scholarly journals Inhibition of coagulation and thrombin-induced platelet activities by a synthetic dodecapeptide modeled on the carboxy-terminus of hirudin

Blood ◽  
1990 ◽  
Vol 75 (2) ◽  
pp. 399-406 ◽  
Author(s):  
JA Jakubowski ◽  
JM Maraganore
Keyword(s):  
Platelet Aggregation ◽  
Antithrombin Iii ◽  
Thromboxane A2 ◽  
Serotonin Release ◽  
Dependent Manner ◽  
Thrombin Time ◽  
Platelet Factor ◽  
Induce Platelet Aggregation ◽  
Apparent Lack

A synthetic, tyrosine-sulfated, dodecapeptide (BG8865) modeled on residues 53–64 of hirudin was found to elevate the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) of human plasma in a dose-dependent manner. The most sensitive assay was the TT, which was prolonged 2 and 3 times control values at 2.2 and 4.1 micrograms/mL hirudin peptide, respectively. The sulfated dodecapeptide exhibited no dependency on antithrombin III as monitored by the APTT in the presence of sheep anti-human antithrombin III antibodies, and its activity was not neutralized by platelet releasates or platelet factor 4. In studies of thrombin-induced platelet activation, the hirudin peptide was found to block aggregation, serotonin release and thromboxane A2 generation. At thrombin concentrations of 0.25 U/mL, the IC50 (concentration resulting in 50% inhibition) for inhibition of platelet aggregation was 0.72 micrograms/mL peptide. Inhibition of TXA2 generation and serotonin release correlated closely with inhibition of aggregation. Using platelets from patients with clinically documented heparin-induced thrombocytopenia anticoagulant doses of heparin were found to induce platelet aggregation and thromboxane A2 generation. In sharp contrast, anticoagulant-equivalent doses of hirudin peptide had no effect on patient platelets, as evidenced by a lack of platelet aggregation and thromboxane A2 generation. These data provide compelling in vitro evidence that the hirudin peptide has several potential advantages over heparin, namely effective inhibition of thrombin-induced platelet activities, co-factor independence, insensitivity to endogenous heparin- neutralizing factors, and an apparent lack of direct or immune-mediated platelet stimulating properties.

scholarly journals Inhibition of coagulation and thrombin-induced platelet activities by a synthetic dodecapeptide modeled on the carboxy-terminus of hirudin

Blood ◽  
1990 ◽  
Vol 75 (2) ◽  
pp. 399-406
Author(s):  
JA Jakubowski ◽  
JM Maraganore
Keyword(s):  
Platelet Aggregation ◽  
Antithrombin Iii ◽  
Thromboxane A2 ◽  
Serotonin Release ◽  
Dependent Manner ◽  
Thrombin Time ◽  
Platelet Factor ◽  
Induce Platelet Aggregation ◽  
Apparent Lack

Abstract A synthetic, tyrosine-sulfated, dodecapeptide (BG8865) modeled on residues 53–64 of hirudin was found to elevate the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) of human plasma in a dose-dependent manner. The most sensitive assay was the TT, which was prolonged 2 and 3 times control values at 2.2 and 4.1 micrograms/mL hirudin peptide, respectively. The sulfated dodecapeptide exhibited no dependency on antithrombin III as monitored by the APTT in the presence of sheep anti-human antithrombin III antibodies, and its activity was not neutralized by platelet releasates or platelet factor 4. In studies of thrombin-induced platelet activation, the hirudin peptide was found to block aggregation, serotonin release and thromboxane A2 generation. At thrombin concentrations of 0.25 U/mL, the IC50 (concentration resulting in 50% inhibition) for inhibition of platelet aggregation was 0.72 micrograms/mL peptide. Inhibition of TXA2 generation and serotonin release correlated closely with inhibition of aggregation. Using platelets from patients with clinically documented heparin-induced thrombocytopenia anticoagulant doses of heparin were found to induce platelet aggregation and thromboxane A2 generation. In sharp contrast, anticoagulant-equivalent doses of hirudin peptide had no effect on patient platelets, as evidenced by a lack of platelet aggregation and thromboxane A2 generation. These data provide compelling in vitro evidence that the hirudin peptide has several potential advantages over heparin, namely effective inhibition of thrombin-induced platelet activities, co-factor independence, insensitivity to endogenous heparin- neutralizing factors, and an apparent lack of direct or immune-mediated platelet stimulating properties.

