Antithrombotic effect of crotalin, a platelet membrane glycoprotein Ib antagonist from venom of Crotalus atrox

A potent platelet glycoprotein Ib (GPIb) antagonist, crotalin, with a molecular weight of 30 kD was purified from the snake venom of Crotalus atrox. Crotalin specifically and dose dependently inhibited aggregation of human washed platelets induced by ristocetin with IC50 of 2.4 microg/mL (83 nmol/L). It was also active in inhibiting ristocetin-induced platelet aggregation of platelet-rich plasma (IC50, 6.3 microg/mL). 125I-crotalin bound to human platelets in a saturable and dose-dependent manner with a kd value of 3.2 +/- 0.1 x 10(-7) mol/L, and its binding site was estimated to be 58,632 +/- 3, 152 per platelet. Its binding was specifically inhibited by a monoclonal antibody, AP1 raised against platelet GPIb. Crotalin significantly prolonged the latent period in triggering platelet aggregation caused by low concentration of thrombin (0.03 U/mL), and inhibited thromboxane B2 formation of platelets stimulated either by ristocetin plus von Willebrand factor (vWF), or by thrombin (0.03 U/mL). When crotalin was intravenously (IV) administered to mice at 100 to 300 microg/kg, a dose-dependent prolongation on tail bleeding time was observed. The duration of crotalin in prolonging tail bleeding time lasted for 4 hours as crotalin was given at 300 microg/kg. In addition, its in vivo antithrombotic activity was evidenced by prolonging the latent period in inducing platelet-rich thrombus formation by irradiating the mesenteric venules of the fluorescein sodium-treated mice. When administered IV at 100 to 300 microg/kg, crotalin dose dependently prolonged the time lapse in inducing platelet-rich thrombus formation. In conclusion, crotalin specifically inhibited vWF-induced platelet agglutination in the presence of ristocetin because crotalin selectively bound to platelet surface receptor-glycoprotein Ib, resulting in the blockade of the interaction of vWF with platelet membrane GPIb. In addition, crotalin is a potent antithrombotic agent because it pronouncedly blocked platelet plug formation in vivo.     

Platelet aggregation induced by a monoclonal antibody to the A1 domain of von Willebrand factor

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of alphaIIb beta3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to alphaIIb beta3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds-1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds-1, DSP was increased from 5.9% +/- 3.5% in the absence of vWF to 32.7% +/- 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to alphaIIb beta3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds-1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to alphaIIbbeta3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab')2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcgamma receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.     

Platelet-vessel wall interactions in thrombosis and restenosis role of von Willebrand factor

As a consequence of vessel wall injury, subendothelial matrix and collagen fibers are exposed to the flowing blood. Circulating platelets adhere to these structures and initiate arrest of blood flow. Subendothelial von Willebrand Factor (vWF) plays an important role in mediating platelet adhesion to the injured site, at least in the arterial circulation, characterized by sufficiently elevated shear forces to allow a critical conformation change in vWF, enabling an interaction between the vWF domain A1 and the vWF receptor on the platelet, the GPIb/IX complex. In vitro, in the absence of shear forces, non-physiological mediators are required to induce vWF binding to GPIb. Analysis of the mechanism according to which ristocetin induces vWF binding to GPIb revealed that 2 dimers of ristocetin simultaneously bind to vWF and GPIb, thus forming a quaternary complex in which repulsive negative charges are neutralized by the positively charged ristocetin. The interaction of vWF with its vascular receptor, i.e. collagen VI, which was isolated from human placenta and the extracellular matrix from lung fibroblasts, showed that vWF binds to collagen VI entirely via its A1 domain, i.e. via the domain that binds to GPIb. Also, vWF binding to intact extracellular matrices occurs to matrix associated collagen VI via the vWF A1 domain. By using a combination of 2 specific monoclonal anti-vWF antibodies, it was possible to induce conformational changes in WF that exposed the binding sequences in the A1 domain for GPIb. Thus, in the absence of shear forces, specific vWF binding to GPIb could be induced in the absence of any further mediators. This increased vWF binding to GPIb was sufficient to induce vWF dependent platelet aggregation, although as a consequence of Fc binding to the platelet Fc receptor, platelet activation also occurred via this pathway. Thus, general conformational changes in vWF suffice to expose the relevant amino acid sequences in the A1 domain that enable binding to GPIb. The collagen binding protein calin, isolated from the saliva of the medicinal leech, not only blocks platelet binding to collagen but also inhibited vWF binding. Thus this protein was able to inhibit both the vWF independent and vWF dependent platelet adhesion to various collagens, but much less the platelet binding to endothelial extracellular matrices, that contain matrix anchored vWF. In vivo anti-thrombotic studies in the hamster showed that the vWF antagonist aurin tricarboxylc acid was a more potent inhibitor of arterial thrombosis than of venous thrombosis, confirming the in vivo role of vWF during thrombus formation. Following vessel wall damage and thrombus formation, the neointima that formed in the hamster carotid artery developed more rapidly than in other models, and its formation partially responded to reported inhibitors of restenosis. The combination of cardiovascular drugs with complementary modes of action, such as G4120 (inhibitor of platelet GPIIb/IIIa and smooth muscle cell alpha(v) beta(3)) and quinapril (potent vascular ACE inhibitor) prevented neointima formation to about 70%, i.e. better than with any treatment separately.     

