Structural changes in blood clots during

compression-decompression cycling

We used confocal and scanning electron microscopy to determine how PPP, PRP and whole blood clots responded to cycling of compression-decompression loads, and to see what the structural bases for these mechanical behaviors are. At first, I studied PPP clots at the same degree of compression under two different rates of compression, 10 µm/sec and 100

µm/sec. At the maximum extent of compression, about 400 optical sections were collected for each clot using a confocal microscope. In response to compression, the fibrin network became divided into two regions with distinctive structural characteristics, a densified re-

Figure 6.3: Changes in the structure of PPP and PRP clots after the first cycle of compression-decompression as observed by scanning electron microscopy. The clots were

compressed 2X at a rate of 10 µm/sec and decompressed at the same rate. (A) PPP clot

before compression. (B) Zoomed area from panel (A); (C) PPP clot after first cycle of compression-decompression. (D) Zoomed area from panel (C). (E) PRP clot before com- pression. (F) Zoomed area from panel (E). (G) PRP clot after first cycle of compression; (H) Zoomed area from panel (G). White arrowheads point to fibrin bundles; black arrowheads point to broken ends of fibers. Magnification bar = 10µm.

gion adjacent to the top plate of the rheometer, where the compression was applied and a rarefied region below with a much lower fiber density (Figure 6.2A-C). Those changes depend on the rate of compression, such that the size of the densified region is larger and denser at the high rate of compression. To quantify the densified region, three-dimensional reconstructions of optical sections were carried out. The intensity profile was measured for all XY planes along the Z axis and plotted as a function of distance from the top of the clot, where compression started (Figure 6.2D). The densified area was identified by changes

of fluorescence intensity along the direction of compression. I found that a high rate of compression resulted in a denser network, which is reflected by the almost 2-fold higher maximum intensity than at a low rate of compression. In addition, increasing the rate of compression resulted in propagation of higher intensities (due to densification) deeper into the clot along the direction of compression. The boundary between the densified and rar- efied region was sharper at the low rate of compression (Figure 6.2D). In terms of network structure, densification means buckling, bending, criss-crossing and bundling of fibers, as well as alignment of fibers in the plane perpendicular to the stressKim et al.(2016). Those structural changes happened during the compressive part of the stress strain cycle (Figure 6.1(a-c)). During the linear elastic response from the beginning of compression to the first inflection point (Figure 6.1(b)), mostly bending and rotation of fibers occurred. During the next portion of the curve (Figure 6.1(b-c)), as compression proceeds, the distance between fibers was decreasing, which resulted in buckling, criss-crossing, bundling and reorientation of fibers in the network, such that they became aligned perpendicular to the direction of the compression (Figure 6.2A-C). As a result of those structural changes, the stiffness of the network decreased (Section 6.2.6). Some structural changes that occurred during com- pression were reversible while others were irreversible. The first part of the decompression curve was a non-linear elastic response that happens when the compressive strain changed less than 0.05, up to the first inflection point during decompression (Figure 6.1(b)). At this point, most of the reversible changes, such as bending and rotation, which happened during the linear response of compression, returned to their original form during decompression. As strain continued to decrease, the stress decreased due to network rearrangement via dissociation of fiber-fiber junctions that arose from contacts during compression. These rearrangements resulted in energy dissipation, and the second cycle of compression started with a clot with some structures still modified.

To determine how the network changed in detail after the first cycle of compression- decompression, scanning electron microscopy was performed. It is very clear that after the first compression-decompression cycle, the network of PPP clots was rearranged (SEM Fig- ure 6.3A-D). After the first cycle, the thickness of fibers increased for the clots as a result of both criss-crossing and bundling of fibers during compression. However, not all fibers stuck to each other either in criss-crossing structures or in bundles, and were dissociated after decompression was completed. In addition, many fibers ends were observed for PPP clots,

probably as a result of fiber breakage. Those changes in clot structure were responsible for the increasing stiffness of the clot with each cycle. The next sets of experiments were carried out with PRP clots. I found that PRP clots responded on compression-decompression cycles generally in a similar manner as PPP clots. Originally, PRP clots have a denser fibrin net- work compared to PPP clots, which means shorter distances between fibers and smaller pore size. Due to those differences, more bundling and criss-crossing were observed in PRP clots (SEM Figure 6.3E-H) than in PPP clots. Those structural differences resulted in higher stiffness after the first compression-decompression cycle (Section 6.1.3). There were simi- larities and differences in the responses of whole blood clots on compression-decompression cycling, as compared to PPP and PRP clots. The same densified and rarefied regions were observed in the fibrin network. The differences are likely due to clot composition, since a major component of whole blood clots are RBCs that make up about 40% of the clot volume. As I observed during compression-decompression cycling, RBCs move out dur- ing compression and move back into the clots during decompression (Supplemental video). When I measured the hematocrit of the blood, and hence the hematocrit of the clot before compression, and the hematocrit of the serum and RBCs expelled from the clot at the point when compression was completed but decompression had not yet started, surprisingly the hematocrit was the same, meaning that the RBCs were expelled from the clot in the same proportion as the serum. Furthermore, all the RBCs that were forced out of the clot during compression returned during decompression. To follow the structural changes of whole blood clots during compression and decompression, clots were examined by confocal microscopy. I found that RBCs, while initially randomly distributed throughout the clot, were rearranged non-randomly during the compressive part of the cycle. They were pushed down in the direction of compression into the rarefied phase, as well as being forced out with the liquid. As a result of this redistribution, more RBCs were observed in the bottom of the clot in the rarefied region and on the periphery of clot near the edges of the rheometer plates, while there were few RBCs in the densified region, which was mostly fibrin network (Figure 6.8C).

Interestingly, the shape of RBCs in the compressed clot changed as a result of compres- sion, such that no biconcave cells were observed in the compressed clot. In addition, many fibrin fibers in the densified zone were oriented perpendicular to the direction of compres- sion, as observed for PPP and PRP clots. When the restraints keeping the clots compressed

were removed and the clots were allowed to relax for an hour before being imaged in the confocal microscope, I observed that some liquid with RBCs moved back into the clot. This passive decompression corresponds to the linear response up to the first inflection point (Figure 6.1(c-d)). Interestingly, after passive decompression, most RBCs were polyhedro- cytes (Figure 6.8E, F). To see in detail the structural changes in whole blood clots after the first compression-decompression cycle, those clots were examined by scanning electron microscopy, with observations consistent with those from confocal microscopy. Some irre- versible changes occurred, e.g. the fibrin network was rearranged with thicker fibers and smaller pore sizes than before compression (Supplemental Figure 2B, D, F). Furthermore, most of the RBCs present after the first compression-decompression cycle were polyhedral in shape (Supplemental Figure 2B). Also, on the periphery of the clot at the edges of the rheometer plates, RBCs were a more prominent component, as a result of the compression- decompression cycle.