Effects of chlorogenic acid,caffeine and coffee on components of the purinergic system of streptozotocin-induced diabetic rats

We evaluated the effect of chlorogenic acid (CGA), caffeine (CA) and coffee (CF) on components of the purinergic system from the cerebral cortex and platelets of streptozotocin-induced diabetic rats. Animals were divided into eight groups: control animals treated with (I) water (WT), (II) CGA (5 mg/kg), (III) CA (15 mg/kg) and (IV) CF (0.5 g/kg), and diabetic animals treated with (V) WT, (VI) CGA (5 mg/kg), (VII) CA (15 mg/kg) and (VIII) CF (0.5 g/kg). Our results showed an increase (173%) in adenosine monophosphate (AMP) hydrolysis in the cerebral cortex of diabetic rats. In addition, CF treatment increased adenosine diphosphate (ADP) and AMP hydrolysis in group VIII synaptosomes. Platelets showed an increase in ectonucleotidase activity in group V, and all treatments reduced the increase in adenosine triphosphate and ADP hydrolysis. Furthermore, there was an increase in platelet aggregation of 72% in the diabetic rats, and CGA and CF treatment reduced platelet aggregation by nearly 60% when compared to diabetic rats. In this context, we can suggest that CGA and CF treatment should be considered a therapeutic and scientific target to be investigated in diseases associated with hyperglycemia.     

Effect of sarpogrelate on altered STZ-diabetes induced cardiovascular responses to 5-hydroxytryptamine in rats

Sarpogrelate, a specific 5-HT_2A receptor antagonist is reported to produce a number of beneficial cardiovascular effects in diabetes mellitus. In the present investigation we have studied the effects of sarpogrelate on 5-HT receptors in heart and platelets in streptozotocin (STZ)-diabetic rats. Diabetes was induced by a single tail vein injection of STZ (45 mg/kg) and sarpogrelate (1 mg/kg, i.p.) was administered daily for 6 weeks. Injection of STZ produced significant loss of body weight, polyphagia, polydypsia, hyperglycemia, hypoinsulinemia, hypertension and bradycardia. Treatment with sarpogrelate significantly lowered fasting glucose levels with corresponding increase in insulin levels. It also significantly prevented STZ-induced polydypsia, hyperphagia, hypertension, and bradycardia but not the loss of body weight. 5-HT produced dose-dependent positive inotropic effect that was found to be decreased significantly in STZ-diabetic rats. Hearts obtained from sarpogrelate treated diabetic rats did not show any decrease in responsiveness to 5-HT. Relative platelet aggregation per se was found to be higher in STZ-diabetic rats as compared to control and this was significantly prevented by sarpogrelate treatment. 5-HT produced a dose-dependent increase in platelet aggregation in non-diabetic and sarpogrelate treated diabetic rats. However, 5-HT failed to produce any increase in platelet aggregation in untreated diabetic rats. Our data suggest that STZ-induced diabetes may produce down-regulation of cardiac 5-HT_2A receptors and increased platelet aggregation. Treatment with sarpogrelate seems to prevent STZ-induced down-regulation of 5-HT receptors and increase in platelet activity in diabetic rats.     

应激性高血糖与急性自发性脑出血患者术后并发症和早期预后的相关性研究

目的:探讨应激性高血糖与自发性脑出血患者术后并发症及早期预后的关系。方法:回顾性分析我院收治的自发性脑出血患者358例,根据入院时血糖水平、糖化血红蛋白(HbAlc)及既往有无糖尿病史分为血糖正常组(96例)、应激性高血糖组(107例)及糖尿病组(155例),记录和比较各组入院时的血糖、格拉斯哥昏迷评分(GCS)、平均出血量及入院后30 d时各组的术后并发症发生情况、格拉斯哥预后评分(GOS)的差异。结果:糖尿病组入院时血糖水平、平均出血量、重型患者所占比率、脑出血破入脑室、颅内再出血、颅内感染、肺部感染、尿路感染及上消化道出血发生率、GOS分级植物状态或死亡发生率均明显高于应激性高血糖组(P0.05),GOS分级良好率低于应激性高血糖组(P0.05);而应激性高血糖组入院时血糖水平、平均出血量、重型患者所占比率、脑出血破入脑室、颅内再出血发生率、GOS分级植物状态或死亡发生率均明显高于血糖正常组(P0.05)。结论:自发性脑出血患者入院时应激性高血糖与患者的病情显著相关,可加重急性脑出血的不良预后。     

