Deya Cherpokova

Gray platelet syndrome and defective thrombo-inflammation in Nbeal2-deficient mice

Platelets are anuclear organelle-rich cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity. The major platelet organelles, α-granules, release proteins that participate in thrombus formation and hemostasis. Proteins stored in α-granules are also thought to play a role in inflammation and wound healing, but their functional significance in vivo is unknown. Mutations in NBEAL2 have been linked to gray platelet syndrome (GPS), a rare bleeding disorder characterized by macrothrombocytopenia, with platelets lacking α-granules. Here we show that Nbeal2-knockout mice display the characteristics of human GPS, with defective α-granule biogenesis in MKs and their absence from platelets. Nbeal2 deficiency did not affect MK differentiation and proplatelet formation in vitro or platelet life span in vivo. Nbeal2-deficient platelets displayed impaired adhesion, aggregation, and coagulant activity ex vivo that translated into defective arterial thrombus formation and protection from thrombo-inflammatory brain infarction following focal cerebral ischemia. In a model of excisional skin wound repair, Nbeal2-deficient mice exhibited impaired development of functional granulation tissue due to severely reduced differentiation of myofibroblasts in the absence of α-granule secretion. This study demonstrates that platelet α-granule constituents are critically required not only for hemostasis but also thrombosis, acute thrombo-inflammatory disease states, and tissue reconstitution after injury.

Defective tubulin organization and proplatelet formation in murine megakaryocytes lacking Rac1 and Cdc42

Blood platelets are anuclear cell fragments that are essential for blood clotting. Platelets are produced by bone marrow megakaryocytes (MKs), which extend protrusions, or so-called proplatelets, into bone marrow sinusoids. Proplatelet formation requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases, such as RhoA, Rac1, and Cdc42, are important regulators of cytoskeletal rearrangements in platelets; however, the specific roles of these proteins during platelet production have not been established. Using conditional knockout mice, we show here that Rac1 and Cdc42 possess redundant functions in platelet production and function. In contrast to a single-deficiency of either protein, a double-deficiency of Rac1 and Cdc42 in MKs resulted in macrothrombocytopenia, abnormal platelet morphology, and impaired platelet function. Double-deficient bone marrow MKs matured normally in vivo but displayed highly abnormal morphology and uncontrolled fragmentation. Consistently, a lack of Rac1/Cdc42 virtually abrogated proplatelet formation in vitro. Strikingly, this phenotype was associated with severely defective tubulin organization, whereas actin assembly and structure were barely affected. Together, these results suggest that the combined action of Rac1 and Cdc42 is crucial for platelet production, particularly by regulating microtubule dynamics.

Peptidylarginine deiminase 4 promotes age-related organ fibrosis

Aging promotes inflammation, a process contributing to fibrosis and decline in organ function. The release of neutrophil extracellular traps (NETs [NETosis]), orchestrated by peptidylarginine deiminase 4 (PAD4), damages organs in acute inflammatory models. We determined that NETosis is more prevalent in aged mice and investigated the role of PAD4/NETs in age-related organ fibrosis. Reduction in fibrosis was seen in the hearts and lungs of aged PAD4−/− mice compared with wild-type (WT) mice. An increase in left ventricular interstitial collagen deposition and a decline in systolic and diastolic function were present only in WT mice, and not in PAD4−/− mice. In an experimental model of cardiac fibrosis, cardiac pressure overload induced NETosis and significant platelet recruitment in WT but not PAD4−/− myocardium. DNase 1 was given to assess the effects of extracellular chromatin. PAD4 deficiency or DNase 1 similarly protected hearts from fibrosis. We propose a role for NETs in cardiac fibrosis and conclude that PAD4 regulates age-related organ fibrosis and dysfunction.

Critical off-target effects of the widely used Rac1 inhibitors NSC23766 and EHT1864 in mouse platelets

Platelet aggregation at sites of vascular injury is essential for normal hemostasis, but may also cause pathologic vessel occlusion. Rho GTPases are molecular switches that regulate essential cellular processes, and they have pivotal functions in the cardiovascular system. Rac1 is an important regulator of platelet cytoskeletal reorganization, and contributes to platelet activation. Rac1 inhibitors are thought to be beneficial in a wide range of therapeutic settings, and have therefore been tested in vivo for a variety of disorders. Two small‐molecule inhibitors, NSC23766 and EHT1864, have been characterized in different cell types, demonstrating high specificity for Rac1 and Rac, respectively.

