Association of polymorphisms of platelet membrane integrins αIIbβ3 (HPA-1b/PlA2) and α2β1 (α2807TT) with premature myocardial infarction

Conflicting results of an association of the human platelet antigen 1b (HPA-1b/PlA2), localized on the beta-subunit of the integrin alpha(IIb)beta3, and the alpha(2)807TT genotype of the integrin alpha2beta1 with coronary atherosclerosis and myocardial infarction have been reported. Both platelet receptor polymorphisms were genotyped in 3261 patients who had undergone coronary angiography, including 1175 survivors of a myocardial infarction, 1211 individuals with coronary artery disease but no history of myocardial infarction, and 571 control patients without angiographic coronary artery disease, and in 793 blood donors. In a case-control design, the prevalence of HPA-1b and alpha(2)807TT genotypes did not differ significantly between the patient groups with coronary artery disease or myocardial infarction and patient controls or blood donors. By contrast, using a multivariate case-only design, it was found that the median age of onset of myocardial infarction was 5.2 years earlier (P = 0.006) in carriers of the HPA-1b allele and 6.3 years earlier (P = 0.006) in carriers of the alpha(2)807TT genotype in the 264 survivors of myocardial infarction of recent onset with one- or two-vessel coronary artery disease. A significant interaction with the conventional risk factors hypercholesterolemia, smoking, diabetes, hypertension, and hyperfibrinogenemia was excluded. Human platelet antigen 1b and alpha(2)807TT are associated with premature myocardial infarction but not with coronary artery disease, suggesting a role of distinct integrin genotypes for increased platelet thrombogenicity. This association requires confirmation in follow-up studies.     

Differential activation of β1-, β2- and β3-adrenoceptors by catecholamines in white and brown adipocytes

Summary— This review summarizes the experiments performed by various groups to determine how the activation of the different β-adrenoceptors (β-ARs) is ordinated when they are present in the same fat cell and involved in the same biological event. When expressed after the transfection of their genes in Chinese hamster ovary cell (CHO cells), β₁- and β₂-ARs present a higher affinity for catecholamines than β₃-ARs. In vitro, the lipolytic effect induced by low concentrations of catecholamines in dog and rat white fat cells is due to the selective activation of β₁- and/or β₂-ARs. Higher concentrations only are able to activate β₃-ARs. Similar results have been obtained in rat brown adipocytes. On the other hand, the lipolytic effect of catecholamines in human and primate adipocytes does not involve a β₃-AR component whatever the concentration used. In vivo experiments in the dog have also shown that lipomobilization induced by low doses of isoprenaline only involved β₁- and β₂-AR activation, this effect being blocked by β₁-/ β₂-antagonist pretreatment. However, in the same blockade conditions, perfusion of a 10-fold higher dose of isoprenaline revealed a β₃-AR contribution in the lipomobilizing effect. These data showed that brown and white adipocyte β₃-ARs possess a lower affinity for catecholamines than β₁- and β₂-ARs and are only recruited by high concentrations of the amines. Thus, even if the β₃-AR plays an indisputable role in the white and brown adipose tissue of some species, its physiological status and its expression are still subject to debate in human white fat cells.     

α2-Antiplasmin is involved in the production of transforming growth factor β1 and fibrosis

BACKGROUND: Fibrotic disease occurs in most tissues. Transforming growth factor (TGF)-beta is the major inducer of fibrosis. The fibrinolytic system is considered to play an important role in the degradation of extracellular matrices. However, the detailed mechanism of how this system affects fibrosis remains unclear. METHODS AND RESULTS: We examined experimental fibrosis in mice with a deficiency of alpha(2)-antiplasmin (alpha2AP), which is a potent and specific plasmin inhibitor. We found that the lack of alpha2AP attenuated bleomycin-induced TGF-beta(1) synthesis and fibrosis. In addition, the production of TGF-beta(1) from the explanted fibroblasts of alpha2AP(-/-) mice decreased dramatically as compared to that in wild-type mice. Moreover, we found that alpha2AP specifically induces the production of TGF-beta(1) in fibroblasts. CONCLUSION: The lack of alpha2AP attenuated TGF-beta(1) synthesis, thereby resulting in attenuated fibrosis. This is the first report to describe the crucial role that alpha2AP plays in TGF-beta(1) synthesis during the process of fibrosis. Our results provide new insights into the role of alpha2AP in fibrosis.     

