The effect of a single nucleotide polymorphism on human alpha 2-antiplasmin activity

Abstract

The primary inhibitor of plasmin, alpha(2)-antiplasmin (alpha(2)AP), is secreted by the liver into plasma with Met as the amino-terminus. During circulation, Met-alpha(2)AP is cleaved by antiplasmin-cleaving enzyme (APCE), yielding Asn-alpha(2)AP, which is crosslinked into fibrin approximately 13 times faster than Met-alpha(2)AP. The Met-alpha(2)AP gene codes for either Arg or Trp as the sixth amino acid, with both polymorphic forms found in human plasma samples. We determined the Arg6Trp genotype frequency in a healthy population and its effects on Met-alpha(2)AP cleavage and fibrinolysis. Genotype frequencies were RR 62.5%, RW 34.0%, and WW 3.5%. The polymorphism related to the percentage of Met-alpha(2)AP in plasma was WW (56.4%), RW (40.6%), and RR (23.6%). WW plasma tended to have shorter lysis times than RR and RW plasmas. APCE cleaved purified Met-alpha(2)AP(Arg6) approximately 8-fold faster than Met-alpha(2)AP(Trp6), which is reflected in Asn-alpha(2)AP/Met-alpha(2)AP ratios with time in RR, RW, and WW plasmas. Removal of APCE from plasma abrogated cleavage of Met-alpha(2)AP. We conclude that the Arg6Trp polymorphism is functionally significant, as it clearly affects conversion of Met-alpha(2)AP to Asn-alpha(2)AP, and thereby, the rate of alpha(2)AP incorporation into fibrin. Therefore, the Arg6Trp polymorphism may play a significant role in governing the long-term deposition/removal of intravascular fibrin.

Figure 1

Met-α₂AP as percent of total α₂AP and by genotype. α₂AP was purified from each plasma of persons with RR (n = 15), RW (n = 15), and WW (n = 5) genotypes and amino-terminal sequences determined by Edman degradation. Percent Met-α₂AP was calculated from picomole recoveries of Met and Asn in the first cycle.

Figure 2

Plasma APCE levels in a normal population and partitioned by genotype. APCE levels in plasma samples were determined by ELISA. (A) Histogram of APCE concentrations in a normal population (n = 109). (B) APCE levels by Met-α₂AP genotype.

Figure 3

APCE cleavage of polymorphic forms of Met-α₂AP. Equal amounts (40 μg) of purified Met-α₂AP(Arg6) and Met-α₂AP(Trp6) were digested by APCE. After stopping the reaction at selected times, samples were assessed by electrospray mass spectrometry for the quantity of the amino-terminal 12-amino acid peptide produced from each Met-α₂AP form.

Figure 4

Effect of Met-α₂AP genotype on generation of Asn-α₂AP in plasma with time. A plasma sample from a person of the RR, RW, or WW genotype was incubated at 29°C; at selected times α₂AP was purified from each sample and subjected to amino-terminal sequence analysis. The ratio of Asn-α₂AP/Met-α₂AP was calculated from picomole recoveries of Met and Asn in the first cycle.

Figure 5

Plasma clot lysis times (PCLT) by Met-α₂AP genotype. PCLTs were determined on plasma samples from RR, RW, and WW persons. (A) PCLT values were divided by genotype and plotted as mean ± SEM for the total population, men only and women only. (B) Percentage of plasmas that did not lyse (n = 14) compared to percentage of total population (n = 200) within each genotype.

Figure 6

Effect of APCE removal on conversion of Met-α₂-AP to Asn-α₂-AP with time. Plasma was drawn from a person of RR Met-α₂AP genotype and divided into 3 aliquots. One aliquot was mixed with an APCE F19 mAb (F19) bound to chromatography beads and incubated at 4°C to remove APCE. The second aliquot was incubated at 4°C with a nonspecific rabbit α-goat Ab (RAG) bound to beads. The third aliquot (RR) received no treatment. After removal of beads, each sample was incubated at 29°C, and Asn-α₂AP/Met-α₂AP ratios determined at selected times. In addition, F19-bound beads and RAG-bound beads were boiled with SDS to remove antibody-bound protein. Samples were electrophoresed on 10% Bis-Tris SDS-PAGE gels and blotted to nitrocellulose. APCE (97 kDa) was identified by Western blotting using a goat Ab to its amino-terminal region and visualized with a chemiluminescent substrate.