Effect of SR121566A, a Potent GP IIb-IIIa Antagonist, on the HIT Serum/heparin-Induced Platelet Mediated Activation of Human Endothelial Cells

1998 ◽  
Vol 80 (08) ◽  
pp. 326-331 ◽  
Author(s):  
Pierre Savi ◽  
Walter Jeske ◽  
Jeanine Walenga ◽  
Jean-Marc Herbert
Keyword(s):  
Endothelial Cells ◽  
Platelet Aggregation ◽  
Umbilical Vein ◽  
Common Adverse Effect ◽  
Surface Protein ◽  
Heparin Therapy ◽  
Serotonin Release ◽  
Dependent Manner ◽  
Platelet Factor

SummaryHeparin-induced thrombocytopenia (HIT) is a common adverse effect of heparin therapy that carries a risk of serious thrombotic events. This condition is caused by platelet aggregation, which is mediated by anti-heparin/platelet factor 4 antibodies. Sera from patients with HIT in the presence of platelets, induced the expression of E-selectin, VCAM, ICAM-1 and tissue factor and the release of IL1β, IL6, TNFα and PAI-1 by human umbilical vein endothelial cells (HUVECs) in vitro and initiated platelet adhesion to activated HUVECs. These effects which occurred in a time-dependent manner were significant in the first 1-2 h of incubation and reached a maximum after 6 to 9 h. The GP IIb-IIIa receptor antagonist SR121566A which has been shown to block platelet aggregation induced by a wide variety of agonists including HIT serum/heparin, reduced in a dose-dependent manner the HIT serum/heparin-induced, platelet mediated expression and release of the above mentioned proteins. The IC50 for inhibition of HIT serum/ heparin-induced platelet dependent HUVEC activation by SR121566A was approximately 10-20 nM. ADP, but not serotonin release, also appeared to be involved as apyrase and ATPγS blocked platelet-dependent, HIT serum/heparin-induced cell surface protein expression and cytokine release by HUVECs. Increased platelet adherence to HIT serum/heparin-activated HUVECs was inhibited by SR121566A and, to a lesser extent, by apyrase and ATPγS, showing that platelet activation and release was at the origin of the HIT serum/heparin-induced expression of these proteins by HUVECs.Thus, sera from patients with HIT induced the expression of adhesive and coagulation proteins and the release of cytokines by HUVECs through the activation of platelets which occurred in a GP IIb-IIIa-dependent manner, a process that could be selectively blocked by SR121566A.

In Vitro and In Vivo Effects of Adrenaline and Phentolamine on Platelet Function

1978 ◽  
Vol 40 (02) ◽  
pp. 428-438 ◽  
Author(s):  
Oreste Ponari ◽  
Emilio Civardi ◽  
Alessandro Megha ◽  
Mario Pini ◽  
Raffaele Poti’ ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Threshold Concentration ◽  
Two Phase ◽  
Platelet Factor ◽  
Induce Platelet Aggregation ◽  
In Vivo Effects ◽  
In Vivo Administration

Summary In vitro and in vivo effects of adrenaline (ADR) on platelet aggregation, on platelet factor 3 (PF3) availability and on platelet factor 4 (PF4) release were studied in man. Inhibitory action of an alpha-blocker, phentolamine (PHEN) was investigated in the same conditions.The threshold concentration (TC) of ADR inducing the typical two-phase response in aggregation tests when added to platelet-rich plasma (PRP) varied in different pools of plasma, but always induced an evident PF4 release and increased PF3 availability. A further increase in both parameters was obtained with higher concentrations but without any significant dose/response correlation.Adding PHEN alone to PRP did not induce platelet aggregation or modify PF4 release induced by stirring, but it reduced PF3 availability. On the other hand, PHEN prevented the effects of ADR in different platelet tests, at appropriate concentrations.Intravenous infusion of ADR lowered the TC, and increased PF3 availability and PF4 release. In vivo administration of PHEN, in contrast, increased TC and reduced PF3 availability, while PF4 remained unchanged.

A Comparative Study of the Effects of Adrenoceptor Antagonists on Platelet Aggregation and Thromboxane Generation

1985 ◽  
Vol 54 (02) ◽  
pp. 480-484 ◽  
Author(s):  
I A Greer ◽  
J J Walker ◽  
M McLaren ◽  
A A Calder ◽  
C D Forbes
Keyword(s):  
Platelet Aggregation ◽  
Vascular Disease ◽  
Platelet Rich Plasma ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Wide Range ◽  
The Right

SummaryPlatelet aggregation and thromboxane A2 have been implicated in the pathogenesis of several forms of vascular disease. The aim of this study was to determine the effect of a wide range of adrenoceptor antagonists on platelet aggregation, and thromboxane A2 production, from normal human platelet rich plasma in vitro. Labetalol, pindolol and propranolol inhibited platelet aggregation to collagen in a dose dependent manner. Increasing the concentration of collagen “shifted” the dose response curve to the right. These 3 drugs also significantly inhibited thromboxane A2 generation in response to collagen but not to arachidonic acid. This effect was independent of any inhibitory effect of these drugs on platelet aggregation, and occurred at a drug concentration close to that obtained in vivo. Atenolol, metoprolol, prazosin and timolol were similarly assessed but had no effect on either platelet aggregation or thromboxane A2 generation. This ability of labetalol, pindolol, and propranolol to inhibit platelet aggregation and thromboxane generation, may be of clinical benefit in view of the increasing evidence implicating thromboxane A2 in the pathogenesis of vascular disease.

