The radioisotope labelling of thrombocytes using a monoclonal antibody against membrane glycoproteins IIb-IIIa and the measurement of thrombocyte adhesion/aggregation on the substrate surface]

A new method for platelet labeling based on binding of monoclonal antibody to human platelets has been suggested in this study. Monoclonal antibody VM16a against membrane glycoproteins IIb-IIIa was labeled by 125I and then incubated with platelets. About 70% of added antibody was bound when it was used at the concentrations corresponding to the linear part of the concentration curve (0.5 and 1.0 micrograms/ml). Due to high efficiency of binding 125I-VM16a-labeled platelets were used for the measurement of adhesion/aggregation to the substrate in platelet-rich plasma without washing of the free label. Experiments with washed platelets double labeled with 51Cr and 125I-VM 6a showed high correlation between the data obtained with both labels. The method of platelet labeling has been applied for the assessment of drug action on platelet adhesion/aggregation. Measurements were performed in platelet-rich plasma and adhesion/aggregation was stimulated by ADP and analogue of thromboxane A2, U46619. It was shown/that antianginal drug trapidil strongly inhibited and antiatherogenic drug probucol did not affect platelet adhesion/aggregation stimulated by both agonists.     

Platelet adhesion receptors and (patho)physiological thrombus formation

In thrombus formation associated with hemostasis or thrombotic disease, blood platelets first undergo a rapid transition from a circulating state to an adherent state, followed by activation and aggregation. Under flow conditions in the bloodstream, this process potentially involves platelet-platelet, platelet-endothelium, platelet-subendothelial matrix, and platelet-leukocyte interactions. Specific adhesion receptors on platelets mediate these interactions, by engaging counter-receptors on other cells, or noncellular ligands in the plasma or matrix. The glycoprotein (GP) Ib-IX-V complex on platelets initiates adhesion at high shear stress by binding the adhesive ligand, von Willebrand Factor (vWF). GP Ib-IX-V may also mediate platelet-endothelium or platelet-leukocyte adhesion, by recognition of P-selectin or Mac-1, respectively. Other membrane glycoproteins, such as the collagen receptor GP VI, may trigger platelet activation at low shear rates. Engagement of GP Ib-IX-V or GP VI leads ultimately to platelet aggregation mediated by the integrin, alphaIIbbeta3 (GP IIb-IIIa). This review will focus on recent advances in understanding structure-activity relationships of GP Ib-IX-V, its role in initiating thrombus formation, and its emerging relationships with other vascular cell adhesion receptors.     

Antidote-controlled platelet inhibition targeting von Willebrand factor with aptamers

Thrombus formation is initiated by platelets and leads to cardiovascular, cerebrovascular, and peripheral vascular disease, the leading causes of morbidity and mortality in the Western world. A number of antiplatelet drugs have improved clinical outcomes for thrombosis patients. However, their expanded use, especially in surgery, is limited by hemorrhage. Here, we describe an antiplatelet agent that can have its activity controlled by a matched antidote. We demonstrate that an RNA aptamer targeting von Willebrand factor (VWF) can potently inhibit VWF-mediated platelet adhesion and aggregation. By targeting this important adhesion step, we show that the aptamer molecule can inhibit platelet aggregation in PFA-100 and ristocetin-induced platelet aggregation assays. Furthermore, we show that a rationally designed antidote molecule can reverse the effects of the aptamer molecule, restoring platelet function quickly and effectively over a clinically relevant period. This aptamer-antidote pair represents a reversible antiplatelet agent inhibiting a platelet specific pathway. Furthermore, it is an important step towards creating safer drugs in clinics through the utilization of an antidote molecule.     

The peptide Glu-His-Ile-Pro-Ala binds fibrinogen and inhibits platelet aggregation and adhesion to fibrinogen and vitronectin.

Antiplatelet agents are clinically useful as antithrombotic entities. The importance of antiplatelet agents led us to design, synthesize, and characterize a new antiplatelet peptide. This peptide is a presumptive mimic of a ligand binding site on the platelet fibrinogen receptor. Unlike peptides related to Arg-Gly-Asp-Ser and His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val that bind to the fibrinogen receptor, this peptide binds to fibrinogen. The anticomplementarity hypothesis was used to design this presumptive peptide mimic of the vitronectin binding site on the fibrinogen receptor, glycoprotein IIb/IIIa complexes. The resulting peptide (Glu-His-Ile-Pro-Ala) has the characteristics of a fibrinogen binding site mimic: It binds fibrinogen and inhibits both the adhesion of platelets to fibrinogen and platelet aggregation. The peptide also inhibits the adhesion of platelets to vitronectin. The antiplatelet activity of this mimic peptide was dependent on its amino acid sequence, since closely related analogues were either inactive or less active inhibitors of platelet function than the original peptide. These results demonstrate that the peptide Glu-His-Ile-Pro-Ala has the characteristics expected of a mimic of a glycoprotein IIb/IIIa ligand binding site.     

