Increased impact of GPIb receptors on platelet adhesion under flow in severe COVID-19

Graphical abstract

In recent years, coronavirus disease 2019 (COVID-19) has become a global challenge. Early on, during the pandemic, it became obvious that thrombotic complications are a major cause of morbidity and mortality in COVID-19. The subsequent use of anticoagulants in acute COVID-19 treatment has significantly improved the outcomes (Schulman et al. 2022). Nevertheless, pathological mechanisms causing COVID-19 associated coagulopathy (CAC) are still poorly understood due to their multifaceted nature. Investigation of these underlying mechanisms is of great importance as it may lead to further improvement of treatment strategies and therapeutic approaches (Page and Ariëns 2021).

One of the current concepts concerning the driving mechanisms of COVID-19 pathogenesis is the reciprocal mutual amplification of innate immunity and coagulation system (Conway et al. 2022). A combination of these processes is referred to as immunothrombosis and/or thromboinflammation, and it plays an important role in multiple clinical settings including acute respiratory distress syndrome and sepsis. One of the links between thrombotic and inflammatory processes is the von Willebrand Factor (vWF) known for promoting a prothrombotic state in inflammatory-induced conditions. A vast body of evidence indicates that vWF levels are increased multifold in COVID-19 patients (Favaloro et al. 2021). Moreover, an increased vWF concentration shows a strong correlation with disease severity and poor prognosis (Xu et al. 2022). At the same time, vWF-cleaving protease-ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) activity shows a relative deficiency in severe COVID-19 patients (Favaloro et al. 2021). The decrease in ADAMTS-13 activity in COVID-19 patients is less radical than in the case of thrombotic thrombocytopenic purpura (TTP), but it still shows clear evidence for imbalance of the vWF-ADAMTS-13 axis in COVID-19 (Mancini et al. 2021; Joly et al. 2021).

vWF is a major player in primary haemostasis. In particular, it regulates platelet adhesion under flow. Damaged by SARS-CoV-2 endothelium can release ultra-large and high-molecular-weight multimers of vWF into the circulating blood. The decreased activity of ADAMTS-13 does not allow for the timely degradation of ultra-large vWF multimeters. Accumulated multimers can be unfolded and activated by mildly increased shear rates, exposing numerous binding sites for Glycoprotein Ib (GPIb) receptors of platelets. Interaction of GPIb with unfolded vWF multimers initiates platelet adhesion and subsequently activates multiple signaling pathways leading to further amplification of platelet adhesion and clot formation. Moreover, GPIb also interacts with leukocytes via their integrin CD11b/CD18 (Mac-1) amplifying even further clot growth and participating in so-called throboinflammation processes (Denorme et al. 2019).

Knowing the present imbalance of the vWF/ADAMTS-13 axis in COVID-19, one might expect platelet adhesion to be altered in such case as well. In this brief report, we present new data concerning platelet adhesion in severe COVID-19 patients. We also have analyzed the influence of GPIb on the adhesion process in COVID-19 patients. The main observation of our study is that, while platelet adhesion at high-shear rates is not altered much, the effect of the GPIb receptor on the adhesion process is substantially increased in COVID-19 patients.

A group of 17 consecutive patients with severe COVID-19 pneumonia admitted to the intensive care unit (ICU) from January to February of 2021 was included in this study. None of the patients were vaccinated. The study was approved by the Ethics Committee of the National Medical Research Centre of Cardiology. Written informed consent was obtained from the patient or the first next of kin. A group of healthy volunteers (n = 17) was used as a control. Healthy controls were not matched with patients with severe COVID-19. Platelet adhesion was assessed in a flow-through in vitro test system described previously (Gabbasov et al. 2021). Briefly, whole blood was perfused over a fibrinogen-coated surface at the shear rate of 1300 s⁻¹ for 15 min, and platelet adhesion was registered in real time by detection of scattered laser light. Blood samples were anticoagulated with D-phenylalanyl-L-prolyl-L-arginine chloromethylketone (PPACK) in a final concentration of 100 µmol/L. Prior to the perfusion experiment, 5 μmol/L ADP was added to achieve uniform activation of platelets in different samples. For each patient/volunteer, two separate measurements were performed: with and without the addition of a monoclonal antibody (mAb) against GPIb. Blood samples were preincubated for 3 min with 20 µg/mL of mAb AK2 generously gifted by Prof. A.V. Mazurov (Berndt et al. 1988). The difference in platelet adhesion between those two measurements was calculated in percent (%) from the level of adhesion observed without the addition of mAb.

