Several animal species are used to test various cardiovascular implants. Differences in platelet function and coagulation systems between human and animal models have long been suggested but have not been well characterized, mainly because of methodologic limitations. For example, coagulation assays sensitive to in vivo occurrences of thrombus formation, such as F1.2 and D-dimer in humans, are not measurable in animals. Antibodies used in humans to detect the platelet surface expression of GMP-140 with flow cytometry, and the receptors for fibronectin, thrombospondin, and von Willebrand factor cannot be used in sheep and calves because of a lack of species cross-reactivity. 1 Recently, we reported the results of a comparative study of platelet function among different animal species as measured by whole blood platelet aggregometry (WPA), in which we found that human, canine, and bovine platelets vary markedly in their responses to various agonists. 2
To further characterize the differences of platelet and coagulation function in different animal species, the Xylum Clot Signature Analyzer (CSA) (Xylum Corporation, Scarsdale, NY) was used in the present study. The CSA is a newly developed blood coagulation assay system that can simultaneously measure platelet and coagulation parameters under controlled flow conditions. 3 Furthermore, this measurement is done with fresh, whole blood without any anticoagulant, chemical, or immunologic agent. The measurement of platelet function and coagulation under flow is deemed much more relevant to in vivo thrombus formation than measurement with conventional in vitro methods. The purposes of this study were to characterize and compare platelets and the coagulation systems of four animal species, i.e., human, calf, sheep, and dog, utilizing this CSA system. The data were also compared with platelet responses to exogenous aggregates, as measured by Chrono-Log (Havertown, PA) WPA.
Materials and Methods
Clot Signature Analyzer
The details of the CSA have been described elsewhere. 3 Three parameters are measured simultaneously: (1) platelet mediated hemostasis time (PHT), the time for platelets to occlude holes “punched” in a blood conduit; (2) collagen induced thrombus formation time (CITF) as blood flows in a channel containing a collagen fiber; and (3) clotting time (CT). These parameters are measured under flow at shear rates of > 10,000, 6,200, and 300 S−1, respectively. The CSA consists of two polyethylene perfusion channels, the “punch” and “collagen” channels. In the punch channel, blood flow is driven by a “liquid piston” through tubing that is pierced by a 150 μm needle, causing an immediate drop in the pathway pressure from ≈ 65 mm Hg to ≈ 10 mm Hg. Because the diameter of this punch hole is significantly smaller than that of the tubing, the shear rate of blood leaking through this punch hole is high, exceeding 10,000 S−1. An increased shear rate in the punch hole induces platelet activation and aggregation. As the platelet aggregates occlude the punch hole, the pressure in the tubing recovers with time. PHT indicates the interval between punch and 90% recovery of pressure. The flow of the blood within the tubing continues as the activation of coagulation proteins generates fibrin. The build-up of this fibrin clot causes cessation of luminal blood flow in the punch channel. CT is the time between the start of blood draw and the time coagulation causes cessation of flow. Simultaneously, in the collagen channel, blood is perfused over a collagen fiber. Platelets adhere, activate, and aggregate onto this thrombogenic surface, building a thrombus that eventually occludes the tubing, resulting in a measured decrease in pressure. CITF is the time required for the pressure in the collagen channel to decrease halfway to the fully occluded pressure. Blood samples for the CSA were drawn in two separate 3 ml syringes, which were prewarmed to 37° C. An initial 5 ml of blood was drawn and discarded to prevent possible sample contamination from tissue thromboplastin. The two 3 ml syringes were inserted into the CSA cassette within 2 minutes of being drawn. The CSA apparatus is portable and uses fresh whole blood, allowing quick bedside monitoring of platelet and coagulation function.
