Differences in platelet aggregometers to study platelet function and coagulation dysregulation in xenotransplantation

Abstract

Xenotransplantation (i.e. cross-species transplantation) using genetically-engineered pig organs could be a limitless source to solve the shortage of organs and tissues worldwide. However, despite prolonged survival in preclinical pig-to-nonhuman primate xenotransplantation trials, interspecies coagulation dysregulation remains to be overcome in order to achieve continuous long-term success. Different platelet aggregometry methods have been previously used to study the coagulation dysregulation with wild-type and genetically-engineered pig cells, including the impact of possible treatment options. Amongst these methods, while thromboelastography and rotational thromboelastometry measures the change in viscoelasticity, optical aggregometry measures the change in opacity. Recently, impedance aggregometry has been used to measure changes in platelet aggregation in electrical conductance, providing more information to our understaning of coagulation dysregulation in xenotransplantation compared to previous methods. The present study reviews the merits and differences of the above-mentioned platelet aggregometers in xenotransplantation research.

1. INTRODUCTION

There is a devastating imbalance between organ donation and organ demand worldwide (1). Xenotransplantation (i.e. cross-spieces transplantation, e.g. pig-to-human) has potential to be an infinite organ source for the people who are in need of transplantation (2). However, some major obtacles need to be overcome before moving xenotransplantation to the routine clinical practice. Interspecies coagulation dysregulation is one of these hurdles which limits efforts for clinical xenotransplantation trials (2). Thrombotic microangiopathy in the graft and consumptive coagulopathy in the recipient are two major phenomena of coagulation dysregulation in xenotransplantation research (3).

Measuring the coagulation parameters pre- and post-transplant, and especially during the xenotransplantation surgeries, as occurs in the clinical setting, would greatly advance the understanding of the pathophysiology in coagulation dysregulation, and help to further develop novel treatment strategies. To our knowledge, using an instrument to measure coagulative properties during the transplantation procedure was not introduced in xenotransplantation research until mid-1990s. Nevertheless, in the past three decades, different platelet aggregometers (PAs) have been employed in xenotransplantation research to better understand coagulation dysregulation (4–7). PAs mainly measure the characteristics of aggregation and thrombolysis by using the variable parameters, such as (i) viscoelasticity, (ii) optical density, and (iii) electrical conductivity of the coagulating samples (8). PAs are widely used in the clinic as a point of care testing of coagulation to monitor the critically ill patients, the haemostatic treatments, post-operative patients, and particularly trauma patients. (9–11)

In the present review, we sought to understand the pitfalls and limitations of four different PAs that were used in xenotransplantation research, including (i) thromboelastography (TEG), (ii) rotational thromboelastometry (ROTEM),(iii) optical aggregometry (OPA), and (iv) impedance aggregometry (IPA). We also briefly compared other methodologiesthat have been used to study coagulation dysregulation in xenotransplantation, such as flow cytometry and enzyme-linked immunosorbent assay (ELISA), which were also used to test platelet function in xenotransplantation research (12–15).

2. PLATELET AGGREGOMETERS IN XENOTRANSPLANTATION

Main adventages and disadvantages of all four PAs are reported in Table 1.

Table 1:

Platelet aggregometers in xenotransplantation.

Method	Advantages	Disadvantages	References
Thromboelastography (TEG)	Inexpensive. Bedside usability. Provides a real-time functional evaluation. High PPV.	Low NPV. Extreme dependence on manual procedures. Non-reliable. Requires fresh whole blood.	4*, 20*, 21*, 45, 46, 47, 48, 49
Rotational thromboelastometry (ROTEM)	Inexpensive. Bedside usability. Provides a real-time functional evaluation. High PPV. Automatic pipetting system. Rapid and precise in post-op monitoring.	Low NPV. Requires fresh whole blood.	5*, 24*, 45, 48, 49, 50*, 51
Optical aggregometry (OPA)	Provides insights about different pathways of platelet aggregation with using different agonists.	Works with PRP only. Collected samples need to be processed. Large volume sample needed. Hemolysis and low platelet count influence the results. Expensive and time consuming.	6*, 8, 27*, 28*, 29*, 30*, 31*, 32*, 33*, 34*, 35*, 36*
Impedance aggregometry (IPA)	Provides insights about different pathways of platelet aggregation with using different agonists. Works with whole blood and PRP. Collected samples can be used directly. Reliable. Can work with low platelet counts.	Electrodes are very sensitive tools and should be cleaned carefully.	7*, 8, 39*, 40*, 41*, 42*

^*=The references that related to xenotransplantation research. PPV = Positive predictive value. NPV = Negative predictive value. PRP = Platelet rich plasma.

