Recombinant FVIII and FVIIa Added to Flowing Hemophiliac Blood Co-Localize with Platelet Aggregates Adherent to a Collagen Surface, Promoting Local Fibrin Deposition in Wake-like Patterns Around the Aggregates

Mural platelet thrombi are considered to facilitate hemostasis in hemophilia via rFVIII/ rFVIIa binding to platelet membranes with enhancement of local fibrin deposition.  We therefore sought to show that Factors VIII and VIIa co-localize to platelet membranes of adherent platelet aggregates and promote local deposition of fibrin.  We collected blood from 9 patients with severe hemophilia A just prior to a prophylactic dose of rFVIII, and from 8 normals, into a plastic tube containing 5 U/ml FC low MW heparin, 1.75 µg/ml of the Tab (anti-CD41) mo antibody, and 1.0 μg/ml of ALEXA 555-conjugated rabbit anti-mouse secondary antibody, plus 0, 11, 33 or 100 ng/ml rFVIII-Oregon green.  Similar approaches were used for rFVIIa or fibrin.  Fibrin was detected using the  monoclonal antibody 59D8 directed against human β fibrin, together with the above Alexa555-conjugated rabbit anti-mouse secondary antibody, and co-localized to platelet aggregate surfaces using 10 µM quinicrine dihydrochloride.  Blood at 37° C was withdrawn at 270 sec-1 through a flow chamber, over 150 µm glass cover slips with preadsorbed microfibrillar collagen, using epifluorescence video microscopy and digital image analysis (MetaMorph Premier software).   Added rFVIII or rFVIIa co-localized (Fig. 1) with platelet membranes and increased with rFVIII concentration.  With hemophiliac blood and 100 ng/ml rFVIII, percent co-localization at 7 min was 52.6 ± 7.7 (N=4).  Platelet aggregate growth rate, image pixels/min from time 3 to 9 min, was 26% lower in hemophiliac blood, but increased (p = 0.0001) 2.34 ± 0.62 (6)-fold with rFVIII, and (p = 0.0005) 1.91 ± 0.56 (6)-fold with 30 nM rFVIIa (Fig. 2).  These increases essentially restored (or only slightly augmented) platelet deposition to levels observed in normal blood.  In contrast, the effect on local fibrin deposition was profound:  fibrin deposition was delayed by 3-5 min in normal blood, but by >13 min in hemophiliac blood.  Addition of rFVIII or rFVIIa shortened the latter time to 7-9 min.  Increases in platelet aggregate growth rate, following a lag phase in growth rate, paralleled the onset of fibrin formation.  Fibrin appeared as comet-shaped deposits (comet head 20-25 µm in diameter) centered over each platelet aggregate (3-10 µm), the comet tail trailing 60-75 µm downstream (Fig. 3).  In flowing whole blood in real time, these data show that rFVIII and rFVIIa co-localize with platelet membranes of activated, surface-adherent platelet aggregates, and support fibrin deposition in the slow-flow wakes immediately surrounding the platelet aggregates.