Parvathy Sasikumar

Platelet protein disulfide isomerase is required for thrombus formation but not for hemostasis in mice.

Protein disulfide isomerase (PDI) derived from intravascular cells is required for thrombus formation. However, it remains unclear whether platelet PDI contributes to the process. Using platelet-specific PDI-deficient mice, we demonstrate that PDI-null platelets have defects in aggregation and adenosine triphosphate secretion induced by thrombin, collagen, and adenosine diphosphate. Such defects were rescued by wild-type but not mutant PDI, indicating that the isomerase activity of platelet surface PDI is critical for the regulatory effect. PDI-deficient platelets expressed increased levels of intracellular ER protein 57 (ERp57) and ERp72. Platelet PDI regulated αIIbβ3 integrin activation but not P-selectin exposure, Ca(2+) mobilization, β3-talin1 interaction, or platelet spreading on immobilized fibrinogen. Inhibition of ERp57 further diminished αIIbβ3 integrin activation and aggregation of activated PDI-deficient platelets, suggesting distinct roles of PDI and ERp57 in platelet functions. We found that platelet PDI is important for thrombus formation on collagen-coated surfaces under shear. Intravital microscopy demonstrates that platelet PDI is important for platelet accumulation but not initial adhesion and fibrin generation following laser-induced arteriolar injury. Tail bleeding time in platelet-specific PDI-deficient mice were not significantly increased. Our results provide important evidence that platelet PDI is essential for thrombus formation but not for hemostasis in mice.

Gap Junctions and Connexin Hemichannels Underpin Hemostasis and Thrombosis

Background— Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair. Methods and Results— We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses before platelet-platelet contact and reduced laser-induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion, and clot retraction, indicating an important role for connexin37 hemichannels and gap junctions in platelet thrombus function. Conclusions— Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of hemostasis and thrombosis and represent potential therapeutic targets.

The platelet‐surface thiol isomerase enzyme ERp57 modulates platelet function

Summary.  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. Aim: 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.

LXR as a novel antithrombotic target

Liver X receptors (LXRs) are transcription factors involved in the regulation of cholesterol homeostasis. LXR ligands have athero-protective properties independent of their effects on cholesterol metabolism. Platelets are involved in the initiation of atherosclerosis and despite being anucleate express nuclear receptors. We hypothesized that the athero-protective effects of LXR ligands could be in part mediated through platelets and therefore explored the potential role of LXR in platelets. Our results show that LXR-β is present in human platelets and the LXR ligands, GW3965 and T0901317, modulated nongenomically platelet aggregation stimulated by a range of agonists. GW3965 caused LXR to associate with signaling components proximal to the collagen receptor, GPVI, suggesting a potential mechanism of LXR action in platelets that leads to diminished platelet responses. Activation of platelets at sites of atherosclerotic lesions results in thrombosis preceding myocardial infarction and stroke. Using an in vivo model of thrombosis in mice, we show that GW3965 has antithrombotic effects, reducing the size and the stability of thrombi. The athero-protective effects of GW3965, together with its novel antiplatelet/thrombotic effects, indicate LXR as a potential target for prevention of athero-thrombotic disease.

Antithrombotic actions of statins involve PECAM-1 signaling

Statins are widely prescribed cholesterol-lowering drugs that are a first-line treatment of coronary artery disease and atherosclerosis, reducing the incidence of thrombotic events such as myocardial infarction and stroke. Statins have been shown to reduce platelet activation, although the mechanism(s) through which this occurs is unclear. Because several of the characteristic effects of statins on platelets are shared with those elicited by the inhibitory platelet adhesion receptor PECAM-1 (platelet endothelial cell adhesion molecule-1), we investigated a potential connection between the influence of statins on platelet function and PECAM-1 signaling. Statins were found to inhibit a range of platelet functional responses and thrombus formation in vitro and in vivo. Notably, these effects of statins on platelet function in vitro and in vivo were diminished in PECAM-1(-/-) platelets. Activation of PECAM-1 signaling results in its tyrosine phosphorylation, the recruitment and activation of tyrosine phosphatase SHP-2, the subsequent binding of phosphoinositol 3-kinase (PI3K), and diminished PI3K signaling. Statins resulted in the stimulation of these events, leading to the inhibition of Akt activation. Together, these data provide evidence for a fundamental role of PECAM-1 in the inhibitory effects of statins on platelet activation, which may explain some of the pleiotropic actions of these drugs.