Effects of warfarin and l-carnitine on hemostatic function and oxidative stress in streptozotocin-induced diabetic rats

Diabetes mellitus (DM) is a complex progressive disease characterized by hyperglycemia and a high risk of atherothrombotic disorders affecting the coronary, cerebral, and peripheral arterial trees. Oxidative stress is reported in diabetic patients. We investigated the hemostatic functions and oxidative stress in streptozotocin (STZ)-induced diabetic rats and the effects of warfarin and l-carnitine on those parameters. Forty male Sprague–Dawley rats were divided into four groups: control, DM, and DM received warfarin or l-carnitine. In all rats, blood glucose, insulin, hemoglobin A1c (HbA1c), fibrinogen, factor VII (FVII), plasminogen activator inhibitor-1 (PAI-1), fibrin degradation products (FDP), protein C, antithrombin III (ATIII), malondialdehydes (MDA), and antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione) were measured. Also, prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation time, and platelet aggregation were evaluated. In diabetic rats, plasma glucose, HbA1c, MDA, fibrinogen, FVII, FDP, PAI-1, and platelet aggregation increased while insulin, PT, aPTT, coagulation time, protein C, ATIII, and antioxidants decreased. Warfarin administration to diabetic rats decreased FVII and FDP and increased PT, aPTT, and coagulation time with no effect on MDA, antioxidants, PAI-1, protein C, ATIII, and platelet aggregation. On the other hand, l-carnitine decreased fibrinogen, FVII, FDP, PAI-1, MDA, and platelet aggregation and increased PT, aPTT, coagulation time, protein C, ATIII, and antioxidants in diabetic rats. Therefore, we concluded that hyperglycemia plays an important role in hypercoagulation state and oxidative stress in STZ-induced DM. While l-carnitine improves oxidative stress and decreases the hypercoagulation state in DM, warfarin normalizes the hypercoagulation state with no effect on oxidative stress.     

Rac1 对糖尿病大鼠脑缺血后血管新生的影响机制的研究

目的:通过观察糖尿病大鼠脑缺血后,脑组织内Rac1表达的变化及其对VEGF、SDF-1表达的影响,探讨不同血糖水平时Rac1对脑缺血后血管新生的影响机制。方法:腹腔注射链脲霉素制作糖尿病大鼠脑缺血模型,尾静脉注射Rac1抑制剂NSC23766,通过western blotting法检测脑组织中总Rac1、VEGF和SDF-1表达;通过GST-pulldown法检测脑组织中活性Rac1表达。结果:在糖尿病大鼠脑缺血模型中,VEGF和SDF-1的表达随着血糖水平的升高而下降;脑组织内Rac1蛋白表达随着血糖水平升高而减少;抑制Rac1活性后,脑组织中VEGF表达均下降但SDF-1的表达反而增加。结论:在糖尿病大鼠脑缺血模型中,高血糖可抑制脑组织Rac1、VEGF和SDF-1的水平,该抑制作用随着血糖升高而增加。抑制Rac1水平可抑制VEGF表达但并未抑制SDF-1水平,提示高血糖可能通过抑制Rac1导致VEGF表达下降,从而影响血管新生。     

Antithrombotic effect of onion in streptozotocin-induced diabetic rat

In the present study, we investigated whether onion has antithrombotic effect in streptozotocin (STZ)-induced diabetic rats. In diabetic rats, serum thromboxane B(2) (TXB(2)) level was elevated compared to that in normal, and this elevation in diabetes was significantly inhibited by treatment with onion (0.5 g/ml/kg/day, i.p.) for 4 weeks. In normal rats, the serum TXB(2) level remained unaltered after the treatment with onion. To investigate in vitro effect of onion, we examined its effect on TXB(2) formation, platelet aggregation and arachidonic acid (AA)-release in platelets from diabetic and normal rats. Onion showed a significant inhibitory effect on collagen- or AA-induced TXB(2) formation with greater potency in diabetic platelets than in normal. Similarly, more potent inhibitory effects of onion in diabetes were observed in collagen- or AA-induced platelet aggregation and collagen-induced AA release response. In conclusion, these results suggest that onion can produce more beneficial antithrombotic effect in diabetes.     