Increased blood plasma hydrolysis of acetylsalicylic acid in type 2 diabetic patients: a role of plasma esterases

Hydrolysis of acetylsalicylic acid (ASA, aspirin), an antiplatelet drug commonly used in the prevention of stroke and myocardial infarction, seems to play a crucial role in its pharmacological action. Thirty-eight healthy volunteers and 38 type 2 diabetic patients were enrolled to test the hypothesis that the enhanced plasma degradation and lowered bioavailability of ASA in diabetic patients is associated with the attenuation of platelet response. Aspirin esterase activities were tested at pH 7.4 and 5.5. A significantly higher overall aspirin esterase activity was noted at pH 7.4 in the diabetic patients (P<0.003), corresponding to faster ASA hydrolysis (P<0.006). This increased activity was attributable to butyrylcholinesterase and probably to albumin, because it was effectively inhibited by eserine and 4-bis-nitrophenyl phosphate (P<0.01). No significant differences between control and diabetic subjects were found at pH 5.5 in either enzymatic activities or ASA hydrolysis rates. The enhanced plasma ASA degradation in diabetic subjects was significantly associated with the refractoriness of blood platelets to ASA (P<0.05) and modulated by plasma cholesterol (P<0.01). No direct effects of plasma pH or albumin were observed. In conclusion, higher aspirin esterase activity contributes to the lowered response of diabetic platelets to ASA-mediated antiplatelet therapy.     

Novel antiplatelet and antithrombotic activities of essential oil from Lavandula hybrida Reverchon “grosso”

Lavender extracts are known to produce several mild effects at central and peripheral level. However, no studies are so far available about the potential effects of lavender essential oil on the hemostatic system. In this work, we demonstrated antiplatelet properties of lavender oil towards platelet aggregation induced by arachidonic acid, U46619, collagen and ADP (IC₅₀=51, 84, 191 and 640 μg/ml, respectively) on guinea-pig platelet rich plasma (PRP) and its ability to destabilize clot retraction (IC₅₀=149 μg/ml) induced by thrombin on rat PRP.

Furthermore, antithrombotic properties were studied in an in vivo model of pulmonary thromboembolism induced by intravenous injection of a collagen–epinephrine mixture in mice subacutely treated with lavender oil. In this model, lavender oil (100 mg/kg/day os for 5 days) significantly reduced thrombotic events without inducing prohemorrhagic complications at variance with acetylsalicylic acid used as reference drug. Finally, main components of the oil were studied in vitro in order to assess their antiplatelet effects, but none of them possessed an activity comparable to the oil itself. These results provide the first experimental evidence of lavender oil's antiplatelet/antithrombotic properties which could be due to a synergistic effect of its components.     

Comparison of verapamil and nifedipine in thrombosis models

Calcium blockers and calmodulin antagonists have been reported to inhibit the aggregation of blood platelets in vitro. In the present study, the effects of two calcium blockers, verapamil and nifedipine, were compared in several rodent thrombosis models. In rat and mouse platelet-rich plasma, preincubation with either verapamil or nifedipine had a dose-dependent inhibitory effect on collagen-induced aggregation (P less than 0.01). The concentration required for 50% inhibition of rat platelet aggregation was 0.91 X 10(-4) M for verapamil and 1.77 X 10(-4) M for nifedipine. In in vivo thrombosis models in mice, acute pretreatment with nifedipine had a significant, dose-dependent protective effect (P less than 0.05). At a dose of 500 micrograms/kg, nifedipine inhibited thrombotic sudden death provoked by arachidonic acid, a thromboxane agonist (U46619), or a combination of collagen and epinephrine. In vivo platelet depletion induced by U46619 was also inhibited by this calcium blocker. Thus, nifedipine is protective against a variety of thrombotic stimuli, and its antiplatelet aggregatory effect apparently extends to the in vivo situation. In contrast, no in vivo antithrombotic activity was observed for verapamil. Two additional calcium blockers, perhexilene and diltiazem, and three calmodulin antagonists, W-7, chlorpromazine, and trifluoperazine, were also tested in the U46619-induced thrombotic sudden death model. Of these, only diltiazem (5 and 10 mg/kg) had an acute protective effect.     

