A low molecular weight platelet inhibitor of factor XIa: purification, characterization, and possible role in blood coagulation.
A low molecular weight platelet inhibitor of factor XIa (PIXI) has been purified 250-fold from releasates of washed and stimulated human platelets. Molecular weight estimates of 8400 and 8500 were determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, although a second band of Mr 5000 was present upon electrophoresis. The inhibitor does not appear to be one of the platelet-specific, heparin-binding proteins, since it neither bound to nor was affected by heparin. An amount of PIXI which inhibited by 50% factor XIa cleavage of the chromogenic substrate S2366 (Pyr-Glu-Pro-Arg-pNA-2H2O) only slightly inhibited (5-9%) factor XIIa, plasma kallikrein, plasmin, and activated protein C and did not inhibit factor Xa, thrombin, tPA, or trypsin, suggesting specificity for factor XIa. Kinetic analyses of the effect of PIXI on factor XIa activity demonstrated mixed-type, noncompetitive inhibition of S2366 cleavage and of factor IX activation with Ki's of 7 x 10(-8) and 3.8 x 10(-9) M, respectively. Immunoblot analysis showed that PIXI is not the inhibitory domain of protease nexin II, a potent inhibitor of factor XIa also secreted from platelets. Amino acid analysis showed that PIXI has no cysteine residues and, therefore, is not a Kunitz-type inhibitor. PIXI can prevent stable complex formation between alpha 1-protease inhibitor and factor XIa light chain as demonstrated by SDS-polyacrylamide gel electrophoresis. The inhibition by PIXI of factor XIa-catalyzed activation of factor IX and its capacity to prevent factor XIa inactivation by alpha 1-protease inhibitor, combined with the specificity of PIXI for factor XIa among serine proteases found in blood, suggest a role for PIXI in the regulation of intrinsic coagulation.
Cronlund
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Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Walsh
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D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001777:Blood Coagulation / Q000502:physiology*; D001792:Blood Platelets / Q000737:chemistry*; D002417:Cattle; D002845:Chromatography; D002863:Chromogenic Compounds / Q000378:metabolism; D015945:Factor XIa / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000097:blood* / Q000737:chemistry / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000378:metabolism
D016564:Amyloid beta-Protein Precursor; D002863:Chromogenic Compounds; D011480:Protease Inhibitors; D000515:alpha 1-Antitrypsin; D015945:Factor XIa