Eelo Gitz

CLEC-2 expression is maintained on activated platelets and on platelet microparticles

The C-type lectin-like receptor CLEC-2 mediates platelet activation through a hem-immunoreceptor tyrosine-based activation motif (hemITAM). CLEC-2 initiates a Src- and Syk-dependent signaling cascade that is closely related to that of the 2 platelet ITAM receptors: glycoprotein (GP)VI and FcγRIIa. Activation of either of the ITAM receptors induces shedding of GPVI and proteolysis of the ITAM domain in FcγRIIa. In the present study, we generated monoclonal antibodies against human CLEC-2 and used these to measure CLEC-2 expression on resting and stimulated platelets and on other hematopoietic cells. We show that CLEC-2 is restricted to platelets with an average copy number of ∼2000 per cell and that activation of CLEC-2 induces proteolytic cleavage of GPVI and FcγRIIa but not of itself. We further show that CLEC-2 and GPVI are expressed on CD41+ microparticles in megakaryocyte cultures and in platelet-rich plasma, which are predominantly derived from megakaryocytes in healthy donors, whereas microparticles derived from activated platelets only express CLEC-2. Patients with rheumatoid arthritis, an inflammatory disease associated with increased microparticle production, had raised plasma levels of microparticles that expressed CLEC-2 but not GPVI. Thus, CLEC-2, unlike platelet ITAM receptors, is not regulated by proteolysis and can be used to monitor platelet-derived microparticles.

Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators.

Flierl et al. show that phosphorothioate (PS) oligonucleotides activate platelets via interacting with the collagen receptor GPVI. As PS backbone modification is currently used for nucleotide-based drug candidates, the findings suggest that this widely used method may present a risk to patients in the form of arterial thrombosis.

Syk and Src Family Kinases Regulate C-type Lectin Receptor 2 (CLEC-2)-mediated Clustering of Podoplanin and Platelet Adhesion to Lymphatic Endothelial Cells

Background: The interaction of platelet CLEC-2 with Podoplanin is critical for development of the lymphatics. Results: CLEC-2 forms a central cluster upon engagement with Podoplanin, which clusters Podoplanin. Clustering is dependent on Syk and is critical for adhesion. Conclusion: Clustering regulates the interaction of platelets with lymphatic endothelial cells. Significance: These findings account for the similar lymphatic phenotype of CLEC-2- and Syk-deficient mice. The interaction of C-type lectin receptor 2 (CLEC-2) on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signaling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signaling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the center of the platelet to form a single structure. Fluorescence lifetime imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilized Podoplanin using direct stochastic optical reconstruction microscopy. These findings provide mechanistic insight by which CLEC-2 signaling promotes adhesion to Podoplanin and regulation of Podoplanin signaling, thereby contributing to lymphatic vasculature development.

Improved platelet survival after cold storage by prevention of glycoprotein Ibα clustering in lipid rafts

Background Storing platelets for transfusion at room temperature increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance after transfusion, initiated by changes in glycoprotein Ibα, the receptor for von Willebrand factor. Design and Methods: We examined the change in glycoprotein Ibα distribution using Förster resonance energy transfer by time-gated fluorescence lifetime imaging microscopy. Results Cold storage induced deglycosylation of glycoprotein Ibα ectodomain, exposing N-acetyl-Dglucosamine residues, which sequestered with GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Ibα formed clusters upon binding of 14-3-3ζ adaptor proteins to its cytoplasmic tail, a process accompanied by mitochondrial injury and phosphatidyl serine exposure. Cold storage left glycoprotein Ibα surface expression unchanged and although glycoprotein V decreased, the fall did not affect glycoprotein Ibα clustering. Prevention of glycoprotein Ibα clustering by blockade of deglycosylation and 14-3-3ζ translocation increased the survival of cold-stored platelets to above the levels of platelets stored at room temperature without compromising hemostatic functions. Conclusions We conclude that glycoprotein Ibα translocates to lipid rafts upon cold-induced deglycosylation and forms clusters by associating with 14-3-3ζ. Interference with these steps provides a means to enable cold storage of platelet concentrates in the near future.

Arachidonic acid depletion extends survival of cold-stored platelets by interfering with the [glycoprotein Ibα – 14-3-3ζ] association

Background Cold storage of platelets reduces bacterial growth and preserves their hemostatic properties better than current procedures do. However, storage at 0°C induces [14-3-3ζ-glycoprotein Ibα] association, 14-3-3ζ release from phospho-Bad, Bad activation and apoptosis. Design and Methods We investigated whether arachidonic acid, which also binds 14-3-3ζ, contributes to coldinduced apoptosis. Results Cold storage activated P38-mitogen-activated protein kinase and released arachidonic acid, which accumulated due to cold inactivation of cyclooxygenase-1/thromboxane synthase. Accumulated arachidonic acid released 14-3-3ζ from phospho-Bad and decreased the mitochondrial membrane potential, which are steps in the induction of apoptosis. Addition of arachidonic acid did the same and its depletion made platelets resistant to cold-induced apoptosis. Incubation with biotin-arachidonic acid revealed formation of an [arachidonic acid-14-3-3ζ-glycoprotein Ibα] complex. Indomethacin promoted complex formation by accumulating arachidonic acid and released 14-3-3ζ from cyclo-oxygenase-1. Arachidonic acid depletion prevented the cold-induced reduction of platelet survival in mice. Conclusions We conclude that cold storage induced apoptosis through an [arachidonic acid-14-3-3ζ-glycoprotein Ibα] complex, which released 14-3-3ζ from Bad in an arachidonic acid-dependent manner. Although arachidonic acid depletion reduced agonist-induced thromboxane A2 formation and aggregation, arachidonic acid repletion restored these functions, opening ways to reduce apoptosis during storage without compromising hemostatic functions post-transfusion.

