Antibody-induced procoagulant platelets in severe COVID-19 infection.

The pathophysiology of COVID-19-associated thrombosis seems to be multifactorial. We hypothesized that COVID-19 is accompanied by procoagulant platelets with subsequent alteration of the coagulation system. We investigated depolarization of mitochondrial inner transmembrane potential (ΔΨm), cytosolic calcium (Ca2+) concentration, and phosphatidylserine (PS) externalization. Platelets from COVID-19 patients in the intensive care unit (ICU; n = 21) showed higher ΔΨm depolarization, cytosolic Ca2+, and PS externalization compared with healthy controls (n = 18) and non-ICU COVID-19 patients (n = 4). Moreover, significant higher cytosolic Ca2+ and PS were observed compared with a septic ICU control group (ICU control; n = 5). In the ICU control group, cytosolic Ca2+ and PS externalization were comparable with healthy controls, with an increase in ΔΨm depolarization. Sera from COVID-19 patients in the ICU induced a significant increase in apoptosis markers (ΔΨm depolarization, cytosolic Ca2+, and PS externalization) compared with healthy volunteers and septic ICU controls. Interestingly, immunoglobulin G fractions from COVID-19 patients induced an Fcγ receptor IIA-dependent platelet apoptosis (ΔΨm depolarization, cytosolic Ca2+, and PS externalization). Enhanced PS externalization in platelets from COVID-19 patients in the ICU was associated with increased sequential organ failure assessment score (r = 0.5635) and D-dimer (r = 0.4473). Most importantly, patients with thrombosis had significantly higher PS externalization compared with those without. The strong correlations between markers for apoptosic and procoagulant platelets and D-dimer levels, as well as the incidence of thrombosis, may indicate that antibody-mediated procoagulant platelets potentially contributes to sustained increased thromboembolic risk in ICU COVID-19 patients.

Platelet glycoprotein VI aids in local immunity during pneumonia-derived sepsis caused by gram-negative bacteria.

Platelet collagen receptor glycoprotein VI (GPVI) and podoplanin receptor C-type lectin-like receptor 2 (CLEC2) are receptors implicated in platelet activation that both signal via an immunoreceptor tyrosine-based activation motif. Platelets are necessary for host defense and prevention of hemorrhage during sepsis, but the role of platelet GPVI and CLEC2 herein is unknown. To investigate this, we infected mice depleted of platelet GPVI or CLEC2 by antibody treatment or GPVI-/- mice with the common human sepsis pathogen Klebsiella pneumoniae via the airways to induce pneumonia-derived sepsis. The GPVI ligand collagen and the CLEC2 ligand podoplanin were constitutively present in the lung, whereas the GPVI ligands fibrin and histone were induced during pneumonia. During late-stage infection, both mice depleted of GPVI and GPVI-/- mice showed increased bacterial growth in lungs, and GPVI-/- mice also showed increased bacterial growth in distant body sites. Despite higher bacterial loads, GPVI-depleted mice showed reduced platelet numbers, platelet activation, and platelet-leukocyte complex formation in the bronchoalveolar space. Consistently, in human whole blood, GPVI stimulation of platelets increased platelet-leukocyte complex formation and leukocyte activation, which was accompanied by enhanced phagocytosis of Klebsiella GPVI-depleted mice showed increased lung hemorrhage during infection, but not to the extent observed in platelet-depleted mice, and lung bleeding was not significantly different between GPVI-/- and wild-type mice. CLEC2 depletion did not affect any of the responses during pneumonia. These results suggest that platelet GPVI, but not CLEC2, contributes to local host defense during pneumonia-derived sepsis by enhancing leukocyte function.

The Coffin-Lowry syndrome-associated protein RSK2 regulates neurite outgrowth through phosphorylation of phospholipase D1 (PLD1) and synthesis of phosphatidic acid.

More than 80 human X-linked genes have been associated with mental retardation and deficits in learning and memory. However, most of the identified mutations induce limited morphological alterations in brain organization and the molecular bases underlying neuronal clinical features remain elusive. We show here that neurons cultured from mice lacking ribosomal S6 kinase 2 (Rsk2), a model for the Coffin-Lowry syndrome (CLS), exhibit a significant delay in growth in a similar way to that shown by neurons cultured from phospholipase D1 (Pld1) knock-out mice. We found that gene silencing of Pld1 or Rsk2 as well as acute pharmacological inhibition of PLD1 or RSK2 in PC12 cells strongly impaired neuronal growth factor (NGF)-induced neurite outgrowth. Expression of a phosphomimetic PLD1 mutant rescued the inhibition of neurite outgrowth in PC12 cells silenced for RSK2, revealing that PLD1 is a major target for RSK2 in neurite formation. NGF-triggered RSK2-dependent phosphorylation of PLD1 led to its activation and the synthesis of phosphatidic acid at sites of neurite growth. Additionally, total internal reflection fluorescence microscopy experiments revealed that RSK2 and PLD1 positively control fusion of tetanus neurotoxin insensitive vesicle-associated membrane protein (TiVAMP)/VAMP-7 vesicles at sites of neurite outgrowth. We propose that the loss of function mutations in RSK2 that leads to CLS and neuronal deficits are related to defects in neuronal growth due to impaired RSK2-dependent PLD1 activity resulting in a reduced vesicle fusion rate and membrane supply.

Effect of infusing rat monoclonal antibodies to the murine GPIb-IX-V complex on platelet and megakaryocyte morphology in mice.

In the Bernard-Soulier syndrome, the absence of GPIb-IX-V leads to thrombocytopenia and giant platelets. In autoimmune thrombocytopenia in man, anti-platelet antibodies are associated with changes in megakaryocyte (MK) morphology and platelet size heterogeneity. We have compared the ultrastructural changes in mature MK following the infusion of rat monoclonal antibodies (MoAbs) to different epitopes of the murine GPIb-IX-V complex in mice. Blood and marrow samples were examined during both the acute thrombocytopenic phase and during the recovery phase. A MoAb to GPV induced neither thrombocytopenia nor changes in platelet morphology. During the acute thrombocytopenic phase with anti-GPIbalpha MoAbs, the size of residual platelets was heterogeneous and included large forms and platelets with few granules. During recovery, platelet size heterogeneity continued, and some platelets showed signs of activation. But only rare platelets were giant forms with ultrastructural defects resembling BSS. Megakaryocytopoiesis during acute thrombocytopenia was already abnormal, with some mature cells often showing vacuoles and an irregular development of the demarcation membrane system which varied in extent. These changes continued into the recovery phase. The anti-GPV MoAb had no effect on MK. Thus, anti-platelet antibodies can induce a different medullary response even when binding to the same receptor.