Whole Blood Platelet Aggregometry
For WPA, 9 ml of blood was drawn into a 10 ml syringe that contained 1 ml of 3.8% sodium citrate. The sample was immediately transferred to a 15 ml Falcon conical plastic tube. All procedures were conducted in accordance with the Chrono-Log Diagnostic Protocol. The whole blood sample, 0.45 ml, was diluted with an equal amount of 0.9% NaCl solution. A 0.1 ml aliquot of luciferin-luciferase reagent was added, and 30 to 60 seconds later the agonist, either 1 μg of collagen or 10 μM of adenosine diphosphate (ADP), was added. All reagents were purchased from Chrono-Log (Havertown, PA). WPA was recorded continuously for 10 minutes, and the maximum change in impedance was recorded as the WPA value for the agonist used. The dose response of animal platelets to these agonists has been reported elsewhere. 2
For platelet counts, a 3 ml blood sample was taken and tested in the Division of Laboratory Medicine at the Cleveland Clinic Foundation (CCF) using an automated blood analyzer (NE-ALPHE/SP1, Sysmax Corp., Kobe, Japan).
CSA data from 204 normal humans were provided by Xylum Corporation. At the CCF, CSA measurements were obtained from 19 healthy Holstein calves (bovine), 7 normal Suffolk sheep (ovine), and 10 healthy mongrel dogs (canine). No subject had received any antiplatelet or anticoagulant medications. In both bovine and ovine species, multiple measurements were obtained from individual animals to examine the intermeasurement variation of the parameters. For WPA and platelet counts, data were quoted from our own database compiled at the CCF.
Results are expressed as means ± standard deviation. Analysis was made by t-test, with α set at 0.05, and all tests were two-tailed.
The results of CSA measurements from four different animal species are shown in Table 1. Table 2 summarizes the p values for interspecies comparison. Table 3 shows the coefficient of variation (CV, %) in CSA parameters for each animal species.
As shown in Table 1, the PHT value was lowest in dogs (61.5 ± 46.6 sec), followed by sheep, humans, and calves. PHT in dogs was markedly different from that in other species, indicating that canine platelets are most responsive to shear induced platelet activation. Humans and sheep showed similar values (241.1 ± 107.4 sec and 230.4 ± 120.2 sec) with no statistical difference (p = 0.37). PHT was most prolonged in calves, suggesting that bovine platelets are less sensitive to shear induced platelet activation. It should be noted, however, that PHT values show a high CV in all species (44.5% to 75.7%) when compared with those for CT (12.6% to 18.8%) and CITF (14.5% to 25.2%) (Table 3). The variation is largest in dogs (75.8%), followed by sheep (52.2%), calves (45.4%), and humans (44.5%). To investigate whether this high variation is due to high individual differences or intermeasurement variation, five measurements were obtained from five individual calves and five sheep (Table 4). The results showed significant intermeasurement variations, even within each individual animal in both species. The CV for PHT ranged from 34.3 to 63.8% (avg. 45.9%) in calves, and from 8.8 to 61.9% (avg. 36.0%) in sheep.
CT was shortest in dogs (901.1 ± 169.2 sec), followed by calves (1,162.3 ± 145.9 sec), sheep (1,163.3 ± 206.5 sec), and humans (1,290.8 ± 235.2 sec), again indicating that dog blood is more easily clottable under flow than other species. The difference between humans and calves is statistically significant (Table 2). The intermeasurement CV for calves and sheep did not differ and was 18.6% and 19.0%, respectively (Table 4). These values are quite similar to CVs shown in CT values for interspecies comparison (12.6% for calves and 17.8% for sheep) (Table 3).
Dogs showed the lowest CITF value, followed by sheep, calves, and humans, indicating that animal platelets are more responsive when exposed to collagen, although the difference between calves and humans was not statistically significant (p = 0.064). The CV for intermeasurement variation for calves and sheep (22.7% and 23.4%, respectively) did not show significant differences from those for the interspecies variation (20.4% and 23.4%, respectively) (Table 3).
The results from WPA and platelet counts for each animal species are shown in Table 5.
Collagen Induced WPA.
