The Effect of an Arg/Trp Polymorphism in α2-Antiplasmin on Fibrinolysis.

Abstract

α2-antiplasmin (α2AP) is a serine proteinase inhibitor with plasmin as its primary target. Plasmin plays a critical role in fibrin proteolysis and tissue remodeling and is highly regulated to prevent excessive proteolysis. α2AP is cleaved by antiplasmin-cleaving enzyme (APCE) between Pro12-Asn13 to yield a shortened version with Asn as the amino-terminus. Asn-α2AP is crosslinked into fibrin ~13 X faster than the full-length Met-α2AP. Clot lysis is slowed in direct proportion to the ratio of Asn-α2AP to Met-α2AP present during fibrin formation. The cloned gene for Met-α2AP contained either a CGG codon resulting in Arg as the sixth amino acid or TGG, resulting in Trp at that position. Both polymorphic forms of Met-α2AP, Arg6 and Trp6, have been found in human plasma. In the present study we determined the genotype frequency of the polymorphism and whether it affected cleavage of Met-α2AP to Asn-α2AP, and ultimately whether fibrinolytic rates were affected by one polymorphic form as opposed to the other. In our normal population, the genotype frequencies were found to be RR 60.9%; RW 35.8%; WW 3.4%. The R allele had a frequency of 78.8% and the W allele had a frequency of 21.2%. A primate DNA panel revealed that the polymorphism follows an evolutionary split with Old World monkeys having the WW genotype and Great Apes having the RR genotype. We found that the polymorphism affected the ratio of Asn-α2AP to Met-α2AP in plasma with WW having the highest percentage of Met-α2AP (56.4%), RR having the least (23.6%) and RW falling in between (40.6%). We examined whether this was due to Met-α2AP(Arg6) being a better substrate for APCE than Met-α2AP(Trp6). When purified α2AP(Arg6) or α2AP(Trp6) were digested with APCE, comparisons of reaction rates based on linear regression analysis of early time points revealed that APCE cleaved Met-α2AP(Arg6) ~8-fold faster that Met-α2AP(Trp6). This phenomenon was confirmed in whole plasma, by incubating plasma from subjects with either the RR, RW or WW genotype and determining the Asn-α2AP/Met-α2AP ratio at 0, 24 and 48 hours. When APCE was removed from the plasma by a specific antibody bound to chromatography beads, the ratio of Asn-α2AP to Met-α2AP did not change over the same time period. Since Asn-α2AP/Met-α2AP ratios are in direct proportion to clot lysis times, we determined whether the polymorphism also impacts and correlates with correspondent plasma clot lysis times. The first tertile of plasma clot lysis times contained all of the WW persons; the longer lysis times in the second and third tertiles contain only RR and RW persons. We conclude that the Arg6Trp polymorphism is functionally significant in that it clearly affects conversion of Met-α2AP to Asn-α2AP, and as a consequence, the rate of Asn-α2AP incorporation into fibrin. Based on these data, the Arg6Trp polymorphism may play a significant role in governing the long-term deposition/removal of intravascular fibrin.