Platelet Aggregation In Plasma Research Articles

CARD12: Shear Stress Renders Redistribution of P2Y12 and PAR-1 Receptors from Platelets to Microparticles: A Cue to Limited Effectiveness of Antiplatelet Therapy in MCS

Background: Platelet exposure to hypershear stress generated by mechanical circulatory support (MCS) results in platelet dysfunction, thrombosis, and bleeding coagulopathy. Antiplatelet agents, targeting ADP-evoked P2Y12 and thrombin-evoked PAR1 platelet receptors, have limited effectiveness reducing MCS thrombosis, rather favoring bleeding. Shear-mediated platelet dysfunction is associated with the downregulation of platelet adhesion receptors, decreased aggregation response, and generation of platelet-derived microparticles (PDMPs). We tested the hypothesis that MCS shear stress alters the surface expression of agonist receptors P2Y12 and PAR1 on platelets and PDMPs, thus affecting platelet aggregation response induced by ADP, collagen, and TRAP-6. Methods: Human platelets were exposed to shear stress (30-70 dynes/cm2, 10’) or sonication; PDMPs were isolated via differential centrifugation. P2Y12, PAR1, & αIIbβ3 expression on platelets and PDMPs was visualized by immunostaining and multi-colored flow cytometry. Platelets were distinguished from PDMPs by forward/side scatter vs. standard fluorescent nanobeads SPHEROTM. Platelet aggregation in plasma induced by ADP, collagen, and TRAP-6 was assessed by optical aggregometry. Results: Platelet exposure to increasing shear stress and sonication resulted in a decrease in P2Y12+ and PAR1+ platelet populations (Fig. 1A&B). The baseline surface density of P2Y12 on platelets was very low and slightly increased after 70 dyne/cm2 shear (Fig. 1C). While also low, PAR1 density on platelets tended to decrease with increasing shear stress (Fig. 1D). Shear stress induced generation of P2Y12+ and PAR1+ PDMPs, accounting for 3.5±0.6% and 4.3±0.5% after 70 dyne/cm2 shear, respectively. Remarkably, P2Y12 and PAR1 surface density on PDMPs was between 7 and 10-fold higher than on platelets (Fig. 1C&D). When added to intact platelets, PDMPs significantly decreased platelet aggregation in plasma induced by ADP, collagen, and TRAP-6. Conclusions: Elevated shear stress promotes generation of PDMPs carrying increased amounts of P2Y12 and PAR1 on their surface; the surface density of these receptors on sheared platelets was also affected. Sheared PDMPs reduced platelet aggregation likely acting as competitive inhibitors imposing steric difficulties for platelets to aggregate and thus favoring MCS bleeding phenotype and limiting the effectiveness of antiplatelet agents.

Nanoparticle size and surface charge determine effects of PAMAM dendrimers on human platelets in vitro.

Blood platelets are essential in maintaining hemostasis. Various materials can activate platelets and cause them to aggregate. Platelet aggregation in vitro is often used as a marker for materials' thrombogenic properties, and studying nanomaterial interaction with platelets is an important step toward understanding their hematocompatibility. Here we report evaluation of 12 formulations of PAMAM dendrimers varying in size and surface charge. Using a cell counter based method, light transmission aggregometry and scanning electron microscopy, we show that only large cationic dendrimers, but not anionic, neutral or small cationic dendrimers, induce aggregation of human platelets in plasma in vitro. The aggregation caused by large cationic dendrimers was proportional to the number of surface amines. The observed aggregation was not associated with membrane microparticle release, and was insensitive to a variety of chemical and biological inhibitors known to interfere with various pathways of platelet activation. Taken in context with previously reported studies, our data suggest that large cationic PAMAM dendrimers induce platelet aggregation through disruption of membrane integrity.

