Hijacking coagulation using platelets

One-third of COVID-19 patients in ICUs develop thrombotic complications leading to high morbidity

Hijacking coagulation using platelets

In the one year since SARS-CoV-2 (COVID-19) arrived on the scene, the virus has precipitated nothing short of a serious global health crisis. One of the virus’ less-obvious dimensions has been its impact on the human cardiovascular system.

Over the past year, COVID-19 has been associated with several critical cardiovascular complications. So much so that even patients with no history of cardiovascular disease seem to be at risk of developing cardiovascular complications as a result of COVID-19. Indeed, patients with severe COVID-19 infection commonly experience thrombotic disorders with elevated D-dimer, sepsis, disseminated intravascular coagulation (DIC) with high mortality rates.

Several large-scale studies have shown that 18.8%-36.2% of COVID-19 patients show thrombocytopenia at the time of admission into hospital and in cumulative, 31% of ICU patients experience thrombotic complications. Even though there is overwhelming evidence of a hypercoagulable state in COVID-19 patients, the molecular events and underlying mechanisms still remain largely a mystery.

The role of platelets

It is well known that platelets play an important role in thrombosis and hemostasis. During infection, activated platelets adhere to the sub-endothelium and their hyperactivity can result in thrombus formation, leading to arterial ischemia and pulmonary embolisms. Many viral infections, such as hepatitis C (HCV), Ebola, human immunodeficiency virus (HIV), dengue virus (DV) and influenza, directly lead to the activation of platelets triggering uncontrolled coagulation cascades, resulting in lung injury. We know that SARS-CoV-2 uses a spike protein to bind to ACE2 receptors on the host cell for entry and multiplication, but we have little insight into how this influences platelets.

In this regard, Zhang et al. (J of Hematology & Oncology (2020) 13:120) recently demonstrated a very important mechanism in which SARS-CoV-2 spike proteins induce platelet activation and thrombosis. Their study included 201 healthy volunteers and 589 patients (422 COVID-19 positive and 167 non-COVID-19). Severe and critically severe COVID-19 patients showed abnormal platelet count, plateletcrit and increased mean platelet volume (MPV) and platelet distribution width (PDW). Abnormalities were also noticed on various parameters such as prothrombin time (PT), international normalized ratio (INR), activated thromboplastin time (APTT), D-dimer, fibrinogen degradation products (FDPs) and prothrombin time activity (PTA) compared to healthy subjects.

Viral RNA & platelet hyperactivity

Besides increased MPV, higher levels of integrin αIIbβ3 activation (PAC-1 binding) and P-selectin (CD62P) expression were also observed, especially in critically ill COVID-19 patients. Out of 241 COVID-19 patients, 15 were severely and critically ill with RNA positivity for SARS-COV-2 in the blood. These patients have higher integrin αIIbβ3 activation and P-selectin expression on platelets, compared with those with undetectable viral RNA in the blood. Furthermore, studies have also found that SARS-COV-2 RNA-positive PPP (platelet-poor plasma) enhanced platelet aggregation, compared with SARS-COV-2 RNA negative PPP and healthy PPP. These results suggest that the presence of SARS-CoV-2 viral RNA in the blood is an indicator of platelet hyperactivity. SARS-CoV-2 promoted ACE2 internalization and degradation in a time-dependent manner in platelets, indicating that once the platelets are activated, ACE2 is degraded. Studies also suggest that the S1 subunit of the spike protein interacts with the ACE2 receptor, and higher levels of spike S1 subunits (but not S2) induce platelet aggregation via integrin αIIbβ3 activation and P-selectin expression. At the molecular level, important phosphorylation markers such as Erk, p38 and JNK — which are associated with the MAPK pathway — were found to be hyperphosphorylated in COVID-19 patients than in control subjects.

Interestingly, transgenic mice bearing human ACE2 (hACE2) injected intravenously with spike S1 subunit of SARS-COV-2 showed increased thrombus formation in comparison to uninjected or wild type mice. Platelets activated with S1 protein had increased coagulation factors such as Factor V and XIII in α granules. S1 protein interaction with platelets can also stimulate a large variety of inflammatory cytokines as has been observed in COVID-19 patients. The presence of spike S1 subunit has also been associated with high levels of PF4 and TNFα, along with increased aggregation of leukocyte-platelet (CD45+ CD41+), monocyte-platelet (CD14+ CD41+) and neutrophil-platelets (CD65+ CD41+). All this suggests that anti-spike treatments can become a potential way of overcoming this pandemic.

Peptide approach

Initial experimental data using decoy peptides, which act as ACE2 receptors,  has shown that very low-dose peptides can inhibit the binding or SARS-CoV-2 spike S1 protein with ACE2 and block viral activity. Such therapeutic approaches not only block the virus from entering into host cells but can also block several thrombotic events which follow post-infection. It is well understood that several viruses indirectly activate platelets during an infection by creating an inflammatory microenvironment and vascular endothelial dysfunction. However, there is now evidence that SARS-CoV-2 spike S1 protein can directly interact with platelets as is seen cases such as that of encephalomyocarditis virus via TLR7, rotavirus with GPIa/IIa, hantavirus and adenovirus through GPIIb/IIIa.

Another important member in the coagulation pathway is the tissue factor (TF) in platelets, which is a physiological activator of the extrinsic coagulation pathway and plays an important role in the hemostatic protection of vital organs such as the lung, brain and heart. TF is a transmembrane glycoprotein essentially present in the subendothelial vessel wall under normal physiological conditions. In a normal, healthy individual, TF levels are low and are supposed to be non-functional as it is highly procoagulant. However, any disruption to the vessel wall immediately results in upregulation of TF, which has a high affinity to factors VII and VIIa. SARS-CoV-2 spike S1 subunit interaction with the ACE2 receptors on platelets or on monocytes can lead to increased levels of TF and promote deregulated thrombosis. In case of some other infections like dengue virus, the infection activates the fibrinolysis pathway by degrading fibrinogen and thus promotes secondary activation of the pro-coagulant expression of TF. In the case of Ebola, TF expression is stimulated in macrophages, inducing abnormal coagulation.

SARS-CoV-2 associated coagulation mechanisms are still under investigation, but it is important for us to understand that this virus can have more than one way of derailing the normal functions of the body. A multipronged approach to understanding molecular events is crucial for tackling this pandemic as early as possible.

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