2.1. Thromboelastography (TEG)

The main principle of TEG, established by Hartert, is measuring the change in viscoelastic properties of a whole blood sample through the coagulation process (16). It is widely used in a clinical setting, especially in trauma and critical care patients (17–18). Mechanical properties of TEG are presented in Figure 1. As the blood sample clots, the changes in viscoelasticity transmit more movement from the cup to the pin. The opposite occurs during fibrinolysis. These changes in the transmitted rotation are converted into electrical signals via a mechanical-electrical transducer, which is connected with the torsion wire (18). Ultimately, a computer generates a graph and numerical output using these electrical signals (19).

Figure 1: A demonstration of thromboelastography (TEG).

During the test, the cup rotates at a certain speed through an arc of 4°45′, back and forth, and viscoelastic properties of whole blood are measured.

Several groups used TEG to investigate coagulation dysregulation in preclinical xenotransplantation (Table 1). Badet et al. showed the effect of coagulation process on hyperacute rejection of mouse islets incubated with human whole blood (4). In this ex vivo study, islet destruction was correlated with clotting and platelet consumption (4). Chen et al. used TEG to profile and compare the reference values of Guizhou minipigs against those of humans, observing significant differences (20). In a more recent study, Abicht et al. used TEG to investigate the possible benefits of using a complement C3 inhibitor (Cp40) for future clinical xenotransplantation (21). Wild-type (WT) pig hearts were ‘ex-vivo’ perfused with either diluted human blood or diluted human blood with Cp40, and pig blood and thrombus formation was measured by TEG (21). TEG did not show differences among autologous, treated, and untreated groups, but the drug (Cp40) was found to be promising for complement-mediated reaction (21).

2.2. Rotation Thromboelastometry (ROTEM)

ROTEM resembles TEG in terms of working principle and its mechanical system, with two essential differences in the detection system and rotating object (Figure 2). In ROTEM, movement is generated by the pin, not the metal block (22). As the blood sample clots, the increase in viscoelasticity strengthens the connection between the pin and the metal block. Since the metal block is stable, coagulation interferes with the movement of the pin (22). This interference generates a stimulation that is perceived by an optical detector system (23).

Figure 2: A demonstration of rotational thromboelastometry (ROTEM).

During the test, the cup rotates back and forth at a 4°75′ arc. The movement is generated by the pin, not by the metal block. ROTEM also measures viscoelasticity of whole blood, as TEG.

The Padua University Group employed ROTEM to determine the reference values for thromboelastometry in cynomolgus monkeys to compare with that of humans (5). They found that primates have a ROTEM profile that reflects higher coagulability than humans, suggesting more challenges if cynomolgus monkeys were to be used for clinical xenotransplantation (5). They also found that ROTEM works with good precision (5). In another study by the same group, ROTEM was used to evaluate coagulopathy in primate kidney xenotransplant recipients, using three different types of genetically-engineered pigs as donors (24). The authors suggested that ROTEM analyzer could be a useful and convenient tool to assess consumptive coagulopathy in pig-to-monkey kidney xenotransplantation (24).

2.3. Optical Aggregometry (OPA)

OPA, first described by Born for the quantification of platelet aggregation, is the most widely-used PA in xenotransplantation (25). OPA is a turbidimetric method that works best with platelet-rich plasma (PRP) (Figure 3). Light transmission is relatively lower in non-aggregated PRP. Platelet aggregation and clumping thrust aside the part of the sample that is opaquer (platelets), which concomitantly increases the amount of detected light beams by the photocell sensor (26). The amount of perceived light beams is proportional to the amount of aggregation (26). Different type of aggregation stimulants, such as calcium, arachidonic acid, thrombin, thromboxane, ristocetin, adenosine diphosphate (ADP), collagen (COL), von Willebrand Factor (vWF), thrombin receptor activating peptide-6 (TR1AP), and activation of PAR-4 thrombin receptor subtype (TR4AP) can be used to activate platelets and study different pathways of platelet aggregation (3, 7, 8).

Figure 3: A demonstration of optical aggregometry (OPA).

OPA measures platelet aggregation in a platelet-rich plasma via turbidimetric method. Various types of platelet activators can be used to stimutalte platelet aggregation.

Candinas and Lesnikoski, et al. observed prolonged survival in guinea pig-to-rat heart xenotransplantation by using a platelet glycoprotein (GP) IIb/IIIa receptor antagonist (27). Using WT porcine aortic endothelial cells and human PRP prepared with washed platelets, Robson et al. found that the complement system was essential for platelet aggregation and that thrombin generation played a significant role in platelet aggregation (6). In a similar study using porcine peripheral blood mononuclear cells and human PRP prepared with unwashed platelets, Benatuil et al. demonstrated that complement activation was not the cause of platelet aggregation, but was responsible for thrombin generation (28).

