In the presence of phospholipids, glycosaminoglycans potentiate factor Xa-mediated protein C activation by modulating factor Xa activity.
Although the thrombin/thrombomodulin complex is considered the physiological activator of protein C, factor Xa (f.Xa) can also activate protein C in a reaction that is potentiated by glycosaminoglycans. To explore this phenomenon, we first examined the effect of glycosaminoglycans of varying degrees of sulfation on the kinetics of protein C activation by f.Xa in the presence of Ca2+ and phosphatidylcholine-phosphatidylserine vesicles (PCPS). Heparin increased the rate of protein C activation by f.Xa by 4-fold. In contrast, N-desulfated heparin had no effect on activation, whereas dextran sulfate, which is more sulfated than heparin, increased catalytic efficiency 21-fold. These data suggest that the capacity of glycosaminoglycans to catalyze protein C activation by f.Xa depends on their degree of sulfation. The affinities of individual glycosaminoglycans for protein C and f.Xa were measured in the absence or presence of PCPS by monitoring changes in extrinsic fluorescence when fluorescein-labeled f.Xa or protein C was titrated with the various glycosaminoglycans. Heparin binds protein C with low affinity in the absence or presence of PCPS. In contrast, the affinity of heparin for f.Xa is 86-fold higher in the presence of PCPS compared to that in the absence of PCPS. Similar results were obtained using surface plasmon resonance. These findings suggest that a high affinity glycosaminoglycan binding site is exposed when f.Xa binds to PCPS. The observation that heparin promotes f.Xa-mediated activation of prethrombin 1 only in the presence of phospholipid suggests that glycosaminoglycan binding modulates the active site of f.Xa. This study reveals that when f.Xa interacts with anionic phospholipids, glycosaminoglycans bind f.Xa more tightly, allosterically modulate its active site, and enhance its capacity to activate protein C.
McRae
Simon J
SJ
Department of Medicine, McMaster University, 1280 Main Street West, Hamilton Ontario L8S 4L8, Canada.
Stafford
Alan R
AR
Fredenburgh
James C
JC
Weitz
Jeffrey I
JI
D004792:Enzyme Precursors / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000494:pharmacology*; D006493:Heparin / Q000031:analogs & derivatives; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D010713:Phosphatidylcholines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D010743:Phospholipids / Q000502:physiology*; D011486:Protein C / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism; D013917:Thrombin / Q000502:physiology
D004792:Enzyme Precursors; D006025:Glycosaminoglycans; D008081:Liposomes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D011486:Protein C; D011516:Prothrombin; D006493:Heparin; C012007:prethrombins; D013917:Thrombin; D015951:Factor Xa