Induction of insulin resistance by the adipokines resistin, leptin, plasminogen activator inhibitor-1 and retinol binding protein 4 in human megakaryocytes.

Background In normal platelets, insulin inhibits agonist-induced Ca2+ mobilization by raising cyclic AMP. Platelet from patients with type 2 diabetes are resistant to insulin and show increased Ca2+ mobilization, aggregation and procoagulant activity. We searched for the cause of this insulin resistance. Design and Methods Platelets, the megakaryocytic cell line CHRF-288-11 and primary megakaryocytes were incubated with adipokines and with plasma from individuals with a disturbed adipokine profile. Thrombin-induced Ca2+ mobilization and signaling through the insulin receptor and insulin receptor substrate 1 were measured. Abnormalities induced by adipokines were compared with abnormalities found in platelets from patients with type 2 diabetes. Results Resistin, leptin, plasminogen activator inhibitor-1 and retinol binding protein 4 left platelets unchanged but induced insulin resistance in CHRF-288-11 cells. Interleukin-6, tumor necrosis factor-α and visfatin had no effect. These results were confirmed in primary megakaryocytes. Contact with adipokines for 2 hours disturbed insulin receptor substrate 1 Ser307-phosphorylation, while contact for 72 hours caused insulin receptor substrate 1 degradation. Plasma with a disturbed adipokine profile also made CHRF-288-11 cells insulin-resistant. Platelets from patients with type 2 diabetes showed decreased insulin receptor substrate 1 expression. Conclusions Adipokines resistin, leptin, plasminogen activator-1 and retinol binding protein 4 disturb insulin receptor substrate 1 activity and expression in megakaryocytes. This might be a cause of the insulin resistance observed in platelets from patients with type 2 diabetes.

Patient autoantibodies induce platelet destruction signals via raft-associated glycoprotein Ibα and Fc RIIa in immune thrombocytopenia

Immune thrombocytopenia (ITP) is an acquired immune-mediated disorder characterized by thrombocytopenia in the absence of an underlying cause.1 The patho-physiology of ITP is multifactorial and includes the development of autoantibodies that trigger abnormal thrombopoiesis, enhanced platelet destruction, complement activation and T-cell mediated effects.1–3 Platelet autoantibodies are detected in approximately 50% of patients4 and generally target the fibrinogen receptor αIIbβ3 or the receptor for von Willebrand factor (VWF), the glycoprotein (GP) Ib-V-IX complex. Anti-αIIbβ3 antibodies (70–80% of cases) are thought to induce thrombocytopenia through increased platelet clearance by Fcγ receptor-bearing macrophages. Autoantibodies against GPIb-V-IX (20–40% of cases) often induce a more severe fall in platelet count5 that is less responsive to standard therapies, such as intravenous immunoglobulin G (IVIG).6 Thrombocytopenia induced by GPIb-V-IX autoantibodies has not been characterized in great detail. Some monoclonal antibodies against GPIbα are known to induce platelet activation7 that may lead to accelerated platelet destruction in ITP patients8 with autoantibodies against this receptor. Here we report how an autoantibody against GPIbα, obtained from a patient with ITP, induces recognition signals for macrophages through interplay between glycoprotein Ibα and the low affinity IgG receptor FcγRIIa in lipid rafts.

Platelet interaction with von Willebrand factor is enhanced by shear-induced clustering of glycoprotein Ibα

Initial platelet arrest at the exposed arterial vessel wall is mediated through glycoprotein Ibα binding to the A1 domain of von Willebrand factor. This interaction occurs at sites of elevated shear force, and strengthens upon increasing hydrodynamic drag. The increased interaction requires shear-dependent exposure of the von Willebrand factor A1 domain, but the contribution of glycoprotein Ibα remains ill defined. We have previously found that glycoprotein Ibα forms clusters upon platelet cooling and hypothesized that such a property enhances the interaction with von Willebrand factor under physiological conditions. We analyzed the distribution of glycoprotein Ibα with Förster resonance energy transfer using time-gated fluorescence lifetime imaging microscopy. Perfusion at a shear rate of 1,600 s−1 induced glycoprotein Ibα clusters on platelets adhered to von Willebrand factor, while clustering did not require von Willebrand factor contact at 10,000 s−1. Shear-induced clustering was reversible, not accompanied by granule release or αIIbβ3 activation and improved glycoprotein Ibα-dependent platelet interaction with von Willebrand factor. Clustering required glycoprotein Ibα translocation to lipid rafts and critically depended on arachidonic acid-mediated binding of 14-3-3ζ to its cytoplasmic tail. This newly identified mechanism emphasizes the ability of platelets to respond to mechanical force and provides new insights into how changes in hemodynamics influence arterial thrombus formation.