Persistent clotting protein pathology in Long COVID/PASC is accompanied by increased levels of antiplasmin, 2021, Pretorius et al

The Pretorius study is interesting - I'm trying to understand it. I haven't read the whole paper yet.

They looked at the blood of

• healthy volunteers (13)
• people with acute Covid (15)
• people with type 2 diabetes (10)
• patients with Long Covid (11)

The definition of Long Covid was

2 months is a pretty short time, especially as some of these patients were very sick with acute Covid-19 (6 were hospitalised and given oxygen, one of those was on a ventilator). Maybe some of the symptoms attributed to Long Covid are really just the result of recovering from having a significant illness?

And they did not look at the blood of people who recovered from Covid-19 and who do not have Long Covid symptoms. So, we can't be sure that anything that is found in the Long Covid patients would not also be found in other people who had Covid-19 not long ago and don't have ongoing symptoms.




In the results section there is a section about
Platelet poor plasma (PPP): Amyloid fibrin(ogen) protein and anomalous clotting in platelet poor plasma samples, before and after two trypsin digestion protocols

They say that they have previously shown that platelet poor plasma from healthy controls and type 2 diabetes patients have significantly less 'anomalous Microclots' than that from people with Covid-19. They use a marker, thioflavin, to bind to the clots. And they say that they have found that people with Long Covid have a similar amount of microclots as the people with acute Covid-19.

So in Figure 3, they are looking at platelet poor plasma (PPP) before trypsin was added to digest the microclots. Pictures A and B are from one individual who donated a blood sample when healthy, and then went on to get Covid-19 and developed Long Covid symptoms. And C is pictures from other Long Covid patients.






Then, they add trypsin, to, they say, 'digest', the microclots. And that seems to work in the platelet poor serum of the controls and diabetes patients (Pictures A and B in Figure 4). But it doesn't work in platelet poor serum of the Covid-19 patients (Pictures C in Figure 4), or of the Long Covid patients (Figure 5).

 

So, then the authors go on to look at what is in these resistant clots. They applied trypsin again, this time successfully digesting the clots. And then they looked for proteins in the dissolved clot liquid. Table 1 is interesting - they certainly found significant differences between the proteins of the Covid-19, Long Covid and control samples. They reported the results as fold changes (so 1 means the same amount, 2 means twice the amount). All of the differences have p-values indicating very good significance.

Between acute Covid -19 and control samples
Von Willebrand Factor - 4.5 x control level
Complement component C4b - 4.2 x control level
C-reactive protein - 18.7 x control level

Between Long Covid and Control samples
Von Willebrand Factor - 10.2 x control level
Complement component C4b - not in the table
C-reactive protein - 11.2 x control level
Coagulation factor XIII A chain - 6.9 x control level
Plasminogen - 3 x control level
Fibrinogen alpha chain - 4.1 x control level
a2 Antiplasmin (a2AP) - 8.0 x control level
Serum Amyloid (SAA4) - 17.5 x control level
Complement component C7 - 20 x control level

Between Long Covid and acute Covid-19 samples
Plasminogen - 2.3 x acute Covid-19 level
Fibrinogen beta chain - 2.8 x acute Covid-19 level
Coagulation factor XIII B - 2.7 x acute Covid-19 level
Fibrinogen alpha chain - 3.1 x acute Covid-19 level
Complement component C6 - 7.5 x acute Covid-19 level
a2 Antiplasmin (a2AP) - 9.2 x acute Covid-19 level
Complement factor 1 - 25 x acute Covid-19 level

This looks pretty interesting to me. It's not often we see really big, statistically valid differences. I really want to see this study replicated, looking at Long Covid (>6 months) versus controls who have had Covid-19 about the same number of months ago. And of course in ME/CFS too.

 

The other thing the authors talk about is
Platelet pathology as viewed with fluorescence microscopy

They say that they and others have found that platelets are hyperactivated in type 2 diabetes. And that they have recently found this hyperactivation in acute Covid-19.

They say that they have found the same in Long Covid. Not only are the platelets activated, but they are also clumping.

An activated platelet seems to be one that is doing what needs to be done when there is a wound: forming a clot to stop the bleeding. The authors used two different fluorescent markers that attach to the surface of the platelet when it is activated.

Figure 2 gives some images. A and B are controls; C and D are Covid-19; E, F G and H are all Long Covid. all of the pictures are at the same level of magnification.




In the discussion, the authors mention Von Willebrand Factor (VWF) as causing the activation. VWF seems to act specifically to cause the platelet to stick to the wall of a blood vessel.

So, the authors suggest there are two things going on:
1. the hyper-activation and clumping of the platelets, and
2. as discussed in the previous two posts, the amyloid microclots

 

We have a discussion thread for consideration of the Microclots hypothesis here:
The micro-clot finding in Long Covid — implications for the possible aetiology of ME/CFS