Autoproteolysis or Plasmin-mediated Cleavage of Factor Xaα Exposes a Plasminogen Binding Site and Inhibits Coagulation

Edward L.G Pryzdial,Garry E Kessler

doi:10.1074/jbc.271.28.16614

Abstract

Blood coagulation factor Xa (FXa) has recently been shown to function as a plasminogen receptor in the presence of procoagulant phospholipid (phosphatidylserine; PS) and Ca2+. In the current work, the possible effect of autoproteolytic and plasmin-mediated cleavage of FXa on complex formation with plasminogen was investigated. 125I-plasminogen binding to derivatives of FXa electrotransferred to polyvinylidene difluoride revealed that the autoproteolytic conversion of FXaalpha to FXabeta was required for the expression of a plasminogen binding site. In the presence of PS and Ca2+, plasmin was shown to convert FXaalpha to a FXabeta-like species at least 3 orders of magnitude faster than the autoproteolytic mechanism. This also resulted in the exposure of a plasminogen binding site. Further processing by plasmin generated a fragment (33 kDa) due to cleavage at Gly331 in the FXa heavy chain. Production of this species enhanced apparent plasminogen binding compared with FXabeta and resulted in the loss of FXa amidolytic and clotting activity. In the absence of either PS or Ca2+, the plasmin-mediated fragmentation of FXaalpha was altered to include a FXabeta-like molecule and a species (40 kDa) with intact beta-heavy chain disulfide linked to a COOH-terminal fragment of the light chain starting at Tyr44. Neither of these products was observed to interact with plasminogen. The 40-kDa species had amidolytic activity comparable with FXaalpha but inhibited clotting activity. Cumulatively the data provide the first evidence for a functional difference between the FXa subforms and suggest a mechanism where autoproteolysis and plasmin-mediated cleavage modulate the function of FXaalpha from a procoagulant enzyme to a profibrinolytic plasminogen receptor.

Highlights

factor Xa (FXa) biochemistry is well studied, a question that still remains concerns the functional significance of an autoproteolytic step that excises a 4-kDa glycopeptide (␤-peptide) from the heavy subunit COOH terminus [7,8,9]
To determine the effect of FXa␣ autoproteolysis on the interaction with plasminogen, similar ligand blotting experiments were conducted. In this system ligand blotting is advantageous because the relative amount of plasminogen associated with discrete proteolytic products of FXa␣ in a mixture can be directly analyzed due to the resolution offered by electrophoresis
In the absence of PCPS binding, subform conversion was insignificant, and as a result expression of new plasminogen binding sites was not evident. This demonstrated that structural changes in FXa␣ over the course of the experiment that are indiscernible by SDS-PAGE do not account for the observed plasminogen association

Summary

Introduction

FXa biochemistry is well studied, a question that still remains concerns the functional significance of an autoproteolytic step that excises a 4-kDa glycopeptide (␤-peptide) from the heavy subunit COOH terminus [7,8,9]. In the absence of either PS or Ca2؉, the plasmin-mediated fragmentation of FXa␣ was altered to include a FXa␤-like molecule and a species (40 kDa) with intact ␤-heavy chain disulfide linked to a COOH-terminal fragment of the light chain starting at Tyr44 Neither of these products was observed to interact with plasminogen. FXa was observed to accelerate the generation of the fibrinolytic enzyme plasmin by tissue plasminogen activator (tPA) [13] This was found to involve an interaction with the precursor, plasminogen [13], which is known from other work to require Lys at the COOH terminus of the receptor [14, 15]. Cleavage of the FXa cofactor, FVa, by plasmin has been ob-

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