Synthetic peptide from the V3 loop consensus motif with a potent anti-HIV activity inhibits ristocetin-mediated vWF-GPIb interaction

The V3 loop consensus motif. Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile (HIV-1 IIIB), inhibits an interaction of HIV with CD4-positive lymphocytes. Recently, both proline-rich peptides and peptides containing proline-glycine loops (beta-turns) form a complex with ristocetin dimers. These peptides interact with ristocetin-loaded platelet membrane glycoprotein (GP) Ib and act as inhibitors of von Willebrand factor (vWF)-GPIb interaction by preventing the subsequent formation of ristocetin dimer bridges. The Pro-Gly sequence is also present in the V3 loop consensus motif, Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile (HIV-1 IIIB). In this report, we have evaluated the effect of the HIV-1 IIIB peptide on vWF binding to GPIb. This peptide only inhibited vWF binding to GPIb as well as platelet aggregation in the presence of ristocetin while it had no effect on botrocetin-mediated vWF interaction with platelets. The peptide inhibited a binding of anti-vWF monoclonal antibody (RG-46) to immobilized vWF. Furthermore, ristocetin inhibited the binding of HIV-1 IIIB peptide to immobilized CXC-chemokine receptor-4 (CXCR-4) peptide. These results indicate that ristocetin may prevent HIV infection and would be useful a tool to understand the mechanism of HIV tissue tropism and infection.     

Galactose-specific lectin from Viscum album as a mediator of aggregation and priming of human platelets

Galactose-specific lectin from Viscum album (VAA) was found to induce aggregation of human platelets in a dose- and sugar-dependent manner. Small nonaggregating concentrations of VAA primed the response of platelets to known aggregants (ADP, arachidonic acid, thrombin, ristocetin, and A23187). VAA-induced platelet aggregation was completely reversible by addition of the sugar inhibitor lactose and the platelets from disrupted aggregates maintained the response to other aggregants. The lectin-induced aggregation of washed platelets was more resistant to metabolic inhibitors than thrombin- or arachidonic acid-dependent cell interaction. In contrast to the related galactose-specific lectin from Ricinus communis and the soy bean agglutinin, the lectin did not aggregate liposomes prepared from total platelet lipids, indicating different affinities of aggregation-mediating lectins to platelet glycolipids.     

Effect of type IIB von Willebrand disease mutation Arg(545)Cys on platelet glycoprotein Ib binding--studies with recombinant von Willebrand factor

Type IIB von Willebrand disease (vWD) is characterized by a selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma due to their abnormally enhanced reactivity with platelets. Several missense mutations in the platelet glycoprotein Ib (GPIb) binding domain of vWF were recently characterized that cause type IIB vWD. The effect of type IIB mutation Arg(545)Cys on vWF binding to platelet GPIb was studied using recombinant wild type (rvWFWT) and mutant rvWFR545C expressed in COS-7 cells. In the absence of ristocetin, 50% of rvWFR545C bound spontaneously to platelet GPIb and the binding increased to 70% in the presence of 0.2 mg/ml ristocetin; rvWFWT did not bind significantly under either condition. Botrocetin-induced binding of rvWFR545C was only slightly increased compared to rvWFWT. These data demonstrate that the Arg(545)Cys mutation increases the affinity of vWF for GPIb, resulting in the characteristics gain-of-function type IIB vWD phenotype.     

