Inhibition of microsurgical thrombosis by the platelet glycoprotein IIb/IIIa antagonist SR121566A

Despite major improvements in tools and significant refinements of techniques, microsurgical anastomosis still carries a significant risk of failure due to microvascular thrombosis. The key to improving the success of microvascular surgery may lie in the pharmacologic control of thrombus formation. Central to pathologic arterial thrombosis are platelets. Glycoprotein IIb/IIIa is a highly abundant platelet surface receptor that plays a major role in platelet aggregation by binding platelets to each other through the coagulation factor fibrinogen. To explore the ability of antithrombotic agents to prevent microvascular thrombosis, a rabbit ear artery model was used in which a standardized arterial injury results in predictable thrombus formation. This model was used to examine whether SR121566A, a specific and potent glycoprotein IIb/IIIa inhibitor, can successfully prevent microsurgical thrombosis.Using a coded, double-blind experimental design, 20 rabbits (40 arteries) were assigned to four treatment groups: (1) saline injection (n = 10), (2) acetylsalicylic acid 10 mg/kg (n = 10), (3) heparin 0.5 mg/kg bolus with subsequent intermittent boluses of 0.25 mg/kg every 30 minutes (n = 10), and (4) SR121566A 2 mg/kg bolus (n = 10). After vessel damage and clamp release, arteries were assessed for patency at 5, 30, and 120 minutes by the Acland refill test. Coagulation assays, in vivo bleeding times, and ex vivo platelet aggregation studies were also conducted. Scanning electron microscopy was used to examine mural thrombus composition.A significant, fourfold increase in vessel patency following administration of SR121566A over saline control (80 percent versus 20 percent patency, respectively, at 35 minutes after reperfusion, p < 0.01) was noted. This was correlated with marked inhibition of ex vivo platelet aggregation. This antiplatelet treatment did not prolong coagulation assays (mean international normalized ratio: saline, 0.66 +/- 0.04; SR121566A, 0.64 +/- 0.03; mean thromboplastin time: saline, 19.63 +/- 0.67; SR121566A, 17.87 +/- 3.27) and bleeding times (mean bleeding time: saline, 42 +/- 4; SR121566A, 48 +/- 6). Scanning electron microscopy demonstrated extensive platelet and fibrin deposition in control vessel thrombi. In contrast, thrombi from SR121566A-treated vessels demonstrated predominance of fibrin with few platelets when examined under scanning electron microscopy.Administration of SR121566A was associated with a significant increase in vessel patency, without deleterious effects on coagulation assays or bleeding times. The increase in vessel patency was correlated with inhibition of platelet aggregation and decreased platelet deposition, as demonstrated by scanning electron microscopy. Glycoprotein IIb/IIIa antagonists represent a new class of anti-platelet agents that may be suited for inhibiting microsurgical thrombosis. This study supports further investigation into the use of these agents in microsurgery.     

The anti-platelet approach targeting the fibrinogen ligand of the GPIIB/IIIa receptor.

Activation of the platelet surface receptor GPIIb/IIIa is the final pathway of platelet aggregation, regardless of the initiating stimulus. RGD analogues, peptidomimetics and monoclonal antibodies to GPIIb/IIIa have been developed targeting the blockage of the receptor and inhibition of the fibrinogen binding. However, the intrinsic activating effect of GPIIb/IIIa blockers is widely discussed as one potential contributing factor for the disappointing outcome of trials with GPIIb/IIIa inhibitors. An alternative method for thrombus prevention could be the use of specific fibrinogen blockers since they will act at the final step of the platelet aggregation and are expected to leave the receptor unaffected. To achieve this target the design of the fibrinogen ligands could be based on (i) sequences derived from GPIIb/IIIa ligand binding sites, and (ii) sequences complementary to RGD and/or to fibrinogen gamma-chain. The available information, which could be used as a starting point for developing potent fibrinogen ligands, is reviewed.     