Inhibition of Human Platelet Functions by Verapamil

1981 ◽  
Vol 45 (02) ◽  
pp. 158-161 ◽  
Author(s):  
Y Ikeda ◽  
M Kikuchi ◽  
K Toyama ◽  
K Watanabe ◽  
Y Ando
Keyword(s):  
Platelet Aggregation ◽  
Calcium Ionophore ◽  
Serotonin Release ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Dependent Inhibition ◽  
Platelet Functions

SummaryThe effects of verapamil, a coronary vasodilator, on platelet functions were studied.Platelet aggregation induced by ADP, epinephrine or collagen was inhibited by verapamil in vitro. Calcium ionophore A23187-induced platelet aggregation was also inhibited by verapamil in a concentration dependent manner. In washed platelets, verapamil caused a dose-dependent inhibition of serotonin release induced either by thrombin or A23187 in the absence of extracellular calcium. Addition of 1 mM CaCl2 with A23187 or thrombin partially overcame this inhibition. Addition of 1 mM CaCl2 in the absence of verapamil had no effect on thrombin- or A23187-induced secretion. When verapamil was administered to the healthy volunteers at the dosage commonly used, inhibition of platelet aggregation was observed 2 hrs after the drug ingestion. It is of great interest that verapamil potentiated the anti-aggregating activity of prostacyclin in vitro.Our results may suggest a potential role for verapamil in the treatment of thrombotic disorders.

Platelet Aggregation and Serotonin Release, Coagulation, Fibrinolysis, Antiplasmin, Factor XIII, and Antithrombin III in Acute Transmural Non+Complicated Myo-Cardial Infarction

1979 ◽  
Author(s):  
J. Bjerre Knudsen ◽  
O. Amtorp ◽  
K. Skagen ◽  
J. Gormsen
Keyword(s):  
Platelet Aggregation ◽  
Factor Viii ◽  
Factor Xiii ◽  
Antithrombin Iii ◽  
Peak Level ◽  
Serotonin Release ◽  
Myocardial Infarctions ◽  
Normal Range ◽  
Cardial Infarction

Platelet aggregation in vitro (ADP and adrenalin). Serotonin release, APTT, Factor VIII, Factor XIII, FE-a, plasma ethanol gelation teat, euglobulinclotlysiatime, antiplaeain and antithrombin III were estimated in 12 patients with acute transmural nob-complicated myocardial infarctions receiving no drugs with any known influence on the actual parameters. Distinct, uniform, changes were round. Initially distinct decrease in aggregation and release was present, changing within a week into increased aggregability, as estimated by threshold concentrations, and increased release function. The platelets remained “hyperactive” in above 50% at the discharge. Positive gelation test appeared after 1-2 days with peak level day 5, became afterwards negative in all. The fibrinolytic activity was within normal range day 1-2. It decreased rapidly with lowest activity on day 5-6 contemporarily with a peak activity of antiplasmin. Afterwards it increased slowly, but was still low in around 50% at the discharge. Factor VIII activity increased significantly with peak day 5-6. Factor XIII decreased biologically with lowest activity on day 5-6, increased to normal levels, Antithrombin III remained unchanged, in upper part of normal range. Thus, changes in platelet patterns are demonstrable at the onset of the infarction, while the other parameters develop later.

The Effect of a Medicinal Chinese Herb on Platelet Function

1982 ◽  
Vol 48 (03) ◽  
pp. 301-306 ◽  
Author(s):  
Z Wang ◽  
J M Roberts ◽  
P G Grant ◽  
R W Colman ◽  
A D Schreiber
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Platelet Rich Plasma ◽  
Chinese Herb ◽  
Serotonin Release ◽  
Dependent Manner ◽  
Heat Stable ◽  
Artery Disease ◽  
Dose Dependent

SummaryWe investigated the effect of the Chinese herb Injectio Salvia Miltiorrhizae (ISM) on human platelet function in vitro. ISM inhibited platelet aggregation and serotonin release induced by either ADP or epinephrine in a dose dependent manner. This effect of ISM was observed with both gel-filtered platelets (ID50 = 8–30 μg ISM/ml gel-filtered platelets) and platelets in plasma (ID50 = 400–900 μg ISM/ml of platelet-rich plasma). The active molecule(s) in ISM was heat stable, resistant to acid, base and proteolysis and fractionated on Sephadex 6-25 at MW ~ 280. ISM did not interact with the platelet α-adrenergic receptor, but increased cAMP in intact platelets. The results are consistent with the concept that ISM inhibition of platelet aggregation and release is mediated by an increase in platelet cAMP. The exact mechanism whereby ISM increases platelet cAMP appears to be that of inhibition of cyclic AMP phosphodiesterase. The effect of ISM on platelet function is one mechanism which might explain the therapeutic effect of ISM in experimental and clinical coronary artery disease.

