Massive image-based single-cell profiling reveals high levels of circulating platelet aggregates in patients with COVID-19

A characteristic clinical feature of COVID-19 is the frequent incidence of microvascular thrombosis. In fact, COVID-19 autopsy reports have shown widespread thrombotic microangiopathy characterized by extensive diffuse microthrombi within peripheral capillaries and arterioles in lungs, hearts, and other organs, resulting in multiorgan failure. However, the underlying process of COVID-19-associated microvascular thrombosis remains elusive due to the lack of tools to statistically examine platelet aggregation (i.e., the initiation of microthrombus formation) in detail. Here we report the landscape of circulating platelet aggregates in COVID-19 obtained by massive single-cell image-based profiling and temporal monitoring of the blood of COVID-19 patients (n = 110). Surprisingly, our analysis of the big image data shows the anomalous presence of excessive platelet aggregates in nearly 90% of all COVID-19 patients. Furthermore, results indicate strong links between the concentration of platelet aggregates and the severity, mortality, respiratory condition, and vascular endothelial dysfunction level of COVID-19 patients.

Microlyse; a thrombolytic agent that targets VWF for clearance of microvascular thrombosis

Thrombotic microangiopathies are hallmarked by attacks of disseminated microvascular thrombosis. In thrombotic thrombocytopenic purpura (TTP), this is caused by a rise in thrombogenic ultra-large von Willebrand factor (VWF) multimers because of ADAMTS13 deficiency. We previously reported that systemic plasminogen activation is therapeutic in a TTP mouse model. In contrast to its natural activators (i.e. tPA and uPA), plasminogen can directly bind to VWF. For optimal efficacy and safety, we aimed to focus and accelerate plasminogen activation at sites of microvascular occlusion. We here describe the development and characterization of Microlyse, a fusion protein consisting of a high-affinity VHH targeting the CT/CK domain of VWF and the protease domain of uPA, for localized plasminogen activation on microthrombi. Microlyse triggers targeted destruction of platelet-VWF complexes by plasmin on activated endothelial cells and in agglutination studies. At equal molar concentrations, Microlyse degrades microthrombi 7-fold more rapidly than blockade of platelet-VWF interactions with a bivalent humanized VHH (caplacizumab*). Finally, Microlyse attenuates thrombocytopenia and tissue damage (reflected by increased plasma lactate dehydrogenase activity, as well as PAI-1 and fibrinogen levels) more efficiently than caplacizumab* in an ADAMTS13-/- mouse model of TTP, without affecting hemostasis in a tail-clip bleeding model. These findings show that targeted thrombolysis of VWF by Microlyse is an effective strategy for the treatment of TTP and might hold value for other forms of VWF-driven thrombotic disease.

Mice lacking PECAM-1 and Ceacam1 have an aberrant platelet and thrombus phenotype

The immunoglobulin (Ig)–immunoreceptor tyrosine–based inhibitory motif (ITIM) bearing receptors, PECAM-1 and CEACAM1 have been shown net negative regulators of platelet-collagen interactions and hemi-ITAM signalling pathways. In this study, a double knockout (DKO) mouse was developed with deleted PECAM-1 and CEACAM1 to study their combined contribution in platelet activation by glycoprotein VI, C-type lectin-like receptor 2 (CLEC-2), protease activated receptor PAR-4, ADP purinergic receptors and thromboxane receptor TP A2 pathways. Additionally, their collective contribution was examined in thrombus formation under high shear and microvascular thrombosis using in vivo models. DKO platelets responded normally to ADP purinergic receptors and TP A2 pathway. However, DKO platelets released significantly higher amounts of P-selectin compared to hyper-responsive Pecam-1-/- or Ceacam1-/- versus wild-type (WT) upon stimulation with collagen related peptide or rhodocytin. Contrastingly, DKO platelets released increased amounts of P-selectin upon stimulation with PAR-4 agonist peptide or thrombin but not Pecam-1-/-, Ceacam1-/- or WT platelets. Blockade of phospholipase C (PLC) or Rho A kinase revealed that DKO platelets enhanced alpha granule release via PAR-4/Gαq/PLC signalling without crosstalk with Src/Syk or G12/13 signalling pathways. This DKO model showed a significant increase in thrombus formation compared to the hyper-responsive Ceacam1-/- or Pecam-1-/- versus WT phenotype. DKO platelets have similar glycoprotein surface expression compared to Pecam-1-/-, Ceacam1-/- and WT platelets. PECAM-1 and CEACAM1 work in concert to negatively regulate hemiITAM signalling, platelet-collagen interactions and PAR-4 Gαq protein coupled signalling pathways. Both PECAM-1 and CEACAM1 are required for negative regulation of platelet activation and microvascular thrombosis in vivo.