Fine structural and functional consequences of deglycosylation of the platelet adhesion receptor GPIb-IX (CD 42b)

To investigate the role of the glycosylation of the platelet receptor glycoprotein Ib (GPIb, CD 42b), platelets and purified GPIb were deglycosylated by neuraminidase, O- and N-glycosidases. N-deglycosylation and neuraminic-acid cleavage had little effect on ristocetin and botrocetin-induced platelet agglutination. However, O-deglycosylation reduced the response by approximately 50%, and total deglycosylation (the combination of all three glycosidases) fully abolished the response to ristocetin. Interestingly, binding of von Willebrand Factor (vWF) to purified GPIb in the presence of ristocetin and botrocetin in a standardized microtiter plate assay was not altered by partial or even by total deglycosylation. Electron microscopy indicated that the normally stretched approximately 50 nm long molecule was approximately 32 nm after N-deglycosylation, approximately 20 nm after O-deglycosylation, and reduced in a approximately 15 nm long collapse by total deglycosylation. These results suggest that deglycosylation has major structural impacts on GPIb, strongly impairing platelet-vWF interactions; however, vWF binding to isolated GPIb remains unaffected.     

Enhanced shear-induced platelet aggregation in acute myocardial infarction

BACKGROUND: Experiments under controlled flow conditions indicate that the binding of von Willebrand factor (vWF) to platelet glycoprotein (GP) Ibalpha and integrin alphaIIbbeta3 (GP IIb/IIIa complex) is crucial for aggregation at elevated shear rates. We have tested how the plasma of patients with acute myocardial infarction affects this process. METHODS AND RESULTS: Citrated plasma was obtained from 18 patients with acute myocardial infarction within 6 hours from the onset of symptoms and from 26 control subjects with chest pain syndrome without evidence of ischemia. Aggregation of normal platelets at high shear rates was significantly greater in the presence of patient than control plasma and was inhibited by both anti-GP Ibalpha and anti-alphaIIbbeta3 monoclonal antibodies. The observed values (mean+/-SD) were 47.6+/-17.8% versus 30.1+/-9.9% at 10 800 s-1 (P<0.01) and 32.9+/-14.1% versus 17.5+/-9.5% at 7200 s-1 (P<0.01), respectively, and were positively correlated with plasma vWF antigen levels and ristocetin cofactor activities. In contrast, at the lower shear rate of 1200 s-1, aggregation was similar in the presence of control or patient plasma and was not inhibited by the anti-GP Ibalpha antibody. Both vWF antigen and platelet aggregation decreased 2 weeks after the onset of myocardial infarction. CONCLUSIONS: Shear-induced platelet aggregation is enhanced in plasma in the presence of acute myocardial infarction, apparently as a result of increased vWF concentration. This may contribute to the onset of acute coronary artery thrombosis and early reocclusion after reperfusion treatment.     

Activation of human platelets by the membrane-expressed A1 domain of von Willebrand factor

Platelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into primates. This pathological process could be mediated, at least in part, by aberrant interactions of von Willebrand Factor (vWF) associated with the donor vasculature with host platelets. Unlike human vWF, native porcine vWF (pvWF) interacts with human GPIb independently of shear stress or nonphysiological stimuli, eg, ristocetin. We therefore contrasted the potential of isolated human and porcine vWF-A1-domains to interact with human platelets in vitro. Both human and porcine vWF-A1-domains expressed as glycosyl phosphatidylinositol-linked FLAG fusion proteins on COS-7 cells induced GPIb-dependent aggregation and intracellular Ca++ uptake of platelets, independent of both the remainder of the vWF protein and additional modifying factors. Porcine A1-domains were more potent than human homologues, and in addition ristocetin could boost platelet aggregation only with the human A1-domain. Putative conformational changes in the porcine A1-domain could result in the heightened, ristocetin-independent interactions observed with human platelets and may be of importance for xenograft survival.     

