Extracellular creatine kinase may modulate purinergic signalling

Abstract

Extracellular purine nucleotides and nucleosides including ADP and ATP regulate a wide array of physiological processes including platelet aggregation, vasomotor responses and inflammation through specific purinergic receptors. In the recent years, a strong association has been reported between circulating cytoplasmic-type creatine kinase and adverse clinical outcomes such as major bleeding, hypertension and obesity. Therefore, it is proposed that extracellular CK may modulate purinergic signalling through its ADP binding and/or ATP-generating effect.

Introduction

Extracellular nucleotides and nucleosides originating through lytic and non-lytic processes from blood cells, the vessel wall, nerves and tissues may act as short-term or long-term signalling molecules via specific purinergic receptors, to regulate a wide array of physiological functions including neurotransmission, platelet aggregation, vasomotor responses, inflammation and trophic responses. The large body of evidence on this signalling system has been exquisitely reviewed by several authors [1–6]. In brief, this highly conserved signalling system governs a functional step-wise interaction between extracellular purines and an interconnected network of ectoenzymes that may generate as well as degrade purine and pyrimidine nucleotides and nucleosides, to provide tight control over the duration and the magnitude of the responses. Specific ligand-gated ionotropic purine P2X receptors (subtypes P2X1–7) are activated by ATP, while G protein coupled metabotropic P2Y receptors have main ligand preferences in the purine molecules ADP (P2Y₁); UTP, ATP (P2Y₂); UTP (P2Y₄); UDP (P2Y₆); ATP, NAD⁺ (P2Y₁₁); ADP (P2Y₁₂ and P2Y₁₃) and UDP (P2Y₁₄). Finally, the G protein-coupled receptor P1, with subtypes A₁, A_2A, A_2B and A₃, binds adenosine [1–6] and potentially AMP (subtype A₁) [7]. The interconversion of these purinergic agonists is catalysed by different enzyme families, ecto-nucleoside triphosphate diphosphohydrolases that hydrolyse nucleoside tri- and diphosphates into nucleoside monophosphates, ecto-5′-nucleotidase that converts AMP to adenosine, ecto-adenosine deaminase that converts adenosine into inosine, and nucleotidases that hydrolyse other substrates aside nucleoside tri-, di-, and monophosphates. The ectonucleotide pyrophosphatase/phosphodiesterase family also hydrolyses dinucleoside polyphosphates, ADP ribose and other substrates, but not AMP, while alkaline phosphatases also degrade other phosphate monoesters [1–5]. More recently, ATP-generating enzymes appeared to be relevant for extracellular purine signalling, including Ecto-F1FoATP synthase/F1 ATPase, ecto-adenylate kinase and ectonucleoside diphosphokinase (Table 1) [1, 5, 6]. In this paper, it is proposed that extracellular cytoplasmic-type creatine kinase (CK, EC, 2.7.3.2) is a fourth main ATP-generating enzyme associated with purinergic signalling.

Table 1.

Purines with main receptors and ectoenzyme interconversion pathways

Purine	Receptor	Ectoenzyme1–8‡	EC	Interconversion║
ATP (NTP) and ADP (NDP)	P2X1–7 P2Y2 P2Y11 (ATP) P2Y1 P2Y12 P2Y13 (ADP)	Ecto-nucleoside triphosphate diphosphohydrolases	3.6.1.5	ATP= > ADP= > AMP
		Ecto-nucleotide pyrophosphatase/phosphodiesterases	3.1.4.1	ATP= > ADP= > AMP
		Ecto-alkaline phosphatases	3.1.3.1	ATP= > ADP= > AMP
		Ecto-F1FoATP synthase/F1 ATPase	7.1.2.2	ADP < =>ATP
		Ecto-adenylate kinase	2.7.4.3	2ADP < =>ATP + AMP
		Ecto-nucleoside diphosphokinase	2.7.4.6	NTP + NDP < =>NDP + NTP
		(Creatine kinase)§	2.7.3.2	ADP < =>ATP¶
AMP (NMP)	P1*	Ecto-5′-nucleotidase	3.1.3.5	AMP= > adenosine
Adenosine	P1†	Ecto-adenosine deaminase	3.5.4.4	Adenosine= > inosine

EC International Union of Biochemistry and Molecular Biology’s Enzyme Commission numbering system

*A₁, Rittiner et al. [7]

†A₁, A_2A, A_2B and A₃

‡Several enzymes also broadly acting on non-adenine (N) purine and pyrimidine compounds and polyphosphates, as reviewed in detail by Burnstock, Zimmermann, Yegutkin, and others [1–8]

§Proposed in this paper.

