Platelet-Derived Beta Thromboglobulin: A Potential Novel Activator of Coagulation Factor X
Authors: Egan Karl , Ma Hui , Kevane Barry , Lennon Áine , Neary Elaine , Wynne Kieran , Allen Seamus , O'Shea Susan , O Kennedy Richard , Ní Áinle Fionnuala 
Published: October 2019

Abstract
Background
During clotting, factors IXa and VIIa participate in the activation of Factor X, leading to thrombin generation. This process occurs on the surface of activated platelets. Activated platelets release an array of proteins that can modulate thrombin generation. This includes platelet factor 4 (PF4), a chemokine that modulates coagulation by promoting protein C activation and by attenuating the anticoagulant activity of activated protein C. PF4 shares significant homology with another protein released in high concentration by platelets, β-thromboglobulin (βTG). The physiological effects of βTG, including effects on blood coagulation, are poorly characterized. The aim of this study was to assess the effect of βTG on blood coagulation.
Methods
The effect of purified human βTG on coagulation was determined using the prothrombin time (PT), activated partial thromboplastin time (APTT), calibrated automated thrombography (4µM phospholipids ± 1pM tissue factor (TF)) and chromogenic FX and prothrombin activation assays. Mass spectrometry and surface plasmon resonance (SPR) were used to assess the composition of the purified βTG preparation and to measure protein-protein interactions respectively.
Results
Mass spectrometry confirmed the absence of TF, FVIIa, FVIIIa, FIXa, FVa, and FXa in the βTG preparation. In normal pooled plasma, βTG dose-dependently increased the rate and extent of TF stimulated thrombin generation. In the absence of βTG, the lagtime was 9±1 min, which was shortened in a dose-dependent manner upon incubation with βTG (100µg/ml; 5±1 min, p<0.05). Incubation with βTG (50µg/ml) also shortened the APTT (35±1 to 25±1 secs, p<0.05) and reduced the lagtime to thrombin generation (26±5 to 7±1 min, p<0.05) and increased the peak thrombin generation (60±30 to 97±23nM, p<0.05) in the absence of an exogenous TF stimulus. Immunodepleted plasmas and inhibitory antibodies were used to determine the underlying mechanism of action. In FVIII-deficient plasma, thrombin generation was not observed in the absence of an exogenous TF stimulus. However, upon incubation with βTG (50µg/ml), thrombin generation was observed and peak thrombin generation increased from 1±1 to 75±12nM IIa (p<0.05). βTG also shortened the APTT in FVIII-deficient plasma from 131±8 to 103±14 secs (50µg/ml, p<0.05). The procoagulant effect of βTG in FVIII-deficient plasma was not inhibited by TF or FVIIa inhibitory antibodies, suggesting that the effect was independent of the intrinsic tenase complex, TF or FVIIa. Interestingly, homologous PF4 did not induce thrombin generation in FVIII-deficient plasma (peak thrombin generation was 75±12nM v 0±0nM IIa upon incubation with 50µg/ml βTG and PF4 respectively). The procoagulant effect of βTG persisted when thrombin generation was independent of FV activation (supplementation of FV-deficient plasma with FVa). In contrast, the effect was not observed when thrombin generation was independent of FX activation (supplementation of FX-deficient plasma with FXa). Collectively, these data raised the possibility that βTG may modulate FX activation. To investigate this hypothesis, chromogenic FX activation was measured. Cleavage of a FXa-specific chromogenic substrate was observed upon incubation of βTG with FX, suggesting a direct effect of βTG upon FX activation. No measurable chromogenic substrate cleavage was observed upon incubation with βTG or FX alone. In contrast, βTG did not induce prothrombin activation, measured using a thrombin-specific chromogenic substrate. Using SPR, βTG was found to bind directly to immobilised FX (KD 1.36±0.7x10-7 M). The kinetics of the interaction between βTG and FX was lower than that between FIXa and FX (KD 4.25±1.90 × 10-9 M).
Conclusion
In conclusion, we have identified that βTG is a potential novel platelet-derived activator of coagulation FX. Moreover, βTG is capable of inducing thrombin generation in immunodepleted FVIII and FIX-deficient plasma, a finding that may be of translational relevance to patients with haemophilia.

Disclosures
Ní Áinle: Actelion Pharma: Research Funding; Leo Pharma: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.