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.     

Effects of high-altitude chronic hypoxia on platelet α2-receptors in man

During chronic high-altitude (HA) exposure, basal and exercise-induced noradrenaline (NA) increases do not parallel blood pressure (BP) changes observed; unlike β-adrenergic receptors, to our knowledge no data are available on α-receptors. We studied platelet α₂- and leucocyte β-receptors and basal catecholamine levels in 11 trained climbers before and after they had spent a 15-day period at a height of over 4400 m. In six of the climbers we also evaluated catecholamines after maximal bicycle ergometer exercise. After chronic high-altitude exposure, a significant decrease was found in platelet α₂-receptor density and affinity [ B _max from 92.6 ± 6.7 to 54.6 ± 4.2 fmol mg⁻¹ protein ( P  < 0.001) and K _D from 1.271 ± 0.034 to 1.724 ± 0.077 nmol L⁻¹ ( P  < 0.05)], although no changes to β-receptors were observed. No changes were found in basal pre- and post-expedition NA and adrenaline (A), and there was only a slight decrease in post-expedition NA after maximal exercise. Our results suggest that prolonged exposure to hypoxia induces a down-regulation of α₂-receptors, which may be a contributory factor in the regulation of the physiological vascular response to acclimatization.     

β2-glycoprotein I inhibits vascular endothelial growth factor and basic fibroblast growth factor induced angiogenesis through its amino terminal domain

Summary.  Background:  Beta-2 glycoprotein I (β₂GPI) is a plasma glycoprotein which interacts with various proteins of the coagulation and fibrinolysis system. β₂GPI has recently been shown to have anti-angiogenic properties. Objectives:  We undertook this study to investigate the specific domain of β₂GPI involved in the anti-angiogenic function and its effect on downstream signaling of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Methods:  Various preparations of β₂GPI were used on human umbilical vein endothelial cells (HUVECs) in the absence or presence of VEGF and bFGF. The effect on HUVECs' proliferation, migration and tubule formation in Matrigel matrix was investigated. The effect of β₂GPI on the mRNA expression of VEGF receptors and phosphorylation of signaling molecules was also studied. Results:  β₂GPI is shown in this study to be an anti-angiogenic molecule in vitro by inhibiting VEGF and bFGF-induced proliferation, migration and papillary-like tubule formation of HUVECs. This inhibition was achieved by native, proteolytically clipped and domain deletion mutants, domain I-IV (DI-IV) but not domain II-V (DII-V) of β₂GPI. Native β₂GPI was found to downregulate the expression of the VEGF receptor KDR/Flk-1 on endothelial cells and to block the phosphorylation of VEGF's downstream effector molecules in the MAPK/ERK and PI3K/Akt/GSK3β pathways. Conclusions:  These results indicate that β₂GPI has anti-angiogenic functions which depend on the presence of domain I. This anti-angiogenic activity may have important implications for the therapeutic manipulation of angiogenesis in various disease states.     

Multiple ways to switch platelet integrins on and off

Summary.  In the classical concept of platelet integrin activation, it is considered that unidirectional conformational changes of α_IIbβ₃ and α₂β₁ regulate the adhesiveness of platelets for fibrin(ogen) and collagen, respectively. Here, we summarize recent evidence that these conformational changes: (i) can also occur in the reverse direction; and (ii) are not independent events. Platelet stimulation through the P2Y₁₂ receptors provokes only transient α_IIbβ₃ activation via signaling routes involving phosphoinositide 3-kinases and Rap1b. Furthermore, α_IIbβ₃ can be secondarily inactivated in platelets with prolonged high Ca²⁺ rises, which expose phosphatidylserine and bind coagulation factors. Thus, platelet stimulation with strong agonists (collagen and thrombin) also results in transient integrin activation. Integrin α₂β₁ is found to be activated by a mechanism that is directly linked to α_IIbβ₃ activation. Integrin α₂β₁ can adopt different activation states, depending on the trigger. Conclusively, reversibility and synchrony of platelet integrin activation are newly identified mechanisms to restrict thrombus growth and to allow optimal coagulation factor binding. Back-shifting of activated integrins towards their resting state may be a novel goal of antithrombotic medication.     

