Dual Mechanism of Integrin alpha(IIb)beta(3) Closure in Procoagulant Platelets

Aggregation of platelets via activated integrin α(IIb)β(3) is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that α(IIb)β(3) inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the β(3) chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed α(IIb)β(3) inactivation. Integrin α(IIb)β(3) inactivation was unchanged in platelets from Capn1(−/−) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced α(IIb)β(3) inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(−/−) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and α(IIb)β(3) inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of α(IIb)β(3) inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation

Factor XII Regulates the Pathological Process of Thrombus Formation on Ruptured Plaques

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. Plaque rupture after ultrasound treatment evoked immediate formation of subocclusive thrombi in the carotid arteries of Apoe(-/-) mice, which became unstable in the presence of structurally different FXIIa inhibitors. In contrast, inhibition of FVIIa reduced thrombus size at a more initial stage without affecting embolization. Genetic deficiency in FXII (human and mouse) or FXI (mouse) reduced ex vivo whole-blood thrombus and fibrin formation on immobilized plaque homogenates. Localization studies by confocal microscopy indicated that FXIIa bound to thrombi and fibrin particularly in luminal-exposed thrombus areas. The FVIIa- and FXIIa-triggered coagulation pathways have distinct but complementary roles in atherothrombus formation. The tissue factor-FVIIa pathway contributes to initial thrombus buildup, whereas FXIIa bound to thrombi ensures thrombus stabilit

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

xii 122 ha

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

In patients with dysfunctions of the Ca2+ 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 in nine rare patients, including relatives, with confirmed genetic mutations of ORAI1, STIM1 or FERMT3, platelet responsiveness by multi-parameter assessment of whole blood thrombus formation under high-shear flow conditions. In platelets isolated from 5 out of 6 patients with ORAI1 or STIM1 mutations, store-operated Ca2+ entry (SOCE) was (in)completely defective compared to control platelets. Parameters of platelet adhesion and aggregation on collagen microspots were impaired for 4/6 patients, in part related to a low platelet count. For 4 patients, platelet adhesion/aggregation and procoagulant activity on VWF/rhodocytin and VWF/fibrinogen microspots were impaired, independently of platelet count and partly correlated with SOCE deficiency. Measurement of thrombus formation at low shear rate confirmed a larger impairment of platelet functionality in the ORAI1 patients than in the STIM1 patient. For 3 patients/relatives with a FERMT3 mutation, all parameters of thrombus formation were strongly reduced regardless of the microspot. Bone marrow transplantation, required by two patients, resulted in overall improvement of platelet function. We concluded that multiparameter assessment of whole-blood thrombus formation, in a surface-dependent way, can detect: (i) additive effects of low platelet count and impaired platelet functionality; (ii) aberrant ORAI1-mediated Ca2+ entry; (iii) differences in platelet activation between patients carrying the same ORAI1 mutation; (iv) severe platelet function impairment linked to a FERMT3 mutation and bleeding history

Hemocytometric characteristics of COVID-19 patients with and without cytokine Storm syndrome on the Sysmex XN-10 hematology analyzer

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. From March 1st till May 16th, 2020, all patients admitted to our hospital with respiratory complaints and suspected for COVID-19 were included (n=1,140 of whom n=533 COVID-19 positive). The hemocytometric parameters of immunocompetent cells in peripheral blood (neutrophils [NE], lymphocytes [LY] and monocytes [MO]) obtained upon admission to the emergency department (ED) of COVID-19 positive patients were compared with those of the COVID-19 negative ones. Moreover, patients with CSS (n=169) were compared with COVID-19 positive patients without CSS, as well as with COVID-19 negative ones. In addition to a significant reduction in leukocytes, thrombocytes and absolute neutrophils, it appeared that lymphocytes-forward scatter (LY-FSC), and reactive lymphocytes (RE-LYMPHO)/leukocytes were higher in COVID-19-positive than negative patients. At the moment of presentation, COVID-19 positive patients with CSS had different neutrophils-side fluorescence (NE-SFL), neutrophils-forward scatter (NE-FSC), LY-FSC, RE-LYMPHO/lymphocytes, antibody-synthesizing (AS)-LYMPHOs, high fluorescence lymphocytes (HFLC), MO-SSC, MO-SFL, and Reactive (RE)-MONOs. Finally, absolute eosinophils, basophils, lymphocytes, monocytes and MO-FSC were lower in patients with CSS. Hemocytometric parameters indicative of changes in immunocompetent peripheral blood cells and measured at admission to the ED were associated with COVID-19 with and without CSS

Hyperreactivity of Junctional Adhesion Molecule A-Deficient Platelets Accelerates Atherosclerosis in Hyperlipidemic Mice

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. This gain-of-function was specifically exploited to investigate the role of platelet hyperreactivity in plaque development. JAM-A-deficient platelets showed increased aggregation and cellular and sarcoma tyrosine-protein kinase activation. On αIIbβ3 ligation, JAM-A was shown to be dephosphorylated, which could be prevented by protein tyrosine phosphatase nonreceptor type 1 inhibition. Mice with or without platelet-specific (tr)JAM-A-deficiency in an apolipoprotein e (apoe(-/-)) background were fed a high-fat diet. After ≤12 weeks of diet, trJAM-A(-/-)apoe-/- mice showed increased aortic plaque formation when compared with trJAM-A(+/+) apoe(-/-) controls, and these differences were most evident at early time points. At 2 weeks, the plaques of the trJAM-A(-/-) apoe(-/-) animals revealed increased macrophage, T cell, and smooth muscle cell content. Interestingly, plasma levels of chemokines CC chemokine ligand 5 and CXC-chemokine ligand 4 were increased in the trJAM-A(-/-) apoe(-/-)mice, and JAM-A-deficient platelets showed increased binding to monocytes and neutrophils. Whole-blood perfusion experiments and intravital microscopy revealed increased recruitment of platelets and monocytes to the inflamed endothelium in blood of trJAM-A(-/-) apoe(-/-)mice. Notably, these proinflammatory effects of JAM-A-deficient platelets could be abolished by the inhibition of αIIbβ3 signaling in vitro. Deletion of JAM-A causes a gain-of-function in platelets, with lower activation thresholds and increased inflammatory activities. This leads to an increase of plaque formation, particularly in early stages of the diseas

Platelet CD40L Modulates Thrombus Growth Via Phosphatidylinositol 3-Kinase beta, and Not Via CD40 and I kappa B Kinase alpha

To investigate the roles and signaling pathways of CD40L and CD40 in platelet-platelet interactions and thrombus formation under conditions relevant for atherothrombosis. 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. In all genotypes, addition of CD40L to the blood enhanced the growth of dense thrombi on plaques and collagen. Similarly, CD40L enhanced glycoprotein VI-induced platelet aggregation, even with platelets deficient in CD40. This potentiation was antagonized in Pik3cb(R/R) platelets or by inhibiting phosphatidylinositol 3-kinase β (PI3Kβ). Addition of CD40L also enhanced collagen-induced Akt phosphorylation, which was again antagonized by absence or inhibition of PI3Kβ. Finally, platelets from Chuk1(A/A)Apoe(-/-) mice deficient in IκB kinase α (IKKα), implicated in CD40 signaling to nuclear factor (NF) κB, showed unchanged responses to CD40L in aggregation or thrombus formation. Under atherogenic conditions, CD40L enhances collagen-induced platelet-platelet interactions by supporting integrin αIIbβ3 activation, secretion and thrombus growth via PI3Kβ, but not via CD40 and IKKα/NFκB. This role of CD40L exceeds the no more than modest role of CD40 in thrombus formatio