With 1 μg collagen as an agonist, human (30.93 ± 6.31 ohm) and dog (27.72 ± 7.12 ohm) platelets appear to be more responsive than sheep (20.22 ± 7.30 ohm) and calf platelets (14.88 ± 7.73 ohm). There was no significant difference between human and dog data (p = 0.10); however, the difference between humans and sheep or calves was significant (Table 6).
ADP Induced WPA.
Aggregation response was highest in dogs followed by sheep, calves, and humans, showing a quite different response pattern when compared with the collagen induced response (Table 5). ADP induced aggregation was significantly less in humans than in other species. No significant difference was noted between sheep and calve or sheep and dog values (Table 6).
The platelet count was significantly higher in calves and sheep than in dogs and humans (Tables 5 and 6). There was no correlation between platelet count and platelet aggregability as measured by CSA and WPA.
Platelets and the coagulation system are essential for hemostasis, and their interaction is the determining factor in blood’s compatibility with cardiovascular implants. In evaluating such devices using various animal models, it is important to recognize species differences for proper extrapolation of the results of animal experiments to humans. A comparison of platelet and coagulation function in different animal species using the literature has limitations in that the methods, agents, and doses used in multiple studies are different. In our previous study, WPA was used to characterize and compare the platelet agreeability in humans, dogs, and calves. 2 A significant difference was noted among these species. Human and canine platelets responded to all six agonists used: collagen, ADP, ristocetin, epinephrine, arachidonic acid (AA), and thrombin. Bovine platelets, however, responded only to collagen, ADP, and thrombin, not to ristocetin, epinephrine, or AA, reflecting the differences in activation pathways. Furthermore, as shown in Table 5 of the present study, the degree of responsiveness among these species was different with different agonists. With collagen, human platelets showed the strongest response, followed by dogs, sheep, and calves. In contrast, when stimulated with ADP, dog platelets responded most strongly, followed by sheep, calves, and humans. At this time, it is not clear whether the response to a particular agonist reflects better in vivo behavior of platelets, especially when they are exposed to unphysiologic flow or foreign surfaces. The effect of sodium citrate, the anticoagulant of choice for both impedance and light transmittance platelet aggregometry, on platelet function needs careful consideration because chelation of calcium ion is known to affect platelet aggregation under both low and high shear stresses. 4
To further characterize the species differences of platelets, CSA, which became available recently, was used in the present study. One advantage of this new technology is that neither agonist nor anticoagulant is required. In addition, not only platelet function but also coagulation function is measurable simultaneously. Among the four species compared, all three CSA time parameters are shortest in dogs, suggesting that platelet response to both shear stress and collagen, and overall coagulation, are most responsive in this species. This finding is supported by results obtained from WPA, which demonstrated that canine platelets show the strongest response to ADP and the second strongest to collagen. Previous studies, including our own study, have indicated the enhanced responsiveness of canine platelets to ADP or foreign surfaces when compared with human platelets. 2,5,6 Greisler et al. 6 noted the presence of two different canine groups in terms of the responsiveness of platelets when tested with turbidimetry with ADP. Among 70 dogs tested, 26% were aggregators and 51% were nonaggregators. They demonstrated that the patency rate of vascular grafts was higher in nonaggregators (85%) as compared with aggregators (68%). The present study and the results from our previous studies with WPA did not show any distinctly different groupings in dogs. The difference may be attributed to the difference in methods used and/or the rather small number of animals used in our studies. Nonetheless, dogs are most commonly used in the field of vascular graft research. 6–9 The extrapolation of the results obtained from the canine model to the human is to be done with caution because of the hyperresponsiveness of canine platelets. PHT and CT in sheep and humans showed no statistical difference (Table 2). These results may suggest that sheep are an acceptable animal model for testing the blood compatibility of devices. Bovine platelets are less responsive to shear induced aggregation than are human platelets, although overall clotting under flow in this species appears to be accentuated more than in humans.
