Differences in human and sheep platelet adherence,aggregation and activation induced by glass beads in a modified chandler loop-system

Abstract

In human cardiovascular research, sheep in particular are used as a large animal model in addition to pigs. In these animals, medical products, developed and tested for human medical purposes, are almost exclusively used in interventional studies. Therefore, the extent to which platelets from human and ovine blood differ in terms of adherence, aggregation and activation after a 4- or 8-minutes exposure to glass was investigated. Testing was performed with platelet-rich plasma (PRP) and a modified chandler loop-system, with 4- and 8-minute blood-material exposure times corresponding to 20 and 40 test cycles, respectively, through the entire silicone tube loop of the test system.

In sheep and human PRP, contact with the silicone tubing resulted in a decrease in platelet count after 4 minutes and 20 test cycles, respectively. Four more minutes (20 additional test cycles) caused a further decrease of the platelet count only in sheep PRP. When the silicon tube was partly filled with glass beads, these effects were more pronounced and stronger in sheep then in human PRP.

The mean platelet volume, which was used as parameter for platelet aggregation, did not change over time in human PRP without glass exposure. With glass exposure in human and sheep PRP the mean platelet volume increased within 40 test cycles, but this increase was stronger in sheep than in human PRP.

Regarding activation behavior, the activation markers CD62P and CD63 were detectable only in < 30% (sheep) and < 45% (human) of platelets, whereas after 8 min of glass exposure, the proportion of CD62P⁺ and CD63⁺ cells was more increased than before only in sheep. These results indicate that ovine platelets adhere more strongly to glass and show stronger aggregation behavior after glass contact than human platelets, but that ovine and human platelets differ only slightly in activability by glass.

Keywords

Sheep platelet activation platelet volume CD42a CD61 CD62P CD63

1 Introduction

Medical devices are increasingly being used in animals [1] including Class IIb and III medical devices. For example, in pulmonary stenosis, one of the most common congenital heart diseases in dogs, catheter systems for pulmonary stenosis ballooning are successfully used [2 –4]. A persistent Ductus arteriosus botalli in dogs can be successfully closed with coils [5], and cardiac pacemakers have already been implanted in dogs [6, 7], but also in pigs [8], horses [9, 10] and donkeys [11]. Medical devices used for these purposes were generally developed for applications in humans, and were tested for interaction with human blood as part of the safety testing in a conformity assessment procedure (see e.g. [12]). In the EU, however, there are no rules on market approval for the use of medical devices in veterinary medicine, so that these products can be used in animals without further testing [13]. However, there are numerous hints that animal blood differs significantly from human blood, particularly with regard to the interaction with materials. For example, human platelets differ from animal platelets in volume [14], in activation by various materials (pyrolytic carbon, PE, silicone [15]), in the availability of the GP IIb/IIIa receptor [16], and in adhesiveness to biomaterials [17].

Based on these differences, the question arises to what extent the results from studies with human blood are transferable to animal blood. Since in the cardiovascular field sheep are used as animal models [18], we investigated under dynamic testing conditions in a modified chandler loop-system [19, 20] whether platelet counts, mean platelet volumes (MPV), and platelet activation differ when human and sheep blood are exposed to glass as a thrombogenic material.

2 Materials and methods

2.1 Humans and animals

Human blood samples were from healthy adults (2 male, 1 female) who consented to blood collection on a voluntary basis. For each participant, a recovery period of 1 week was observed between each donation.

The blood samples taken from the animals were approved by the Lower Saxony State Office for Consumer Protection and Food Safety (approval number 33.8-42502-05-20A513) and were sourced from two adult sheep of the breed Bentheimer Landschaf which were housed under conventional holoxenic housing conditions and cared for according to the guidelines of the European Societies of Laboratory Animal Sciences. Animals were without clinical signs of disease and were monitored by whole blood analysis and general examination by an experienced veterinarian on each day of testing.

2.2 Blood sampling and dynamic test model

For each test 83 ml blood were sourced from the external jugular vein using an 18 G sized cannula and nine blood collection systems filled with 1 ml of 0.106 molaric tri-sodium citrate each (S-Monovette^® 10 ml 9NC) to prevent spontaneous coagulation. There were at least seven days between blood collections.