The roles of glucagon and adrenal epinephrine in mediating hyperglycemia induced by third cerebroventricular injection of bombesin

The roles of glucagon and adrenal epinephrine in mediating bombesin-induced central hyperglycemia were further studied in anesthetized rats. Bombesin (10(-9) mol) injected into the third cerebral ventricle produced an increase in plasma concentrations of glucose, glucagon, and epinephrine. Prior bilateral adrenalectomy completely prevented the hyperglucagonemic and hyperglycemic responses to third cerebral ventricle injection of bombesin. These results support the view that bombesin-induced increases in plasma glucose and glucagon are fully dependent on adrenal epinephrine secretion. Furthermore, during constant intravenous infusion of somatostatin, the hyperglycemic response to third cerebral ventricle injection of bombesin was not significantly influenced despite complete inhibition of the increase in plasma glucagon. Therefore, it is suggested that bombesin-induced central hyperglycemia is mainly mediated by epinephrine itself rather than via epinephrine-stimulated glucagon secretion.     

Human factor XII binding to the glycoprotein Ib-IX-V complex inhibits thrombin-induced platelet aggregation

Factor XII deficiency has been postulated to be a risk factor for thrombosis suggesting that factor XII is an antithrombotic protein. The biochemical mechanism leading to this clinical observation is unknown. We have previously reported high molecular weight kininogen (HK) inhibition of thrombin-induced platelet aggregation by binding to the platelet glycoprotein (GP) Ib-IX-V complex. Although factor XII will bind to the intact platelet through GP Ibalpha (glycocalicin) without activation, we now report that factor XIIa (0. 37 microm), but not factor XII zymogen, is required for the inhibition of thrombin-induced platelet aggregation. Factor XIIa had no significant effect on SFLLRN-induced platelet aggregation. Moreover, an antibody to the thrombin site on protease-activated receptor-1 failed to block factor XII binding to platelets. Inhibition of thrombin-induced platelet aggregation was demonstrated with factor XIIa but not with factor XII zymogen or factor XIIf, indicating that the conformational exposure of the heavy chain following proteolytic activation is required for inhibition. However, inactivation of the catalytic activity of factor XIIa did not affect the inhibition of thrombin-induced platelet aggregation. Factor XII showed displacement of biotin-labeled HK (30 nm) binding to gel-filtered platelets and, at concentrations of 50 nm, was able to block 50% of the HK binding, suggesting involvement of the GP Ib complex. Antibodies to GP Ib and GP IX, which inhibited HK binding to platelets, did not block factor XII binding. However, using a biosensor, which monitors protein-protein interactions, both HK and factor XII bind to GP Ibalpha. Factor XII may serve to regulate thrombin binding to the GP Ib receptor by co-localizing with HK, to control the extent of platelet aggregation in vivo.     

Zinc deficiency in rats decreases thrombin-stimulated platelet aggregation by lowering protein kinase C activity secondary to impaired calcium uptake

Aggregation of rat platelets, when stimulated by adenosine diphosphate (ADP) or fluoride, is impaired by zinc deficiency, and the defect is associated with a decreased uptake of external Ca²⁺. Zinc deficiency also impairs the aggregatory response of platelets to phorbol myristate acetate (PMA), an activator of protein kinase C, but low zinc status decreases the PMA response only when calcium is added to the external medium. The purpose of this study was to determine the role of protein kinase C in rat platelet function and its relationship to the zinc deficiency pathology observed in platelets stimulated by thrombin (THR). The percent of maximal aggregation and the concentration of cytosolic-free Ca²⁺ were measured in washed platelets stimulated by THR and PMA. For the protein kinase C experiments platelets were obtained from rats fed a grain-based diet, and for the thrombin experiments they were from rats fed purified diets. In the latter experiments, immature male rats were fed for 2 weeks a low zinc diet (<1 mg/kg) ad libitum or a zinc adequate (100 mg/kg) diet either ad libitum or pair-fed. Zinc deficiency impaired the aggregation of platelets stimulated by 0.045 U/mL of THR by approximately 40%, and the external calcium uptake (0.03 U/mL of THR) was decreased by approximately 30%. Staurosporine, a protein kinase C inhibitor, decreased thrombin-induced aggregation in a concentration-dependent manner, but it had no effect on the external calcium uptake. While PMA had a synergistic effect with thrombin in the stimulation of platelet aggregation, it actually decreased the cytosolic-free calcium response to thrombin. It is concluded that zinc deficiency impairs thrombin-stimulated platelet aggregation and calcium uptake and that protein kinase C activity is essential for rat platelet aggregation. Protein kinase C does not stimulate calcium uptake and must act downstream of the calcium uptake defect. A model of rat platelet activation is presented depicting impaired Ca²⁺ uptake as the primary defect in zinc deficiency.     