The physical exchange of factor VIII (FVIII) between von Willebrand factor and activated platelets and the effect of the FVIII B-domain on platelet binding

Normal hemostasis proceeds through the assembly of coagulant complexes on a lipid surface derived from activated platelets. The activation complex assembly is governed by multiple factors including the binding constants (Kd) of the coagulant factors for the lipid surface. The formation of the tenase complex requires delivery of factor VIII (FVIII) to the activated lipid surface by von Willebrand factor (vWF). Using electrophoretic quasi-elastic light scattering (ELS), we have examined the interaction of FVIII in the presence and absence of vWF with both resting and activated gel-filtered human platelets. Resting platelets do not bind FVIII. Platelets activated by thrombin, epinephrine, or SFLLRN, but not ADP or collagen, bind unactivated FVIII if vWF is not present. In the absence of vWF, unactivated FVIII binds to activated platelets with a Kd of 10.4 nM. B-domain deleted FVIII binds to activated platelets with a Kd of 5.1 nM. Thrombin -activated FVIII (FVIIIa) binds to activated platelets with a Kd of 1.7 nM. The activation of FVIII while bound to the platelet surface can be monitored as a function of time. In the presence of vWF, binding of unactivated FVIII to activated platelets was inhibited, but not the binding of FVIIIa. Displacement of bound unactivated FVIII from the platelet surface occurs when vWF is added to the FVIII-platelet complex. The binding of FVIII to activated platelets is affected by the B-domain, the state of FVIII activation, and the presence of soluble vWF and proceeds as a multistep process. FVIII binding by activated platelets is not affected by platelet gpIIb/IIIa or by platelet vWF.     

Aggregation efficiency of activated normal or fixed platelets in a simple shear field: effect of shear and fibrinogen occupancy

Shear rate can affect protein adsorption and platelet aggregation by regulating both the collision frequency and the capture efficiency (alpha). These effects were evaluated in well defined shear field in a micro-couette for shear rate G = 10 - 1000 s-1. The rate of protein binding was independent of G, shown for adsorption of albumin to latex beads and PAC1 to activated platelets. The initial aggregation rate for ADP-activated platelets in citrated platelet-rich plasma followed second order kinetics at the initial platelet concentrations between 20,000 and 60,000/microliters. alpha values, which dropped nearly fivefold for a 10-fold increase in G, were approximately proportional to G-1, contrary to a minor drop predicted by the theory that includes protein cross-bridging. Varying ADP concentration did not change alpha of maximally activated platelet subpopulations, suggesting that aggregation between unactivated and activated platelets is negligible. Directly blocking the unoccupied but activated GPIIb-IIIa receptors without affecting pre-bound Fg on "RGD"-activated, fixed platelets (AFP) by GRGDSP or Ro 43-5054 eliminated aggregation, suggesting that cross-bridging of GPIIb-IIIa on adjacent platelets by fibrinogen mediates aggregation. Alpha for AFP remained maximal (approximately 0.24) over 25-75% Fg occupancy, otherwise decreasing rapidly, with a half-maximum occurring at around 2% occupancy, suggesting that very few bound Fg were required to cause significant aggregation.     

Role of chondroitin 4-sulphate as a receptor for polycation induced human platelet aggregation

1. Proteoglycans provide negatively charged sites on the surface of platelets, leukocytes and endothelial cells. Since chondroitin 4-sulphate is the main proteoglycan present on the platelet surface, the role of this molecule in mediating the activation of human platelets by polylysine was studied. 2. Platelets were desensitized with phorbol 12-myristate 13-acetate (PMA, 10 nM) 5 min before the addition of polylysine to platelet-rich plasma (PRP). Changes in the intracellular Ca2+ concentration were measured in fura2-am (2 microM) loaded platelets and protein phosphorylation was assessed by autoradiography of the electrophoretic profile obtained from [32P]-phosphate labelled platelets. The release of dense granule contents was measured in [14C]-5-hydroxytryptamine loaded platelets and the synthesis of thromboxane (TXA2) was assessed by radioimmunoassay. Surface chondroitin 4-sulphate proteoglycan was degraded by incubating platelets with different concentrations of chondroitinase AC (3 min, 37 degrees C). The amount of chondroitin 4-sulphate remaining in the platelets was then quantified after proteolysis and agarose gel electrophoresis. 3. The addition of PMA to PRP before polylysine inhibited the aggregation by 88 +/- 18% (n = 3). Staurosporine (1 microM, 5 min) prevented the PMA-induced inhibition. Chondroitinase AC (4 pu ml-1 to 400 muu ml-1, 3 min) abolished the polylysine-induced aggregation in PRP but caused only a discrete inhibition of ADP-induced aggregation. The concentration of chrondroitin 4-sulphate in PRP (0.96 +/- 0.2 microgram/10(8) platelets, n = 3) and in washed platelets (WP; 0.35 +/- 0.1 microgram/10(8) platelets, n = 3) was significantly reduced following incubation with chondroitinase AC (PRP = 0.63 +/- 0.1 microgram/10(8) platelets and WP = 0.08 +/- 0.06 microgram/10(8) platelets). 4. Washed platelets had a significantly lower concentration of chondroitin 4-sulphate than platelets in PRP. The addition of polylysine to WP induced a rapid increase in light transmission which was not accompanied by TXA2 synthesis or the release of dense granule contents. This effect was not inhibited by sodium nitroprusside (SNP), iloprost, EDTA or the peptide RGDS. This event was accompanied by the discrete phosphorylation of plekstrin and myosin light chain, which were inhibited by staurosporine (10 microM, 10 min). The hydrolysis of platelet surface chondroitin 4-sulphate strongly reduced the polylysine-induced phosphorylation. 5. Our results indicate that polylysine activates platelets through a specific receptor which could be the proteoglycan chondroitin 4-sulphate present on the platelet membrane.     

