So if the spike protein in Covid causes disease itself?
Does that saying anything about the spike in the vaccines?
For the douchebags who will attack...
I am not making any claims... I am investigating...
Does that saying anything about the spike in the vaccines?
For the douchebags who will attack...
I am not making any claims... I am investigating...
https://portlandpress.com/bioscirep...RS-CoV-2-spike-protein-S1-induces-fibrin-ogen
SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19
Discussion
In this laboratory analysis, we provide evidence that spike protein does indeed play a major role in hypercoagulability seen in COVID-19 patients. It causes anomalous clotting in both purified fluorescent fibrinogen and in PPP from healthy individuals, where the nature of the clots were shown to be amyloid (ThT as our amyloid dye of choice). An interesting observation was that these dense deposits were noted both in smears exposed to spike protein, and when thrombin was added. The addition of thrombin causes purified (Alexa Fluor™ 488) fibrinogen to polymerize into fibrin networks. Typically, these networks are net-like (Figure 3A). In the presence of spike protein, the structure changed to form dense clot deposits (Figure 3B). These deposits were seen in our fluorescent fibrin(ogen) model and PPP from healthy individuals exposed to spike protein. In healthy PPP exposed to spike protein, followed by incubation with ThT, there was a significant increase in anomalous clots with an amyloid nature, (Figure 4D), when compared with the healthy PPP. In the current paper, we did not analyze blood samples and clotting propensity of PPP from other patient cohorts, e.g. those with bacterial pneumonia or other acute viral diseases. However, our group (and others) have previously studied blood from HIV patients, where significant hypercoagulation in this patient cohort was noted [36–39]. We have also, recently, reported significant hypercoagulation in patients suffering from long COVID/PASC [40].
Spike protein also caused major ultrastructural changes in WB (as viewed with the SEM), where platelet hyperactivation was noted (Figure 6C,D). Increased in spontaneously formed fibrin network, as well as anomalous clot formation were also observed in SEM micrographs (Figure 6E–H). Interestingly, extensive spontaneous fibrin network formation was noted, without the addition of thrombin. This is in line with results that were recently published, where we showed similar ultrastructure in blood smears form COVID-19 positive patients. In these patients, platelet hyperactivation, anomalous clotting with amyloid signal, and spontaneous fibrin fiber formation were also observed [6,7].
With the microfluidics flow system, clots were formed, by infusing the entire microchannel with thrombin, thus simulating a hypercoagulable state, where endothelial damage was extensive. Given that the flow channel was made entirely of plastic and was devoid of any endothelial cells, the main component under investigation was the PPP (mostly fibrinogen protein) itself, which, in the case of the COVID-19 samples, may have contained downstream effects of some endothelial changes that would give rise to the hypercoagulable state that is characteristic of the disease. The flow setup used in the present study could not directly account for endothelial changes but nonetheless demonstrated that COVID-19 also results in changes in the clotting profile of the PPP. This was evident in the rapid rate of thrombin consumption and fibrin formation in COVID-19 clots, and also in the nature of the PPP clots that were formed.
The clots that were observed in the healthy PPP with added spike protein, were of particular interest as they demonstrated a bridge between healthy PPP clots and COVID-19 clots. As described in the ‘Results’ section, the healthy PPP clots were relatively small and orderly, while COVID-19 PPP clots were large, disorderly masses that formed rapidly and disrupted PPP flow in the channel. The healthy PPP clots with added spike protein, were a combination of the two, demonstrating disorderly clumped clot areas, coexisting with laminar fibrous PPP clots (which were larger than the healthy PPP clots). This intermediate state may arise from a number of factors, including the interaction of other biological actors which were absent from the flow setup and the time of exposure to spike protein. Further investigations would be beneficial for understanding the clotting mechanisms that are altered in the presence of spike protein.
One of the obvious differences, which was inadvertently observed while trying to clean the channels with high-speed water flow (i.e. by mechanical means), was the ease of healthy PPP and healthy PPP with added spike protein clot dissolution. However, there was a complete failure to dislodge or disturb COVID-19 PPP clots from the channels. Given the clot lysis and dissolution is a complex interplay between biochemical and biophysical factors, investigation of the effects of different therapeutic agents could elucidate this phenomenon [41]. The flow protocol used in this study would be a useful platform for testing different treatments for clinical application. A further limitation of this exploration is the use of PPP in investigating clot formation at a scale appropriate to the microvasculature. While the protocol enables the study of fibrin microclots, which are of interest in COVID-19, it excludes the influence of RBCs, which are known to heavily influence the non-Newtonian flow behavior of blood at that scale [42]. The inaccuracy of the flow regime arising from this exclusion and from the variability of viscosity introduces error into the results. Nonetheless, the inclusion of flow an appropriate spatial scale has enabled us to observe COVID-19 PPP clot formation over space and time, under dynamic conditions, and has given insights which would otherwise prove difficult to glean.
A further avenue for exploration would include examining clot stability and removal for other patients with acute inflammatory responses arising from acute infections. Longstaff and co-workers found increased fibrinolytic resistance in inflammatory conditions arising from acute infection [43]. Examining this phenomenon in our microfluidic setup would be beneficial.
Given that microclots can block microcapillaries and thereby inhibit oxygen exchange, we have recently also studied plasma samples, using proteomics results from Long COVID/PASC, T2DM, with acute COVID-19 and compared results with plasma samples from healthy individuals. Interestingly, plasma from T2DM and form healthy individuals, immediately digested fully after a first trypsinization step, however, persistent microclots remained in the plasma samples from Long COVID/PASC and from acute COVID-19 samples, still contained large anomalous (amyloid) deposits (microclots) [40]. After a second trypsinization, the persistent pellet deposits were solubilized. We detected various inflammatory molecules that are substantially increased in both the supernatant and trapped in the solubilized pellet deposits of acute COVID-19 and Long COVID/PASC, versus the equivalent volume of fully digested fluid of the control samples and T2DM [40]. Of particular interest was a substantial increase in α(2)-antiplasmin (α2AP), various fibrinogen chains in both acute COVID-19 and Long COVID/PASC digested microclots. A comparison between healthy plasma and acute COVID-19 solubilized clots also showed a significant increase in coagulation factor XIII A chain, VWF Complement component C7 and CRP [40].
In the current study, mass spectrometry confirmed that spike protein causes structural changes to β and γ fibrin(ogen), complement 3, and prothrombin. These proteins become less resistant to trypsinization and changes the conformation, in such a way that there is a significant difference in peptide structure before and after spike protein addition. The current results therefore confirm results we have found in our recent proteomics analysis [40].
Our recent data suggest that there is an increase in (2)-antiplasmin inside the microclots of both acute COVID-19 and Long COVID, and we also note pathophysiology in the fibrinogen chains. In the present paper, we could induce fibrinogen chain pathology, after adding spike protein to healthy plasma. To result in an increase in molecules like α2-antiplasmin and VWF (and others), in patients with acute COVID-19 and also those with Long COVID/PASC, many physiological pathways should be activated. See Figure 9 (adjusted from [40, 44–46]) for a visual representation of the coagulation pathway and where it may be affected by S1.