Differential role of protein kinase C delta isoform in agonist-induced dense granule secretion in human platelets

Several platelet agonists, including thrombin, collagen, and thromboxane A(2), cause dense granule release independently of thromboxane generation. Because protein kinase C (PKC) isoforms are implicated in platelet secretion, we investigated the role of individual PKC isoforms in platelet dense granule release. PKCdelta was phosphorylated in a time-dependent manner that coincided with dense granule release in response to protease-activated receptor-activating peptides SFLLRN and AYPGKF in human platelets. Only agonists that caused platelet dense granule secretion activated PKCdelta. SFLLRN- or AYPGKF-induced dense granule release and PKCdelta phosphorylation occurred at the same respective agonist concentration. Furthermore, AYPGKF and SFLLRN-induced dense granule release was blocked by rottlerin, a PKCdelta selective inhibitor. In contrast, convulxin-induced dense granule secretion was potentiated by rottlerin but was abolished by Go6976, a classical PKC isoform inhibitor. However, SFLLRN-induced dense granule release was unaffected in the presence of Go6976. Finally, rottlerin did not affect SFLLRN-induced platelet aggregation, even in the presence of dimethyl-BAPTA, indicating that PKCdelta has no role in platelet fibrinogen receptor activation. We conclude that PKCdelta and the classical PKC isoforms play a differential role in platelet dense granule release mediated by protease-activated receptors and glycoprotein VI. Furthermore, PKCdelta plays a positive role in protease-activated receptor-mediated dense granule secretion, whereas it functions as a negative regulator downstream of glycoprotein VI signaling.     

Effect of long-term and short-term diabetes on the parathyroid hormone sensitive rat renal adenylate cyclase: correlation with vitamin D metabolism

In short-term experiments, male Wistar rats were made diabetic for 10 days with a single injection of streptozotocin (65 mg/kg body weight). One group of diabetic rats was treated with insulin for 3 days prior to sacrifice. In long-term experiments, vitamin D replete or vitamin D depleted rats were made diabetic for 6 weeks. Criteria for diabetes were loss of weight, glycosuria (Tes-Tape), and hyperglycemia. In long-term diabetic rats the activity of renal mitochondrial 25-hydroxyvitamin D3 (25-(OH)D3) 1 alpha-hydroxylase was significantly decreased and that of 25-(OH)D3 24-hydroxylase increased. However, the parathyroid hormone (PTH) sensitive renal adenylate cyclase activity of diabetic rats was not different from that of the nondiabetic rats in either the vitamin D replete group or the vitamin D depleted group. On the other hand, the PTH-sensitive renal adenylate cyclase activity was significantly higher in short-term diabetic rats than in control and insulin-treated rats. These differences were observed at doses of 10(-8) to 10(-5) M of PTH. This study has demonstrated for the first time that there are differences in the PTH-sensitive adenylate cyclase response between long-term and short-term diabetic rats. The hypersensitivity to PTH of the renal adenylate cyclase observed in short-term diabetic rats probably represents a response to insulin deficiency during the early development of diabetes mellitus in the rats.     