Human signaling protein 14-3-3zeta interacts with platelet glycoprotein Ib subunits Ibalpha and Ibbeta

The initiation of primary hemostasis is mediated by interaction of the platelet glycoprotein Ib (GPIb) surface receptor and its arterial subendothelial von Willebrand factor (vWF) ligand. The intracellular signaling immediately following GPIb receptor occupancy connecting the adhesive event to platelet activation and aggregation has not been well characterized. The 14-3-3 proteins are a 27- to 30-kD ubiquitous protein family with diverse biologic roles, including functional modulation of several prominent signaling proteins. We used the yeast two-hybrid system and confocal microscopy to characterize the recently described interaction between GPIb and platelet 14-3-3zeta, and provide evidence for the potential signaling role of this protein. Two-hybrid interactions suggest that platelet 14-3-3zeta associates with the cytoplasmic domain of GPIb subunits Ibalpha and Ibbeta in transformed yeast cells. The 14-3-3 interaction with GPIbbeta may be partly mediated through the latter's phosphorylated serine 166 residue as its mutagenesis results in 20% to 40% reduced interaction. There was 51% to 59% reduced interaction between GPIb and three 14-3-3zeta deletion mutants compared with full-length 14-3-3zeta, suggesting that either the N-terminal dimerization or membrane-binding domains or more than one noncontiguous 14-3-3zeta element may be required for optimal GPIb interaction. Confocal studies of platelets and a megakaryocyte cell line provided additional evidence for interaction of 14-3-3zeta with GPIbalpha and GPIbbeta. We also found that, similar to the signaling mediators phosphatidylinositol 3-kinase and Src, platelet cytoskeletal 14-3-3zeta content is increased following vWF and ristocetin stimulation. We suggest that platelet 14-3-3zeta interacts with GPIbalpha and Ibbeta, that this interaction may be partly mediated through phosphoserine recognition, and that 14-3-3zeta cytoskeletal translocation may serve as a GPIb post-receptor occupancy signaling event.     

Apolipoprotein(a) enhances platelet responses to the thrombin receptor-activating peptide SFLLRN