Ropivacaine: A review of its pharmacology and clinical use

Ropivacaine is a long-acting amide local anaesthetic agent and first produced as a pure enantiomer. It produces effects similar to other local anaesthetics via reversible inhibition of sodium ion influx in nerve fibres. Ropivacaine is less lipophilic than bupivacaine and is less likely to penetrate large myelinated motor fibres, resulting in a relatively reduced motor blockade. Thus, ropivacaine has a greater degree of motor sensory differentiation, which could be useful when motor blockade is undesirable. The reduced lipophilicity is also associated with decreased potential for central nervous system toxicity and cardiotoxicity. The drug displays linear and dose proportional pharmacokinetics (up to 80 mg administered intravenously). It is metabolised extensively in the liver and excreted in urine. The present article details the clinical applications of ropivacaine and its current place as a local anaesthetic in the group.

Calcium-dependent platelet aggregation through the interaction between phosphatidylserine and LOX-1

Aims Platelet aggregation at the injured surface of blood vessels plays a crucial role in arterial thrombosis. Platelet aggregation in plasma has been analysed in the presence of calcium chelator to avoid coagulation. Here, we describe a novel mechanism of platelet aggregation that involves calcium-dependent binding between phosphatidylserine (PS) and lectin-like oxidized LDL receptor-1 (LOX-1), and the contribution of platelet LOX-1 to arterial thrombosis. Methods and results In vitro , LOX-1 gene deletion and anti-LOX-1 antibody suppressed the aggregation of platelets from mice and humans, respectively, in the absence of calcium-chelating citrate, but not in the presence of citrate. LOX-1 blockade and annexin V, in parallel, suppressed platelet aggregation and shifted the population of aggregates from large to small in size. Simultaneous application of anti-LOX-1 antibody and annexin V did not exert additive suppressive effects on aggregation. On activation, platelets exposed LOX-1 and PS and bound to a PS-coated surface in an LOX-1-dependent manner. In vivo , both LOX-1 deletion and anti-LOX-1 antibody significantly suppressed thrombus formation in mice and rats, respectively. Furthermore, platelet transfusion from LOX-1 knockout to wild-type mice still showed the suppressive effects of LOX-1 gene deletion, suggesting that platelet LOX-1 promoted thrombus formation. Conclusion LOX-1 blockade could be a novel approach to prevent platelet aggregation and thrombosis.

Platelet Aggregation Is Inhibited by Nitrite Reduction to Nitric Oxide In Blood

AbstractAbstract 3189Nitrite is now recognized as a circulating and storage form of nitric oxide (NO), whose biological effect is largely mediated by its chemical reduction to NO via the nitrite reductase activity of heme-containing proteins and other enzymatic reactions. NO activates soluble guanylyl cyclase to produce cGMP, leading to vasodilation that facilitates tissue oxygen supply, but which is also known to strongly inhibit platelet aggregation. Although the vasorelaxation effect of nitrite has recently been clarified, the effect of nitrite, especially at physiologic levels, on platelet function has not been well studied. We hypothesized that platelet inhibition by nitrite could occur under the conditions known to promote its reduction to NO, such as the presence of deoxygenated erythrocytes. We found that nitrite up to 100 μM had no effect on platelet aggregation in plasma; however, in the presence of erythrocytes inhibition of platelet aggregation by nitrite was observed, which was dependent on hematocrit. At 20% hematocrit, nitrite at physiologic concentration (0.1 mM) significantly inhibited platelet aggregation induced by either ADP or collagen. In contrast, DEANONOate, an NO donor, at low concentrations inhibited platelet aggregation in plasma, but its effect was abolished by erythrocytes at even 1% hematocrit. C-PTIO, a membrane impermeable NO scavenger, completely inhibited the effect of nitrite plus erythrocytes on platelets, suggesting that nitrite was converted to NO in or on the surface of the erythrocytes and then entered the platelets. Furthermore, deoxygenation increased the inhibitory effect of nitrite plus erythrocytes on platelets as demonstrated by a decrease of P-selectin expression (a marker of platelet degranulation) and a corresponding increase in cGMP levels in the platelets. We also found negative correlations between platelet aggregation induced by ADP and nitrite concentrations in whole blood and erythrocyte suspensions from various individuals. These results suggest an essential role of endogenous nitrite as a source of NO, produced by erythrocytes and perhaps other cells in the vasculature, in modulating platelet activity in the circulation. Although nitrite alone at physiological concentrations does not have a direct inhibitory effect on platelets, nitrite plus erythrocytes, which simulate the normal circulating blood, inhibit platelet aggregation and the effect is promoted by deoxygenation. The net effect is modulated by the tendency of oxyhemoglobin in erythrocytes to destroy NO and will likely depend on relative concentrations and the kinetics of the competing reactions, including the mechanisms by which NO activity may leave the erythrocyte. We suggest that these reactions may contribute to differences in the mechanisms of hemostasis in arterial and venous blood and might be the basis of pharmacological approaches to preventing thrombosis. Disclosures:Schechter:National Institutes of Health: I am a co-inventor of a patent for the use of nitrites in the treatment of cardiovascular diseases., Patents & Royalties.

