Functional redundancy between RAP1 isoforms in murine platelet production and function.

RAP GTPases, important regulators of cellular adhesion, are abundant signaling molecules in the platelet/megakaryocytic lineage. However, mice lacking the predominant isoform, RAP1B, display a partial platelet integrin activation defect and have a normal platelet count, suggesting the existence of a RAP1-independent pathway to integrin activation in platelets and a negligible role for RAP GTPases in megakaryocyte biology. To determine the importance of individual RAP isoforms on platelet production and on platelet activation at sites of mechanical injury or vascular leakage, we generated mice with megakaryocyte-specific deletion (mKO) of Rap1a and/or Rap1b Interestingly, Rap1a/b-mKO mice displayed a marked macrothrombocytopenia due to impaired proplatelet formation by megakaryocytes. In platelets, RAP isoforms had redundant and isoform-specific functions. Deletion of RAP1B, but not RAP1A, significantly reduced α-granule secretion and activation of the cytoskeleton regulator RAC1. Both isoforms significantly contributed to thromboxane A2 generation and the inside-out activation of platelet integrins. Combined deficiency of RAP1A and RAP1B markedly impaired platelet aggregation, spreading, and clot retraction. Consistently, thrombus formation in physiological flow conditions was abolished in Rap1a/b-mKO, but not Rap1a-mKO or Rap1b-mKO, platelets. Rap1a/b-mKO mice were strongly protected from experimental thrombosis and exhibited a severe defect in hemostasis after mechanical injury. Surprisingly, Rap1a/b-mKO platelets were indistinguishable from controls in their ability to prevent blood-lymphatic mixing during development and hemorrhage at sites of inflammation. In summary, our studies demonstrate an essential role for RAP1 signaling in platelet integrin activation and a critical role in platelet production. Although important for hemostatic/thrombotic plug formation, platelet RAP1 signaling is dispensable for vascular integrity during development and inflammation.

Small GTPase Rap1A/B Is Required for Lymphatic Development and Adrenomedullin-Induced Stabilization of Lymphatic Endothelial Junctions.

Objective- Maintenance of lymphatic permeability is essential for normal lymphatic function during adulthood, but the precise signaling pathways that control lymphatic junctions during development are not fully elucidated. The Gs-coupled AM (adrenomedullin) signaling pathway is required for embryonic lymphangiogenesis and the maintenance of lymphatic junctions during adulthood. Thus, we sought to elucidate the downstream effectors mediating junctional stabilization in lymphatic endothelial cells. Approach and Results- We knocked-down both Rap1A and Rap1B isoforms in human neonatal dermal lymphatic cells (human lymphatic endothelial cells) and genetically deleted the mRap1 gene in lymphatic endothelial cells by producing 2 independent, conditional Rap1a/b knockout mouse lines. Rap1A/B knockdown caused disrupted junctional formation with hyperpermeability and impaired AM-induced lymphatic junctional tightening, as well as rescue of histamine-induced junctional disruption. Less than 60% of lymphatic- Rap1a/b knockout embryos survived to E13.5 exhibiting interstitial edema, blood-filled lymphatics, disrupted lymphovenous valves, and defective lymphangiogenesis. Consistently, inducible lymphatic- Rap1a/b deletion in adult animals prevented AM-rescue of histamine-induced lymphatic leakage and dilation. Conclusions- Rap1 (Ras-related protein) serves as the dominant effector downstream of AM to stabilize lymphatic junctions. Rap1 is required for maintaining lymphatic permeability and driving normal lymphatic development.

RAP GTPases and platelet integrin signaling.