1,2,4-triazolo[3,4-a]pyridine as a novel, constrained template for fibrinogen receptor (GPIIb/IIIa) antagonists

Conformationally constrained analogues of the GPIIb/IIIa antagonist elarofiban (RWJ-53308) have been synthesized and biologically evaluated. The 1,2,4-triazolo[3,4-a]pyridine scaffold provided potent antagonists with favorable pharmacodynamic and pharmacokinetic attributes in dogs. Compounds 12a and 13a exhibited enhancements in oral bioavailability, t(1/2), and ex vivo duration of action (inhibition of ADP-induced platelet aggregation) relative to elarofiban.     

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

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

Purification and characterization of a new RGD/KGD-containing dimeric disintegrin, piscivostatin, from the venom of Agkistrodon piscivorus piscivorus: the unique effect of piscivostatin on platelet aggregation

Piscivostatin, a novel dimeric disintegrin containing Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD) sequences, was isolated from the venom of Agkistrodon piscivorus piscivorus. The molecule consisted of two chains designated as the alpha and beta chains, comprising 65 and 68 amino acid residues, respectively. Piscivostatin had two binding motifs recognized by platelet glycoprotein IIb/IIIa (GPIIb/IIIa), and the biological activity of dimeric disintegrin piscivostatin toward platelet aggregation differed from those of other monomeric disintegrins such as trimestatin and echistatin. We measured platelet aggregation by the laser light scattering method during the process of ADP-induced platelet aggregation. Both dimeric and monomeric disintegrins inhibited the formation of small (9 to 25 microm in diameter), medium-sized and large aggregates (25 to 70 microm in diameter) in a dose-dependent manner. The platelet aggregates disaggregated after reaching a maximal number on either treatment with ADP alone or monomeric disintegrin/ADP. However, the small aggregates did not disaggregate on treatment with piscivostatin/ADP even when applied over time. When washed platelets were incubated with an anti-GPIIb/IIIa monoclonal antibody, PT25-2, which induces conformational changes of GPIIb/IIIa to a form accessible to fibrinogen and other adhesion proteins without platelet activation, piscivostatin induced a platelet shape change alone with no aggregate formation. The present study indicated that piscivostatin has two unique contradictory activities; acting as a double inhibitor of platelet aggregation and platelet aggregate dissociation.     

Platelet talin is phosphorylated by calyculin A

Calyculin A and okadaic acid, potent and cell permeable inhibitors of type 1 and type 2A protein phosphatases, inhibit platelet aggregation and secretion. However, the relationship between phosphatase inhibition and inhibition of platelet function is not well understood. We found that in unstimulated platelets, talin (P235) was phosphorylated at threonine residues by calyculin A. Furthermore, the extent of talin phosphorylation by calyculin A was closely correlated with its inhibition of thrombin-induced platelet aggregation. Since the binding of talin to platelet glycoprotein IIb/IIIa complex has been shown to be affected by its phosphorylation, these results suggest that type 1 and/or type 2A protein phosphatases may play a role in the regulation of membrane-cytoskeleton interaction through dephosphorylation of talin.     

Lack of influence of the COX inhibitors metamizol and diclofenac on platelet GPIIb/IIIa and P-selectin expression in vitro

BACKGROUND: The effect of non-steroidal anti-inflammatory drugs (NSAIDs) for reduced platelet aggregation and thromboxane A2 synthesis has been well documented. However, the influence on platelet function is not fully explained. Aim of this study was to examine the influence of the COX-1 inhibiting NSAIDs, diclofenac and metamizol on platelet activation and leukocyte-platelet complexes, in vitro. Surface expression of GPIIb/IIIa and P-selectin on platelets, and the percentage of platelet-leukocyte complexes were investigated. METHODS: Whole blood was incubated with three different concentrations of diclofenac and metamizol for 5 and 30 minutes, followed by activation with TRAP-6 and ADP. Rates of GPIIb/IIIa and P-selectin expression, and the percentage of platelet-leukocyte complexes were analyzed by a flow-cytometric assay. RESULTS: There were no significant differences in the expression of GPIIb/IIIa and P-selectin, and in the formation of platelet-leukocyte complexes after activation with ADP and TRAP-6, regarding both the time of incubation and the concentrations of diclofenac and metamizol. CONCLUSIONS: Accordingly, the inhibitory effect of diclofenac and metamizol on platelet aggregation is not related to a reduced surface expression of P-selectin and GPIIb/IIIa on platelets.     

