Evidence of the Intrinsic Coagulation Pathway and Contact System Activation in Sickle Cell Patients During Steady State

A chronic hypercoagulable state and increased risk for venous thromboembolism are prominent features of sickle cell disease (SCD). This hypercoagulable state is mediated by activation of both the intrinsic and extrinsic coagulation pathways. There is growing interest in targeting components of the intrinsic coagulation pathway (factor XII and XI) to prevent thrombotic disorders, primarily because this goal may be attainable without incurring any significant bleeding risk. In addition, FXIIa can also activate plasma prekallikrein to kallikrein (PK), which initiates the contact pathway. We have previously reported that FXIIa contributes to the prothrombotic state and vascular inflammation in sickle mice. In this study we investigated if the intrinsic and contact pathways are activated in SCD patients. Fifty four African American outpatients with SCD and twenty three healthy African‐Americans were recruited. The study was approved by the University of North Carolina’s Institutional Review Board for human subjects. Platelet‐poor plasma was prepared from blood drawn into 3.2% sodium citrate (9:1). To detect activation of FXII, FXI, FIX and PK we have developed sandwich ELISAs to quantitate plasma levels of complexes formed between active forms of these proteases and their natural inhibitor C1 esterase inhibitor (C1). We used mouse monoclonal antibodies specific to the proteases to capture the complexes and a sheep polyclonal antibody to recognize C1. The polyclonal antibody is biotinylated, and detected with streptavidin‐horseradish peroxidase. Protein standards are generated by combining ~6μM purified proteases with ~12μM recombinant C1 for 18 hours at 37C, and quenched with 25μM PPACK. These standards are diluted to achieve the working linear range of the assay. Compared to healthy controls, plasma levels of FXIIa‐C1 (1.07±0.19 vs 2.33±0.29nM; mean ±SEM), FXIa‐C1 (0.47±0.09 vs 0.98±0.10nM) and FIXa‐C1 (0.84±0.13 vs1.43±0.16nM) were all significantly elevated in SCD patients (p<0.05 for all groups). Similar to that plasma levels of PK‐C1 complexes were also elevated in SCD patients (12.50±1.06 vs 16.74±1.05; p<0.05). In the SCD patient group, highly significant correlations were observed between activation of FIX and activation of both upstream components of the intrinsic pathway (r=0.91; p<0.0001 for FXIIa and r=0.91; p<0.0001 for FXIa). Interestingly, a significant correction was also observed between FIX‐C1 and PK‐C1 complexes (r=0.64; p<0.0001). Our data strongly suggests that both the intrinsic coagulation pathway and contact system are activated and contribute to the chronic hypercoagulable state observed during steady state in SCD patients. The contribution of these two pathways to the pathology of SCD should be further investigated. Support or Funding Information The authors wish to acknowledge support from NIH grants 1UO1HL117659 and R01HL142604