A novel anticoagulant purified from fish protein hydrolysate inhibits factor XIIa and platelet aggregation

A novel fish protein having anticoagulant and antiplatelet properties was enzymatically extracted from the marine fish, yellowfin sole (Limanda aspera) and purified to homogeneity producing an overall purification fold of 206.6. MALDI-TOF mass spectroscopic and SDS-PAGE analysis identified the purified protein as 12.01 kDa single-chain monomeric protein. It inhibited the activated coagulation factor XII (FXIIa) by forming an inactive complex regardless of Zn2+ mediation, and was named, yellowfin sole anticoagulant protein (YAP). In addition, YAP act to antagonize platelet membrane glycoprotein integrin, to arrest platelet aggregation. However, YAP was not able to block the adhesion of platelets to collagen, which mediate via major collagen receptors, GPIa/IIa on platelet membrane. Furthermore, YAP did not possess plasminogen activator-like activity to activate fibrinolysis. In fact, our findings indicate that YAP binds with FXIIa and platelet membrane integrins to inhibit thrombosis in vitro.     

Inhibitory mechanisms of metallothionein on platelet aggregation in in vitro and platelet plug formation in in vivo experiments

Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that contains heavy metals such as cadmium and zinc. The biological function of MT in platelets is not yet understood. Therefore, the aim of this study was to systematically examine the inhibitory mechanisms of metallothionein in platelet aggregation. In this study, metallothionein concentration-dependently (1-8 microM) inhibited platelet aggregation in human platelets stimulated by agonists. Metallothionein (4 and 8 microM) inhibited phosphoinositide breakdown in [3H]-inositol-labeled platelets, intracellular Ca+2 mobilization in Fura-2 AM-loaded platelets, and thromboxane A2 formation stimulated by collagen. In addition, metallothionein (4 and 8 microM) significantly increased the formation of cyclic GMP but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (100 nM). This phosphorylation was markedly inhibited by metallothionein (4 and 8 microM) in phosphorus-32-labeled platelets. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Metallothionein (6 microg/g) significantly prolonged the latency period for inducing platelet plug formation in mesenteric venules. These results indicate that the antiplatelet activity of metallothionein may involve the following pathways: (1) metallothionein may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of intracellular Ca+2 mobilization; (ii) Metallothionein also activated the formation of cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. The results strongly indicate that metallothionein provides protection against thromboembolism.     

Preparation and platelet aggregating activity of I-labeled ristocetin

The antibiotic ristocetin is a glycoproteoid isolated from the culture media of Nocardia lurida N.R.R.L. 2430 whose structural features have yet to be resolved (1–3). The antibiotic has been studied both for its antibiotic properties (4–6) and, more recently, for its inducement of platelet aggregation in humans, both in vivo and in vitro (7–9). This latter phenomena resulted in the discontinued use of ristocetin in antibiotic therapy (10), but has provided a useful tool for studying the bleeding disorder, von Willebrand's disease. The disease is genetic in origin and is characterized by the lack of a functional plasma protein believed to be required for the adhesion of platelets to vascular epithelia during primary thrombotic events (11). This plasma protein, termed von Willebrand's factor, is also necessary to support ristocetin-induced platelet aggregation of human platelets (12). Thus, ristocetin has found increasing use not only in facilitating the diagnosis of von Willebrand's disease but also in characterizing the plasma protein (13).     

Effect of soluble fibrin on the blood coagulation process and platelets aggregation

The accumulation of soluble fibrin (SF) in the blood plasma causes acceleration of the final stage of blood coagulation. It increases functional activity of a hemostasis system platelet link, that is the precondition of thrombotic complication. Accumulation of SF in the blood plasma is accompanied by proportional reduction of coagulation time in ancistron and thrombin time tests, and also the intensification of platelets aggregation process. A conclusion was drawn that for early diagnostics of the DIC-syndrom it is expedient to carry out complex estimation of the hemostasis system with obligatory definition of the blood SF content, performance of ancistron and thrombin time tests, and also study of platelets aggregation.     