A novel naphthalimide derivative reduces platelet activation and thrombus formation via suppressing GPVI

Naphthalimide derivatives have multiple biological activities, including antitumour and anti-inflammatory activities. We previously synthesized several naphthalimide derivatives; of them, compound 5 was found to exert the strongest inhibitory effect on human DNA topoisomerase II activity. However, the effects of naphthalimide derivatives on platelet activation have not yet been investigated. Therefore, the mechanism underlying the antiplatelet activity of compound 5 was determined in this study. The data revealed that compound 5 (5–10 μM) inhibited collagen- and convulxin- but not thrombin- or U46619-mediated platelet aggregation, suggesting that compound 5 is more sensitive to the inhibition of glycoprotein VI (GPVI) signalling. Indeed, compound 5 could inhibit the phosphorylation of signalling molecules downstream of GPVI, followed by the inhibition of calcium mobilization, granule release and GPIIb/IIIa activation. Moreover, compound 5 prevented pulmonary embolism and prolonged the occlusion time, but tended to prolong the bleeding time, indicating that it can prevent thrombus formation but may increase bleeding risk. This study is the first to demonstrate that the naphthalimide derivative compound 5 exerts antiplatelet and antithrombotic effects. Future studies should modify compound 5 to synthesize more potent and efficient antiplatelet agents while minimizing bleeding risk, which may offer a therapeutic potential for cardiovascular diseases.     

Variations of glycoprotein IIb–IIIa (αIIb/β3-integrin) content in healthy donors. The influence on platelet aggregation activity and efficacy of aspirin action

The effects of content of a fibrinogen receptor, glycoprotein (GP) IIb–IIIa (αIIb/β3-integrin), GP IIIa genetic polymorphism (substitution Leu33Pro), and fibrinogen concentration in blood plasma on platelet aggregation activity have been investigated in a group of healthy volunteers. In 35 examined donors the GP IIb–IIIa content on platelet surface varied from 40 to 71 × 10³ per platelet. Repeated measurements revealed that the GP IIb–IIIa content coefficient of variation was 9.5%, and deviations from mean levels did not exceed 20%. The level and the rate of platelet aggregation induced by ADP (1.25–20 μM) correlated with GP IIb–IIIa number (r from 0.315 to 0.591) and were higher in the group of donors with high in comparison with low GP IIb–IIIa content (>60 and (40–50) × 10^?3 per platelet, respectively). Aspirin, the inhibitor of thromboxane A₂ synthesis, partially suppressed ADP-induced platelet aggregation. The level of residual aggregation in the presence of aspirin also correlated with GP IIb–IIIa content and increased in subjects with high receptor content. Parameters of ADP-induced aggregation did not differ in donors with genotypes GP IIIa Pro33(?) (Leu33Leu33, n = 20) and Pro33(+) (Leu33Pro33, n = 13, and Pro33Pro33, n = 2) genotype. GP IIb–IIIa content was also not affected by GP IIIa polymorphism. No significant correlations were found between the level and rate of platelet aggregation and fibrinogen concentration in blood plasma. The data obtained indicate that the effects of variations of GP IIb–IIIa content on platelet aggregation are higher than GP IIIa Leu33Pro polymorphism and variations of fibrinogen concentration. High GP IIb–IIIa content is associated with increased platelet aggregation activity and decreased efficacy of aggregation inhibition by aspirin.     

