Suppressive Role of Tissue Factor Pathway Inhibitor-α in Platelet-Dependent Fibrin Formation under Flow Is Restricted to Low Procoagulant Strength

Abstract

Tissue factor pathway inhibitor-alpha (TFPI-α) is a Kunitz-type serine protease inhibitor, which suppresses coagulation by inhibiting the tissue factor (TF)/factor VIIa complex as well as factor Xa. In static plasma-phospholipid systems, TFPI-α thus suppresses both factor Xa and thrombin generation. In this article, we used a microfluidics approach to investigate how TFPI-α regulates fibrin clot formation in platelet thrombi at low wall shear rate. We therefore hypothesized that the anticoagulant effect of TFPI-α in plasma is a function of the local procoagulant strength-defined as the magnitude of thrombin generation under flow, due to local activities of TF/factor VIIa and factor Xa. To test this hypothesis, we modulated local coagulation by microspot coating of flow channels with 0 to 100 pM TF/collagen, or by using blood from patients with haemophilia A or B. For blood or plasma from healthy subjects, blocking of TFPI-α enhanced fibrin formation, extending from a platelet thrombus, under flow only at <2 pM coated TF. This enhancement was paralleled by an increased thrombin generation. For mouse plasma, genetic deficiency in TFPI enhanced fibrin formation under flow also at 0 pM TF microspots. On the other hand, using blood from haemophilia A or B patients, TFPI-α antagonism markedly enhanced fibrin formation at microspots with up to 100 pM coated TF. We conclude that, under flow, TFPI-α is capable to antagonize fibrin formation in a manner dependent on and restricted by local TF/factor VIIa and factor Xa activities.

Localized endothelial‐based control of platelet aggregation and coagulation under flow: A proof‐of‐principle vessel‐on‐a‐chip study

“…When binding thrombin, thrombomodulin converts protein C into APC in a protein S‐dependent manner, thus resulting in inactivation of the coagulation factors Va and VIIIa, and thereby preventing the formation of fibrin. In our microfluidics approach, this anticoagulant effect of thrombomodulin on HUVEC was found to be restricted to conditions of low TF, in a similar way as we have reported before for the anticoagulant effect of plasmatic TFPI . An explanation for this is that at high TF levels, the amounts of thrombin formed under flow are too high to be targeted the coagulation factor‐inactivating thrombomodulin.…”

“…Herein, first perfusion with anticoagulated whole blood allowed to monitor collagen‐dependent platelet activation via glycoprotein VI; and a second perfusion with recalcified plasma served to induce TF‐triggered thrombin generation and ensuing fibrin clot formation. This clotting process appeared to rely on the flow rate, the coagulation factors VIII, IX and X, and was controlled by plasma‐derived TFPI . For the present paper, we converted this method into a “thrombogenic” vessel‐on‐a‐chip model by growing a discontinuous layer of endothelial cells on top of the collagen/TF surface.…”

“…After washing with sterile phosphate‐buffered saline, co‐coating was performed with TF (75 µL of 500 pmol/L, Dade‐Behring, Breda, The Netherlands), unless indicated otherwise. This gave an estimated coating density of 15 ng/mm 2 collagen and 90 × 10 6 molecules/mm 2 TF . Subsequently, HUVEC (around 100 000 cells/slide, passage 4‐6) were seeded on the coated slides, and cultured until a coverage of 40% to 60% was obtained, thus representing a discontinuous endothelial layer.…”

“…Using the endothelial‐covered microfluidic chambers, a two‐step protocol was followed to monitor platelet deposition and ensuing coagulation activation, by adapting a procedure described earlier . Immediately before flow perfusion, the cellular nuclei were stained for 10 minutes with Hoechst 33342 (1 µg/mL, Molecular Probes, Waltham, MA).…”

“…After a total of 10 minutes of plasma perfusion, z ‐stacks of endothelial cells, platelets (DiOC 6 labeled) and fibrin (AF546 labeled) were taken with 1‐µm steps. Time to first fibrin formation was assessed from recorded images, by observing the presence of fluorescent fibrin fibers, and checked from differential subtracted images, as described in detail previously . For the analysis of fibrin formation, images were thresholded to assess the highly fluorescent fibers only, thus eliminating a residual fluorescence from noncleaved fibrinogen binding to platelets .…”