Suppressed ORAI1-STIM1-dependent Ca entry by protein kinase C isoforms regulating platelet procoagulant activity.

Agonist-induced rises in cytosolic Ca control most platelet responses in thrombosis and hemostasis. In human platelets, we earlier demonstrated that the ORAI1-STIM1 pathway is a major component of extracellular Ca entry, in particular when induced via the ITAM-linked collagen receptor, glycoprotein VI (GPVI). In the present article, using functionally defective platelets from patients with a loss-of-function mutation in ORAI1 or STIM1, we show that Ca entry induced by the endoplasmic reticulum ATPase inhibitor, thapsigargin, fully relies on this pathway.

Hemocytometric characteristics of COVID-19 patients with and without cytokine storm syndrome on the sysmex XN-10 hematology analyzer.

Objectives: COVID-19 is an ongoing global pandemic. There is an urgent need for identification and understanding of clinical and laboratory parameters related to progression towards a severe and fatal form of this illness, often preceded by a so-called cytokine-storm syndrome (CSS). Therefore, we explored the hemocytometric characteristics of COVID-19 patients in relation to the deteriorating clinical condition CSS, using the Sysmex XN-10 hematology analyzer.

Impaired iloprost-induced platelet inhibition and phosphoproteome changes in patients with confirmed pseudohypoparathyroidism type Ia, linked to genetic mutations in GNAS.

Patients diagnosed with pseudohypoparathyroidism type Ia (PHP Ia) suffer from hormonal resistance and abnormal postural features, in a condition classified as Albright hereditary osteodystrophy (AHO) syndrome. This syndrome is linked to a maternally inherited mutation in the GNAS complex locus, encoding for the GTPase subunit Gsα. Here, we investigated how platelet phenotype and omics analysis can assist in the often difficult diagnosis.

A synthesis approach of mouse studies to identify genes and proteins in arterial thrombosis and bleeding.

Antithrombotic therapies reduce cardiovascular diseases by preventing arterial thrombosis and thromboembolism, but at expense of increased bleeding risks. Arterial thrombosis studies using genetically modified mice have been invaluable for identification of new molecular targets. Because of low sample sizes and heterogeneity in approaches or methodologies, a formal meta-analysis to compare studies of mice with single-gene defects encountered major limitations.

Variable impairment of platelet functions in patients with severe, genetically linked immune deficiencies.

In patients with dysfunctions of the Ca channel ORAI1, stromal interaction molecule 1 (STIM1) or integrin-regulating kindlin-3 (FERMT3), severe immunodeficiency is frequently linked to abnormal platelet activity. In this paper, we studied platelet responsiveness by multiparameter assessment of whole blood thrombus formation under high-shear flow conditions in 9 patients, including relatives, with confirmed rare genetic mutations of ORAI1, STIM1 or FERMT3. In platelets isolated from 5 out of 6 patients with ORAI1 or STIM1 mutations, store-operated Ca entry (SOCE) was either completely or partially defective compared to control platelets.

Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition.

Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbβ3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy.

Combined Quantification of the Global Proteome, Phosphoproteome, and Proteolytic Cleavage to Characterize Altered Platelet Functions in the Human Scott Syndrome.

The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets.

Sunitinib uptake inhibits platelet function in cancer patients.

Background: Sunitinib is an oral tyrosine kinase inhibitor used for cancer treatment. Patients treated with sunitinib are at higher bleeding risk. As tyrosine kinases are essential for platelet signalling, the effects of sunitinib on platelet function in vitro and in cancer patients on treatment were investigated.

Coated platelets function in platelet-dependent fibrin formation via integrin αIIbβ3 and transglutaminase factor XIII.

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays.

Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage.

Scott syndrome is a rare bleeding disorder, characterized by altered Ca(2+)-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano)6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca(2+)-dependent KCa3.

Coordinated Membrane Ballooning and Procoagulant Spreading in Human Platelets.

Background: Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic response. It remains unclear, however, how and why platelets generate these structures.

Platelet CD40L Modulates Thrombus Growth Via Phosphatidylinositol 3-Kinase β, and Not Via CD40 and IκB Kinase α.

Objective: To investigate the roles and signaling pathways of CD40L and CD40 in platelet-platelet interactions and thrombus formation under conditions relevant for atherothrombosis.

Approach And Results: Platelets from mice prone to atherosclerosis lacking CD40L (Cd40lg(-/-)Apoe(-/-)) showed diminished αIIbβ3 activation and α-granule secretion in response to glycoprotein VI stimulation, whereas these responses of CD40-deficient platelets (Cd40(-/-)Apoe(-/-)) were not decreased. Using blood from Cd40lg(-/-)Apoe(-/-) and Cd40(-/-)Apoe(-/-) mice, the glycoprotein VI-dependent formation of dense thrombi was impaired on atherosclerotic plaque material or on collagen, in comparison with Apoe(-/-) blood.

