven antibodies were screened for their ability to inhibit ERp72 activity and the most potent inhibitory antibody (anti-ERp72) selected for further testing in platelet functional assays. Results and conclusions: Anti-ERp72 inhibited platelet aggregation, granule secretion, calcium mobilisation and integrin activation revealing an important role for extracellular ERp72 in the regulation of platelet activation. Consistent with this, infusion of anti-ERp72 into mice protected against thrombosis

Reversal of stress fibre formation by Nitric Oxide mediated RhoA inhibition leads to reduction in the height of preformed thrombi

Evidence has emerged to suggest that thrombi are dynamic structures with distinct areas of differing platelet activation and inhibition. We hypothesised that Nitric oxide (NO), a platelet inhibitor, can modulate the actin cytoskeleton reversing platelet spreading, and therefore reduce the capability of thrombi to withstand a high shear environment. Our data demonstrates that GSNO, DEANONOate, and a PKG-activating cGMP analogue reversed stress fibre formation and increased actin nodule formation in adherent platelets. This effect is sGC dependent and independent of ADP and thromboxanes. Stress fibre formation is a RhoA dependent process and NO induced RhoA inhibition, however, it did not phosphorylate RhoA at ser188 in spread platelets. Interestingly NO and PGI2 synergise to reverse stress fibre formation at physiologically relevant concentrations. Analysis of high shear conditions indicated that platelets activated on fibrinogen, induced stress fibre formation, which was reversed by GSNO treatment. Furthermore, preformed thrombi on collagen post perfused with GSNO had a 30% reduction in thrombus height in comparison to the control. This study demonstrates that NO can reverse key platelet functions after their initial activation and identifies a novel mechanism for controlling excessive thrombosis

Elucidating the role of Staphylococcus epidermidis serine-aspartate repeat protein G in platelet activation.

BACKGROUND: Staphylococcus epidermidis is a commensal of the human skin that has been implicated in infective endocarditis and infections involving implanted medical devices. S. epidermidis induces platelet aggregation by an unknown mechanism. The fibrinogen-binding protein serine-aspartate repeat protein G (SdrG) is present in 67-91% of clinical strains. OBJECTIVES: To determine whether SdrG plays a role in platelet activation, and if so to investigate the role of fibrinogen in this mechanism. METHODS: SdrG was expressed in a surrogate host, Lactococcus lactis, in order to investigate its role in the absence of other staphylococcal components. Platelet adhesion and platelet aggregation assays were employed. RESULTS: L. lactis expressing SdrG stimulated platelet aggregation (lag time: 2.9 +/- 0.5 min), whereas the L. lactis control did not. L. lactis SdrG-induced aggregation was inhibited by alpha(IIb)beta3 antagonists and aspirin. Aggregation was dependent on both fibrinogen and IgG, and the platelet IgG receptor FcgammaRIIa. Preincubation of the bacteria with Bbeta-chain fibrinopeptide inhibited aggregation (delaying the lag time six-fold), suggesting that fibrinogen acts as a bridging molecule. Platelets adhered to L. lactis SdrG in the absence of fibrinogen. Adhesion was inhibited by alpha(IIb)beta3 antagonists, suggesting that this direct interaction involves alpha(IIb)beta3. Investigation using purified fragments of SdrG revealed a direct interaction with the B-domains. Adhesion to the A-domain involved both a fibrinogen and an IgG bridge. CONCLUSION: SdrG alone is sufficient to support platelet adhesion and aggregation through both direct and indirect mechanisms

