Attachment of Platelets During Flow

To test the adhesion of the platelets to the substrate under flow, the experiment was conducted with the flow rates and concentrations as later used during the measurements. The attachment and possible contraction was monitored over 1.5 h.

At the end of each recording, the last image of the attached platelets was taken and all spread cells were evaluated as to their time point of attachment and whether they contracted. This analysis gave answers to three important questions:

1. Do the platelets attach under flow?

2. If so, when do they attach?

Chapter 7 RESULTS

Figure 7.12.: Starting at the gel surface at 25 µm, 28 images in height were taken according to the sketch in A. The rst image was taken directly on the surface (red solid line) and than varied in height to produce equidistant images (red dashed lines). The results for 300µL/h (B) and 700 µL/h (C) are shown here. The rst row denotes the actual measured velocity values with their median. Subsequently, for each height, the mean velocity was calculated. From the measured, averaged values (crosses), a parabola was tted (red line) using Matlab, shown in the middle row. The theoretical velocity prole determined with Comsol can be seen in the lower row. Here, the crosses mark the actually calculated values by Comsol and the red parabola t was determined with Matlab. The dierence in the maximal velocity between the simulated and measured velocity is less than 5 %. The image is taken from Hanke et al. [36].

Blood Platelets Under Flow Conditions 7.3 3. Do the platelets that attach also contract?

The second question was eminent to decide whether a recording time of 1.5 h was reasonable or had to be shortened or lengthened.

In Fig. 7.13 A, an example of an attachment test is seen. For this particular ex-ample, the flow rate was set to 300 µL/h and the last image of the recording is shown. All platelets that adhered and contracted were put into three categories:

cells that attached as clusters, platelets that adhered separately but had other cells attaching to them at a later time point, thus forming clusters, and platelets that remained single during the entire recording process. For the measurements to de-termine the contraction force of a platelet, the first group was discarded, cells in the second group were only analysable until another cell attached to the original platelet while the last group was analysable in its entirety. Additionally, for this test, the approximate time of attachment was noted for all platelets. The exam-ple presented here corresponds to an average situation observed in terms of the attachment rate. While all tests conducted for flow rates between 300µL/h and 700µL/h saw some degree of adhesion, the rate varies greatly, from 4 to nearly 40 cells or cell clusters after 1.5 h of recording. At the same time, as can already be assessed from this example, out of all adhered platelets, the amount of clus-tering platelets were equally as often observed as single, contracting platelets. In Fig. 7.13B, the relative attachment rate according to the time point of adhesion for all experiments is shown, a total of 202 platelets and platelet clusters taken from 5 independent experiment days. We observed that the majority of platelets attached between 5 min and 35 min after recording started. In the following 15 min inter-val, the attachment rate remained near constant before decaying further. To ensure that the bulk of platelets were recorded for about 30 min, a recording time of an hour was sufficient. We still chose to record for 1.5 h. The reason here was the aforementioned partially low attachment rate. By increasing the recording time for another 30 min, we increased the probability to record more platelets as we also included the group adhering at the small plateau found between 35 min and 50 min in case of a reduced attachment rate.

Apart from the already mentioned flow rates, a higher flow rate of 1000 µL/h was also tested. All of the lower flow rates worked well concerning the attachment and contraction as presented above, however, the highest flow rate did not show any adhesion. The only attachment observed was found if an air bubble developed near the recording site which redirected the flow, effectively reducing the velocity.

An example of the attachment due to the change in flow direction is shown in

Chapter 7 RESULTS

0 10 20 30 40 50 60 70 80 90 t (min)

0.16 0.12 0.08 0.04

re la ti ve a tt a ch me n t

B A

Figure 7.13.: A An example of an attachment test taken with a ow rate of 300µL/h. Green boxes mark platelets that attach and contract as a single cell. Blue boxes are platelets that attach as single cells and contract before other platelets attach to them. Red boxes denote contracting clots. All cells are marked as to their attachment time point relative to the start of recording. The streaks within the image are bypassing cells. The total recording time were 1.5 h. B The time points of attachments of all experiments combined. Most platelets attached between 5 min and 35 min after recording started. Between 35 min and 50 min, the attachment rate remains the same before decaying further.

Blood Platelets Under Flow Conditions 7.3

Figure 7.14.: A time series of a recording at 1000µL/h. Here, the ow direction changed during recording, denoted in the image by the blue arrow. At about 38 min, an obstacle outside the frame of recording redirected the ow, after which a cell attached at about 43 min (red arrow).

At 52 min of recording, the ow has changed its direction by nearly 90^◦. The direction of the ow was derived from the entire recording by studying the traces of the by-passing cells.

Fig. 7.14. This observation was previously reported in a precursor of the used flow chamber by C. Ranke [96], where the flow needed to be stopped to facilitate attachment. Hence, only the aforementioned three flow rates, 300µL/h, 500µL/h and 700 µL/h, were employed to study the contraction of platelets under low shear rates.