Beta3-adrenoceptors in the cardiovascular system

Summary— Behind the classic beta₁, and beta₂-adrenoceptors, recent molecular and pharmacological studies have described a new receptor, called the beta₃-adrenoceptor, in various mammalian tissues (brown and white adipose tissue, digestive smooth muscle). Few authors have investigated the putative existence of the beta₃-adrenoceptor in the cardiovascular system. This paper reviews the available data. In vitro studies show that beta₃-adrenoceptor agonists (BRL 37344, CGP 12177) induce a relaxation of fragments of rat carotid artery which is not antagonized by propranolol. In dogs, these drugs elicit a decrease in blood pressure due to peripheral vasodilation and an increase in heart rate which is of baroreflex origin. The vasodilating effects are mainly observed in cutaneous and adipose tissue vessels and cannot be explained by any known transductional mechanism. Activation of this vascular β₃-adrenoceptor requires higher doses of catecholamines than for β₁- or β₂-adrenoceptors. In humans, the cardiovascular effects of beta₃-adrenoceptor agonists are explained by the activation of beta₁- or beta₂ (and not beta₃-)-adrenoceptors. These studies suggest the presence of vascular (but not cardiac) beta₃-adrenoceptors in dogs. In other species, including man, the presence of such cardiac β₃-adrenoceptors remains to be resolved. Their physiological relevance remains unknown.     

A simple method to discriminate between β2-glycoprotein I- and prothrombin-dependent lupus anticoagulants

Summary.  Lupus anticoagulants (LAC) are a heterogeneous group of autoantibodies that prolong phospholipid-dependent clotting assays. The autoantibodies that cause LAC activity are predominantly directed against β₂-glycoprotein I (β₂GPI) or prothrombin. In the present study, we describe a method to differentiate between LAC caused by antibodies directed against β₂GPI or prothrombin. Monoclonal antibodies, affinity purified patient antibodies, and selected patient samples were used to show that in an aPTT-based clotting assay (PTT-LA; Diagnostica Stago), the use of cardiolipin vesicles in the neutralization procedure discriminates between β₂GPI- or prothrombin-dependent LAC activities. Addition of cardiolipin vesicles shortened the prolonged clotting time caused by anti-β₂GPI antibodies with LAC activity, whereas this procedure further prolonged clotting times caused by antiprothrombin antibodies with LAC activity. In contrast, addition of phosphatidylcholine/phosphatidylserine vesicles corrected prolonged clotting times caused by either anti-β₂GPI or antiprothrombin antibodies with LAC activity. The effects of cardiolipin (CL) on β₂GPI-induced LAC activity were specific for contact activation mediated clotting assays. Possible explanations for these findings are the relatively high affinity of β₂GPI for cardiolipin, as determined by surface plasmon resonance analysis, and inhibition by anti-β₂GPI antibodies of the CL-induced prolongation of the PTT-LA.     

Association of the antagonism of von Willebrand factor but not fibrinogen by platelet αIIbβ3 antagonists with prolongation of bleeding time

Summary.  Background:  The α _IIb β ₃ antagonists inhibit platelet aggregation and are used as antithrombotic agents for cardiothrombotic disease. The present study investigates the correlation of inhibition of fibrinogen and von Willebrand factor (VWF) binding by α _IIb β ₃ antagonists with the inhibition of platelet aggregation and prolongation of bleeding time (BT). Methods:  Inhibition of fibrinogen and VWF binding were assessed in a purified α _IIb β ₃-binding assay. As an in vitro cell-based assay, platelet aggregation and VWF-mediated adhesion studies were performed using human platelets. In vivo effects on BT were measured using a template device in dogs at the same time as an ex vivo aggregation study was performed. Results:   In vitro studies demonstrated that the antiaggregatory effects of α _IIb β ₃ antagonists correlate with their inhibition of fibrinogen binding, but not VWF. Interestingly, the effects of α _IIb β ₃ antagonists on BT could be differentiated from the inhibition of platelet aggregation. Furthermore, this differentiation was strongly correlated with the different inhibitory potencies between fibrinogen and VWF binding, as well as that between VWF-mediated adhesion and aggregation. Conclusions:  Our study provides novel evidence showing that the inhibitory effect of α _IIb β ₃ antagonists on VWF, but not fibrinogen binding, correlates with their ability to prolong BT.     