Fructose-1,6-bisphosphate inhibits in vitro and ex vivo platelet aggregation induced by ADP and ameliorates coagulation alterations in experimental sepsis in rats

Sepsis is a systemic response to an infection that leads to a generalized inflammatory reaction. There is an intimate relationship between procoagulant and proinflammatory activities, and coagulation abnormalities are common in septic patients. Pharmaceutical studies have focused to the development of substances that act on coagulation abnormalities and on the link between coagulation and inflammation. Fructose-1,6-bisphosphate (FBP) is a high-energy glycolitic metabolite that in the past two decades has been shown therapeutic effects in great number of pathological situations, including sepsis. The aims of this study were to assess the effects of FBP on platelet aggregation in vitro and ex vivo in healthy and septic rats and evaluate the use of FBP as a treatment for thrombocytopenia and coagulation abnormalities in abdominal sepsis in rat. FBP inhibited platelet aggregation (P < 0.001) in vitro in healthy rats from the smallest dose tested, 2.5 mM, in a dose-dependent manner. The mean effective dose calculated was 10.6 mM. The highest dose tested, 40 mM, completely inhibited platelet aggregation (P < 0.001) induced by ADP. Platelet aggregation in plasma from septic rats was inhibited only with higher doses of FBP, starting from 20 mM (P < 0.001). The calculated mean effective dose was 19.3 mM. Ex vivo platelet aggregation in septic rats was significantly lower (P < 0.05) than healthy rats and the treatment with FBP, at the dose of 2 g/kg, diminished the platelet aggregation at the extension of 27% (P < 0.001), suggesting that FBP is a potent platelet aggregation inhibitor in vivo. Moreover, treatment with FBP 2 g/kg prevented thrombocytopenia (P < 0.001), prolongation of prothrombin and partial thromboplastin time (P < 0.001), but not fibrinogen, in septic rats. The most important findings in this study are that FBP is a potent platelet aggregation inhibitor, in vitro and ex vivo. It presents protective effects on coagulation abnormalities, which can represent a treatment against DIC. The mechanisms for these effects remain under investigation.

The in-vitro effects of E5555, a protease-activated receptor (PAR)-1 antagonist, on platelet biomarkers in healthy volunteers and patients with coronary artery disease