Juglone prevents human platelet aggregation through inhibiting Akt and protein disulfide isomerase

Background/PurposeJuglone, a natural compound widely found in Juglandaceae plants, has been suggested as a potential drug candidate for treating cancer, inflammation, and diabetic vascular complications. In the present study, the antiplatelet effect and underlying mechanisms of juglone were investigated for the first time.Study design/methodsHuman platelet aggregation and activation were measured by turbidimetric aggregometry, flow cytometry, and Western blotting. In vitro antithrombotic activity of juglone was assessed using collagen-coated flow chambers under whole-blood flow conditions. The effect of juglone on protein disulfide isomerase (PDI) activity was determined by the dieosin glutathione disulfide assay.ResultsJuglone (1 – 5 μM) inhibited platelet aggregation and glycoprotein (GP) IIb/IIIa activation caused by various agonists. In a whole blood flow chamber system, juglone reduced thrombus formation on collagen-coated surfaces under arterial shear rates. Juglone abolished intracellular Ca²⁺ elevation and protein kinase C activation caused by collagen, but had no significant effect on that induced by G protein-coupled receptor agonists. In contrast, Akt activation caused by various agonists were inhibited in juglone-treated platelets. Additionally, juglone showed inhibitory effects on both recombinant human PDI and platelet surface PDI at concentrations similar to those needed to prevent platelet aggregation.ConclusionJuglone exhibits potent in vitro antiplatelet and antithrombotic effects that are associated with inhibition of Akt activation and platelet surface PDI activity.     

Stereoisomeric relationships among anti-inflammatory activity, inhibition of platelet aggregation, and inhibition of prostaglandin synthetase

This study compares the affects of a new non-steroidal anti-inflammatory drug, d,l-6-chloro-alpha-methyl-carbazole-2-acetic acid, its enantiomers, and indomethacin on platelet aggregation, prostaglandin synthetase, adjuvant arthritis, gastric ulceration and arachidonic acid induced diarrhea. In the adjuvant arthritic rat, doses producing anti-inflammatory activity were similar for all compounds with the exception of the l-isomer which was much less active. On the other hand, indomethacin was 10 to 25 times more potent with regard to inhibition of platelet aggregation, inhibition of prostaglandin synthetase, inhibition of arachidonic acid induced diarrhea, and induction of gastric ulceration than the racemate and its isomers. Such divergence of potencies suggests that the racemate, unlike indomethacin, would have no affect on platelet aggregation and, hence, produce no prolongation of bleeding time at doses possessing anti-inflammatory activity. The data also suggest that the racemate and d-isomer have greater specificity toward anti-arthritic activity and are less ulcerogenic than indomethacin. The d-isomer apparently is the more active component of the racemate in all the systems tested since: (a) the d-isomer has 2 to 3 times the inhibitory potency of the racemate and (b) the l-isomer, at high dosages or high concentrations had considerably less affect. Comparison of potencies relative to inhibition of platelet aggregation and of prostaglandin synthetase, are quite close; therefore, mechanistically, the anti-aggregatory affects of these drugs, or lack thereof, may be related to inhibition of prostaglandin synthetase.     

Roles for fibrinogen, immunoglobulin and complement in platelet activation promoted by Staphylococcus aureus clumping factor A

Staphylococcus aureus is an important cause of infective endocarditis (IE) in patients without a history of prior heart valve damage. The ability to stimulate the activation of resting platelets and their subsequent aggregation is regarded as an important virulence factor of bacteria that cause IE. Clumping factor A is the dominant surface protein responsible for platelet activation by S. aureus cells in the stationary phase of growth. This study used Lactococcus lactis as a surrogate host to study the mechanism of ClfA-promoted platelet activation. Expression of ClfA from a nisin-inducible promoter demonstrated that a minimum level of surface-expressed ClfA was required. Using platelets that were purified from plasma, the requirement for both bound fibrinogen and immunoglobulin was demonstrated. The immunoglobulin G (IgG) requirement is consistent with the potent inhibition of platelet activation by a monoclonal antibody specific for the platelet FcgammaRIIa receptor. Furthermore the IgG must contain antibodies specific for the ClfA A domain. A model is proposed whereby bacterial cells armed with a sufficient number of surface-bound fibrinogen molecules can engage resting platelet glycoprotein GPIIb/IIIa, aided by bound IgG molecules, which encourages the clustering of FcgammaRIIa receptors. This can trigger activation of signal transduction leading to activation of GPIIb/IIIa and aggregation of platelets. In addition, analysis of a mutant of ClfA totally lacking the ability to bind fibrinogen revealed a second, although less efficient, mechanism of platelet activation. The fibrinogen-independent pathway required IgG and complement deposition to trigger platelet aggregation.     