Integrin-linked kinase regulates the rate of platelet activation and is essential for the formation of stable thrombi

Integrin‐linked kinase (ILK) and its associated complex of proteins are involved in many cellular activation processes, including cell adhesion and integrin signaling. We have previously demonstrated that mice with induced platelet ILK deficiency show reduced platelet activation and aggregation, but only a minor bleeding defect. Here, we explore this apparent disparity between the cellular and hemostatic phenotypes.

Farnesoid X Receptor and Its Ligands Inhibit the Function of Platelets

Objective—Although initially seemingly paradoxical because of the lack of nucleus, platelets possess many transcription factors that regulate their function through DNA-independent mechanisms. These include the farnesoid X receptor (FXR), a member of the superfamily of ligand-activated transcription factors, that has been identified as a bile acid receptor. In this study, we show that FXR is present in human platelets and FXR ligands, GW4064 and 6&agr;-ethyl-chenodeoxycholic acid, modulate platelet activation nongenomically. Approach and Results—FXR ligands inhibited the activation of platelets in response to stimulation of collagen or thrombin receptors, resulting in diminished intracellular calcium mobilization, secretion, fibrinogen binding, and aggregation. Exposure to FXR ligands also reduced integrin &agr;IIb&bgr;3 outside-in signaling and thereby reduced the ability of platelets to spread and to stimulate clot retraction. FXR function in platelets was found to be associated with the modulation of cyclic guanosine monophosphate levels in platelets and associated downstream inhibitory signaling. Platelets from FXR-deficient mice were refractory to the actions of FXR agonists on platelet function and cyclic nucleotide signaling, firmly linking the nongenomic actions of these ligands to the FXR. Conclusions—This study provides support for the ability of FXR ligands to modulate platelet activation. The atheroprotective effects of GW4064, with its novel antiplatelet effects, indicate FXR as a potential target for the prevention of atherothrombotic disease.

RXR ligands negatively regulate thrombosis and hemostasis

Objective— Platelets have been found to express intracellular nuclear receptors including the retinoid X receptors (RXR&agr; and RXR&bgr;). Treatment of platelets with ligands of RXR has been shown to inhibit platelet responses to ADP and thromboxane A2; however, the effects on responses to other platelet agonists and the underlying mechanism have not been fully characterized. Approach and Results— The effect of 9-cis-retinoic acid, docosahexaenoic acid and methoprene acid on collagen receptor (glycoprotein VI [GPVI]) agonists and thrombin-stimulated platelet function; including aggregation, granule secretion, integrin activation, calcium mobilization, integrin &agr;IIb&bgr;3 outside-in signaling and thrombus formation in vitro and in vivo were determined. Treatment of platelets with RXR ligands resulted in attenuation of platelet functional responses after stimulation by GPVI agonists or thrombin and inhibition of integrin &agr;IIb&bgr;3 outside-in signaling. Treatment with 9-cis-retinoic acid caused inhibition of thrombus formation in vitro and an impairment of thrombosis and hemostasis in vivo. Both RXR ligands stimulated protein kinase A activation, measured by VASP S157 phosphorylation, that was found to be dependent on both cAMP and nuclear factor &kgr;-light-chain-enhancer of activated B cell activity. Conclusions— This study identifies a widespread, negative regulatory role for RXR in the regulation of platelet functional responses and thrombus formation and describes novel events that lead to the upregulation of protein kinase A, a known negative regulator of many aspects of platelet function. This mechanism may offer a possible explanation for the cardioprotective effects described in vivo after treatment with RXR ligands.

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

Essentials ERp72 is a thiol isomerase enzyme. ERp72 levels increase at the platelet surface during platelet activation. We generated a humanized monoclonal antibody which blocks ERp72 enzyme activity (anti‐ERp72). Anti‐ERp72 inhibits platelet functional responses and thrombosis.

The chaperone protein HSP47: a platelet collagen binding protein that contributes to thrombosis and haemostasis

Essentials Heat shock protein 47 (HSP47), a collagen specific chaperone is present on the platelet surface. Collagen mediated platelet function was reduced following blockade or deletion of HSP47. GPVI receptor regulated signalling was reduced in HSP47 deficient platelets. Platelet HSP47 tethers to exposed collagen thus modulating thrombosis and hemostasis.