E5555 is a potent protease-activated receptor (PAR-1) antagonist targeting the G-coupled receptor and modulating thrombin-platelet-endothelial interactions. The drug is currently being tested in phase II trials in patients with coronary artery disease (CAD) and has potential antithrombotic and anti-inflammatory benefits. We investigated the in-vitro effects of E5555 on platelet function beyond PAR-1 blockade in healthy volunteers and CAD patients treated with aspirin (ASA) with or without clopidogrel. Conventional aggregation induced by 5 microM ADP, 1 microg/ml collagen, 10 microM TRAP, whole blood aggregation with 1 microg/ml collagen, and expression of 14 intact, and TRAP-stimulated receptors by flow cytometry were utilised to assess platelet activity after preincubation with escalating concentrations of E5555 (20 ng/ml, 50 ng/ml, and 100 ng/ml) in healthy volunteers, CAD patients treated with ASA, and CAD patients treated with ASA and clopidogrel combination (n=10, for each group). E5555 inhibited a number of platelet biomarkers. Platelet inhibition was usually moderate, present already at 20 ng/ml, and was not seemingly dose-dependent without TRAP stimulation. E5555 caused 10-15% inhibition of ADP- and collagen-induced platelet aggregation in plasma, but not in whole blood. TRAP-induced aggregation was inhibited almost completely. PECAM-I, GP IIb/IIIa antigen, and activity with PAC-1, GPIb, thrombospondin, vitronectin receptor expression, and formation of platelet-monocyte aggregates were also significantly reduced by E5555. TRAP stimulation caused dose-dependent effects between 20 and 50 ng/ml E5555 doses. P-selectin, LAMP-1, LAMP, and CD40-ligand were not affected by E5555. In conclusion, E5555 in vitro moderately but significantly inhibits platelet activity beyond PAR-1 blockade. Antiplatelet potency of ASA alone, and the combination of ASA and clopidogrel may be enhanced by E5555 providing rationale for their synergistic use. Selective blockade of platelet receptors suggests unique antiplatelet properties of E5555 as a potential addition to current antithrombotic regimens.

Evidence for the involvement of 5-HT 2A receptors in mild mesenteric ischemia/reperfusion dysfunctions in mice

In this study, the involvement of 5-HT 2A receptors on mesenteric ischemia–reperfusion injury was examined in mice. Intestinal ischemia produced by 45 min occlusion of superior mesenteric artery was followed by 24 h reperfusion (I/R). The 5-HT 2A selective antagonist, ketanserin (0.5 mg kg −1) or the 5-HT 2A agonist DOI (0.25 mg kg −1) was intravenously administered before ischemia and 8 h after the beginning of reperfusion. The effects were compared with those obtained in sham operated animals (S). Ketanserin prevented the upper gastrointestinal transit delay induced by I/R ( P < 0.01), protected intestine from leukocyte recruitment as indicated by jejunal myeloperoxidase activity ( P < 0.05) and reverted Evans Blue extravasation elicited by I/R in lung, colon and jejunum ( P < 0.05). On the other hand, 5-HT 2A activation by DOI mimicked the effects of I/R in S mice prolonging small intestine transit ( P < 0.05) and enhancing neutrophil accumulation in jejunal tissues ( P < 0.05). Furthermore, the reduction of ADP-induced platelet aggregation in plasma of I/R mice was prevented by ketanserin treatment. All together, these findings support the critical involvement of 5-HT 2A receptor subtype in mediating the damage induced by mesenteric I/R in mice.

Porphyromonas gingivalis‐induced platelet aggregation in plasma depends on Hgp44 adhesin but not Rgp proteinase

Evidence from recent epidemiological studies suggests a link between periodontal infections and increased risk of atherosclerosis and related cardiovascular and cerebrovascular events in human subjects. One of the major pathogens of periodontitis, Porphyromonas gingivalis, has the ability to aggregate human platelets in platelet-rich plasma (PRP). Mechanism of P. gingivalis-induced platelet aggregation in PRP was investigated. Proteinase inhibitors toward Arg-gingipain (Rgp) and Lys-gingipain (Kgp) did not suppress P. gingivalis-induced platelet aggregation in PRP, whereas the Rgp inhibitor markedly inhibited P. gingivalis-induced platelet aggregation using washed platelets. Mutant analysis revealed that P. gingivalis-induced platelet aggregation in PRP depended on Rgp-, Kgp- and haemagglutinin A (HagA)-encoding genes that intragenically coded for adhesins such as Hgp44. Hgp44 adhesin on the bacterial cell surface, which was processed by Rgp and Kgp proteinases, was essential for P. gingivalis-induced platelet aggregation in PRP. P. gingivalis cell-reactive IgG in plasma, and FcgammaRIIa receptor and to a lesser extent GPIbalpha receptor on platelets were found to be a prerequisite for P. gingivalis-induced platelet aggregation in PRP. These results reveal a novel mechanism of platelet aggregation by P. gingivalis.