Bm-TFF2, a trefoil factor protein with platelet activation activity from frog Bombina maxima skin secretions

In mammals, trefoil factor family (TFF) proteins are involved in mucosal maintenance and repair, and they are also implicated in tumor suppression and cancer progression. A novel two domain TFF protein from frog Bombina maxima skin secretions (Bm-TFF2) has been purified and cloned. It activated human platelets in a dose-dependent manner and activation of integrin alpha(IIb)beta(3) was involved. Aspirin and apyrase did not largely reduce platelet response to Bm-TFF2 (a 30% inhibition), indicating that the aggregation is not substantially dependent on ADP and thromboxane A2 autocrine feedback. Elimination of external Ca(2+) with EGTA did not influence the platelet aggregation induced by Bm-TFF2, meanwhile a strong calcium signal (cytoplasmic Ca(2+) release) was detected, suggesting that activation of phospholipase C (PLC) is involved. Subsequent immunoblotting revealed that, unlike in platelets activated by stejnulxin (a glycoprotein VI agonist), PLCgamma2 was not phosphorylated in platelets activated by Bm-TFF2. FITC-labeled Bm-TFF2 bound to platelet membranes. Bm-TFF2 is the first TFF protein reported to possess human platelet activation activity.     

Inhibition of human tumor cell induced platelet aggregation by antibodies to platelet glycoproteins Ib and IIb/IIIa

Tumor cell induced platelet aggregation was shown to be inhibited in a dose dependent manner by preincubation of human platelets with antibodies to platelet glycoprotein Ib and the IIb/IIIa complex. Combination of antibody to Ib and antibody to the IIb/IIIa complex at concentrations which produced half maximal inhibition of platelet aggregation alone caused complete inhibition of tumor cell induced platelet aggregation. Antibodies to platelet glycoproteins Ib and the IIb/IIIa complex also inhibited platelet synthesis of thromboxane A2, but not synthesis of 12-hydroxyeicosatrienoic acid. Inhibition of tumor cell induced platelet aggregation with antibodies against platelet glycoproteins suggests a role for these glycoproteins in tumor cell-platelet interactions and possibly platelet facilitated tumor cell metastasis.     

Effects of Timing of Dexamethasone Treatment on the Outcome of Collagenase-Induced Intracerebral Hematoma in Rats

The effect of timing in providing dexamethasone treatment after intracerebral hematoma was evaluated in rats with hematoma induced by a subcortical collagenase injection. Male Sprague–Dawley rats (n = 30; body weight, 185 to 230 g) received dexamethasone (1 mg/kg) intraperitoneally at 2 h, 4 h, or 6 h (1 group per time point) after intracerebral collagenase injection, with another dose (1 mg/kg) administered at 24 h after collagenase injection. Neurologic examinations and rotarod treadmill tests were used to evaluate motor behavior before and at 24 and 48 h after intracerebral injection. Rats were euthanized after the last behavioral test. Brains were evaluated for hematoma size, number of penumbral necrotic neurons, neutrophils within the hematoma, and astrocytic response. Compared with the control and other treatment groups, rats treated with dexamethasone at 2 and 24 h after intracerebral collagenase injection scored significantly better on neurologic exams and rotarod tests. Hematoma volume was significantly smaller in all treated groups than in the control group but did not differ between treatment groups. Fewer neutrophils were seen in the perihematoma region of all treated rats compared with controls, but the number of necrotic neurons was decreased significantly only in the group treated with dexamethasone at 2 and 24 h. These results indicate that a 1-mg/kg dose of dexamethasone is beneficial for treatment of intracerebral hemorrhage, particularly if administered early after the hemorrhagic insult.Traumatic cerebral hemorrhages, which in veterinary patients are caused mainly by automobile accidents and falls, are diagnostic and therapeutic challenges.¹⁵ Animal models have played an important role in elucidating the cascade of cellular and biochemical events occurring after traumatic brain injury.⁷ These models have helped to elucidate various aspects of the pathogenesis and treatment of intracranial hemorrhaging. Among these, the collagenase-induced intracerebral hematoma model¹⁴ is highly reproducible and shows many characteristics of the intracerebral hemorrhagic process in mammals. In addition, the short- and long-term histologic and behavioral changes associated with this model have been evaluated.^5,6 Compared with the blood infusion model, the collagenase model causes greater primary injury that occurs distal to the hematoma, and neurologic deficits resolve less rapidly over time, making the collagenase model more appealing for long-term studies.¹²The efficacy of corticosteroids for the treatment of brain hemorrhages has been evaluated in different experimental studies with conflicting results.^3,8-11 Administration of corticosteroids at 1 h after hematoma induction is beneficial for the treatment of this condition.^10,11,16 Although these studies have promising results, the interval between trauma and treatment might greatly influence the response to corticosteroid treatment. The aim of this study was to assess the motor performance and histopathology associated with dexamethasone treatment at 2, 4, and 6 h after hematoma induction in the intracranial collagenase rat model. A second dexamethasone dose was administered 24 h after collagenase injection.     

Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans

To facilitate feeding, certain hematophagous invertebrates possess inhibitors of collagen-induced platelet aggregation in their saliva. However, their mechanisms of action have not been fully elucidated. Here, we describe two major salivary proteins, triplatin-1 and -2, from the assassin bug, Triatoma infestans, which inhibited platelet aggregation induced by collagen but not by other agents including ADP, arachidonic acid, U46619 and thrombin. Furthermore, these triplatins also inhibited platelet aggregation induced by collagen-related peptide, a specific agonist of the major collagen-signaling receptor glycoprotein (GP)VI. Moreover, triplatin-1 inhibited Fc receptor gamma-chain phosphorylation induced by collagen, which is the first step of GPVI-mediated signaling. These results strongly suggest that triplatins target GPVI and inhibit signal transduction necessary for platelet activation by collagen. This is the first report on the mechanism of action of collagen-induced platelet aggregation inhibitors from hematophagus invertebrates.     

Lipid-induced beta-cell dysfunction in vivo in models of progressive beta-cell failure

We determined the effect of 48-h elevation of plasma free fatty acids (FFA) on insulin secretion during hyperglycemic clamps in control female Wistar rats (group a) and in the following female rat models of progressive beta-cell dysfunction: lean Zucker diabetic fatty (ZDF) rats, both wild-type (group b) and heterozygous for the fa mutation in the leptin receptor gene (group c); obese (fa/fa) Zucker rats (nonprediabetic; group d); obese prediabetic (fa/fa) ZDF rats (group e); and obese (fa/fa) diabetic ZDF rats (group f). FFA induced insulin resistance in all groups but increased C-peptide levels (index of absolute insulin secretion) only in obese prediabetic ZDF rats. Insulin secretion corrected for insulin sensitivity using a hyperbolic or power relationship (disposition index or compensation index, respectively, both indexes of beta-cell function) was decreased by FFA. The decrease was greater in normoglycemic heterozygous lean ZDF rats than in Wistar controls. In obese "prediabetic" ZDF rats with mild hyperglycemia, the FFA-induced decrease in beta-cell function was no greater than that in obese Zucker rats. However, in overtly diabetic obese ZDF rats, FFA further impaired beta-cell function. In conclusion, 1) the FFA-induced impairment in beta-cell function is accentuated in the presence of a single copy of a mutated leptin receptor gene, independent of hyperglycemia. 2) In prediabetic ZDF rats with mild hyperglycemia, lipotoxicity is not accentuated, as the beta-cell mounts a partial compensatory response for FFA-induced insulin resistance. 3) This compensation is lost in diabetic rats with more marked hyperglycemia and loss of glucose sensing.     

Characterization of platelet abnormalities of Tester Moriyama (TM) rats with storage pool deficiency

Platelet abnormalities of Tester Moriyama (TM) rats, which have prolonged bleeding time with normal platelet count, were characterized by comparison with those of fawn-hooded (FH) rats with platelet storage pool deficiency (SPD). Morphologically, the dense granules were virtually lacking in platelets from TM and FH rats. Platelets from TM and FH rats aggregated in response to adenosine diphosphate (ADP), but failed to have secondary aggregation. In contrast, platelet aggregation was completely absent in response to 1 to 20 micrograms of collagen/ml, although partial aggregation was observed at the higher dosage of 50 micrograms/ml. Normal amounts of platelet membrane glycoproteins IIb/IIIa were expressed in TM and FH rats, but platelet adenosine triphosphate (ATP) and ADP contents were lower than those in platelets from control Wistar rats. Platelet ATP-to-ADP ratio of TM and FH rats was significantly higher than that of Wistar rats. Serotonin content in platelets from TM and FH rats was 20 to 25% that of Wistar rat platelets. These results suggested that platelet abnormalities of TM rats are a typical characteristic of platelet SPD and are similar to those of FH rats, which are genetically different from TM rats. Therefore, TM rats may serve as a useful animal model for the study of platelet SPD.     

Peroxiredoxin II Is an Antioxidant Enzyme That Negatively Regulates Collagen-stimulated Platelet Function

Collagen-induced platelet signaling is mediated by binding to the primary receptor glycoprotein VI (GPVI). Reactive oxygen species produced in response to collagen have been found to be responsible for the propagation of GPVI signaling pathways in platelets. Therefore, it has been suggested that antioxidant enzymes could down-regulate GPVI-stimulated platelet activation. Although the antioxidant enzyme peroxiredoxin II (PrxII) has emerged as having a role in negatively regulating signaling through various receptors by eliminating H₂O₂ generated upon receptor stimulation, the function of PrxII in collagen-stimulated platelets is not known. We tested the hypothesis that PrxII negatively regulates collagen-stimulated platelet activation. We analyzed PrxII-deficient murine platelets. PrxII deficiency enhanced GPVI-mediated platelet activation through the defective elimination of H₂O₂ and the impaired protection of SH2 domain-containing tyrosine phosphatase 2 (SHP-2) against oxidative inactivation, which resulted in increased tyrosine phosphorylation of key components for the GPVI signaling cascade, including Syk, Btk, and phospholipase Cγ2. Interestingly, PrxII-mediated antioxidative protection of SHP-2 appeared to occur in the lipid rafts. PrxII-deficient platelets exhibited increased adhesion and aggregation upon collagen stimulation. Furthermore, in vivo experiments demonstrated that PrxII deficiency facilitated platelet-dependent thrombus formation in injured carotid arteries. This study reveals that PrxII functions as a protective antioxidant enzyme against collagen-stimulated platelet activation and platelet-dependent thrombosis.     

Aegyptin, a novel mosquito salivary gland protein, specifically binds to collagen and prevents its interaction with platelet glycoprotein VI, integrin alpha2beta1, and von Willebrand factor

Blood-sucking arthropods have evolved a number of inhibitors of platelet aggregation and blood coagulation. In this study we have molecularly and functionally characterized aegyptin, a member of the family of 30-kDa salivary allergens from Aedes aegypti, whose function remained elusive thus far. Aegyptin displays a unique sequence characterized by glycine, glutamic acid, and aspartic acid repeats and was shown to specifically block collagen-induced human platelet aggregation and granule secretion. Plasmon resonance experiments demonstrate that aegyptin binds to collagen types I-V (K(d) approximately 1 nm) but does not interact with vitronectin, fibronectin, laminin, fibrinogen, and von Willebrand factor (vWf). In addition, aegyptin attenuates platelet adhesion to soluble or fibrillar collagen. Furthermore, aegyptin inhibits vWf interaction with collagen type III under static conditions and completely blocks platelet adhesion to collagen under flow conditions at high shear rates. Notably, aegyptin prevents collagen but not convulxin binding to recombinant glycoprotein VI. These findings suggest that aegyptin recognizes specific binding sites for glycoprotein VI, integrin alpha2beta1, and vWf, thereby preventing collagen interaction with its three major ligands. Aegyptin is a novel tool to study collagen-platelet interaction and a prototype for development of molecules with antithrombotic properties.     

Protein kinase C-theta in platelet activation

Members of the protein kinase C (PKC) family of serine/threonine kinases have been implicated in several physiological processes regulating the activation response of platelets. They are involved in processes leading to granule secretion, integrin activation, platelet aggregation and spreading, and procoagulation. The protein kinase C θ (PKCθ) isoform, which was originally identified in T lymphocytes, is also expressed at relatively high levels in platelets, wherein it is involved in the regulation of hemostasis and thrombosis. Recent studies suggest a role for PKCθ in protease-activated receptor (PAR)-, glycoprotein VI (GPVI) receptor- and glycoprotein α(IIb)β(3) integrin receptor-linked signal transduction pathways. The present review focuses on the latest observations relevant to the role of PKCθ in platelet activation.