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.     

Transforming growth factor β1 modulates angiotensin induced calcium influx in vascular smooth muscle

Abstract. The modulatory effects of transforming growth factor β₁ (TGF β₁) on the angiotensin II (Ang II)-induced increase in cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). [Ca²⁺]_i in VSMC was measured using the fluorescent dye fura-2. When TGF β₁ was applied 30 s prior to Ang II, the Ang II-induced [Ca²⁺]_i increase was significantly enhanced in VSMC from SHR ( P < 0.05 compared to control), whereas after the preincubation with TGF β₁ for 30 min, the Ang II-induced [Ca²⁺]_i increase was significantly reduced in VSMC from both strains. Using the manganese-quenching technique, it was confirmed that short-term exposure to TGF β₁ enhanced the Ang II-induced trans-plasma-membrane calcium influx in SHR. The inhibition of protein kinase C by calphostin C abolished the stimulatory effect of TGF β₁ on the Ang II-induced [Ca²⁺]_i increase. It is concluded that TGF β₁ modulates the Ang II-induced calcium handling in VSMC.     

Serum β2-microglobulin at remission and relapse in patients with multiple myeloma

Abstract. Serum β₂-microglobulin (S-β₂m) was determined before treatment in fifty patients with multiple myeloma (MM), twenty-two of which had pure Bence Jones (BJ) myeloma. S-β₂m was related to clinical stage before, but not after, a correction of S-β₂m values for co-existent raised S-creatinine values (> 106 μmol 1^-1)- However, S-β₂m as well as corrected β₂m was a parameter of prognostic value. The median expected survival time was 14 months at S-β₂m values > 8·0 mg 1^-1 and 56 months at values<3·5 mg 1^-1. A response to treatment was associated with a decrease of S-β₂m and a stationary course with unchanged values, whereas a relapse or progressive disease was connected with an increase. In β₂m producers', i.e. patients with a clear initial high S-β₂m, serial determinations are of value for monitoring patients with MM. In particular, this is the case in BJ myelomas, as they lack a quantifiable serum M-component. With respect to β₂m no difference was found between BJ and other patients with MM.     

New evidence that serum β2-microglobulin behaves as a biological marker of bone remodelling in women

Abstract. Having observed that serum β ₂-microglobulin concentration correlates with serum tartrate-resistant acid phosphatase (TRAP) concentration in postmenopausal osteoporosis, and that metacarpal endosteal diameter is dependent on bone resorption, we correlated the two biochemical parameters with the radiographic parameter to determine if β ₂-microglobulin behaves like a biological marker of bone remodelling. In 105 women (mean age 68±4 years) consisting of 60 normal postmenopausal women and 55 osteoporotic postmenopausal women, there was a significant positive correlation between metacarpal endosteal diameter and these two biochemical values ( r = 0.66 with β ₂-microglobulin and r = 0.68 with TRAP in the osteoporotic postmenopausal women: r = 0.48 with β ₂-microglobulin and r = 0.56 with TRAP in the normal postmenopausal women: P < 0.001 for all comparisons). All three measurements were significantly higher ( P < 0.001) in the osteoporotic postmenopausal women than in the normal postmenopausal women. These findings show that serum β ₂-microglobulin behaves like a biological marker of remodelling.     

