Formation of Prothrombinase Complex on Human Platelets Stimulated by Gingipain-R

Objectives: The prothrombinase complex consists of FVa, FXa, Ca, and phospholipids supplied by the activated platelet surface. We sought to determine if the prothrombinase complex forms on the surface of human platelets activated by gingipain-R (RgpA) from Porphyromonas gingivalis.

Methods: Both membrane vesicles (MV) and RgpA were used in these studies. RgpA was purified from membrane vesicles by hydroxyapatite chromatography. RgpA consists of a complex made up of a 50 kDa catalytic peptide and a 44 kDa hemmaglutinin peptide as observed on SDS-PAGE. Platelet rich plasma (PRP) was isolated from anticoagualted whole blood that was obtained from healthy volunteers. Gel filtered platelets (GFP) were prepared from PRP by Sepharose 2B chromatography to rid the platelets of plasma proteins. GFP were suspended in Tyrodes buffer containing apyrase (0.01 U/ml) and acetysalicylic acid (1 mM). GFP (10⁶ platelets/mL), were activated by either MV or RgpA (5-40 µg) in the presence of FVa, FXa, and calcium. After the addition of prothrombin (1.5 µM), thrombin formation on the platelet surface was measured spectrophotometrically (OD = 405 nm) by the cleavage of the chromogenic substrate N-benzoyl-phe-val-arg-p-nitroanilide (arg-pNa). Controls were performed with MV and RgpA in the presence of GFP,FVa, FXa, and Ca but without prothrombin, in order to account for the cleavage of arg-pNa by RgpA. Data was recorded as units of amidase acitivty.

Results: Both MV and RgpA cleaved arg-pNa in a dose-dependent manner. Prothrombin caused both a leftward shift and an increase in E_max of both the MV and RgpA dose-response curve.

Conclusion: Activation of platelets by RgpA causes thrombin generation via assembly of the prothrombinase complex on the platelet surface.