Polyhedrocytes: Compressed Polyhedral Erythrocytes In Contracted Blood Clots and Thrombi
Authors: Weisel John W. , Lebedeva Tatiana , Nagaswami Chandrasekaran , Hayes Vincent M. , Massefski Walter , Litvinov Rustem I. , Rauova Lubica , Lowery Thomas J. , Cines Douglas B. 
Published: October 2019

Background
Contraction of blood clots is necessary for hemostasis, wound healing and to restore flow past obstructive thrombi. However, little has been known about the structure of contracted clots and mechanisms of contraction. Erythrocytes, biconcave cells that are highly deformable to allow their passage through the microvasculature, are abundant in venous thrombi, and to a lesser extent in arterial thrombi. Erythrocytes promote hemostasis, but their participation in clot contraction has not been reported. Here we study the mechanisms of clot contraction and the roles of erythrocytes, platelets and fibrin, and show that erythrocyte shape change into compressed polyhedrocytes allows tight packing consistent with the major function of clots to stem bleeding.

Methods
Whole blood was clotted by recalcification and addition of thrombin or kaolin, while following the process of clotting, including contraction, with a new technique using T2 magnetic resonance. We examined the structure and composition of contracted whole blood clots by scanning electron microscopy and confocal light microscopy.

Results
Contracted clots display a remarkable structure, with a close-packed, tessellated array (or mosaic tiling of space) of compressed polyhedral erythrocytes (called polyhedrocytes) on the interior and a meshwork of fibrin and platelet aggregates on the exterior. Little fibin and few platelets were found on the interior of the contracted clots. The same results were obtained with both thrombin and kaolin as activators of clotting and also with reconstituted human blood and clots prepared from mouse blood. Confocal microscopy of hydrated clots confirms the results of scanning electron microscopy. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a closely packed polyhedral array. To assess the density of packing of the polyhedral erythrocytes, we replaced the water surrounding the clots with D2O and observed by T2 magnetic resonance that hydrogen/deuterium exchange for the contracted clots was very slow, consistent with their very tightly packed, almost impermeable structure. The same polyhedrocyte structures were observed from in vivo thrombi aspirated by cardiologists from the coronary arteries of ST-elevation myocardial infarction patients.

Summary/Conclusions
We have observed a previously undiscovered, naturally occurring erythrocyte function and morphology, closely packed polyhedra, in contracted clots and thrombi, and an unexpected spatial redistribution of platelets and fibrin that occurs during contraction. Clot contraction is an essential part of hemostasis, since both human genetic disorders of platelet myosin IIA and megakaryocyte myosin IIA-knock out mice show a bleeding phenotype. These observations on contracted clots imply that they are stiff, rigid structures that can form an impermeable, watertight seal. On the one hand, contraction of clots within the vasculature may relieve obstruction of blood vessels and allow recanalization, especially in the venous system. On the other hand, these results account for long-standing clinical observations that fibrinolysis is greatly prolonged following clot contraction, since perfusion or diffusion of lytic enzymes into these tightly packed polyhedral erythrocytes would be nearly impossible. These results suggest a vital role for erythrocytes and clot contraction in hemostasis and wound healing.

Disclosures:
No relevant conflicts of interest to declare.