TRPM7 Kinase Controls Calcium Responses in Arterial Thrombosis and Stroke in Mice

Objective: TRPM7 (transient receptor potential cation channel, subfamily M, member 7) is a ubiquitously expressed bifunctional protein comprising a transient receptor potential channel segment linked to a cytosolic alpha-type serine/threonine protein kinase domain. TRPM7 forms a constitutively active Mg2+ and Ca2+ permeable channel, which regulates diverse cellular processes in both healthy and diseased conditions, but the physiological role of TRPM7 kinase remains largely unknown. Approach and Results: Here we show that point mutation in TRPM7 kinase domain deleting the kinase activity in mice (Trpm7(R/R)) causes a marked signaling defect in platelets. Trpm7(R/R) platelets showed an impaired PIP2 (phosphatidylinositol-4,5-bisphosphate) metabolism and consequently reduced Ca2+ mobilization in response to stimulation of the major platelet receptors GPVI (glycoprotein VI), CLEC-2 (C-type lectin-like receptor), and PAR (protease-activated receptor). Altered phosphorylation of Syk (spleen tyrosine kinase) and phospholipase C gamma 2 and beta 3 accounted for these global platelet activation defects. In addition, direct activation of STIM1 (stromal interaction molecule 1) with thapsigargin revealed a defective store-operated Ca2+ entry mechanism in the mutant platelets. These defects translated into an impaired platelet aggregate formation under flow and protection of the mice from arterial thrombosis and ischemic stroke in vivo. Conclusions: Our results identify TRPM7 kinase as a key modulator of phospholipase C signaling and store-operated Ca2+ entry in platelets. The protection of Trpm7(R/R) mice from acute ischemic disease without developing intracranial hemorrhage indicates that TRPM7 kinase might be a promising antithrombotic target

Partially Defective Store Operated Calcium Entry and Hem(ITAM) Signaling in Platelets of Serotonin Transporter Deficient Mice

Background Serotonin (5-hydroxytryptamin, 5-HT) is an indolamine platelet agonist, biochemically derived from tryptophan. 5-HT is secreted from the enterochromaffin cells into the gastrointestinal tract and blood. Blood 5-HT has been proposed to regulate hemostasis by acting as a vasoconstrictor and by triggering platelet signaling through 5-HT receptor 2A (5HTR2A). Although platelets do not synthetize 5-HT, they take 5-HT up from the blood and store it in their dense granules which are secreted upon platelet activation. Objective To identify the molecular composite of the 5-HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke. Methods: 5-HT transporter knockout mice (5Htt$^{-/-}$) were analyzed in different in vitro and in vivo assays and in a model of ischemic stroke. Results In 5Htt$^{-/-}$ platelets, 5-HT uptake from the blood was completely abolished and agonist-induced Ca2+ influx through store operated Ca$^{2+}$ entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) were reduced. These observed in vitro defects in 5Htt$^{-/-}$ platelets could be normalized by the addition of exogenous 5-HT. Moreover, reduced 5-HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt$^{-/-}$ mice. Surprisingly, in the transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke 5Htt$^{-/-}$ mice showed nearly normal infarct volume and the neurological outcome was comparable to control mice. Conclusion Although secreted platelet 5-HT does not appear to play a crucial role in the development of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and plays an important role in thrombus stabilization

Abolished 5-HT uptake in <i>5Htt</i>^<i>-/-</i> platelets.

<p>Defective (hem)ITAM induced integrin activation, α-granule release and aggregation responses in <i>5Htt</i>^{<b><i>-/-</i></b>} platelets. (A) Platelet count and (B) size measured by FACS analysis or with a hematology analyzer (Sysmex). (C) Measurement of released platelet 5-HT before and after agonist dependent activation. 5-HT ELISA was performed with washed platelets of <i>Wt</i> and <i>5Htt</i>^<i>-/-</i> mice (n.d.: not detectable). Total 5-HT concentration was quantified in platelet lysates. (D) 5-HT concentration was measured in blood plasma with a 5-HT ELISA performed with platelet poor plasma (PPP) of <i>Wt</i> and <i>5Htt</i>^<i>-/-</i> mice. (E) 5-HIAA and (F) melatonin concentration was measured in urine and plasma samples of <i>Wt</i> and <i>5Htt</i>^<i>-/-</i>, respectively with a 5-HIAA and melatonin ELISA. (G) Spreading of <i>Wt</i> and <i>5Htt</i>^<i>-/-</i> platelets on a fibrinogen coated surface in the presence of thrombin. Washed platelets of <i>Wt</i> and <i>5Htt</i>^<i>-/-</i> mice were allowed to spread for 5, 15 and 30 min after stimulation with 0.01 U/mL thrombin. Statistical evaluation of the percentage of spread platelets at different spreading stages. 1: roundish; 2: only filopodia; 3: filopodia and lamellipodia; 4: fully spread. (H) Flow cytometric analysis of <i>Wt</i> and <i>5Htt</i>^<i>-/-</i> platelets. Integrin αIIbβ3 activation (upper panel) and degranulation (lower panel) in response to the indicated agonists were measured on a FACSCalibur. Results presented as MFI ± SD (I) Aggregation responses of <i>5Htt</i>^<i>-/-</i> platelets (grey line) compared to <i>Wt</i> platelets (black line) measured by change in light transmission upon activation with indicated agonists. ADP measurements were performed in platelet rich plasma (PRP), whereas all others were performed in washed platelets (Thr: thrombin; U46.: stable thromboxane A₂ analogue U46619; CRP: collagen-related peptide; Coll: HORM collagen; Rhd: rhodocytin; CVX: convulxin).</p