Platelets are highly specialized cells that continuously patrol the vasculature to ensure its integrity (hemostasis). At sites of vascular injury, they are able to respond to trace amounts of agonists and to rapidly transition from an anti-adhesive/patrolling to an adhesive state (integrin inside-out activation) required for hemostatic plug formation. Pathological conditions that disturb the balance in the underlying signaling processes can lead to unwanted platelet activation (thrombosis) or to an increased bleeding risk. The small GTPases of the RAP subfamily, highly expressed in platelets, are critical regulators of cell adhesion, cytoskeleton remodeling, and MAP kinase signaling. Studies by our group and others demonstrate that RAP GTPases, in particular RAP1A and RAP1B, are the key molecular switches that turn on platelet activation/adhesiveness at sites of injury. In this review, we will summarize major findings on the role of RAP GTPases in platelet biology with a focus on the signaling pathways leading to the conversion of integrins to a high-affinity state.

Small GTPases in megakaryocyte and platelet biology.

Review series that provides a state-of-the-art overview of the role of small GTPases in megakaryocyte and platelet biology. While the focus of the reviews is on recent advances in the area of basic science, the clinical relevance of alterations in small GTPase signaling for platelet count and function is also discussed.

Glucocorticoids impair platelet thromboxane biosynthesis in community-acquired pneumonia.

Previous reports suggest that community-acquired pneumonia (CAP) is associated with an enhanced risk of myocardial infarction (MI) and that enhanced platelet activation may play a role. Aims of this study were to investigate if urinary excretion of 11-dehydro-thromboxane (Tx) B2, a reliable marker of platelet activation in vivo, was elevated in CAP and whether glucocorticoid administration reduced platelet activation. Three-hundred patients hospitalized for CAP were recruited and followed-up until discharge. Within the first 2 days from admission, urinary 11-dehydro-TxB2 and serum levels of methylprednisolone and betamethasone were measured. 11-Dehydro-TxB2 was also measured in a control group of 150 outpatients, matched for age, sex, and comorbidities. Finally, in-vitro studies were performed to assess if glucocorticoids affected platelet activation, at the same range of concentration found in the peripheral circulation of CAP patients treated with glucocorticoids. Compared to controls, CAP patients showed significantly higher levels of 11-dehydro-TxB2 (110 [69-151] vs. 163 [130-225] pg/mg creatinine; p < 0.001). During the in-hospital stay, 31 patients experienced MI (10%). A COX regression analysis showed that 11-dehydro-TxB2 independently predicted MI (p = .005). CAP patients treated with glucocorticoids showed significantly lower levels of 11-dehydro-TxB2 compared to untreated ones (147 [120-201] vs. 176 [143-250] pg/mg creatinine; p < 0.001). In vitro, glucocorticoids-treated platelets showed a dose-dependent decrease of ADP-induced platelet aggregation, TxB2 production, cPLA2 phosphorylation and arachidonic acid release from the platelet membrane. In conclusion, platelet TxB2 is overproduced in CAP patients and may be implicated in MI occurrence. Glucocorticoids reduce platelet release of TxB2 in vitro and urinary excretion of 11-dehydro-TxB2 in vivo and may be a novel tool to decrease platelet activation in this setting.

A talin mutant that impairs talin-integrin binding in platelets decelerates αIIbß3 activation without pathological bleeding.

Tight regulation of integrin affinity is critical for hemostasis. A final step of integrin activation is talin binding to 2 sites within the integrin ß cytoplasmic domain. Binding of talin to a membrane-distal NPxY sequence facilitates a second, weaker interaction of talin with an integrin membrane-proximal region (MPR) that is critical for integrin activation. To test the functional significance of these distinct interactions on platelet function in vivo, we generated knock-in mice expressing talin1 mutants with impaired capacity to interact with the ß3 integrin MPR (L325R) or NPLY sequence (W359A). Both talin1(L325R) and talin1(W359A) mice were protected from experimental thrombosis. Talin1(L325R) mice, but not talin(W359A) mice, exhibited a severe bleeding phenotype. Activation of αIIbß3 was completely blocked in talin1(L325R) platelets, whereas activation was reduced by approximately 50% in talin1(W359A) platelets. Quantitative biochemical measurements detected talin1(W359A) binding to ß3 integrin, albeit with a 2.9-fold lower affinity than wild-type talin1. The rate of αIIbß3 activation was slower in talin1(W359A) platelets, which consequently delayed aggregation under static conditions and reduced thrombus formation under physiological flow conditions. Together our data indicate that reduction of talin-ß3 integrin binding affinity results in decelerated αIIbß3 integrin activation and protection from arterial thrombosis without pathological bleeding.

Platelet ITAM signaling.

PURPOSE OF REVIEW: G protein-coupled receptors (GPCRs) like PAR1/4 and P2Y12 have long been known for their critical role in hemostasis. In contrast, deficiency in the immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors glycoprotein (GP)VI or C-type lectin-like receptor (CLEC)-2 is associated with only a mild bleeding diathesis in humans and mice. This review summarizes recent developments on the physiological importance of platelet ITAM signaling as well as the molecular mechanisms facilitating this signaling pathway. RECENT FINDINGS: Genetic experiments identified a critical role for platelet CLEC-2 signaling in the formation of lymphatic vessels during development. Similarly, signaling by both GPVI and CLEC-2, but not GPCRs, is required for the maintenance of vascular integrity at sites of inflammation in the adult. The molecular mechanisms underlying ITAM signaling in platelets continue to be refined. SUMMARY: Platelet ITAM signaling plays a key role for the maintenance of vascular integrity in development and the adult. This novel form of hemostasis differs from hemostasis at sites of vascular injury in that it does not depend on major platelet adhesion receptors or GPCR signaling.

The Src family kinases and protein kinase C synergize to mediate Gq-dependent platelet activation.

The Src family kinases (SFKs) play essential roles in collagen- and von Willebrand factor (VWF)-mediated platelet activation. However, the roles of SFKs in G protein-coupled receptor-mediated platelet activation and the molecular mechanisms whereby SFKs are activated by G protein-coupled receptor stimulation are not fully understood. Here we show that the thrombin receptor protease-activated receptor 4 agonist peptide AYPGKF elicited SFK phosphorylation in P2Y(12) deficient platelets but stimulated minimal SFK phosphorylation in platelets lacking G(q). We have previously shown that thrombin-induced SFK phosphorylation was inhibited by the calcium chelator 5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (dimethyl-BAPTA). The calcium ionophore A23187 induced SFK phosphorylation in both wild-type and G(q) deficient platelets. Together, these results indicate that SFK phosphorylation in response to thrombin receptor stimulation is downstream from G(q)/Ca(2+) signaling. Moreover, A23187-induced thromboxane A(2) synthesis, platelet aggregation, and secretion were inhibited by preincubation of platelets with a selective SFK inhibitor, PP2. AYPGKF-induced thromboxane A(2) production in wild-type and P2Y(12) deficient platelets was abolished by PP2, and AYPGKF-mediated P-selectin expression, integrin α(IIb)ß(3) activation, and aggregation of P2Y(12) deficient platelets were partially inhibited by the PKC inhibitor Ro-31-8220, PP2, dimethyl-BAPTA, or LY294002, but were abolished by Ro-31-8220 plus PP2, dimethyl-BAPTA, or LY294002. These data indicate that Ca(2+)/SFKs/PI3K and PKC represent two alternative signaling pathways mediating G(q)-dependent platelet activation.

CalDAG-GEFI deficiency protects mice in a novel model of Fcγ RIIA-mediated thrombosis and thrombocytopenia.

