All patients (n = 40) were diagnosed with SARS-CoV-2 infection as confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) testing of nasopharyngeal specimens at the time of hospital admission. All patients admitted to the hospital were in critical condition and required mechanical ventilation from the first day of hospitalization. Enoxaparin-based anticoagulant therapy and antiviral therapy with tocilizumab, lopinavir, and ritonavir were administered. For bacterial infections, patients were treated with intravenous antibiotics, vancomycin, clarithromycin, and azithromycin. None of the patients had been vaccinated. Medical and laboratory records were reviewed to obtain the demographic, clinical, and laboratory data. The study protocol was carried out in accordance with the Declaration of Helsinki and was approved by the Scientific, Ethics, and Biosafety Committees of INER (B05-24).

Postmortem biopsies were obtained from 40 individuals who died between April 15, 2020, and August 20, 2021. The autopsies were initiated between three and five hours after the subject's estimated time of death. The procedure was performed using minimally invasive techniques. Lung samples (n=40) were obtained via a unilateral thoracotomy. For the kidney samples (n=16), a Tru-Cut needle (20 G X15 cm) was used for biopsy, and the tissues were subsequently fixed in neutral-buffered formalin for 24 hours before processing. Paraffin-embedded sections (3-μm thickness) were stained with hematoxylin and eosin (H&E) to detect pathological changes. Phosphotungstic acid-hematoxylin (PTAH), a specific method for fibrin staining, was used to demonstrate the presence of fibrin in the thrombi. Immunohistochemical staining was performed using the peroxidase method with the following antibodies: anti-CD61 (2f2, Bio SB) for platelets, anti-von Willebrand Factor (VWF) (GTX26994; Genetex), and anti-factor VIII (Biocare Medical CP 039) for procoagulant proteins. Diaminobenzidine was used as a substrate for color development. Tissue sections were counterstained with Harry's hematoxylin and observed by two experienced pneumopathologists. In cases of discrepancies, the material was reviewed jointly to reach a final decision.

Microscopic images were captured using a DFC425 C color camera (Leica Microsystems Inc., Wetzlar, Germany) mounted on a Leica DMLB optical microscope and processed using the Leica Application Suite v software version 3.6.0 (Leica Microsystems Inc.).

Quantitative variables were analyzed using one-way analysis of variance followed by Tukey's test for comparisons among three groups, and two-tailed t-tests for comparisons between two groups. Qualitative variables were analyzed using Fisher's exact test. Statistical significance was set at p < 0.05. Statistical analysis was performed with the package available online at https://www.socscistatistics.com/tests/.

The study population comprised 40 subjects, including 8 females and 32 males, with mean ages of 62.6 ± 9.7 years and 57.9 ± 15.5 years, respectively. All the patients were infected with SARS-CoV-2. Fibrin thrombi were identified in 33 out of 40 patients (82.5%), and bacterial co-infections were detected in 33/40 (70%) of the cases (Table 1). The most frequently isolated microorganisms included Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus epidermidis and Stenotrophomonas maltophilia.

The most common comorbidities in the population were systemic arterial hypertension, diabetes mellitus, acute kidney injury, and obesity (Table 1).

In light of the study's primary objective, which was to examine the structural pattern of fibrin in thrombi, patients with thrombi were classified into two groups, designated as the "Star-shaped pattern" and the "Reticular pattern" (Table 2), based on the observed fibrin structure in their thrombi. The analysis of the PaO₂/FiO₂ ratio and oxygen saturation (SatO₂) revealed no significant differences between the two groups (Table 2). Coagulation parameters, including prothrombin time, activated partial thromboplastin time, thrombin time, and D-dimer levels, were elevated above their respective reference values. However, no statistically significant differences were observed between the groups (Table 2). However, analysis of inflammatory indicator parameters indicated that the group of patients with thrombi exhibiting a star-shaped fibrin structure exhibited higher levels of C-reactive protein and neutrophil-to-lymphocyte ratio than the group of patients with a reticular fibrin pattern. Nevertheless, only CRP concentration exhibited a statistically significant difference (Table 2).

The lung tissue showed a patchy pattern, including areas with minimal and severe damage, separated by the interlobular septum. In areas exhibiting severe damage, interstitial thickening, edema, and inflammatory infiltrates, comprising neutrophils and lymphocytes, were observed. In contrast, regions exhibiting minimal damage displayed a paucity of the previously listed histopathological findings (Figure 1A). Hyaline membranes, pneumocyte detachment, intra-alveolar fibrin deposition, and alveolar macrophages were observed in the alveolar lumen in both areas regardless of the severity of the injury (Figure 1A and Figure 2A). The presence of thrombi was documented in both the arterioles (29/33) and pulmonary capillaries (19/33). In general, the pulmonary thrombi showed a characteristic reticular fibrin pattern (Figure 2C-D). However, in addition to this fibrin pattern, in 4/33 (12%) cases, the fibrin scaffold of the thrombus was observed to have a different arrangement than that of the classic reticular structure. Since we did not find any references describing this pattern, we call it "star-shaped fibrin pattern." Thrombi with star-shaped fibrin structures were observed in the pulmonary arterioles and capillaries (Figure 2E-F), predominantly in areas of severe lung injury (Figure 1C-E). Intra-alveolar fibrin deposits were observed in 24 of the 33 cases, but no star-shaped structures were observed, regardless of the severity of lung injury (Figure 2A-B). PTAH staining of the star-shaped structures demonstrated that they were composed of fibrin (Figure 2E-F and Figure 3A). Similarly, immunohistochemical staining with CD61 demonstrated the presence of platelet clumps (Figure 3B), which expressed the procoagulant proteins VWF (Figure. 3C) and Factor VIII (Figure 3D). Erythrocytes were not observed in fibrin structures.

Histopathological examination of renal tissue revealed tubular degenerative changes and glomerular damage. Fibrin thrombi were identified in 12/16 (75%) analyzed cases. These thrombi were predominantly located in peritubular (10/12) and glomerular (8/12) capillaries. The fibrin structure observed in renal thrombi was mostly fibrillary. However, in 4/12 (33%) cases, star-shaped fibrin structures were found in the glomerular and peritubular capillaries (Figure 3E-F). Collectively, thrombi with a star-shaped fibrin structure were observed in 7/40 autopsies (27.5%), with four cases located in the lungs and four in the kidneys. Of these, only one patient had this fibrin conformation in both organs.

The star-shaped fibrin structure is a conglomerate of discrete fibrin units, each consisting of a spheroid core, with fine fibrin needles radiating from it. The core had an irregular size with an average diameter of 4 µm. The overall size of the structure ranged from 12 to 15 µm. Thrombi with this fibrin pattern were observed more frequently in blood vessels located in areas of severe lung damage (1.04 ± 0.7 vessels/mm^²) compared to areas of minimal damage (0.33 ± 0.3 vessels/mm^²) (p = 0.012). In areas of minimal damage, this fibrin pattern was observed only in paraseptal pulmonary capillaries adjacent to areas of severe damage.

The study protocol was reviewed and approved by the Research Ethics Committee of the National Institute of Respiratory Diseases, Ismael Cosio Villegas, Mexico City, Mexico (approval number - B05-24). Written informed consent was obtained from each patient's relatives to perform autopsies.

This study was not supported by any sponsors or funders.

M, M-F., C, L-R., and JS, L-G - Conception, Design, and Formal Analysis. E, B-V - Performance of the microbiological test.

S, S-T, D, A-C, F, B-M - Acquisition and curation of data as well as execution of the research methodology. All authors contributed to the initial draft and subsequent revisions, and approved the final version of the manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding authors.