Within 30 minutes, the citrate-anticoagulated whole blood was centrifuged at 120 G for 15 min and platelet-rich plasma (PRP) was carefully removed and placed in plastic test tubes. For the tests a sterilized modified chandler loop-system [20] was filled with the PRP for fluorescence-activated cell sorting (FACS) analysis of the platelets (MACSQant, Milteny), and for platelet number and platelet volume analysis (Procyte DX^®, IDEXX). In brief, the modified chandler loop-system consisted of two separate medical-grade silicone tubes with a non-thrombogenic inner surface, which were mounted to a closed loop using two three-way valves made of polyvinylidene fluoride (PVDF). The closed tube was filled with the PRP via one of both three-way valves. The second three-way valve ensured that the air in the system could escape. After insertion of the pump tube into a roller pump a defined flow was maintained (7.5 ml/h). Filled only with PRP the system was regarded as the negative-reference-system (non-thrombogenic control). For tests under thrombogenic conditions the tube system was partly filled with glass beads (OD 0.75–1.0 mm). The glass beads (n = 1,084; calculated total surface 2,221 mm²) were prevented to follow the blood by a medical-grade V2A-steel-based mesh (mesh width 0.5 mm), which was integrated in the tube.

Directly after the initial filling of the tube system with PRP was completed, baseline values were determined. PRP volumes removed were always replaced isovolemically by autologous PRP. Platelet analysis was performed after the PRP within the closed tubing had circulated through the total tube system 20 or 40 times. This corresponded to a circulation time of 4 or 8 min.

2.3 Platelet count and mean platelet volume

The platelets that were free to move in the tube system and that did not become adherent to inner surfaces of the tube system respectively were analyzed in terms of total number and mean volume (as parameter for platelet aggregation). These parameters were measured using an automated hematology analyzer (ProCyte Dx^®, IDEXX, Germany).

2.4 FACS analysis

Platelet activation was evaluated using flow cytometry (MACSQuant^® Analyzer, Miltenyi, Germany). Platelets were defined as CD42a positive cells within the PRP-sample. To assess the platelet activation state the platelet markers CD61 (= GPIIIa), CD62P (= P-selectin), and CD63 (= GP53) were used.

In preliminary concentration tests the antibodies used (recombinant human IgG1 antibodies; PE-conjugated anti-CD42a, FITC-conjugated anti-CD61, APC-conjugated anti-CD62P, PE-Vio 771-conjugated anti-CD63; Miltenyi, Germany) were titrated to the endpoint so that measurements could always be conducted at saturation level, thereby excluding any dependence between the fluorescent intensity and the concentration of the antibodies.

2.5 Statistics

Data analysis was performed with SPSS Statistics 26 using descriptive statistic parameters (means and standard deviations) and t-test for comparison of mean values of dependent samples. PRP samples were gained from each sheep/human six times; each PRP sample was measured three times when it has passed through a modified chandler loop-system 20 or 40 times.

3 Results

Without glass contact, in human PRP the count of non-adherent platelets was only slightly decreased after 20 test cycles (p < 0.05, Fig. 1). Additional 20 test cycles (40 in summary) without glass contact caused no further change in the count of human non-adherent platelets.

Fig. 1

Total number of non-adherent platelets from platelet rich plasma (PRP) of adult sheep (n = 2) and humans (n = 3); each PRP sample was measured three times when it has passed 20 or 40 times through a modified chandler loop-system filled with glass beads; means and standard deviation; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

With glass exposure the count of human non-adherent platelets was decreased after 20 test cycles (p < 0.001) and additionally after 40 test cycles (p < 0.05).

Sheep platelets showed a significantly stronger response with and without glass contact. Without glass contact in sheep PRP, the number of non-adherent platelets decreased by about 33.5 % (p < 0.001) already after 20 test cycles, and showed a further decrease after additional 20 test cycles (p < 0.001).

With glass contact the decrease of the sheep platelet count after 20 test cycles was almost twice as high (–60.9 %, p < 0.001) as without glass contact. Also after 20 additional test cycles (40 in summary) with glass contact the sheep platelet count showed a further decrease, which was much stronger than without glass contact (–52.1 %, p < 0.001).

