The phospholipase A1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation

Abstract

Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purification of a phospholipase A(1) (PLA(1)) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate (FP)-biotin affinity labeling and proteomics characterization that identified acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA(1). Addition of this recombinant PLA(1) significantly increased the production of sn-2-esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl-sn-glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn-1 to the sn-2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1) Activated platelets release PLA(1); 2) PLA(1) generates a pool of sn-2 lysophospholipids; 3) These newly generated sn-2 lysophospholipids undergo acyl migration to yield sn-1 lysophospholipids, which are the preferred substrates of ATX; and 4) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids.

Fig. 1.

ATX is required for LPA production in activated platelet supernatant. Supernatant from thrombin-activated human platelets was incubated in the absence or presence of recombinant human ATX with 10 µg PC 18:2 (A), 10 µg PS 18:1 (B), 1 µg NBD-PC 18:1/12:0 (C), or 1 µg NBD-PS 18:1/12:0 (D) supplemented with 250 µM BSA and 135 mM NaCl in the presence or absence of 200 nM ATX at 37°C for 1 h. Generation of the corresponding LPC and LPA species was quantified using LC/MS (A and B) or TLC (C and D). In the absence of added ATX, very little LPA was produced. In sharp contrast, LPC was abundantly generated by a PLA activity. Addition of ATX lead to a 9.5-fold increase in the amount of LPA generated from LPC and a 2-fold increase in the amount of LPA generated from LPS, with a concomitant decrease in the LPC or LPS. When the unnatural NBD-PC/PS analogs were used, essentially the same findings were noted, validating the usability of these fluorescent analogs for monitoring LPA production. Bars are the mean of three independent determinations ± SD.

Fig. 2.

FP-RH binds to PLA₁, inhibiting activity and NBD-LPS production. Incremental amounts of FP-RH were incubated with NBD-PS and a phospholipase A1 active Butyl-Sepharose fraction (A). The production of NBD-LPS decreased with increasing amounts of FP-RH. Expression of serine hydrolase transcripts in human platelets (B). RT-PCR for LYPLA-I/APT1, MGLL, PS-PLA₁, and LCAT was performed using two different gene-specific primer pairs (denoted 1 and 2) using mRNA isolated from human purified platelets. Only LYPLA-I/APT1 and MGLL amplification yielded PCR products of the expected size, indicating the expression of these enzymes in human platelets. These results were confirmed with platelets from five other donors of both sexes. MGLL does not possess PLA activity (C and D). When incubated with NBD-PS, no LPS formed, which indicated MGLL does not possess PLA activity (C). Human recombinant MGLL was shown to have MGL activity to prove functionality (D). Bars are the mean of three independent determinations ± SD.

Fig. 3.

Human recombinant LYPLA-I/APT1 with and without a GST tag were purified for analysis (A). Coomassie Blue-stained SDS-PAGE gels show the protein profiles of the GST-tagged LYPLA-I/APT1 protein and the thrombin-cleaved LYPLA-I/APT1 protein (5 µg each). Human recombinant LYPLA-I/APT1 must be unlabeled for functionality (B). PLA activity assay was performed on both GST-tagged LYPLA-I/APT1 and thrombin-cleaved untagged LYPLA-I/APT1 (0.5 µg each). No activity was found when the GST tag was present. The thrombin-cleaved version of LYPLA-I/APT1 had abundant PLA1 activity when incubated with NBD-PC in the presence of BSA. Bars are the mean of three independent determinations ± SD.

Fig. 4.

Human recombinant LYPLA-I/APT1 acts first as PLA₁, and then has lysophospholipase activity when incubated with NBD-PC (A). Purified human recombinant LYPLA-I/APT1 was incubated with NBD-PC and BSA for 0, 1, 2.5, 5, 10, 20, and 30 min. NBD-LPC was generated immediately, indicating PLA₁ activity of the enzyme. After the initial generation of NBD-LPC, NBD-FA was generated, indicating a lysophospholipase activity of the enzyme with a simultaneous decrease in the starting substrate. Human recombinant LYPLA-I/APT1 did not cleave LPA, and therefore, did not contribute to the breakdown of LPA (B). LYPLA-I/APT1 was incubated with LPA 18:1 in the presence of BSA for 1 h. The amount of LPA did not decrease during incubation, indicating that LPA is not a substrate for LYPLA-I/APT1. Bars are the mean of three independent determinations ± SD.

Fig. 5.

Human recombinant LYPLA-I/APT1 increases the amount of LPA 18:2 and LPA 20:4 in a time-dependent manner. Physiologically activated plasma and plasma with heparin additive were incubated with or without human recombinant LYPLA-I/APT1 for 24 h. LPA 18:2 and LPA 20:4 in activated plasma began increasing after 3 h and increased greatly at 24 h. In contrast, in heparinized plasma LPA 18:2 and LPA 20:4 showed no increase, except in the sample with LYPLA-I/APT1 at 24 h.

Fig. 6.

Human recombinant LYPLA-I/APT1 increases the amount of LPA 18:2 (A), LPA 20:4 (B), LPC 18:2 (C), and LPC 20:4 (D) produced in physiologically activated plasma. Physiologically activated plasma was incubated with or without human recombinant LYPLA-I/APT1 for 24 h. LPA 18:2 and LPA 20:4 increased in 24 h compared to the amount present in plasma immediately after blood draw. Production of LPA and LPC substantially increased with the addition of LYPLA-I/APT1, showing that LYPLA-I/APT1 plays a role in the production of LPA during blood coagulation. Results are from three female donors and four male donors (A-C) or one female donor and three male donors (D). Open symbols represent female donors, and closed symbols represent male donors. Identical shapes throughout indicate same donor. P < 0.05, 24 h activated plasma with LYPLA-I versus without LYPLA-I analyzed by paired t-test.

Fig. 7.

Comparison of the cleavage of sn-1 with sn-2 lyso-PAF 18:1 by ATX. A linear segment of time versus amount of product was plotted for each substrate concentration (31.25 µM-2 mM) to determine initial velocity. Substrate concentration versus initial velocity was plotted. All absorbance values were converted to µM of resorufin produced to report as actual product. Bars are the mean of three independent determinations ± SEM.