To the Editor,
Thrombotic complications are common features of coronavirus disease 2019 (COVID-19) but the underlying pathogenesis is not fully elucidated yet. It has been observed that the spike protein (SP), namely the protruding membrane protein of SARS-CoV-2, may activate the coagulation cascade by binding Angiotensin-converting enzyme 2 (ACE2) directly on platelets and/or endothelial cells.1 Additionally, the isolated circulating SP may induce an hypercoagulability status by directly interacting with fibrin/fibrinogen.2 Noteworthy, free SP fragments have been found in plasma of COVID-19 patients.3 SARS-CoV-2 has been detected rarely in thrombi retrieved from brain arteries of acute ischemic stroke (AIS) patients 4 and more frequently in those retrieved from coronary arteries of acute myocardial infarct (AMI) patients.5 A few data on SP detection in retrieved thrombi from stroke patients have been reported.6. We aimed to investigate the possible evidence of isolated SP in clots retrieved by mechanical thrombectomy of COVID-19 patients with AIS and AMI.
The study was conducted on patients admitted to the Emergency Department of Policlinico Umberto I hospital, University of Rome La Sapienza, from March 2020 to April 2021. Among a series of consecutive adult patients with large vessel occlusion (LVO) related AIS or with AMI and a concomitant diagnosis of COVID-19, we retrospectively selected patients with retrieved thrombus available after endovascular treatment for histological analysis. The diagnosis of COVID-19 was based on the positive results of SARS-CoV-2 on real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis of nasopharyngeal swab specimens. We used as control, thrombi retrieved from patients with LVO-AIS not affected by COVID-19. The collected thrombi were immediately fixed in 10% formalin and embedded in paraffin. Sections were stained with Hematoxylin and Eosin. Immunohistochemical staining was performed using two different antibodies: SARS-CoV-2 SP (rabbit polyclonal anti-SARS-CoV-2 SP - Cell Signaling Technology, Boston, MA, USA, cat. #56996, dil. 1:100) and Nucleocapsid protein (NP) (monoclonal anti SARS/SARS CoV-2 (B46F) – Invitrogen, Rockford USA, MA1-7404, dil. 1:200). The positive control consisted of a COVID-19 lung section. A double immunofluorescence was performed to co-localize platelets with SARS-CoV-2 SP, using the primary antibodies, anti-CD61 (Monoclonal Mouse Anti-Human CD61, Platelet Glycoprotein IIIa/APC, Clone Y2/51, dil. 1:100) and anti-SARS-CoV-2 SP, visualized, respectively, with Goat anti-Mouse Alexa Fluor 594 (dil. 1:300) and donkey anti-rabbit Alexa Fluor 488 (dil. 1:300) (Thermo Fisher). The nuclei were stained with DAPI. Morphologic and immunohistochemical findings were assessed by two of the authors (GD & ML).
SARS-CoV-2 RNA extraction from clots was carried out with using Total purification RNA kit (Norgen Biotek Corp.), according to the manufacturer’s instructions. Viral RNA was amplified using a real time RT-PCR system (FTD SARS-CoV-2 test, Siemens Healthineers) for the qualitative detection of SARS-CoV-2 RNA, as previously described.7
We enrolled four COVID-19 positive (COVID-19+) patients: three with LVO-AIS (mean age: 67 [± 11]; 3 males) (one out of three patients was also treated with intravenous thrombolysis) and one affected by AMI (43 years old, male). All COVID-19+ patients had lung ground-glass opacity on pulmonary CT scan. We included a control group of four LVO-AIS without SARS-CoV-2 infection (COVID-19-) (mean age: 69 [± 11]; 3 males), three out of whom received intravenous thrombolysis.
The relative amount of platelets and fibrin/red blood cells did not significantly differ from COVID-19+ thrombi and controls. COVID-19+ thrombi retrieved from cerebral arteries showed mild positivity for SP whereas SP immunostaining was more marked in the COVID-19+ thrombus retrieved from anterior descending coronary artery (Figure, Panel 1 A,C). Neither cerebral nor coronary artery thrombi showed positive cells for NP (Figure, Panel 1 B,D). As for comparison, Figure Panel 2 reports representative immunohistochemical staining for SP and NP which was positive for both (E and F, respectively) in the lung of a patient affected by COVID-19 (positive control) and negative (G and H, respectively) in a thrombus retrieved from the middle cerebral artery of a patient not affected by COVID-19 (negative control).
Finally, to characterize the cellular population expressing SP we performed a double-immunostaining with antibody against CD61 and we found that most of the SP co-localized with platelets (Figure, Panel 3).
No SARS-CoV-2 RNA could be identified in three COVID+ thrombi analyzed with RT-PCR.
In conclusion, present data confirm the hypothesis that free SP besides the whole virus, may be the trigger of platelet activation and clot formation in COVID-19. To best of our knowledge only one other group has recently looked at the presence of SP in retrieved thrombi from 6 AIS patients, however, with negative results.6 The different kind of anti-SP antibodies used (monoclonal versus polyclonal in our study) as well as the different burden of COVID-19 on stroke pathogenesis may be plausible explanations. In addition, a possibly diverse genetically-determined ACE2 receptor expression on platelets and endothelial cells, could also justify the different chance to find SP on clots.