The platelet-surface thiol isomerase enzyme ERp57 modulates platelet function

Background: Thiol isomerases are a family of endoplasmic reticulum enzymes which orchestrate redox-based modifications of protein disulphide bonds. Previous studies have identified important roles for the thiol isomerases PDI and ERp5 in the regulation of normal platelet function. Objectives: Recently, we demonstrated the presence of a further five thiol isomerases at the platelet surface. In this report we aim to report the role of one of these enzymes - ERp57 in the regulation of platelet function. Methods/Results: Using enzyme activity function blocking antibodies, we demonstrate a role for ERp57 in platelet aggregation, dense granule secretion, fibrinogen binding, calcium mobilisation and thrombus formation under arterial conditions. In addition to the effects of ERp57 on isolated platelets, we observe the presence of ERp57 in the developing thrombus in vivo. Furthermore the inhibition of ERp57 function was found to reduce laser-injury induced arterial thrombus formation in a murine model of thrombosis. Conclusions: These data suggest that ERp57 is important for normal platelet function and opens up the possibility that the regulation of platelet function by a range of cell surface thiol isomerases may represent a broad paradigm for the regulation of haemostasis and thrombosis

Regulation of ADAMTS13 by proteolitic enzymes

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The platelet laminin receptor : discovery of a 67kDA receptor for laminin on the membranes of human platelets : characterisation and isolation

Previous work on the binding of resting platelets to the basement membrane glycoprotein, laminin, has identified the Ic/IIa integrin c01aplex (CD49f/CD29), also known as VLA-6, as the receptor. There exists however, another protein with a molecular weight of 67kDa, that mediates this function on other cells. It is abundantly expressed on the membranes of breast cancer cells, where it plays a key role in both the localisation at, and penetration of vascular beds, by metastases. The objectives of this study were: * The development of a micro-titre assay similar to those used in previous studies, standardised and calibrated to characterize the adhesion of unstimulated normal human platelets to laminin-coated surfaces. * To determine the effect on adhesion of platelet activation, enzymatic surface-glycoprotein removal, antibodies to specific receptors and interaction with other adhesive proteins known to bind to platelet membranes. * To establish the in vivo relevance of the experimental findings, by the assay of adhesion of glycoprotein IIb/IIIa-deficient platelets of two patients with Glanzmann's Thrombasthenia. These studies serve d to distinguish specific binding sites for laminin from the known surface receptors of platelets. The methodology used to isolate laminin receptors from the membranes of breast carcinoma cells was then applied to platelet concentrates. Membranes were obtained by centrifuging the ultrasonic lysate of a unit of platelets. These were solubilized and passed over a laminin-Sepharose column. The bound components were eluted and identified by means of SDS-gel electrophoresis, after which a concentrate was tested for laminin binding by means of dot-blot methodology. The principle contribution of this work is the finding of a 67kDa receptor for laminin on the surface membranes of platelets. The combination of the various approaches applied to characterise the adhesion of platelets to laminin, show that this is a specific, Mg²⁺-dependent process, inhibited by Ca²⁺ and not enhanced by platelet activation. Adhesion was decreased by proteolysis with trypsin and chymotrypsin, showing that the adhesion is mediated by a surface glycoprotein. Proteolysis with the Serratia marcescens metalloprotease, which cleaves off glycoprotein lb, did not affect adhesion, proving that this well-known receptor for platelet adhesion is not involved in the adhesion. The receptors GPIV and glycocalicin were also excluded, as the presence of antibodies to these receptors had no effect. Prior incubation with fibrinogen or von Willebrand factor, which binds to specific receptors on the platelet membrane, inhibited adhesion, most likely due to spatial interference with the receptor site for laminin. The presence of the tetrapeptide recognised by the membrane receptors for many adhesive proteins, RGDS, at concentrations of up to 1mM, had no effect. The platelets of the two subjects with Glanzmann's Thrombasthenia adhered normally, definitively ruling out the involvement of GPIIb/IIIa, which is absent from these platelets. The isolation process recovered a membrane component from the laminin-Sepharose column with an elution pattern identical to that for the well characterised 67kDa receptor for laminin on the surface of breast carcinoma cells. They have the same molecular weights in both the reduced (67kDa) and non-reduced (53kDa) states. Blot identification demonstrated laminin binding by the eluate. In the last part of the work, collaborative studies using more sophisticated methodology have confirmed that platelet receptors for laminin play a role in their adhesion to living tissue. Anti-laminin Fab antibodies significantly decreased the adhesion when whole blood was perfused over isolated rabbit aortic segments. That these receptors are identical to the 67kDa receptor of breast carcinoma cells was shown by the specific, high affinity binding of antibodies directed at the carcinoma receptors to the surface of platelets when examined by flow cytometry. In addition, they inhibit platelet adhesion by 50-60% in the micro-titre assay. It is proposed that both the VLA-6 and the 67kDa receptors are required for platelet adhesion to laminin, possibly as a two-stage process, similar to the systems for adhesion to von Willebrand factor, where binding is initially to GPIb, followed by binding to GPIIb/IIIa. The possible relevance of this receptor in the pathophysiology of the metastatic process is discussed

GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets.