Alwayn et al. tested different drugs to show their antiaggregant activity in baboons (29). The combination of heparin and eptifibatide was found to be the most beneficial for inhibiting thrombosis after xenotransplantation (29). In subsequent studies attempting to create a hematopoietic chimera pig-to-baboon model, eptifibatide was repeatedly successful at attenuating platelet aggregation (30, 31).

The combination of aspirin and clopidogrel has been used to inhibit platelet aggregation in heart xenotransplantation from human CD46 transgenic pigs to baboons. OPA results confirmed the antiaggregant effects in the treated group, however no significant impact was detected on the length of xenograft survival (32). Dwyer et al. generated human CD39 transgenic mice, which decreased platelet aggregation without bleeding symptoms. This study demonstrated the importance and possible utility of human CD39 transgene in minimizing xenograft-related thrombosis (33). Chen et al. evaluated the anti-aggregant effect of TMVA (a snake C-type lectin-like protein from Trimeresurus mucrosquamatus venom), a novel GPIb-binding protein, in a guinea pig-to-rat cardiac xenotransplantation model. The drug effectively inhibited platelet microthrombi formation and fibrin deposition and prolonged xenograft survival (34).

In a comparative study, the pig and human platelet GPIbα sequence were compared, revealing 67% similarities (35). In the same study, ristocetin-induced OPA demonstrated much more aggregation with pig PRP than with human PRP, which could be partly attributed to species-specific structural aspects of von Willebrand Factor (vWF) (35). Peng et al. studied the inducibility of platelet aggregation in baboons by Gal-positive and Gal-negative (α1,3-galactosyltransferase gene knockout, GTKO) pig endothelial cells, and found that platelet aggregation was preventable using anti-aMb2 integrin antibody, eptifibatide, or aurintricarboxylic acid (36).

2.4. Impedance Aggregometry (IMA)

Although some of the basic principles of IMA are derived from OPA, the main working principle, developed by Cardinal and Flower, is completely different (37). In IMA, as the platelets aggregate onto electrodes, the electrical conductance between the electrodes falls (Figure 4). The characteristics of this fall (timing, speed, amplitude) determine the coagulative properties of the sample (38). The platelet aggregation stimulants for OPA (calcium, arachidonic acid, thrombin, thromboxane, ristocetin, ADP, COL, vWF, TR1AP, and TR4AP) can be used for IMA as well.

Figure 4: A demonstration of impedance aggregometry (IMA).

In IMA, platelets aggregate onto electrodes and electrical conductance between electrodes diminishes. A stir bar stirs to activate paltelets. Various platelet activators can be used to study different pathways in platelet aggregation.

A study by Iwase et al. compared platelet aggregation in whole blood from three primate species, such as humans, baboons, and cynomolgus monkeys (39). In the study, Iwase et al demonstrated that the IMA is a reliable method to assess in vitro platelet activation and aggregation of primates (39). Subsequently, the same gorup compared human whole blood aggregation profiles when co-incubated with aortic endothelial cells from humans, WT pigs, and different genetically-engineered pigs to asses the impact of genetic modifications on platelet aggregation and coagulation (40). The study was particularly important since it studied, for the first time, the impact of different human genes (coagulation genes [thrombomodulin, endothelial cell protein c receptor] and complement inhibitory genes [CD46, CD55]) expressed on porcine cells, whether they were effective to ameliorate platelet aggregation in pig-to-human xenotransplantation model. In fact, the study by Iwase et al showed less aggregation with genetically-engineered pig cells than with WT pig cells. It also detailed human platelet aggregation response to up to 3-gene genetically-engineered pigs (40).

Recently, Ponschab et al. used IMA to evaluate the aggregation response to different platelet activators (ADP, COL, TR1AP, TR4AP) in humans and baboons (7). Their study showed an overall reduced aggregation response with all aggregation activators in baboons except TR1AP, which was found to be ineffective for baboons (7). In fact, Li et al. showed a concordance between extracellular histone levels and consumptive coagulopathy or infection in baboons that received pig xenografts, showing that interspecies coagulation dysregulation is an important barrier to overcome (41).

