Platelet-Delivered Factor VIII (FVIII) Decreases Thrombus Stability.

Abstract

FVIII is normally not expressed by developing megakaryocytes. We have previously developed a transgenic mice in whom human B-domainless FVIII (hbdFVIII) was expressed specifically within developing megakaryocytes and stored in α-granules. This platelet (p) FVIII was especially potent in correcting bleeding in a FeCl3 carotid artery injury model. In FVIIInull mice, ~5% pFVIII plasma equivalency was as effective as a >25% plasma correction in normalizing thrombosis in this model. One concern with our observation was whether this enhanced efficacy would lead to an arterial prothrombotic state. We, therefore studied the details of pFVIII-dependent thrombosis using the cremaster arteriole/venule laser injury model, simultaneously monitoring platelet accumulation and fibrin clot growth at the site of injury (>30 injuries per study). Baseline studies in FVIIInull mice show that these mice had a decrease in platelet plug growth to ~50% of WT mice after either arteriole or venule injury. Fibrin accumulation was decreased after arteriole injury to ~20% of WT and was barely detectable after venule injury at ~3% of WT. Time to onset of fibrin clot formation on the arteriole side was delayed from the normal 30 sec to >50 secs and on the venule side from the normal of 40 secs to >60 secs. Stepwise improvement in platelet plug size, and size and time to onset of fibrin clot was seen with increasing amounts of hbdFVIII infusions. pFVIII/FVIIInull mice had decreased platelet plug formation compared to WT mice on both the arterial side (~10% of WT) and venule side (~25%). Fibrin accumulation was similar to the FVIIInull animals after arterial injury, but time to onset of clot was normalized. On the venule side, fibrin accumulation was 2–3 times that of the FVIIInull mice and again time to onset was normalized. Individual films of the pFVIII/FVIIInull suggested that these animals showed more clot instability than WT mice or FVIIInull mice. Indeed, quantitative analysis of downstream embolization showed that the pFVIII/FVIIInull mice had a significant increase in detectable emboli per 3 min study compared to WT mice (7.3 vs. 5.0 arterial and 6.3 vs. 0.5 venule, ns arterial and p<0.05 venule), but not compared to FVIIInull mice with or without a 25% hbdFVIII correction. Importantly, the size of the average emboli (in relative light units) in the pFVIII/FVIIInull mice was increased 10-fold (112 ± 224 arteriole and 145 ± 339 venule) vs. WT (11.6 ± 12.9 arteriole and 14.5 ± 13.1 venule, each p <0.0001) and vs. FVIIInull mice after a 25% correction (9.6 ± 15.6 arteriole and 24.2 ± 30.9 venule, each p<0.001). Compared to the FVIIInull mice, the average embolus was significantly (p<0.001) larger only on the venule side (74.1 ± 108 arteriole and 18.8 ± 12.2 venule). Thus, FVIIInull mice have a clear defect in cremaster laser injury studies affecting venule more than arterial fibrin formation with a concomitant defects in platelet plug formation. pFVIII improves venule more than arteriole fibrin clot formation but only to a level equivalent to the pFVIII content. There was no improvement in platelet plug formation, but there was enhanced thrombus embolization. These data suggest that pFVIII alters the details of thrombus structure with associated decrease in clot stability, especially on the venous side, suggesting that platelet-delivered FVIII may be associated with an increased risk of embolization.