Platelet activation via Fcγ receptor IIA (FcγRIIA) is a critical event in immune-mediated thrombocytopenia and thrombosis syndromes (ITT). We recently identified signaling by the guanine nucleotide exchange factor CalDAG-GEFI and the adenosine diphosphate receptor P2Y12 as independent pathways leading to Rap1 small GTPase activation and platelet aggregation. Here, we evaluated the contribution of CalDAG-GEFI and P2Y12 signaling to platelet activation in ITT. Mice transgenic for the human FcγRIIA (hFcR) and deficient in CalDAG-GEFI(-/-) (hFcR/CDGI(-/-)) were generated. Compared with controls, aggregation of hFcR/CDGI(-/-) platelets or P2Y12 inhibitor-treated hFcR platelets required more than 5-fold and approximately 2-fold higher concentrations of a FcγRIIA stimulating antibody against CD9, respectively. Aggregation and Rap1 activation were abolished in P2Y12 inhibitor-treated hFcR/CDGI(-/-) platelets. For in vivo studies, a novel model for antibody-induced thrombocytopenia and thrombosis was established. FcγRIIA-dependent platelet thrombosis was induced by infusion of Alexa750-labeled antibodies to glycoprotein IX (CD42a), and pulmonary thrombi were detected by near-infrared imaging technology. Anti-GPIX antibodies dose-dependently caused thrombocytopenia and pulmonary thrombosis in hFcR-transgenic but not wild-type mice. CalDAG-GEFI-deficient but not clopidogrel-treated hFcR-transgenic mice were completely protected from ITT. In summary, we established a novel mouse model for ITT, which was used to identify CalDAG-GEFI as a potential new target in the treatment of ITT.

The kinetics of αIIbß3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy.

Two major pathways contribute to Ras-proximate-1-mediated integrin activation in stimulated platelets. Calcium and diacyglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI, RasGRP2) mediates the rapid but reversible activation of integrin αIIbß3, while the adenosine diphosphate receptor P2Y12, the target for antiplatelet drugs like clopidogrel, facilitates delayed but sustained integrin activation. To establish CalDAG-GEFI as a target for antiplatelet therapy, we compared how each pathway contributes to thrombosis and hemostasis in mice. Ex vivo, thrombus formation at arterial or venous shear rates was markedly reduced in CalDAG-GEFI(-/-) blood, even in the presence of exogenous adenosine diphosphate and thromboxane A(2). In vivo, thrombosis was virtually abolished in arterioles and arteries of CalDAG-GEFI(-/-) mice, while small, hemostatically active thrombi formed in venules. Specific deletion of the C1-like domain of CalDAG-GEFI in circulating platelets also led to protection from thrombus formation at arterial flow conditions, while it only marginally increased blood loss in mice. In comparison, thrombi in the micro- and macrovasculature of clopidogrel-treated wild-type mice grew rapidly and frequently embolized but were hemostatically inactive. Together, these data suggest that inhibition of the catalytic or the C1 regulatory domain in CalDAG-GEFI will provide strong protection from athero-thrombotic complications while maintaining a better safety profile than P2Y12 inhibitors like clopidogrel.

Key role of glycoprotein Ib/V/IX and von Willebrand factor in platelet activation-dependent fibrin formation at low shear flow.

A microscopic method was developed to study the role of platelets in fibrin formation. Perfusion of adhered platelets with plasma under coagulating conditions at a low shear rate (250(-1)) resulted in the assembly of a star-like fibrin network at the platelet surface. The focal fibrin formation on platelets was preceded by rises in cytosolic Ca(2+), morphologic changes, and phosphatidylserine exposure. Fibrin formation was slightly affected by α(IIb)ß(3) blockage, but it was greatly delayed and reduced by the following: inhibition of thrombin or platelet activation; interference in the binding of von Willebrand factor (VWF) to glycoprotein Ib/V/IX (GpIb-V-IX); plasma or blood from patients with type 1 von Willebrand disease; and plasma from mice deficient in VWF or the extracellular domain of GpIbα. In this process, the GpIb-binding A1 domain of VWF was similarly effective as full-length VWF. Prestimulation of platelets enhanced the formation of fibrin, which was abrogated by blockage of phosphatidylserine. Together, these results show that, in the presence of thrombin and low shear flow, VWF-induced activation of GpIb-V-IX triggers platelet procoagulant activity and anchorage of a star-like fibrin network. This process can be relevant in hemostasis and the manifestation of von Willebrand disease.

Rap1-Rac1 circuits potentiate platelet activation.