║Only purine molecule reactants are depicted here.

¶Extracellular cytoplasmic-type CK generates ATP

Creatine kinase

CK is part of a highly conserved family of phosphagen guanidino kinases. These enzymes catalyse the reversible transfer of the gamma-phosphoryl group of ATP to a guanidino compound, a reaction that is central to cellular energy metabolism in vertebrates and invertebrates [8–12]. This enzyme system is thought to have evolved very early in metazoan evolution hundreds of millions of years ago, to overcome spatial hindrances in intracellular ATP transport [12]. CK genes are found in Porifera, the most simple members in the ancestral lineage of animals, and although other phosphagen guanidino kinases developed in Metazoa such as arginine kinase, taurocyamine kinase, lombricine kinase and glycocyamine kinase, vertebrates exclusively use creatine kinase [8–12]. The enzyme catalyses the reaction:

$$\mathbf{\text{MgADP}}+\mathbf{\text{Phosphocreatine}}+{\mathbf{H}}^{+}<=>\mathbf{\text{MgATP}}+\mathbf{\text{Creatine}}$$

CK is ubiquitously present in cytoplasm and mitochondria, with high activity levels found in tissues with high and fluctuating energy demands [8–12]. The cytoplasmic enzyme ranks in the top 3 of protein abundance of over 10.000 proteins detected in striated muscle tissue [13]. The CK enzyme system is notably absent in the hepatocyte [14], probably related to hepatic creatine co-synthesis and its release to the circulation.

The CK system is highly compartmentalized, with specific isoenzymes in cytoplasmic and mitochondrial subcellular locations. CK is either homo or heterodimeric in cytoplasmic CK, or homooctameric (tetrahomodimeric) in mitochondrial CK, with a monomer MW of around 43 kDa [15, 16]. Cytosolic CK protein subunits are CK-muscle (M), found mainly in striated muscle and encoded by the CKM gene on human chromosome 19q13.32; and CK-brain (B) found in different tissue types, encoded by the CKB gene on human chromosome 14q32. Mitochondrial CK is the sarcomeric isoenzyme in striated muscle, and the ubiquitous isoenzyme in other tissue encoded for by respectively the nuclear CKMT2 gene on chromosome 5q13.3 and the CKMT1 gene on chromosome 15q15. All CK isoenzymes contain a highly conserved catalytic cysteine domain. Furthermore, cytoplasmic CK-B and CK-M cDNA share a 78% nucleotide sequence identity and 79% predicted amino acid sequence identities, while the human CKMT1 and CKMT2 cDNA share a 73% nucleotide and 80% predicted amino acid sequence identities, and < 66% identity with the cytoplasmic CK, as summarized recently [17]. The main difference between cytoplasmic and mitochondrial CK is that the latter enzyme, located in the peripheral mitochondrial intramembrane space and the intracristae space, functions to catalyse the formation of phosphocreatine from ATP, which is generated by F1FoATP synthase in the mitochondrial matrix and transported to the intermembrane space by adenine nucleotide translocase. Phosphocreatine is delivered to the cytosol via the outer membrane voltage-dependent anion channel [9, 10] The cytoplasmic CK reaction favours ADP binding and ATP production from phosphocreatine [8–10, 15, 16]. A significant fraction of cytoplasmic CK is soluble. However, at subcellular locations of ATP utilization, CK is tightly bound in the immediate proximity of ATPases, including plasma membrane Na⁺/K⁺-ATPase, sarco/endoplasmic reticulum membrane Ca²⁺-ATPase and myosin ATPase at the M-line of myofibrils. Here, CK kinetically favours the ATPase reaction through rapid regeneration of ATP in situ from phosphocreatine and ADP, thereby maintaining relatively high [ATP]/[ADP] ratios near these ATPases. The Km of the reaction is around 500 μM for phosphocreatine and 1 mM for creatine [16]. Creatine and phosphocreatine are smaller molecules than ADP or ATP, with no or a small negative charge, less binding to proteins, and therefore greater diffusivity in the intact cell. In a functional coupling to ATP and ADP metabolism, energy in the form of phosphocreatine is thought to be “shuttled” between mitochondrial CK at ATP producing sites and cytoplasmic CK bound at energy-consuming sites. Rapidly available energy is “stored” as cytosolic phosphocreatine near ATPases in greater quantities than ATP, and the CK system has a greater and faster ATP regenerating capacity than glycolysis and oxidative phosphorylation together. Thus, the intracellular CK system functions as a temporal and spatial energy buffer. Other metabolic functions of this central regulatory enzyme of energy metabolism include proton buffering and the indirect regulation of glycogenolysis, glycolysis, insulin resistance and mitochondrial activity [8–18].