The platelet volumes, which were measured as a parameter for platelet aggregation, ranged between 11.1 and 11.3 fl in humans and between 8.0 and 9.2 fl in sheep. In human PRP there was no change in the mean platelet volume with and without glass exposure after the first 20 test cycles. Only after additional 20 test cycles with glass contact an increase in mean platelet volume was detectable (p < 0.01, Fig. 2).

Fig. 2

Volume of non-adherent platelets from platelet rich plasma (PRP) of adult sheep (n = 2) and humans (n = 3); each PRP sample was measured three times when it has passed 20 or 40 times through a modified chandler loop-system filled with glass beads; means and standard deviation; ^*p < 0.05, ^**p < 0.01.

In sheep PRP the mean platelet volume already increased without glass contact after the initial 20 test cycles (p < 0.05) and additionally after further 20 test cycles (p < 0.05). Also with glass contact there was an increase of the mean platelet volume over time (p < 0.01).

The proportion of platelets (CD42a⁺ cells) within the PRP samples, that were also positive for CD61, remained constant over the time of 20 or 40 test cycles evident whether or not the PRP was exposed to glass (see Fig. 3).

Fig. 3

Percentage of CD42a⁺/CD61⁺, CD42a⁺/CD62⁺ and CD42a⁺/CD63⁺ cells in platelet rich plasma (PRP) of adult sheep (n = 2) and humans (n = 3); each PRP sample was measured three times when it has passed 20 or 40 times through a modified chandler loop-system filled with glass beads; means and standard deviation; ^*p < 0.05.

The proportion of CD42a⁺ cells for which the markers CD62P and CD63 indicated activation was markedly lower in sheep and human PRP. It ranged in sheep PRP between 15.2 % (CD62P⁺ cells w/o glass contact at t₀) and 42.5 % (CD63⁺ cells after 40 test cycles with glass contact), and in human PRP between 10.9 % (CD63⁺ cells w/o glass contact at t₀) and 27.5 % (CD62P⁺ cells after 40 test cycles with glass contact) of the CD42a⁺ cells.

Exposure to glass resulted in sheep PRP after 40 test cycles (A) in a 1.9-fold increase (from 15.8 % to 30.8 %, p < 0.05) in platelets, which were positive for the degranulation marker CD62P, and (B) in a 2.2-fold increase (from 19.5 % to 42.5 %, p > 0.05) in platelets, which were positive for the activation marker CD63. Within human PRP, the proportion of CD62P⁺ and CD63⁺ cells proved to be independent of the number of test cycles and of exposure to glass.

4 Discussion

The purpose of the study was to investigate whether there are differences in activation and adherence of human and sheep platelets in a modified chandler loop-system when exposed to glass for up to 8 min in the test system. Thereby species-specific platelet behavior was noted.

Immediately after blood collection the platelet counts were within the physiological reference ranges of human and sheep blood [21, 22]. In sheep PRP, contact with the silicone-based tubing system of the modified chandler loop-system resulted in a strong reduction in platelet count. In addition, an increase of the mean platelet volume and platelet aggregation respectively after only 4 min respecting 20 circulations through the total length of the tubing system was measured, whereas platelet count in human PRP was unchanged at the same time. Moreover, the platelet count in human PRP showed only a stronger decrease and the mean platelet volume an increase when (A) the tubing was prefilled with glass beads and when (B) the human PRP was exposed to the glass-filled tubing for at least 8 min. It can be assumed that the reduction in platelet count is mainly due to adherence of platelets to the inner wall of the tubing and to the glass beads, and the increase of the mean platelet volume mainly due to platelet aggregation. This suggests that human platelets were less adherent and formed less aggregates than sheep platelets.