Patient characteristics are summarized in Table 1. It can be clearly seen that in our group of patients, the vWF/ADAMTS-13 axis was heavily imbalanced. We have observed a massive increase in vWF levels (median 488%, interquartile range (IQR) 389%–676%) and a pronounced deficiency of ADAMTS-13 activity (median 21%, IQR 18.5%–31.5%). These observations are in line with previous studies including ICU patients with COVID-19 (Favaloro et al. 2021). It is worth mentioning that ADAMTS-13 activity in all the patients was above the 10% level indicative of TTP. At the same time, the decrease of ADAMTS-13 activity was relatively high in comparison to other previously reported studies, which might be attributed to the severity of the illness in our cohort of ICU patients (Fig. 1). Raised levels of D-dimer and C-reactive protein were also observed (see Table 1).

Results of platelet adhesion measurements are shown in Figs. 2A and 2B. No difference was observed in platelet adhesion between the COVID group and the non-COVID group (median 42.1 arbitrary units (a.u.) (IQR 23.0–70.3) vs 40.7 a.u. (IQR 31.9–56.9), P = 0.73, Mann-Whitney U test). In experiments with the addition of anti-GPIb mAb, a reduced platelet adhesion was observed in both groups: 9.4 a.u. (IQR 5.2–25.0) in the COVID-19 group and 29.1 a.u. (IQR 13.1–47.6) in the control group. Inhibition of platelet adhesion by anti-GPIb mAb was much more pronounced in the COVID-19 group (Fig. 2C). Relative inhibition of platelet adhesion by anti-GPIb mAb was 64% (IQR 48%–84%) and 45% (IQR 21%–50%) in COVID-19 and control groups, correspondingly (P = 0.0046, Mann-Whitney U test). In other words, the influence of GPIb on platelet adhesion was increased in severe COVID-19 patients in comparison to healthy controls.

Interestingly, an excess of vWF in severe COVID-19 patients did not lead to enhanced platelet adhesion in our experiments. The question of why there is no increase in platelet adhesion while very high levels of vWF are present in severe COVID-19 patients remains unclear. This observation seems rather counterintuitive. At the same time, a similar result concerning platelet adhesion under flow in a cohort of severe COVID-19 patients was reported in a recent study (Tacquard et al. 2023). Though experimental conditions were somewhat different, they also observed no increase of vWF-mediated platelet adhesion under high shear rates and even reported a non-significant tendency towards reduced platelet adhesion.

One might assume that excess of vWF is compensated by a defective adhesive function of platelets from patients with severe COVID-19, for instance by a reduced expression of platelet receptors. For today the data available on this subject in the literature is somewhat contradictory. While indicate a reduction in the surface expression of GPIIb/IIIa and GPIb in severe COVID-19 platelets, an earlier work (Tacquard et al. 2023), on the contrary, states significantly higher expression of both GPIIb/IIIa and GPIb in COVID-19 patients. There is also a chance that platelet receptor expression depends on the severity of COVID-19. This subject needs to be thoroughly analyzed in future studies. It also should be taken into account that both our study and the French study (Tacquard et al. 2023) are limited by a small number of included patients.

There are several limitations of the present study that need to be mentioned. First, a significant limitation of our work is the limited cohort of patients, which was not age/sex-matched with the control group. Although the results obtained in our study were statistically significant, their generalization on the overall population may require further investigation in a broader cohort of patients. Also, we didn’t test the patients for specific variants of SARS-CoV-2. Our study was performed during the second wave of COVID-19, therefore we suppose that the patients had the original SARS CoV-2 variant. There is a possibility that various SARS-CoV-2 variants may differ in their influence on platelet adhesion. It would be interesting to analyze the impact of different variants of SARS-CoV-2 on the effects described, but this is beyond the scope of the present study.