Intracellular CK and cardiovascular disease

The ATP-regenerating cytoplasmic CK system is considered to be the final common effector of intracellular pathways that promote vascular and cardiac contractility, sodium retention and hypertension [8, 19–23] Through binding of ADP and rapid conversion into ATP, cytoplasmic CK is thought to enhance the metabolic support and function of intracellular ATPases involved in pressor responses, with high CK activity leading to a greater flux through the CK reaction, greater sodium retention in the kidney (Na⁺/K⁺-ATPase), enhanced vascular contractility (Ca²⁺- and myosin ATPase) and higher blood pressure levels [8, 19–23]. Intracellular CK has shown to be constitutively high in animal models of hypertension, as well as in different tissues of hypertension-prone subgroups of the population, including in resistance arteries, where it is strongly associated with blood pressure [8, 17]. In addition, relatively high sarcoplasmic CK activity and predominance of fast muscle fibres are associated with greater weight gain upon overeating, ascribed to the metabolic effects of CK, which promotes insulin resistance and obesity [8, 24, 25]. Importantly, reduction of the flux through the cytoplasmic CK reaction by the specific competitive creatine transporter and cytoplasmic CK inhibitor beta-guanidinopropionic acid (currently in Phase 0 tolerance studies in humans) [26] reduces blood pressure and body weight, supporting the notion that CK is involved in hypertension, obesity and cardiovascular disease [8, 27]

Extracellular CK

Although the intracellular function of CK is well studied, there is a paucity of data on the possible functionality of extracellular (including pericellular and circulating) CK. Merely seen as a diagnostic enzyme for decades, plasma estimations of cytoplasma-derived CK are increasingly considered obsolete in different fields of medicine [28–30]. However, it is hereby proposed that extracellular CK is relevant, bioactive and may modulate purinergic signalling.

In healthy persons, cytoplasmic CK is released to the peri- and extracellular space and reaches the circulation through the lymphatic system [8, 22, 31]. Currently, available evidence indicates that extracellular CK is predominantly soluble cytoplasmic CK MM and MB [8, 19, 22]. CK BB is also released, but has a relatively short T_1/2 compared with the other two cytoplasmic isoenzymes, and this contributes to its relatively low constitutive plasma levels in the absence of disease [8, 19]. Mitochondrial CK expression and activity is modest compared with cytoplasmic CK and rarely occurs in plasma [8, 17, 19]. The mechanism by which CK is released from cells is unclear, but in physiological circumstances at rest, the release is proportional to the intracellular CK concentration [8, 22, 31]. Under resting conditions, relatively high plasma CK activities are found in men, in obese persons and in persons of African ancestry, population subgroups with high intracellular CK activity [8, 20, 22–25, 32, 33]. In addition, plasma CK increases during physical exercise, mainly due to enhanced lymphatic flow [8, 20, 22, 31]. After moderate physical exercise, plasma CK is typically increased to several hundred IU/L during a few days [8]. During eccentric exercise (where the muscle contracts and lengthens at the same time, such as stepping exercise), skeletal muscle tissue is damaged, and plasma CK may be highly elevated, up to 10.000 IU/L or more, during up to 10 days [8, 34]. The most dramatic cellular efflux of CK, ADP, ATP and phosphocreatine occurs after frank damage to high CK tissues such as in myocardial infarction or rhabdomyolysis, where CK levels may exceed 50.000 IU/L [8, 29]. It is proposed that through its catalytic and ADP scavenging action, extracellular CK may increase extracellular [ATP]/[ADP] ratios, affecting ATP and ADP-dependent processes in the peri- and extracellular space and in the circulation.