Activation of GPVI by collagen is regulated by α2β1 and secondary mediators

Summary.  We have compared the roles of adenosine diphosphate (ADP), thromboxanes and the integrin α₂β₁ in the activation of washed platelets by collagen in the presence of the α_IIbβ₃ antagonist lotrafiban. The stimulation of protein tyrosine phosphorylation by a collagen suspension is markedly delayed in the presence of the above inhibitors but shows substantial recovery with time. In comparison, activation of phospholipase C (PLC), Ca²⁺ elevation and dense granule secretion are more severely suppressed by the above inhibitors. α₂β₁ blockade has a slightly greater inhibitory effect on all of the above responses than a combination of ADP receptor antagonists and cyclooxygenase inhibitor. Platelets exposed to a collagen monolayer show robust elevation of Ca²⁺ that is delayed in the presence of the above inhibitors and which is accompanied by α-granule secretion. These results demonstrate that secondary mediators and α₂β₁ modulate collagen-induced intracellular signaling but have negligible effect on GPVI signaling induced by the specific agonist convulxin. This work supports the postulate that the major role of α₂β₁ is to increase the avidity of collagen for the platelet surface and by doing so enhance activation of GPVI. Therefore we propose an important role of secondary mediators in collagen-induced signaling is the indirect regulation of GPVI signaling via activation of α₂β₁.     

Involvement of the β3 E749ATSTFTN756 region in stabilizing integrin αIIbβ3-ligand interaction

Summary. Platelet integrin α_IIbβ₃ must be activated via intracellular mechanisms before it binds soluble ligands, and it is thought to be activated at its extracellular site by surface‐bound ligands. Integrin activation is associated with rearrangement of the cytoskeleton and phosphorylation of proteins that become localized in focal contacts. In these processes, the cytoplasmic tail of the β‐subunit plays a central role. We introduced peptides homologous to the E⁷⁴⁹ATSTFTN⁷⁵⁶ domain (E–N peptide) and the T⁷⁵⁵NITYRGT⁷⁶² domain (T–T peptide) of β₃ in streptolysin O‐permeabilized platelets and analyzed the initial interaction with soluble fibronectin, fibrinogen and PAC‐1 after stimulation with thrombin. E–N peptide left the initial binding of fibronectin intact but interfered with stable receptor occupancy. E–N peptide also inhibited fibrinogen binding, thereby reducing the formation of large aggregates. Strikingly, E–N peptide did not disturb the binding of PAC‐1, which is known to reflect activation of the integrin. E–N peptide also inhibited tyrosine phosphorylation of focal adhesion kinase, a response known to be dependent on α_IIbβ₃. T–T peptide did not affect these processes. In a model for outside‐in integrin activation, E–N peptide disrupted the binding of CHO cells expressing α_IIbβ₃ to surface‐bound ligand. Again, T–T peptide had no effect. We conclude that the E⁷⁴⁹ATSTFTN⁷⁵⁶ region of the β₃‐tail stabilizes the binding of soluble and surface‐bound ligand to integrin α_IIbβ₃ via a mechanism that involves the phosphorylation of FAK.     

Cytoplasmic regions of the β3 subunit of integrin αIIbβ3 involved in platelet adhesion on fibrinogen under flow conditions

Summary.  Platelet adhesion to surface-bound fibrinogen depends on integrin α_IIbβ₃. In the present study, we investigated the role of the regions ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T of the β₃ cytoplasmic tail in the regulation of platelet adhesion under flow conditions, by introducing peptide mimetics in platelets. Introduction of peptide EATSTFTN (E–N) increased surface coverage by 35%, an effect caused by 25% more adhesion. In contrast, peptide TNITYRGT (T–T) decreased surface coverage by 16%, as a result of 25% less adhesion. An S→P substitution in the E–N peptide, thereby mimicking a mutation in Glanzmann's thrombasthenia, abolished the effect of E–N. A suboptimal concentration of cytochalasin D is known to enhance ligand binding to α_IIbβ₃ in platelet suspensions. Under flow, cytochalasin D (1 µmol L⁻¹) induced 50% more platelet adhesion, with a strong reduction in platelet spreading. Both peptides opposed the increase in adhesion by cytochalasin D and partly (E–N) and completely (T–T) restored platelet spreading. Thus, the ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T regions of β₃ contribute to the regulation of αIIbβ3 anchorage to the cytoskeleton and platelet spreading to an adhesive surface.     

Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated α2β1 activation

Summary. Background: The platelet α₂β₁ integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that α₂β₁ function can be activated via inside‐out signaling, similar to the prototypical platelet integrin α_IIbβ₃. However, signaling molecules that regulate α₂β₁ activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates α_IIbβ₃ activation. Objectives: We hypothesized that Rap1b positively regulates α₂β₁ during agonist‐induced platelet activation. Methods: To test whether Rap1b activates α₂β₁ downstream of glycoprotein (GP)VI or other platelet receptors, we stimulated platelets purified from Rap1b^?/? or wild‐type mice with diverse agonists and measured α₂β₁ activation using fluorescein isothiocyanate‐labeled monomeric collagen. We also examined the role of Rap1b in outside‐in signaling pathways by analyzing adhesion and spreading of Rap1b^?/? or wild‐type platelets on monomeric, immobilized collagen. Finally, we monitored the activation status of related Rap GTPases to detect changes in signaling pathways potentially associated with Rap1b‐mediated events. Results: Rap1b^?/? platelets displayed comparable ADP‐induced or thrombin‐induced α₂β₁ activation as wild‐type platelets, but reduced convulxin‐dependent α₂β₁ activation. Rap1b^?/? platelets exhibited increased spreading on immobilized collagen but similar adhesion to immobilized collagen compared to wild‐type platelets. Rap1b^?/? platelets also showed Rap1a and Rap2 activation upon agonist stimulation, possibly revealing functional compensation among Rap family members. Conclusions: Rap1b is required for maximal GPVI‐induced but not ADP‐induced activation of α₂β₁ in murine platelets.     

The suppressive effect of sphingosine 1-phosphate on monocyte-endothelium adhesion may be mediated by the rearrangement of the endothelial integrins α5β1 and αvβ3

BACKGROUND: Sphingosine 1-phosphate (S1P), known to play important roles in vascular biology, is a bioactive lysophospholipid mediator that maintains endothelial integrity via its cell-surface receptors (S1Ps). In this in vitro study, we aimed to examine the role of S1P in monocyte-endothelium adhesion, which is an important event in the pathophysiology of atherosclerosis. METHODS AND RESULTS: S1P pretreatment of human umbilical vein endothelial cells (ECs), but not U937 cells, effectively suppressed U937-EC adhesion independently from the expression of adhesion molecules, namely ICAM-1, VCAM-1, and E-selectin. This S1P-induced suppressive effect was inhibited by the blockage of S1P(1) and S1P(3) receptors and the specific inhibitors of G(i) protein, Src family proteins, phosphatidylinositol 3-kinase, and Rac1, indicating involvement of these key downstream pathways. Moreover, the RGD peptide and antibodies, which neutralize adhesion via alpha(5)beta(1) and alpha(v)beta(3), effectively inhibited U937-EC adhesion with a degree similar to S1P pretreatment. Both an adhesion assay and flow-cytometric analysis demonstrated that U937 cells adhered through integrins alpha(5)beta(1) and alpha(v)beta(3) expressed on the apical surface of monolayer ECs, and S1P shifted the localization of these integrins from the apical surface to the basal surface. CONCLUSIONS: From the present results, we propose that S1P may contribute to the maintenance of vascular integrity and the regulation of atherogenesis through the rearrangement of endothelial integrins.     