It is also worth noting that the use of fibrinogen-coated slides as an adhesive substrate in our study was a forced measure due to our temporal constraints and technical limitations at the time of the study. To our knowledge, vWF itself does directly bind to fibrinogen. We assume that the initial stage of platelet adhesion in our experimental model is mediated through platelet–fibrinogen interactions. At the same time, further growth of the platelet aggregate on the adhesive surface is governed by the formation of vWF strands and their interactions with GPIb and other platelet receptors. That is why we consider our setup suitable for analysis of the impact of vWF and GPIb on platelet adhesion in flowing blood. Of course, it would be very interesting to compare our results to a more direct approach, for instance, the one using collagen/vWF-coated slides.

An interesting observation made in the present study is the increased effect of GPIb on platelet adhesion in severe COVID-19 patients. Inhibition of platelet adhesion by anti-GPIb mAb was much more pronounced in the COVID-19 group compared to healthy controls. To our knowledge, similar results have not been reported previously. Explanation of this effect will for sure require further in-depth investigation. The following hypotheses might be tentatively suggested. Firstly, one may suppose that platelets of COVID-19 patients have a shifted balance in the expression of different platelet receptors involved in platelet adhesion under shear flow. Relatively increased expression of GPIb could potentially explain the effect observed. However, a separate series of thorough experiments is required before we can state or disprove this, because, as we have already mentioned, the data available for today is somewhat contradictory (Bongiovanni et al. 2021; Tacquard et al. 2023). A second possible hypothesis is a higher occupancy of GPIb complex by ligands such as SARS-CoV-2 spike protein. Further research is needed to clarify the influence of competing ligands to GPIb on platelet adhesion in COVID-19.

It is also worth noting that GPIb is known to be a major factor in thromboinflammatory processes in different pathological conditions including, for example, stroke (Denorme et al. 2019). Moreover, leukocyte integrin Mac-1 (also known as CD11b/CD18) directly interacts with platelet receptor GPIb promoting thrombosis (Wang et al. 2017). A couple of most recent studies indicate upregulation of Mac-1 (Hottz et al. 2022; Rieder et al. 2023) and increase of circulating platelet-neutrophil complexes in COVID-19 (Rieder et al. 2023). Taken together, these data suggest that GPIb might play a significant role in COVID-19 pathogenesis.

In conclusion, this study demonstrates that the contribution of GPIb to platelet adhesion was significantly altered in patients with severe COVID-19. Further investigations are necessary to determine the pathogenic mechanisms that produced this change.

References

[1] Berndt MC, Du X, Booth WJ (1988). Ristocetin-dependent reconstitution of binding of von Willebrand factor to purified human platelet membrane glycoprotein Ib-IX complex. Biochemistry 27: 633-640

[2] Bongiovanni D , Klug M , Lazareva O , Weidlich S , Biasi M , Ursu S , Warth S , Buske C , Lukas M , Spinner C. D , Scheidt MV , Condorelli G , Baumbach J , Laugwitz KL , List M , Bernlochner I . SARS-CoV-2 infection is associated with a pro-thrombotic platelet phenotype. Cell Death Dis, 2021, 12: 50

[3] Conway EM , Mackman N , Warren RQ , Wolberg AS , Mosnier LO , Campbell RA , Gralinski LE , Rondina MT , van de Veerdonk FL , Hoffmeister KM , Griffin JH , Nugent D , Moon K , Morrissey JH . Understanding COVID-19-associated coagulopathy. Nat Rev Immunol, 2022, 22: 639–649

[4] Denorme F , Vanhoorelbeke K , De Meyer SF . von Willebrand factor and platelet glycoprotein Ib: a thromboinflammatory axis in stroke. Front Immunol, 2019, 10: 2884