Extracellular CK reduces platelet aggregation

The important role of the ADP-binding enzyme CK in purinergic signalling is evident in the inhibition of platelet aggregation. ADP is central to platelet activation and aggregation [1–3, 29, 35–38]. Activation of the P2Y₁ receptor leads to phospholipase C activation and transient platelet aggregation, while P2Y₁₂ receptor activation results in glycoprotein receptor IIb/IIIa (integrin (I)-αIIbβ3) activation and firm platelet aggregation [1–3, 29, 35–38]. Furthermore, through its action on P2Y₁₂ receptors, ADP is thought to be an essential co-stimulus for all known platelet-agonists [37].

High concentrations CK had been used in vitro to reduce ADP and platelet aggregation [8]. CK is thought to reduce ADP-dependent platelet activation and aggregation through ADP scavenging and binding action and/or to the conversion of ADP into ATP, in the presence of phosphocreatine. In line with this, the experimental use of a supra-physiological dose of external phosphocreatine injected intravenously was associated with reduced platelet aggregation [38]. However, catalytic activity of peri-, extracellular or circulating CK was not considered to be clinically relevant until recently, when dedicated studies indicated that even within the physiological range of activity, circulating CK may attenuate platelet aggregation [8].

Experiments in human volunteers

Human exogenous creatine kinase in the range observed at rest and after exercise (500 to 4000 IU/L, phosphocreatine 5 mM) was added to plasma obtained from 7 healthy women of European ancestry, (mean age 23.1 years, SE 0.8), with relatively low endogenous plasma CK activity (45 to 128 IU). This induced a dose-dependent reduction to complete inhibition of ADP-induced platelet aggregation at CK 4000 IU/L [8]. In addition, in 9 healthy men of European and African ancestry (mean age 27.9 years, SE 3.3), endogenous plasma creatine kinase (115 to 859 IU/L, median 358) was inversely associated with ADP-induced platelet aggregation (Spearman’s rank correlation coefficient, − 0.6; p < 0.05). In one volunteer with an endogenous creatine kinase level of 4664 IU/L after exercise, ADP-induced platelet aggregation was completely abolished. The volunteer had no symptoms or signs of rhabdomyolysis. Plasma CK activity reduced to 258 IU/L after 7 days of rest, with a concomitant normalization of ADP-induced platelet aggregation. Platelet count was within the normal range in all volunteers (151 to 257 × 10⁹/L, median 203), and there was no abnormality at physical or laboratory examinations and no intrinsic platelet disorders; activated partial thromboplastin time and prothrombin time were within the normal range, with a mean duration of respectively 27.0 (SE 0.5) and 11.3 (SE 0.2) seconds [8]. To address the clinical relevance of these pilot findings, the association between CK and bleeding was further studied in patients with acute coronary syndromes (ACS), who generally have highly elevated extracellular CK activity [29, 35].