Plasma protein regulation of platelet function and metabolism

Summary This reviews summarizes our evidence suggesting that the plasma protein environment influences platelet aggregation potential and metabolic activity.Cationic proteins are capable of restoring the aggregation potential of washed human platelets. The aggregation restoring effect of gamma globulin is inhibited by more anionic proteins in subfractions of Cohn fraction IV and fractions V and VI. Artificial enhancement of the net negative charge of plasma proteins through acylation produces derivatives capable of inhibiting platelet aggregation in platelet rich plasma.The oxygen consumption of washed human platelets is lower than in platelet rich plasma while the lactate production is identical. Autologous plasma, albumin or IgG immunoglobulin restores the oxygen consumption of washed platelets to values comparable to those obtained for platelet rich plasma, while the lactate production is unaffected. Fibrinogen or IgA myeloma protein increases the lactate production, but not the oxygen consumption. Cyclic AMP levels are considerably lower in washed platelets than in platelet rich plasma. Gamma globulin and albumin causes a further decrease, which is progressive with time. Fibrinogen causes no change in platelet cyclic AMP content.It is suggested that these observations may in part be explained by the equilibrium between anionic and cationic proteins in the platelet microenvironment.This hypothesis appears applicable in certain clinical situations.     

Primary platelet adhesion receptors

Thrombotic diseases such as heart attack and stroke remain a major health concern in the Western world despite existing anti-thrombotic drugs. Current studies are revealing structure-function relationships of primary platelet adhesion receptors mediating adhesion, activation and aggregation, and the molecular mechanisms underlying platelet thrombus formation. Platelet adhesion is relevant not only to thrombotic disease, but there is increasing evidence of a specific role for platelets in vascular processes such as inflammation and atherogenesis. This review focuses on recent advances in understanding the molecular basis for platelet thrombus formation, in particular the receptors, glycoprotein (GP)Ib-IX-V and GPVI, that initiate platelet adhesion and activation at high shear stress.     

Synthesis and biological evaluation of some dibenzofuran-piperazine derivatives

In the present paper, a novel series of dibenzofuran-piperazine derivatives were synthesized via the treatment of N-(2-methoxy-3-dibenzofuranyl)-2-chloroacetamide with substituted piperazine derivatives. The chemical structures of the compounds were elucidated by ¹H NMR, ¹³C NMR, mass spectral data; elemental analysis and HPLC analysis. Each derivative was evaluated for antiplatelet activity and anticholinesterase activity. Compound 2?m with 2-furoyl moiety exhibited high percentage inhibition as much as standard drug aspirin on arachidonic acid (AA)-induced platelet aggregation. None of the compounds presented significant inhibitor effect on collagen-induced platelet aggregation. Furthermore, the anticholinesterase activity of the compounds was determined and they did not show promising inhibitor activity compared with standard drug donepezil.     

Norcantharidin,a clinical used chemotherapeutic agent,acts as a powerful inhibitor by interfering with fibrinogen–integrin αIIbβ3 binding in human platelets

During platelet activation, fibrinogen binds to its specific platelet receptor, integrin α_IIbβ₃, thus completing the final common pathway for platelet aggregation. Norcantharidin (NCTD) is a promising anticancer agent in China from medicinal insect blister beetle. In this study, we provided the evidence to demonstrate NCTD (0.1–1.0 μM) possesses very powerful antiplatelet activity in human platelets; nevertheless, it had no effects on surface P‐selectin expression and only slight inhibition on ATP‐release reaction in activated platelets. Moreover, NCTD markedly hindered integrin α_IIbβ₃ activation by interfering with the binding of FITC‐labelled PAC‐1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin β₃, Src and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin α_IIbβ₃‐mediated outside‐in signalling in human platelets. Besides, NCTD substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. In conclusion, NCTD has dual activities, it can be a chemotherapeutic agent for cancer treatment, and the other side it possesses powerful antiplatelet activity for treating thromboembolic disorders.     

Changes in the cyclic nucleotide level and the inhibition of human thrombocyte aggregation in 3-hydroxypyridine exposure

The effect of six 3-oxypiridine derivatives (at a concentration of 10(-3) and 5 X 10(-3) M) on cyclic nucleotide level in human platelets and platelet aggregation was studied. Five 3-oxypiridine derivatives were shown to depress platelet aggregation, four of them causing the increase in cAMP platelet level. The correlation between antiaggregation activity of 3-oxypiridine derivatives and their ability to rise cyclic nucleotide level in human platelets is discussed.     