Aggregation of human platelets in plasma by porcine blood cells in vitro is probably mediated by thrombin generation.

The infusion of pig progenitor cells into baboons is associated with a thrombotic microangiopathy probably related to the interaction of these cells with the baboon endothelial cells and platelets. We have shown previously that pig peripheral blood mononuclear cells (p-PBMC), are able to activate the human coagulation cascade as they are able to generate thrombin when added to defibrinated plasma. In this work, we have tested the interaction of p-PBMC with human platelets to assess the capacity of p-PBMC to cause platelet aggregation and the possible role of complement activation in this aggregation. Human platelet aggregation assays, using collagen (1 or 2 microg/ml), were performed with platelets in platelet-rich plasma (PRP) or platelets washed by filtration. PRP or washed platelets were also incubated with p-PBMC or human PBMC (h-PBMC) at several concentrations and aggregation was measured. The effect of Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA), an inhibitor of thrombin, was studied on platelet aggregation caused by the pig cells. Complement activation was measured by deposition of fragment c derived from C3 splitting (C3c) on pig cells incubated with citrated platelet poor plasma (PPP). When human PRP was incubated with p-PBMC, aggregation was a consistent event quantitatively similar to that induced by collagen. No aggregation of washed platelets was observed when these were incubated with p-PBMC or h-PBMC. Aggregation of human platelets in PRP, induced by p-PBMC, was inhibited when DAPA (100 microm) was added to the incubation mixture (23%), indicating that the thrombin inhibitor blocked the capacity of p-PBMC to aggregate human platelets. No deposition of C3c fragments on p-PBMC was detected when the porcine cells were incubated for up to 20 min with citrated PPP. The fact is that p-PBMC induces human platelet aggregation in plasma being thrombin generation a likely explanation for this observation. Our data suggest that, in the system assayed, complement activation is not a cause of platelet aggregation. These findings are relevant for the clarification of the reported thrombotic microangiopathy complicating the intravenous infusion of pig cells in primates in attempts to induce pig tolerance in baboons.

Roles of Asp54 and Asp213 in Ca 2+ utilization by soluble human CD39/ecto-nucleotidase

Soluble human CD39 (solCD39) rapidly metabolizes nucleotides, especially ADP released from activated platelets, thereby inhibiting further platelet activation and recruitment. Using alanine substitution mutagenesis, we established a functional role for aspartates D54 and D213 in solCD39. Kinetic analyses of D54A and D213A indicated decreased K m s of the mutants, compared to wild type, for the cofactor calcium and for the substrates ADP and ATP. These decreases in calcium and nucleotide affinity of the mutants were accompanied by increases in their rate of catalysis. The decreased affinity of the mutants for calcium was responsible for their diminished ability to reverse platelet aggregation in plasma anticoagulated with citrate, a known calcium chelator. Their ADPase activity in the presence of citrated plasma was also decreased, although this could be overcome with excess calcium. Thus, aspartates 54 and 213 are involved in calcium utilization and potentially involved in cation coordination with substrate in the catalytic pocket of solCD39.