G protein-coupled receptors (GPCRs) mediate the majority of platelet activation in response to agonists. However, questions remain regarding the mechanisms that provide negative feedback toward activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6-/- mice are larger than in wild-type (WT) controls during the early stages of thrombus formation, with a rapid increase in platelet accumulation at the site of injury. GRK6-/- platelets have increased platelet activation, but in an agonist-selective manner. Responses to PAR4 agonist or adenosine 5\u27-diphosphate stimulation in GRK6-/- platelets are increased compared with WT littermates, whereas the response to thromboxane A2 (TxA2) is normal. Underlying these changes in GRK6-/- platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that human platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase in the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Taken together, these data show that GRK6 regulates the hemostatic response to injury through PAR- and P2Y12-mediated effects, helping to limit the rate of platelet activation during thrombus growth and prevent inappropriate platelet activation

A comprehensive evaluation of the activity and selectivity profile of ligands for RGD-binding integrins

Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC50-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins avß3, avß5, avß6, avß8, a5ß1, aIIbß3, using homogenous ELISA-like solid phase binding assay.Postprint (published version

Proteases of the neutrophil membrane represent an alternative fibrinolytic pathway to that mediated by plasmin

The cellular components of the blood, which become associated with fibrin through specific cellular adhesive processes, play a significant role in the breakdown of fibrin. Fibrinolysis by elastase and cathepsin G, enzymes present within the azurophilic granules of the neutrophil, has previously been shown. Recent studies have demonstrated neutrophil-mediated fibrinogenolysis by a membrane-associated protease which suggests that proteases connected with the neutrophil membrane might also be capable of clot dissolution. Investigations showed that neutrophil-mediated clot lysis was effected by a membrane-associated serine protease that can be dissociated by SDS-PAGE to bands that migrate to apparent molecular weights of 501 kDa, 398 kDa, 316 kDa, 245 kDa and 209 kDa. This degradation was distinct from that produced by plasmin, neutrophil lysosomal enzymes and purified human neutrophil elastase and enhanced the action of plasmin in clot solubilization. Preincubation of neutrophils with monoclonal antibodies directed against the CD 11 c/CD 18 integrin was able to significantly inhibit neutrophil membrane-dependent fibrinolytic activity. Upregulation of enzyme activity occurred following association of fibrin substrate with the cell membrane and was dependent on the activation of cellular kinases, in particular protein kinase C. Fibrin products generated by neutrophil membrane proteolytic activity were found to possess biological activity. The low molecular weight peptides effected substantial inhibition of thrombin-induced platelet aggregation while the presence of the higher molecular weight material could partially overcome platelet-induced resistance to plasmic lysis. No modulation of platelet-mediated fibrin clot retraction was observed using these same fibrin products. Neutrophil lysosomal enzyme activity was shown to further degrade the end products of plasmic fibrin degradation into low molecular weight material, followed by reassembly of higher molecular weight products in a process dependent on calcium and factor XIII. The reformed products have a similar molecular weight to those produced by plasmic lysis of fibrin, as well as a putative crosslinked site. However, the isoelectric point of these reformed products indicates they are distinctly different from plasmin-derived fibrin products. These reassembled products were recognized by a monoclonal antibody raised against D-dimer. Processing by neutrophils of the end products of plasmic fibrin degradation may have the potential for modulating the immune response as well as compromising the predictive value of tests measuring D-dimer, used as a laboratory marker of a number of thromboembolic disorders encountered in clinical practice