Inhibition of Thrombin-Neutralizing Activity of Antithrombin III by Steroid Hormones

1982 ◽  
Vol 47 (02) ◽  
pp. 157-161 ◽  
Author(s):  
H Nagasawa ◽  
B K Kim ◽  
M Steiner ◽  
M G Baldini
Keyword(s):  
Platelet Aggregation ◽  
Antithrombin Iii ◽  
Steroid Hormone ◽  
Human Platelets ◽  
Dependent Manner ◽  
At Iii ◽  
High Doses ◽  
Dose Dependent ◽  
Inhibition Of Thrombin

SummaryEstrogens in high doses have been shown to inhibit, in vitro, the thrombin-neutralizing action of antithrombin III (AT III). In this study we investigate the effect of estrogens on AT III in greater detail. To increase the sensitivity of measurement of AT III activity in the absence of heparin, we have developed an assay system utilizing human platelets, AT III and thrombin. The two proteins derived from human plasma were prepared in high purity. Platelet aggregation was induced by approximately 0.02 NIH U of thrombin. AT III was added in amounts that suppressed 95% of the aggregation-inducing effect of thrombin. Estrogens blocked the thrombin-neutralizing effect of AT III in dose-dependent manner. This effect was shown to be specific for AT III. Neither aggregability of platelets nor aggregating effect of thrombin were affected by the steroid hormone. Evidence for binding of estrogen to AT III was obtained from changes in intrinsic fluorescence of AT III. Activity of AT III was also reduced in increasing order of effectiveness by cholesterol, cortisone, testosterone and progesterone. Our studies suggest a direct effect of estrogens and other steroids on AT III, altering its specific neutralization of thrombin.

BINDING OF FIBRINOGEN TO PLATELET GLYCOPROTEIN (GP) IIb/IIIa IS CRUCIAL FOR SHEAR-INDUCED PLATELET AGGREGATION

1987 ◽  
Author(s):  
Y Ikeda ◽  
M Murata ◽  
Y Araki ◽  
M Yamamoto ◽  
K Watanabe ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Shear Rate ◽  
Platelet Rich Plasma ◽  
Serotonin Release ◽  
Platelet Glycoprotein ◽  
Dependent Manner ◽  
Particle Counts ◽  
Induced Aggregation

It is well known that human platelets can aggregate in vitro under certain shear stress without adding aggregating inducers. However, the mechanism of this shear-induced platelet aggregation has not been clarified yet. In this paper, we have investigated the role of fibrinogen and GP IIb/IIIa in shear-induced platelet aggregation. Citrated human platelet-rich plasma (PRP) was subjected to controlled shear stress levels in a polycarbonate cone and plate viscometer at 37°C for 2 minutes. After shearing the particle count was measured by an electronic particle counter. Particles with sizes from 3 to 20 μ cum were considered as single platelets. In unsheared PRP most of the particles were single platelets, but platelet doublets and platelet fragments larger than 3 μ cum were also counted. After exposure to shear rate of 3,600 - 9,000 sec−1 , the particle counts were decreased in a shear rate dependent manner, while LDH leakage from platelets was not significantly increased and 3H-serotonin release was 2-7%. Scanning electronmicroscopy clearly showed the presence of large platelet aggregates when the particle counts were decreased. Platelets from two patients with thrombasthenia and one patient with afibrinogenemia, however, failed to aggregate at a shear rate of 9,000 sec−1. Shear-induced aggregation was inhibited by monoclonal antibody to GPIIb/IIIa (1 μg/ml) and synthetic peptide, Arg-Gly-Asp-Ser, (1 mM). When fibrinogen was added to PRP from a patient with afibrinogenemia, shear-induced aggregation became evident as seen in normal platelets. Apyrase and hirudin showed no effect on shear-induced aggregation. Indomethacin (100 μM) and TXA2 synthetase inhibitor, OKY-046 (100 μM) markedly inhibited aggregation, while TXA2 competitive inhibitor, ONO-3708 (100 μM) exhibited only partial inhibition.Our results indicate that binding of fibrinogen to GPIIb/lIIa is also crucial for shear-induced platelet aggregation and that the exposure of fibrinogen receptor on GPIIb/IIIa may partially depend upon TXA2 synthesis in platelets.

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