Mechanism Underlying a Role for Factor XIII (FXIII) Polymorphism in Sickle Cell Disease-Associated Priapism

Background: The pathophysiology of priapism in sickle cell disease (SCD) is poorly understood. While blood stasis is essential to tumescence, most research in SCD-associated priapism to date has focused on the potential role of abnormal signaling pathways. We have previously observed that a coding sequence single nucleotide polymorphism (rs5988) of the transglutaminase factor XIII gene (FXIII) is strongly associated with SCD priapism, with an odds ratio of 2.52 [C.I. 1.27 -5.03] for the risk genotype (G/G, expressing only FXIII E652) vs the most common non-risk genotype (G/C, expressing both FXIII E652 and FXIII Q652). We therefore explored the effect of the rs5988 polymorphism on various aspects of FXIII function.

Methods: Recombinant FXIII (rFXIII) E652 and rFXIII Q652 were expressed by 293 kidney cells and isolated from serum-free tissue culture supernatant. Before use in assays, activation of rFXIII by thrombin was confirmed by generating activated rFXIII (rFXIIIa) with thrombin (10 U/ml), followed SDS-PAGE, western blotting for FXIII, and densitometry for quantitation. Whole blood samples and plasmas were obtained from previously genotyped subjects with SCD under an IRB-approved protocol. Plasma FXIII and rFXIII transglutaminase activity was measured by the ability to catalyze 5-(biotinamido)pentylamine incorporation into a suitable substrate. Plasma FXIII antigen was assayed by ELISA. Clot contraction was measured after tissue factor-initiated clotting of recalcified whole blood. Clot resistance to lysis was measured after exposure to tissue plasminogen activator (tPA).

Results: Transglutaminase activity of each rFXIII was measured using fibrinogen and fibronectin as substrates. At 20 minutes, rFXIIIa E652 showed 1.44-fold more transglutaminase (crosslinking) activity toward fibrin(ogen) than rFXIIIa Q652 (p=0.027), and a nonsignificant trend toward more activity (1.32-fold, p=0.079) toward fibronectin. Kinetic assays also showed that rFXIII E652 had significantly greater activity toward both matrices (p=0.006 and 0.012, respectively), suggesting the risk genotype (homozygosity for the G allele) enhances fibrin(ogen) and/or fibronectin crosslinking. FXIII activity in the plasma of 18 genotyped SCD patients (3 CC, 7 GC, 8 GG) demonstrated a consistent, but not significant, trend toward increased FXIII activity with increasing presence of G alleles (80.01% CC, 98.90% CG, and 107.20% GG). Although results were not adjusted for genotype at other loci reported to affect FXIII expression, there was no significant difference in FXIII antigen among genotypes. Compared to contracted whole blood clots from patients with only one or no risk alleles, clots from SCD patients with two copies of the risk allele (GG, expressing only FXIII E652) did not differ in either RBC retention within clots or clot mass (weight). Moreover, inhibition of transglutaminase activity with T101 significantly increased RBC release and decreased clot weight to a similar degree in contracted clots from patients with either the GC or GG genotype. In clots formed from FXIII-deficient plasma supplemented with rFXIIIa E652 or rFXIIIa Q652 and ABO-compatible donor RBCs, clots containing rFXIIIa E652 were 14% more resistant to lysis than clots containing rFXIIIa Q652 (p=0.016). Parallel studies with SCD patient plasma samples also showed that clots containing only FXIII E652 were more resistant to lysis than clots containing both FXIII E652 and FXIII Q652(p=0.0001).

Conclusions: These data suggest the FXIII rs5988 polymorphism does not alter protein expression or clot contraction but may regulate clot stability via slightly increased transglutaminase activity and enhanced resistance to lysis. These effects may predispose patients to formation of microclots during tumescence, thus impairing blood egress and increasing risk of priapism. Further studies should be conducted to determine if anticoagulation or fibrinolytic treatments are viable preventative or treatment strategies for SCD patients with the risk FXIIIGG genotype and recurrent priapism.