Platelet FV Deficiency Restores Survival of TFPI Null Mice Independently of Plasma FV

Introduction: Platelets are essential for the hemostatic process, releasing "just in time" prothrombotic molecules, such as factor V (FV) and prothrombin to sites of vascular injury. Paradoxically, platelets also carry potent antithrombotic molecules, including tissue factor pathway inhibitor (TFPI), which may prevent hemostatic platelet plugs from expanding into vascular occluding thrombi. The TFPI gene undergoes alternative splicing to produce several isoforms, including TFPIα and TFPIβ. Platelets are the primary location of TFPIα in the murine circulation. TFPIα has a basic 9 amino acid sequence in its C-terminal region nearly identical to a basic sequence in the FV B domain. The TFPIα basic region tightly binds to an acidic region of the FV B domain. This binding takes place only when the FV basic region is absent, as occurs following proteolysis by factor Xa (FXa), in some forms of FV naturally present in platelet α-granules, or in genetically altered forms of FV, such as FV-short (associated with East Texas bleeding disorder). This interaction provides an essential exosite for TFPIα inhibition of the prothrombinase complex (FXa-FVa) assembled with forms of FVa retaining the B domain acidic region. Since prothrombinase assembles on the surface of activated platelets, we hypothesized that inhibition of prothrombinase via the platelet TFPIα-platelet FV interaction is a physiologically important biochemical mechanism for the inhibiting early stages of coagulation. We probed this hypothesis using TFPI K1 deleted (TFPI^-/-), FV deleted (F5^-/-) and FV transgenic (F5^Tg) mice to uncover whether specifically reducing or removing platelet FV would rescue TFPI^-/- mice from embryonic lethality.

Methods/Results: We performed crossbreeding experiments using F5^-/- mice carrying liver- or platelet-specific transgenes. F5^LiverTg produces 50% plasma FV with no FV in platelets, whereas F5^PltTg produces 3% platelet FV with no FV in plasma. F5^-/- F5^LiverTgand F5^-/- F5^PltTgmice were bred into TFPI^+/- mice. Offspring from this cross were bred to determine the effects of the liver or platelet transgene on the survival of mice lacking TFPI.F5^-/- is embryonic lethal in mice. F5^-/- mice are rescued by transgenes expressing either liver-specific FV or platelet specific FV. When we bred F5^+/- TFPI^+/- x F5^+/- F5^LiverTg TFPI^+/-, neither F5^-/- TFPI^-/- nor F5^+/- TFPI^-/- mice without the liver transgene were viable. Liver transgene expression did not rescue F5^+/- TFPI^-/- mice, however, it produced grossly normal pups lacking TFPI (F5^-/- F5^LiverTg TFPI^-/-) that lived to adulthood with no overt phenotypes. We further characterized this phenotype by crossbreeding F5^+/- TFPI^+/- x F5^-/- F5^LiverTg TFPI^-/-. In this cross, F5^-/- F5^LiverTg TFPI^-/- pups survived with ~80% penetrance (45 of 234 total mice, p<5.0 x10^-12). Next, we bred F5^+/- TFPI^+/- x F5^+/- F5^PltTgTFPI^+/-. Platelet transgene expression also produced grossly normal pups lacking TFPI (F5^-/- F5^PltTg TFPI^-/-) that lived to adulthood with no overt phenotypes. Further crossbreeding, as described for the liver transgene, revealed a similar ~80% survival of F5^-/- F5^PltTg TFPI^-/- pups (56 of 291 total mice, p<9.0 x10^-18). Both male and female platelet and liver specific F5^-/- F5^Tg TFPI^-/- were fertile, and mothers supported pups until weaning.

Conclusions: Neither total loss of FV nor concerted 50% reduction in plasma and platelet FV rescued TFPI^-/- embryonic lethality, indicating that TFPI anticoagulant activity is not simply counterbalanced by FV procoagulant activity (or vice versa) during embryogenesis. Remarkably, either the total lack of platelet FV with 50% normal plasma FV, or the total lack of plasma FV with 3% normal platelet FV, resulted in the nearly complete rescue of F5^-/- TFPI^-/- mice with pups surviving to adulthood without grossly observable pathology. These observations indicate that severe reduction of platelet FV, independent of the plasma FV concentration, produces this extraordinary rescue of TFPI^-/- embryonic lethality. Collectively, these findings expose the critical role for platelet TFPIα in regulating procoagulant activity of prothrombinase assembled with platelet FV. This platelet FV-platelet TFPIα interaction highlights the important biological activity of pro and anticoagulant proteins located within platelets in regulating hemostatic balance during murine development and into adulthood.