Antiplatelet Effects of Sodium Nitroprusside in Flowing Human Blood: Studies under Normoxic and Hypoxic Conditions

We explored the ability of sodium nitroprusside to modify adhesive and cohesive function of platelets in flowing blood, under normoxic and hypoxic conditions. Aliquots of both untreated and sodium nitroprusside–treated blood were prepared for studies of: (1) platelet aggregation in plasma; (2) erythrocyte deformability; (3) platelet interaction with damaged subendothelium, by using a well-defined perfusion system; and (4) blood gasometry in the perfused samples. Results showed that sodium nitroprusside–treated blood always showed a totally inhibited arachidonic acid–induced platelet aggregation in plasma, as well as significantly increased erythrocyte deformability (0.44±0.09 up to 0.66±0.05; p<0.05). However, treatment with sodium nitroprusside did not modify the pattern of platelet interaction with subendothelium (percentage of contact, adhesion, thrombus, and covered surface) with respect to untreated blood, under any of the shear rates used (300, 800, and 1800 seconds −1), although it significantly reduced the height of thrombi (9.8±0.4 vs. 8.3±0.4 μm; p<0.05). Hypoxic conditions did not have a noticeable effect in modifying antiplatelet effects of sodium nitroprusside. Additionally, the presence of sodium nitroprusside impaired the normal oxygenation of the blood during perfusion. pO 2 in control untreated samples rose from 40.3±5.0 mm Hg perfusions to 100.4±12.5 mm Hg but remained at 66.3±6.3 mm Hg in sodium nitroprusside–treated blood ( p<0.05). Our results did not show a significant effect of sodium nitroprusside in the modulation of platelet interaction with subendothelium. The marginal reduction in the thrombi height could be related to rheological interference of increased erythrocyte deformability.

Aggregation of blood platelets in static magnetic fields

We investigated the effects of intense magnetic fields on the blood platelet aggregation process with and without static magnetic fields of up to 14 T. A rabbit plasma and collagen mixture was used as the model system for a wounded blood vessel. Platelet aggregation was activated by the stimulation of acid soluble collagen. The platelet aggregates in strong magnetic fields were larger than the aggregates in an ambient field. An optical transmission of blood plasma during platelet aggregation also indicated that strong magnetic fields enhanced blood platelet aggregation in plasma.

Effect of high-density lipoproteins on ADP-induced platelet aggregation in plasma

Autooxidized high-density lipoproteins (HDL2) inhibit ADP-induced platelet aggregation in platelet-rich plasma. Platelet aggregation in the presence of native HDL2 and HDL3 and autooxidized HDL3 does not differ from the control (plasma with buffer). A conclusion is made on the important role of autooxidized HDL2 as a thrombogenesis-inhibiting factor in atherosclerosis.

Effect of Ca2+ on GP IIb-IIIa interactions with integrilin: enhanced GP IIb-IIIa binding and inhibition of platelet aggregation by reductions in the concentration of ionized calcium in plasma anticoagulated with citrate.

Integrilin (eptifibatide), a potent inhibitor of the fibrinogen binding function of GP IIb-llla, has been shown to reduce the thrombotic complications of angioplasty and of acute coronary syndromes. The present study was designed to determine whether the reduced Ca2+ concentrations in plasma anticoagulated with citrate affect Integrilin binding to GP IIb-IIIa and the ex vivo pharmacodynamic measurements for this drug. Lower concentrations of Integrilin were found to inhibit platelet aggregation in plasma anticoagulated with citrate (for ADP, mean+/-SD IC(50)=140+/-40 nmol/L, n=6; Ca2+ =40 to 50 micromol/L) than with PPACK (IC(50)=570+/-70 nmol/L, P<.0001, n=6; Ca2+ approximately 1 mmol/L). Chelation of Ca2+ with EDTA or citrate caused a similar degree of enhancement in the inhibitory activity of Integrilin. Measurements of D3 LIBS epitope expression showed that the enhanced inhibitory activity was caused by enhanced GP IIb-IIIa occupancy by Integrilin. Citrate anticoagulation decreased the amounts of Integrilin required to inhibit the binding of PAC1, a monoclonal antibody that mimics the GP IIb-IIIa binding activity of fibrinogen. Reduced Ca2+ also increased Integrilin inhibition of the binding of biotinylated fibrinogen to purified, immobilized GP IIb-IIIa. These data suggest that citrate anticoagulation removes Ca2+ from GP IIb-IIIa and enhances the apparent inhibitory activity of Integrilin. This finding indicates that the inhibitory activity of Integrilin is overestimated in blood samples collected with citrate, suggesting that it may be possible to achieve greater antithrombotic efficacy beyond that observed in clinical trials to date with Integrilin.