Extracellular CK is associated with major bleeding after acute coronary syndromes

An acute reduction in platelet aggregability had been reported after ST-elevation myocardial infarction (STEMI) in earlier studies. However, the cause of this was not clear [39]. The observation has recently been linked to the large quantities of CK, ATP and phosphocreatine that enter the peri- and extracellular space and the circulation after acute myocardial necrosis [29, 35, 40]. It was proposed that CK acts as a “tissue factor” that inhibits platelet activation through ADP scavenging action or conversion into ATP [29, 35]. This might serve to reduce excessive thrombus formation, sustain the microcirculation and reduce further (ischaemic) tissue damage [29, 35]. Other ectonucleotidases may also degrade ADP to AMP, or convert it into ATP (Table 1), but CK is one of the most abundant cellular proteins, far in excess of AK in most tissues [8–10], with massive amounts of CK occurring in the peri- and extracellular space as well as in the circulation after tissue damage, in particular of muscle and brain tissue. Extracellular CK might reduce ADP-dependent platelet-aggregation, in particular with the concomitant use of antithrombotic or thrombolytic drugs, as is common after myocardial infarction [29, 35]. The potential association between extracellular CK and bleeding was studied in post-hoc analyses of two large multi-centre randomized controlled clinical trials in patients with ACS [29, 35].

In the Thrombolysis in Myocardial Infarction Study Group phase II trial that addressed the efficacy of angioplasty following intravenous recombinant tissue-type plasminogen activator (rt-PA), 3339 patients with STEMI were studied (82% men, 88% of European ancestry, mean age 57 years, SE 0.2). Peak plasma CK levels, ranging between 16 and 55,890 IU/L (mean 2389 IU/L; SE 41), were reached within 8 h in 51% of the patients (93% within 24 h). Thereafter, with a peak at day 2, adjudicated fatal/non-fatal bleeding occurred in 30% of the patients (respectively 26% in the low vs 34% in the high CK tertile) and bleeding/all-cause mortality in 35% (29% in the low, vs 40% in the high CK tertile).

Multivariable binary logistic regression analysis suggested an independent association between CK and bleeding outcomes, with a 2 to 4-fold increase in odds for bleeding (and ACM) compared with non-bleeding (and survival) per log CK increase, when holding the other clinical and laboratory variables constant. The C-index of the regression models including CK, adjusted for age, sex and treatment variables was 0.74 (95% CI, 0.70 to 0.78) for major fatal or non-fatal bleeding; and 0.70 (0.67 to 0.74) for bleeding or all-cause mortality [29].

In a second study, the Thrombolysis in Myocardial Ischemia 3B trial, rt-PA (35 to 80 mg) vs placebo and early catheterization vs conservative management were compared in patients with non-ST segment elevation acute coronary syndromes (NSTE-ACS). This analysis in 1473 patients (66% men, 80% of European ancestry, mean age 59 years, SE 0.3) also indicated the presence of an independent association and temporal relationship between peak CK and bleeding. Peak CK was lower than in STEMI patients, as expected in NSTE-ACS, ranging between 15 and 19,045 IU/L. With elaborate preventive measures against bleeding in this trial, including drastic dose reduction of thrombolytic and antithrombotic drugs compared with the phase II trial, and rigorous exclusion of high risk-patients, major bleeding occurred in 2.0% of the patients (mean age 65 years, SE 1.3).

However, CK remained a main predictor of bleeding. Mean CK was 1015 (SE 319) IU/L (or 6 times the upper reference limit, URL) in patients with major bleeding vs 439 (SE 23) IU/L (2*URL) in patients without major bleeding (with URLs varying among participating study sites). Multivariable binary logistic regression analysis suggested an independent association between peak CK and major bleeding (and death), with per log CK increase a 3- to 4-fold increase in odds for non-fatal or fatal major bleeding or hospital death, compared with non-bleeding and survival. The C-index of the regression model for major bleeding including CK was 0.80 [35].

The data from these trials suggest that proportionate to its plasma activity, the ADP-binding enzyme CK is strongly and independently associated with non-fatal and fatal major bleeding during treatment for acute coronary syndromes [29, 35]. The association between ADP-dependent platelet aggregation and CK was not directly assessed in these studies, but the observations are biologically plausible, given the highly elevated CK activity after onset of the ACS, the strong ADP-binding capacity of this enzyme, the attenuated platelet aggregation observed after acute myocardial infarction and the CK-dependent reduction in ADP-dependent platelet aggregation [8, 29, 35, 38, 39].