Stereoisomeric relationships among anti-inflammatory activity, inhibition of platelet aggregation, and inhibition of prostaglandin synthetase

This study compares the affects of a new non-steroidal anti-inflammatory drug, d,1-6-chloro-alpha-methyl-carbazole-2-acetic acid, its enantiomers, and indomethacin on platelet aggregation, prostaglandin synthetase, adjuvant arthritis, gastric ulceration and arachidonic acid induced diarrhea. In the adjuvant arthritic rat, doses producing anti-inflammatory activity were similar for all compounds with the exception of the I-isomer which was much less active. On the other hand, indomethacin was 10 to 25 times more potent with regard to inhibition of platelet aggregation, inhibition of prostaglandin synthetase, inhibition of arachidonic acid induced diarrhea, and induction of gastric ulceration than the racemate and its isomers. Such divergence of potencies suggests that the racemate, unlike indomethacin, would have no affect on platelet aggregation and, hence, produce no prolongation of bleeding time at doses possessing anti-inflammatory activity. The data also suggest that the racemate and d-isomer have greater specificity toward anti-arthritic activity and are less ulcerogenic than indomethacin. The d-isomer apparently is the more active component of the racemate in all the systems tested since: (a) the d-isomer has 2 to 3 times the inhibitory potency of the racemate and (b) the I-isomer, at high dosages or high concentrations had considerably less affect. Comparison of potencies relative to inhibition of platelet aggregation and of prostaglandin synthetase, are quite close; therefore, mechanistically, the anti-aggregatory affects of these drugs, or lack thereof, may be related to inhibition of prostaglandin synthetase.     

Inhibition of platelet function by abciximab or high-dose tirofiban in patients with STEMI undergoing primary PCI: a randomised trial

BackgroundIn patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary PCI, few data exist on the magnitude of platelet activation, aggregation and dosing of glycoprotein (GP) IIb/IIIa receptor inhibitors. Methods Sixty STEMI patients were randomised to abciximab, to high-dose tirofiban or to no additional GP IIb/IIIa inhibitor treatment. Platelet activation (P-selectin expression) was measured using flow cytometry and the level of inhibition of platelet aggregation was assessed using the Plateletworks assay. Additionally, the PFA-100 with the collagen/adenosine-diphosphate cartridge (CADP) was used to compare the levels of platelet inhibition. All measurements were performed at baseline (T₀), immediately after (T₁), 30 minutes (T₂), 60 minutes (T₃) and 120 minutes (T₄) after primary PCI. Results The level of platelet activation in both GP IIb/IIIa receptor inhibitor treated groups was significantly lower compared with the control group at all time points after primary PCI (p=0.04). Also the administration of the currently recommended dose of abciximab resulted in significantly lower levels of inhibition of aggregation compared with high-dose tirofiban (p<0.0001). In addition, the CADP closure times were significantly prolonged in both GP IIb/IIIa inhibitor treated groups compared with the control group at time points T1 (p=0.006) and T4 (p<0.0001). Conclusion The administration of high-dose tirofiban resulted in a significantly higher inhibition of platelet aggregation compared with the currently recommended dose of abciximab. Large clinical trials are needed to assess whether this laboratory superiority of high-dose tirofiban translates into higher clinical efficacy. (Neth Heart J 2007;15:375-81.)     

