CK2ß regulates thrombopoiesis and Ca2+-triggered platelet activation in arterial thrombosis.

Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory ß-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2ß (ck2ß-/- ) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2ß-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2ß-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor-dependent intracellular calcium (Ca2+) release. Presumably due to a blunted increase in the concentration of cytosolic Ca2+, activation-dependent increases of α and dense-granule secretion and integrin αIIbß3 activation, and aggregation were abrogated in ck2ß-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2ß-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2ß-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2ßfl/fl mice. The present observations reveal CK2ß as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo.

Proplatelet formation is selectively inhibited by collagen type I through Syk-independent GPVI signaling.

Collagen receptors GPVI (also known as GP6) and integrin α2ß1 are highly expressed on blood platelets and megakaryocytes, their immediate precursors. After vessel injury, subendothelial collagen becomes exposed and induces platelet activation to prevent blood loss. Collagen types I and IV are thought to have opposite effects on platelet biogenesis, directing proplatelet formation (PPF) towards the blood vessels to prevent premature release within the marrow cavity. We used megakaryocytes lacking collagen receptors or treated megakaryocytes with blocking antibodies, and could demonstrate that collagen-I-mediated inhibition of PPF is specifically controlled by GPVI. Other collagen types competed for binding and diminished the inhibitory signal, which was entirely dependent on receptor-proximal Src family kinases, whereas Syk and LAT were dispensable. Adhesion assays indicate that megakaryocyte binding to collagens is mediated by α2ß1, and that collagen IV at the vascular niche might displace collagen I from megakaryocytes and thus contribute to prevention of premature platelet release into the marrow cavity and thereby directionally promote PPF at the vasculature.

A novel two-nucleotide deletion in HPS6 affects mepacrine uptake and platelet dense granule secretion in a family with Hermansky-Pudlak syndrome.

BACKGROUND: Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disease characterized by oculocutaneous albinism and platelet dysfunction. We report on a novel HPS6 homozygous frameshift variant (c.1919_1920delTC; p.Val640Glyfs*29) in a nonconsanguineous Caucasian family with two affected siblings (index patients) who presented with oculocutaneous albinism at birth and a mild bleeding phenotype during childhood and adolescence. PROCEDURE: Genetic analysis was conducted by panel-based next-generation sequencing (NGS) and Sanger sequencing. Platelets of the index patients, their parents, and the unaffected sister were then comprehensively evaluated by luminoaggregometry, whole blood flow cytometry, immunoblotting, immunofluorescence, and transmission electron microscopy. RESULTS: The homozygous frameshift variant in HPS6 gene detected by panel-based NGS and its segregation in the family was confirmed by Sanger sequencing. Flow cytometric analysis of the patients' platelets revealed a substantially decreased mepacrine uptake and release upon activation with a thrombin receptor agonist. Electron microscopy of resting platelets confirmed diminished dense granule content and enhanced vacuolization. Reduced release of adenosine triphosphate and CD63 neoexposition upon activation indicated not only a lack of dense granule content, but even an impairment of dense granule release. CONCLUSIONS: Our results demonstrate that the novel loss-of-function variant in the HPS6 subunit of biogenesis of lysosome-related organelles complex 2 is pathologic and leads to a reduced platelet dense granules and their release. The findings are compatible with an impaired platelet function and hence an enhanced bleeding risk. In future, a valid genotype-phenotype correlation may translate into best supportive care, especially regarding elective surgery or trauma management.

Impact of Itga2-Gp6-double collagen receptor deficient mice for bone marrow megakaryocytes and platelets.

The two main collagen receptors on platelets, GPVI and integrin α2ß1, play an important role for the recognition of exposed collagen at sites of vessel injury, which leads to platelet activation and subsequently stable thrombus formation. Both receptors are already expressed on megakaryocytes, the platelet forming cells within the bone marrow. Megakaryocytes are in permanent contact with collagen filaments in the marrow cavity and at the basal lamina of sinusoids without obvious preactivation. The role of both collagen receptors for megakaryocyte maturation and thrombopoiesis is still poorly understood. To investigate the function of both collagen receptors, we generated mice that are double deficient for Gp6 and Itga2. Flow cytometric analyses revealed that the deficiency of both receptors had no impact on platelet number and led to the expected lack in GPVI responsiveness. Integrin activation and degranulation ability was comparable to wildtype mice. By immunofluorescence microscopy, we could demonstrate that both wildtype and double-deficient megakaryocytes were overall normally distributed within the bone marrow. We found megakaryocyte count and size to be normal, the localization within the bone marrow, the degree of maturation, as well as their association to sinusoids were also unaltered. However, the contact of megakaryocytes to collagen type I filaments was decreased at sinusoids compared to wildtype mice, while the interaction to type IV collagen was unaffected. Our results imply that GPVI and α2ß1 have no influence on the localization of megakaryocytes within the bone marrow, their association to the sinusoids or their maturation. The decreased contact of megakaryocytes to collagen type I might at least partially explain the unaltered platelet phenotype in these mice, since proplatelet formation is mediated by these receptors and their interaction to collagen. It is rather likely that other compensatory signaling pathways and receptors play a role that needs to be elucidated.

Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B.

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.

Thrombopoiesis is spatially regulated by the bone marrow vasculature.

In mammals, megakaryocytes (MKs) in the bone marrow (BM) produce blood platelets, required for hemostasis and thrombosis. MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Through imaging of MKs in the intact BM, here we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. By combining in vivo two-photon microscopy and in situ light-sheet fluorescence microscopy with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche. As MKs do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts.Megakaryocyte maturation is thought to occur as the cells migrate from a vessel-distant (endosteal) niche to the vessel within the bone. Here, the authors show that megakaryocytes represent largely sessile cells in close contact with the vasculature and homogeneously distributed in the bone marrow.