OBJECTIVE: The goal of this study was to investigate the potential crosstalk between Rap1 and Rac1, 2 small GTPases central to platelet activation, particularly downstream of the collagen receptor GPVI. METHODS AND RESULTS: We compared the activation response of platelets with impaired Rap signaling (double knock-out; deficient in both the guanine nucleotide exchange factor, CalDAG-GEFI, and the Gi-coupled receptor for ADP, P2Y12), to that of wild-type platelets treated with a small-molecule Rac inhibitor, EHT 1864 (wild-type /EHT). We found that Rac1 is sequentially activated downstream of Rap1 on stimulation via GPVI. In return, Rac1 provides important feedback for both CalDAG-GEFI- and P2Y12-dependent activation of Rap1. When analyzing platelet responses controlled by Rac1, we observed (1) impaired lamellipodia formation, clot retraction, and granule release in both double knock-out and EHT 1864-treated wild-type platelets; and (2) reduced calcium store release in EHT 1864-treated wild-type but not double knock-out platelets. Consistent with the latter finding, we identified 2 pools of Rac1, one activated immediately downstream of GPVI and 1 activated downstream of Rap1. CONCLUSIONS: We demonstrate important crosstalk between Rap1 and Rac1 downstream of GPVI. Whereas Rap1 signaling directly controls sustained Rac1 activation, Rac1 affects CalDAG-GEFI- and P2Y12-dependent Rap1 activation via its role in calcium mobilization and granule/ADP release, respectively.

Opposite Effects of mRNA-Based and Adenovirus-Vectored SARS-CoV-2 Vaccines on Regulatory T Cells: A Pilot Study.

New-generation mRNA and adenovirus vectored vaccines against SARS-CoV-2 spike protein are endowed with immunogenic, inflammatory and immunomodulatory properties. Recently, BioNTech developed a noninflammatory tolerogenic mRNA vaccine (MOGm1Ψ) that induces in mice robust expansion of antigen-specific regulatory T (Treg) cells. The Pfizer/BioNTech BNT162b2 mRNA vaccine against SARS-CoV-2 is identical to MOGm1Ψ except for the lipid carrier, which differs for containing lipid nanoparticles rather than lipoplex. Here we report that vaccination with BNT162b2 led to an increase in the frequency and absolute count of CD4posCD25highCD127low putative Treg cells; in sharp contrast, vaccination with the adenovirus-vectored ChAdOx1 nCoV-19 vaccine led to a significant decrease of CD4posCD25high cells. This pilot study is very preliminary, suffers from important limitations and, frustratingly, very hardly can be refined in Italy because of the >90% vaccination coverage. Thus, the provocative perspective that BNT162b2 and MOGm1Ψ may share the capacity to promote expansion of Treg cells deserves confirmatory studies in other settings.

Dual stimulation by autoantigen and CpG fosters the proliferation of exhausted rheumatoid factor-specific CD21low B cells in hepatitis C virus-cured mixed cryoglobulinemia.

Introduction: Hepatitis C virus (HCV) causes mixed cryoglobulinemia (MC) by driving clonal expansion of B cells expressing B cell receptors (BCRs), often encoded by the VH1-69 variable gene, endowed with both rheumatoid factor (RF) and anti-HCV specificity. These cells display an atypical CD21low phenotype and functional exhaustion evidenced by unresponsiveness to BCR and Toll-like receptor 9 (TLR9) stimuli. Although antiviral therapy is effective on MC vasculitis, pathogenic B cell clones persist long thereafter and can cause virus-independent disease relapses. Methods: Clonal B cells from patients with HCV-associated type 2 MC or healthy donors were stimulated with CpG or heath-aggregated IgG (as surrogate immune complexes) alone or in combination; proliferation and differentiation were then evaluated by flow cytometry. Phosphorylation of AKT and of the p65 NF-kB subunit were measured by flow cytometry. TLR9 was quantified by qPCR and by intracellular flow cytometry, and MyD88 isoforms were analyzed using RT-PCR. Discussion: We found that dual triggering with autoantigen and CpG restored the capacity of exhausted VH1-69pos B cells to proliferate. The signaling mechanism for this BCR/TLR9 crosstalk remains elusive, since TLR9 mRNA and protein as well as MyD88 mRNA were normally expressed and CpG-induced phosphorylation of p65 NF-kB was intact in MC clonal B cells, whereas BCR-induced p65 NF-kB phosphorylation was impaired and PI3K/Akt signaling was intact. Our findings indicate that autoantigen and CpG of microbial or cellular origin may unite to foster persistence of pathogenic RF B cells in HCV-cured MC patients. BCR/TLR9 crosstalk might represent a more general mechanism enhancing systemic autoimmunity by the rescue of exhausted autoreactive CD21low B cells.