These changes of the platelet count contradict results of a study by Goodman et al. [15]. He investigated how the adherence of sheep platelets differs from that of human platelets on various polymers including silicone. It was observed that sheep platelets adhered to silicone in much lower numbers than human platelets. Since Goodman used Suffolk sheep (n = 2) and a crossbred between a Suffolk and a Dorset sheep for his study rather than Bentheim sheep, the differences to our values may be due in part to breed differences. This hypothesis is supported by the results of a study by Pelagalli et al. [16]. They investigated the adhesion of platelets from different species and from humans to immobilized autologous fibrinogen. They found that sheep platelets, in contrast to human platelets, were able to adhere to autologous fibrinogen only after platelet activation by adenosine-5-phosphate. Grabowski et al. [23] also found significant species-specific differences in the ability of platelets to adhere to foreign surfaces. They compared platelets of several animal species with those of humans in a dynamic closed loop model with respect to their ability to adhere to a cellulose-based hemodialysis membrane. After 10 min of blood flow at a shear rate of 986/sec, they found that < 100/mm² ovine and human platelets, respectively, had adhered to the surface of the membrane. In the tests with platelets from dogs and rabbits, this value was several times higher (dog 27,400±4,600; rabbit 78,400±6,400).

These results are supported by our own investigations. Only a small proportion of 30–45 % of human and ovine platelets (CD61⁺ and CD42a⁺ cells) expressed the activation markers CD62P and CD63 after 4 or 8 min of exposure to the silicone tubing with or without glass. CD62P, also referred to as platelet surface P-Selectin, is expressed on α-granule membranes inside of resting platelets, and is translocated to the platelet surface after activation. Hence, a P-selectin-specific monoclonal antibody binds only to activated platelets after an α-granule connects to the cell membrane and secretes its contents [24]. CD63 (= GP53), also known as lysosomal membrane associated glycoprotein 3 (LAMP3), was first described in granules of resting platelets. Only on activated platelets, CD63 can also be found on the surface membrane [25].

Because the platelets examined by FACS were those suspended in PRP, this number of activated or CD62P- and CD63-positive cells cannot be used to infer the total number of activated platelets. Irrespective of this, however, the results clearly show that neither in the human PRP nor in that from sheep was the four-minute exposure to the tube system filled with glass beads sufficient to significantly increase the proportion of activated or CD62P⁺ and CD63⁺ platelets. Only after 8 min of circulation of the ovine PRP in the tubing system filled with glass beads a increase in the proportion of activated platelets was found. However, part of this increase was probably due to the mechanical action of the roller pump. Jung et al. [26] studied the thrombogenicity of a coronary stent using the same dynamic test system as the one presented here. They found an increase in CD62P⁺ platelets even in the tests in which the PRP was not exposed to the stents and attributed this to mechanical stress on the platelets by the roller pump. Based on this and on our results, we hypothesize that human platelets are less responsive to mechanical stress than those of sheep.

Beside of species-specific differences also test settings had an impact on the results, even if these effects cannot be regarded as the main cause of the species-specific differences, but rather as modulators of the observed effects. In the study by Steven L. Goodman [15], the platelets were exposed to the polymers for 45 min, whereas in the study presented here the exposure time had a maximum of 8 min. Goodman also used a static model, whereas we used a dynamic model in which shear forces were applied, which could have influenced the adherence of the platelets. In addition, the different roughness of the foreign surfaces used probably also played a role [27 –29]. Hecker and Scandrett [30] investigated how the roughness of polyvinyl chloride affects the thrombogenicity of the material. They investigated thrombus formation on tubes of different roughness (OD 3.0 mm) in 40 adult M erino sheep after insertion the tubes into the saphenous vein and the aortic artery. They found a positive correlation between roughness and thrombus formation.

5 Conclusion

It was shown that adherence, aggregation and activation behavior differed only slightly between human and ovine platelets after a 4–8 min lasting glass exposure time. However, the results showed that sheep platelets are more adherent to glass, form more aggregates after glass contact and can be more activated by glass than human platelets. This has to be respected in calculation of the anticoagulative therapy which accompanies cardiovascular interventional studies in sheep, especially if medical products with glass components (e. g. glass fiber optics) are used, which come in contact with blood.

Footnotes

Acknowledgments

We thank Dr. Alexandra von Altrock, Dr. Theresa Maria Punsmann, Klaus Schlotter, and Thorsten Waßmann, staff members of the Clinic for Swine and Small Ruminants, Forensic Medicine and Ambulatory Service, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany, for performing the blood collections, housing, and care of the animals.

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