Factor VIIa-mediated tenase function on activated platelets under flow.
BACKGROUND
Tissue factor (TF) and/or active factor (F)VIIa may be stored inside resting platelets.


OBJECTIVE
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.
Institute for Medicine and Engineering, Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, USA.
M S
MS
Goel
S L
SL
Diamond
D000818:Animals; D001665:Binding Sites; D001777:Blood Coagulation; D001792:Blood Platelets / Q000378:metabolism; D002469:Cell Separation; D002850:Chromatography, Gel; D003435:Crotalid Venoms / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D015942:Factor VIIa / Q000096:biosynthesis / Q000737:chemistry*; D005337:Fibrin / Q000737:chemistry; D005340:Fibrinogen / Q000378:metabolism; D006403:Hematologic Tests; D006801:Humans; D037181:Lectins, C-Type; D008055:Lipids / Q000737:chemistry; D051379:Mice; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D015539:Platelet Activation; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000096:biosynthesis / Q000378:metabolism; D013997:Time Factors
D003435:Crotalid Venoms; D037181:Lectins, C-Type; D008055:Lipids; D009363:Neoplasm Proteins; C028890:convulxin; D005337:Fibrin; D005340:Fibrinogen; D013925:Thromboplastin; D015942:Factor VIIa; D013917:Thrombin; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant