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.

In mammals, megakaryocytes (MKs) in the bone marrow (BM) produce blood platelets, required for hemostasis and thrombosis. MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Through imaging of MKs in the intact BM, here we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. By combining in vivo two-photon microscopy and in situ light-sheet fluorescence microscopy with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche. As MKs do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts.

Background: Heritable bleeding and platelet disorders (BPD) are heterogeneous and frequently have an unknown genetic basis. The BRIDGE-BPD study aims to discover new causal genes for BPD by high throughput sequencing using cluster analyses based on improved and standardised deep, multi-system phenotyping of cases. Methods: We report a new approach in which the clinical and laboratory characteristics of BPD cases are annotated with adapted Human Phenotype Ontology (HPO) terms. Cluster analyses are then used to characterise groups of cases with similar HPO terms and variants in the same genes. Results: We show that 60% of index cases with heritable BPD enrolled at 10 European or US centres were annotated with HPO terms indicating abnormalities in organ systems other than blood or blood-forming tissues, particularly the nervous system. Cases within pedigrees clustered closely together on the bases of their HPO-coded phenotypes, as did cases sharing several clinically suspected syndromic disorders. Cases subsequently found to harbour variants in ACTN1 also clustered closely, even though diagnosis of this recently described disorder was not possible using only the clinical and laboratory data available to the enrolling clinician. Conclusions: These findings validate our novel HPO-based phenotype clustering methodology for known BPD, thus providing a new discovery tool for BPD of unknown genetic basis. This approach will also be relevant for other rare diseases with significant genetic heterogeneity.

Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.

Collagen receptors GPVI (also known as GP6) and integrin α2β1 are highly expressed on blood platelets and megakaryocytes, their immediate precursors. After vessel injury, subendothelial collagen becomes exposed and induces platelet activation to prevent blood loss. Collagen types I and IV are thought to have opposite effects on platelet biogenesis, directing proplatelet formation (PPF) towards the blood vessels to prevent premature release within the marrow cavity. We used megakaryocytes lacking collagen receptors or treated megakaryocytes with blocking antibodies, and could demonstrate that collagen-I-mediated inhibition of PPF is specifically controlled by GPVI. Other collagen types competed for binding and diminished the inhibitory signal, which was entirely dependent on receptor-proximal Src family kinases, whereas Syk and LAT were dispensable. Adhesion assays indicate that megakaryocyte binding to collagens is mediated by α2β1, and that collagen IV at the vascular niche might displace collagen I from megakaryocytes and thus contribute to prevention of premature platelet release into the marrow cavity and thereby directionally promote PPF at the vasculature.