Platelet integrin αIIbβ3: activation mechanisms

Integrin alpha(IIb)beta(3) plays a critical role in platelet aggregation, a central response in hemostasis and thrombosis. This function of alpha(IIb)beta(3) depends upon a transition from a resting to an activated state such that it acquires the capacity to bind soluble ligands. Diverse platelet agonists alter the cytoplasmic domain of alpha(IIb)beta(3) and initiate a conformational change that traverses the transmembrane region and ultimately triggers rearrangements in the extracellular domain to permit ligand binding. The membrane-proximal regions of alpha(IIb) and beta(3) cytoplasmic tails, together with the transmembrane segments of the subunits, contact each other to form a complex which restrains the integrin in the resting state. It is unclasping of this complex that induces integrin activation. This clasping/unclasping process is influenced by multiple cytoplasmic tail binding partners. Among them, talin appears to be a critical trigger of alpha(IIb)beta(3) activation, but other binding partners, which function as activators or suppressors, are likely to act as co-regulators of integrin activation.     

Platelet α2- and leucocyte β2-adrenoceptors in phaeochromocytoma: effect of tumour removal

Platelet alpha 2- and leucocyte beta 2-adrenoceptors (as well as noradrenaline and adrenaline plasma levels) were studied in five patients with confirmed phaeochromocytoma using tritiated yohimbine and iodated cyanopindolol, respectively, before and after tumour removal. Patients with phaeochromocytoma had a lower leucocyte iodated cyanopindolol binding than control subjects, but no change in platelet tritiated yohimbine binding. Plasma catecholamine levels were higher than controls. Tumour removal induced a return to normal values of leucocyte iodated cyanopindolol binding and a decrease in plasma catecholamine levels. These results underline the potential interest of leucocyte beta-adrenoceptor quantification in the diagnosis and the follow-up of phaeochromocytoma. Pharmacologically, they show that down-regulation occurs in vivo for leucocyte beta 2-adrenoceptors but not for platelet alpha 2-adrenoceptors.     

MMP-2 regulates human platelet activation by interacting with integrin αIIbβ3

Summary. Background: Human platelets contain matrix metalloproteinases (MMPs) that are secreted during platelet activation. Platelet MMPs have been implicated in the regulation of cellular activation and aggregation. Although the proaggregatory effect of MMP‐2 has been demonstrated, the functional mechanism is not clearly understood. Objectives: This work was carried out in order to elucidate the biochemical mechanism of MMP‐2‐associated platelet activation and aggregation. Methods: MMP‐2 binding to the platelet surface was analyzed by flow cytometry. The cell surface target of MMP‐2 was identified in thrombin receptor‐activating peptide‐stimulated platelets by immunoprecipitation, Western blotting and fluorescence microscopy. A recombinant hemopexin‐like domain was used to characterize the nature of MMP‐2 binding to the platelet surface. The functional significance of MMP‐2 in platelet activation was investigated by quantitative measurements of the activation markers P‐selectin (CD62P) and active α_IIbβ₃. The role of MMP‐2 in platelet aggregation was analyzed with an aggregometer. Results: ProMMP‐2 binds to integrin α_IIbβ₃ in stimulated platelets in which proMMP‐2 is converted into MMP‐2. Fibrinogen was able to replace the α_IIbβ₃‐bound MMP‐2. The molecular interaction of MMP‐2 and integrin α_IIbβ₃ was abrogated by the recombinant human hemopexin‐like domain of MMP‐2, leading to reduced cell surface expression of activation markers CD62P and active α_IIbβ₃, and resulting in suppressed platelet aggregation. Conclusion: This work clearly demonstrates that platelet activation and aggregation is regulated by MMP‐2 that specifically interacts with integrin α_IIbβ₃. The C‐terminal hemopexin‐like domain of MMP‐2 is an essential element for binding to α_IIbβ₃.     

