Graphical abstractDipyridamole bound to the SARS-CoV-2 protease Mpro after identified via the virtual screening and bioassay validation, and thus suppressed viral replication in vitro. As a result, dipyridamole supplementation was associated with significantly decreased concentrations of D-dimers, increased lymphocyte and platelet recovery in the circulation, and markedly improved clinical outcomes in comparison to the control patients.Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can cause acute respiratory distress syndrome, hypercoagulability, hypertension, and multiorgan dysfunction.Effective antivirals with safe clinical profile are urgently needed to improve the overall prognosis. In an analysis of a randomly collected cohort of 124 patients with Corona Virus Disease 2019 , we found that hypercoagulability as indicated by elevated concentrations of D-dimers was associated with disease severity. By virtual screening of a U.S. Food and Drug Administration (FDA) approved drug library, we identified an anticoagulation agent dipyridamole (DIP) in silico, which suppressed SARS-CoV-2 replication in vitro. In a proof-of-concept trial involving 31 patients with COVID-19, DIP supplementation was associated with significantly decreased concentrations of D-dimers (P<0.05), increased lymphocyte and platelet recovery in the circulation, and markedly improved clinical outcomes in comparison to the control patients. In particular, all 8 of the DIP-treated severely ill patients showed remarkable improvement: 7 patients (87.5%) achieved clinical cure and were discharged from the hospitals while the remaining 1 patient (12.5%) was in clinical remission.

Background: Platelet-neutrophil interactions contribute to vascular occlusion and tissue damage in thromboinflammatory disease. Platelet glycoprotein Ibα (GPIbα), a key receptor for the cell-cell interaction, is believed to be constitutively active for ligand binding. Here, we established the role of platelet-derived protein disulfide isomerase (PDI) in reducing the allosteric disulfide bonds in GPIbα and enhancing the ligand-binding activity under thromboinflammatory conditions. Methods: Bioinformatic analysis identified 2 potential allosteric disulfide bonds in GPIbα. Agglutination assays, flow cytometry, surface plasmon resonance analysis, a protein-protein docking model, proximity ligation assays, and mass spectrometry were used to demonstrate a direct interaction between PDI and GPIbα and to determine a role for PDI in regulating GPIbα function and platelet-neutrophil interactions. Also, real-time microscopy and animal disease models were used to study the pathophysiological role of PDI-GPIbα signaling under thromboinflammatory conditions. Results: Deletion or inhibition of platelet PDI significantly reduced GPIbα-mediated platelet agglutination. Studies using PDI-null platelets and recombinant PDI or Anfibatide, a clinical-stage GPIbα inhibitor, revealed that the oxidoreductase activity of platelet surface–bound PDI was required for the ligand-binding function of GPIbα. PDI directly bound to the extracellular domain of GPIbα on the platelet surface and reduced the Cys4-Cys17 and Cys209-Cys248 disulfide bonds. Real-time microscopy with platelet-specific PDI conditional knockout and sickle cell disease mice demonstrated that PDI-regulated GPIbα function was essential for platelet-neutrophil interactions and vascular occlusion under thromboinflammatory conditions. Studies using a mouse model of ischemia/reperfusion–induced stroke indicated that PDI-GPIbα signaling played a crucial role in tissue damage. Conclusions: Our results demonstrate that PDI-facilitated cleavage of the allosteric disulfide bonds tightly regulates GPIbα function, promoting platelet-neutrophil interactions, vascular occlusion, and tissue damage under thromboinflammatory conditions.

The human coronavirus HCoV-19 infection can cause acute respiratory distress syndrome 47 (ARDS), hypercoagulability, hypertension, extrapulmonary multiorgan dysfunction. Effective 48 antiviral and anti-coagulation agents with safe clinical profiles are urgently needed to improve the 49 overall prognosis. We screened an FDA approved drug library and found that an anticoagulant agent 50 dipyridamole (DIP) suppressed HCoV-19 replication at an EC50 of 100 nM in vitro. It also elicited 51 potent type I interferon responses and ameliorated lung pathology in a viral pneumonia model. In 52 analysis of twelve HCoV-19 infected patients with prophylactic anti-coagulation therapy, we found 53 that DIP supplementation was associated with significantly increased platelet and lymphocyte counts 54 and decreased D-dimer levels in comparison to control patients. Two weeks after initiation of DIP 55 treatment, 3 of the 6 severe cases (60%) and all 4 of the mild cases (100%) were discharged from the 56 hospital. One critically ill patient with extremely high levels of D-dimer and lymphopenia at the time 57 of receiving DIP passed away. All other patients were in clinical remission. In summary, HCoV-19 58 infected patients could potentially benefit from DIP adjunctive therapy by reducing viral replication, 59 suppressing hypercoagulability and enhancing immune recovery. Larger scale clinical trials of DIP 60 are needed to validate these therapeutic effects.61 62

Protein disulfide isomerase (PDI) participates in the pathogenesis of numerous diseases. Increasing evidence indicates that intravascular cell-derived PDI plays an important role in the initiation and progression of cardiovascular diseases, including thrombosis and vascular inflammation. Recent studies with PDI conditional knockout mice have advanced our understanding of the function of cell-specific PDI in disease processes. Furthermore, the identification and development of novel small-molecule PDI inhibitors has led into a new era of PDI research that transitioned from the bench to bedside. In this review, we will discuss recent findings on the regulatory role of PDI in cardiovascular disease.