GPVI inhibition by glenzocimab synergistically inhibits atherosclerotic plaque-induced platelet activation when combined with conventional dual antiplatelet therapy

Introduction Aspirin and a potent platelet P2Y12 inhibitor, such as prasugrel or ticagrelor, are not always sufficient to prevent thrombus formation in patients with ST-elevation MI (STEMI), leading to “slow flow” or “no reflow” effects after stenting. GPIIb/IIIa inhibitors, such as eptifibatide, may help in this setting, but are not used routinely due to their bleeding risk. GPVI has critical roles in thrombosis and a minimal role in haemostasis. Here we tested whether depletion of GPVI has effects on thrombus formation after MI in an animal model and investigated the effects of a novel platelet GPVI inhibitor, glenzocimab (a Fab fragment of a monoclonal antibody), on platelet activation and thrombus formation when combined with aspirin and ticagrelor. Methods We used intravital microscopy in a murine model of ST-elevation myocardial infarction and ischaemia-reperfusion injury to investigate microvascular thrombosis. We investigated the antithrombotic effects of adding glenzocimab (previously known as ACT017) to blood from healthy donors and 20 patients with ACS treated with aspirin and ticagrelor. We compared the effect of glenzocimab with the GPIIb/IIIa inhibitor eptifibatide ex-vivo. We stimulated platelets with collagen and atherosclerotic plaque material that was sourced from patients undergoing carotid endarterectomy. We investigated effects on platelet aggregation, spreading, signalling, adhesion, thrombin generation, thrombus formation and clot stability ex vivo. Results Genetic depletion of GPVI in an animal model of myocardial infarction reduced microvascular thrombosis. Ex vivo, aspirin and ticagrelor partially inhibited atherosclerotic plaque-induced platelet aggregation (assessed by multiple electrode aggregometry) by 48% compared to control (34±3 vs. 65±4 U; P<0.001; Figure 1). Atherosclerotic plaque-induced platelet aggregation, adhesion, secretion and activation were critically dependent on platelet GPVI activation and were potently inhibited by glenzocimab. Glenzocimab alone reduced atherosclerotic plaque-induced platelet aggregation by 75% compared to control (16±4 vs. 65±4 U; P<0.001; Figure 1) and by over 95% when combined with aspirin and ticagrelor (3±1 vs 65±4 U; P<0.001; Figure 1). Furthermore, glenzocimab provided multiple synergistic antithrombotic effects when added to the blood of aspirin and ticagrelor-treated patients with ACS ex vivo. Glenzocimab and the GPIIb/IIIa inhibitor, eptifibatide, had many similar antithrombotic effects but glenzocimab had less effect on mechanisms of general haemostasis compared to eptifibatide, as assessed by ROTEM (Figure 2). Conclusions The addition of glenzocimab to aspirin and ticagrelor provides synergistic inhibition of multiple critical mechanisms of atherothrombosis. Glenzocimab and the GPIIb/IIIa inhibitor, eptifibatide, share many similar antithrombotic effects, although glenzocimab has less impact on mechanisms involved in haemostasis compared to eptifibatide. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Academy of Medical Sciences UK Clinical Lecturer Starter GrantRoyal Embassy of Saudi Arabia

Intraoperative Vasopressor Usage in Free Tissue Transfer: Should We Be Worried?

Background The role of vasopressors has long been a subject of debate in microsurgery. Conventional wisdom dictates the avoidance of vasopressor use, due to concerns such as peripheral vasoconstriction, inducing vasospasm of the anastomoses, and leading to failure in perfusion. It has since become common practice in some centers to avoid intraoperative vasopressor use during free tissue transfer surgery. Recent studies have suggested that this traditional view may not be supported by clinical evidence. However, none of these studies have separated vasopressor use by method of administration. Methods We conducted a retrospective review of our experience of vasopressor use in free flap surgery at a single high-volume center. The outcome measures were flap failure, flap-related complications and overall postoperative complications (reported using the Clavien–Dindo classification). Groups were compared using Chi-square or Fisher's Exact test where appropriate. Results A total of 777 cases in 717 patients were identified. 59.1% of these had vasopressors administered intraoperatively. The overall failure rate was 2.2%, with 9.8% experienced flap-related complications. There was no difference in flap loss when vasopressors were administered, but an increased rate of microvascular thrombosis was noted (p = 0.003). Continuous administration of vasopressors was associated with reduced venous congestion, whereas intermittent boluses increased risk of microvascular thrombosis. Conclusion Our study confirms previous findings that intraoperative vasopressor use in free flap surgery is not associated with increased failure rate. Administering vasopressors continuously may be safer than via repeated boluses.

VENOUS GANGRENE - AN INSTITUTIONAL EXPERIENCE

Venous gangrene represents the most severe manifestation following deep venous thrombosis (DVT), and presents as acral ischaemic necrosis. The pathophysiology is driven by an acquired coagulopathic state leading to microvascular thrombosis outcomes are generally poor. This study was aimed at analysing the outcomes in these rare group of patients who presented with venous gangrene at a single institution during a two year period.

COVID-19 Thrombosis: Bridging the Old and New Concepts

The coronavirus disease 2019 (COVID-19) pandemic is an evolving condition in the absence of established treatment and vaccines. The few autopsy studies on COVID-19 patients suggested the presence of pulmonary microvascular thrombosis. Hence, it is imperative to understand the pathobiology of thrombus formation and speculate the therapeutic goals in combating COVID-19. This paper focuses on a holistic approach by integrating the previous concepts and current concepts of thrombosis to better understand the pathogenesis of thrombosis.