Hyperreactivity of junctional adhesion molecule A-deficient platelets accelerates atherosclerosis in hyperlipidemic mice.

Rationale: Besides their essential role in hemostasis, platelets also have functions in inflammation. In platelets, junctional adhesion molecule (JAM)-A was previously identified as an inhibitor of integrin αIIbβ3-mediated outside-in signaling and its genetic knockdown resulted in hyperreactivity.

Objective: This gain-of-function was specifically exploited to investigate the role of platelet hyperreactivity in plaque development.

Factor XII regulates the pathological process of thrombus formation on ruptured plaques.

Objective: Atherothrombosis is the main cause of myocardial infarction and ischemic stroke. Although the extrinsic (tissue factor-factor VIIa [FVIIa]) pathway is considered as a major trigger of coagulation in atherothrombosis, the role of the intrinsic coagulation pathway via coagulation FXII herein is unknown. Here, we studied the roles of the extrinsic and intrinsic coagulation pathways in thrombus formation on atherosclerotic plaques both in vivo and ex vivo.

The effects of arterial flow on platelet activation, thrombus growth, and stabilization.

Injury of an arterial vessel wall acutely triggers a multifaceted process of thrombus formation, which is dictated by the high-shear flow conditions in the artery. In this overview, we describe how the classical concept of arterial thrombus formation and vascular occlusion, driven by platelet activation and fibrin formation, can be extended and fine-tuned. This has become possible because of recent insight into the mechanisms of: (i) platelet-vessel wall and platelet-platelet communication, (ii) autocrine platelet activation, and (iii) platelet-coagulation interactions, in relation to blood flow dynamics.

Calcium-activated and apoptotic phospholipid scrambling induced by Ano6 can occur independently of Ano6 ion currents.

Immune cells and platelets maintain plasma membrane phospholipid asymmetry. Upon activation, this asymmetry is disrupted by phospholipid scrambling (PS), which is a major step during activation of immune cells, hemostasis and apoptosis. Anoctamin 6 (Ano6; TMEM16F) causes chloride (Cl(-)) and cation currents and is required for Ca(2+)-dependent PS.

Dual mechanism of integrin αIIbβ3 closure in procoagulant platelets.

Background: Inactivation of integrin αIIbβ3 reverses platelet aggregate formation upon coagulation.

Results And Conclusion: Platelets from patient (Scott) and mouse (Capn1(-/-) and Ppif(-/-)) blood reveal a dual mechanism of αIIbβ3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling.

Significance: These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets.

Both TMEM16F-dependent and TMEM16F-independent pathways contribute to phosphatidylserine exposure in platelet apoptosis and platelet activation.

Scott syndrome, a bleeding disorder caused by defective phospholipid scrambling, has been associated with mutations in the TMEM16F gene. The role of TMEM16F in apoptosis- or agonist-induced phosphatidylserine (PS) exposure was studied in platelets from a Scott syndrome patient and control subjects. Whereas stimulation of control platelets with the BH3-mimetic ABT737 resulted in 2 distinct fractions with moderate and high PS exposure, the high PS-exposing fraction was markedly delayed in Scott platelets.

Platelet-based coagulation: different populations, different functions.

Platelets in a thrombus interact with (anti)coagulation factors and support blood coagulation. In the concept of cell-based control of coagulation, three different roles of platelets can be distinguished: control of thrombin generation, support of fibrin formation, and regulation of fibrin clot retraction. Here, we postulate that different populations of platelets with distinct surface properties are involved in these coagulant functions.

G-protein βγ subunits in vasorelaxing and anti-endothelinergic effects of calcitonin gene-related peptide.

Background And Purpose: Calcitonin gene-related peptide (CGRP) has been proposed to relax vascular smooth muscle cells (VSMC) via cAMP and can promote dissociation of endothelin-1 (ET-1) from ET(A) receptors. The latter is not mimicked by other stimuli of adenylate cyclases. Therefore, we evaluated the involvement of G-protein βγ subunits (Gβγ) in the arterial effects of CGRP receptor stimulation.

Calcitonin gene-related peptide terminates long-lasting vasopressor responses to endothelin 1 in vivo.

Slow dissociation of endothelin 1 from its endothelin A receptors is responsible for the long-lasting vasoconstrictor effects of the peptide. We showed recently that calcitonin gene-related peptide selectively terminates long-lasting contractile responses to endothelin 1 in isolated rat mesenteric arteries. Here we assessed whether the antiendothelinergic effect of calcitonin gene-related peptide is vascular bed specific and may terminate long-lasting pressor responses to exogenous and locally produced endothelin 1 in vivo.