CIB1 deficiency results in impaired thrombosis: the potential role of CIB1 in outside-in signaling through integrin αIIbβ3

Summary.  Background: Agonist-induced inside-out signaling activates platelet integrin α_IIbβ₃, rendering it to bind plasma fibrinogen (Fg). Fg binding induces outside-in signaling that culminates in platelet aggregation, leading to physiological hemostasis and pathological thrombosis. How outside-in signaling through α_IIbβ₃ regulates hemostasis and thrombosis is not well understood. We have previously shown that CIB1 is involved in regulating α_IIbβ₃ function. Objective: To determine the in vivo role of CIB1 in the process of hemostasis and thrombosis. Methods and Results: Genetic ablation of Cib1 significantly increased mouse tail bleeding time. Greater than 50% of the Cib1 null mice showed a rebleeding phenotype. Time taken for complete occlusion of carotid artery upon 10% FeCl₃-induced injury was significantly delayed in the absence of Cib1. This was also associated with unstable thrombus formation. The inside-out signaling appears normal as ADP-, collagen- and PAR4 peptide-induced aggregation and fibrinogen binding was unaffected. The absence of Cib1 also affected the ability of platelets to spread on immobilized Fg, but not filopodia formation. Spreading could be restored in Cib1 null platelets by the addition of exogenous ADP. Outside-in signaling-dependent tyrosine phosphorylation of the integrin β₃ subunit was significantly reduced in the absence of Cib1 as determined by Western blot analysis. Conclusion: Using gene knockout mice, we show for the first time that lack of Cib1 results in impaired thrombosis. CIB1 regulates these processes by affecting platelet spreading, but not platelet filopodia formation. These in vivo and in vitro results clearly show that CIB1 is a key regulator of thrombosis.     

Platelet receptor recognition and cross-talk in collagen-induced activation of platelets

Summary.  Comprehensive mapping of protein-binding sites within human collagen III has allowed the recognition motifs for integrin α₂β₁ and VWF A3 domain to be identified. Glycoprotein VI-binding sites are understood, although less well defined. This information, together with recent developments in understanding collagen fiber architecture, and crystal structures of the receptor collagen-binding domains, allows a coherent model for the interaction of collagen with the platelet surface to be developed. This complements our understanding of the orchestration of receptor presentation by membrane microdomains, such that the polyvalent collagen surface may stabilize signaling complexes within the heterogeneous receptor composition of the lipid raft. The ensuing interactions lead to the convergence of signals from each of the adhesive receptors, mediated by FcR γ-chain and/or FcγRIIa, leading to concerted and co-operative platelet activation. Each receptor has a shear-dependent role, VWF/GpIb essential at high shear, and α₂β₁ at low and intermediate shear, whilst GpVI provides core signals that contribute to enhanced integrin affinity, tighter binding to collagen and consequent platelet activation.     

Anti-Migraine Drug Interactions with Cloned Human 5-Hydroxytryptamine 1 , Receptor Subtypes

SYNOPSIS
Multiple 5-hydroxytryptamine (5-HT) receptors have been identified in humans. A subgroup of these receptors (designated 5-HT ₁ receptors) have been hypothesized to be involved in the mechanism of action of acute anti-migraine drugs. At present, this hypothesis cannot be tested directly in human tissues due to technical limitations. However, recent molecular biological advances have allowed for the development of assays employing cloned human 5-HT ₁ receptors expressed in cells by DNA transfection. This study analyzed the ability of ergotamine, dihydroergotamine, 5-HT and sumatriptan to interact with the four known human 5-HT ₁ receptor subtypes. The four acute anti-migraine agents interacted with all 4 human 5-HT ₁ receptor subtypes with less than 1 μM affinity. However, drug affinities for the human 5-HT _1B and 5-HT _1D receptors correlate most closely with the rank order of clinical dosages used to treat a migraine attack. Therefore, these data indicate that human 5-HT _1B and/or 5-HT _1D receptors are likely to mediate the therapeutic efficacy of acute anti-migraine drugs.     