The H1-histamine antagonist dithiaden inhibits human platelet function in vitro

The H1-histamine receptor antagonist Dithaden® inhibited in a dose-dependent manner, human platelet aggregation in vitro that was induced with stimuli in the following rank order of potency: thrombin > A23187 > adrenaline > ADP. The aggregation of platelets in plasma induced with adrenaline was inhibited by Dithiaden (DIT) (both the first and the second phase) and the onset of the second phase was prolonged significantly. In a concentration-dependent manner DIT inhibited thrombin- and calcium ionophore A23187-induced [3H]arachi-donic acid liberation from, and peroxidation (measured as malondialdehyde formation) of membrane phospholipids. The same effect of DIT was found for the inhibition of thromboxane B2 generation. It is suggested that DIT does not inhibit stimulated platelets at specific histamine receptors sites but rather at the phospholipase A2 and thromboxane synthase level. Results from in vitro experiments should be verified in vivo.

The H1-histamine antagonist dithiaden inhibits human platelet function in vitro

The H-histamine receptor antagonist Dithaden inhibited in a dose-dependent manner, human platelet 1 aggregation in vitro that was induced with stimuli in the following rank order of potency: thrombin > A23187 > adrenaline > ADP. The aggregation of platelets in plasma induced with adrenaline was inhibited by Dithiaden (DIT) (both the first and the second phase) and the onset of the second phase was prolonged significantly. In a concentration-dependent manner DIT inhibited thrombin- and calcium ionophore A23187-induced {3H}arachidonic acid liberation from, and peroxidation (measured as malondialdehyde formation) of membrane phospholipids. The same effect of DIT was found for the inhibition of thromboxane B suggested that DIT does not inhibit stimulated platelets at specific histamine receptors sites but rather at the phospholipase A and thromboxane synthase level. Results from in vitro experiments should be verified in 2 vivo . generation. It is 2.

The glycoprotein IIb/IIIa antagonist c7E3 inhibits platelet aggregation in the presence of heparin-associated antibodies.

Heparin-associated antibodies (HAAb), in the presence of heparin, can cause platelet activation and aggregation. The purpose of this study was to assess whether a platelet glycoprotein (GP) IIb/IIIa receptor antagonist, c7E3, would inhibit platelet aggregation in the presence of HAAb. If aggregation is inhibited by c7E3, enzyme-linked immunosorbent assays (ELISA) would be done to determine whether c7E3 interfered with the binding of heparin and the HAAb. HAAb-positive plasmas from 21 patients (determined by platelet aggregation assays) were studied. Normal donor platelet-rich plasmas (PRP) were incubated (1 minute) with either saline solution or 3 microg/ml of c7E3. Platelet-poor plasma from patients with HAAb and one of three sources of heparin (25 microl, 10 U/ml; porcine heparin, bovine heparin, and low molecular weight heparin [enoxaparin]) were added to the PRP mixture. Aggregation was determined using a platelet aggregometer by measuring time to aggregation, the slope of the aggregation curve, and the percent change in optical density. Platelet aggregation occured in 100%, 100%, and 95% of the saline solution incubations exposed to porcine heparin, bovine heparin, and enoxaparin, respectively. Incubation with c7E3 caused 100% inhibition of platelet aggregation in plasma exposed to porcine heparin, bovine heparin, and enoxaparin. The optical density curves obtained from the ELISA, which were dependent on the binding of HAAb to heparin, were not significantly different when c7E3 was compared to buffer alone. The GP IIb/IIIa receptor antagonist, c7E3, inhibits HAAb-induced platelet aggregation via a mechanism that does not appear to interfere with the binding between heparin and HAAb. Clinical trials are warranted to assess whether GP IIb/IIIa antagonists may allow patients with HAAb to safely receive heparin.