Extracellular CK and blood pressure

CK is associated with blood pressure in populations across the world [20–22]. The association was first reported in a random sample of the population of Amsterdam, the Netherlands, consisting of 1444 citizens (503 of European, 292 of South Asian, 580 of African and 69 of other ancestry) aged 34 to 60 years, with an increase in systolic and diastolic pressure of respectively 8.0 (95% CI, 3.3 to 12.7) and 4.7 (95% CI, 1.9 to 7.5) mm Hg per log resting plasma CK increase after adjustment for age, sex, body mass index and ethnicity [22]. Thereafter, the association of CK with blood pressure was found across different, mainly smaller studies worldwide [21], and recently in a large population sample of the multi-ethnic, cross-sectional Healthy Life in an Urban Setting (HELIUS) study (n = 14,937, mean age 43.3, SD 12.9, mean resting plasma CK (SD) 129.0 (74.4). In univariable regression analysis, blood pressure increase per log plasma CK increase was 20.2 (95% CI, 18.9 to 21.4) mm Hg for SBP and 13.0 (12.2 to 13.7) for DBP, and this was similar across subgroups by age, sex, BMI strata, European, Asian, West-African and Mediterranean ancestry [20]. The isoenzyme distribution of the enzyme showed no abnormality with high blood pressure, which precludes myocardial damage [19, 20, 22]. Furthermore, resting plasma CK was associated with obesity [20, 24], with an increase in waist circumference of 9 cm (95% CI, 5 to 12) cm and in body mass index of 4 (95% CI, 2 to 5) kg/m² per log CK increase, after adjustment for age, sex, ethnicity, educational level, physical activity and plasma creatinine [24].

The association of circulating CK with blood pressure and obesity has hitherto been ascribed to the intracellular effect of constitutive or induced CK, metabolically supporting Ca²⁺ATPase, myosin ATPase and Na⁺/K⁺-ATPase [8]. This is thought to result in greater cardiovascular contractility, peripheral resistance and renal sodium retention, while in skeletal muscle, a more glycolytic and insulin-resistant phenotype is induced [8, 20, 22, 24, 25, 27]. In addition, the existing evidence indicates that enhanced creatine synthesis with high activity of the CK system attenuates the bioavailability of L-Arginine for NO synthesis, as creatine and nitric oxide are both synthesized from this semi-essential amino acid [8, 20, 22]. In line with this, high vascular CK activity is associated with reduced NO-dependent vasodilation, and this might contribute to the higher blood pressure levels and NO-dependent dysfunction observed with high CK [8, 20, 22].

The high levels of circulating CK with these conditions were hitherto viewed upon as an epiphenomenon, a surrogate measure of high intracellular CK activity [8, 20–22]. However, it is apparent from the effect of CK on platelet aggregation that cytoplasmic-type extracellular CK might increase the free (unbound) extracellular [ATP]/[ADP] ratio through its ADP binding action and/or its potential to generate ATP [8, 29, 35, 38]. Extracellular ATP is intimately involved in regulation of the vasomotor responses, hypertension, inflammation and lipid metabolism, among other functions [1–6]. It is well known that erythrocytes undergoing shear stress release ATP, leading to endothelium-dependent vasodilation [1–3]. On the other hand, sympathetic nerve stimulation and purinergic signalling with extracellular ATP involving vascular smooth muscle may lead to vasoconstriction, in particular under conditions of endothelial dysfunction [1–6]. ATP in the pericellular space may occur after cell damage or non-lytic pathways including synthesis by ATP-generating ecto-enzymes such as ecto-F1FoATP synthase/F1 ATPase, ecto-adenylate kinase and ecto-nucleoside diphosphokinase [5, 6, 41]. Pannexin-1 hemichannels are thought to contribute to purinergic signalling through the release of small signalling molecules including ATP to the extracellular space that are implied in vasomotor function, endothelial proliferation, angiogenesis, lipid metabolism, atherosclerosis and obesity [6, 41–46]. Furthermore, extracellular ATP is thought to serve as a “danger signal” to the immune system, leading to P2 receptor-mediated purinergic signalling and the initiation of inflammation [1–3, 5, 6, 44–46]. ATP binding to the P2X7 receptor leads to activation of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) and the formation of the “NLRP3 inflammasome”, a cytosolic multiprotein complex assembled in response to danger or damage associated molecules and other forms of cellular stress. The inflammasome activates caspase-1, which cleaves the inactive precursor forms of cytokines including IL-1β into their pro-inflammatory active forms that initiate, regulate and maintain inflammation. Inflammasomes may also induce pyroptosis and are implicated in a wide range of renal, cardiovascular, neurological and psychiatric diseases, as well as in the low grade inflammation in hypertension, obesity, metabolic syndrome, atherosclerosis and type 2 diabetes [42, 47]. Therefore, through extracellular ATP-generation, CK may contribute to these processes, but currently there are no data beyond the strong association of circulating CK with relevant clinical outcomes to substantiate this. The presented findings may stimulate further studies on the potential contribution of this highly conserved enzyme system to extracellular purinergic signalling in the generation of blood pressure and other cardiometabolic conditions.

