Genetic Absence of Integrin alpha2b Improves Survival of Tissue Factor Pathway Inhibitor Null Mice but Results in Hydrocephalus Formation

Introduction: Genetic deletion of Tissue Factor Pathway Inhibitor (TFPI) exon 4, encoding its Kunitz 1 (K1) domain, results in complete intrauterine lethality (PMID 9242522). TFPI K1 null mice (Tfpi^-/-) are born live if Tissue Factor expression is reduced or in the complete absence of Protease Activated Receptor-4 (Par4), and these exhibit a normal life span without overt signs of thrombosis (PMID 15598816, 25954015). Based on these data, it has been postulated that modulation of thrombin-dependent platelet activation by TFPI is essential for survival. Platelet activation results in a number of downstream events. Of these, platelet aggregation via the integrin receptor αIIbβ₃ is considered to have a key role in hemostasis and could participate in thrombotic pathology in the absence of TFPI. Binding of αIIbβ₃ to its ligands also mediates critical interactions of platelets with endothelial cells, leukocytes and other cell types. In this work, we have investigated whether modulation of platelet activity via genetic absence of integrin receptor αIIbβ₃ confers protection and allows generation of adult Tfpi^-/- mice.

Methods: Tfpi^+/- αIIb^-/- mice were generated by breeding Tfpi^+/- and integrin αIIb^-/- mice and identified by PCR-based genotyping of tissues obtained by tail biopsies. Tfpi^+/- αIIb^-/- intercrosses served as the experimental cross. Pups were genotyped at the time of wean, around 4 weeks of age. In some experiments, surgeries were performed to analyze pregnancies at 18.5 days post coitum (dpc). Embryos and placentae were observed under the dissecting microscope and any phenotypic abnormalities were noted. Presence of heart beats and limb movements were used to identify live embryos. Embryos and placentae were fixed in zinc-formalin and embedded in paraffin for sectioning and histological analysis. T1 weighted Magnetic Resonance Images were acquired on a 9.4T scanner to measure cerebral ventricle sizes of Tfpi^-/- αIIb^-/- and littermate control mice. Ventricular regions of interest (ROI) were drawn on each image slice from which total ventricular volume was computed. These mice were later perfused with 4% paraformaldehyde for collection of organs and histological analysis.

Results: We analyzed 122 pups from intercrosses of Tfpi^+/- αIIb^-/- mice and observed a genetic distribution 39 Tfpi^+/+, 77 Tfpi^+/- and 6 Tfpi^-/- (25% or 31 Tfpi^-/- were expected, 5% observed, 95% CI 1.8 to 10.4%). Thus, genetic absence of αIIb results in incomplete rescue of Tfpi^-/- mice (P< 0.000002, Χ² GOF) with only ~20% surviving past embryonic development to 4 weeks of age. These data contrast with 40% Tfpi^-/- offspring surviving in the absence of Par4 (Tfpi^+/- Par4^-/- intercrosses: 23 Tfpi^+/+, 39 Tfpi^+/- and 8 Tfpi^-/-; PMID 25954015). Thus, the absence of αIIb is much less effective than the absence of Par4 in allowing early survival of TFPI null mice (P < 1.9E-09; Χ² test of independence).

We compared survival of Tfpi^-/- αIIb^-/- offspring close to term of pregnancy (18.5 dpc) and at 4 weeks of age. TFPI null embryos were found at reduced frequency at 18.5 dpc (12 Tfpi^+/+, 21 Tfpi^+/- and 5 Tfpi^-/-; 25% Tfpi^-/- expected, 13% observed, 95% CI 4.4 to 28.1%), but even fewer survived the trauma of birth (P<0.0004, Χ² test of independence).

Of the 6 Tfpi^-/- αIIb^-/- pups found at 4 weeks of age, 3 died by 9 weeks of age. Dome shaped heads indicative of hydrocephalus or histological evidence of hydrocephalus was noted in the pups that died. Surviving mice were observed for 7 months to 1 year of age and imaged with MRI. Comparison of ventricular volumes between Tfpi^-/- mice and Tfpi^+/- controls demonstrated a higher volume in Tfpi^-/- mice (39.3 ± 18.3 versus 5.3 ± 2.2 mm³; P=0.08). Both Tfpi^-/- and Tfpi^+/- mice were αIIb^-/- in this experiment. Thus, αIIb in not involved in hydrocephalus formation. Hydrocephalus formation in Tfpi^-/- mice was confirmed through serial histological sections of the brain (Figure 1).

Conclusions: Our data demonstrates that genetic absence of αIIb improves survival of TFPI null mice, but to a much lesser extent than the genetic absence of Par4. Thus, the critical role of Par4 in the demise of TFPI null mice is unlikely to be primarily through excessive platelet aggregation. We further show that TFPI null pups exhibit varying degrees of hydrocephalus formation. While the mechanism of hydrocephalus formation in the absence of TFPI remains unclear, our results demonstrate a critical role of TFPI in the brain.

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