Novel Initiation Mechanism of Blood Coagulation by Intravascular Tissue Factor.
Authors: Engelmann Bernd 
Published: October 2019

Abstract
Bernd Engelmann Vascular Biology and Hemostasis, Inst. of Clinical Chemistry, Ludwig-Maximilians-Universität, Munich, Germany Following addition of fibrillar collagen to whole blood in order to mimick the starting process of hemostasis, we unexpectedly observed that tissue factor (TF), the central initiator of coagulation, was exposed within 3–5 min in association with CD15 and CD14 positive blood cells. A series of experiments revealed that the TF presentation was restricted to conjugates of neutrophils (and monocytes) with platelets. To verify the source of the TF, isolated neutrophils and platelets were evaluated for the presence of TF. Using a double sandwich Elisa, the washed platelets were found to contain TF. Conversely, TF was undetectable in the neutrophils. When searching for the intraplatelet location of TF by immunoelectron microscopy (IEM), TF was observed to reside in the alpha-granules and in the surface connected system. No TF was present in the cytoplasma and the dense granules. In response to activation, platelet TF was translocated to the cell surface by fusion of the alpha-granules with the plasma membrane. The externalized TF was found to cluster on platelet filopodia. Inspection of rapidly isolated buffy coat preparations confirmed the absence of TF from the neutrophils. Stimulation of TF-dependent factor Xa formation by the activated platelets was markedly amplified by the isolated neutrophils. This required neutrophil-platelet conjugate formation, as evident from inhibition by antibodies targeting PSGL-1 and CD18. To assess whether the TF triggered coagulation was connected to the platelet recruitment, we evaluated the participation of the ADP system. Disrupting the interaction of ADP with its platelet receptors P2Y12 and P2Y1 suppressed the TF activity in the neutrophil-platelet conjugates. Since the TF exposing filopodia represent preferential sites for the formation of microparticles (MP), we isolated the total pool of circulating MP from whole blood, known to be mainly derived from the platelets. Then, the MP were separated by cell sorting. In MP positive for the platelet specific CD42b, TF could be detected and quantified by western blotting and Elisa. Moderate increases in MP number excessively stimulated blood based TF activity in the presence of platelets and in whole blood. Since activated platelets are known to secrete tissue factor pathway inhibitor (TFPI), an anti-TFPI antibody targeting the Kunitz-2 domain of TFPI was included into the suspensions of the activated platelets. Thereby the TF activity of the isolated platelets was enforced, while the activity in the presence of the neutrophils remained unaffected, suggesting that TFPI partially masks the functional competence of the platelet TF. The potential contribution of platelet-collagen interactions for the activation of coagulation in vivo was analyzed by injecting collagen into the venous blood of mice. Local fibrin formation was documented in pulmonary vessels by EM, and systemic thrombin generation was revealed by increased thrombin-antithrombin complexes. In mice deficient for the P2Y1 ADP receptor, the thrombin generation was markedly reduced, indicating a basic role for the platelet-triggered coagulation during thrombus growth. In conclusion, the intravascular tissue factor enables the entire coagulation system to proceed on the plasma membrane of a single blood component, the surface of the activated platelets. Consequently the coagulation start can be regulated within the platelet aggregate, allowing fibrin formation to be flexibly adjusted to the size of the thrombus and the duration of its development.