Several studies have demonstrated that platelets can interact with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and consequently undergo programmed cell death, extracellular vesicle release, and increased platelet reactivity in coronavirus disease 2019 (COVID-19) patients in response to low doses of α-thrombin.[1] [2] [3] Such hyper-sensitivity could be partially due to increased mitogen-activated protein kinase (MAPK) signaling pathway activation, protein kinase C delta (PKCδ) phosphorylation, and thromboxane synthesis.[1] [2] These posttranslational modifications are often triggered following activation of one or more receptors on the platelet surface.

GPIbα (CD42b) in the GPIb-IX-V receptor complex is the major binding site for α-thrombin associated with platelets, and through this function may support procoagulant activities and contribute to platelet activation and aggregation.[4] Importantly, blocking α-thrombin binding to the N-terminal region of GPIbα (His1-Glu282) can be achieved by a blocking antibody (SZ2) that selectively inhibits the α-thrombin binding site on GPIbα.[4] According to recent findings, GPIbα is the receptor through which SARS-CoV-2 spike protein binds to platelets as well as activates their increased expression of ligands.[5] However, the role of GPIbα in the α-thrombin-induced platelet hyper-responsiveness observed during SARS-CoV-2 infection is not well understood.

At the low enzyme concentration, we identified α-thrombin/GPIbα interaction as a novel mechanism triggering the hyper-reactivity observed in COVID-19 patients. Our findings showed that during SARS-CoV-2 infection, pretreatment of platelets with human anti-GPIbα antibody (SZ2) prevents platelet hyper-aggregation and degranulation. Interestingly, we found that platelets derived from patients with Bernard–Soulier syndrome (BSS) and who are infected with SARS-CoV-2 are not hyper-reactive. We thus have identified low α-thrombin–GPIbα interaction as a novel prothrombotic pathway which triggers the formation of procoagulant platelets in COVID-19 patients.

COVID-19 patients (n = 10) and COVID-19 patients with BSS (n = 3) who were admitted to the Cheikh Zaid Hospital (Rabat, Morocco) were included in this prospective, observational study conducted from December 7, 2020 to January 12, 2022. Patients with BSS have a homozygous mutation on GP9 which prevents GPIbα expression on platelets. All patients with COVID-19 were studied (on average) at 5.8 ± 1.2 hours after receiving a nasopharyngeal swab that showed positivity for SARS-CoV-2. Sex- and age-matched (age: 47.5, interquartile range: 38–52.4, 50% female) healthy blood donors (n = 10) were used as controls. None of the patients or healthy volunteers were treated with antithrombotic drugs (either premorbidly or for the treatment of their COVID-19 infection), and that could affect platelet functions or coagulation. All human blood studies were approved by the Local Ethics Committee of Cheikh Zaid Hospital, Rabat, Morocco; Project: CEFCZ/PR/2020-PR04. Informed consent was obtained from each subject and all experiments were conducted according to the principles set out in the Declaration of Helsinki.

Washed platelets were prepared as previously described.[1] Markers of platelet activation, α-granule release (CD62P or P-selectin expression) ([Fig. 1Ai, Aii]) and dense granule secretion, as assessed by ATP release ([Fig. 1Aiii]) and loss of mepacrine fluorescence ([Fig. 1Aiv]), were significantly enhanced in the presence of sub-threshold concentrations of α-thrombin in platelets from COVID-19 patients. We sought to investigate the contribution of GPIbα to the regulation of platelet hyper-activation. As shown in [Fig. 1A(i–iv)], this process was prevented following pretreatment of platelets with a selective human anti-GPIbα antibody (SZ2), whereas a control immunoglobulin G (IgG) antibody had no effect. This suggests that SARS-CoV-2 triggered platelet degranulation in an α-thrombin- and GPIbα-dependent manner. This is consistent with a report that GPIbα is the major α-thrombin binding site on the platelet surface with evidence for a high affinity site within its extracellular domain adjacent to the von Willebrand factor (VWF) binding site.[6] Also, at a higher α-thrombin concentration, the protease-activated receptors (PAR1 and PAR4) likely become significantly engaged and further enhance degranulation and extent of platelet hyper-activation and aggregation.[7]