Identification and characterization of α1- and β2-adrenergic receptors in human liver

Adrenergic receptors were identified in healthy human hepatic tissue from thirty-nine subjects undergoing elective abdominal surgery by using the specific alpha 1-antagonist [3H]-prazosin and the beta adrenergic antagonist [3H]-dihydroalprenolol ([3H]-DHA). [3H]-prazosin binding to plasma membranes was rapid, of high affinity, saturable and stereospecific with a maximal binding capacity (Bmax) of 74.1 +/- 5.5 fmol mg-1 of protein. The displacement curve for (-)-norepinephrine was better explained by a one-site binding and after addition of GTP 0.1 mM the curve was not right-shifted, suggesting the majority of alpha receptors in healthy human liver are of the alpha 1 subtype and not linked to a GTP-binding protein. [3H]-DHA binding to liver plasma membranes was also rapid, of high affinity, saturable and stereospecific with a Bmax 96.5 +/- 10.3 fmol mg-1 of protein of receptors. Computer aided analysis of the displacement curve of ICI 118,551, a subtype selective beta 2-antagonist (IC50 = 62 +/- 2 nM), indicated a one-site binding, thus, showing that beta adrenergic receptors are of the beta 2 subtype. The displacement curve of [3H]-DHA for (-)-isoproterenol was right shifted by GTP indicating that beta 2 adrenergic receptors are linked to a GTP-binding protein in human liver. These results indicate that alpha 1- and beta 2-receptors co-exist in human liver but only beta 2-receptors are linked to a GTP-binding protein.     

β3-adrenoceptors and intestinal motility

Summary— Early substantial evidence of the low susceptibility to β-adrenoceptor antagonists of non α-adrenergic responses reducing gut motility and tone was reluctantly accepted as indicating a third β-receptor subtype different from the β₁ and β₂. This applied likewise to lipolysis until new selective “lipolytic” β-agonists poorly effective at established β-receptors were introduced. Shortly afterwards these “lipolytic” as well as certain newer and even more selective β-adrenoceptor agonists were shown to be potent inhibitors of intestinal motility. The latter are the “gut-specific” phenylethanolaminotetralins whose availability as pure isomers attested to the stringent stereochemical requirements for selectivity at non-β₁, non-β₂ β-adrenoceptors. Acceptance of the functionally based concept of a β₃-adrenoceptor was boosted on structural grounds by molecular biology studies. Sequence analysis indicated the existence in humans and rodents of genes coding for a third subtype of β-receptor that, when expressed in transfected heterologous cells, had a pharmacological profile distinct from the previously established subtypes. Finally, aryloxypropanolaminotetralins have been prepared as the first selective antagonists of β₃-adrenoceptors, thus providing unambiguous conclusive evidence of the distinctive functional features of those abundant in the rat colon. The therapeutic potential in gastroenterology of the newer compounds targetable on the β₃-adrenoceptor is suggested by their potent intestinal action in vivo in animal models without any of the cardiovascular or other unwanted effects of conventional β₃-adrenoceptor agonists and antagonists, and by the clinically confirmed importance of β-adrenergic control of motor function throughout the alimentary canal. However, open questions include the incidence of species-related differences in β₃-adrenoceptors, and as yet there are no data on gastrointestinal functions in humans under the influence of drugs designed to act selectively at these receptors.     

Interaction of β2-glycoprotein I with members of the low density lipoprotein receptor family

The antiphospholipid syndrome (APS) is a non-inflammatory autoimmune disease characterized by arterial and/or venous thrombosis and/or pregnancy morbidity in the presence of autoantibodies that recognize beta2-glycoprotein I (beta2GPI) bound to phospholipids. We have previously demonstrated that dimerization of beta2GPI by autoantibodies induces platelet activation, involving the platelet receptor apolipoprotein E receptor 2' (apoER2') a receptor belonging to the low-density lipoprotein receptor (LDL-R) family. Here, we show that dimeric beta2GPI, but not monomeric beta2GPI, interacts with four other LDL-R family members: the LDL-R related protein (LRP), megalin, the LDL-R and the very-low density lipoprotein receptor (VLDL-R). Interaction between dimeric beta2GPI and LDL-R, apoER2' and VLDL-R was best described with a one-site binding model (half-maximal binding; approximately 20 nm for apoER2' and VLDL-R and approximately 300 nm for LDL-R), whereas the interaction between dimeric beta2GPI and LRP or megalin was best described with a two-site binding model, representing a high- (approximately 3 nm) and a low-affinity site (approximately 0.2 microm). Binding to all receptors tested was unaffected by a tryptophane to serine (W316S) substitution in domain V of beta2GPI, which is known to disrupt the phospholipid binding site of beta2GPI. Also deletion of domain I or II left the interaction with the receptors unaffected. Deletion of domain V, however, significantly decreased the affinity for the receptors. In conclusion, our data show that dimeric beta2GPI can interact with different LDL-R family members. This interaction is dependent on a binding site within domain V of beta2GPI, which does not overlap with the phospholipid-binding site within domain V.     