Laropiprant Attenuates EP3 and TP Prostanoid Receptor-Mediated Thrombus Formation

The use of the lipid lowering agent niacin is hampered by a frequent flush response which is largely mediated by prostaglandin (PG) D(2). Therefore, concomitant administration of the D-type prostanoid (DP) receptor antagonist laropiprant has been proposed to be a useful approach in preventing niacin-induced flush. However, antagonizing PGD(2), which is a potent inhibitor of platelet aggregation, might pose the risk of atherothrombotic events in cardiovascular disease. In fact, we found that in vitro treatment of platelets with laropiprant prevented the inhibitory effects of PGD(2) on platelet function, i.e. platelet aggregation, Ca(2+) flux, P-selectin expression, activation of glycoprotein IIb/IIIa and thrombus formation. In contrast, laropiprant did not prevent the inhibitory effects of acetylsalicylic acid or niacin on thrombus formation. At higher concentrations, laropiprant by itself attenuated platelet activation induced by thromboxane (TP) and E-type prostanoid (EP)-3 receptor stimulation, as demonstrated in assays of platelet aggregation, Ca(2+) flux, P-selectin expression, and activation of glycoprotein IIb/IIIa. Inhibition of platelet function exerted by EP4 or I-type prostanoid (IP) receptors was not affected by laropiprant. These in vitro data suggest that niacin/laropiprant for the treatment of dyslipidemias might have a beneficial profile with respect to platelet function and thrombotic events in vascular disease.     

Effect of raw versus boiled aqueous extract of garlic and onion on platelet aggregation.

The effects of aqueous extracts of raw and boiled garlic and onions were studied in vitro on the collagen-induced platelet aggregation using rabbit and human platelet-rich plasma. A dose dependant inhibition of rabbit platelet aggregation was observed with garlic. Onion also showed dose-dependent inhibitory effects on the collagen-induced platelet aggregation but this inhibition was of a lesser magnitude compared to garlic when related to dose. The concentration required for 50% inhibition of the platelet aggregation for garlic was calculated to be approximately 6.6 mg ml(-1) plasma, whereas the concentration for onion was 90 mg ml(-1) plasma. Boiled garlic and onion extracts showed a reduced inhibitory effect on platelet aggregation. Garlic but not onion significantly inhibits human platelet aggregation in a dose-dependent fashion. The potency of garlic in inhibiting the collagen-induced platelet aggregation is approximately similar to that of rabbit platelets (8.8 mg ml(-1) produced 50% inhibition of platelet aggregation). The results of this study show that garlic is about 13 times more potent than onion in inhibiting platelet aggregation and suggest that garlic and onion could be more potent inhibitors of blood platelet aggregation if consumed in raw than in cooked or boiled form.     

An antiplatelet peptide, gabonin, from Bitis gabonica snake venom.

Interaction of fibrinogen with its receptors (glycoprotein IIb/IIIa complex) on platelet membranes leads to platelet aggregation. By means of gel filtration, CM-Sephadex C-50, and reverse-phase HPLC, an antiplatelet peptide, gabonin, was purified from the venom of Bitis gabonica. The purified protein migrates as a 21,100-Da polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and as a 11,000-Da peptide in the presence of beta-mercaptoethanol, indicating that gabonin is a disulfide-linked dimer. It is a polypeptide consisting of about 84 amino acid residues, rich in Asp, Pro, and half-cystine. Gabonin dose-dependently inhibited human platelet aggregation stimulated by ADP, collagen, U46619, or thrombin in preparations of platelet-rich plasma and platelet suspension (IC50 = 340-1600 nM). It also blocked platelet aggregation of whole blood. However, it apparently did not affect the initial shape change and only slightly reduced ATP release caused by aggregation agonists. Gabonin did not inhibit the rise of cytosolic calcium in Quin-2-loaded platelets stimulated by thrombin. In addition, gabonin dose-dependently inhibited fibrinogen-induced aggregation of elastase-treated platelets. In conclusion, gabonin inhibits platelet aggregation mainly through the blockade of fibrinogen binding toward fibrinogen receptors of the activated platelets.     

