Soluble phospholipids enhance factor Xa-catalyzed prothrombin activation in solution.
Acidic phospholipids play an important but incompletely understood role in prothrombin activation. Here we report the effect of short-chain phosphatidylserine (dicaproylphosphatidylserine, C6PS) and the corresponding phosphatidylglycerol (C6PG) and phosphatidylcholine (C6PC) derivatives on the rate of prothrombin activation by factor Xa. The critical micellar concentrations of these short-chained phospholipids have been determined under a variety of conditions that we used for kinetic and structural studies. Under conditions for which these lipids exist in a soluble form, the results demonstrate that: (i) the rate of human prothrombin activation by human factor Xa was enhanced in a calcium-dependent fashion up to 60-fold by addition of C6PS, roughly 20% of the optimal enhancement seen with bovine phosphatidylserine/palmitoyloleoylphosphatidylcholine (25/75 PS/POPC) membranes; (ii) C6PS inhibited the rate of hydrolysis of synthetic factor Xa substrate (S-2765), an effect that was mimicked, but at much lower lipid concentrations, by PS/POPC membranes; (iii) there was no enhancement of prothrombin activation and much less inhibition of hydrolysis of S-2765 by factor Xa in the presence of C6PG or C6PC; and (iv) the thermal denaturation of prothrombin was altered in a calcium-independent but dose-dependent fashion by either C6PS or C6PG. These results have been interpreted in terms of the existence of (a) specific PS binding site(s) on factor Xa (Kd approximately 73 microM) that regulate(s) the activity of this serine protease. Our results do not rule out the possibility that the rate of prothrombin activation is also influenced by a weaker, calcium-independent, and less specific acidic lipid binding site on prothrombin, the occupancy of which results in conformational changes in this protein. The results clearly suggest that PS binding regulates the rate of prothrombin activation.
Koppaka
V
V
Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill 27599-7260, USA.
Wang
J
J
Banerjee
M
M
Lentz
B R
BR
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D015951:Factor Xa / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008823:Micelles; D009691:Nucleic Acid Denaturation; D010743:Phospholipids / Q000494:pharmacology*; D011516:Prothrombin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012996:Solutions; D013329:Structure-Activity Relationship
D008823:Micelles; D010743:Phospholipids; D012996:Solutions; D011516:Prothrombin; D015951:Factor Xa; D002118:Calcium