The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin α2β1

BACKGROUND: Activation of the platelet integrin alpha 2 beta 1 is closely regulated due to the high thrombogenicity of its ligand. As a beta 1 interacting kinase, ILK represents a candidate intracellular regulator of alpha 2 beta 1 in human platelets. OBJECTIVES: We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha 2 beta 1 activation in HEL cells, a megakaryocytic cell line. METHODS: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta 1-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha 2 beta 1 function assessed by overexpression studies in HEL cells. RESULTS: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta 1-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha 2 beta 1-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha 2 beta 1. CONCLUSIONS: Our findings that ILK regulates alpha 2 beta 1 in HEL cells, is activated in platelets and associates with beta 1-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.     

A role for PKCθ in outside-in αIIbβ3 signaling

Fibrinogen binding to platelets triggers alpha(IIb)beta3-dependent outside-in signals that promote actin rearrangements and cell spreading. Studies with chemical inhibitors or activators have implicated protein kinase C (PKC) in alpha(IIb)beta3 function. However, the role of individual PKC isoforms is poorly understood. Biochemical and genetic approaches were used to determine whether PKCtheta is involved in alpha(IIb)beta3 signaling. PKCtheta was constitutively associated with alpha(IIb)beta3 in human and murine platelets. Fibrinogen binding to alpha(IIb)beta3 stimulated the association of PKCtheta with tyrosine kinases Btk and Syk, and tyrosine phosphorylation of PKCtheta, Btk and the actin regulator, Wiskott-Aldrich syndrome protein (WASP). Mouse platelets deficient in PKCtheta or Btk failed to spread on fibrinogen. Furthermore, PKCtheta was required for phosphorylation of WASP-interacting protein on Ser-488, an event that has been linked to WASP activation of the Arp2/3 complex and actin polymerization in lymphocytes. Neither PKCtheta nor Btk were required for agonist-induced inside-out signaling and fibrinogen binding to alpha(IIb)beta3. Thus, PKCtheta is a newly identified, essential member of a dynamic outside-in signaling complex that includes Btk and that couples alpha(IIb)beta3 to the actin cytoskeleton.     

α1 and α2-adrenergic control of large and small coronary arteries during exercise in conscious dogs under β-blockade

Summary— The aim of this study was to determine the relative roles of α₁-and α₂-adrenoceptors at the level of large epicardial and small resistance coronary arteries when sympathetic tone is increased by exercise in conscious dogs. The responses of left circumflex coronary artery diameter and blood flow were investigated at rest and during graded treadmill exercise (5, 10 and 12 km/h) in six chronically instrumented dogs during control conditions (saline) and after administration of propranolol (1 mg/kg) either alone or in combination with either prazosin (50 μg/kg), or idazoxan (300 μg/kg), or the association of prazosin + idazoxan (same doses). In control conditions, graded treadmill exercise resulted in a progressive increase in coronary artery diameter (+ 3.8 ± 0.6% from 3479 ± 80 μm) and in a decrease in coronary vascular resistance (- 46.0 ± 4.5% from 8.49 ± 1.51 mmHg/cm/s). Propranolol significantly constricted large (- 4.4 ± 0.6% from 3486 ± 87 μm) and limited dilation of small coronary arteries during exercise. These coronary effects of propranolol remained unchanged following additional α₂-adrenoceptor blockade by idazoxan but were abolished following α₁-adrenoceptor blockade by prazosin, given either alone or combined with idazoxan. Thus, α₁- but not α₂-adrenoceptors are responsible for propranolol-induced constriction of large coronary arteries and limitation of small coronary arteries dilation during exercise in conscious dogs.