Elevated levels of lipoprotein(a) [Lp(a)] are correlated with an increased risk of atherosclerotic disease. We examined the effect of recombinant apolipoprotein(a) [r-apo(a)] and Lp(a) on responses of washed human platelets, prelabeled in the dense granules with [14C]serotonin and suspended in Tyrode's solution, to ADP and the thrombin receptor-activating peptide SFLLRN. No effect of the 17 kringle (K), 12K, or 6K r-apo(a) derivatives (at concentrations of 0.35 and 0.7 micromol/L) or Lp(a) (up to 0.1 micromol/L) on primary ADP-induced platelet aggregation was observed. In contrast, weak platelet responses stimulated by 7.5 micromol/L SFLLRN were significantly enhanced by the r-apo(a) derivatives; eg, 0.7 micromol/L 17K r-apo(a) increased aggregation from 15+/-4% to 58+/-6%, release of [14C]serotonin from 9+/-3% to 36+/-6%, and formation of thromboxane A2, measured as its stable metabolite thromboxane B2, from 7+/-1 to 29+/-5 ng/10(9) platelets (n=3; P<0.04 to 0.015). Significant enhancement of aggregation and release of granule contents was observed at a concentration of 17K r-apo(a) as low as 0.175 micromol/L. Purified Lp(a) (0.25 to 0.1 micromol/L) also enhanced SFLLRN-induced aggregation and release in a dose-dependent manner. Although plasminogen (0.7 and 1.5 micromol/L) and low density lipoprotein (0.025 to 0.1 micromol/L) both exhibited potentiating effects on SFLLRN-mediated platelet aggregation, the magnitude of the responses was less than that observed with either the r-apo(a) derivatives or Lp(a). The enhanced responses of platelets via the protease-activated receptor- thrombin receptor in the presence of Lp(a) may contribute to the increased risk of thromboembolic complications of atherosclerosis associated with this lipoprotein.     

Modulatory effect of 8-iso-PGE2 on platelets

1. 8-Iso-PGE2 induced either reversible or irreversible aggregation of platelets in human platelet-rich plasma (PRP) or in the suspension of washed platelets (WP). The values of EC50 for irreversible aggregation in PRP and WP were 4 and 2 microM, respectively. 2. In rabbit PRP, 8-iso-PGE2 (0.1-100 microM) itself did not induce or induced only reversible aggregation. 3. 8-Iso-PGE2 (0.1-20 microM) potentiated adenosine diphosphate-(ADP) induced platelet aggregation in both human and rabbit. The same effect also was found for adrenaline-induced platelet aggregation in rabbit. 4. The lower concentrations (0.2-0.5 microM) of 8-iso-PGE2 decreased, and higher concentrations (1-2 microM) increased platelet aggregating factor- (PAF) induced aggregation in human PRP. In rabbit PRP, 8-iso-PGE2 (0.02-200 microM) had only a decreasing effect on PAF-induced aggregation. 5. The results suggest that low concentrations of 8-iso-PGE2 can amplify or weaken platelet aggregation induced by various aggregatory agents.     

Platelet sialic acid and platelet survival after aggregation by ADP

Some investigators have reported recently that platelet surface sialic acid is decreased during ADP-induced aggregation, whereas others have reported an increase. Since removal of sialic acid from the platelet surface shortens platelet survival, we have determined the survival of platelets that have been aggregatad by ADP. We have also measured the amount of sialic acid in the suspending fluid of platelets after ADP-induced aggregation. ADP-induced aggregation did not cause the loss of sialic acid from rabbit platelets (which do not undergo a release reaction in response to ADP) nor from washed human platelets in a medium containing physiologic concentrations of calcium in which granule contents are not released. In a medium without added calcium, ADP caused the release of 14C-serotonin (42.5% +/- 3%) from human platelets, but less than 4% of the sialic-acid-containing material was released. It seems likely that little of the releasable sialic acid of platelets is in the dense granules or the alpha-granules. Thrombin (5 U/ml) released 90.0% +/- 3.4% of the serotonin from human platelets but only 20.6% +/- 7.4% of the total sialic-acid-containing material. Neuraminidase removed 42.3% of the total sialic acid, presumably from the platelet surface. Rabbit platelets that had been aggregated by ADP and deaggregated survived normally when returned to the circulation. This observation also provides evidence that they had not lost membrane sialic acid during aggregation and deaggregation.     

A fifty percent reduction of platelet surface glycoprotein Ib does not affect platelet adhesion under flow conditions

Glycoprotein (GP) Ib is an adhesion receptor on the platelet surface that binds to von Willebrand Factor (vWF). vWF becomes attached to collagens and other adhesive proteins that become exposed when the vessel wall is damaged. Several investigators have shown that during cardiopulmonary bypass (CPB) surgery and also during platelet activation in vitro by thrombin or thrombin receptor activating peptide (TRAP) GPIb disappears from the platelet surface. Such a disappearance is presumed to lead to a decreased adhesive capacity. In the present study, we show that a 65% decrease in platelet surface expression of GPIb, due to stimulation of platelets in Orgaran anticoagulated whole blood with 15 micromol/L TRAP, had no effect on platelet adhesion to both collagen type III and the extracellular matrix (ECM) of human umbilical vein endothelial cells under flow conditions in a single-pass perfusion system. In contrast to adhesion, ristocetin-induced platelet agglutination was highly dependent on the presence of GPIb. Immunoelectron microscopic studies showed that GPIb almost immediately returned to the platelet surface once platelets had attached to collagen. In a subsequent series of experiments, we showed that when less than 50% of GPIb was blocked by an inhibitory monoclonal antibody against GPIb (6D1), platelet adhesion under flow conditions remained unaffected.     

