Role of coagulation factors in cerebral venous sinus and cerebral microvascular thrombosis

Abstract

Objective: The objective of this study was to define the relative contributions of three major pro- and anti-coagulation pathways (heparin-antithrombin, protein C, and tissue factor (TF)) in the thrombogenic responses that occur in large and small vessels of the brain.

Methods: Cerebral venous sinus thrombosis was induced by topical application of FeCl3 on the superior sagittal sinus, while photoactivation of fluorescein was used to induce thrombus formation in cerebral microvessels. Heparin, activated protein C (APC), and antibodies to either APC or TF were used to assess thrombogenesis in wild-type mice. Mutant mice that overexpress the endothelial protein C receptor (EPCR-tg) or with TF deficiency in Tie2-expressing endothelial cells (LTFE) were also used.

Results: Thrombus formation in the superior sagittal sinus of wild-type mice was attenuated by heparin and in EPCR-tg mice, while treatment with the APC antibodies enhanced thrombogenesis. Arteriolar thrombosis was largely unresponsive to the interventions studied. However, in cerebral venules, thrombosis was inhibited by heparin and in EPCR-tg mice. TF antibody treatment also inhibited venular thrombosis, with a similar attenuation noted in LTFE mice.

Conclusion: Thrombin promotes while the APC pathway blunts thrombus formation in an experimental model of cerebral venous sinus thrombosis. TF involvement is more evident in cerebral microvascular thrombogenesis, with endothelial cell-associated TF mediating this response in venules, but not arterioles.

Figure 1. Targets of action of genetic and pharmacological interventions used to probe the role of components of the coagulation and anti-coagulation pathways

TF, activated factor VII (VIIa), activated factor V (FVa) and activated factor X (FXa) complex is derived from extrinsic pathway, while platelet factor 3 (PF3), activated factor VIII (FVIIIa), FVa, and FXa complex are derived from the intrinsic pathway. These complexes (prothombin activating complex (PAC); surrounded by broken lined square) generate thrombin from prothrombin. TF act as an initial activator after vessel injury after binding FVIIa. LTFE mice do not contain TF in endothelium and hematopoietic cells. Protein C is activated with through the interactions of thrombomodulin (TM), protein S and EPCR binding. APC inactivates FVa and FVIIIa, which is a component of PAC. Heparin acts as an enhancer of antithrombin, which blocks both thrombin activity and other coagulation factors to generate fibrin.

Figure 2. Measurement of thrombosis in the superior sagittal sinus (SSS)

(Panel A) view through the cranial window; P designates the FeCl₃ soaked filter paper lying on the posterior segment of the SSS, A designates the filter paper lying on the anterior segment. Panel B depicts an angiogram of a flow cessation determination in the posterior segment of the SSS. Panel C is an angiogram of the plugging in the anterior segment of SSS after exposure to 40% FeCl₃.

Figure 3. Effects of heparin on thrombin formation in the superior sagittal sinus (SSS) and cerebral venules

Panel A: WT (n = 10), WT + hep (n = 9). Panel B: WT (n = 10), WT + hep (n = 7). *designates p<0.05 versus WT; **. P<0.01 versus WT.

Figure 4. Role of the protein C pathway in thrombus formation within the superior sagittal sinus (SSS) and cerebral venules

Panel A, B, C: WT (n = 10), WT + APC (n = 10), WT + APC Ab (n = 7), EPCR-tg (n = 8). Panel D, E, F: WT (n = 10), WT + APC (n = 7), WT + APC Ab (n = 5), EPCR-tg (n = 11). * designates p<0.05 versus WT; **. P<0.01 versus WT.

Figure 5. Effects of tissue factor (TF) immunoneutralization on thrombus formation in the superior sagittal sinus (SSS), venules, and arterioles

Panel A: WT (n = 10), WT + TF Ab (n = 5). Panel B, C: WT (n = 10), WT + TF Ab (n = 6). * designates p<0.05 versus WT; **. P<0.01 versus WT.

Figure 6. Effect of endothelial tissue factor deficiency on thrombus formation in cerebral arterioles and venules

Panel A and B: Cre^-/- (n = 6), LTFE (n = 7). *designates p<0.05 versus Cre^-/-.