Platelet receptors for the Streptococcus sanguis adhesin and aggregation-associated antigens are distinguished by anti-idiotypical monoclonal antibodies.

Platelets aggregate in response to an adhesin and the platelet aggregation-associated protein (PAAP) expressed on the cell surfaces of certain strains of Streptococcus sanguis. We sought to identify the corresponding PAAP receptor and accessory adhesin binding sites on platelets. Since the adhesion(s) of S. sanguis for platelets has not been characterized, an anti-idiotype (anti-id) murine monoclonal antibody (MAb2) strategy was developed. First, MAb1s that distinguished the adhesin and PAAP antigens on the surface of S. sanguis I 133-79 were selected. Fab fragments of MAb1.2 (immunoglobulin G2b [IgG2b]; 70 pmol) reacted with 5 x 10(7) cells of S. sanguis to completely inhibit the aggregation of human platelets in plasma. Under similar conditions, MAb1.1 (IgG1) inhibited the adhesion of S. sanguis cells to platelets by a maximum of 34%, with a comparatively small effect on platelet aggregation. Together, these two MAb1s inhibited S. sanguis-platelet adhesion by 63%. In Western immunoblots, both MAb1s reacted with S. sanguis 133-79 87- and 150-kDa surface proteins and MAb1.2 also reacted with purified type I collagen. The hybridomas producing MAb1.1 and MAb1.2 were then injected into BALB/c mice. Enlarged spleens were harvested, and a panel of MAb2 hybridomas was prepared. To identify anti-ids against the specific MAb1s, the MAb2 panel was screened by enzyme-linked immunosorbent assay for reaction with rabbit polyclonal IgG antibodies against the 87- and 150-kDa antigens. The reactions between the specific rabbit antibodies and anti-ids were inhibited by the 87- and 150-kDa antigens. When preincubated with platelets, MAb2.1 (counterpart of MAb1.1) inhibited adhesion to platelets maximally by 46% and MAb2.2 (anti-MAb1.2) inhibited adhesion to platelets maximally by 35%. Together, both MAb2s inhibited the adhesion of S. sanguis to platelets by 81%. MAb2.2 also inhibited induction of platelet aggregation. MAb2.2 immunoprecipitated a biotinylated platelet membrane antigen of 170 kDa (unreduced); MAb2.1 precipitated membrane antigens of 175- and 230-kDa (unreduced). Therefore, platelet binding sites and the receptor for the S. sanguis adhesin and PAAP, respectively, are distinguished by the anti-id MAb2s.

Antihistaminic drug bromadryl and stimulated platelet aggregation

The H1-receptor antagonist bromadryl dose-dependently inhibited stimulated rat platelet aggregation in vitro. Bromadryl was 10 times more effective on the secondary aggregation of thrombin-stimulated platelets compared with its inhibition of ADP-stimulated primary platelet aggregation in plasma. The inhibition of aggregation was accompanied by a dose-dependent inhibition of thrombin-stimulated malondialdehyde formation and thromboxane B2 production. The results indicate that bromadryl may interfere intracellularly with membrane phospholipid peroxidation and the arachidonic acid metabolism of stimulated platelets. Bromadryl, like other cationic amphiphilic drugs, may inhibit stimulated platelet functions by decreasing the stimulus-induced activation of phospholipase A2. Our results support the possible interference of bromadryl with histamine as an intraplatelet messenger responsible for aggregation.