Recent studies suggest that Tissue factor (TF)-initiated pathway can directly and rapidly yield FVIIIa-FIXa intrinsic tenase complex prior to coagulation amplification by thrombin (PMID 28729433). FVIII may also mediate TF/FVIIa-dependent FXa generation in the initiation phase of coagulation (PMID 30756344). These new insights prompted us to evaluate whether FVIII plays an important role in generating the minimum level of thrombin required for platelet activation in vivo.
We used two distinct murine models of platelet- and Par4-mediated pathology to address this question. We have previously shown that mice homozygous for a hypomorphic thrombomodulin allele (ThbdPro/Pro) are aborted in pregnancies of FV Leiden (FVQ/Q) females before day 12.5 of gestation (Table 1, row 1; PMID 23325830). Par4-activation on maternal platelets plays a key role in fetal death in this model; its absence allows embryonic development and restores Mendelian frequency of live ThbdPro/Pro embryos found at term (Table 1, row 2; PMID 17438064). We examined the effect of reduced TF or genetic deletion of FVIII on embryonic survival of ThbdPro/Pro embryos. We demonstrate that inhibition of TF or the absence of FVIII, each significantly protect the fetus from maternal platelet-mediated death (Table 1, rows 3 & 4). Treatment of the mother with anti-TF antibody 1H1 resulted in 11 ThbdPro/Pro embryos (30 analyzed, 50% or 15 expected; 37% observed, 95% CI 20 to 56%). In the absence of FVIII, 10 ThbdPro/Pro embryos were observed (30 analyzed, 50% or 15 expected; 37% observed, 95% CI 20 to 56%). The outcome of TF inhibition was comparable to the absence of FVIII (P=0.79 Χ2 test of independence). In a second model, Endothelial Protein C Receptor null mice (ProcR-/-) die in midgestation (Table 2, row 1), but reducing TF expression restores Mendelian frequency of ProcR-/- pups (PMID 15956290). We find that ProcR-/- embryos survive past midgestation if the mother is injected with platelet depleting antibodies. Genetic absence of Par4 allows ProcR-/- mice to survive embryonic development (Table 2, row 2; 25% or 18 ProcR-/- neonates expected, 13 observed, 95% CI 10.3 to 29.7%). We investigated the role of FVIII in the midgestational demise of ProcR-/- mice. We demonstrate that genetic absence of FVIII also protects ProcR-/- mice from intrauterine demise (Table 2, row 3; 25% or 12 out of 48 ProcR-/- expected, 12 observed, 95% CI 13.6 to 29.6%). Similar to TF reduction, the absence of FVIII restores Mendelian frequency of ProcR-/- embryos at term. Thus, the outcome from the genetic absence of FVIII was comparable to the absence of Par4 (P=0.4 Χ2 test of independence). In a third model, we have established that epiblast restricted deletion results in normal Mendelian frequency of thrombomodulin-deficient embryos (Meox2Cretg Thbdδ/δ) at term, but about 1/3rd survive the trauma of birth. Most surviving mice later succumb to severe tail and limb thrombosis and several exhibit circling behavior indicative of stroke. Genetic absence of Par4 does not improve perinatal survival, but protects thrombomodulin-deficient mice from stroke and thrombotic disease, significantly improving adult survival. We tested whether genetic absence of FVIII affords protection similar to the absence of Par4. We report that similar to Par4-/- Meox2Cretg Thbdδ/δ mice, FVIII-/- Meox2Cretg Thbdδ/δ mice do not exhibit thrombosis or stroke (current age range 2 to 8 months). Thus, the absence of FVIII is protective in Par4-mediated thrombosis and stroke observed in thrombomodulin-deficient mice.
These studies establish an essential role of FVIII in Par4-mediated pathologies. They strongly support the notion that FVIII activation is required for sufficient accumulation of thrombin for platelet activation. Notably, these are models of physiological thrombosis or platelet-mediated fetal death, without the use of artificial injury or insult. All three models that we describe lack a fully functional system for generating activated protein C (aPC). Our results suggest that inactivation of FVIII by aPC may be necessary to tightly regulate thrombin-mediated platelet activation. Reduced platelet activation observed in hemophilia A mice, albeit in the context of prevention of thrombosis, also lends support to the notion that defects in primary hemostasis due to reduced platelet activity could be an important component of bleeding diathesis in hemophilia A patients.
Disclosures
No relevant conflicts of interest to declare.
In Silico Mutation and Binding Studies of Human FVIIa γ-Carboxyglutamic Acid-Domain to Endothelial Protein C Receptor: A Molecular Dynamics Simulation Approach