Platelet Gi protein Gαi2 is an essential mediator of thrombo-inflammatory organ damage in mice.

Significance Platelet activation is crucial for hemostasis and thrombosis but also contributes to inflammation and progression of tissue damage following ischemia/reperfusion injury. Here we demonstrate that platelet activation through the Gi protein Gαi2 not only controls hemostatic responses but also thrombo-inflammatory tissue damage following cerebral and cardiac ischemia. Our report on a dual role of Gi proteins in platelet function opens new options for pharmaco-therapeutic strategies fighting ischemic diseases such as heart attack and stroke. Platelets are crucial for hemostasis and thrombosis and exacerbate tissue injury following ischemia and reperfusion. Important regulators of platelet function are G proteins controlled by seven transmembrane receptors. The Gi protein Gαi2 mediates platelet activation in vitro, but its in vivo role in hemostasis, arterial thrombosis, and postischemic infarct progression remains to be determined. Here we show that mice lacking Gαi2 exhibit prolonged tail-bleeding times and markedly impaired thrombus formation and stability in different models of arterial thrombosis. We thus generated mice selectively lacking Gαi2 in megakaryocytes and platelets (Gnai2fl/fl/PF4-Cre mice) and found bleeding defects comparable to those in global Gαi2-deficient mice. To examine the impact of platelet Gαi2 in postischemic thrombo-inflammatory infarct progression, Gnai2fl/fl/PF4-Cre mice were subjected to experimental models of cerebral and myocardial ischemia/reperfusion injury. In the model of transient middle cerebral artery occlusion stroke Gnai2fl/fl/PF4-Cre mice developed significantly smaller brain infarcts and fewer neurological deficits than littermate controls. Following myocardial ischemia, Gnai2fl/fl/PF4-Cre mice showed dramatically reduced reperfusion injury which correlated with diminished formation of the ADP-dependent platelet neutrophil complex. In conclusion, our data provide definitive evidence that platelet Gαi2 not only controls hemostatic and thrombotic responses but also is critical for the development of ischemia/reperfusion injury in vivo.

SLAP/SLAP2 prevent excessive platelet (hem)ITAM signaling in thrombosis and ischemic stroke in mice

Glycoprotein VI and C-type lectin-like receptor 2 are essential platelet activating receptors in hemostasis and thrombo-inflammatory disease, which signal through a (hem)immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway. The adapter molecules Src-like adapter proteins (SLAP and SLAP2) are involved in the regulation of immune cell surface expression and signaling, but their function in platelets is unknown. In this study, we show that platelets expressed both SLAP isoforms and that overexpression of either protein in a heterologous cell line almost completely inhibited glycoprotein VI and C-type lectin-like receptor 2 signaling. In mice, single deficiency of SLAP or SLAP2 had only moderate effects on platelet function, whereas double deficiency of both adapters resulted in markedly increased signal transduction, integrin activation, granule release, aggregation, procoagulant activity, and thrombin generation in response to (hem)ITAM-coupled, but not G protein-coupled, receptor activation. In vivo, constitutive SLAP/SLAP2 knockout mice displayed accelerated occlusive arterial thrombus formation and a dramatically worsened outcome after focal cerebral ischemia. This was attributed to the absence of both adapter proteins in platelets, as demonstrated by adoptive transfer of Slap(-/-)/Slap2(-/-) platelets into wild-type mice. Our results establish SLAP and SLAP2 as critical inhibitors of platelet (hem)ITAM signaling in the setting of arterial thrombosis and ischemic stroke.

A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis

Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.

Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms

Thrombosis in myeloproliferative neoplasms is associated with increased NETosis that can be targeted with JAK inhibitors. A NET increase in thrombosis Excessive thrombosis is a major cause of complications in cancer, including myeloproliferative neoplasms. Neutrophil extracellular traps (NETs) are structures formed from DNA expelled by activated neutrophils in infection, autoimmune disease, or cancer, and these can be associated with thrombosis. Wolach et al. discovered that increased activity of a protein called JAK2 is associated with NET formation in myeloproliferative neoplasms and that inhibiting it with ruxolitinib, a drug that targets JAK2, decreases thrombosis in a mouse model. Moreover, in a data set from more than 10,000 people without myeloproliferative neoplasms, detection of a clonal population of blood cells with overactive JAK2 correlated with a risk of thrombotic events, suggesting a broader relevance for this finding beyond the setting of cancer. Thrombosis is a major cause of morbidity and mortality in Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs), clonal disorders of hematopoiesis characterized by activated Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signaling. Neutrophil extracellular trap (NET) formation, a component of innate immunity, has been linked to thrombosis. We demonstrate that neutrophils from patients with MPNs are primed for NET formation, an effect blunted by pharmacological inhibition of JAK signaling. Mice with conditional knock-in of Jak2V617F, the most common molecular driver of MPN, have an increased propensity for NET formation and thrombosis. Inhibition of JAK-STAT signaling with the clinically available JAK2 inhibitor ruxolitinib abrogated NET formation and reduced thrombosis in a deep vein stenosis murine model. We further show that expression of PAD4, a protein required for NET formation, is increased in JAK2V617F-expressing neutrophils and that PAD4 is required for Jak2V617F-driven NET formation and thrombosis in vivo. Finally, in a population study of more than 10,000 individuals without a known myeloid disorder, JAK2V617F-positive clonal hematopoiesis was associated with an increased incidence of thrombosis. In aggregate, our results link JAK2V617F expression to NET formation and thrombosis and suggest that JAK2 inhibition may reduce thrombosis in MPNs through cell-intrinsic effects on neutrophil function.

Targeting platelet receptors in thrombotic and thrombo-inflammatory disorders

Platelet activation at sites of vascular injury is critical for the formation of a hemostatic plug which limits excessive blood loss, but also represents a major pathomechanism of ischemic cardio- and cerebrovascular diseases. Although currently available antiplatelet therapies have proved beneficial in preventing the recurrence of vascular events, their adverse effects on primary hemostasis emphasize the necessity to identify and characterize novel pharmacological targets for platelet inhibition. Increasing experimental evidence has suggested that several major platelet surface receptors which regulate initial steps of platelet adhesion and activation may become promising new targets for antiplatelet drugs due to their involvement in thrombotic and thrombo-inflammatory signaling cascades. This review summarizes recent developments in understanding the function of glycoprotein (GP)Ib, GPVI and the C-type lectin-like receptor 2 (CLEC-2) in hemostasis, arterial thrombosis and thrombo-inflammation and will discuss the suitability of the receptors as novel targets to treat these diseases in humans.

GPVI signaling is compromised in newly formed young platelets after acute thrombocytopenia in mice

At sites of vascular injury, exposed subendothelial collagens trigger platelet activation and thrombus formation by interacting with the immunoreceptor tyrosine-based activation motif (ITAM)-coupled glycoprotein VI (GPVI) on the platelet surface. Platelets are derived from the cytoplasm of megakaryocytes (MKs), which extend large proplatelets into bone marrow (BM) sinusoids that are then released into the bloodstream, where final platelet sizing and maturation occurs. The mechanisms that prevent activation of MKs and forming proplatelets in the collagen-rich BM environment remain largely elusive. Here, we demonstrate that newly formed young platelets (NFYPs) released after antibody-mediated thrombocytopenia in mice display a severe and highly selective signaling defect downstream of GPVI resulting in impaired collagen-dependent activation and thrombus formation in vitro and in vivo. The diminished GPVI signaling in NFYPs is linked to reduced phosphorylation of key downstream signaling proteins, including Syk, LAT, and phospholipase Cγ2, whereas the G protein-coupled receptor and C-type lectin-like receptor 2 signaling pathways remained unaffected. This GPVI signaling defect was overcome once the platelet counts were restored to normal in the circulation. Overall, these results indicate that the GPVI-ITAM signaling machinery in NFYPs after antibody-mediated thrombocytopenia only becomes fully functional in the blood circulation.