Hypergravity results in human platelet hyperactivity

Thrombotic diseases or fatalities have been reported to occasionally occur under conditions of hypergravity although the mechanism is still unclear. To investigate the effect of hypergravity on platelets that are the primary players in thrombus formation, platelet rich plasma (PRP) or washed platelets were exposed to hypergravity at 8 G for 15 minutes. No platelet aggregation was induced by 8 G alone, whereas ristocetin or collagen-induced platelet aggregation was significantly increased. The number of platelets adherent to immobilized fibrinogen and the area of platelets spreading on von Willbrand factor (VWF) matrix were increased simultaneously. Flow cytometry assay indicated that integrin αIIbß3 was partially activated in 8 Gexposed platelets, but there was no significant difference in P-selectin surface expression between platelets treated with 8 G and 1 G control. The results indicate that hypergravity leads to human platelet hyperactivity, but fails to incur essential platelet activation events, suggesting a novel mechanism for thrombotic diseases occurring under hypergravitional conditions.     

Peptide-decorated liposomes promote arrest and aggregation of activated platelets under flow on vascular injury relevant protein surfaces in vitro

Platelet-mimetic synthetic hemostats are highly attractive in transfusion medicine. To this end, past research reports have described particles that either amplify platelet aggregation or mimic platelet adhesion. However, a construct design that effectively combines both functionalities has not been reported. Here we describe the design of a liposomal construct simultaneously surface-decorated with three peptides (a vWF-binding peptide (VBP), a collagen-binding peptide (CBP), and an active platelet clustering cyclic-RGD (cRGD) peptide), that can integrate platelet-mimetic dual hemostatic activities of adhesion and aggregation. We first demonstrate that surface-immobilized cRGD-liposomes are capable of aggregating activated platelets onto themselves. Subsequently, we demonstrate that hetero-multivalent liposomes bearing VBP, CBP, and cRGD, when introduced in flow with ≈ 20,000 activated platelets per microliter, are capable of adhering to vWF/collagen surfaces and promoting the recruitment/aggregation of platelets onto themselves. We envision that optimizing this construct can lead to a highly refined synthetic hemostat design for potential application in transfusion medicine.     

Antiplatelet,antioxidative, and anti-inflammatory effects of hydroquinone

Platelets play crucial roles in thrombosis and hemostasis through platelet activation and aggregation that are crucial in cardiovascular diseases. Hydroquinone (HQ) and its derivatives are present in many dermatological creams, paints, motor fuels, air, microorganisms, and plant products like wheat bread, fruit, coffee, and red wine. The effect of HQ on humans is not clear. In this study, we found that HQ (>25 μM) inhibited arachidonic acid (AA)-induced platelet aggregation. HQ suppressed AA-induced thromboxane B2 production of platelets. HQ (>10 μM) also attenuated ex vivo platelet-rich plasma aggregation. HQ prevented the interleukin (IL)-1β-induced 8-isoprostane, and PGE2 production, but not IL-8 production of pulp cells. These results indicate that HQ may have an antiplatelet effect via inhibition of thromboxane production. HQ has antioxidative and anti-inflammatory effects, and possible inhibition of COX. Exposure and consumption of HQ-containing products, food or drugs may have antiplatelet, antioxidative, and anti-inflammatory effects.     

Chitosan enhances platelet adhesion and aggregation

In this study, chitosan (MW=50,000) was tested for its enhancing platelet activity in rabbit platelet suspensions and the possible mechanisms involved were further investigated. Our results showed that after initial (5 min) and long-term (30 min) contact of platelets with chitosan, the platelet adhesion to chitosan-coated microtiter plates was dose-dependently increased compared to that of solvent control. Similarly, chitosan also dose-dependently increased the platelet aggregation and the intracellular free Ca(2+) rise of Fura-2-AM loaded platelets. Additionally, in the presence of FITC-labeled anti-CD41/CD61, chitosan significantly enhanced the expression of platelet glycoprotein IIb/IIIa complex assayed by a flow cytometer. It is concluded that chitosan is an effective inducer for platelet adhesion and aggregation and the mechanisms of action of chitosan may be associated, at least partly, with the increasing [Ca(2+)](i) mobilization and enhancing expression of GPIIb/IIIa complex on platelet membrane surfaces.