Pro-Coagulant Lipids in Physiological Ratios Found in the Activated Platelet Membrane Do Not Impact Clot Structure or Fibrinolysis in Purified Assays

Abstract

Purpose: A central role for the pro-coagulant membrane comprising aminophospholipids (aPL) and enzymatically oxidized phospholipids (eoxPL) in promoting hemostasis via interaction with coagulation factor Gla domains is well established. However, little is known about their interactions with the fibrinolytic pathway, their ability to alter clot structure or to support the activated protein C (APC) pathway. Previous studies used membrane liposome compositions that differ from those expected physiologically and/or generated inconsistent findings. To address this, pro-coagulant membranes comprising physiological proportions of aPL and eoxPL will be tested for their ability to support fibrinolysis using standard assays.

Methods: The impact of phospholipids on clot structure and clot lysis was tested using absorbance-based assays. To investigate the impact of PS or eoxPL on fibrinolysis, plasmin was monitored chromogenically, and clot dissolution measured in a purified lysis system activated by tissue plasminogen activator or urokinase. To determine the impact of eoxPL on APC/protein S, FVa was incubated with APC (± protein S) in a purified prothrombinase assay.

Results: At the concentrations of lipids tested in our study, PS did not significantly impact clot structure or fibrinolysis. Similarly, eoxPL did not impact either fibrinolysis or activity of APC/Protein S.

Conclusion: Using liposome compositions that approximate activated blood cells, we found that the pro-coagulant membrane is unlikely to influence either clot structure or fibrinolytic activity directly, beyond its well characterized role in supporting Gla dependent coagulation factors and the actions of platelet associated proteins/receptors.

Keywords: activated protein C; fibrin; phosphatidylserine; phospholipids; plasmin; protein S.

Figure 1

Overview of the coagulation cascade including fibrinolysis and activated protein C pathways, highlighting phospholipid interactions.

Figure 2

Liposomes containing physiological amounts of PS do not alter thrombin activity or structure of clots formed by purified thrombin cleavage of fibrinogen. Panels (A and B). PS did not impact the activity of 0.25 NIH/mL (A and B) thrombin. Thrombin activity was measured by the metabolism of its chromogenic substrate S-2238 (0.56 mM) in the presence of liposomes (60 µM) containing varying proportions of PS as outlined in methods (n = 3, mean ± SD). Panels (C and D). PS did not impact the structure of clots formed from fibrinogen (2 mg/mL) and 0.25 NIH/mL (C and D) thrombin. Clot structure was measured by the maximum absorbance (405 nm) of clots formed in the presence of liposomes (60 µM) containing a varying proportion of PS as outlined in methods (n = 3, mean ± SD). Left panels; representative traces, right panels; summary data. Statistics performed using one-way ANOVA. Composition of liposomes was as follows: 0% PS (50% DSPC, 30% SOPE, 20% SOPC), 5% PS (50% DSPC, 30% SOPE, 15% SOPC, 5% SOPS), 10% PS (50% DSPC, 30% SOPE, 10% SOPC, 10% SOPS) 20% PS (50% DSPC, 30% SOPE, 20% SOPS).

Figure 3

Pro-coagulant lipids have little or no impact on the activity of plasmin or its formation from plasminogen by u-PA or t-PA. Panels (A and B). Pro-coagulant liposomes did not impact the activity of plasmin. Plasmin (12.5 nM) activity was measured by the metabolism of its chromogenic substrate S-2251 (500 µM) in the presence of varying concentrations of pro-coagulant liposomes (0–20 µM) as outlined in methods (n = 3, mean ± SD). Panels (C and D) Liposomes did not alter lysis time of purified fibrin clots by plasminogen activated by t-PA. Lysis time was measured as the time from half maximal absorbance (405 nm) during clot formation to half maximal absorbance during the lysis of fibrin clots (1.25 mg/mL fibrinogen) formed by thrombin (0.25 NIH/mL) in the presence of CaCl₂ (5 mM) where plasminogen (0.25 µM) was activated by t-PA (20 pM) as outlined in methods (n = 3, mean ± SD). Panel (C) shows liposomes at 4 µM. Panels (E and F) Liposomes did not alter lysis time of purified fibrin clots by plasminogen activated by u-PA, apart from at one single concentration. Lysis time was measured as the time from half maximal absorbance (405 nm) during clot formation to half maximal absorbance during the lysis of fibrin clots (1.25 mg/mL fibrinogen) formed by thrombin (0.25 NIH/mL) in the presence of CaCl₂ (5 mM) where plasminogen (0.25 µM) was activated by u-PA (600 pM) as outlined in methods (n = 3, mean ± SD). Panel (E) shows liposomes at 10 µM. Statistics were performed using one-way ANOVA with Tukeys post-hoc test (* p<0.05, ** p<0.01, *** p<0.001). Composition of liposomes was as follows: PC/PE (65% DSPC, 20% SOPE, 10% SAPE, 5% SOPC), PC/PE/PS (65% DSPC, 20% SOPE, 10% SAPE, 5% SOPS), PC/PE/PS (15-HETE-PE) (65% DSPC, 20% SOPE, 10% 15-HETE-PE, 5% SOPS).

Figure 4

Liposomes containing 15-HETE-PE and 15-HETE-PC do not enhance activated protein C (APC) or protein S activities. Liposomes (20 µM, containing eoxPL as listed in Table 1), FVa (5 mM), CaCl₂, (5 mM), APC (0.25 nM) ± protein S (50 nM) were incubated at 37°C for 5 minutes, APC then inactivated with p-APMSF before remaining FVa activity measured using a prothrombinase assay (n = 3, mean ± SD). Results are recorded as a percentage of remaining FVa activity compared with controls containing no APC or protein S (ie 100% FVa activity). Blanks that included no FVa were also tested to account for the level of FXa activity in the absence of FVa and these values were deducted from all other values. No significant differences between liposome types were detected, either in the presence or absence of protein S (tested by one-way ANOVA). Composition of liposomes was as follows: PC/PE/PS (55% DSPC, 10% SAPE, 20% SOPE, 10% SAPC, 5% SOPS), PC/PE/PS (15-HETE-PE) (55% DSPC, 10% 15-HETE-PE, 20% SOPE, 10% SAPC, 5% SOPS), PC/PE/PS (15-HETE-PC) (55% DSPC, 10% SAPE, 20% SOPE, 10% 15-HETE-PC, 5% SOPS).