More recently, our group employed IMA to test human platelet aggregation profiles to genetically-engineered pig cells (42). The study was the most extensive analysis to date showing many important findings. (i) It detailed the human platelet coagulation response to 20 different types of porcine endothelial cells, including genetically-engineered pig cells with up to 9-gene modifications (most complex and advanced genetic modification reported in the literature). (ii) Using porcine aortic endothelial cell, lung microvascular endothelial cell, and liver sinusoidal endothelial cells from the same pig donor, we demonstrated that the coagulation response is an organ-specific response (42). Our findings extend the recent evidence of tissue-specific endothelial cells displaying distinct organ-specific barrier properties, angiogenic potential, metabolic rate, and support of organ functionality with organ-specific coagulation profiles (43, 44). (iii) Finally, we showed that newly produced genetically-engineered pigs were associated with very low platelet aggregation, consistent across different human blood donors, extending implications of selecting a pig genotype for early xenotransplantation clinical trials (42).

3. OTHER METHODS

Other than above-mentoned PAs, ELISA and flow cytometry-based methods were also employed in xenotransplantation to detect platelet aggregation and activation. In ELISA based methods, beta-thromboglobulin (platelet activation), prothrombin fragments 1+2 (coagulation activation), thromboxane B2 (platelet activation and aggregation), vWF (coagulation activation) and platelet activating factor (platelet activation) were used as a marker in different studies (12, 13, 52–56).

In flow cytometry-based methods, antibodies such as anti-human CD41 (GPIIb/IIIa, α subunit), anti-human CD62P (P-selectin), anti-human CD42a (GP IX), and anti-human PAC-1 (GPIIb/IIIa, α_IIbβ₃) were used to detect platelet activation and aggregation (14, 15, 54, 57, 58). While storability of samples is a great advantage of ELISA and flow cytometry methods, the time spent, dependence of quality of the kit and manual procedures (operator-dependence) are disadvantages.

Other methods which were not previously used but could serve as alternative methods in xenotransplantation research are platelet function analyzers (PFA-100, PFA-200), VerifyNow, Plateletworks, and IMPACT-R. PFAs measure the cessation of whole blood flow in a capillary-like tube that is caused by aggregates (59). High-dependence on vWF is the most important disadvantage (59). VerifyNow, previously known as rapid platelet function analyzer or Ultegra, measures the change of optical density in a whole blood flowing capillary-like tube containing fibrinogen coated beads (60). The main usage of VerifyNow is monitoring the antiplatelet therapies (60). Plateletworks system detects the difference in platelet numbers in between an EDTA tube and citrate tube both filled with whole blood (61). Although its simplicity and speed could work for the benefit of bedside using, it doesn’t give any specific information about aggregation process. The IMPACT-R device constitutes a cone plate forming a shear stress and whole blood that mimics the arterial flow on the plate (62). Platelets adhere and aggregate on the surface of the plate and an image analyser quantifies the amount of adherence and expresses the results as percentage (62).

4. CONCLUSIONS AND FUTURE DIRECTIONS

With current advances in genetic engineering of pigs (63), the prolonged survival has now been achieved in preclinical xenotransplantation trials (64, 65). However, interspecies coagulation dysregulation (e.g. increased platelet aggregation) continues to be one of the major barriers for long-term success, which is necessary to overcome before the initiation of clinical trials. PAs are useful tools for monitoring platelet aggregation and coagulation dysregulation in xenotransplantation. More recently, IMA showed better potential to further study platelet aggreagation using different chemical stimulants to delinate the coagulation pathway responses. We believe that IMA could work best for in vitro studies in xenotransplantation research given its less dependence on manual procedures over OPA. However, for the in vivo studies (i.e. clinical xenotransplantation trials), ROTEM or TEG might be preferable due to their broad usage in the clinical setting.

Future studies are required to understand the impact of specific gene modifications (insertion of human gene(s) or deletion of pig gene(s), or in combination) on spefic cogulation pathways. Better understanding of pros and cons of different PAs will immensely improve our therapeutic options to overcome interspecies coagulation dysregulation and therefore success in clinical xenotransplantation.

ABBREVIATIONS

ADP

Adenosine diphosphate

COL

Collagen

ELISA

Enzyme-linked immunosorbent assay

GP

Glycoprotein

GTKO

α−1,3-galactosyl transferase gene-knockout

IMA

Impedance aggregometry

OPA

Optical aggregometry

PAs

Platelet aggregometers

PFAs

Platelet function analyzers

PRP

Platelet rich plasma

ROTEM

Rotational Thromboelastometry

TEG

Thromboelastography

TR1AP

Thrombin receptor activating peptide-6

TR4AP

Thrombin-receptor-4-activating-peptide

vWF

von Willebrand factor

α-Gal

Galactose-α1,3-galactose