Phospholipase A Activates Hemostasis

Abstract

Background Phospholipases A 2 (PLA 2 ) are aggressive enzymes that can destroy phospholipids of cell membranes. The resulting cell fragments trigger the kallikrein—mediated contact phase of coagulation. The aim of the present study was to expose citrated whole blood to PLA 2 and to quantify thrombin generation in recalcified plasma. Methods Normal citrated blood was exposed to bovine pancreatic or snake PLA 2 , lipopolysaccharide (LPS), or zymosan A for 30–45 min (RT). After centrifugation the plasma samples were recalcified (10 + 1) with 250 mM CaCl 2 in the recalcified coagulation activity assay (RECA). After 0–45 min coagulation reaction time (CRT at 37°C) 1.6 M arginine (final test concentration) was added to stop hemostasis activation and to depolymerize non-crosslinked fibrin. The generated thrombin activity was chromogenically determined. Results 100 ng/ml bovine pancreatic or snake PLA 2 generates about 0.2–0.8 IU/ml thrombin after 15 min CRT. This thrombin generation is similar as that induced by 200 ng/ml LPS or 20 μg/ml zymosan A. Up to 60 ng/ml bovine pancreatic PLA 2 the generated thrombin activity is proportional to the PLA 2 activity used; 1 μg/ml PLA 2 induces much less thrombin, but PLA 2 at 10 μg/ml again results into thrombin generation of 0.1–3 IU/ml at 10–15 min CRT. As control, in pooled normal citrated plasma there is no significant change in thrombin generation when exposed to up to 10 μg/ml bovine pancreatic PLA 2 . Discussion Elevated plasmatic PLA 2 activities (occurring e.g. in trauma, pancreatitis, or sepsis) activate the blood hemostasis system resulting in pathologic disseminated intravascular coagulation (PDIC). It is suggested to diagnose these life threatening states as early as possible, screening all patients for plasmatic thrombin activity. Abbreviations ΔA/t, absorbance increase per time; CRT, coagulation reaction time (37°C); CS-IIa, chromogenic substrate HD-cyclohexylglycyl-alanyl-arginyl-paranitroanilide for thrombin; F-well, flat bottom well Polysorp ® ; IU, international units; LPS, lipopolysaccharide; mA, milli absorbance units; PDIC, pathologic disseminated intravascular coagulation; PLA 2 , phospholipase A 2 ; RECA, recalcified coagulation activity assay; RT, room temperature; sPLA 2 , secretory PLA 2 ; U, units; ZyA, zymosan A.