Summary

Emerging evidence indicates that extracellular CK is relevant for the extracellular purinergic signalling system that regulates a wide range of functions including vasomotor activity, platelet aggregation and inflammation, using ATP, ADP and other purines as signalling molecules. The abundantly present cytoplasmic-type CK enzyme binds ADP and promotes ATP regeneration from phosphocreatine. Through a hitherto unknown mechanism, cytoplasmic dimeric CK is proportionally released to the extracellular space in healthy persons, with major efflux occurring after tissue damage. Plasma CK is strongly associated with hypertension, obesity and a reduction in platelet aggregation. Furthermore, in two large trials, plasma CK was independently associated with a 2 to 4-fold increase in odds of major bleeding during treatment for acute coronary syndromes, compared with non-bleeding. Therefore, it is postulated that extracellular CK through its ADP scavenging action and/or its catalytic activity in ATP generation may affect extracellular [ATP]/[ADP] ratios and modulate purinergic signalling (Fig. 1). The strong association between circulating CK and relevant clinical parameters warrant further studies to address the extracellular effects of this highly conserved enzyme, and explore the potential therapeutic effects of CK inhibition on perturbations in extracellular purine signalling and clinical outcomes.

Fig. 1.

Highly schematic overview of the proposed role of extracellular creatine kinase (CK) in purinergic signalling. CK is proportionally released to the extracellular space physiologically and after tissue damage together with ATP, ADP and CrP [8]. ATP may also be released in response to stressors through pannexin hemichannels (PNX), while ecto-F1FoATP synthase/F1 ATPase (ATP synthase), ecto-adenylate kinase (AK) and ecto-nucleoside diphosphokinase (NDPK) may synthesize extracellular ATP [5, 6, 41]. ATP may induce vasodilation in response to shear stress or hypoxia, but also promote vasoconstriction, smooth muscle proliferation and inflammation [1–3]. In addition, extracellular ATP is a potential “danger signal”, implied in lipid metabolism, atherosclerosis and obesity [5, 6, 41–47]. Extracellular ATP is rapidly degraded by ectonucluotidases to AMP and adenosine (ADO), which inhibits inflammatory responses, vasoconstriction and smooth muscle proliferation [1–6]. ADP is central to platelet activation. Extracellular cytoplasmic-type CK is proposed to increase extracellular [ATP]/[ADP] ratios through ADP scavenging and/or ATP synthesis (generating creatine, not depicted). The enzyme may thus reduce ADP-dependent platelet aggregation, promote bleeding, and affect vascular tone, lipid metabolism, and inflammation. P2X, P2Y, P1, purinergic receptors. Red T line, inhibition; black dashed line, emerging evidence on AMP as P1 receptor agonist [7]

Compliance with ethical standards

Conflict of interest

LMB is an inventor on patent WO/2012/138226, an “open” non-restrictive patent request filed and published as “prior art” to protect the freedom of researchers to operate and share their innovative ideas on CK and CK inhibition without licence or payment.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.