Adherence to the Mediterranean Diet in Preventing Major Cardiovascular Events in Patients with Ischemic Heart Disease: The EVA Study.

BACKGROUND: Adherence to healthy dietary patterns, such as the Mediterranean diet (Med-diet), is recommended for the maintenance of cardiovascular health. The determinants for adherence to Med-diet and its importance in secondary cardiovascular disease prevention are still unclear. The aim of the study was to evaluate the influence of sex- and psycho-socio-cultural (i.e., gender-related) factors on Med-diet adherence and its role in preventing major cardiovascular events (MACEs) in patients with ischemic heart disease (IHD). METHODS: Med-diet adherence was evaluated among 503 consecutive adults with IHD. MACEs were collected during a long-term follow-up. RESULTS: Male Bem Sex-Role Inventory score (i.e., male personality traits) and physical functional capacity were associated with higher adherence, while cohabitation with a smoker and physical inactivity with poorer adherence. During a median follow-up of 22 months, 48 participants experienced MACEs (17.5%, 8.1%, and 3.9% of patients with low, medium, and high adherence, respectively; p = 0.016). At multivariate Cox--regression analysis, a greater adherence remained inversely associated with MACEs (HR: 0.49; 95% CI: 0.29-0.82; p = 0.006) after adjusting for confounding factors. CONCLUSION: The study suggests that gender-related factors have a role in maintaining a healthy dietary pattern. Improving Med-diet adherence may lower the risk of recurring cardiovascular events.

Distinct platelet crosstalk with adaptive and innate immune cells after adenoviral and mRNA vaccination against SARS-CoV-2.

BACKGROUND: Genetic-based COVID-19 vaccines have proved to be highly effective in reducing the risk of hospitalization and death. Because they were first distributed in a large-scale population, the adenoviral-based vaccines were linked to a very rare thrombosis with thrombocytopenia syndrome, and the interplay between platelets and vaccinations increasingly gained attention. OBJECTIVES: The objective of this article was to study the crosstalk between platelets and the vaccine-induced immune response. METHODS: We prospectively enrolled young healthy volunteers who received the mRNA-based vaccine, BNT162b2 (n = 15), or the adenovirus-based vaccine, AZD1222 (n = 25) and studied their short-term platelet and immune response before and after vaccine injections. In a separate cohort, we retrospectively analyzed the effect of aspirin on the antibody response 1 and 5 months after BNT162b2 vaccination. RESULTS: Here, we show that a faster antibody response to either vaccine is associated with the formation of platelet aggregates with marginal zone-like B cells, a subset geared to bridge the temporal gap between innate and adaptive immunities. However, although the mRNA-based vaccine is associated with a more gradual and tolerogenic response that fosters the crosstalk between platelets and adaptive immunity, the adenovirus-based vaccine, the less immunogenic of the 2, evokes an antiviral-like response during which the platelets are cleared and less likely to cooperate with B cells. Moreover, subjects taking aspirin (n = 56) display lower antibody levels after BNT162b2 vaccination compared with matched individuals. CONCLUSION: Platelets are a component of the innate immune pathways that promote the B-cell response after vaccination. Future studies on the platelet-immune crosstalk post-immunization will improve the safety, efficacy, and strategic administration of next-generation vaccines.