Abstract
Blood coagulation mechanisms play key roles in health and disease. Pilot studies using selected human plasmas showed the potential associations of plasma skeletal muscle myosin (SkM) isoforms and phenotypes with pulmonary embolism and thrombin generation, suggesting SkM may contribute to blood coagulation reactions in plasma. Here we report that ex vivo studies of the coagulability of fresh flowing human blood over SkM-coated surfaces showed that an anti-factor XI (FXI) mAb, but not an anti-tissue factor mAb, inhibited clot formation, indicating that FXI is an essential contributor for the normal observed procoagulant response of blood during its exposure to immobilized SkM. This raised the question of whether procoagulant SkM's requirement for FXI involves direct or indirect effects on FXI.
To assess direct interactions between SkM and FXI, Bio-Layer Interferometry (BLI) (Octet Red system) was used to record kinetics for binding of soluble FXI to immobilized SkM. BLI data showed that FXI bound to SkM with a Kd of 0.2 nM (k on= 2.92x10 6 M -1s -1 and k off=9.25x10 -3 s -1) (Fig. 1A). In contrast, prekallikrein (PK) did not bind to the SkM (Fig.1A), indicating the specificity of SkM for binding FXI. The anti-FXI mAb1A6, which recognizes the Apple (A)3 domain of FXI, potently inhibited binding of FXI to immobilized SkM, implying SkM binds the FXI A3 domain.
Studies using purified clotting factors were made to identify which FXI-related activities might be affected by SkM. When FXI activation by thrombin was evaluated under conditions where polyphosphate (PolyP) 100-mer and 700-mer enhance FXI activation, SkM concentration-dependently enhanced FXI activation by thrombin (Fig. 1B). Whereas alkaline phosphatase destroyed PolyP's ability to stimulate FXI activation by thrombin, it did not cause a reduction of SkM's ability to enhance FXI activation, indicating SkM's activity is independent of PolyP-like sequences in SkM. Small unilamellar phospholipid vesicles (20% phosphatidylserine (PS) / 80% phosphatidylcholine) did not affect FXI activation by thrombin; furthermore, reagents that neutralize procoagulant PS, i.e., lactadherin, annexin V, and phospholipase A2, did not affect SkM's enhancement of FXI activation by thrombin, indicating that this activity is not due to anionic phospholipids linked to SkM.
The effects of SkM on FXI autoactivation and FXI activation by FXIIa were evaluated. As is well known, PolyP and some other anionic reagents, e.g., nucleic acid polymers, enhance not only FXI activation by thrombin but also FXI autoactivation and FXI activation by FXIIa. However, SkM did not significantly affect FXI autoactivation or FXI activation by factor XIIa, further emphasizing that SkM's enhancement of FXI activation by thrombin is not due to any PolyP-like compounds and that it is a unique property of procoagulant SkM. This also suggests that SkM has a unique mechanism for its procoagulant activity on FXI activation which is limited to the thrombin positive feedback loop.
To evaluate further the basis for interactions between FXI and SkM, we employed FXI- PK chimeras because BLI binding studies showed that, in contrast to FXI, PK did not bind to SkM. Recombinant FXI proteins in which each of the four A domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding A domain from PK and were used to identify the site of factor XI to interact with SkM for FXI activation by thrombin. The FXI chimera with the substitution of the PKA1 domain was not activated by thrombin, which is consistent with the fact that the FXI A1 domain is an interactive site for thrombin. Thrombin activation of the two FXI chimeras (FXI/PKA3 and FXI/PKA4) with substitutions of either the A3 or A4 domains was not enhanced by SkM, whereas substitution of the A2 domain (FXI/PKA2) did not reduce the enhancement of activation by thrombin compared to wild type FXI. Furthermore, mAb1A6, which recognizes the A3 domain and which inhibited the prothrombotic activity of fresh blood flowing over a SkM-coated surface, potently inhibited FXI binding to SkM in BLI studies. These data strongly suggest that functional interaction sites on FXI for SkM involve the A3 and A4 domains of FXI.
In summary, we found that SkM's ex vivo procoagulant activity requires FXI, that SkM enhances FXI activation by thrombin and this requires FXI's A3 and A4 domains, and that SkM's high affinity binding of FXI requires the FXI A3 domain (Fig. 1C).
Figure 1 Figure 1.
Disclosures
Gruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment. Gailani: Anthos Therapeutics: Consultancy; Aronora: Membership on an entity's Board of Directors or advisory committees; Bayer Pharma: Consultancy; Bristol Myer Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees.
Ex vivo evaluation of blood coagulation on endothelial glycocalyx-inspired surfaces using thromboelastography