Turnover in humans of β2-microglobulin: the constant chain of HLA-antigens

Abstract The turnover of β₂-microglobulin, the common subunit of the HLA antigens, has been examined in normal subjects and in some patients with kidney disorders, multiple myeloma and rheumatoid arthritis. All patients displayed elevated serum levels of β₂-microglobulin. The plasma disappearance curve of ¹²⁵I-β₂-microglobulin demonstrated that the protein has a rapid turnover (11/2 = 21 h; range 1 1–2–8 h) in normal persons and in patients with a normal glomerular filtration rate. In patients with kidney disorders the impaired renal filtration prolonged the turnover time and led to elevated serum levels of β₂-microglobulin.
Simultaneous measurements of ¹²⁵-β₂-microglobulin in serum and urine allowed estimations of the β₂-microglobulin net reabsorption in the renal tubuli. Two patients with renal disease reabsorbed 84% and 89%, respectively, of the β₂-microglobulin filtered in the glomeruli. In normal persons the net reabsorption is close to 100%.
In patients with normal kidney function increased serum levels of β₂-microglobulin seem to be due to an increased synthetic rate of the protein as the elimination rate is normal. HLA antigen heavy chains in serum are present in smaller amounts than β₂-microglobulin. The present data, therefore, suggest an imba-lanced synthesis of the two chains.     

Differential response of hypertrophied rat hearts to various α1-adrenoceptor agonists

Summary— With respect to the heart, the prolonged existence of hypertension, both in man and in experimental animals is predominantly characterized by an increase in left ventricular myocardial mass. In this process, the autonomic nervous system plays an important role. Although endogenous catecholamine stimulation of the heart is mainly exerted via the β-adrenoceptors, in several mammalian species, the stimulation of cardiac α-adrenoceptors also mediates positive inotropic actions. We investigated the functional responses of isolated hypertrophied hearts taken from spontaneously hypertensive rats (SHR) and rats with an induced aortic stenosis (ASR) to various α₁-adrenoceptor agonists and compared them with those from age matched Wistar Kyoto (WKY) and "sham" operated controls. Accordingly, we studied the functional response to: methoxamine (α₁), cirazoline (α₁) and phenylephrine (α₁ > β₁). The inotropic response to the α₁-adrenoceptor agonists cirazoline and methoxamine proved to be significantly weaker in hypertrophied hearts from SHR and ASR than in non-hypertrophied hearts from WHY and "sham" operated controls ( p < 0.05). The inotropic response to phenylephrine remained intact in hypertrophied myocardial tissue. However, it was significantly reduced when the hearts were pre-treated with the intracellular Ca²⁺-antagonists ryanodine and TMB-8. These findings show that the mechanism of sarcolemmal Ca²⁺ release, activated by phenylephrine, is still intact in the hypertrophied myocardial cell. In conclusion, these data show that cardiac hypertrophy, be it of genetical or mechanical origin, leads to a reduced response of the isolated heart to α₁-adrenoceptor stimulation.     

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.     

Alzheimer amyloid β-peptides exhibit ionophore-like properties in human erythrocytes

Abstract. There is growing evidence that the amyloid β-peptide (β₁_₄₀) is involved in the aetiology of Alzheimer's disease also implicating an altered calcium homeostasis of affected cells. Beta₁_₄₀ has been proposed to form calcium channels in synthetic bilayer membranes [1]. We wanted to investigate in the present study whether β₁-₄₀ (or fragments thereof) could act as ionophores in a biological membrane like the one in human erythrocytes. Incubation of the cells for 2h and 4h at 37°C together with 6μmolL^-1 of β₁-₄₀ or of fragments β₁_₂₈and β₂₅-₃₅, resulted in a significantly decreased energy charge qualitatively similar to the one obtained by a known calcium ionophore (A 23187, 0.05μmolL^-1). Moreover, β₁_₄₀ and its two fragments induced a significant alteration of ⁴⁵Ca permeability in human red blood cells of the same type as the one achieved by the calcium ionophore. The ionophoric action of β₁_₄₀ and its two fragments may lead to an increase of the intracellular calcium ion concentration, in turn resulting in enhanced Ca²⁺-ATPase activity and a decrease in energy charge. This may be valid also for neuronal plasma membranes and could, therefore, be a possible aetiological mechanism in Alzheimer's disease.