[5] Favaloro EJ , Henry BM , Lippi G . Increased VWF and decreased ADAMTS-13 in COVID-19: creating a milieu for (micro)thrombosis. Semin Thromb Hemost, 2021, 47: 400–418

[6] Gabbasov ZA , Avtaeva YN , Melnikov IS , Okhota SD , Caprnda M , Mozos I , Prosecky R , Rodrigo L , Kruzliak P , Zozulya NI . Kinetics of platelet adhesion to a fibrinogen‐coated surface in whole blood under flow conditions. J Clin Lab Anal, 2021, 35(9): e23939

[7] Hottz ED , Martins-Gonçalves R , Palhinha L , Azevedo-Quintanilha IG , De Campos MM , Sacramento CQ , Temerozo JR , Soares VC , Dias SSG , Teixeira L , Castro Í , Righy C , Souza TML , Kurtz P , Andrade BB , Nakaya HI , Monteiro RQ , Bozza FA , Bozza PT . Platelet-monocyte interaction amplifies thromboinflammation through tissue factor signaling in COVID-19. Blood Adv, 2022, 6(17): 5085–5099

[8] Joly BS , Darmon M , Dekimpe C , Dupont T , Dumas G , Yvin E , Beranger N , Vanhoorelbeke K , Azoulay E , Veyradier A . Imbalance of von Willebrand factor and ADAMTS13 axis is rather a biomarker of strong inflammation and endothelial damage than a cause of thrombotic process in critically ill COVID‐19 patients. J Thromb Haemost, 2021, 19(9): 2193–2198

[9] Mancini I , Baronciani L , Artoni A , Colpani P , Biganzoli M , Cozzi G , Novembrino C , Anzoletti MB , De Zan V , Pagliari MT , Gualtierotti R , Aliberti S , Panigada M , Grasselli G , Blasi F , Peyvandi F . The ADAMTS13‐von Willebrand factor axis in COVID‐19 patients. J Thromb Haemost, 2021, 19(2): 513–521

[10] Page EM , Ariëns RAS . Mechanisms of thrombosis and cardiovascular complications in COVID-19. Thromb Res, 2021, 200: 1–8

[11] Rieder M , Baldus N , Stallmann D , Jeserich M , Goller I , Wirth L , Pollmeier L , Hofmann M , Bode C , Busch H-J , Schmid B , Gauchel N , Scharf RE , Duerschmied D , Lother A , Krauel K . Early SARS-CoV-2 infection: Platelet-neutrophil complexes and platelet function. Res Pract Thromb Haemost, 2023, 7(1): 100025

[12] Schulman S , Sholzberg M , Spyropoulos AC , Zarychanski R , Resnick HE , Bradbury CA , Broxmeyer L , Connors JM , Falanga A , Iba T , Kaatz S , Levy JH , Middeldorp S , Minichiello T , Ramacciotti E , Samama CM , Thachil J; International Society on Thrombosis . ISTH guidelines for antithrombotic treatment in COVID‐19. J Thromb Haemost, 2022, 20(10): 2214–2225

[13] Tacquard C , Mouriaux C , Delabranche X , Bourdon C , Eckly A , Magnenat S , Sattler L , Gachet C , Michel P , Hechler B , Mangin PH . Platelet dysfunction and thrombus instability in flow conditions in patients with severe COVID-19. Thromb Res, 2023, 221: 137–148

[14] Wang Y , Gao H , Shi C , Erhardt PW , Pavlovsky A , Soloviev DA , Bledzka K , Ustinov V , Zhu L , Qin J , Munday AD , Lopez J , Plow E , Simon DI . Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα. Nat Commun, 2017, 8: 15559

[15] Xu X , Feng Y , Jia Y , Zhang X , Li L , Bai X , Jiao L . Prognostic value of von Willebrand factor and ADAMTS13 in patients with COVID-19: a systematic review and meta-analysis. Thromb Res, 2022, 218: 83–98

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The Author(s) 2026. Published by Higher Education Press. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0)