Characterization and platelet inhibitory activity of bitistatin, a potent arginine-glycine-aspartic acid-containing peptide from the venom of the viper Bitis arietans

A platelet aggregation inhibitory protein, bitistatin, was isolated from the venom of the puff adder Bitis arietans. This protein is a single-chain peptide containing 83 amino acids and 7 disulfide bonds. Bitistatin contains the sequence arginine-glycine-aspartic acid and shows considerable homology to two previously described snake venom platelet aggregation inhibitors, trigramin and echistatin. Bitistatin inhibited human and canine platelet aggregation initiated by 10 microM ADP in vitro with IC50 values of 237 +/- 13 and 28 +/- 3 nM, respectively. In order to assess the antithrombotic potential of bitistatin, a canine model of platelet-dependent coronary thrombus formation was utilized. Injection of bitistatin at 10-100 micrograms/kg (0.7-7 nmol/kg, intravenously (i.v.] resulted in dose-dependent inhibition of both platelet aggregation ex vivo and platelet-dependent cyclical flow reductions. The effective dose to inhibit cyclical flow reductions was 30 micrograms/kg, i.v. A higher dose of bitistatin (100 micrograms/kg, i.v.) inhibited cyclical flow reductions for 160 +/- 29 min as well as attenuated ex vivo platelet aggregation. Bitistatin at 100 micrograms/kg, i.v. prolonged the bleeding time 4 x normal at 15 min post-administration but returned to normal at 3 h. Thus, in a canine model of in vivo platelet aggregation, bitistatin is an effective antiplatelet agent to inhibit periodic cyclical flow reductions. Bitistatin also exhibits reversible effects of ex vivo platelet aggregation as well as on bleeding time.     

Conformational study of cyclo(1,5)-Ac-Pen-Arg-Gly-Asp-Cys-NH2 in water by NMR and molecular dynamics.

Cyclo(1,5)-Ac-Pen-Arg-Gly-Asp-Cys-NH2 (3) is a potent inhibitor of platelet aggregation. Nuclear magnetic resonance (NMR) and restrained molecular dynamics were used to study the conformations of 3. Elucidation of RGD conformations in 3 will increase the understanding of interaction between the RGD-sequence with GPIIb/IIIa.     

pH and magnesium alter 45calcium binding to platelets at sites other than glycoproteins I or IIb/IIIa

Calcium is a cofactor of human platelet aggregation. Moreover a direct correlation between the ability of platelets to bind this divalent cation and to aggregate has been demonstrated. Since magnesium can substitute for calcium in supporting aggregation, especially in the presence of low calcium concentrations, and platelet aggregation is inhibited at low pH, the present study was designed to examine the effects of magnesium and low pH on 45calcium binding to human platelets, and to determine whether such effects might be associated with calcium binding to glycoproteins I (GPI) or IIb/IIIa (GPIIb/IIIa), the putative fibrinogen receptor. 45Calcium binding to aspirin-treated platelets that had been depleted of surface-associated calcium by brief exposure to EDTA was evaluated. Magnesium (5-10 mM) or a change in hydrogen ion concentration to decrease the pH from 7.5 to 6.0 was found to inhibit the binding of 45calcium to platelets from healthy donors by 34 +/- 6 and 32 +/- 8% (mean +/- SD, n = 13), respectively. Similar results were obtained with platelets incubated with chymotrypsin to selectively remove GPI or platelets from a patient with the Bernard Soulier Syndrome, congenitally deficient in GPI. In contrast, calcium binding to platelets from two patients with thrombasthenia, lacking GPIIb/IIIa, was reduced 49 +/- 6% and 42 +/- 8% (n = 4) by magnesium and hydrogen ions, respectively. This apparently increased inhibition was attributed to the combined effects of an overall decrease (approximately 50%) in calcium binding to thrombasthenic platelets compared with that in control platelets, and a similar absolute reduction in calcium binding in the presence of magnesium and/or hydrogen ions. No additional inhibition of 45calcium binding was noted in the presence of magnesium and at low pH, indicating that magnesium and hydrogen ions may affect the same platelet membrane binding sites. The data suggest that although modulation of platelet aggregation by magnesium and pH is accompanied by changes in platelet-associated calcium, calcium binding to the three major platelet membrane glycoproteins, GPI, IIb, and IIIa is unaffected.