Inhibition of rat platelet aggregation by mycalolide-B, a novel inhibitor of actin polymerization with a different mechanism of action from cytochalasin-D

In vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 microM) but was inhibited by higher concentrations (3 and 10 microM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 microM). Potentiation of ADP-induced aggregation by MB (0.3 microM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 microM). In contrast, cytochalasin-D (CD) at 3 microM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 microg/ml) or ADP (10 microM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 microM) and CD (3 microM) abolished both ADP (10 microM)- and collagen (3 microg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 microM) had no effects on intracellular Ca2+ concentrations in ADP (10 microM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 microM)(was inhibited by MB (0.3-3 microM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 microM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.     

Difference of (Ca2+)i movements in platelets stimulated by thrombin and TRAP: the involvement of alpha(IIb)beta3-mediated TXA2 synthesis

This study investigated the difference of [Ca2+]i movement in platelets in response to thrombin and TRAP. The involvement of alpha(IIb)beta3 in this signaling was also studied. Stimulation of platelets with thrombin at 0.03 U/ml caused platelet aggregation and a two-peak increase in [Ca2+]i. The second peak of [Ca2+]i, but not the first peak was abolished by the inhibition of platelet aggregation with alpha(IIb)beta3 antagonists or by scavenging endogenous ADP with apyrase. A cyclooxygenase inhibitor, aspirin, and a TXA2 receptor antagonist, BM 13505, also abolished the second peak of [Ca2+]i but not the first peak, although these regents did not inhibit aggregation. Under the same assay conditions, measurement of TXB2 demonstrated that alpha(IIb)beta3 antagonists and aspirin almost completely inhibited the production of TXB2. In contrast to thrombin-stimulation, TRAP caused only a single peak of [Ca2+]i even in the presence of platelet aggregation, and a high level of [Ca2+]i increase was needed for the induction of platelet aggregation. The inhibition of aggregation with alpha(IIb)beta3 antagonists had no effect on [Ca2+]i change and TXB2 production induced by TRAP. Inhibition studies using anti-GPIb antibodies suggested that GPIb may be involved in the thrombin response, but not in the TRAP. Our findings suggest that low dose thrombin causes a different [Ca2+]i response and TXA2 producing signal from TRAP. Endogenous ADP release and fibrinogen binding to alpha(IIb)beta3 are responsible for the synthesis of TXA2 which results in the induction of the second peak of [Ca2+]i in low thrombin- but not TRAP-stimulated platelets.     

The clinical course of asymptomatic mesenteric arterial stenosis

The activation of rabbit platelets by rabbit plasma clots, and the inhibition of clot-associated thrombin by heparin:antithrombin III, recombinant hirudin (rHV2Lys47) and argatroban, a low molecular weight thrombin inhibitor, was studied. Plasma clots caused the aggregation of platelets suspended in a plasma-free medium as assessed by single platelet counting, and by scanning electron microscopy (platelet aggregates present on the clot surface). Platelet aggregation, induced by clot-associated thrombin, was inhibited by argatroban with an IC50) of 14 +/- 3 nM compared to an IC50) of 12 +/- 2 nM when human thrombin in solution titrated to give the same decrease in the platelet count as plasma clots was used. rHV2Lys47 also inhibited aggregation induced by clot-associated thrombin with an IC50 of 1.6 +/- 0.4 nM compared to 1.6 +/- 0.5 nM with thrombin in solution. Heparin was less active against clot-associated thrombin (IC50) = 69 +/- 9 mU/ml) than against thrombin in solution (IC50 = 15 +/- 5 mU/ml). This study shows that plasma clot-bound thrombin activates platelets and that direct-acting thrombin inhibitors such as argatroban and rHV2Lys47 are more effective than heparin:antithrombin III in inhibiting this phenomenon.