Background: Tissue factor (TF) and/or active factor (F)VIIa may be stored inside resting platelets.
Objectives: The objective of this study was to examine if platelets, following activation of GPVI, could support tenase and prothrombinase activity without any exogenously added tissue factor.
Methods: Thrombin (IIa) formation on gel-filtered platelets with added factors or the clotting of platelet-free plasma (PFP) or platelet-rich plasma (PRP) supplemented with corn trypsin inhibitor (CTI) (to inhibit factor XIIa) was studied in well plate assays with a fluorogenic thrombin substrate or in flow assays by fibrin visualization.
Results: Pretreatment of convulxin (CVX)-stimulated, fibrinogen-adherent, gel-filtered platelets with anti-TF, anti-FVII/VIIa, or 1 nm PPACK [inhibitor of FVIIa, factor XIa and factor (F)IIa] delayed fibrin deposition on platelets perfused with PFP/CTI at 62.5 s(-1). Anti-TF or anti-FVII/VIIa also attenuated thrombin generation in plate assays using recalcified PRP/CTI treated with CVX. Anti-TF or anti-FVII/VIIa (but not inhibited factor IXa) delayed the burst in thrombin production by gel-filtered platelets suspended in prothrombin and CVX by 14 min and 40 min, respectively. Anti-FVII/VIIa completely eliminated thrombin generation on fibrinogen-adherent, gel-filtered platelets pretreated with 10 micro m PPACK and 10 micro m EGR-CK [inhibitor of factor (F)Xa], rinsed, and then supplemented with CVX, prothrombin, and FX. Addition of anionic phospholipid to PFP/CTI or to a mixture of prothrombin, FX, and recVIIa was not sufficient to generate detectable tenase activity. Lastly, isolated, unactivated neutrophils suspended in FX, FII and recVIIa supported a very low level of thrombin generation sensitive to antagonism of P-selectin, CD18, and TF.
Conclusions: Activated platelets supported tenase and prothrombinase activity by elevating the function or level of FVIIa and exposing active FVIIa or FVIIa-cofactor(s), distinct from anionic lipid, that may be, in part, TF.