Altered collagen-induced adhesion and aggregate formation of <i>5Htt</i>^<i>-/-</i> platelets under flow.

<p>(A) Whole blood from <i>Wt</i> or <i>5Htt</i>^<i>-/-</i> mice was perfused over a collagen coated surface (0.2 mg/mL) at a shear rate of 1000 s^-1. Representative brightfield (BF) and fluorescence (DyLight 488 conjugated anti-GPIX) images of aggregate formation on collagen after 4 minutes of perfusion time are shown. Mean surface coverage (%) ± SD and relative thrombus volume expressed as integrated fluorescence intensity (IFI) ± SD in both <i>Wt</i> and <i>5Htt</i>^<i>-/-</i> mice. (B) Impaired procoagulant activity of blood samples from <i>5Htt</i>^<i>-/-</i> mice. PS exposure was detected by Annexin-V-Dylight 488 under similar flow conditions as described above. (C) Restored adhesion and aggregate formation of <i>5Htt</i>^<i>-/-</i> platelets by co-infused 5-HT on a collagen coated surface under flow. Heparinized whole blood of either <i>Wt</i> or <i>5Htt</i>^<i>-/-</i> mice was perfused with 10 μM 5-HT over a collagen coated surface (0.2 mg/mL) at a shear rate of 1000 s^-1.</p

Store operated Ca²⁺ entry is reduced in <i>5Htt</i>^<i>-/-</i> platelets.

<p>(A) Normal Ca²⁺ store release (upper panel), but reduced Ca²⁺ response to platelet agonists in the presence of extracellular CaCl₂ (lower panel) in <i>5Htt</i>^<i>-/-</i> platelets. Fura-2-loaded <i>Wt</i> (black bars) or <i>5Htt</i>^<i>-/-</i> (grey bars) platelets were stimulated with the indicated agonists in calcium-free medium or in the presence of extracellular 1 mM CaCl₂, and [Ca²⁺]_i was monitored by fluorimetry. Representative measurements and maximal Δ[Ca²⁺]_i ± standard deviation (SD) are shown. (B) Platelets were labeled and stimulated under similar conditions as Fig 4A in the presence of 10 μM 5-HT. Representative measurements and maximal Δ[Ca²⁺]_i ± standard deviation (SD) are shown. (C) Unaltered cytoplasmic Ca²⁺ level in resting platelets and TG induced Ca²⁺ store release in <i>5Htt</i>^<i>-/-</i> platelets. (D) Reduced TG induced SOCE in <i>5Htt</i>^<i>-/-</i> platelets. SOCE was measured in fura-2-loaded platelets stimulated with 0.1 or 0.05 μM TG for 5 min followed by the addition of 1 mM extracellular CaCl₂. Representative Ca²⁺ curves were indicated (black line: <i>Wt</i>; gray line: <i>5Htt</i>^<i>-/-</i>). Maximal Δ[Ca²⁺]_i of SOCE were quantified. Data are mean ± SD.</p

Altered hemostasis and thrombus formation in <i>5Htt</i>^<i>-/-</i> mice.