Similarly, we assessed whether the α-thrombin–GPIbα axis plays a role in the hyper-aggregation of platelets from COVID-19 patients. As anticipated, priming, but not high concentration of α-thrombin-induced aggregation, was significantly inhibited in platelets that were pretreated with a human anti-GPIb antibody (SZ2) (Beckman Coulter, sodium azide free), as compared with COVID-19 and to COVID-19 control IgG groups ([Fig. 1Bi, Bii]). Thus, specific inhibition of the high-affinity binding site of α-thrombin on GPIbα was found to inhibit potentiated washed human platelet secretion and aggregation response, indicating that the α-thrombin/GPIbα axis can potentiate platelet function in the presence of suboptimal α-thrombin concentrations.

To further assert our finding based on the mouse-anti-human antibody, clone SZ2, that targets the anionic/sulfated tyrosine sequence 269 to 282 of GPIbα, we performed a similar experiment using a mouse anti-human antibody, clone VM16d (Abcam, dialyzed against phosphate-buffered saline in our laboratory to remove sodium azide), that maps the C-terminal flanking sequence 226 to 268 of GPIbα.[8] Our results show that specific blockade of the thrombin-binding site on GPIbα, by either SZ2 or VM16d ([Fig. 1Biii]) blocking monoclonal antibodies, prevented the potentiation of thrombin-induced platelet aggregation in COVID-19 patients.

To confirm our pharmacological-based approach, the key role for GPIbα was assessed using platelets isolated from COVID-19 patients with BSS (deficiency in GPIb-V-IX complex causing an absence of GPIb expression). Platelets from these patients were found to have markedly diminished aggregation in response to low concentrations of α-thrombin compared with COVID-19 patients without BSS ([Fig. 1Biii, Biv]). Indeed, SARS-CoV-2 infection failed to trigger platelet hyper-aggregation in patients with BSS suggesting that GPIbα positively regulates platelet aggregation downstream of α-thrombin in COVID-19 patients ([Fig. 1C]).

BSS is characterized by prolonged bleeding time, thrombocytopenia,[9] [10] and giant platelets lacking the surface membrane glycoprotein GPIb of the GPIb-IX-V complex.[10] [11] [12] The GPIb-IX-V complex is a platelet-specific adhesion-signaling complex, and it consists of GPIbα linked to GPIbβ via a disulfide bond and to GPIX and GPV noncovalently. Here, GPIbα is the major ligand-binding subunit and binds thrombin and the adhesive ligand VWF.[8] Given that platelets of BSS patients hardly express GPIb, BSS platelets lack GP1b-specific response to α-thrombin. Similarly, SZ2, an anti-GPIbα monoclonal antibody, is known to inhibit VWF binding to platelet and platelet aggregation.[13] Thus, both SZ2 antibody-treated platelets and platelets of BSS lack GP1bα function and GP1bα-driven response to α-thrombin. Other factors may contribute to the diminished response of BSS platelets to thrombin and SARS-CoV-2 infection, including the additional loss of GPV and GPIX. The loss of surface-membrane glycoprotein in BSS platelets has been closely linked to in vitro and in vivo functional impairment.[9] [10]

Our finding was supported by a recent study[14] that worked on S100A8/A9, also known as “calprotectin” or “MRP8/14,” an alarmin primarily secreted by activated myeloid cells with antimicrobial, proinflammatory, and prothrombotic properties. This research group identified the S100A8/A9-GPIbα axis as a novel targetable prothrombotic pathway inducing procoagulant platelets and fibrin formation, in particular in diseases associated with high levels of S100A8/A9, such as COVID-19.

Taken together, GPIbα interaction with low concentration α-thrombin is a potential contributor to the formation of procoagulant platelets in COVID-19 patients. Such interaction is probably only one aspect of many that likely regulate platelet activity in COVID-19 patients. These observations provide a valuable foundation for understanding the disease pathophysiology and to identify a new target for treatment options.