Platelet Fibrinogen Binding Research Articles

Conformational Studies of the 313-320 and 313-332 Peptide Fragments Derived from the αIIb Subunit of Integrin Receptor with Molecular Dynamics Simulations

The peptide sequence YMESRADRKLAEVGRVYLFL, derived from 313-332 region of the αIIb, has been identified as a potent inhibitor of platelet aggregation and fibrinogen binding to αIIbβ3. More detailed studies have revealed that the Y313MESRADR320 sequence is the shortest octapeptide with strong inhibitory activity. This work provides insight of the solution conformation of these peptides, by performing extensive molecular dynamics simulations of 100 ns. The 8mer peptide has no stable conformation in water while the 20mer peptide retains a relative conformational stability. Analysis of side chain orientation of the RAD fragment revealed the synplanar arrangement of guanidinium and β-carboxylic groups providing a framework for explaining the similar biological activity of the two peptides, despite their differences in sequence and conformation.

Effect of a Synthetic Peptide Corresponding to Residues 313 to 320 of the αIIbSubunit of the Human Platelet Integrin αIIbβ3on Carotid Artery Thrombosis in Rabbits

The platelet integrin receptor alpha(IIb)beta(3) plays a critical role in thrombosis. We have shown previously that the octapeptide YMESRADR, corresponding to sequences 313 to 320 of the human alpha(IIb) subunit, inhibits human platelet activation and fibrinogen binding to alpha(IIb)beta(3), possibly interacting with the ligand. We investigated the effect of YMESRADR on electrically induced carotid artery thrombosis in New Zealand white rabbits. Peptide was administered via the femoral vein, starting 60 min before and continuing for 90 min after the electrical stimulation. Carotid blood flow was monitored for 90 min after the electrical stimulation. The peptide effects on platelet aggregation, in vitro and ex vivo, and on various coagulation, bleeding, and hemostatic parameters were evaluated. YMESRADR significantly inhibited rabbit platelet aggregation in vitro in a dose-dependent manner. It is important that peptide administration in vivo, at doses ranging from 3 to 15 mg/kg, prolonged the duration of the patency of the carotid artery, and no artery occlusion was observed until the end of the study (90 min after electrical stimulation). Furthermore, YMESRADR administration reduced platelet aggregation ex vivo and thrombus weight; however, these reductions reached statistical significance, compared with the control group, at the peptide doses of 12 and 15 mg/kg. YMESRADR did not affect any coagulation parameter studied and the hemostatic response observed in control animals. Thus, YMESRADR represents a novel antiplatelet agent that can inhibit thrombus formation effectively and carotid artery occlusion without causing hemorrhagic complications in a rabbit model of arterial thrombosis.

Pro-thrombotic changes in platelet, endothelial and coagulation function following haemodialysis

Purpose: Patients on hemodialysis (HD) have an increased risk of thrombotic events, including myocardial infarction and vascular access thrombosis. The study hypothesis is that a single session of dialysis leads to platelet, endothelial & coagulation activation. Our aim is to determine the effect of a single HD session on prothrombotic vascular biomarkers before and after a single session of hemodialysis. Methods: Blood samples were taken from the vascular access of 55 patients immediately before and after a hemodialysis session. Platelet function was assessed by (1) flow cytometric measurement of P-selectin expression and fibrinogen binding ± ADP stimulation, (2) Ultegra rapid platelet function assay (RPFA) using the agonists thrombin receptor activating peptide (TRAP) and arachidonic acid (AA), (3) soluble P-selectin, and (4) soluble CD40L. Coagulation (thrombin-antithrombin III [TAT] and D-dimer), endothelial von Willebrand factor (vWF) and high sensitivity C-Reactive protein (hsCRP) were assessed by ELISA. Results: Unfractionated heparin was given to all patients during dialysis and 30 patients (55%) were on antiplatelet agents. Post-hemodialysis there were significant increases in unstimulated platelet P-selectin (p=.037), stimulated P-selectin (p<.001), soluble P-selectin (p<.001) and soluble CD40L (p=.036). Stimulated platelet fibrinogen binding was increased post-hemodialysis (p<.001) but unstimulated fibrinogen binding was unchanged. TRAP- (p<.001) and AA-(p=.009) stimulated aggregation were reduced post-hemodialysis. There were increases post-hemodialysis in TAT (p<.001), D-dimer (p<.001), vWF (p<.001) and hsCRP (p=.011). Conclusion: This study has shown that despite heparin therapy, a single session of HD induced increases in platelet, endothelial, and coagulation activation. More effective medical strategies to reduce the prothrombotic state of patients on hemodialysis should be investigated.

Effect of sex difference on platelet adhesion, spreading and aggregate formation under flow

There are clear but poorly understood differences in the etiology and prognosis of thrombotic diseases in men and women. Due to the fact that platelets play a central role in the formation of occlusive thrombi in atherosclerotic coronary arteries, previous studies have examined whether sex differences exist for platelets, and have obtained conflicting results. Additionally, due to the increased use of genetically modified mouse models to explore the molecular mechanisms underlying platelet activation and thrombotic disorders, it is critical to determine if sex is a confounding variable. Our study of the role of sex differences in platelet function was designed to utilise purified platelets from inbred paired female/male littermates in order to minimise genetic and environmental variability. In the current study, we demonstrate that platelet adhesion to and spreading on immobilised fibrinogen, thrombin or collagen was equivalent for both female and male mouse platelets. The ability of the soluble agonist thrombin or convulxin to potentiate platelet P-selectin exposure, fibrinogen binding, or adhesion and spreading on immobilised fibrinogen was equivalent for both female and male mouse platelets. Our data show that an equivalent degree of platelet adhesion and aggregation on collagen or fibrinogen under shear flow was observed for both female and male mouse platelets. In conclusion, our data would argue against an intrinsic difference for female mouse platelets in regulating the major functional platelet responses: platelet adhesion, spreading, or aggregation under flow.

Inhibition of platelet activation by peptide analogs of the β3-intracellular domain of platelet integrin αIIbβ3 conjugated to the cell-penetrating peptide Tat(48–60)

Activation of the platelet integrin-receptor αIIbβ3 is the final pathway of platelet aggregation, regardless of the initiating stimulus. Many studies suggest that there are several cytoplasmic proteins such as talin and β3-endonexin that bind to N744PLY747 and N756ITY759 motif of the β3 cytoplasmic tail and play the major role in the receptor activation. In this study, we investigated the role of the membrane distal region of human β3 cytoplasmic tail and specifically the N743NPLYKEA750 and T755NITYRGT762 sequence that contains an NXXY motif, in platelet aggregation, secretion, αIIbβ3 activation (PAC-1 binding) and fibrinogen binding. We synthesized two peptides corresponding to the above sequences as well as their conjugates with the Tat(48–60) cell-penetrating peptide. The capability of conjugates to penetrate the platelet membrane was investigated with confocal laser scanning microscopy using carboxyfluorescein (CF)-labeled peptides. Our results showed that the conjugated with the Tat(48–60) sequence peptides penetrate the platelet membrane and inhibit platelet aggregation in both PRP and washed platelets in a dose-dependent manner. The Tat-β3743–750 conjugate exhibited similar inhibitory activity in PRP and in washed platelets whereas the Tat-β3755–762 conjugate was more potent inhibitor of aggregation in washed platelets than in PRP. Both conjugated peptides were also able to inhibit P-selectin membrane expression as well as PAC-1 and fibrinogen binding to the platelets, the Tat-β3755–762 conjugate being more potent than Tat-β3743–750. The Tat(48–60) peptide and the peptides β3743–750 and β3755–762, which were not conjugated to the Tat(48–60) sequence, did not exhibit any inhibitory effect on the above parameters. In conclusion, the present study shows for the first time that the peptide analogs of the intracellular domain of the β3 subunit β3743–750 and β3755–762 conjugated to the cell-penetrating peptide Tat(48–60) are capable of penetrating the platelet membrane and expressing biological activity by inhibiting the activation of αIIbβ3, the fibrinogen binding to the activated receptor as well as platelet aggregation. Further studies are necessary to support whether such conjugated peptides may be useful tools for the development of potent antiplatelet agents acting intracellularly through the platelet integrin αIIbβ3.

HES 130/0.4 impairs haemostasis and stimulates pro-inflammatory blood platelet function

IntroductionHydroxyethyl starch (HES) solutions are widely used for volume replacement therapy but are also known to compromise coagulation, impair renal function and increase long-term mortality. To test the hypotheses that HES 130/0.4 has fewer adverse effects than HES 200/0.5 and exerts anti-inflammatory properties, we compared the effects of HES 130/0.4, HES 200/0.5 and saline on in vitro haemostasis and pro-inflammatory platelet function.MethodsWhole blood samples from healthy volunteers were mixed with 6% HES 130/0.4, 10% HES 200/0.5, or normal saline to achieve a final haemodilution rate of 10% or 40%. Haemostatic capacity was characterised by thromboelastography (ROTEM) and measurement for FXIIIa activity. Platelet activation and pro-inflammatory platelet functions were characterised by flow cytometry measuring the platelet activation marker CD62P and binding of fibrinogen to platelets as well as the formation of heterotypic platelet-leukocyte conjugates.ResultsCompared with saline, HES 130/0.4 dose-dependently impaired formation and firmness of the fibrin clot but did not affect the fibrin crosslinking activity of FXIIIa. At 40% but not at 10% haemodilution rate, HES 200/0.5 also increased platelet fibrinogen binding and both HES solutions increased expression of CD62P, the main receptor for platelet-leukocyte adhesion. HES 130/0.4 but not HES 200/0.5 increased formation of platelet-neutrophil conjugates and, to a lesser degree, platelet-monocyte conjugates.ConclusionsOur data demonstrate that HES 130/0.4 has similar adverse effects as HES 200/0.5. In particular, both types of HES impair coagulation capacity and stimulate, rather than attenuate, pro-inflammatory platelet function.

Effectiveness of Reloading to Overcome Clopidogrel Nonresponsiveness in Patients With Acute Myocardial Infarction

Whether increasing doses of clopidogrel can overcome nonresponsiveness was evaluated. Clopidogrel nonresponsiveness was found in up to 25% of treated patients and was associated with worse prognosis in patients with acute coronary syndrome and patients undergoing coronary intervention. Adenosine diphosphate (ADP)-induced platelet aggregation was prospectively determined on day 4 of acute myocardial infarction in 200 consecutive patients, who received clopidogrel 300 mg as a loading dose and 75 mg/day thereafter. Thirty patients (15%) had ADP-induced platelet aggregation >or=80% using light transmittance aggregometry and were considered clopidogrel nonresponders. Nonresponders were reloaded with clopidogrel 600 mg, followed by 150 mg/day for 4 weeks. A 75-mg/day dose was resumed thereafter. ADP-induced platelet aggregation was reassessed 4 hours after reloading and biweekly for 10 weeks. Flow cytometry was used to determine platelet P-selectin expression and fibrinogen binding before and 4 hours after reloading. ADP-induced platelet aggregation significantly decreased 4 hours after reloading (from 83 +/- 6% to 56 +/- 14%; p <0.01). The decrease in platelet aggregation was maintained throughout the 4-week doubled maintenance dose. After resuming a maintenance dose of 75 mg/day, ADP-induced platelet aggregation returned to 66 +/- 12% (p <0.001), and 5 patients (17%) had ADP-induced platelet aggregation >or=80%. Flow cytometry showed a significant decrease in P-selectin expression (from 37 +/- 16% to 26 +/- 13%; p <0.01) and fibrinogen binding (from 84 +/- 7% to 70 +/- 13%; p <0.01) in ADP-stimulated platelets 4 hours after reloading. In conclusion, clopidogrel reloading and increased maintenance dose may overcome clopidogrel nonresponsiveness in patients with acute myocardial infarction.

Conjugation to Hyperbranched Polyglycerols Improves RGD-Mediated Inhibition of Platelet Function in Vitro

RGD (arginine-glycine-aspartic acid) is a known peptide sequence that binds platelet integrin GPIIbIIIa and disrupts platelet-fibrinogen binding and platelet cross-linking during thrombosis. RGD peptides are unsuitable for clinical applications due to their high 50% inhibitory concentration (IC50) and low in vivo residence times. We addressed these issues by conjugating RGD peptides to biocompatible macromolecular carriers: hyperbranched polyglycerols (HPG) via divinyl sulfone. The GPIIbIIIa binding activity of RGD was maintained after conjugation and the effectiveness of the HPG-RGD conjugate was dependent upon molecular weight and the number of RGD peptides attached to each HPG molecule. These polyvalent inhibitors of platelet aggregation decreased the IC50 of RGD in an inverse linear manner based on the number of RGD peptides per HPG. Since HPG-RGD conjugates do not cause platelet activation by degranulation and certain substitution ratios do not increase fibrinogen binding to resting platelets, HPG-RGD may serve as a model for a novel class of antithrombotics.

RGD-Substituted High Molecular Weight Hyper-Branched Polyglycerols (HPG) Are Effective Platelet Inhibitors.

Peptides containing Arg-Gly-Asp (RGD) sequences are known to bind to integrins which mediate cell adhesion and therefore have been utilized in applications such as antithrombotics and tissue engineering. Although RGD and related peptides show promise, their unfavourable pharmacokinetic profiles and susceptibility to in vivo proteolysis hinder their clinical usefulness. Peptide-polymer conjugates can address one of these challenges by extending the peptide's residence in plasma. Hyperbranched polyglycerols (HPGs) are biocompatible polyether polyols with very long plasma circulation half-lives (t1/2 ∼ 60h) and as such are ideally suited to be carriers in such conjugates. HPGs of three different molecular weights were conjugated with RGD peptides at various substitution levels. Some of the terminal hydroxyl groups of polyglycerols were converted to vinyl sulfone groups which were subsequently utilized to couple cysteine terminated RGD peptides. The following conjugates were made: 515 kDa with substitution levels of 100:1, 500:1 and 1000:1; 100 kDa with substitution levels of 100:1 and 1000:1; and 3 kDa with substitution levels of 1:1, and 10:1. RGD-coupled HPG inhibited fibrinogen binding to platelet glycoprotein IIb-IIIa as detected by flow cytometry using anti-fibrinogen. Compared to free RGD (Ic50 = 5 x 10-5 M), inhibition of fibrinogen binding increased with increasing HPG molecular weight and increasing RGD-substitution for the high MW conjugates: Ic50 for the 515 kDa conjugates were 6.3 x 10−8 M for the 100:1 substitution, 4.7 x 10-8 M for the 500:1 substitution, and 4.1 x 10−8 M for 1000:1 substitution; Ic50 for the 100 kDa conjugates were 7.0 x 10−7 M for the 100:1 substitution and 1.2 x 10−7 M for the 1000:1 substitution. Ic50 for the 3 kDa conjugates were 2 x 10−5 M for the 1:1 substitution, 2 x 10−4 M for 10:1 substitution. Similarly, platelet function, as demonstrated by MnCl2-initated aggregation was inhibited in a dose- and molecular weight-dependent manner by HPG-RGD conjugates. Platelet aggregate formation and aggregate size were confirmed by microscopy. However, unsubstituted HPG had no effect on platelet fibrinogen binding and neither conjugated nor unconjugated HPG increased platelet CD62 surface expression. Trypsin digestion (but not SBTI-treated trypsin) removed the inhibitory activity of the conjugates and thus confirmed that both fibrinogen binding and aggregation-inhibition were dependent on the RGD peptide sequence. Such multi-determinant peptide-HPG conjugates represent a new approach to the development of antithrombotic drugs.

Platelet PAR1 receptor density—Correlation to platelet activation response and changes in exposure after platelet activation

IntroductionA polymorphism (-14 A/T) affecting PAR1 expression on the platelet surface has recently been identified. A two-fold variation in receptor density, which correlated with the platelet response to PAR1-activating peptide (PAR1-AP), has been reported. Materials and methodsWe used flow cytometry to measure the correlation between the number of PAR1 receptors and platelet activation. We also measured the changes in receptor exposure after platelet activation with PAR1-AP, ADP, PAR4-AP or a collagen-related peptide (CRP). ResultsIn our study, the PAR1 receptor number varied almost four-fold, from 547 to 2063 copies/platelet (mean±S.D. 1276±320, n=70). The number of PAR1 receptors on resting platelets correlated to platelet fibrinogen binding and P-selectin expression following platelet activation with PAR1-AP (r2=0.30, p<0.01 and r2=0.15, p<0.05, respectively, n=36). The correlation was not improved by exclusion of the ADP-component from the PAR1-AP-induced response. We found a trend, but no statistically significant differences in PAR1 receptor number and platelet reactivity between A/A individuals and T/A or T/T individuals. Ex vivo activation with PAR1-AP decreased PAR1 surface exposure to 71±19% of the exposure on resting platelets (mean±S.D., p<0.01, n=19), while activation by ADP, PAR4-AP or CRP significantly increased the exposure, to 151±27%, 120±21% and 138±25%, respectively (n=11, 11 and 10). ConclusionsThis study shows a large variation in PAR1 receptor number in healthy individuals, a variation correlated to the platelet activation response. We found a significant reduction in PAR1 surface exposure after adding PAR1-AP, while activation with ADP, PAR4-AP or CRP increased the exposure.

Prevalence of reduced fibrinogen binding to platelets in a population of Thoroughbreds

To measure the frequency and magnitude of reduced fibrinogen binding in a population of horses from a Thoroughbred breeding farm. 444 Thoroughbred horses, 1 to 27 years old, including 316 females, 72 geldings, and 56 sexually intact males. Blood was collected from horses into tubes containingacid citrate dextrose adenine, and washed platelets were examined by use of flow cytometry for their ability to bind fibrinogen. Data regarding fibrinogen binding to activated platelets were normally distributed, with nearly identical amounts of variation regardless of sex. In 3 horses, fibrinogen binding to platelets was reduced from 67.6% to 83.4%, compared with normal platelets, which indicated an inability of platelets to aggregate in response to thrombin (0.1 U/mL). Platelet fibrinogen binding of the affected horses identified in this study was characteristic of a reported heritable bleeding disorder in which the reduction in fibrinogen binding correlated with prolonged bleeding times in template bleeding assays. The bleeding disorder is distinct from Glanzmann thrombasthenia, in which platelets fail to bind fibrinogen because of lack of alphallb-beta3 integrin on their surface. The prevalence of affected horses within the small sample population studied here (0.7% [n = 3]) is considerably higher than the prevalence of bleeding disorders within more genetically diverse groups.

Parnaparin, a low-molecular-weight heparin, prevents P-selectindependent formation of platelet-leukocyte aggregates in human whole blood

Parnaparin, a low-molecular-weight heparin (LMWH), prevents platelet activation and interaction with polymorphonuclear leukocyte (PMN) in a washed cell system. The in-vitro effect of parnaparin was studied here on platelet-PMN aggregates formed with more physiologic approaches in whole blood, in parallel with unfractionated heparin and enoxaparin, another LMWH. Citrated blood from healthy subjects was stimulated: i) from passage through the "Platelet Function Analyzer" (PFA-100), a device that exposes blood to standardized high shear flow through collagen/ADP cartridges; ii) by collagen and ADP (2 and 50 mug/ml, respectively) added in combination under stirring in an aggregometer cuvette; iii) with recombinant Tissue Factor, to generate thrombin concentrations able to activate platelets without inducing blood clotting, or iv) the Thrombin Receptor Activating Peptide-6 (TRAP-6). Platelet P-selectin and platelet-PMN aggregates were measured by flow cytometry upon stimulation of blood. Fibrinogen binding to platelets and markers of PMN activation were also detected. Platelet P-selectin expression and platelet-PMN aggregate formation were induced in all four activation conditions tested. Parnaparin prevented in a concentration-dependent manner (0.3-0.8 IUaXa/ml) the expression of P-selectin and the formation of mixed aggregates, while the two reference heparin preparations had a much weaker effect. Platelet fibrinogen binding and PMN activation markers (fibrinogen binding, CD11b and CD40) were also prevented by parnaparin. These data extend in more physiological systems of platelet activation, the anti-inflammatory profile of parnaparin, previously reported in washed cells. The greater effect of parnaparin, as compared to the reference heparins, could be due to chemico-physical differences possibly unrelated to their anticoagulant effect.

Preconditioning ischemia attenuates molecular indices of platelet activation-aggregation.

Previous studies have shown that ischemic preconditioning (PC) not only limits infarct size, but also improves arterial patency in models of recurrent thrombosis. We hypothesize that this enhanced patency is presumably because of a PC-induced attenuation of platelet-mediated thrombosis. However, there is, at present, no direct evidence that PC acts on the platelets per se and favorably down-regulates platelet reactivity. Our goal was to test the concept that PC ischemia attenuates molecular indices of platelet activation-aggregation. Anesthetized dogs were randomly assigned to receive 10 min of PC ischemia followed by 10 min of reperfusion or a time-matched control period. Spontaneous recurrent coronary thrombosis was then initiated in all dogs by injury + stenosis of the left anterior descending coronary artery. Coronary flow was monitored for 3 h poststenosis, and molecular indices of platelet activation-aggregation were quantified by whole blood flow cytometry. Coronary patency was, as expected, better-maintained following injury + stenosis in the PC group vs. controls (53% +/- 5%* vs. 23% +/- 5% of baseline flow, respectively; *P < 0.05). Moreover, PC was accompanied by: (i) a significant down-regulation of platelet-fibrinogen binding and formation of neutrophil-platelet aggregates (112% +/- 14%* vs. 177% +/- 21% and 107% +/- 8%* vs. 155% +/- 19% of baseline values in PC vs. control groups); and (ii) a trend towards a reduction in platelet P-selectin expression (148% +/- 12% vs. 190% +/- 21% of baseline; *P < 0.05 and P = 0.09 vs. control). These data provide novel, direct evidence in support of the concept that ischemic PC attenuates molecular indices of platelet activation-aggregation.

High glucose levels enhance platelet activation: involvement of multiple mechanisms

Diabetes mellitus (DM) and hyperglycaemia are associated with platelet activation. The present study was designed to investigate how high glucose levels influence platelet function. Fasting human blood was incubated with different concentrations of D-glucose (5, 15 and 30 mmol/l) and other sugars without or with in vitro stimuli. Platelet activation was monitored by whole blood flow cytometry. High glucose levels enhanced adenosine diphosphate (ADP)- and thrombin receptor-activating peptide (TRAP)-induced platelet P-selectin expression, and TRAP-induced platelet fibrinogen binding. Similar effects were seen with 30 mmol/l L-glucose, sucrose and galactose. Hyperglycaemia also increased TRAP-induced platelet-leucocyte aggregation. Protein kinase C (PKC) blockade did not counteract the enhancement of platelet P-selectin expression, but abolished the enhancement of TRAP-induced platelet fibrinogen binding by hyperglycaemia. Superoxide anion scavenging by superoxide dismutase (SOD) attenuated the hyperglycaemic enhancement of platelet P-selectin expression, but did not counteract the enhancement of TRAP-induced platelet fibrinogen binding. Hyperglycaemia did not alter platelet intracellular calcium responses to agonist stimulation. Blockade of cyclo-oxygenase (COX), phosphotidylinositol-3 (PI3) kinase, or nitric oxide synthase, or the addition of insulin did not influence the effect of hyperglycaemia. In conclusion, high glucose levels enhanced platelet reactivity to agonist stimulation through elevated osmolality. This occurred via superoxide anion production, which enhanced platelet P-selectin expression (secretion), and PKC signalling, which enhanced TRAP-induced fibrinogen binding (aggregablity).

Investigation of the role of adjacent amino acids to the 313–320 sequence of the αIIb subunit on platelet activation and fibrinogen binding to αIIbβ3

The platelet integrin receptor αIIbβ3 plays a critical role in thrombosis and haemostasis by mediating interactions between platelets and several ligands, primarily fibrinogen. We have previously shown that the synthetic peptide YMESRADRKLAEVGRVYLFL corresponding to residues 313–332 of αIIb, is a potent inhibitor of platelet aggregation and fibrinogen binding to αIIbβ3, interacting with fibrinogen rather than the receptor. Furthermore, we have demonstrated that the biological activities of the above peptide are due to the sequence YMESRADR, which corresponds to residues 313–320. By using new synthetic peptide analogues we investigated the structural characteristics responsible for the biological activity of YMESRADR as well the possible influence of the adjacent amino acids on the peptide's biological potency. According to our results, the synthetic octapeptide YMESRADR, is a potent inhibitor of platelet aggregation and P-selectin expression. Furthermore, YMESRADR inhibits fibrinogen binding but it does not significantly influence the binding of PAC-1 to ADP-activated platelets. The inhibitory potency of YMESRADR was gradually diminished by deleting the YMES sequence from the amino terminus and prolonging the carboxyl terminus of this peptide with the KLAE sequence. Extension of YMESRADR towards the amino terminus with the GAPL sequence (GAPLYMESRADR) does not modify the biological activity of YMESRADR. Furthermore, extension of GAPLYMESRADR at its carboxy terminus with the KLAE sequence (GAPLYMESRADRKLAE) significantly diminished its biological potency. Substitution of E315 with D significantly enhances antiaggregatory potency and completely abolishes the inhibitory effect on P-selectin expression. Importantly, the D315-containing peptides inhibit to a similar extent both fibrinogen and PAC-1 binding to activated αIIbβ3 in contrast to the E315-containing peptide which only inhibits fibrinogen binding. In conclusion, the present study suggests that the YMESRADR sequence 313–320 of αIIb, is an important functional region of the insert connecting the β2 and β3 antiparallel β-strands of the W5 blade of the αIIb subunit. Structural changes significantly modify the biological properties of this region.

A role for the thiol isomerase protein ERP5 in platelet function

AbstractFormation and rearrangement of disulfide bonds during the correct folding of nascent proteins is modulated by a family of enzymes known as thiol isomerases, which include protein disulfide isomerase (PDI), endoplasmic reticulum protein 5 (ERP5), and ERP57. Recent evidence supports an alternative role for this family of proteins on the surface of cells, where they are involved in receptor remodeling and recognition. In platelets, blocking PDI with inhibitory antibodies inhibits a number of platelet activation pathways, including aggregation, secretion, and fibrinogen binding. Analysis of human platelet membrane fractions identified the presence of the thiol isomerase protein ERP5. Further study showed that ERP5 is resident mainly on platelet intracellular membranes, although it is rapidly recruited to the cell surface in response to a range of platelet agonists. Blocking cell-surface ERP5 using inhibitory antibodies leads to a decrease in platelet aggregation in response to agonists, and a decrease in fibrinogen binding and P-selectin exposure. It is possible that this is based on the disruption of integrin function, as we observed that ERP5 becomes physically associated with the integrin β3 subunit during platelet stimulation. These results provide new insights into the involvement of thiol isomerases and regulation of platelet activation.

Randomized clinical trial of the antiplatelet effects of aspirin–clopidogrel combination versus aspirin alone after lower limb angioplasty

There is a high risk of reocclusion after successful lower limb angioplasty. Platelets play a central role in this process. The aim of this study was to investigate the antiplatelet effect of a combination of aspirin and clopidogrel compared with aspirin alone in patients with claudication undergoing endovascular revascularization. This was a double-blind randomized placebo-controlled trial. Some 132 patients were randomized to clopidogrel and aspirin or placebo and aspirin, with a loading dose 12 h before endovascular intervention. Flow cytometric measurements of platelet fibrinogen binding and P-selectin expression were taken as measures of platelet function at baseline, 12 h after the loading dose, and 1 h, 24 h and 30 days after intervention. Within 12 h of the loading dose, platelet activation in the clopidogrel group had decreased (P-selectin by 27.3 per cent, P = 0.017; fibrinogen binding by 34.7 per cent, P = 0.024; stimulated fibrinogen binding by 49.2 per cent, P < 0.001). No change was observed in the placebo group. Platelet function in the clopidogrel group was significantly suppressed compared with baseline at 1 h, 24 h and 30 days after endovascular intervention (stimulated fibrinogen binding by 53.9, 51.7 and 57.2 per cent respectively; all P < 0.001). A combination of clopidogrel and aspirin inhibited platelet function more than aspirin alone in patients with claudication before and after angioplasty.

Influence of extracellular calcium on single platelet activation as measured by whole blood flow cytometry

The influence of extracellular calcium concentrations ([Ca 2+]) on single platelet activation and platelet aggregation was evaluated. Platelet fibrinogen binding and P-selectin expression were monitored by whole blood flow cytometry in the presence of EDTA or 0.125, 1.25, 2.5, 5, or 10 mM [Ca 2+] and in the absence or presence of the thromboxane A 2 (TXA 2) blockade. Platelet aggregation was measured in citrated and hirudinized platelet-rich plasma (PRP). Platelet fibrinogen binding was slightly increased at≥2.5 mM [Ca 2+] in unstimulated samples. ADP-induced platelet fibrinogen binding was, however, higher at 0.125 mM, but lower at 5 and 10 mM [Ca 2+], as compared to 1.25 mM [Ca 2+]. Platelet P-selectin expression was not affected by extracellular [Ca 2+], except mild increases of ADP-induced platelet P-selectin expression in the presence of EDTA. TXA 2 blockade by ICI 192.605 influenced above flow cytometric analyses little. Using Born aggregometry, ADP induced more intense platelet aggregation in citrated PRP than in hirudinized PRP. TXA 2 blockade did not affect platelet aggregation in hirudinized PRP, but reduced aggregation in citrated PRP to ∼85% of that in hirudinized samples. ADP also induced a more marked and sustained elevation of intracellar [Ca 2+] in the presence of extracellular [Ca 2+]. Thus, extracellular [Ca 2+] has little influence on flow cytometric analysis of platelet P-selectin expression. High [Ca 2+] enhances spontaneous platelet fibrinogen binding, but reduces ADP-induced platelet fibrinogen binding, while low [Ca 2+] enhances ADP-induced platelet fibrinogen binding. Physiological [Ca 2+] supports more intense platelet aggregation when effect of artificial TXA 2 synthesis is blocked.

Contrast medium attenuates platelet activation and platelet-leukocyte cross–talk

The influence of ionic and non-ionic contrast media (CM) on platelet and leukocyte activation and platelet-leukocyte crosstalk was investigated in hirudinized whole blood. The blood was incubated with and without the ionic CM ioxaglate and the nonionic CM iodixanol at 37 degrees C for 5 min, without or with stirring. Platelet and leukocyte activation and platelet-leukocyte aggregation were measured using whole blood flow cytometry. When blood samples were pre-incubated in the presence of 2%, 5%, and 10% of CM without stirring, both ioxaglate and iodixanol had little effect on unstimulated samples, but dose-independently decreased 1 microM ADP-induced platelet P-selectin expression and fibrinogen binding, and thus platelet-leukocyte aggregate formation. Ioxaglate had little effect on leukocyte CD11b expression, whilst iodixanol slightly enhanced resting and N-formyl-methionyl-leucyl-phenylalanine (fMLP; 0.1 microM)-stimulated leukocyte CD11b expression. Blood samples were also incubated with stirring to investigate the impact of CM (5% of ioxaglate or iodixanol) on platelet-leukocyte cross-talk. Collagen induced marked platelet activation and platelet-leukocyte aggregation, and subsequently elevated leukocyte CD11b expression. The latter was attenuated by ioxaglate and iodixanol, and was accompanied by reduced platelet-leukocyte aggregation. In conclusion, the CM ioxaglate and iodixanol attenuate platelet activation and platelet-leukocyte cross-talk. Inhibitory effects of the contrast agents on this cross-talk are apparently exerted by reducing heterotypic conjugation, and may be beneficial in connection with PCI.

Effects of Estrogen Replacement Therapies on Mouse Platelet Funcion.

Clinical trials of hormone replacement therapy (HRT) have demonstrated an increase in stroke and myocardial infarction despite the beneficial changes in LDL and HDL cholesterols, and it has been suggested that HRT may adversely affect thrombosis and/or inflammation. Platelets participate in both of these processes and play a central role in thrombosis formation in high shear arterial vasculature. The effects of HRT on platelet function have been controversially reported, probably due to different hormone regimes used (e.g. different estrogenic compounds, different dosages and routes of administration). We used a murine model of menopause to test the hypothesis that HRT alters platelet reactivity. Bilaterally ovariectomized female littermates were treated with various estrogen regimes or matching placebo for 21 days. Three estrogen replacement therapy (ERT) regimes were studied: 1) oral conjugated equine estrogen (CEE) vs. placebo (n=5 pairs), 2) oral 17-beta estradiol (E2) vs. placebo (n=4 pairs), and 3) subcutaneously implanted E2 (SQ E2) vs. placebo (n=8 pairs). Washed platelets were used to focus on intrinsic platelet reactivity in the absence of plasma coagulation factors. Platelet reactivity was assessed by flow cytometric quantification of agonist-induced fibrinogen binding. Oral CEE, but not oral E2 enhanced the platelet response to collagen-related-peptide (CRP) (P<0.001 and P=0.87 for oral CEE and oral E2, respectively). This enhancement of platelet function was agonist specific since neither oral CEE (P=0.862) nor oral E2 (P=0.917) affected the platelet response to thrombin. We also tested whether the route of hormone administration affected platelet function. SQ E2 induced a condition different from that of oral E2: the platelet response to thrombin was enhanced (P=0.028), but only to low (<10 mU/ml), not high concentrations of thrombin. In contrast to oral E2, SQ E2 caused a reduction in CRP-induced platelet fibrinogen binding (P=0.030). Platelet surface expression of GPVI was reduced in response to SQ E2 (P<0.001), and the level of GPVI correlated with CRP-induced platelet fibrinogen binding (P<0.0001). In conclusion: 1) the effects of different estrogen replacement therapies on platelet function differ significantly, perhaps explaining some of the conflicts in the literature, 2) the effects of ERT on platelet function are dependent upon the ERT preparations and routes, 3) the effects of ERT on platelet function are agonist and agonist dose-dependent, and 4) SQ E2 induces lower platelet GPVI expression, most likely explaining the reduced response to CRP. A potential practical consequence of our findings is that oral E2 may be less prothrombotic than either oral CEE or transdermal E2.