Skeletal muscle myosin promotes coagulation by binding factor XI via its A3 domain and enhancing thrombin-induced factor XI activation

Abstract

Skeletal muscle myosin (SkM) has been shown to possess procoagulant activity; however, the mechanisms of this coagulation-enhancing activity involving plasma coagulation pathways and factors are incompletely understood. Here, we discovered direct interactions between immobilized SkM and coagulation factor XI (FXI) using biolayer interferometry (K_d = 0.2 nM). In contrast, we show that prekallikrein, a FXI homolog, did not bind to SkM, reflecting the specificity of SkM for FXI binding. We also found that the anti-FXI monoclonal antibody, mAb 1A6, which recognizes the Apple (A) 3 domain of FXI, potently inhibited binding of FXI to immobilized SkM, implying that SkM binds FXI A3 domain. In addition, we show that SkM enhanced FXI activation by thrombin in a concentration-dependent manner. We further used recombinant FXI chimeric proteins in which each of the four A domains of the heavy chain (designated A1 through A4) was individually replaced with the corresponding A domain from prekallikrein to investigate SkM-mediated enhancement of thrombin-induced FXI activation. These results indicated that activation of two FXI chimeras with substitutions of either the A3 domains or A4 domains was not enhanced by SkM, whereas substitution of the A2 domain did not reduce the thrombin-induced activation compared with wildtype FXI. These data strongly suggest that functional interaction sites on FXI for SkM involve the A3 and A4 domains. Thus, this study is the first to reveal and support the novel intrinsic blood coagulation pathway concept that the procoagulant mechanisms of SkM include FXI binding and enhancement of FXI activation by thrombin.

Keywords: coagulation factor; factor XI; myosin; skeletal muscle myosin; thrombin.

Figure 1

Purified FXIand the endogenous plasma FXIbound to immobilized SkMin solid phase microtiterplate–bindingassays. SkM or BSA was immobilized on 96-well plate in sodium bicarbonate, pH 9.3. After blocking with 0.5% BSA, aliquots (0.2 ml) containing various concentrations of purified FXI (A) or of diluted pooled plasma (B) in 30 mM Hepes buffer, pH 7.4, containing 130 mM NaCl were incubated on the immobilized SkM for 2 h at room temperature. Then, after washing the wells, the bound FXI was exposed to anti-FXI monoclonal antibody (AHXI-5061), which, after a washing step, was detected with the HRP-conjugated goat antimouse antibody as described in the Experimental procedures section. The bound endogenous plasma FXI in (B) was calculated using the curve shown in (A). Each value represents the mean (±SD) of triplicate determinations. BSA, bovine serum albumin; FXI, factor XI; HRP, horseradish peroxidase; SkM, skeletal muscle myosin.

Figure 2

FXI binds with high affinity to immobilized SkM. Binding studies using biolayer interferometry (BLI) were done with FXI or prekallikrein (PK) that were in 30 mM Hepes, pH 7.4, 0.01% Tween-20 and 0.1% PEG, and 50 mM NaCl at 30 °C. SkM or BSA was immobilized onto the Octet Red Amine Reactive Second-Generation Biosensor surface. A, sensorgrams depict the binding of FXI or PK to immobilized SkM (concentrations from top to bottom: 25, 13, 6, 3 nM for SkM and 25 nM for PK). B, sensorgrams depict the binding of 12.5 nM FXI in the presence or the absence of 100 μg/ml of anti-FXI antibody (Ab), namely mAb 1A6 (18). BSA, bovine serum albumin; FXI, factor XI; SkM, skeletal muscle myosin.

Figure 3

SkM enhances FXI activation by thrombin but not FXI autoactivation or FXI activation by alpha-factor XIIa.A–C, for FXI activation by thrombin, (A) FXI was activated by IIa for various times in the presence of 50 nM SkM or (B) for 30 min at 37 °C with various concentrations of SkM or (C) with varying concentrations of PolyPs (100-mer [closed circle] or 700-mer [open circle]). D and E, for the effects of various reagents on SkM enhancement of FXI activation by thrombin, (D) FXI activation was determined in the presence of 50 nM SkM or 4 μM PolyP (100-mer) for 30 min at 37 °C and in the presence (open triangle) or the absence (open circle) of alkaline phosphatase (ALP). E, first, FXI activation by thrombin in the absence (“none”) or the presence of 4 μM PC/PS (80%/20% w/w) phospholipid vesicles was determined; second, FXI activation by thrombin for 30 min at 37 °C in the presence of 50 nM SkM and in the absence (“none”) or, as indicated, in the presence of annexin V (AnnV) (20 nM final), lactadherin (Lact) (20 nM, final), or phospholipase A2 (PLA2) (20 nM, final) was determined. The reactions were quenched using hirudin (2 ATU, final) to inhibit thrombin, and FXIa amidolytic activity was measured. F, activation of FXI/prekallikrein (PK) chimeras by thrombin: Recombinant (r) wt FXI (rFXI) (filled circle) or rFXI/PK Apple domain chimeras (FXI/PKA1 [open square], FXI/PKA2 [open triangle], FXI/PKA3 [filled triangle], or FXI/PKA4 [filled diamond]) were activated by thrombin in the presence of various concentrations (0–100 nM) of SkM. SkM-dependent FXIa generation after 30 min at 37 °C was determined as described in the Experimental procedures section, and FXIa levels are shown after the subtraction of the low level of FXIa generated by thrombin in the absence of SkM. G, FXI autoactivation and (H) FXI activation by alpha-factor XIIa (FXIIaα) were measured as described in the Experimental procedures section. Each value represents the mean (±SD) of triplicate determinations. FXI, factor XI; PC, phosphatidylcholine; PolyP, polyphosphate; PS, phosphatidylserine; SkM, skeletal muscle myosin.