In our previous study, the optimal dosage of agonists for comparison of different animal species was 10 μM ADP and 1 μg collagen. 2 The responsiveness of platelets, as measured by CSA and WPA, was in the following order: PHT, dogs > sheep> humans > calves; CITF, dogs > sheep > calves > humans; collagen induced WPA, humans > dogs > sheep > calves; ADP induced WPA, dogs > sheep > calves > humans. A clear explanation for these differences cannot be given at this time. It may be that CSA does not clearly distinguish platelet aggregation and fibrin clot formation and is less platelet specific. On the other hand, WPA reflects the platelet response to a single chemical agent with an unphysiologically high concentration. Nonetheless, it seems reasonable to conclude that flow induced platelet responsiveness differs from chemically induced platelet aggregation.
Goodman 10 recently compared the number of platelets and morphology of surface-adhered platelets on various foreign surfaces under static in vitro conditions, reporting that ovine platelets were much less reactive than human platelets. Because porcine platelet responses were generally similar to those of humans, it was stated that swine are a useful predictor of in vivo platelet-biomaterial interaction in humans. Leach and Thorburn 11 compared collagen induced thromboxane release from platelets of different species. They found that rabbit, human, canine, and porcine platelets produced significantly greater amounts of TXB2 than ovine platelets. The results of the present study on collagen induced WPA support this observation (Tables 5 and 6). The ADP induced WPA of ovine platelets, however, did not show any difference between canine and bovine platelets, which are more responsive than human platelets (Tables 5 and 6). Furthermore, when compared with CSA, ovine blood did not differ from human blood in either PHT or CT; however, CITF indicates that ovine platelets are more reactive to collagen (Tables 1 and 2). The behavior of platelets seems quite different when studied under flow and static conditions.
In addition to the advantages mentioned above, CSA can measure all parameters within 30 minutes of blood sampling and at the bedside of patients, making this technology attractive in the clinical setting. 12 We are currently evaluating the CSA parameters before and after mechanical heart valve implantation in animal experiments to determine whether these parameters are sensitive enough to detect changes in platelet and coagulation systems that are known to occur with mechanical heart valve implantation. 13
One observation made in this study was the rather high variability of PHT values in all animal species tested. The values ranged from 31 to 617 seconds in 204 humans, from 23 to 167 seconds in 10 dogs, from 95 to 675 seconds in 19 calves, and from 101 to 466 seconds in 7 sheep. Igawa et al.14 also observed a high (49%) CV of PHT, corresponding well with our value of 44.5% in humans (Table 3). They also observed that the sensitivity of PHT for detecting changes in bleeding time with various doses of aspirin was no better than the conventional method. The cause of the high intermeasurement variability of PHT remains unclear; however, it may be attributed to the method by which it is measured. 15 As described in Materials and Methods, this method requires several conditions, such as temperature of the perfused blood, constant perfusion pressure, and accurate reproducibility when making punch holes. Even small variations of any of these conditions might cause a wide variance in PHT. When PHT is used to monitor the coagulation status of patients, care must be taken because PHT may not be sensitive enough to detect activation of the coagulation system and platelet function purportedly induced by cardiovascular implants. 1 Further study is warranted.
Normal platelet counts in these animal species differ significantly (Tables 5 and 6). In both WPA and CSA, the platelet number is not adjusted. This may account for the differences observed in these parameters. There was, however, no correlation found between platelet count and the CSA data (data not shown). It may be argued that the CSA measurement can provide comprehensive information on platelet and coagulation functions under conditions similar to those of the cardiovascular system in vivo. The CSA technology may be a preferred method for characterization of interspecies differences of blood and for evaluation of changes in platelets and coagulation systems following cardiovascular device implantation.
The authors are grateful for the opportunity provided by Xylum Corporation (Scarsdale, NY) and to Drs. Maxim Soloviev and Oleg Melenikov for their participation in this experiment. The technical support of Carol Culler, Larry Oliver, Ron Mallick, Andrew Lee, Kent Wika, and Jaesoon Choi is also appreciated.
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