<p>(A) Prolonged tail bleeding times of <i>5Htt</i>^<i>-/-</i> mice. Each symbol represents one animal. (B-C) Impaired occlusion time of <i>5Htt</i>^<i>-/-</i> mice in models of arterial thrombosis. The abdominal aorta was injured by firm compression with forceps and blood flow was monitored by an ultrasonic flow probe until complete vessel occlusion occurred or for 30 min (B). Time to stable vessel occlusion of <i>Wt</i> and <i>5Htt</i>^<i>-/-</i> mice was determined. Each symbol represents one animal.(C) The right carotid artery was injured by the application of a FeCl₃ soaked filter paper and blood flow was monitored for 30 min using a Doppler flow probe. Each symbol represents one animal. (D) Ischemic stroke development in <i>5Htt</i>^<i>-/-</i> mice using the transient middle cerebral artery occlusion (tMCAO) model with 60 min. Representative images of coronal brain sections (left) stained with 2,3,5-triphenyltetrazolium chloride after 24 hours are shown. Infarct volume was measured by planimetry 24 h after (right panel). (E) Bederson score and (F) grip test were determined 24 h after tMCAO. Each symbol represents one animal. Results represent mean ± SD. (G) Number of infiltrated leukocytes in the ischemic brain of <i>Wt</i> and <i>5Htt</i>^<i>-/-</i> mice. Representative pictures of Ly6B.2 immunostaining show similar number of leukocytes in both <i>Wt</i> and <i>5Htt</i>^<i>-/-</i> ischemic brains. (H) Proposed regulatory role of SOCE in serotonergic system in platelets. During platelet agonist dependent Ca²⁺ store depletion, STIM1 binds Ora1 and modulates SOCE. Enhanced Orai1 activity supports degranulation and 5-HT secretion. Secreted platelet 5-HT further amplifies Ca²⁺ store depletion through 5HTR2A-Gq-PLCβ signaling. At the same time, 5-HT uptake is inhibited by SOCE to keep secreted platelet 5-HT in the extracellular milieu. The functional link between Orai1 mediated SOCE and 5HTT is so far unknown. Abbreviations: phospholipase C (PLC), inositol trisphosphate receptor (IP₃R), inositol trisphosphate (IP₃), 5-HT transporter (5HTT), serotonin (5-HT), serotonin receptor (5HTR2A).</p

Defective (hem)ITAM signaling is rescued by addition of extracellular 5-HT.

<p>(A) Flow cytometric analysis of <i>Wt</i> and <i>5Htt</i>^<i>-/-</i> platelets in the presence of 10 μM 5-HT. Agonists and concentrations are indicated. Integrin αIIbβ3 activation was detected by JON/A-PE (upper panel) and degranulation was detected by anti-P-selectin-FITC as a marker of α-granule secretion (lower panel). Results are MFI ± SD. (B) Aggregation responses of <i>5Htt</i>^<i>-/-</i> platelets (grey line) compared to <i>Wt</i> platelets (black line) in the presence of 10 μM 5-HT upon activation with indicated agonists. Measurements were performed in washed platelets. Light transmission was recorded on a Fibrintimer 4-channel aggregometer and representative aggregation traces of at least 3 individual experiments are depicted. (Rest: resting platelets, Thr: thrombin, U46: U46619, a stable thromboxane A₂ analogue, CRP: collagen-related peptide; CVX: convulxin, Rhd: rhodocytin, Coll.: HORM collagen).</p

MAGT1 Deficiency Dysregulates Platelet Cation Homeostasis and Accelerates Arterial Thrombosis and Ischemic Stroke in Mice.

Background: MAGT1 (magnesium transporter 1) is a subunit of the oligosaccharide protein complex with thiol-disulfide oxidoreductase activity, supporting the process of N-glycosylation. MAGT1 deficiency was detected in human patients with X-linked immunodeficiency with magnesium defect syndrome and congenital disorders of glycosylation, resulting in decreased cation responses in lymphocytes, thereby inhibiting the immune response against viral infections. Curative hematopoietic stem cell transplantation of patients with X-linked immunodeficiency with magnesium defect causes fatal bleeding and thrombotic complications.Methods: We studied the role of MAGT1 deficiency in platelet function in relation to arterial thrombosis and hemostasis using several in vitro experimental settings and in vivo models of arterial thrombosis and transient middle cerebral artery occlusion model of ischemic stroke.Results: MAGT1-deficient mice (Magt1-/y) displayed accelerated occlusive arterial thrombus formation in vivo, a shortened bleeding time, and profound brain damage upon focal cerebral ischemia. These defects resulted in increased calcium influx and enhanced second wave mediator release, which further reinforced platelet reactivity and aggregation responses. Supplementation of MgCl2 or pharmacological blockade of TRPC6 (transient receptor potential cation channel, subfamily C, member 6) channels, but not inhibition of store-operated calcium entry, normalized the aggregation responses of Magt1-/y platelets to the control level. GP (glycoprotein) VI activation of Magt1-/y platelets resulted in hyperphosphorylation of Syk (spleen tyrosine kinase), LAT (linker for activation of T cells), and PLC (phospholipase C) γ2, whereas the inhibitory loop regulated by PKC (protein kinase C) was impaired. A hyperaggregation response to the GP VI agonist was confirmed in human platelets isolated from a MAGT1-deficient (X-linked immunodeficiency with magnesium defect) patient. Haploinsufficiency of TRPC6 in Magt1-/y mice could normalize GP VI signaling, platelet aggregation, and thrombus formation in vivo.Conclusions: These results suggest that MAGT1 and TRPC6 are functionally linked. Therefore, deficiency or impaired functionality of MAGT1 could be a potential risk factor for arterial thrombosis and stroke